Enterotoxigenic escherichia coli colonization factor CS2-CS3 expression in attenuated shigella live vectors

Abstract

A broadly protective Shigella-ETEC vaccine includes components to cover multiple Shigella species and serotypes as well as multiple different colonization factors of ETEC. ETEC colonization factors CS2 and CS3 are among the most prevalent CFs found on ETEC isolates associated with diarrhea. These two factors are important components of a vaccine that can confer broad protection against ETEC. High level expression of these heterologous antigens in Shigella live vectors results in stronger immune responses. This specification describes the genetic engineering of DNA sequences encoding upstream sequences, including promoter and ribosome binding sites, that results in increased expression of CS2 and CS3 in Shigella live vectors and that leads to stronger immune responses when used as a vaccine in animal models.

Description

ENTEROTOXIGENIC ESCHERICHIA COLI COLONIZATION FACTORCS2-CS3 EXPRESSION IN ATTENUATED SHIGELLA LIVE VECTORSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is an international application, which claims the benefit of United States provisional application serial no. 63 / 618,410, filed 8 January 2024, and United States provisional application serial no. 63 / 559,790, filed 29 February 2024. The entire contents of the aforementioned application(s) is / are hereby incorporated by reference as if fully set forth herein.GOVERNMENT FUNDING SUPPORT

[0002] This invention was made with government support under grant no. Al 142725, awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] 1. Field of the Invention

[0004] The invention relates to the general field of medicine, and in particular to vaccines to Shigella spp and enterotoxigenic Escherichia coli.

[0005] 2. Background of the invention

[0006] Enterotoxigenic Escherichia coli (ETEC) causes diarrheal disease in children of less than 5 years of age in developing countries and travelers' diarrhea in persons from industrialized countries who visit developing countries, including military personnel. Clinical isolates of ETEC can produce a heat-labile enterotoxin (LT) that resembles cholera toxin and / or one or more heat-stable enterotoxins (ST) including human ST (STh) and porcine ST (STp). Strains can produce both LT and ST (LT / ST strains) or be ST-only or LT-only. Most ETEC encode colonization factors (CFs) that allow the pathogen to attach to proximal small intestine enterocytes, the critical site of host-pathogen interaction, before expressing enterotoxins that decrease villus tip cell absorption and evoke overt secretion of electrolytes and water by crypt cells.

[0007] The enteropathogens Shigella and ETEC are responsible for a substantial burden of disease in multiple populations within the US and worldwide. Both arc classified as serious threats by the CDC due to multidrug- resistance (MDR), making them high priorities for development of therapeutics and vaccines. The ease and frequency with which these pathogens acquire antimicrobial resistance (AMR) mechanisms and exchange virulence factors which confer increased pathogenic potential render them important emerging threats. Vaccines represent one proven strategy to combat AMR bacterial pathogens. The development of vaccines against these pathogens is a designated NIAID and World health Organization (WHO) priority due to their significant impact on public health which can include deleterious lifelong consequences that negatively impact physical health and cognitive development especially for infants and children.

[0008] Shigella and ETEC have long been recognized as important worldwide pathogens, especially in low resource settings. Recent data from the Global Enteric Multicenter Study (GEMS) identified Shigella and ETEC among the top 4 pathogens causing moderate to severe diarrhea (MSD) in children of less than 5 years of age in 7 sites in Asia and Africa. Shigella was the number 1 pathogen causing MSD in children 12-59 months of age. Moreover, the resulting disruption of intestinal absorptive and barrier functions can lead to malnutrition, growth stunting and impaired cognitive development.

[0009] There are three species of Shigella that are important causes of disease. .S'. flexneri is the most predominant species in LMIC whereas S. sonnei is most prevalent in developed regions and becomes the dominant strain as areas become more developed. .S’, dysenteriae 1 is especially dangerous due to its ability to cause a more severe illness due to the elaboration of Shiga toxin which can cause serious complications.

[0010] Recently, high numbers of cases of MDR Shigella have been documented in the US and other industrialized countries and the alarming spread of MDR strains that move intercontinentally in short periods of time throughout traditionally low risk regions and populations has underscored the need for new interventions. In addition, cases and outbreaks in the US of S. flexneri and S. sonnei expressing Shiga toxin (traditionally only expressed by S. dysenteriae 1) exemplifies the exchange of virulence factors between enterobacteriaceae with resulting increased pathogenic potential. The number of cases of diarrhea due to ETEC in the US is underestimated because ETEC is not differentiated from normal flora E. coli byconventional clinical microbiological techniques. Furthermore, isolation of MDR ETEC, including extended spectrum beta lactamase (ESBL), has been documented. The exchange of virulence factors between E. coli pathovars supports efforts to prevent ETEC infections and their global spread.

[0011] The global burden of Shigella was estimated to include 163 million cases and more than 74,000 deaths each year. The global burden of ETEC is estimated as >400 million cases of diarrhea annually with an estimated 120,000 deaths. More recently, these pathogens have been recognized as causes of considerable disease in the US. Shigella is the third most common enteric bacterial infection in the US causing about 500,000 cases (27,000 drug resistant) per year with 6,000 hospitalizations and 70 deaths. Shigella is easily spread from person to person because of its extremely low infectious dose and, causes infection in populations with compromised hygiene including children in daycare centers and individuals in custodial institutions. ETEC has been estimated to cause up to 10 million episodes of diarrhea each year in travelers, including military personnel. A vaccine that provides broad protection against these pathogens will be a valuable public health tool in multiple populations.

[0012] Three main families of Colonization Factors (CFs) expressed by ETEC cause diarrheal illness or dysentery in humans: CFA / I, CFA / II and CFA / IV. CFA / I is the sole member of that family. CFA / II strains encode coli surface (CS) antigen 3 (CS3) alone or in combination with CS1 or CS2, while CFA / IV strains encode CS6, either alone or in conjunction with CS4 or CS5. CFA / I, CS1, CS2, CS4 and CS5 are rigid fimbriae of diameter of about 6-7 nm; CS3 consists of thin flexible fibrillae 2-3 run in diameter; and CS6 morphology is nondescript.

