In ovo vaccination of genetically modified salmonella vectored vaccines

Abstract

Disclosed are methods for inoculating embryonated avian eggs for conferring protective immune responses against diseases caused by a diversity of bacterial, viral and parasite pathogens. According to certain aspects methods are disclosed that involve administering, in ovo, a safe inoculating dose of a Protective Immunity Enhanced Salmonella Vaccine (PIESV) strain, or derivative thereof, and optionally co-administering a safe inoculating dose of a live self-destructing attenuated adjuvant Salmonella strain (SDAAS), or derivative thereof.

Description

[0001] Attorney Docket No. 10457-621PC0

[0002] in ovo vaccination of genetically modified Salmonella vectored vaccines

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0004] This invention was made with government support under Grant Number(s) 20236701539654, 20206701733237, and 20176701726179, awarded by the U. S. Department of Agriculture's National Institute of Food and Agriculture. The government has certain rights in the invention.

[0005] BACKGROUND

[0006] In the last century poultry meat and eggs became the most consumed sources of protein world-wide. It is estimated that the USA alone produced around 46.2 billion pounds of chicken meat and 109.5 billion eggs in 2022 generating a revenue of $69 billion. Such demand for an affordable, healthy and nutritious source of protein led to development and application of new technologies in animal agriculture that resulted in great improvements in animal health and welfare. In 1956 the average body weight of a broiler at 56 days of age was around two pounds, today a modern broiler can easily reach 6 pounds at 42 days of age with a much better feed conversion. This gain in productivity brought many new challenges to the industry. Birds are now much more susceptible to infection and there is abundant evidence showing that they are a reservoir to many drug-resistant bacteria that can be transmitted to humans through consumption of contaminated meat and eggs. This led regulatory agencies to create and enforce new policies to reduce and ultimately eliminate use of antibiotics as growth promoters in animal agriculture. The CDC has listed Salmonella spp, C. jejuni, E. coli and C. perfringens as important foodborne pathogens transmitted to humans through consumption of contaminated poultry products. In addition, infection of poultry with pathogens such as Eimeria species causing coccidiosis and High Path Avian Influenza (HPAI) viruses cause very significant adverse economic losses.

[0007] Salmonella: Salmonellosis develops different syndromes, including gastroenteritis, enteric fever (typhoid fever), and bacteremia, and as asymptomatic carriage in animals and humans. It is the leading cause of foodborne illness in the U. S.,Attorney Docket No. 10457-621PC0

[0008] with 35% of the hospitalizations and 28% of the deaths. There are approximately 1.03 million cases of non-typhoidal Salmonella each year in the U. S., costing an economic loss of approximately $3.31 billion due to premature mortality, disability, medical and productivity costs, with an annual loss of 16,782 quality-adjusted life years.

[0009] Salmonella has a broad host range and adapts to survive in challenging environments, e.g. up to 16 months in dry feed stored at 25°C. Although a large number of human infections are associated with food animal sources, infections also come from pets, reptiles, fruits, vegetables and other humans. Transmission of Salmonella to humans typically occurs when ingesting foods that are contaminated by animal feces or crosscontaminated from other sources. Poultry and associated products are widely recognized as being among the most important vehicles for human Salmonella infections according to the CDC. With increasing consumption of poultry and poultry products, salmonellosis associated with poultry continues to be a significant public health issue worldwide. The use of improved management practices including biocontainment to preclude access of poultry flocks to insects, rodents and birds and exclusion of animal products from poultry feeds has decreased Salmonella colonization of poultry flocks. The use of vaccines, especially against the Group B and D strains (that were derived from S. Typhimurium and S. Enteritidis) has effectively eliminated transmission of Salmonella in and on eggs to the consumer. However, such vaccines while reducing these serotypes during growth of broilers has led to their selective replacement by Group C strains such as S. Infantis and S. Kentucky. Since Salmonella-vectored vaccines, have the potential to induce protective immunity against Salmonella, we have endeavored in the design of PIESV vectors to enhance induction of immunity against diverse Salmonella serotypes.

[0010] Clostridium perfringens: For many years producers have used antibiotics and ionophores as growth enhancing drugs in animal agriculture. Due to raising concerns regarding transmission of antibiotic-resistant bacteria to humans through the food chain, regulatory agencies implemented policies that limited and / or eliminated use of these drugs in food animal production. Although necessary, this change brought back diseases and pathogens that were once considered eliminated. In the last decade C. perfringens, the causative agent of necrotic enteritis (NE), became the most prevalentAttorney Docket No. 10457-621PC0

[0011] pathogen and the biggest enemy to the broiler industry worldwide, with some cases also being reported in laying hens raised in cage-free systems. In some cases, necrotic enteritis was reported to induce high mortality rates of up to 50%, but the biggest economic losses come from subclinical cases, in which whole operations will have animals underperforming with reduced growth efficiency and lower feed conversion. The high prevalence of this pathogen in broilers results in increased percentages of contaminated carcasses and human outbreaks traced to consumption of chicken have been reported. About 1 million people become infected every year and develop gastroenteritis due to consumption of food items contaminated with C. perfringens. It is estimated that necrotic enteritis cost the poultry industry $2 billion annually worldwide.

[0012] Campylobacter jejuni: C. jejuni is the leading cause of bacterial foodborne gastroenteritis worldwide and is therefore a major public health problem. The CDC recently indicated that C. jejuni is not only among the most common causes of foodborne illnesses in humans with more than 1.5 million cases / year in the USA but also is a leading cause of hospitalizations (over 10,000 annually). Patients infected with C. jejuni often experience watery / bloody diarrhea, abdominal cramps, nausea, and fever. Severe neurological sequelae, bacteremia and other extra-intestinal complications may develop infrequently. The most susceptible individuals are infants, elderly and the immunocompromised. C. jejuni is widespread in food-producing animals, especially in poultry. The majority of human C. jejuni infections are predominantly associated with poor handling and / or consumption of undercooked or raw chicken. The predominant role of poultry in human campylobacteriosis is supported by high prevalence of C. jejuni in both live birds and on carcasses, findings from epidemiological studies, and detection of identical genotypes in both poultry and human infections.

[0013] Escherichia coli: The Avian Pathogenic E. coli (APEC) strains that cause significant disease in poultry are a subset of the Extra Intestinal Pathogenic E. coli (ExPEC) group. In addition to causing disease in poultry, recent studies have demonstrated APEC strains as including those responsible for extra-intestinal infections in humans. Pathogens in the ExPEC group are characterized into specific pathotypes related to the clinical presentation induced in the host and include uropathogenic E. coli (UPEC), neonatal-meningitis E. coli (NMEC) and newborn meningitis (NBM). SeveralAttorney Docket No. 10457-621PC0

[0014] reports have demonstrated that poultry meat can serve as a source of ExPEC, with up to 92.3% of samples testing positive for E. coli and 46.1% of them having virulence factors associated with ExPEC and 15.6% identified as UPEC. In 2010 the CDC released a report to inform the public of the zoonotic risk of ExPECs and their possible transmission through consumption of contaminated chicken meat or mishandling of poultry products. Extensive research has demonstrated that APEC and ExPEC share an array of virulence genes.

[0015] Eimeria: Coccidiosis is an insidious parasitic disease having dire economic consequences for the poultry industry. Seven species of Eimeria parasitize chickens globally (Eimeria acervulina, E. brunetti, E. maxima, E. mitis, E. necatrix, E. praecox, and E. tenella). E. maxima and E. tenella (plus the closely related E. acervulina) are the principal causes of infections in the US. E. maxima is the most problematic species in broiler production because it affects an area of the gut important for nutrient absorption and more importantly is the most critical species for predisposing chickens to necrotic enteritis (NE) caused by C. perfringens. Until recently, almost half of the drugs administered in the poultry industry were to control this disease. The current effort to reduce use of anti-microbial drugs in agriculture has decreased use of coccidiostats. Medication of feed with anticoccidial drugs has thus been replaced by oral administration of virulent or partially attenuated precocious strains of Eimeria as the best means to prevent the more severe consequences of infection. These live vaccines, however, cause mild infections that while inducing immunity have a negative effect on broiler performance. Moreover, use of live Eimeria vaccines leads to seeding of poultry houses with Eimeria oocysts that are capable of causing infection in non-immunized chicks, a problem with current vaccine delivery. Another problem is that these attenuated Eimeria strains can mutate to restore their virulence. The development of an alternative more cost-effective safe efficacious vaccine and a means for its delivery to control the predominant Eimeria species causing disease would thus be very desirable.

[0016] Influenza virus: Influenza virus has been a major cause of pandemics in the last 2 centuries with a high probability to be a causative agent of a future human pandemic. Currently, the high pathogenic H5N1 avian influenza (HPAI) virus is causing a global pandemic in avian species and especially in commercial poultry rearing. Although theAttorney Docket No. 10457-621PC0

[0017] H5N1 virus has been disseminated from Asia globally since 1996 to now result in significant infections of poultry (chickens and turkeys), it's infectivity due to the structure of sialic acid residues on the surface of respiratory epithelial cells which are present in poultry species but absent in most mammals, has provided a modicum of unconcern by our human population. The recent discovery of H5N1 viruses in several mammalian species such as dolphins but more significantly in lactating dairy cows having an adverse effect on milk production and in cats, has awakened human concerns. This concern has been magnified by the occurrence of H5N1 infections reported by CDC in several hundred dairy herds in 16 states in addition to infections of poultry in 48 states resulting in infections in a small number of dairy farm workers and poultry farm workers. This is analogous to the rare but significant infections by the avian H5N1 virus of humans working in close association with infected birds or flocks originally in Hong Kong. Sadly, we started 2025 with the first lethal human case caused by the avian H5N1 virus in the US.

[0018] SUMMARY

[0019] This disclosure describes a safe method for in ovo vaccination of 18-day-old chicken embryos with genetically modified self-destructing Salmonella vectored vaccines to induce protection of chickens (and other poultry types) against diseases caused by a diversity of bacterial, viral and parasite pathogens such as Clostridium perfringens-induced necrotic enteritis, Campylobacter jejuni colonization, influenza virus infection, and Eimeria species induced coccidiosis as non-limiting examples. The features of the self-destructing Protective Immunity Enhanced Salmonella Vaccine (sdPIESV) vectors also serve to enhance induction of innate immunity to increase potential of newly hatched birds to develop immunity to encountered pathogens.

[0020] In one embodiment, provided is a biologically-contained self-destructing genetically-modified bacterial adjuvant and / or vectored vaccine for use for inoculating and vaccinating avian embryonated eggs. The biologically-contained self-destructing genetically-modified bacterial adjuvant and / or vectored vaccine stimulates induction ofAttorney Docket No. 10457-621PC0

[0021] innate and / or acquired immune responses in an avian species. In one example, the bacterial adjuvant and / or vectored vaccine is derived from an antibiotic-sensitive invasive bacterial species, including but not limited to, Salmonella enterica.

[0022] According to a specific embodiment, provided is a biologically-contained self-destructing antibiotic-sensitive genetically-modified bacterial adjuvant derived from Salmonella enterica that possesses either Aa / r, AdadB, AasdA, or Aalr-3 APdadB:: TT araC ParaBAD dadB (or ΔPdadB:: TT rhaRS PrhaBAD dadB), APasdA:: TT araC ParaBAD asdA (or APasdA:: TT rhaRS PrhaBAD asdA) mutations.

[0023] A further embodiment pertains to a biologically-contained, self-destructing, antibiotic-sensitive, genetically-modified bacterial vaccine vector derived from Salmonella enterica that possesses three or more of the following mutations: ΔPmurA:: TT araC ParaBAD murA, ΔasdA, ΔwaaL, ΔpagL:: TT rhaRS PrhaBAD waaL, ΔwbaP, ΔpagL:: TT araC ParaBAD wbaP, Δ(wza-wcaM), ΔrelA, ΔrecF, ΔsifA, ΔendA, ΔsseL, ΔtlpA, ΔrhaDABSR, ΔaraBAD::, ΔPstcA:: PmurA stcA, ΔPsafA:: PmurA safA, ΔompA, ΔPfur:: TT araC ParaBAD fur, ΔmntR, Δ(hin-fljBA), ΔfliC180, and / or Δ(traM-traX):: araC ParaBAD lacI TT.

[0024] Embodiments of biologically-contained self-destructing antibiotic-sensitive genetically-modified bacterial vaccine vectors derived from Salmonella enterica are able to induce cross-protective immunity against Gram negative enteric bacterial pathogens. In a specific example, disclosed is a biologically-contained self-destructing antibioticsensitive genetically-modified bacterial vaccine vector derived from Salmonella enterica that protects against Clostridium perfringens-'mduced necrotic enteritis. In another specific example, disclosed is a biologically-contained self-destructing antibiotic-sensitive genetically-modified bacterial vaccine vector derived from Salmonella enterica that protects against infection and colonization by Campylobacter jejuni. In a further specific example, disclosed is biologically-contained self-destructing antibiotic-sensitive genetically-modified bacterial vaccine vector embodiments derived from Salmonella enterica that protects against infection and colonization by Eimeria species causing coccidiosis. In another example, disclosed is a biologically-contained self-destructing antibiotic-sensitive genetically-modified bacterial vaccine vector derived from Salmonella enterica that protects against infection by and transmission of influenza virus.Attorney Docket No. 10457-621PC0

[0025] BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1. Chromosome map of S. Typhimurium indicating locations of mutated and / or regulated genes.

[0027] Figure 2. pG8R110, pG8R111 and pG8R114 plasmid vectors and pYA4545 DNA vaccine vector used in PIESV strains

[0028] Figure 3. The pG8R389 vector that encodes protective antigens to be synthesized and secreted by the PIESV strain and also serving as a DNA vaccine encoding protective antigens to be synthesized and modified by the vaccinated animal host

[0029] Figure 4. Comparison of hatchability after SDAAS administration in ovo. Hatchability of eggs inoculated at 18 days of incubation with (A) 1x107CFU of Family A strains χ9052, χ12554, χ12700 and χ12793 or (B) 1x104CFU of Family B strains χ12499, χ12548, χ12612 and χ12626. The data presented is a compilation of four independently repeated experiments. Each group has a total of 160 embryonated eggs. Data were analyzed using one-way ANOVA test in Prism - GraphPad 10.0 and no significant difference was found at P <0.05.