[0013] Shigella is a genus of bacteria closely related to E. coli, and causes the diarrheal disease shigellosis in primates. It is one of the leading causes of diarrhea worldwide and is one of the top four pathogens causing moderate-to-severe diarrhea in children in Africa and South Asia. Shigella species are classified as follows: 5. dysenteriae (15 serotypes), S. flexneri (15 serotypes), S', boydii (19 serotypes), and .S'. sonnei (one serotype). Protective immunity against Shigella is directed against the LPS O-antigen and is serotype specific. While only one serotype of .S’. sonnei is required for inclusion in a broadly protective vaccine, multiple serotypes of S. flexneri need to be represented. Vaccination with a mixture of just 3 serotypes, .S'. flexneri 2a, 3a and 6, which express a type- and group-specific antigen found on the other serotypes (except for 7a) has been demonstrated to provide at least partial protection against challenge withheterologous S. flexneri serotypes in an animal model. These four attenuated strains have been engineered to contain mutations in guaBA and sen (and set in .S', flexneri 2a) and shown to be safe, immunogenic and protective against homologous challenge in an animal model.

[0014] Minor putative CFs also exist for which data supporting their role in pathogenesis in humans is less compelling or lacking, although they mediate attachment to human cells in tissue culture. Possible exceptions are CS17 LT-only strains that evoked diarrhea in challenged volunteers. Minor CF antigens CS7, CS12, CS14, CS17, CS19, CS20, CS21 and CS30 have received much attention, while others have also been described including CS8, CS10, CS11, CS13, CS15, CS18 and CS23. The only minor CF statistically associated with diarrhea in the GEMS study was CS14. Addition of a CS14 antigen in a vaccine that includes the major colonization factors extends coverage of the vaccine. Diarrheal diseases caused by ETEC remain a serious problem in the developing world, therefore there remains in the art a need for improved vaccines with a broad coverage of ETEC.

[0015] ETEC vaccines designed to stimulate anti-CF immunity, with or without accompanying antitoxic immunity, are in clinical development. These include purified fimbrial antigens or tip adhesins, inactivated fimbriated ETEC, attenuated ETEC expressing CFs, and bacterial live vectors, such as Shigella, that encode ETEC CFs. Stimulating intestinal secretory IgA antibodies that bind CFs and prevent ETEC from attaching to human small intestine mucosa is generally considered to be fundamental to a successful ETEC vaccine, although some contend that parenteral vaccine-induced serum IgG antibodies that transude onto intestinal mucosa may also prevent diarrhea in humans caused by bacterial enteropathogens. Most ETEC vaccines contain CFA / I, CS1, CS2, CS3, CS5 and CS6 antigens, and some also include CS4, along with an LT toxoid.

[0016] There are no FDA-approved vaccines for Shigella or ETEC at the current time. Vaccines that prevent diarrhea due to these pathogens and its additional sequelae would be of benefit to multiple populations including children in low resource settings, travelers and military personnel from industrialized settings, and at-risk populations in industrialized nations. Current gaps for this vaccine include: a vaccine to prevent enteric diseases in the developing world, an enteric traveler’s vaccine, a vaccine for at-risk populations in the US and other industrialized countries, and a vaccine for use in the case of a natural outbreak. Prevention of disease due toShigella and ETEC by use of an efficacious vaccine would reduce the spread of multi-drug resistant (MDR) strains in all global populations.SUMMARY OF THE INVENTION

[0017] Thus, there exists a need in the art for vaccines effective against Shigella and ETEC. A combined vaccine that targets both enteropathogens would have significant added value and benefit all noted populations and especially young children in low resource settings who are disproportionally affected by these pathogens. Practical advantages of a combined vaccine include lower manufacturing costs of a single vaccine over two separate vaccines, reduced number of vaccines to add to an already crowded vaccination schedule, and an increased acceptance of a vaccine targeting diarrheal disease more broadly than a single pathogen vaccine.

[0018] In particular, the invention relates to a multivalent vaccine composition comprising one or more live attenuated Shigella bacteria which expresses an enterotoxigenic Escherichia coli antigen. Preferably, the Shigella bacteria are selected from one or more of 5. sonnei, S. flexneri, and ,S'. dysenteriae. In certain embodiments, the Shigella bacteria are selected from live attenuated 5. sonnei, live attenuated 5. flexneri serotype 2a, live attenuated S. flexneri serotype 3a, live attenuated S. flexneri serotype 6, live attenuated S. flexneri serotype 1 b, live attenuated 5. flexneri serotype 7a, and any combination thereof. Optionally, the multivalent vaccine composition further comprises live attenuated 5. dysenteriae.

[0019] In preferred embodiments, the multivalent vaccine composition comprises live attenuated 5. sonnei, strain CVD 1233SP; live attenuated S. flexneri serotype 2a, strain CVD 1208S; live attenuated S. flexneri serotype 3a, strain CVD 1213; live attenuated S. flexneri serotype 6, strain CVD 1216; live attenuated S. flexneri serotype lb, strain CVD 1224; live attenuated S. flexneri serotype 7a, strain CVD 1242; and optionally, live attenuated 5. dysenteriae 1, strain CVD 1254.

[0020] In other preferred embodiments, the multivalent vaccine composition wherein the one or more of the Shigella bacteria express one or more enterotoxigenic E. coli (ETEC) antigens selected from the group consisting of CFA / I, CS1, CS2, CS3; CS4, CS5, CS6, CS14, one or more ETEC tip adhesin antigens; LThA2B; or any combination thereof. The multivalent vaccine composition also can contain a Shigella, bacteria that expresses one or more enterotoxigenic E. coli (ETEC) antigens selected from the group consisting of CAF / I, CS1, CS2, CS3; CS4, CS5,CS6, CS14, one or more ETEC tip adhesin antigens; LThA2B; or any combination thereof. Preferred embodiments of the multivalent vaccine composition comprise live attenuated S. sonnei, strain CVD 1233SP, which expresses ETEC antigens CS2 and CS3; live attenuated S. flexneri serotype 2a, strain CVD 1208S, which expresses ETEC antigens CFA / 1 and LTB; live attenuated S. flexneri serotype 3a, strain CVD 1213, which expresses ETEC antigens CS1 and CSS; live attenuated S. flexneri serotype 6, strain CVD 121, which expresses ETEC antigens CS4 and CS6; live attenuated S. flexneri serotype lb, strain CVD 1224, which expresses ETEC antigen CS14; live attenuated S. flexneri serotype 7a, strain CVD 1242, which expresses one or more ETEC tip adhesin antigens; and optionally, live attenuated S. dysenteriae, strain CVD 1254, which expresses ETEC antigens CFA / 1 and LTB.

[0021] In certain preferred embodiments, the multivalent vaccine composition comprises CVD 1233-SP::CS2-CS3-V2 (SEQ ID NO:2).

[0022] In another embodiments, the invention relates to a pharmaceutical composition comprising a multivalent vaccine composition as described herein.

[0023] In addition, certain embodiments of the invention relate to a method of vaccination against diarrheal disease in a subject in need thereof, comprising administering to the subject a multivalent vaccine composition as described above and herein.

[0024] In certain other embodiments, the invention relates to a method of vaccine production, comprising integrating one or more ETEC antigens into the chromosome of a Shigella live vector to produce high levels of expression of the ETEC antigens. Preferably, the Shigella bacteria are selected from one or more of S. sonnei, S. flexneri, and .S', dysenteriae.

[0025] In addition, preferably the operons encoding the one or more ETEC antigens are engineered into the chromosome of .S. sonnei attenuated strain CVD 1233-SP to form CVD 1233-SP::CS2-CS3-V2. The ETEC antigens preferably are selected from CFA / I, CS1, CS4, CS5, CS6, an ETEC tip adhesin, LThA2B, CS2, and CS3. Most preferably, the ETEC antigens are CS2 and CS3.

[0026] In a preferred embodiment, the invention relates to a vaccine composition comprising a live attenuated Shigella sonnei, S. flexneri, or S. dysenteriae bacterial strain that expresses an operon encoding the enterotoxigenic Escherichia coli antigen CS2 and an operon encoding the enterotoxigenic E. coli antigen CS3, wherein the operons encoding CS2 and CS3 are cloned in tandem behind the constitutive mLpp promoter (PmLpp) and include a deletion of 161 base pairsof DNA upstream from the CS2 ATG start codon and the CS2 ATG start codon is engineered about 9bp downstream from PmLpp ribosome binding site.BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Certain embodiments are illustrated by way of example, and not by way of limitation in the figures of the accompanying drawings.

[0028] FIG. 1 presents the DNA sequence upstream of the CS2 ATG start codon in CVD 1233- SP::CS2-CS3-V1 and -V2 (SEQ ID NOs:l and 2).

[0029] FIG. 2 is a western blot showing optimized expression of CS2 and CS3 in CVD 1233- SP::CS2-CS3-V2.

[0030] FIG. 3A, FIG. 3B, and FIG. 3C show pre- and post-immunization serum IgG antibody responses to S. sonnei LPS, CS2 and CS3 in serum from immunized guinea pigs. FIG. 3D, FIG. 3E, and FIG. 3F show pre- and post-immunization mucosal IgA antibody responses for guinea pig tears. This figure shows increased responses in animals immunized with CVD 1233SP::CS2- CS3-V2.

[0031] FIG. 4A and FIG. 4B are a set of bar graphs that present data for sera from CVD 1233- SP::CS2-CS3-V2-vaccinated guinea pigs inhibition of ETEC adherence, CS2 and CS3, respectively.

[0032] FIG. 5 shows stable maintenance of pINV in CVD 1233-SP following growth for about 25 generations in vitro.

[0033] FIG. 6 presents data on invasion, intracellular growth, and plasmid loss in wild type versus stabilized strains in human enteroids.DETAILED DESCRIPTION OF THE INVENTION

[0034] 1. Overview

[0035] The present invention relates to a new vaccine strain in which the operons encoding CS2 and CS3 are cloned in tandem behind the constitutive mLpp promoter (PmLpp) and include a deletion of 161 base pairs of DNA upstream from the CS2 ATG start codon and the CS2 ATG strat codon is engineered in the optimal distance (9bp) downstream from PmLpp ribosome binding site. This conformation allows one to increase and optimize the expression of CS2 and CS3 in Shigella by optimizing the region including the promoter and ribosome binding site upstream ofCS2. This results in increased expression of CS2 and CS3 in Shigella live vectors that leads to a stronger immune response when used as a vaccine.

[0036] In summary, colonization factors are critical antigens and targets for vaccine antigens to induce antibodies that block colonization and disease. Isolates of ETEC express antigenically distinct colonization factors. A vaccine with broad coverage against ETEC therefore should include multiple colonization factor antigens.

[0037] 2. Definitions

[0038] Unless defined otherwise, all technical and scientific terms use herein have the same meaning as commonly understood by one of ordinary skill in the art. Although various methods and materials similar- or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials used are described below. However, the skilled artisan understands that the methods and materials used and described are examples and may not be the only ones suitable for use in the invention. Moreover, as measurements are subject to inherent variability, any temperature, weight, volume, time interval, pH, salinity, molarity or molality, range, concentration, and any other measurements, quantities, or numerical expressions given herein are intended to be approximate and not exact or critical figures unless expressly stated to the contrary.

[0039] In the foregoing specification, the invention has been described with reference to specific embodiments thereof. It will, however, be evident that various modifications and changes may be made thereto without departing from the broader spirit and scope of the invention. The specification and drawings are, accordingly, to be regarded in an illustrative rather than a restrictive sense. Throughout this specification and the claims, unless the context requires otherwise, the word “comprise” and its variations, such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated item, element or step or group of items, elements or steps but not the inclusion of any other item, element, step, or group of items, elements, or steps. Furthermore, the indefinite article “a” or “an” , or step modified by the article.