[0030] Figure 5. Protection induced by Family A strains against S. Typhimurium. Chicks were derived from eggs inoculated at 18 days of incubation with 1 x107CFU of Family A strains χ9052, χ12554, χ12700 and χ12793. At day-of-hatch, birds were challenged with 1x103CFU of S. Typhimurium strain χ12598 by oral administration. Bacterial titers were assessed at different times. The data presented is for 42 days post-challenge. Data were analyzed using Kruskal-Wallis test in Prism - GraphPad 10.0 and is presented with ± standard error of the mean. * significant difference at P < 0.05.

[0031] Figure 6. Protection induced by Family B strains against S. Typhimurium. Chicks were derived from eggs inoculated at 18 days of incubation with 1x104CFU of Family B strains χ12499, χ12548, χ12612 and χ12626. At day-of-hatch, birds were challengedAttorney Docket No. 10457-621PC0

[0032] with 1x103CFU of S. Typhimurium strain χ12598 by oral administration. Bacterial titers were assessed at different times. The data presented is for 42 days post-challenge. Seven chicks per group were euthanized in each time-point. Data were analyzed using Kruskal-Wallis test in Prism - GraphPad 10.0 and is presented with ± standard error of the mean. * significant difference at P < 0.05.

[0033] Figure 7. Determining intestinal colonization after challenge with S. Typhimurium at different times after hatch. Chicks derived from eggs inoculated with 1 x108CFU of Family A strains χ9052 and χ12793 and 1 x105CFU of Family B strains χ12548 and χ12626 were challenged at day-of-hatch with 1x103CFU of S. Typhimurium strain χ3761 by oral inoculation. Animals were euthanized at days 28 and 42 after challenge for titer determination in the bursa of Fabricius and in cecal contents. Seven chicks per group were euthanized at each time. Data were analyzed using Kruskal-Wallis test in Prism -GraphPad 10.0 and are presented with ± standard error of the mean. * significant difference at P< 0.05.

[0034] Figure 8. Lesions in ceca following challenge with wild-type S. Enteritidis. Accumulation of necrotic material in ceca of chicks derived from eggs not inoculated with SDAAS strains and challenged with S. Enteritidis. Similar lesions were observed following S. Heidelberg and S. Typhimurium challenge of chicks hatched from non-SDAAS inoculated eggs.

[0035] Figure 9. Comparative abilities of Family 2 strain x12548 and Family A strain χ12554 with wild-type UK-1 χ3761 to interact with and activate the pattern response regulators NOD1, NOD2, TLR4, TLR5, TLR8 and TLR9 as revealed by using HEK-Blue cells displaying each of the pattern receptors.

[0036] Figure 10. (Left). Map of pG8R256 encoding 5 C. perfringens antigens blaSS plcC-gst-netB-pelBSS fba-dsbASS cbh-ompASS cpeC derived from regulated delayed lysis vector pG8R114; (Right). Production of each antigen in strain χ12341 as measured byAttorney Docket No. 10457-621PC0

[0037] western blot using antibodies to the specific antigens. -, no induction; +, 1 mM IPTG induction.

[0038] Figure 11. Cecal content titers of C. jejuni in chickens challenged 17 days after oral vaccination at day-of-hatch and at 11 days of age. 18 days later they were given a cocktail with 5 different C. jejuni strains and necropsied 14 to 17 days after challenge.

[0039] Figure 12. Map of plasmid pG8R410. Plasmid encoding 5 C. jejuni antigens blaSS cjaA-dsbASS stiK-flaA-ompASS pppA-chpA derived from vector pG8R114.

[0040] Figure 13. Hatchability of eggs inoculated at 18 days of incubation with different SDAAS and PIESV strains. Hatchability of eggs inoculated at 18 days of incubation with Family A strain χ12703 (1X108CFU), Family B strain χ12668 (1X105CFU) and PIESV strains χ12836(pG8R114), χ12836(pG8R410), χ12848(pG8R114) and χ12848(pG8R256) (all 1X106CFU). Each group had 60 embryonated eggs. Data were analyzed using one-way ANOVA test in Prism - GraphPad 10.0 and no significant difference were found at P < 0.05.

[0041] Figure 14. Diagram of plasmid pG8R472 (derived from pG8R389) encoding E. tenella S07 antigen for synthesis and Type 2 secretion and Imp1 and Ama1 encoded on the DNA vaccine portion for synthesis after plasmid transfer to cells in vaccinated chickens.

[0042] Figure 15. Diagram of plasmid pG8R473 (derived from pG8R389) encoding E. maxima S07 antigen for synthesis and Type 2 secretion and Imp1 and Ama1 encoded on the DNA vaccine portion for synthesis after plasmid transfer to cells in vaccinated chickens.

[0043] Figure 16. Diagram of plasmid pG8R419 (derived from pG8R388 that is the same as pG8R389 but lacking the improved bla SS) encoding Ptrc controlled synthesis and secretion of OmpA (using the ompA SS) with fusion inserts encoding synthesis of influenza virus M2e (swine) and M2e (avian) fused to a T3SS for secretion of a fusion of NP to conserved HA T-cell epitopes and a M2e (human) sequence.Attorney Docket No. 10457-621PC0

[0044] Figure 17. The production of M2e and NP in whole cell lysates and in OMPs fractions of Salmonella vectors. Arrows indicate either NP or M2e. Coomassie blue top and western blot bottom. 1. χ12731 (pG8R388), 2. χ12731 (pG8R419), 3. χ12735(pG8R388), 4. χ12735(pG8R419). without IPTG, +, with IPTG.

[0045] Figure 18. Diagram of plasmid pG8R457 (derived from pG8R419) with all the sequences in pG8R419 but with insertion of influenza HA and NA sequences from the mouse adapted H1N1 WSN influenza virus under the control of PCMV for synthesis and glycosylation in cells in vaccinated chickens.

[0046] Figure 19. Diagram of plasmid pG8R-Flu1 that can be derived from pG8R419 with all the sequences in pG8R419 but with insertion of selected influenza HA and NA sequences such as from HPAI H5N1 stains to be under the control of PCMV for synthesis and glycosylation in cells in vaccinated chickens.

[0047] DETAILED DESCRIPTION

[0048] Definitions

[0049] The term “adjuvant” as used here refers to an agent that induces in an inoculated animal host a heightened means to withstand infection and to elicit an improved level of acquired immunity in the host when exposed to an antigen, vaccine or pathogen or part thereof. Adjuvants can also enhance display of natural barriers to infection by pathogens and diminish the ability of pathogens to infect, colonize or cause disease.

[0050] The term “administering” or “administration” of an agent (e.g. self-destructing genetically-modified bacterial adjuvant and / or vectored vaccine) as used herein means providing the agent to a subject using any of the various methods or delivery systems for administering agents or pharmaceutical compositions known to those skilled in the art. Agents described herein may be administered by oral, intradermal, intravenous, intramuscular, intraocular, intranasal, intrapulmonary, intraperitoneal, epidermal, transdermal, subcutaneous, mucosal, or transcutaneous administration. In ovo administration is a specific administration approach defined below.

[0051] The term “administering in ovo" or “in ovo administration,” as used herein, unless otherwise indicated, means administering an agent composition to a bird egg containingAttorney Docket No. 10457-621PC0

[0052] a live, developing embryo by any means of penetrating the shell of the egg and introducing the adjuvant or vaccine composition. Such means of administration include, but are not limited to, in ovo injection of the adjuvant or vaccine composition.

[0053] In certain embodiments, the compositions are administered in the final quarter of egg incubation of the avian subject. Where the subject is a chicken, the final quarter to administer the composition of this invention in ovo would be during the period from day 15 through day 20 of fertile egg incubation, and in particular embodiments, the composition can be administered on day 18 or day 19 of incubation. When the subject is a turkey, the final quarter for administration would be during the period from day 21 through day 28 of incubation and in particular embodiments, the compositions can be administered on day 24 or day 25 of incubation. In other embodiments wherein the subject is a goose, the final quarter of administration would be during the period from day 23 through day 31 of incubation and in particular embodiments, the compositions can be administered on day 28 or day 29 of incubation. In further embodiments wherein the subject is a duck, the final quarter of administration would be during the period from day 21 through day 28 of incubation and in particular embodiments, the compositions can be administered on day 25 or day 26 of incubation.

[0054] For other avian species, the final quarter of incubation and thus the optimal range of days for in ovo administration of a composition of this invention can be determined according to methods well known in the art. For example, a muscovy duck has an incubation period in the range of 33-35 days, a ringneck pheasant has an incubation period of 23-24 days, a Japanese quail has an incubation period of 17-18 days, a bobwhite quail has an incubation period of 23 days, a chuckar partridge has an incubation period of 22-23 days, a guinea has an incubation period of 26-28 days and a peafowl has an incubation period of 28 days. The incubation period is affected by the temperature of incubation. This is further varied by the different body temperatures of species and breeds.

[0055] The term “agent” as used herein refers to either adjuvant and / or vaccine vector.Attorney Docket No. 10457-621PC0

[0056] The term “attenuated” when referring to a PIESV vector strain or live self-destructing attenuated adjuvant Salmonella (SDAAS) strain refers to a strain that comprises one or more attenuating mutations.

[0057] The term “attenuated derivative” refers to a derivative that comprises one or more attenuating mutations.

[0058] The term “attenuating mutation” refers to a mutation that reduces infectivity, virulence, toxicity, induction of disease symptoms, and / or impairment of a subject upon administration of a PIESV strain and / or a live SDAAS strain. Examples of attenuating mutations include those mutations that facilitate lysis in vivo (e.g. impairing synthesis of essential constituents of peptidoglycan layer), reduce or impair synthesis of LPS or other cell-surface components, and one or more mutations that provide auxotrophy (e.g. dependence on an amino acid, purine, pyrimidine, or vitamin for growth).

[0059] The terms “avian” and “avian subjects” or “bird” and “bird subjects” as used herein, are intended to include males and females of any avian or bird species, and in particular are intended to encompass poultry which are commercially raised for eggs, meat or as pets. Accordingly, the terms “avian” and “avian subject” or “bird” and “bird subject” encompass chickens, turkeys, ducks, geese, quail, pheasant, parakeets, parrots, cockatoos, pigeons, cockatiels, ostriches, emus and the like. In particular embodiments, the subject is a chicken or a turkey. Commercial poultry includes broilers and layers, which are raised for meat and egg production, respectively. The avian to be inoculated can be an in ovo, live fetus or embryo or may be a hatched bird, including newly-hatched (i.e., about the first one, two or three days after hatch), adolescent, and adult birds.

[0060] In particular embodiments, the bird is about six-, five-, four-, three-, two- or one-week of age or less. In other representative embodiments, the avian subject is a naive subject, i.e., has not previously been exposed to the antigen against which immunity is desired.

[0061] The term “biological containment” refers to a PIESV or SDAAS strain that undergoes regulated delayed lysis in vivo such that the strain cannot persist in vivo or survive if shed into the environment.Attorney Docket No. 10457-621PC0

[0062] The term “biologically contained plasmid” refers to a plasmid that lacks genetic information to enable its conjugational transfer to another bacterial cell.

[0063] The term “co-administration” or “co-administering” as used herein refers to the administration of an active agent before, concurrently, or after the administration of another active agent such that the biological effects of either agent overlap.

[0064] The terms “comprise,” “have,” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes,” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and also covers other unlisted steps.

[0065] The term “consisting essentially of” when used in conjunction with agent containing compositions described herein refers to a composition comprising an agent and a pharmaceutical carrier and / or an adjuvant without any other immune response enhancing components.

[0066] The term “derivative” in reference to derivatives of a live self-destructing attenuated adjuvant Salmonella strain refers to descendant cells of a live self-destructing attenuated adjuvant Salmonella strain.

[0067] The term “descendant(s)” refers to cells resulting from cell division of a Salmonella cell.

[0068] A “DNA vaccine vector” encodes antigens to be synthesized in a vaccinated animal host after delivery by a PIESV vector strain.

[0069] The term “effective inoculating dose,” as used herein, unless otherwise indicated, means a dose of a PIESV containing composition and / or SDAAS containing composition that is sufficient to induce an innate immune response in the treated birds that is greater than the innate immunity of non-inoculated birds. In the case of birds treated in ovo, an “effective inoculating dose” indicates a dose or doses sufficient for the PIESV strain and / or SDAAS strain to induce an innate immune response in the hatched birds that have been treated in ovo that is greater than the inherent innate immunity of birds that were not inoculated in ovo. An effective inoculating dose in any particular context can be routinelyAttorney Docket No. 10457-621PC0

[0070] determined using methods known in the art. A dose may comprise a single dose or multiple doses administered over a period of time. Generally, an effective inoculating dose comprises one or more doses of the PIESV composition and / or SDAAS composition with sufficient numbers of CFUs so as to achieve the desired level of protection of newly hatched chicks from colonization by and disease from exposure during the first days of life to various pathogens. The individual dose(s) is / are administered in ovo, usually between 17.5 and 19.2 days of chicken egg incubation but will differ if used for other avian species with differing durations for hatching of embryos.

[0071] As used herein, the term “immunogen” refers to an antigen that is recognized as unwanted, undesired, and / or foreign in a subject.

[0072] The term “Innate Immunity” is used here to refer to the natural defenses displayed by an animal host species exposed to a foreign antigen or pathogen and includes natural defense barriers, non-specific phagocytic cells and elicitation of cytokines and chemokines that recruit other cells in the immune system to display natural barriers to infection and / or commence the development of acquired immunity with display of mucosal and systemic antibody and mucosal and internal cellular immunities.

[0073] The term “live self-destructing attenuated adjuvant Salmonella strain” or “SDAAS” refers to a Salmonella strain that possesses one or more mutations that facilitate lysis in vivo (e.g. impairing synthesis of essential constituents of peptidoglycan layer or LPS of the organism), one or more mutations that provide auxotrophy (e.g. dependence on an amino acid or in which synthesis of amino acid is dependent on a sugar); one or more mutations to alter synthesis of flagellar components, one or more mutations to enhance recruitment of innate immunity (e.g. mutations that enhance induction or activation of TLR4, TLR5, TLR8, TLR9, NOD1 and / or NOD2), one or more mutations enhancing DNA degradation in Salmonella cells), and / or one or more mutations that suppress, evade, modulate, eliminate or diminish means of decreasing induction of effective immunogenicity. SDAAS strains described herein are useful to enhance innate immunity when administered at a safe inoculating dose.

[0074] The disclosed self-destructing attenuated adjuvant Salmonella strain, or a derivative thereof, may be prepared and marketed in the form of a suspension or in aAttorney Docket No. 10457-621PC0

[0075] lyophilized form and additionally contains a pharmaceutically acceptable carrier or diluent customary for such compositions. Carriers include stabilizers, preservatives and buffers. Suitable stabilizers are, for example SPGA, carbohydrates (such as sorbitol, mannitol, starch, sucrose, dextran, glutamate or glucose), proteins (such as dried milk, serum albumin or casein) or degradation products thereof. Suitable buffers are for example alkali metal phosphates. Suitable preservatives are thimerosal, Merthiolate and gentamicin. Diluents include water, aqueous buffer (such as buffered saline) and polyols (such as glycerol).

[0076] The term “mutation” refers to a genetic modification of DNA sequences encoding for gene regulation such as promoters (P), protein or RNAgene products and translation and transcription termination (TT) signals. Mutations can be caused by deletions (A) and base pair substitutions and by insertions (:: or Q) into DNA sequences in plasmids or the chromosome. Such mutations cause phenotypic changes in a gene that are independent of the means of gene inactivation. However, to distinguish one mutation from another, “allele: numbers are added after the designated promoter or structural gene in giving the genotype of a particular strain or plasmid. However, for example, the phenotypes of strains with the Aasc 27, asdA33 and asdA34 mutant alleles are all identical in that all strains require diaminopimelic acid to enable peptidoglycan synthesis and growth.

[0077] The term “pathogen” as used herein refers to a bacteria, virus, fungus or parasite that is capable of infecting and / or causing adverse symptoms in an avian subject. Examples of specific pathogens include, but are not limited to, Salmonella spp, Escherichia coli strains, Clostridium spp, Campylobacter spp, Eimeria spp and to influenza virus (e.g. avian influenza virus).

[0078] All microbes including pathogens possess damage-associated molecular patterns (DAMPs) and pathogen associated molecular patterns (PAMPs) (more generally referred to as microbe associated molecular patterns (MAMPs)) that are recognized by pattern recognition receptors on the surface of or internally in host cells to recruit innate immune responses with production of cytokines and chemokines.

[0079] The term “pharmaceutically acceptable carrier” as used herein refers to one or more formulation materials suitable for accomplishing or enhancing the successfulAttorney Docket No. 10457-621PC0

[0080] delivery of the pharmaceutical composition of the agent disclosed herein. As used herein, the term “carrier” refers to a pharmaceutically acceptable solid or liquid filler, diluent or encapsulating material. A water-containing liquid carrier can contain pharmaceutically acceptable additives such as acidifying agents, alkalizing agents, antimicrobial preservatives, antioxidants, buffering agents, chelating agents, complexing agents, solubilizing agents, humectants, solvents, suspending and / or viscosity-increasing agents, tonicity agents, wetting agents or other biocompatible materials. A tabulation of ingredients listed by the above categories, may be found in the U. S. Pharmacopeia National Formulary, 1857-1859, (1990). Examples of liquid carriers include, but are not limited to, water, saline, dextrose, glycerol, ethanol and mixtures thereof.

[0081] The term “Protective Immunity Enhanced Salmonella Vaccine” or “(PIESV)” refers to a vector strain that has been engineered to synthesize and deliver an immunogen. PIESVs described herein are useful as a vaccine when delivered at a safe inoculating dose, in particular when delivered by in ovo administration.

[0082] The term “regulated delayed attenuation” refers to a construction in which the expression of a gene conferring a virulence attribute is regulated by a sugar-dependent process such that the virulence gene is expressed in the presence of a sugar such as but not limited to arabinose or rhamnose supplied during cultivation of the strain and ceases to be expressed in vivo since the sugar is absent to result is the gradual display of attenuation as a consequence of cell division of the PIESV or SDAAS strain in vivo.

[0083] The term “regulated delayed antigen synthesis” refers to a construction in which the expression of a gene specifying synthesis of an antigen is regulated by a sugardependent process controlling synthesis of a repressor protein that governs expression of the antigen-encoding gene such that the repressor gene is expressed in the presence of a sugar supplied during cultivation of the strain and ceases to be expressed in vivo since the sugar is absent to result is the gradual increase in the synthesis of the antigen specified by the antigen-encoding gene as a consequence of cell division of the PIESV strain in vivo.

[0084] The term “regulated delayed lysis” refers to a construction in which the expression of one or more genes specifying synthesis of peptidoglycan precursors such as but notAttorney Docket No. 10457-621PC0

[0085] limited to diaminopimelic acid, D-alanine and muramic acid are regulated by a sugardependent process such that the genes are expressed in the presence of a sugar such as but not limited to arabinose or rhamnose supplied during cultivation of the strain and cease to be expressed in vivo since the sugar is absent to result in lysis as a consequence of cell division of the PIESV or SDAAS strain in vivo. The genes conferring the regulated delayed lysis phenotype may be either chromosomal and / or plasmid encoded.

[0086] The term “regulated delayed lysis plasmid” refers to a construction in which the expression of one or more genes specifying synthesis of peptidoglycan precursors such as but not limited to diaminopimelic acid, D-alanine and muramic acid that are regulated by a sugar-dependent process are located on a plasmid or DNA vaccine vector encoding synthesis of one or more protective antigens.

[0087] The adjuvant and / or vaccine vector according to the invention may be prepared and marketed in the form of a suspension or in a lyophilized form and additionally contains a pharmaceutically acceptable carrier or diluent customary for such compositions. Carriers include stabilizers, preservatives and buffers. Suitable stabilizers are, for example SPGA, carbohydrates (such as sorbitol, mannitol, starch, sucrose, dextran, glutamate or glucose), proteins (such as dried milk, serum albumin or casein) or degradation products thereof. Suitable buffers are for example alkali metal phosphates. Suitable preservatives are thimerosal, Merthiolate and gentamicin. Diluents include water, aqueous buffer (such as buffered saline) and polyols (such as glycerol).

[0088] The term a “safe inoculating dose” comprises an effective inoculating dose that does not reduce hatchability below that level of hatchability observed for non in ovo inoculated embryos and does not impair the health of newly hatched offspring from such in ovo inoculated embryos.

[0089] As used herein, the term “vaccine” refers to an immunogen or a composition comprising an immunogen that elicits an endogenous immune response in a subject (e.g., a human or animal) The endogenous immune response may result in, for example, the switching of a Th1 biased immune response to a Th2 biased immune response, the activation or enhancement of T effector cell responses and / or the reduction of T regulatory cell response, the activation of antigen-specific naive lymphocytes that may then give riseAttorney Docket No. 10457-621PC0

[0090] to antibody-secreting B cells or antigen-specific effector and memory T cells or both, and / or the direct activation of antibody-secreting B cells. Typically, a vaccine provides for protective immunity against a pathogen.

[0091] Overview

[0092] Protective Immunity Enhanced Salmonella Vaccine (PIESV) vectors: We have continually improved our genetically modified Salmonella vaccine vector strains by initially developing a variety of means to attenuate them and to stably maintain plasmid vectors encoding protective antigens in the absence of antibiotic-resistance markers. We then improved the efficacy of these balanced-lethal vector-host constructs by devising regulated delayed attenuation strategies and regulated delayed synthesis of protective antigens that much improved the ability of the vaccines to colonize internal lymphoid tissues after mucosal administration. Lastly, we developed a regulated delayed lysis technology to provide biological containment and an inability of vaccine cells to survive outside of a controlled laboratory growth environment. We now term these vaccine vector strains as self-destructive Protective Immunity Enhanced Salmonella Vaccine (sdPIESV) vectors. Other attributes to further enhance safety and efficacy are described later in this disclosure.

[0093] Innate immunity activators induce early protection. The innate immune system possesses a multitude of germline encoded pattern recognition receptors (PRRs), e.g., toll-like receptors, nucleotide-binding oligomerization domain [NOD]-like receptors and R IG-I-I ike receptors, each recognizing different patterns that are associated with bacterial, viral and fungal infections. Activation of these receptors start a pre-programmed cascade of events that result in rapid activation of the innate immune system. Modulation of these immune responses has been extensively explored as alternatives to prevent and treat many infectious diseases and cancer. It has been shown that pulmonary administration of a phospholipid-conjugated TLR7 ligand protected mice from infection with Bacillus anthracis, Venezuelan equine encephalitis virus and H1N1 influenza virus. It has been reported that administration of CpG oligodeoxynucleotides protected normal and SIV-infected macaques from experimental Leishmania infection. Also, day-of-hatch chicks derived from eggs injected at 18 days of incubation with CpG dinucleotide were protectedAttorney Docket No. 10457-621PC0

[0094] when challenged at day-of-hatch with lethal APEC doses and were partially protected against infection by Avian Infectious Laryngotracheitis Virus. TLR-9 binding to OpG oligonucleotide drives expression of Type-1 interferons through MyD88 signaling, recruiting different immune cells, including macrophages, neutrophils / heterophils and dendritic cells. These results show that administration of PPR agonists can induce an early protective innate immune response against bacterial and viral pathogens using agents that interact with and activate components of the innate immune system.

[0095] Design of Self-Destructing Attenuated Adjuvant Salmonella (SDAAS) strains.

[0096] In previous studies with genetically modified Salmonella vectored vaccines designed to protect against various bacterial, viral and parasite pathogens, we routinely observed that empty vector control constructs not delivering protective antigens gave low-level protective immunity after pathogen challenge that was significantly higher than observed for the buffered saline inoculated controls but less than observed for the constructs delivering protective antigens. We inferred that this was due to an adjuvant effect likely stimulating innate immunity that would enhance the ability of an animal to sometimes mount an adequate level of acquired immunity following challenge to survive. These results were observed in studies with both mice and chickens. We therefore designed Self-Destructing Attenuated Adjuvant Salmonella (SDAAS) strains with augmented abilities to interact with pattern receptors to stimulate innate immunity and tested their safety for inoculation by multiple mucosal and parenteral routes. We also demonstrated that their administration could significantly increase immune responses to subunit antigens (such as ovalbumin) and live attenuated vaccines (such as BCG). Most importantly, we demonstrated the safety and efficacy of many SDAAS strains for in ovo inoculation of 18-day-old chick embryos to stimulate innate immune responses.

[0097] IROMPs as antigens to induce cross-protective immunity against enteric pathogens. Numerous outer membrane proteins (OMPs) made by Gram negative bacteria share antigenic determinants as do the IROMPs-that are required for iron acquisition, an essential important function for pathogen success within an infected animal. We have developed strategies for Salmonella vaccines to display wild-type surface antigenic determinants in vitro and during the initial phase of infection through mucosal surfaces in the immunized host to constitutively synthesize IROMPs afterAttorney Docket No. 10457-621PC0

[0098] reaching internal organs. Immune responses to IROMPs are known to be effective in preventing septicemic infection with enteropathogens, especially E. coli causing colisepticemia in chickens and turkeys. Also, antibodies induced to IROMPs from one Gram negative bacterial species, especially in the Enterobacteriaceae, can recognize IROMPs synthesized by other enteric species and serotypes. The fur gene encodes a repressor that, in the presence of free iron, represses all genes encoding IROMPs. When iron concentrations become low, as is the case in animal host tissues beyond the intestinal wall barrier, the Fur ceases to be synthesized at a high level and one observes increased expression of genes encoding IROMPs and other Fur-regulated genes needed to sequester iron away from the infected animal host, fur mutants are attenuated when fed orally, giving a two to three log higher LDsowhen administered either to mice or day-of-hatch chicks. To achieve a high constitutive synthesis of all components for iron acquisition in vivo including IROMPs, we deleted the furgene promoter (PfUr) and replaced it with a tightly regulated araC ParaBAD activator-promoter so that fur gene expression is solely dependent on the presence of arabinose and is blind to iron concentration. Growth of S. Typhimurium vaccines with a APfur”TT araC ParaBAD fur deletion-insertion mutation in media with a low level of arabinose results in very good colonization of lymphoid tissues, total attenuation at doses of 109CFU and very high levels of induced protective immunity.

[0099] In ovo immunization to produce early protection of newly-hatched-chicks against pathogens. In 1982 Sharma and Burmester showed that in ovo vaccination of 18-day-old chicken embryos was an efficient method to protect day-of-hatch chicks against Marek’s disease and produced better results when chicks derived from immunized eggs were challenged at 3 days of age with a virulent Marek’s Disease Virus. In 1993, the development of modern equipment capable of immunizing thousands of eggs per hour allowed low-cost commercial use of this strategy. Today it is estimated that 20 billion embryonated eggs are immunized every year in the US. Since 1982, many researchers have used this technique to deliver amino acids, antibiotics and more recently, probiotics and components capable of stimulating the immune system to chicken embryos. Despite extensive research conducted by different groups, no one was able to devise a strategy to safely deliver attenuated Salmonella strains or other attenuatedAttorney Docket No. 10457-621PC0

[0100] bacterial pathogens such as those derived from Listeria monocytogenes or APEC strains in ovo.

[0101] The PIESV and SDAAS platforms for safe prevention of infectious diseases.