[0040] As used herein, the term “about” means plus or minus 20 percent of the recited value so that, for example, “about 0.125” means 0.125 ± 0.025, and “about 1.0” means 1.0 ± 0.2. Notwithstanding that the numerical ranges and parameters setting for the broad scope of the invention are approximations, the numerical values set forth in specific non-limiting examplesare reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements at the time of this writing. Furthermore, unless otherwise clear from the context, a numerical value presented herein has an implied precision given by the least significant digit. Moreover, all ranges disclosed herein are to be understood to encompass any and all sub-ranges subsumed therein. For example, a range of "less than 10" can include any and all sub-ranges between (and including) the minimum value of zero and the maximum value of 10, that is, any and all sub-ranges having a minimum value of equal to or greater than zero and a maximum value of equal to or less than 10, e.g., 1 to 4.

[0041] As used herein, the term “treating” and its cognates refers to taking steps to obtain beneficial or desired results, including clinical results, including mitigating, alleviating or ameliorating one or more symptoms of a disease is meant to indicate one or more of the item, element; diminishing the extent of disease; delaying or slowing disease progression; ameliorating and palliating or stabilizing a metric (statistic) of disease. The effect may be prophylactic in terms of completely or partially preventing a conditions or disease or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a condition or disease and / or adverse effect attributable to the condition or disease. “Treatment” refers to the steps taken. It can include any treatment of a condition or disease in a mammal, particularly in a human, and includes: (a) preventing the condition or disease or symptom thereof from occurring in a subject which may be predisposed to the condition or disease but has not yet been diagnosed as having it; (b) inhibiting the condition or disease or symptom thereof, such as, arresting its development; and (c) relieving, alleviating or ameliorating the condition or disease or symptom thereof, such as, for example causing regression of the condition or disease or symptom thereof.

[0042] The term “prophylaxis,” in the context of a disease, includes reducing the severity of a disease, reducing the chance of contracting a disease, and a complete or partial prevention of the disease or its signs and symptoms. As used herein, the term “prophylaxis” and its cognates refers to taking steps to obtain beneficial or desired results, including clinical results, and including steps to obtain total or partial prevention of a disease or condition. Vaccination for prophylaxis generally refers to administration of the composition in order to completely or partially prevent, reduce the symptoms of, severity of, or duration of the illness, including partially or completely inhibiting the condition, or ameliorating the condition or progression ofthe condition. Prophylaxis includes reducing the bacterial infection (bacterial titer), reducing reactions to bacterial toxins associated with the disease or condition, including their symptoms, and reducing the severity or duration of symptoms.

[0043] As used herein, the term “subject” refers to any animal, preferably a mammal, and most preferably a human. Laboratory animals are included in this definition.

[0044] As used herein, the term “subject in need” refers to a subject, including a human patient, who suffers from a diarrheal disease (including dysentery) or is in an environment which might expose the subject to a diarrheal disease. For example, such a subject in need includes, but is not limited to a person of age 0 (newborn) to age 5 residing in an area where diarrheal disease is endemic or a person living in an industrialized country in a higher risk setting such as day care center or custodial institution, or a person living in an industrialized country (including military personnel) who travels to a less developed region where Shigella and / or ETEC are endemic.

[0045] As used herein, the terms “diarrhea” and “diarrheal disease” refer to an episode of three or more loose stools within 24 hours. “Dysentery” refers to diarrhea with the presence of blood and / or mucus in the stool.

[0046] As used herein, the term “therapeutically effective amount” refers to an amount of a therapeutic agent, which, when administered to a subject, has the intended therapeutic effect. A therapeutic effect is an effect that treats the intended disorder or condition, including improving the disease, disorder or condition, or a symptom thereof, including reduction of a symptom or delaying the onset or reoccurrence of the disease, disorder, condition, or symptom. The full therapeutic effect does not necessarily occur by administration of one dose and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations. In the context of a vaccine, a therapeutically effective amount is an amount that reduces the chance of occurrence or reduces the severity of a disease or condition in a subject currently not suffering from the disease or condition; or an amount that produces an immune response to an etiologic agent that is associated with protection against the disease or condition. When administered to a subject currently suffering from the disease or condition, a therapeutically effective amount is an amount that induces an immune response to an etiologic agent that is associated with protection against the disease or condition and ameliorates the disease or condition in the subject.

[0047] As used herein, the term “CF” refers to colonization factor, the most common of whichinclude CFA / I, CS 1 , CS2, CS3, CS3, CS5, and CS6.

[0048] As used herein, the term “CS” refers to coli surface antigen, a group of antigens from ETEC.

[0049] As used herein, the term “CFA” refers to colonization factor antigen, which is found in ETEC isolates. CFA / I, CFA / II, and CFA / IV refer to specific colonization factor antigens I, II, and IV.

[0050] As used herein, the term “ST” refers to heat stable enterotoxins, a group of peptide toxins produced by bacterial strains such as ETEC.

[0051] As used herein, the term “LT” refers to heat labile enterotoxins, a group of toxins (inactivated at high temperatures) that are found in E. coli. LTA2B refers to a construct composed of the A2 subunit of LTh plus the B subunit.

[0052] As used herein, the term “ETEC” refers to enterotoxigenic Escherichia coli, a group of E. coli that produce LT and / or ST toxins that stimulate the intestine, causing them to secrete excessive fluid, leading to diarrhea.

[0053] As used herein, the term “ETEC tip adhesin antigen” refers to antigens from tip adhesins of ETEC. Tip adhesins refer to the tip protein on some ETEC colonization factors.

[0054] As used herein, the term “StxB” refers to the E. coli toxin, Shiga toxin B subunit.

[0055] 3. Embodiments of the Invention

[0056] A. Introduction

[0057] All Shigella spp. contain a large, ~210kb plasmid, pINV, which is essential for virulence because it encodes a Type III secretion system (T3SS), required for host cell invasion and which delivers bacterial protein effectors into human cells. Several plasmid-encoded T3SS effectors, termed Invasion protein antigens (Ipa) elicit host immunity responses following shigellosis and have been implicated in protective responses.