[0102] Our PIESV strains have been shown to deliver protective antigens to induce immunity to a diversity of bacterial, viral and parasite pathogens. These innovative vaccines were designed to effectively colonize internal lymphoid tissues after mucosal administration almost as efficiently as the wild-type virulent Salmonella parent. We achieved this in several ways. Our vaccines synthesize LPS O-antigen during in vitro growth but cease to synthesize LPS in vivo. Thus, vaccine cells become increasingly sensitive to complement-mediated cytotoxicity and phagocytosis after 8 to 10 cell divisions in the absence of continued LPS synthesis. Each cell division in vivo varies from 10 to 30 hours. Another means of regulated delayed attenuation results in lysis of vaccine cells by inability to synthesize the essential peptidoglycan precursor muramic acid whose synthesis is dependent on supply of arabinose during in vitro growth. In vivo, arabinose in an unphosphorylated form is absent and vaccine cells lyse after 8 to 12 cell divisions. Protective antigen delivery by regulated delayed lysis has given superior immune responses compared to delivery without programmed lysis in five different studies. Since synthesis of protective antigens in Salmonella vectored vaccines is a metabolic load that decreases colonizing ability, we developed a regulated delayed synthesis of recombinant antigens by plasmid-encoded codon-optimized sequences. This has increased the induced levels of protective immunity to multiple pathogens. The type of immune response induced can also be altered by inclusion of appropriate deletion mutations. The As / ZA mutation eliminates a means of immunosuppression and enables Salmonella to escape the Sa / mone / / a-containing vesicle (SCV) so that vaccine strains with the regulated delayed lysis attribute can lyse in the cytosol. Synthesized protective antigens are then delivered to the proteasome for Class I presentation to elicit CD8-dependent immune responses. We also conducted and reported studies on the survival of PIESVs in water (with and without chlorination), under desiccation, in sewage, in feces, in whole blood, in sera with and without complement, and in monocyte-derived macrophages. They are also safe when administered to immunodeficient SCID mice. The biocontainment attribute of regulated delayed lysis strains in chicken feces, body fluids, and cells enable the strainAttorney Docket No. 10457-621PC0

[0103] to be safely used in field settings. The SPAAS strains share many similarities with PIESVs but are designed with enhanced attributes to rapidly stimulate activation of the innate immune system. Cell lysis releases peptidoglycan components, DNA, RNA, ATP, flagellin and other PAMPs / DAMPs, which activate the innate immune system through interaction with PRRs.

[0104] Provided below in Table 1 is a list of mutation genotypes and associated phenotypes useful in embodiments described herein.

[0105] Table 1. Mutations and associated phenotypes in S. Typhimurium SDAAS and PIESV strains3It is noted that the genes can be inactivated or deleted in multiple ways to confer the same phenotypic traits. Also, though certain allele numbers are indicated elsewhere herein for certain mutations, reference to a certain allele is not limiting and the mutations can be executed with other alleles.

[0106] Genotype Phenotype

[0107] AaroA encodes the first enzyme in the pathway to synthesize aromatic amino acids and derived vitamins

[0108] AasdA deletes gene for aspartate semialdehyde dehydrogenase essential for synthesis of diaminopimelic acid (DAP) necessary for peptidoglycan synthesis

[0109] APasdA:: TT araC ParaBAD asdA makes synthesis of AsdA dependent on presence of arabinose

[0110] APasdA:: TT rhaRS PrhaBAD asdA makes synthesis of AsdA dependent on presence of rhamnose

[0111] Aa / rand AdadB deletes the genes for two alanine racemases essential for synthesis of D-alanine necessary for peptidoglycan synthesis APdadB:: TT araC ParaBAD dadB makes synthesis of DadB dependent on presence of arabinose

[0112] APdadB:: TT rhaRS PrhaBAD dadB makes synthesis of DadB dependent on presence of rhamnose

[0113] AglmS eliminate gene encoding enzyme to synthesize glucosamine-6-PAttorney Docket No. 10457-621PC0

[0114] APgims:: TT ylR PxyiAB glmS makes synthesis of glucosamine-6-P dependent of presence of xylose

[0115] AnagK eliminates gene for N-acetyl-glucosamine kinase APmurA:: TT araC ParaBAD murA makes synthesis of MurA, the first enzyme in the synthesis of muramic acid, dependent on arabinose in growth medium and ceases synthesis in vivo due to absence of arabinose

[0116] APfUr:: TT araC ParaBAD fur makes synthesis of the Fur repressor protein dependent on arabinose in growth medium that ceases in vivo to result in high-level synthesis of all iron regulated proteins to result in attenuation

[0117] AmntR eliminates gene for repressor MntR that regulates MntR- and some Fur-regulated genes for manganese and iron acquisition, respectively

[0118] / \cya encodes enzyme for adenylate cyclase

[0119] crp encodes adenylate cyclase catabolite repressor protein cysG non-essential gene for cysteine biosynthesis

[0120] Aara / BAD- T deletion of genes to eliminate arabinose catabolism with TT inserted to prevent transcription of downstream genes araCBADWO- T Deletion of all genes in the ara operon

[0121] ArhaBADSR deletion of genes to eliminate rhamnose catabolism ApagP:: PipPIpxE mutation causes regulated delayed in vivo synthesis of the codon- optimized IpxE gene from Francisella tularensis to cause synthesis of the non-toxic adjuvant form of LPS lipid A (MPLA) ApagL and AlpxR eliminates two means by which Salmonella alters LPS components in vivo to decrease recruitment of innate immunity by interaction with TLR4

[0122] eptA prevents addition of ethanolamine to lipid A

[0123] arnT prevents addition of 4-amino-4-deoxy-L-arabinose (L-Ara4N) groups to lipid AAttorney Docket No. 10457-621PC0

[0124] A / / / C deletes gene specifying synthesis of the phase I flagellin FliC AfljB deletes gene specifying synthesis of the phase II flagellin FljB AfliC180 specifies a truncated FliC protein containing TLR5 recognition domain and CD4 epitope

[0125] A(hin-fljBA) locks in expression of gene for phase I FliC flagellin and precludes synthesis of phase II FljB flagellin

[0126] A(agfG-agfC) deletes two operons specifying thin aggregative fimbriae (curli) and an activator for synthesis and export of cellulose and other exopolysaccharides

[0127] AP saf5" PmurA SafA causes constitutive synthesis of Saf fimbriae that facilitate spleen colonization

[0128] APstc:: PmurA stcA causes constitutive synthesis of Stc fimbriae that facilitate spleen colonization

[0129] AompA specifies synthesis of a very prevalent outer membrane protein ApabA & ApabB Encode two enzyme subunits of the enzyme synthesizing p- amino benzoic acid (173, 174)

[0130] Apmi eliminates phosphomannose isomerase that precludes synthesis of GDP-mannose that is needed for LPS O-antigen synthesis AwaaL & ApagL:: TT araC ParaBAD waaL (or ApagL:: TT rhaRS PrhaBAD waaL) make synthesis of the WaaL enzyme that couples O-antigen to the LPS core synthesis dependent on presence of arabinose (or rhamnose)

[0131] wbaP encodes enzyme that couples LPS core to LPS O-antigen A(wza-wcaM) eliminates 20 genes encoding enzymes needed for synthesis of colanic acid, LPS capsular antigen and other polysaccharides to facilitate lysis, enhance immunogenicity and inhibit biofilm formation

[0132] ArelA uncouples growth regulation from a dependence on protein synthesis

[0133] ArelA::araC PBAD / ac / TT and A(traM-traX) araC ParaBAD lad makes synthesis of Lacl that represses gene expression controlled by Ptrc dependent onAttorney Docket No. 10457-621PC0

[0134] presence of arabinose with either inactivation of relA gene or deletion of genes encoding conjugational plasmid transfer in Salmonella virulence plasmid

[0135] ArecA eliminates genetic recombination and responsible for SOS repair ArecF reduces inter- and intra-plasmidic recombination

[0136] AendA deletes gene encoding endonuclease I to prevent degradation of released DNA vaccine

[0137] AsifA enables Salmonella to escape from the SOV to enter the cytosol AsseL eliminates a gene that enables Salmonella to induce pyroptosis AtlpA eliminates a gene that enables Salmonella to induce pyroptosis (traM-traX) eliminates tra genes on Salmonella virulence plasmid to prevent conjugation

[0138] gyrA inactivates gene for DNA gyrase and confers resistance to nalidixic acid

[0139] aA = deletion; TT = transcription terminator; P = promoter

[0140] A list of suicide vectors to introduce each mutation or mutational construction is presented in Table 2.

[0141] Table 2. Suicide vectors for constructing the mutations in Table 1

[0142] Genotype Suicide Vector Marke r A. Deletion and deletion-insertion mutations to facilitate regulated delayed lysis in vivo

[0143] APmurA25:: TT araC ParaB / o murA pYA4686 Cm AasdA33 pYA3736 Cm APasdA55:: TT araC ParaBAD asdA pG8R71 Cm APasdA88:: TT rhaRS PrhaBADi asdA pG8R354 Cm Aalr-3 pYA3667 Cm AdadB4 pYA3668 Cm APdadB66”TT araC ParaBAD c / ac / fi pG8R73 CmAttorney Docket No. 10457-621PC0

[0144] APdadB22”TT rhaRS PrhaBADi dadB pG8R352 Cm AglmSI pG8R509 Cm APgimsi48:: TT xy / R PxyiAB glmS pG8R510 Cm AnagKI pG8R508 Cm A(wza-wcaM)-8 pYA4368 Cm ArelA1123 pYA3679 Cm B. Mutations enabling regulation of genes that might be present on plasmid vectors in conjunction with strains undergoing regulated delayed lysis in vivo ArelA 197::araC ParaBAD / ac / TT pYA4064 Cm AasdA27: TT araC ParaBAD c2 pYA4138 Cm A(traM-traX)-36'.:araC ParaBAD lad pG8R329 Cm TT

[0145] A( traM-traX)-41 waraC ParaBAD lad pG8R397 Cm TT

[0146] C. Mutations conferring attenuation of virulence

[0147] AaroA21419 pYA3600 Cm Acya-27 / 28 pMEG080 Tet Acrp-27 / 28 pMEG084 Tet

[0148] D. Mutations conferring regulated delayed attenuation and over production of iron and manganese-regulated proteins to confer cross-protective immunity

[0149] APfur33:: TT araC ParaBAD fur pYA3722 Cm AmntR28 pYA3975 Cm E. Mutations altering synthesis of LPS components

[0150] Apmi-2426 pYA3546 Tet ApagP8 pYA4288 Cm ApagP81'.'. P\PPlpxE pYA4295 CmAttorney Docket No. 10457-621PC0

[0151] ApagL7 pYA4284 Cm AlpxR9 pYA4287 Cm AarnT6 pYA4286 Cm AeptA4 pYA4283 Cm AwaaL46 pYA4900 Cm AwbaP45 pYA4899 Cm ApagL64:: TT rhaRS PrhaBADi waaL 1 pYA5377 Cm ApagL38: TT rhaRS PrhaBADi waaL2 pG8R296 Cm F. Mutations blocking catabolism of sugars

[0152] AaraBAD65 T pYA4811 Cm ArhaBADSR515 pG8R272 Cm AaraCBAD100:: T pG8R392 Cm G. Mutations altering synthesis of flagellar components

[0153] AfliC180 pYA3729 Cm AfliC2426 pYA3702 Cm AfljB217 pYA3548 Tet A(hin-fljBA)-209 pG8R306 Cm H. Mutations altering synthesis of fimbrial components

[0154] A(agfG-agfC)-999 pYA4941 Cm A Pstc53:: PmurA stcA53 pYA5053 Cm A Psafss:: PmurA safA55 pYA5055 Cm I. Mutation eliminating or altering outer membrane proteins

[0155] AompA11 pYA4757 Tet J. Mutation enhancing effective immunogenicity and enabling escape from SCV

[0156] AsifA26 pYA3716 Cm K. Mutations decreasing / delaying onset of pyroptosisAttorney Docket No. 10457-621PC0

[0157] AsseL116 pYA4621 Cm AtlpA181 pYA4620 Cm L. Mutations altering recombination and / or leading to and preventing degradation of DNA within Salmonella cells

[0158] ArecA62 pYA4680 Cm ArecF126 pYA3886 Cm AendA2311 pYA3652 Cm

[0159] aA = deletion; TT = transcription terminator; P = promoter

[0160] A list of SDAAS strains with genotypes as used in the experimental studies listed in following sections is presented in Table 3 with a list of PIESV strains used listed in Table 4.