[0058] In order to increase expression of CS2 and CS3 in Shigella, rendering the vaccine more immunogenic, this gene region was engineered to include the promoter and ribosome binding site, upstream of CS2. First, the 161 extra base pairs upstream of the CS2 ATG start codon were deleted. Second, the CS2 ATG start codon was engineered to be placed at a distance 9 bp downstream from the mLpp ribosome binding site (see FIG. 1). These modifications resulted in strain CVD 1233-SP::CS2- CS3-V2, which has a consistently high level expression of CS2 andCS3 and was able to induce high levels of serum IgG and mucosal IgA to CS2 and CS3 in vaccinated guinea pigs. The scrum antibodies of the experimental animals were able to inhibit binding of WT ETEC expressing CS2 and CS3 to HT-29 cells.

[0059] Unlike other Shigella, S. sonnei pINV encodes for O-antigen (O-Ag) synthesis for induction of protective immunity. A considerable challenge of working with S. sonnei is a high rate of spontaneous loss of pINV; two orders of magnitude higher than S.flexneri. This presents a serious stumbling block for vaccine development for .S'. sonnei. Spontaneous and unpredictable loss of the T3SS affects the reliability of CHIM studies, which are important for assessing the protective efficacy of vaccines. Furthermore, the efficacy of live attenuated S. sonnei vaccines are adversely affected by loss of pINV leading to loss of key immunogens and impaired invasion.

[0060] In order to increase expression of CS2 and CS3 in Shigella, rendering the bacteria more immunogenic, this gene region was engineered to include the mLpp promoter (in place of the native wild type promoter) and ribosome binding site, upstream of CS2. First, the 161 extra base pairs upstream of the CS2 ATG start codon were deleted. Second, the CS2 ATG start codon was engineered to be placed at a distance 9 bp downstream from the mLpp ribosome binding site. These modifications resulted in strain CVD 1233-SP::CS2- CS3-V2, which has a consistently high level expression of CS2 and CS3 and was able to induce high levels of serum IgG and mucosal IgA to CS2 and CS3 in vaccinated guinea pigs. The serum antibodies of the experimental animals were able to inhibit binding of WT ETEC expressing CS2 and CS3 to HT- 29 cells.

[0061] B. Engineering Strategy

[0062] In this invention, we have optimized the expression of CS2 and CS3 in the Shigella live vector, confirmed high level expression by western blot analysis and demonstrated superior anti- CS2 and anti-CS3 antibody responses in guinea pigs immunized with the optimized vaccine candidate. The strategy used to develop a vaccine that protects against disease caused by Shigella and ETEC is to use live attenuated strains of Shigella to express critical ETEC antigens. The ETEC antigens required for broad protection include colonization factors (CFs) CFA / I, CS1, CS2, CS3, CS4, CS5 and CS6. Antibody responses to ETEC CFs in orally vaccinated individuals can prevent adherence and colonization of ETEC and thus prevent disease. Our method is to use genetic engineering to integrate the genes encoding the ETEC CFs into thechromosome of Shigella live vectors. A strong anti-CF immune response requires high level expression of the antigen in Shigella.

[0063] Initially, the operons encoding CS2 and CS3 were engineered into the chromosome of 5. sonnei attenuated strain CVD 1233-SP to form CVD 1233-SP::CS2-CS3-V1. In this vaccine strain, the operons encoding CS2 and CS3 were cloned in tandem behind the constitutive mLpp promoter (PmLpp) and 161 base pairs of DNA upstream from the CS2 ATG start codon were included. PmT pp drives high level antigen expression in Shigella live vectors. Expression of CS2 and CS3 in CVD 1233-SP::CS2-CS3-V1 was not consistently visible by western blot. See FIG. 2. CVD 1233-SP::CS2-CS3-V1 induced serum IgG and mucosal IgA responses in vaccinated guinea pigs. See FIG. 3. By engineering optimal sequences upstream of CS2 and CS3 in CVD 1233-SP::CS2-CS3-V2, CS2 and CS3 antigen expression was improved (FIG. 2) and antibody responses to CS2 and CS3 were increased (FIG. 3). Furthermore, the antibodies induced by CVD 1233-SP: :CS2-CS3-V2 were able to inhibit binding of wild type ETEC to intestinal cells (see FIG. 4).

[0064] This Shigella-EEEC multivalent vaccine strategy has unique advantages over other R&D candidates. Advantages of a live attenuated Shigella-based vaccine include: 1) the induction of mucosal and systemic immune responses induced by mucosal (oral) immunization that are important for protection; 2) malleability of this system, which allows modification if new antigens are identified that offer superior protective responses; 3) the ease and preference of oral delivery for all populations; 4) the benefit of oral delivery over parenteral for addition to an already crowded parenteral schedule; 5) the convenience and value of a multivalent vaccine formulation that covers 2 important enteric pathogens; and 6) the availability of validated formulation methods for live attenuated Shigella vaccines. The specific advantage of this invention (increased expression of CS2 and CS3) is increased immune responses to these antigens.

[0065] See FIG. 1 for sequence information. FIG. 1 provides the DNA sequence upstream of the CS2 ATG start codon in CVD 1233-SP: :CS2-CS3-V 1 and -V2. The CS2 ATG start codon and part (first 32 bp)of the CS2 coding sequence are in bolded underlined black letters.Upstream CS2 sequence is highlighted in grey. The mLpp promoter (-35 and -10 sequences) are italicized and underlined. The ribosome binding site is italicized, bolded and underlined. The mLpp promoter -10 and -35 sequences are in italicized letters.

[0066] The strategy used to develop a vaccine that protects against disease caused by Shigella and ETEC is to use live attenuated strains of Shigella to express ETEC antigens. The ETEC antigens for broad protection include colonization factors (CFs) CFA / I, CS1, CS2, CS3, CS4, CS5 and CS6. Antibody responses to ETEC CFs in orally vaccinated individuals can prevent adherence and colonization of ETEC and thus prevent disease. Our method is to use genetic engineering to integrate the genes encoding the ETEC CFs into the chromosome of Shigella live vectors. A strong anti-CF immune response requires high level expression of the antigen in Shigella. In this invention, we have optimized the expression of CS2 and CS3 in the Shigella live vector, confirmed high level expression by western blot analysis and demonstrated superior anti-CS2 and anti-CS3 antibody responses in guinea pigs immunized with the optimized vaccine candidate.