[0161] Table 3. List of SDAAS strains constructed and used for studies

[0162]

[0163] and derivations (most rapidly lysing in vivo)

[0164] %9052 Aalr-3 AdadB4 AasdA33 (parent from which other Family A strains are derived) %12554 Aalr-3 AdadB4 AasdA33 AfliC180 A(hin-fljBA)-219 ApagP81:: P\ppIpxE AlpxR9 A

[0165]

[0166] pagL7 %12700 Aalr-3 AdadB4 AasdA33 AfliC180 A(hin-fljBA)-219 ApagP81 P\PPlpxEAIpxR9 ApagL7 AeptA4 AarnT6 AsifA26

[0167] 12703 Aalr-3 AdadB4 AasdA33 AfliC180 A(hin-fljBA)-219 ApagP81 P\PPlpxEAIpxR9 ApagL7 AeptA4 AarnT6 AsifA26 ArecA62

[0168] %12793 Aalr-3 AdadB4 AasdA33 AfliC180 A(hin-fljBA)-219 ApagP81: PPPlpxEAIpxR9 ApagL7 AeptA4 AarnT6 AsifA26 AwbaP45

[0169] %12898 Aalr-3 AdadB4 AasdA33 AglmSI AfliC180 A(hin-fljBA)-219 ApagP81'.: P\PPIpxE AlpxR9 ApagL7 AeptA4 AarnT6 AsifA26 AwbaP45

[0170] / 12899 Aalr-3 AdadB4 AasdA33 AglmSI AfliC180 A(hin-fljBA)-219 ApagP81'. PPPIpxE AlpxR9 ApagL7 AeptA4 AarnT6 AsifA26 AwbaP45 ArecA62Attorney Docket No. 10457-621PC0

[0171]

[0172] B strain and derivations (with regulated delayed lysis attribute) %12499 Aa / r-3 APdadB66" TT araC ParaBAD dadB APasdA55" TT araC ParaBAD asdA (parent from which other Family B strains are derived)

[0173] %12548 Aa / r-3 APdadB66:: TT araC ParaBAD dadB APaSdA55:: TT araC ParaBAD asdA AfliCI 80 A(hin-fljBA)-219 ApagP81 P\pp IpxE ApagL7 AlpxR9

[0174] / 12612 APasdA5s:: TT araC ParaBAD asoM Aa / r-3 APdadB66:: TT araC ParaBAD dadB AfliCI 80 A(hin-fljBA)-219 ApagP81'. Ppp IpxE ApagL7 AlpxR9 AarnT6 AeptA4 AsifA26

[0175] / 12626 APasdA5s:: TT araC ParaBAD asdA Aalr-3 APdadB66:: TT araC ParaBAD dadB AfliCI 80 A(hin-fljBA)-219 ApagP81 Ppp IpxE ApagL7 AlpxR9 AarnT6 AeptA4 AsifA26 AwbaP45 / 12668 APasdA5s:: TT araC ParaBAD asdA Aalr-3 APdadB66:: TT araC ParaBAD dadB AfliC180 ApagP81 P\pp IpxE ApagL7 AlpxR9 A(hin-fljBA)-219 AarnT6 AeptA4 AsifA26 ArecA62 / 12721: APasdA88" TT rhaRS PrhaBADi asdA Aalr-3 APdadB66:: TT araC ParaBAD dadB AfliC180 ApagP81: P\pp IpxE ApagL7 AlpxR9 A(hin-fljBA)-219 AarnT6 AeptA4 AsifA26 AwbaP45 ArhaDABSR515

[0176] 12879 Aa / r-3 Acac / B4APasdA88:: TT rhaRS PrhaBADi asdA AfliC180 A(hin-fljBA)-219 ApagP81" P\pp IpxE AlpxR9 ApagL7 AeptA4 AarnT6 AsifA26 AwbaP45

[0177] / 12883 APasdA8s:: TT rhaRS PrhaBADi asdA Aalr-3 APdadB66:: TT araC PBAD dadB AfliCI 80 A(hin fljBA)-219 ApagP81:: PIPPIpxE ApagL7 AlpxR9 AarnT6 AeptA4 AsifA26 AwbaP45 %12889 APasdA8s:: TT rhaRS PrhaBADi asdA Aalr-3 APdadB66:: TT araC PBAD dadB APgimsi48:: TT ylR PxyiAB glmS AfliCI 80 A(hin fljBA)-219 ApagP81-.-. P\pp IpxE ApagL7 AlpxR9 AarnT6 AeptA4 AsifA26 AwbaP45

[0178] / 12893 APasdA8s:: TT rhaRS PrhaBADi asdA Aalr-3 APdadB66" TT araC PBAD dadB APgimsi48:: TT ylR PxyiAB glmS AnagKI AfliCI 80 A(hin fljBA)-219 ApagP81 P\pp IpxE ApagL7 AlpxR9 AarnT6 AeptA4 AsifA26 AwbaP45

[0179] / 12897 APasdA88:: TT rhaRS PrhaBADi asdA Aalr-3 APdadB66:: TT araC PBAD dadB APgimsi48:: TT xylR PxyiAB glmS AnagKI AfliCI 80 A(hin fljBA)-219 ApagP81 P\pp IpxE ApagL7 AlpxR9 AarnT6 AeptA4 AsifA26 AwbaP45 ArecA62

[0180] Table 4. List of PIESV strains constructed and used for studies

[0181] / 12341 APmurA25:: TT araC PBAD murA AasdA27:: TT araC PBAD C2 A(wza-wcaM)-8 Apmi-2426 ArelAI 97\:araC PBAD / ac / TT ArecFI 26 AsifA26 AwaaL46 ApagL64\\Vf rhaRS PrhaBAD

[0182]

[0183] waaL1Attorney Docket No. 10457-621PC0

[0184] %12702 APmurA25:: TT araC ParaBAD murA asdA33 waaL46 pagL38 T rhaRS PrhaBAD2 waaL2 (wza-wcaM)-8 ArelA 1123 ArecFI 2 AsifA26 AaraBAD65:: TT ArhaDABSR515 ApagP87:: Pipp IpxE AlpxR9 (pSTUK206 A(traM-traX)-41'.:araC ParaBAD / ac / TT) / 12731 APmurA25:: TT araC ParaBAD murA AasdA33 AwaaL46 ApagL38 fTT rhaRS PrhaBAD2 waaL2 A(wza-wcaM)-8 ArelA 1123 ArecFI 26 AsifA26 AendA2113 AsseL 116 A tlpA181 ArhaDABSR515 AaraBAD65:: (pSTUK206 A( raM-traX)-41 -.araC ParaBAD / ac / TT) %12735 PmurA25:: TT araC ParaBAD murA AasdA33 Apmi-2426 AwaaL46 ApagL38:fTT rhaRS PrhaBAD2 waaL2 A(wza-wcaM)-8 ArelA1123 ArecFI 26 AsifA26 AendA2113 AsseL116 AtlpA181 AompA11 ArhaDABSR515 AaraBAD65'.: Tf (pSTUK206 A(traM-traX)-41 araC ParaBAD / ac / TT)

[0185] %12831 APmurA25:: TT araC ParaBAD murA AasdA33 AwaaL46 ApagL38 fT rhaRS PrhaBAD2 waaL2 A(wza-wcaM)-8 ArelA 1123 ArecF12 AsifA26 AaraBAD65:: T ArhaDABSR515 ApagP81 P\pp IpxE AlpxR9 (pSTUK206 A(traM-traX)-41.araC ParaBAD / ac / TT)

[0186] AP safA557PmurA SafA55 AP stc53" PmurA StcA53

[0187] %12836 APmurA25:: TT araC ParaBAD murA AasdA33 A(yvza-wcaM)-8 ArelA1123 ArecFI 26 AsifA26 AwbaP45 ApagL14-.-. Tl' araC ParaBAD wbaPAlpxR9ApagP8AcysG123-.-.pgl-(gne-plgG)-12 AfliC180::cj1433gly21-29 ompA132 cj1433 (2xN-gly) (pSTUK206 A(traM-traX)-41: araC ParaBAD / ac / TT)

[0188] %12848 APmurA25:: TT araC ParaBAD murA AasdA33 AwaaL46 ApagL38:-. TT rhaRS PrhaBAD2 waaL2 A(wza-wcaM)-8 ArelA 1123 ArecFI 26 AsifA26 APfurss:: TT araC ParaBAD fur AmntR28 AaraBAD65-.\Vf ArhaDABSR515 (pSTUK206 A(traM-traX)-41-.:araC ParaBAD

[0189]

[0190] / ac / TT) / 12909 APmurA25:: TT araC ParaBAD murA AasdA33 AwaaL46 ApagL38:: TT rhaRS PrhaBAD2 waal_2 A(wza-wcaM)-8 ArelA1123 ArecFI 26 AsifA26 APtur33:: TT araC ParaBAD fur AmntR28 AaraBAD65-.-. T ArhaDABSR515 APstcA53:: PmurA s / c-453 APsafAss:: PmurA safA55 (pSTUK206 A(traM-traX)-41..araC ParaBAD / ac / TT)

[0191] / 12918 APmurA25:: TT araC ParaBAD murA AasdA33 AwaaL46 ApagL38-.fT rhaRS PrhaBAD2 waaL2 A(wza-wcaM)-8 ArelA1123 ArecFI 26 AsifA26 APtur33:: TT araC ParaBAD fur AmntR28 AaraBAD65 T ArhaDABSR515 A(hin-fljBA)-219 AfliC180 (pSTUK206 A(traM-traXy41::araC ParaBAD / ac / TT)

[0192] / 12963 APmurA25:: TT araC ParaBAD murA AasdA33 AwaaL46 ApagL38" TT rhaRS PrhaBAD2 waaL2 A(wza-wcaM)-8 ArelA 1123 ArecFI 26 AsifA26 AendA2113 AsseL 116 A Up A 181 ArhaDABSR515 AaraBAD65'.'. Tf APstcA53:: PmurA stcA53 APsafAss:: PmurA safA55 (pSTUK206 A(traM-traX)-41-. araC ParaBAD / ac / TT)

[0193] %12964 APmurA25" TT araC ParaBAD murA AasdA33 AwaaL46 ApagL38 T rhaRS PrhaBAD2 waaL2 A( wza-wcaM) - 8 ArelA 1123 ArecFI 26 AsifA26 A endA2113 AsseL 116 A tip A 181Attorney Docket No. 10457-621PC0

[0194] ArhaDABSR515 AaraBAD65 TT APstcA53:: PmurA Sta453 APsafAss:: PmurA safA55 AompA11 (pSTUK206 (traM-traX)-41 -. araC ParaBAD / ac / TT)

[0195] The mutations present in SDAAS and PIESV strains are distributed throughout the Salmonella chromosome (Figure 1), which diminishes the chance loss or safety features for biological containment or for attenuation or for synthesis and delivery of protective antigens by gene transfer from some other bacterial strain.

[0196] Regulated delayed lysis plasmid vectors and DNA vaccine vectors used to encode protective antigens. Vectors with arabinose-regulated expression of the asdA and murA genes encoding unique essential components of the cell wall peptidoglycan layer are required to establish the balanced-lethal vector-host PIESV constructs to confer biological containment to preclude long-term persistence in vivo or survival if released into the environment. We modify the ribosome binding (Shine-Dalgarno) sequences and employ GTG start codons for the asdA and murA genes to decrease levels of synthesis of these enzymes since high-level over expression creates a metabolic imbalance that decreases invasiveness and over attenuates the strain. Representative regulated delayed lysis plasmids are diagramed in Figure 2. The pG8R111 plasmid is designed for the synthesis and delivery of protective antigens that would be toxic to the Salmonella cell if secreted. pG8R114 is designed with a much-improved Type 2 secretion system (T2SS) for synthesis and delivery of antigens that are best secreted. Delivery of these antigens to the periplasmic space augments production of Outer Membrane Vesicles (OMVs) that increase levels of induced protective immunity. The pG8R110 plasmid is designed with an improved T3SS that delivers synthesized protective antigens to the cytosol of host cells that enables Class I presentation and stimulation of CD8-dependant cellular immune responses. This plasmid has a p15A ori and a lower copy number than plasmids with the pBR ori. This is important to achieve low level synthesis of protective antigens which is necessary for inducing cellular immunity. Plasmids similar to those depicted in Figure 2 are available with pSC101 ori (very low-level copy number) and pUC ori (very high copy number) that enhances induction of antibody responses. pYA4545 is a DNA vaccine vector with a eukaryotic promoter to enable synthesis and post-translational modification of protective antigens synthesized in host cells of the vaccinated animal. pYA4545 has nuclear targeting sequences that enables efficient access to theAttorney Docket No. 10457-621PC0

[0197] nucleus and sequences derived from animal DNA viruses that replace plasmid DNA sequences susceptible to host nucleases. pYA4545 also has 34 OpG sequences that enable effective interaction with TRL9 in a diversity of different vertebrate species.

[0198] Expression of encoded protective antigens in PIESV constructs is best achieved by optimizing the codons. In constructs designed to induce high-level antibody responses, codons are selected from those most often used in the 20 Salmonella genes most highly expressed during log-phase growth. We sometimes further enhance synthesis by using A-rich codons for the 2ndand 3rdcodons. A further requirement for optimal expression is to modify the sequence so that the GC content is close to the 52% GC content of Salmonella DNA.

[0199] Intervention to control foodborne pathogens in poultry. Since contaminated poultry products are the major source of human foodborne pathogen infection, early protection of chickens against colonization will be an important strategy to reduce the levels of Salmonella, ExPEC, C. perfringens, and C. jejuni in poultry flocks, which will ultimately lead to lower rates of human foodborne illness. In general, the on-farm control strategies used to reduce the incidence of enteric pathogens in poultry that can be transmitted through the food chain can be broadly divided into two approaches: 1) prevention of flock colonization by use of biosecurity-based interventions, and 2) prevention and / or reduction of colonization by non-biosecurity based measures, vaccination, and / or addition of bacteriocins, bacteriophages, feed additives, and competitive exclusion products. Improving biosecurity on farms has a noticeable effect on lowering the overall flock prevalence of enteric pathogens. However, even the most stringent biosecurity measures do not always have consistent predictable effects on controlling these pathogens and their effectiveness is difficult to assess under commercial settings. In addition, stringent biosecurity measures are cost-prohibitive, hard to maintain, and their effectiveness seems to vary with production systems. Thus cage-free free-range production of chickens expose them to birds flying above, insects and rodents all of which are contrary to bio-security measures. The current main approach to control Salmonella infection in poultry flocks is to use vaccines. There are 3 types available, live attenuated, inactivated and subunit; only the former two are licensed for chickens. Currently administered Salmonella vaccines may take from one to several weeks to produce anAttorney Docket No. 10457-621PC0

[0200] immune response that is restricted to a few serotypes but still often allow infection of birds in the first days or weeks of life. Variable efficacy, persistence, reversion to virulence and lack of cross protection are concerns. Killed vaccines are safe, but they must be delivered by costly injection and require adjuvants to increase efficacy. There is a single live attenuated E. co / / vaccine currently available for use in poultry (Poulvac® E. coli). Despite many attempts by different research groups, no vaccine has proved to be highly efficacious for multiple APEC serotypes. This is why broilers are rarely vaccinated against APEC. Currently there is one licensed vaccine against C. perfringens developed by our group (AVERT® NE) and no licensed vaccines against C. jejuni.

[0201] \Ne have previously investigated potential solutions to these problems. As described above, we have developed new and effective strategies to construct safe efficacious PIESV vector strains with compatible plasmid vectors and DNA vaccines to encode for delivery of protective antigens from heterologous bacterial, viral and parasite pathogens. We have also developed safe efficacious SDAAS strains to successfully deliver DAMPS and PAMPs to stimulate induction of innate immunity to augment induction of acquired immunity. All these PIESV and SDAAS strains have been derived from the highly virulent S. Typhimurium strain UK-1.

[0202] Newly developed PIESV and SDAAS strains and methods of administration by in ovo inoculation of 18-day-old chicken embryos.

[0203] Example 1. General Materials and Methods.