[0067] C. Shigella Species

[0068] Shigella species that are useful with the invention are 5. flexneri, S. sonnei, S. dysenteriae and .S'. boydii. S. flexneri, and .S', sonnei are more preferred Shigella species. The preferred strains of Shigella are strains CVD 1233S, CVD 1208S, CVD 1213, CVD 1216, CVD 1224, CVD 1242, and CVD 1245.

[0069] D. Antigens

[0070] ETEC antigens that can be used in the invention include one or more of CFA / I, CS1, CS2, CS3, CS4, CS5, CS6, CS7, CS8, CS10, CS11, CS12, CS13, CS14, CS15, CS17, CS18, CS19, CS20, CS21, CS23, CS30, LTB, a tip adhesin, Shiga toxin B (StxB) and LT toxoid. Preferred ETEC antigens include one, more than one, or all of (CS)1, CS2, CS3, CS4, CS5, CS6, CS14, Colonization Factor Antigen 1 (CFA / 1), eltB (LTB), a tip adhesin, and Shiga toxin B (StxB).

[0071] E. Vaccines and Vaccine compositions

[0072] The vaccines of the invention are designed to prevent and ameliorate diarrheal disease and dysentery by immunizing a subject in need with a group of antigens associated with bacteria that cause these diarrheal diseases. Vaccines according to some embodiments of this invention contain a live attenuated strain of Shigella flexneri serotype 7a; a live attenuated strain ofShigella flexneri serotype 1b; and enterotoxigenic Escherischia coli surface antigen CS14, which is expressed in one or more of the Shigella strains. Preferably, the vaccine is a multivalent composition that provides broad coverage for the important antigens involved in diarrheal diseases caused by Shigella spp. and E. coli.

[0073] Advantageously, the vaccines are at least pentavalent or hexavalent, combining a mixture of 5-6 live attenuated Shigella strains, one or more of which preferably express an antigen from ETEC. For example, .S'. sonnei, S. flexneri strain 2a, .S'. flexneri strain 3a, .S. flexneri strain 6, S. flexneri strain lb, and S. flexneri strain 7a are mixed together. The vaccine most preferably contains at least S. sonnie, S. flexneri 2a, S. flexneri 3a, and S. flexneri 6. and also contains S. flexneri strain lb, and S. flexneri strain 7a, and S. flexneri strain 6. Preferably, the vaccines provide broad coverage for the Shigella antigens and the common serotypes that arc shown to be important in immunity to diarrheal disease. According to certain embodiments, vaccines according to the invention can be produced with two, three, four, or five Shigella strains, one or more of which express one or more ETEC antigens. In another embodiment, the vaccine also contains a live attenuated strain of .S'. dysenteriae. In addition, the vaccine preferably contains enterotoxigenic Escherichia coli (ETEC) antigens, which are expressed by one or more of the Shigella strains.

[0074] The Shigella strains CVD 1233SP, CVD 1208S, CVD 1213, CVD 1216, CVD 1224, CVD 1242, and CVD 1245 are preferred for embodiments of the invention. See also Tables 1 and 2, below. Other strains may be available to the person of skill in the art.

[0075] The ETEC antigens are expressed in one or more of the Shigella strains in the multivalent vaccine or in all of them. The ETEC antigens useful for the vaccine compositions preferably are selected from one or more of the group consisting of Coli Surface (CS)1, CS2, CS3, CS4, CS5, CS6, CS14, Colonization Factor Antigen 1 (CFA / I), eltB (LTB), a tip adhesin, and Shiga toxin B (StxB). One, any number of, or all of these antigens are expressed in at least one of the Shigella strains in the vaccine composition. In particular, preferably CFA / I, CS1 , CS2, CS3, CD5 and CD6 are present. Although Table 2 indicates particular ETEC antigens expressed in a particular Shigella strain for convenience, the person of skill is aware that any of the Shigella strains used can be engineered according to known methods to express any of the ETEC antigens, as convenient, so the indicated antigens can be expressed in a different strain. Methods for engineering the strains to express a desired ETEC antigen are known in the art andcan be found, for example in Wu et al., Infect. Immun. 79( 12):4912-4922, 201 1 PMC3232646, which is incorporated by reference herein.

[0076] The vaccines are live attenuated strains of Shigella, and therefore preferably have been modified so that virulence is reduced or absent in the human host, while remaining viable (live). Attenuation can be performed by any of the methods known in the art and available to persons of skill. Such methods are described in Wu et al., Infect. Immun. 79(12):4912-4922, 2011 PMC3232646 and Delaine et al., Pathog. Dis. 74(5), 2016 PMC5985478, which are incorporated by reference herein.

[0077] Additionally, the bacterial strains in the vaccine compositions of the invention can contain other modifications or mutations to reduce virulence. In some embodiments, strains of Shigella are attenuated by mutations in guaBA and sen and / or set. In an alternative embodiment, the Shigella can be inactivated (killed) rather than attenuated, or both attenuated and inactivated so that the vaccine comprises a single strain or a mixture of killed strains. This also can be accomplished by any of the methods available in the art, such as heat inactivation, formalin treatment, and the like.

[0078] A certain embodiment of the vaccine compositions of the invention comprises live attenuated 5. sonnei, strain CVD 1233SP, which expresses ETEC antigens CS2 and CS3; live attenuated S. flexneri serotype 2a, strain CVD 1208S, which expresses ETEC antigens CFA / 1 and LTB; live attenuated S. flexneri serotype 3a, strain CVD 1213, which expresses ETEC antigens CS1 and CS5; live attenuated S. flexneri serotype 6, strain CVD 1216, which expresses ETEC antigens CS4 and CS6; live attenuated S.flexneri serotype lb, strain CVD 1224, which expresses ETEC antigen CS14; live attenuated S. flexneri serotype 7a, strain CVD 1242, which expresses one or more ETEC tip adhesin antigens: and optionally, live attenuated S. dysenteriae 1, strain CVD 1254, which expresses ETEC antigens CFA / 1 and LTB.

[0079] In certain embodiments of the invention, the vaccine compositions discussed herein are formulated and administered as a pharmaceutical composition that contains the vaccine and a pharmaceutically acceptable carrier, and optionally one or more additional active agents. Preferably, the pharmaceutical compositions comprise a therapeutically effective amount of vaccine, i.e., an amount to affect the immunological reaction of the subject in respect to the Shigella spp. and / or E. coli.

[0080] A pharmaceutically acceptable carrier refers to any convenient compound or group ofcompounds that is not toxic and that does not destroy or significantly diminish the pharmacological or immunological (vaccine) activity of the agent with which it is formulated. Such pharmaceutically acceptable earners or vehicles encompass any of the standard pharmaceutically accepted solid, liquid, or gaseous carriers known in the art, such as those discussed in the art.

[0081] A suitable carrier depends on the route of administration contemplated for the pharmaceutical composition. Routes of administration are determined by the person of skill according to convenience, the health and condition of the subject to be treated, and the location and stage of the condition to be treated. Such routes can be any route which the practitioner deems to be most effective or convenient using considerations such as the patient, the patient’s general condition, and the specific condition to be treated. The live attenuated, inactivated, or killed vaccines of the invention preferably are administered or delivered by the oral route. Alternatively, the vaccine can be administered intravenously, intramuscularly, intradermally, subcutaneously, nasally, or the like. Oral administration of the vaccine is an advantageous route of administration because it is non-invasive, needing no needle and syringe, or special equipment.

[0082] Therefore, the forms which the pharmaceutical composition can take will include any form suitable or convenient for any of those routes of administration. Such product forms or dosage forms of the vaccine compositions include, but are not limited to: powders or granules for dilution, pre-filled syringes, liquids for injection, suspensions for injection, oral solutions, oral suspensions, oral emulsions, nasal spray solutions, tablets, capsules, caplets, lozenges, dragees, pills, granules, powders for inhalation, vapors, gases, rectal suppositories, vaginal suppositories, creams, lotions, oils, ointments, suspensions, emulsions, lipid vesicles, and the like. Preferably, each dosage form contains an effective amount of vaccine, however an effective dose may require a first dose and one or more subsequent booster doses as is known in the art. Any pharmaceutically acceptable carrier and / or excipient known in the art is contemplated for use with the invention.

[0083] Carriers can include, for example, solvents, diluents, and the like to contain the active ingredient(s) in solid, liquid, or gas form. Common carriers in solid form include starch (e.g., com starch, potato starch, rice starch), celluloses (e.g., microcrystalline cellulose, methylcellulose, and the like), sugars (e.g., lactose, sucrose, glucose, fructose, and the like),clays, minerals (e.g., talc, and the like), gums, waxes, and the like. Common carriers in liquid or semi-liquid form include gels, lipids (e.g., lipid vesicles or nanoparticlcs), oils, polyethylene glycols, glycerin, propylene glycol, emulsifiers, organic solvents (e.g., ethanol), aqueous solvents (e.g., water, saline solutions, electrolyte solutions, lactated saline solutions), suspending agents, and the like.

[0084] Excipients also can include adjuvants, flavorings, preservatives, colorings, taste-masking agents, sweeteners, wetting agents, fillers, dispersants, anti-caking agents, binders, pH adjusters and buffers, lubricants (e.g., magnesium stearate and the like), antibacterial agents or preservatives (e.g., benzyl alcohol, methyl parabens), antioxidants (e.g., ascorbic acid, sodium bisulfite), chelating agents (e.g., EDTA), glidants (e.g., colloidal silicon dioxide), and the like. Extended and sustained release compositions also are contemplated for use with and in the inventive embodiments. Thus, suitable carriers can include any of the ingredients known to achieve a delayed release, extended release or sustained release of the active components.

[0085] The compounds or pharmaceutical compositions containing the compounds can be provided in containers such as sachets, envelopes, blister packs, boxes, ampoules, vials, bottles, pre-filled syringes, bags, sprayers, inhalers, and the like.

[0086] The vaccines of the invention, in certain embodiments, are formulated as a lyophilized vaccine powder (representing a dry mix of the six individual lyophilized vaccines strains), presented in a sachet that represents one dose of vaccine, and optionally packaged with a sachet or other package of buffer powder. In some cases, the vaccine can be provided in 5-dose, or 10- dose packages. In certain formulations of the vaccines, the dose is prepared for oral administration, by suspending the contents of the sachet in water, for example about 100 mL of water, optionally with buffer powder contained in an accompanying sachet. Once reconstituted, the resulting “cocktail” of vaccine strains in buffer solution is delivered orally. Optionally, the sachet containing the vaccine contains suitable buffers so that reconstitution is performed with water alone, or the sachet is reconstituted in a buffer solution.

[0087] This embodiment of vaccine is suitable for a travelers’ vaccine, for distribution to at-risk populations and for distribution in the developing world or to military personnel. Different embodiments of the dosage forms include, but are not limited to, an individual (single use) dose containing the mix of strains and a multi-pack of individual doses containing the mix of strains. Either embodiment can optionally be accompanied by a sachet of buffer powder or a buffersolution. An additional embodiment includes a multi-dose package of individual dosage sachets, such as a 5-dosc or a 10-dosc presentation.

[0088] F. Doses and Regimens

[0089] Treatment regimens include a single administration of the vaccine or a course of an initial administration and one or more “booster” administrations. Preferably, the administrations are determined by medical personnel. The initial administration is given to the subject prior to exposure to conditions where diarrheal diseases are endemic, but may be given after exposure, or after symptoms of diarrhea occur. Booster administrations are given when needed. For example, for travelers or military personnel who are entering an area where dysentery is endemic can take orally two doses ten days apart, advantageously prior to entering the area. Infants in low-to-middle income countries (LMIC) are recommended to take 1 dose at each of 10 weeks, 14 weeks, and 9 months of age. Children can be administered a booster dose at school entry (age 5-6 years), at adolescence, and then later at about age 65 years.