[0204] a. Bacterial strains, media and bacterial growth. All of our SDAAS and PIESV strains for testing in day-of-hatch chicks were derived from the highly virulent S. Typhimurium strain UK-1 (%3761). LB broth and agar will be used as complex media for propagation and plating of Salmonella and APEC strains. Family A SDAAS strains are cultured in LB broth and agar enriched with 50 pg / ml of D-alanine, 50 pg / ml of diaminopimelic acid and for some strain 100 pg / ml N-acetyl glucosamine. Family B SDAAS strains require 0.1% L-arabinose and / or 0.1% L-rhamnose and / or 0.1% D-xylose to be added to LB broth. PIESV strains mostly require addition of 0.1% L-arabinose and 0.1% L-rhamnose to LB broth and agar. Salmonella-Shigella agar will be used to enumerate bacteria isolated from chickens. PIESV and SDAAS strains will be grown toAttorney Docket No. 10457-621PC0

[0205] stationary phase to mimic conditions used in vaccine production. Sequenced and well-characterized C. jejuni strain RM221 isolated from chickens will be used in challenge studies. This strain will be cultured microaerophilically (85% N2, 10% CO2, 5% O2) on Mueller-Hinton (MH) medium at 42°C for 24 h. For C. jejuni isolation from chicken feces and organs, charcoal cefoperazone deoxycholate agar (mCCDA) will be used. Cooked meat broth and fluid thioglycolate will be used for C. perfringens growth, Tryptose Sulfite Cycloserine Agar (TSC) with egg yolk will be used for bacterial titer determination in small intestinal samples. C. perfringens will be cultured at 37°C under anaerobic atmosphere. APEC strains will be cultured in growth media and with the same cultural conditions as for Salmonella strains. SDAASs and PIESVs strains used for in ovo inoculation are listed in Tables 3 and 4. Bacterial strains used in the challenge studies are listed in Table 5. Table 5. Bacterial strains used for challenge studies

[0206] / 3212 wild-type S. Heidelberg

[0207] %3550 wild-type S. Enteritidis

[0208] %3761 wild-type S. Typhimurium UK-1

[0209] %7122 wild-type APEC E. coli gyrA 078: K80: H9

[0210] / 12598 S. Typhimurium UK-1 gyrA183 rpoB2212

[0211] %12599 S. Enteritidis gyrA33 rpoB12

[0212] %12600 S. Heidelberg gyrA183 rpoB13

[0213] C. perfringens CP4P Type A, NetB [+]

[0214] C. jejuni RM1221 Chicken isolate HS:53

[0215] All strains and PIESV constructs are extensively evaluated for correctness of all genotypic and phenotypic traits including sugar-dependent LPS O-antigen synthesis, protective antigen synthesis and lysis. Vaccine constructs are also tested for stability of genotypic and phenotypic traits during 50 generations of growth under permissive growth conditions.Attorney Docket No. 10457-621PC0

[0216] b. Eimeria maxima and tenella. Challenge studies are conducted at USDA facilities in Beltsville, MD and employ methods for oocyst preparation and use for challenge studies have been published. Methods of scoring for lesions and determining feed conversion efficiency and weight gain have also been described.

[0217] c. Influenza virus propagation and use for challenge. We will propagate low path H5N1 and, if approved, high path H5N1 at UF using published methods. The virus will be propagated and titrated in Madin-Darby canine kidney (MDCK) cells cultured in RPMI-1640 (Gibco) containing 2 pg / ml acetyl-trypsin (Sigma). The TCIDso titers will be quantitated on MDCK cells in culture. MDCK cells will be plated in a 96-well plate and infected with serial dilutions, in triplicate, of virus in DMEM containing 1 pg of acetyl trypsin, penicillin, and streptomycin / ml and 1 pg of doxycycline / ml for the samples (146). The cells will be stained with naphthol blue black after 72 h at 37°C, and the cytopathic effect scored visually. A Reed and Muench calculation will be used to determine the 50% tissue culture infective dose (TCIDso).

[0218] Evaluation of levels of protective immunity and reduction in virus shedding will be conducted in chickens vaccinated with the best PIESV constructs delivering H5N1 protective antigens at the University of Minnesota that has ABSL-3 facilities certified for conducting challenge studies with high path H5N1 viruses.

[0219] d. Molecular and genetic procedures. Methods for DNA isolation, restriction enzyme digestion, DNA cloning and use of PCR and real-time PCR for construction and verification of bacterial strains and vectors are standard and methods for generating mutant strains are described in previous publications. Methods for measuring LPS and sensitivity to complement, defensins and antibiotics have been previously described.

[0220] e. Animal experimentation. Studies on SDAASs and PIESVs will be conducted in SPF white leghorn and commercial broiler embryonated eggs, chicks and chickens hatched at UF. Fertilized eggs will be incubated and candled every week thereafter (to remove eggs with dead embryos) until day 18 of incubation. At this time eggs will be inoculated with 5 different doses (1x104CFU up to 1x108CFU) of candidate SDAAS and / or PIESV strains suspended in 20 pl of sterile buffered saline with 0.01% gelatin (BSG). Two control groups will also be used, one with embryonated eggs inoculated withAttorney Docket No. 10457-621PC0

[0221] BSG only and another group not inoculated. In initial studies we establish the optimal dose of newly constructed SDAAS and PIESV strains to allow maximum hatchability without diminishing chick quality upon hatching. In studies to evaluate optimal doses and safety, some chicks will be euthanized 6 to 48 h after hatching following AVMA guidelines for animal euthanasia to determine titers of PIESV and SDAAS strains and conduct histological evaluations to monitor chick health. After determination of best dose(s) in initial studies, chicks derived from inoculated eggs will in some cases be orally vaccinated as day-of-hatch chicks with a PIESV construct. In both cases with and without the second vaccination at day-of-hatch, chicks will be challenged with specific pathogens or oocysts as described below. Food and water will be provided ad libitum 30 min after oral vaccination or challenge. SDAAS and PIESV strains (as well as Salmonella and APEC challenge strains) will be grown in LB broth overnight, sedimented by centrifugation at room temperature and suspended in BSG at densities of 8 X 1O10CFU / ml and decimal dilutions will be performed to allow proper strain doses to be administered in 20 pl into either embryonated eggs and / or day-pf-hatch chicks. C. perfringens CP4 will be grown for 24 h in cooked meat broth and chicks at day-of-hatch (SDAASs) or 21 days old (PIESVs) will be inoculated with 100 pl twice with a 12-hour interval and animals will be fed a high protein diet after challenge. Blood will be collected by wing vein puncture for immunological studies.

[0222] SDAAS strains will be evaluated for induction of early protective innate immune responses that diminish tissue (bursa, liver and spleen) and cecal titers of Salmonella spp., cecal titers of the C. jejuni, small intestine titers of C. perfringens, intestinal colonization, internal organ lesions and mortality of chicks challenged with APEC strains. Long-lasting acquired immune responseswill be evaluated after immunization with PIESV strains and determined by I g Y and IgA production (ELISA) and the reduction or elimination of Salmonella spp., C. jejuni, C. perfringens and APEC persistence in intestinal contents. PIESV strains to protect against Eimeria will be evaluated for induction of IgY and IgA production against Eimeria protective antigens at UP and oocyst challenge studies conducted at Beltsville. Chicks vaccinated with PIESV strains conferring protection against influenza virus will be challenged with tissue culture propagated influenza virus. Animals will be housed in poultry isolators from hatch until euthanasia. All experimentalAttorney Docket No. 10457-621PC0

[0223] work will be conducted in compliance with the regulations and policies of the Animal Welfare Act and the Public Health Service Policy on Humane Care and Use of Laboratory Animals and approved by the UF IACUC.

[0224] f. Monitoring immune responses. ELISA for antibody detection. IgY responses in serum and IgA responses in feces will be measured by indirect ELISA as previously described. Briefly, microtiter plates will be coated with purified recombinant C. jejuni, C. perfringens proteins or PEC / Salmonella spp. LPS and whole cell lysates, washed, and blocked with Sea Block blocking buffer (Pierce, Rockford, IL). Test samples (serum or feces) will be added, the plates washed, and anti-antigen antibodies detected with biotinylated goat anti-chicken IgG or IgA antibody (Bethyl Laboratories, Montgomery, TX). Flow Cytometry for identification of different cell

[0225]

[0226] . Immune cell population are analyzed in chicks derived from eggs inoculated with the most promising SDAAS and PIESV strains at 18 days of incubation and from eggs not inoculated. Blood samples will be collected at day-of-hatch and every 7 days thereafter until animals become 28 days old. Blood samples will be collected using 0.3cc insulin syringes with a needle (31 G) attached. Syringes will be loaded with a small amount of 3.8% sodium citrate solution to prevent blood coagulation. After sample collection, an optimal concentration of the desired primary antibody will be added to 100 pL of anti-coagulated whole blood sample and incubated in the dark for 20 min. Two milliliters of 1X red blood cell lysis solution will be added to the mixture and incubated at room temperature in the dark for 10 min. After incubation the solution will be centrifuged at 350xg for 5 min and the supernatant discarded. The pellet will be washed once with 2 ml of cell staining buffer and centrifuged at 350xg for five min. The supernatant will be discarded and the cell pellet resuspended in residual buffer containing the secondary antibody followed by incubation in the dark for 20 min. The cells will be washed again using cell staining buffer as described above. The cell pellet will then be resuspended in 0.5 ml of fixation buffer. Lastly, flow cytometry will be performed, data collected and analyzed for comparison between groups. The data obtained will be used to follow the duration of the innate immune response by looking at the size of different innate immune cells at different times. If the number of different augmented cell populations does not decrease after a month, we will continue monitoring birds.Attorney Docket No. 10457-621PC0

[0227] g. Statistical analysis. All results will be analyzed using the most appropriate statistical test from the SAS program to evaluate the relative significance or lack thereof of results obtained. In specific cases, we will consult with the staff at the Clinical Translational Science Institute at UF. Using ten chickens per treatment group proposed in this project will yield statistical power > 0.80. In our previous studies we have found that n=10 is an adequate number of birds to obtain statistically significant differences between treatment groups. However, the best strains will be evaluated multiple times to substantiate validity of conclusions.

[0228] Example 2. PIESV strains constructed for use in making genetically modified vaccines to protect against bacterial, viral and parasite pathogens.

[0229] We have constructed many PIESV strains and evaluated them for desired abilities to induce protective immune responses either against Salmonella antigens or those encoded by codon-optimized sequences from other pathogens as specified on plasmid vectors or DNA vaccines present in the constructed PIESV strains. Very often, we compare strains with and without a particular mutation (Table 1) to decide on the best choice. Some strains were constructed to maximize induction of protective immunity against a diversity of pathogens. In other cases, the PIESV strain was designed more specifically to enhance induction of protective immunity to a specific pathogen such as C. jejuni or to enhance induction of cross protective immunity to a diversity of enteric pathogens. In still other situations in which the PIESV strain will deliver a plasmid modified to become a DNA vaccine, the PIESV strain possesses specific mutations to enhance the stability of the DNA vaccine and it's targeting to the nucleus of cells within the vaccinated avian host. In such strains used to induce protective immunity to Eimeria or influenza virus, we designed new plasmid vectors combining attributes of pG8R110 and pG8R114 (Figure 2) to encode antigens to be synthesized and delivered by secretion to the vaccinated avian host combined with attributes of the DNA vaccine vector pYA4545 (Figure 2) to encode protective antigens to be synthesized by the vaccinated avian host and subject to post translational modifications. The pG8R389 vector with all of these attributes is diagramed in Figure 3.Attorney Docket No. 10457-621PC0

[0230] Based on these considerations we list the PIESV strains constructed (Table 4) that will be used in the Examples that follow.

[0231] Example 3. SDAAS strains effective in recruiting innate immunity and safe and efficacious for inoculation into 18-day-old chicken embryos.

[0232] The SDAAS strains are divided into two distinct families: 1) Family A strains are defined by the alr-3 dadB4 asdA33 mutations that prevent production of amino acids essential for synthesis and maintenance of the peptidoglycan layer of the bacterial cell wall, resulting in requirement of DAP and D-alanine in growth media and rapid lysis in the absence of these supplements. 2) Family B strains require L-arabinose for growth due to the APdadB66" TT araC ParaBAD dadB and APasdA55:: TT araC ParaBAD asdA deletion-insertion mutations in which the wild-type asdA and dadB promoters are deleted and replaced by the arabinose regulated promoter. In the presence of arabinose, the asdA and dadB genes are expressed and bacterial cells grow normally, but in its absence of arabinose, which is unavailable in vivo, gene expression ceases resulting in delayed lysis in host tissues as the enzymes encoded by the asdA and dadB genes are diluted as a consequence of cell division to levels that no longer sustain sufficient synthesis of DAP and D-alanine for peptidoglycan synthesis. We have constructed and extensively evaluated two SDAAS strains, %12554 and %12548 and their derivatives designed to enhance induction of innate immunity in inoculated animals to provide early and broad protection against colonization by bacterial pathogens. The adjuvant strains mentioned above have been tested for their safety when inoculated in ovo and compared with the wild-type S. Typhimurium strain UK-1 %3761. As shown in Figure 4 SDAAS strains can be safely delivered in ovo without compromising hatchability. We have also evaluated ability of SDAAS strains to induce improved protective immunity against S. Typhimurium when challenged at day-of-hatch or at 28 days of age (Figures 5, 6 and 7). Surprisingly, the birds that were challenged at day-of-hatch had lower S. Typhimurium titers at 42 days of age compared with the animals challenged at 28 days of age (Figure 7). This suggests that administration of SDAAS strains in ovo facilitates induction of acquired immunity to the challenge strain when animals are exposed early in life. This could be an end-result of animals having higher prevalence of phagocytic cells in the intestinal mucosa,Attorney Docket No. 10457-621PC0

[0233] facilitating antigen presentation. It was also observed in different studies that birds derived from eggs not inoculated with SDAAS strains had lesions in the caeca following Salmonella challenge, with accumulation of caseous / necrotic material (Figure 8). These findings were much less prevalent in birds derived from eggs inoculated with either Family A or Family B strains and challenged with Salmonella. In contrast, samples of liver, spleen and bursa of Fabricius collected from SDAAS inoculated embryos and used for histopathology have demonstrated presence of heterophilic infiltration in the bursa of Fabricius and inflammation of the air sacs and lungs, suggesting migration of immune cells to the respiratory system.

[0234] We demonstrated that the Family A 12554 and Family B %12548 SDAAS strains have increased ability to activate different PRRs in vitro using HEK- Blue™ cells (Figure 9). Recently obtained RNA-seq data from spleen, liver and cecal tonsils of animals derived from eggs inoculated with Family A strain x12793 and Family B strain 12626 demonstrated differential expression of genes involved in immune responses, such DDX1, NOX4, NOD1, IRF4, RNF144B and CD99.

[0235] Based on the results above, we designed and constructed the improved Family A strain %12899 and the Family B strain x12897 with additional modifications making them safer and more immunogenic. The strains tested have the ApagP81 P\PPIpxE deletioninsertion mutation that causes Salmonella to synthesize the adjuvant mono-phosphoryl lipid A due to codon-optimized expression of the Francisella tularensis IpxE gene. We recently added the ApagL7, lpxR9, AeptA4, arnT6 and wbaP45 mutations, each further enhancing activation of TLR4 / TRIF pathway. Deletion of the wbaPgene results in synthesis of a truncated form of LPS that may facilitate induction of acquired immunity against different Salmonella serotypes. Inclusion of recA62 renders strains unable to repair DNA damage, leading to increased DNA degradation to enhance activation of TLR9. The sifA26 mutation renders them capable of escaping the SCV to lyse in the cell cytoplasm allowing better activation of intracellular TLR and NOD receptors and also eliminates one means Salmonella uses to suppress induction of immunity by the infected animal host.Attorney Docket No. 10457-621PC0

[0236] The genotypes of these two new Family A %12899 and Family B %12897 SDAAS strains are presented in Table 3. These improved strains have a triple safety feature, in which growth of the Family A strain %12899 is dependent on presence of diaminopimelic acid, D-alanine and N-Acetyl-D-glucosamine (GIcNAc) and growth of the Family B strain X12897 is dependent on the presence of L-arabinose, L-rhamnose and D-xylose.

[0237] During the course of our diverse studies with testing SDAAS strains for interaction with HEK cells expressing different pattern receptors and for secretion of the TLR5 activator FHC180 and in mice for safety and efficacy to enhance immunity to subunit vaccines such as ovalbumin and live attenuated vaccines such as BCG, we have investigated use and benefit of other mutations. These include Arfc-112, AwaaL46, AaraCBADI 00:: TT, ArhaDABRS515, APstc53:: PmurA stcA53, and APsafA55:: PmurA safA55, which have also been included in SDAAS strains in various combinations.

[0238] Example 4. Construction of improved PIESV strain delivering five protective antigens of Clostridium perfringens to reduce necrotic enteritis and safely administered by in ovo vaccination.

[0239] We previously constructed PIESV vector strain %12341 (Table 4) to deliver two protective C. perfringens antigens encoded on a regulated delayed lysis plasmid to vaccinated broilers. This vaccine administered by course spray to day-of-hatch chicks gave superior protective immunity against C. perfringens causing necrotic enteritis but very poor immunity to challenge with wild-type S. enterica serotypes. Viable vaccine cells were unrecoverable from internal tissues after several days and from ceca after one week following vaccination, a desirable feature to preclude vaccine persistence at time of slaughter. However, the inability of this PIESV construct to protect against Salmonella is most likely due to not persisting for two weeks or more since this has been shown by us and others to be necessary to induce cellular immunity of long duration. Nevertheless, the PIESV strain x12341 delivering plasmid pG8R220, encoding PIcC and GST-NetB protected chickens in a field setting challenged with the highly virulent C. perfringens strain CP4. The x12341 (pG8R220) construct eliminated all mortality, significantly reduced lesion scores and gave the same feed conversion efficiency and weight gain after C.Attorney Docket No. 10457-621PC0

[0240] perfringens challenge compared to unchallenged controls and antibiotic-treated chickens. As a consequence, the %12341(pG8R220) construct was licensed by APHIS and is now marketed as AVERT by Huvepharma.

[0241] We still, however, desired a strain to further enhance protection against C. perfringens-induced necrotic enteritis that would not only protect by blocking activities of the two toxins but would also induce immunities to reduce the colonizing ability of C. perfringens. A further desired attribute would be to induce a higher level of protective immunity to Salmonella serotypes yet not be present on carcasses as slaughter. We first constructed a regulated delayed lysis plasmid vector encoding five C. perfringens protective antigens, including cell surface proteins and secreted toxins, that had been shown to confer individually and in combination protective immunity to prevent NE. In addition, the added antigens were expected to induce immunities to reduce colonization by C. perfringens. All five nucleotide sequences for gene inserts were optimized to use codons preferentially used by Salmonella in highly expressed genes. This plasmid pG8R256 derived from pG8R114 is diagrammed in Figure 10. Four of the five antigens, except the Gst-NetB non-toxic toxin antigen, are secreted by different T2SSs to preclude recombinational deletion. Each antigen is synthesized under the control of Lacl production by the arabinose regulated synthesis of Lacl in a manner to provide a regulated delayed in vivo synthesis of protective antigens. It should be noted that secretion of antigens increases formation of OMVs and thus immunogenicity.

[0242] We then constructed improved PIESV derivative strains to achieve the second objective of improving induced immune protection against Salmonella. The PIESV strains with superior attributes currently being evaluated for conferring high-level protective immunity to C. perfringens-'mdoced necrotic enteritis are %12702 (APmurA2s:: TT araC ParaBAD murA EasdA33 waal_46 pagL38 Tf rhaRS PrhaBAD2 waaL2 {wza-wcaM)-8 relA 1123 recFI 2 EsifA26 araBAD65: TT rhaDABSR515 pagP81:: Pipp IpxE lpxR9 (pSTUK206 (traM-traX)-41 araC ParaBAD / ac / TT) and 12831 (APmurA25:: TT araC ParaBAD murA asdA33 waaL46 pagL38-. f rhaRS PrhaBAD2 waaL2 {wza-wcaM-8 relA1123 recF12 EsifA26 araBAD65 ~n' rhaDABSR515 pagP81 P\PPIpxE ElpxR9 PsafA55:: PmurA safA55 APstc53:: PmurA stcA53 (pSTUK206 (traM-traX)-41-.-.araC ParaBAD laclAttorney Docket No. 10457-621PC0

[0243] TT) each possessing pG8R256. They will be compared with 12341 ( PmurA25:: TT araC PBAD murA AasdA27 T7 araC PBAD c2 A(wza-wcaM)-8 Apmi-2426 relA197-.-.araC PBMJ lad TT ArecF126 AsifA26 AwaaL46 ApagU -.-. T rhaRS PrhaBAD waaL1) construct with pG8R256, which has been demonstrated to be safe for in ovo inoculation into 18-day-old chick embryos (see Example 5).

[0244] The improvements in %12702 and x12831 over x12341 include: the ApagL38 Tl' rhaRS PrhaBAD2 waaL2 substitution for ApagL64:: TT rhaRS PrhaBAD waaL1 to delay the loss in presence of the LPS O-antigen which allows longer persistence prior to onset of lysis; the inclusion of the araBAD65 T and ArhaDABSR515 mutations that also delays lysis by a few in vivo cell divisions; the inclusion of the ApagP81'. P\PPIpxE AlpxR9 mutation that enhances induction of innate immunity via interaction with TLR4; and the substitution of (pSTUK206 A traM-traX)-41 araC ParaBAD lad TT) for ArelA 197 araC PBAD lad TT, which eliminates conjugational ability of the Salmonella virulence plasmid to provide enhanced biological containment while still conferring the regulated delayed antigen synthesis phenotype. x12831 also possess the ability to constitutively synthesize the Saf and Stc fimbrial antigens that facilitates high-level colonization of the spleen to enhance immunogenicity.

[0245] Example 5. Construction of improved PIESV strain delivering five protective antigens of Campylobacter jejuni to reduce colonization of chickens and safely administered by in ovo vaccination.

[0246] We recently examined PIESV delivery of 15 different known and putative protective C. jejuni antigens to broiler chicks to decrease colonization and shedding of C. jejuni following challenge of PIESV vaccinated chickens with a mixture of 5 C. jejuni strains isolated from poultry (Figure 11). These studies used two different PIESV strain genotypes and included repeat studies with individual and mixtures of PIESV strains. Based on the collective results, 5 antigens each causing significant (but incomplete) reduction in C. jejuni colonization and shedding were selected to present in a single PIESV strain. We thus constructed pG8R410 (Figure 12) encoding 5 C. jejuni protective antigens using the strategies used in constructing pG8R256. This plasmid in a speciallyAttorney Docket No. 10457-621PC0

[0247] constructed PIESV strain derived from %12836 (see below) is now commencing trials and other evaluations to hopefully result in an APHIS-licensed vaccine to reduce C. jejuni in poultry and thus lessen C. jejuni transmission through the food chain to humans.

[0248] We constructed the improved strain x12836 (Table 4) ( PmurA25:: TT araC ParaBAD murA AasdA33 A(wza-wcaM)-8 ArelA1123 ArecF126 AsifA26 AwbaP45 ApagL14-.-. TT araC ParaBAD wbaPAlpxR9ApagP8AcysG123"pgl-(gne-plgG)-12 AfliC180::cj1433gly21-29 ompA132 cj1433 (2xN-gly) (pSTUK206 (traM-traX)-41 araC ParaBAD lad TT)) with the 14-gene pg / operon from C. jejuni that encodes the enzymes to synthesize the immuno-protective N-linked glycan attached to many C. jejuni surface proteins and inserted sequences encoding the canonical bacterial N-glycosylation sequence D / E-X-N-Y-T / S present in genes encoding PIESV surface antigens as well as in C. jejuni genes. This modified operon was inserted into a deletion of the cysG gene that does not impair invasiveness or persistence. Importantly, this strain has the AwbaP45 ApagL14 T araC ParaBAD wbaP construction that gradual eliminated the LPS O-antigen in vivo while enabling N-glycosylation since the AwaaL46 pagL38 - rhaRS PrhaBAD2 waaL2 sequences gradually eliminates the waaL encoded enzyme that is essential for attachment on the C. jejuni N-glycan to proteins with the appropriate glycosylation sequence(s).

[0249] Based on findings disclosed in other Examples, %12836 can be modified in multiple ways to further enhance its ability to elicit protective immune responses. The APfUr33:: TT araC ParaBAD fur mutation can be added to enhance ability to induce better cross-protective immunity to Gram negative enteric bacterial pathogens. The AaraBAD65-. TT mutation can be used to increase vaccine cell persistence in vivo by one or two cell divisions. The ApagP8 mutation can be replaced by the ApagP81 P\PPIpxE deletioninsertion mutation to detoxify lipid A and render it an effective activator of TLR4. The APstcAss:: PmurA stcA53 and APsafAss:: PmurA safA55 deletion-insertion mutations can be added to enhance the ability of the vaccine strain to colonize the spleen to further enhance induction of protective immunity.

[0250] We and others have found that in ovo inoculation of 18-day-old embryos with vaccines commercially now used to control Salmonella infections in poultry invariably leadAttorney Docket No. 10457-621PC0

[0251] to much embryo death and poor hatchability. We demonstrated this to be true in using S. Typhimurium UK-1 strains with the AaroA mutation present in many vaccine strains making use of discoveries by Bruce Stocker and colleagues and also with Acya Acrp mutations in the vaccine constructs made by our group. Since all Family B SDAAS strains have a self-destructing regulated delayed lysis in vivo phenotype and are safe for in ovo inoculation into 18-day-old chick embryos, we reasoned that the regulated delayed lysis attributes of our genetically modified PIESV constructs would likewise be well tolerated when used for in ovo inoculation. We present data in Figure 13 that demonstrates this is in fact true. With the hatchability the same for 18-day old embryos inoculated with Family A SDAAS strain %12703, the Family B SDAAS strain %12668 as well as the PIESV constructs %12836(pG8R114) (empty vector control), %12836(pG8R410) (encoding 5 C. jejuni antigens), %12848(pG8R114) (empty vector control) and %12848(pG8R256) (encoding 5 C. perfringens antigens). These results indicate that in ovo administration of either or both SDAAS and PIESV construct strains will be safe with no reduction in hatchability and provide an early induction of protection being developed when chicks hatch and are first exposed to a diversity of pathogens.

[0252] Example 6. Construction of improved PIESV strain to induce cross-protective immunity to bacterial species in the Enterobacteriaceae and protect chickens against infection by APEC strains.

[0253] A key feature of strains to induce cross protective immunity is the in vivo upregulation of genes specifying proteins for the uptake of manganese and especially iron. We have now constructed two improved PIESV strains to induce enhanced cross protective immunity to Gram negative enteric pathogens. These strains x!2909 and 12918, which were both derived from %12848 (Table 4), were shown to be safe and effective for in ovo inoculation of 18-day-old chicken embryos (Figure13) and have the AaraBAD65:: TT and ArhaDABSR515 mutations to eliminate catabolism of arabinose and rhamnose to enable additional cell divisions before commencement of lysis and require lower concentrations of L-arabinose and L-rhamnose in growth media. %12909 has the APstcA53:: PmurASta453and APsafA55:: PmurAsaM55 mutations to cause constitutive synthesis of fimbriae that increase efficient colonization of the spleen to enhance the level ofAttorney Docket No. 10457-621PC0

[0254] immunity. ^12918 has the A(hin-fljBA)-219 and AfliCI 80 mutations to cause continuous secretion of the modified FliC flagellin that does not assemble into flagella and is therefore a most efficient activator of innate immunity by its binding to TLR5. If both strains induce cross protective immune responses better than 12848, we will make a derivative strain with the APstcA53:: PmurA stcA53, APsafAss:: PmurA safA55, A hin-fljBA)-219 and AfliC180 mutations to determine if the beneficial activities are additive.

[0255] Although we expect these strains to be broadly protective against a diversity of Salmonella serotypes, we are especially interested in reducing colonization and infection with APEC strains that cause more costly disease outcomes in the poultry industry. In this regard, Salmonella and APEC strains share homology for a number of IROMPs with 83% homology for IroN, 66% homology for SitD, 90% homology for YncE and 76% homology for FhuA.

[0256] A significant problem in evaluating protective immunity against APEC infections is the need to develop a realistic APEC challenge model that reflects commercial field conditions. Previous evaluations have used subcutaneous infections, thoracic duct challenge infections and caudal air sac challenge infections. These are not likely the means by which APEC strains infect chicks during commercial broiler raising. We therefore are investigating placement of newly hatched chicks on APEC-contaminated litter versus using a coarse spray infection on the day of hatch as well as several days later. We initially evaluate induction of immune protection against the 078 APEC strain X7122 (Table 5). Studies will then be expanded to use 01 and 02 APEC challenge strains with the best challenge model.

[0257] Example 7. Combined use of in ovo inoculation with improved SDAAS strain and vaccination with PIESV strain designed to induce cross protective immunity when either co-administered by in ovo vaccination or used to mucosally vaccinate day-of-hatch chicks to protect chickens against infection by APEC strains.Attorney Docket No. 10457-621PC0

[0258] Further improvement in the level of induced protective immunity against APEC colonization and disease causation can likely be achieved by in ovo inoculation of 18-day-old chick embryos with an efficacious SDAAS Family A strain such as %12703 (or derivatives) or the family B strain %12626 (or derivatives) with and without coadministration of the optimal PIESV strain to induce cross protective immunity to enteric pathogens such as APEC strains. A comparative evaluation will be made by taking chicks hatched from in ovo SDAAS inoculated embryos at day of hatch to vaccinate with an optimal PIESV strain. In all cases the optimal APEC challenge model described above in Example 6 will be used.

[0259] Example 8. Modification of PIESV strain that induces cross-protective immunity to bacterial species in the Enterobacteriaceae to deliver protective APEC antigens to further improve levels of protection against APEC infections and reduce potential for transmission of ExPEC strains through the food chain.

[0260] Our group including former colleagues have continued studies to establish the genetic basis of pathogenicity of APEC strains started some 40 years ago. Much has been learned and the genetic contents of the large virulence plasmids present in APEC strains have been determined. It is interesting that these virulence plasmids possess iron acquisition genes acquired from Salmonella, Shigella and Yersinia strains, which emphasizes the importance of iron acquisition to the virulence displayed by these E. coli strains infecting poultry. In addition, we learned that all APEC strains as well as most E. coli strains encode for the synthesis of a common pilus that is only synthesized and assembled under cultural conditions reflective of in vivo environments. The pilus gene operon has been placed on a low copy pSC101 oh regulated delayed lysis plasmid that is stably maintained in PIESV strains to constitutively synthesize and assemble these common pili. The synthesis and display of this common pilus induces a significant level of protective immunity against APEC challenges. This plasmid will thus be introduced into the optimal PIESV strain inducing cross protective immunity based on the studies in Examples 6 and 7 above.

[0261] If necessary, we previously developed methods for PIESV strains to synthesize and display IROMPs specified by genes in APEC strains as well as in other pathogens.Attorney Docket No. 10457-621PC0

[0262] Overproduction of these I ROMPs is toxic to Salmonella vaccine strains and impedes invasiveness and immunogenicity. This problem has been and can be overcome by placement of genes such as iutA specifying the Shigella I ROMP or pen specifying the Yersinia IROMP either into the chromosome of Salmonella in the cysG gene or on the single copy remnants of the Salmonella pSTUK virulence plasmid pSTUK 206. In addition, there are many other APEC virulence genes that could be included in an improved vaccine against APEC infections.

[0263] Example 9. Construction of improved PIESV strain synthesizing and delivering by secretion protective Eimeria antigens and delivering a DNA vaccine encoding two other protective Eimeria antigens to be synthesized by the vaccinated avian host.

[0264] The parasite Eimeria causing coccidiosis causes significant disease in the poultry industry that result in copious use of drugs of increasing ineffectiveness because of drug resistance necessitating use of live partially attenuated Eimeria vaccine strains that have both negative and positive attributes. We have endeavored to develop Salmonella vectored vaccines to protect against Eimeria for a number of years with partial success in decreasing disease and weight loss. A significant problem is that while some protective Eimeria antigens are not subject to post translational modification and thus can be synthesized and delivered by Salmonella vectored vaccines to elicit significant protective immune responses, other Eimeria antigens are glycosylated by post translational modification and such antigens when synthesized by bacteria such as Salmonella are not glycosylated and induce inferior protective immune responses. As described in Example 2, we have constructed a plasmid pG8R389 that can encode antigens to be synthesized and secreted by the Salmonella vaccine vector but also be a DNA vaccine in which the eukaryotic promoter enables synthesis of encoded antigens in cells of the vaccinated animal host and then subject to post translational modification to produce antigens capable of eliciting protective immune responses.

[0265] Figures 14 and 15 diagram the plasmids pG8R472 and pG8R473, respectively, derived from pG8R389 (Figure 3) that deliver protective antigens derived from sequences from E. tenella and E. maxima, respectively. Since the SO7 antigen is not glycosylated and is synthesized and delivered by secretion by the Salmonella vaccineAttorney Docket No. 10457-621PC0

[0266] vector strain, the codons encoding the SO7 antigen are modified to use the codons used by Salmonella for its most highly expressed genes and done so in a manner to maintain the GC content of the modified genes to be similar to the 52% GC content of Salmonella DNA. On the other hand, the Imp1 and Ama1 sequences from E. tenella and E. maxima are the native sequences since the expression will occur in cells within the vaccinated avian host.

[0267] Delivery of pG8R472 and pG8R473 requires use of a PIESV vector strain designed for effectiveness in both the synthesis and delivery of protective antigens and in protecting and facilitating the delivery of the plasmid to serve as a DNA vaccine targeted to the nucleus of cells in the vaccinated avian host for antigen synthesis and post translational modification. %12731 (Table 4), and its derivative 12963 (APmurA25" TT araC ParaBAD murA AasdA33 waaL46 pagL38 T rhaRS PrhaBAD2 waaL2 A(wza-wcaM)-8 ArelA1123 ArecF126 XsifA26 endA2113 AsseL116 MlpA181 rhaDABSR515 AaraBAD65 APstcA53:: PmurA stcA53 APsafA55” PmurA safA55 (pSTUK206 (traM-traX)-41-. araC ParaBAD / ac / TT), are the strains constructed with these attributes. Both %12731 and x12963 with pG8R472 and pG8R473 have been constructed and subjected to extensive tests to verify genotypic and phenotypic traits including by transfecting the plasmid constructs into various host cell types in culture to measure synthesis of the Imp1 and Ama1 antigens. We also evaluated genetic stability during 50 generations of growth under permissive growth conditions. The use of in ovo inoculation with optimal SDAAS strains will be evaluated to determine whether that elevates levels of protective immunity induced by vaccine administration to prevent disease symptoms after Eimeria oocyst challenge and ensure maximal feed conversion levels and weight gain.

[0268] Example 10. Construction of improved PIESV strain synthesizing and delivering by type 2 secretion multiple protective influenza virus M2e antigens and by type 3 secretion the conserved NP-HA T-cell epitope fusion and a DNA vaccine encoding influenza virus HA and NA antigens to be synthesized by the vaccinated animal host.Attorney Docket No. 10457-621PC0

[0269] There is a significant need for a potential universal vaccine to protect against influenza virus infections that is inexpensive to produce and deliver and easily modified depending upon the HA and NA antigen types circulating in influenza viruses infecting avian, companion, farm and wildlife animal and human hosts. We have thus designed and constructed a genetically modified Salmonella vectored vaccine that can be rapidly modified, produced in high quantity by fermentation technology and administered by a diversity of mucosal and parental routes of vaccination. We thus made use of pG8R388, a plasmid almost identical to the pG8R389 vector diagramed in Figure 3, but lacking the improved b / a T2SS, to construct the pG8R419 plasmid diagramed in Figures 16, which uses the very active ompA SS. pG8R419 delivers three different highly conserved M2e sequences derived from swine, avian and human influenza virus strains and with delivery by Type 3 secretion the highly conserved universal influenza A virus NP protein fused to two highly conserved T-cell epitopes derived from the HA antigen to stimulate cellular immunity. The swine and avian M2e encoding sequences have been inserted into the exposed OmpA loop 3 to be displayed on the Salmonella vaccine cell surface - especially after reduced synthesis of the LPS O-antigen that masks display of outer membrane proteins for surveillance by the immune system. Figure 17 provides analysis of regulated synthesis and delivery of the M2e and NP antigens encoded on pG8R419 in %12731 and X12735.

[0270] This pG8R419 plasmid is readily modified by insertion of selected sequences encoding HA and NA antigens such as we have done for the mouse adapted H1N1 WSN influenza virus to generate pG8R457 (Figure 18), which we have evaluated for inducing protective immunity to influenza virus challenge in mice. pG8R419 can likewise be modified by encoding HA and NA antigens from circulating influenza viruses such as the high path avian influenza H5N1 virus to generate a plasmid like pG8R-Flu diagramed in Figure 19. We have also constructed a genetically modified PIESV strain %12964 (Table 4) to be effective in delivering these plasmid constructs to avian, porcine, bovine, wildlife carriers / vectors, and humans. The impact of pre-inoculation or co-administration of an optimal SDAAS strain will be evaluated to see whether that enhances the level of duration of immunity and also diminishes influenza virus transmission.

Claims

Attorney Docket No. 10457-621PC0CLAIMSWhat is claimed is:

1. A method of inoculating embryonated avian eggs, the method comprising administering, in ovo, a safe inoculating dose of a Protective Immunity Enhanced Salmonella Vaccine (PIESV) strain, or derivative thereof, and optionally coadministering a safe inoculating dose of a live self-destructing attenuated adjuvant Salmonella strain (SDAAS), or derivative thereof.

2. The method of claim 1, wherein the PIESV strain is in a composition with a carrier, and the SDAAS is in a composition with a carrier, optionally the PIESV strain and SDAAS strain being in the same composition.

3. The method of claims 1 or 2, wherein the PIESV is a vaccine against an avian pathogen.

4. The method of claim 3, wherein the pathogen comprises a bacterial, viral or parasite pathogen.

5. The method of claim 3, wherein the administering or the co-administering protects against Clostridium perfringens-mduced necrotic enteritis.

6. The method of claim 3, wherein the administering or the co-administering protects against infection and colonization by Campylobacter jejuni.

7. The method of claim 3, wherein the administering or the co-administering protects against infection and colonization by Eimeria species causing coccidiosis.

8. The method of claim 3, wherein the administering or the co-administering protects against infection by and transmission of influenza virus.

9. The method of any of claims 1 -8, wherein administering induces innate immunity in hatched offspring from the inoculated embryonated avian eggs.

10. The method of any of claims 1-9, wherein administering does not reduce hatchability of the inoculated embryonated avian eggs.

11. The method of any of claims 1-10, wherein administering decreases severity of infection of hatched offspring from the inoculated embryonated avian eggs by an avian pathogen.Attorney Docket No. 10457-621PC012. The method of claim 11, wherein the avian pathogen is selected from Clostridium spp, Campylobacter spp., Eimeria spp., or influenza viruses.

13. The method of any of claims 1-12, wherein the SDAAS possesses either Aa / r AdadB AasdA or Aalr-3 APdadB:: TT araCParaBAD dadB (or APdadB:: TT rhaRS PrhaBAD dadB) APaSdA:: TT araC ParaBAD asdA (or APaSdA:: TT rhaRS PrhaBAD asdA) mutations.

14. The method of any of claims 1-12, wherein the PIESV possesses three or more of the following mutations: APmurA:: TT araC ParaBAD murA, AasdA, AwaaL, ApagL fW rhaRS PrhaBAD waaL, AwbaP, ApagL:: T araC ParaBAD wbaP, A(wza-wcaM), ArelA, ArecF, AsifA, AendA, AsseL, AtlpA, ArhaDABSR, AaraBAD T, APstcA:: PmurA stcA, APsafA:: PmurA safA, AompA, APfUr:: TT araC ParaBAD fur, AmntR, A hin-fljBA), AfliC180, AglmS, APgims:: TT xylR PxyiAB glmS, AnagK, or (pSTUK206 (traM-traX)".araC ParaBAD / ac / TT).

15. The method of claim 1, wherein the PIESV strain is selected or derived from X12702, 12831, x12341, %12836, %12848, %12909, %12918, %12963 or %12964.

16. A PIESV selected from x12702, x12831, and x12341.

17. An improved SDAAS strain comprising a genotype set forth in Table 3.

18. An improved x12836 strain.

19. An improved PIESV strain selected from x12909 and x12918.

20. A method comprising co-administering a PIESV and SDAAS embodiment described herein, wherein co-administering comprises in ovo administration.

21. A plasmid selected from plasmids pG8R472 and pG8R473.

22. An SDAAS strain comprising a genotype set forth in Table 3 and with one or more mutations listed in Table 1.

23. A PIESV strain comprising a genotype set forth in Table 4 and with one or more mutations listed in Table 1 to deliver protective antigens of Clostridium perfringens to prevent C. perfringens induced disease.Attorney Docket No. 10457-621PC024. A PIESV strain comprising a genotype set forth in Table 4 and with one or more mutations listed in Table 1 to deliver protective antigens of Campylobacter jejuni to prevent colonization of C. jejuni.

25. A PIESV strain comprising a genotype set forth in Table 4 and with one or more mutations listed in Table 1 to enhance synthesis of antigens and / or deliver antigens of to reduce colonization by enteric pathogens.

26. A PIESV strain comprising a genotype set forth in Table 4 and with one or more mutations listed in Table 1 to deliver protective antigens and / or genes encoding such antigens of Eimeria Spp to prevent coccidiosis.

27. A PIESV strain comprising a genotype set forth in Table 4 and with one or more mutations listed in Table 1 to deliver protective antigens and / or genes encoding such antigens of influenza virus to prevent disease caused by influenza infection.

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