[0090] A dose of vaccine preferably contains about 108colony forming units (CFU) to about 5 x 1010CFU dry attenuated bacteria, or as recommended by medical personnel. Doses may vary from about 107CFU to about 1011CFU, preferably about 108CFU to about 5 x 1010CFU, or about 5 x 108CFU to about 1010CFU or about 108CFU to about 109CFU, or any dose suitable as determined by the practitioner, depending on the size, exposure, general health, age, or other factors.

[0091] G. Subjects

[0092] The vaccine compositions according to the invention can be given to any subject, preferably a primate such as a human, in need. Subjects can include human patients, including children of any age from newborn infant to 18 years (preferably children of less than 5 years old) and adults of any age, including the elderly. Preferably, the subject is in need of vaccination against diarrheal disease. Such a subject in need therefore includes any person from 0 months or less than one month old to the extreme elderly over 100 years old. A use of the vaccines according to embodiments of the invention is administration of the vaccine in mixed populations including young infants and children. However multiple populations, or any subject in need of a broad spectrum mucosal vaccine against the epidemiologically most important Shigella serotypesand toxin and colonization factor types of E. coli (ETEC), can be served by administration of the vaccine. Multiple populations for administration of the vaccine preferably include, but arc not limited to: (1) adult and child travelers who visit less developed countries where these infections are hyperendemic; (2) children and adults in certain high risk populations in developed countries; (3) children aged less than 5 years in developing countries, and (4) for mass immunization of the population to control natural or deliberate outbreaks.

[0093] In summary, embodiments of the vaccine product preferably comprise 6 live, attenuated strains of Shigella each expressing protective antigens from ETEC. This vaccine is intended to prevent diarrhea and dysentery caused by S. flexneri, S. sonnei, and ETEC. A component to protect against .S'. dysenteriae 1 optionally can be added if this strain becomes a public health problem. The vaccine components have been selected from the key serotypes and antigens based on epidemiologic data and knowledge of cross -reacting group epitopes to produce this multivalent vaccine. Preclinical data supports the stability, safety, immunogenicity and protective capacity.

[0094] 4. Examples

[0095] This invention is not limited to the particular processes, compounds, compositions, or mthods described, as these may vary. The terminology used in the description is for the purpose of describing the particular’ versions or embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, the preferred methods, devices, systems, compounds, compositions and materials are now described. All publications mentioned herein, are incorporated by reference in their entirety; nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0096] Example 1: .87?Vaccine Development,

[0097] Live attenuated strains of Shigella were developed as a platform to stably express the most important protective antigens from ETEC to form a vaccine that can target two pathogens in a single formulation. A vaccine that engenders broad protection against two important pathogens has added value for use in multiple populations including children in low resourcesettings, travelers, and military personnel. This is aligned with WHO published Preferred Product Characteristics (PPC) for Shigella and ETEC to facilitate advancement in the US and abroad. One goal of this project is to complete a multivalent formulation composed of live attenuated strains of Shigella that express protective ETEC colonization factor and toxoid antigens. See Table 1, below.

[0098] Table !. Multivalent Shigella-ETEC Vaccines.

[0099] Unique aspects of the CVD vaccines described herein include the combined formulation consisting of 5 strains of live attenuated Shigella, each also expressing important ETEC antigens. Thus, in summary, the specification describes a unique engineering strategy to facilitate high level expression of ETEC CFs CS2 and CS3 in Shigella live vectors.

[0100] Protective immunity against Shigella is directed against the LPS O-antigen and is serotype specific. While only one serotype of 5. sonnei is required for inclusion in a vaccine, multiple serotypes of .S', flexneri are preferred in a vaccine for broad coverage of Shigella isolates in a population. We have identified a combination of 4 serotypes (S. flexneri 2a, 3a, lb and 6) that express type- and / or group-specific antigens found on the remaining serotypes and demonstrated that vaccination with a mixture of these strains, provided at least partial protection against challenge with heterologous .S’, flexneri serotypes in an animal model.

[0101] Additional evidence for cross protection is supplied by serum bactericidal activity (SBA) of antibodies from animals vaccinated with 5. flexneri 2a strain CVD 1208S against multiple serotypes including 5. flexneri 2a, 2b, 4a and 3a. Estimates of the coverage of the pentavalent vaccine (Table 1) is about 72% direct coverage of Shigella isolates and >88% total coverage (including cross-reactive serotypes).

[0102] Example 2: ETEC Vaccine Development.

[0103] ETEC are defined by the presence of heat labile toxin (LT) and / or heat stable toxin (ST) which elicit net secretion of ions and water to result in watery diarrhea. ETEC express colonization factors (CFs) that mediate attachment to the human intestine. Although a multitude of colonization factors have been identified, seven (CFA / I, and CS1 through CS6) are considered major fimbriae identified on the majority of clinical isolates. CS14, was recently identified as the only minor colonization factor significantly associated with MSD among ETEC isolates.

[0104] Immune responses directed against CF antigens have been demonstrated to protect against disease caused by ETEC. The preferred pentavalent vaccine strategy here includes the 7 major and 1 minor colonization factor antigens which would provide coverage of 84.2% of circulating ETEC isolates. Other important ETEC virulence factors have been identified as potential vaccine antigens and are contemplated for inclusion in the vaccine. It is important to pursue multiple promising strategies to identify the most safe, efficacious and practical candidates.

[0105] One prototype strain was tested in human clinical trials in which healthy adult volunteers ingested a single dose of S. flexneri vaccine strain CVD 1208S(pCFA / L LThA2B) at doses ranging from 106-109CFU. In most volunteers the plasmid was lost from the Shigella strain (as assessed by fecal shedding of CVD 1208S without the plasmid). However, in a small subset of vaccinees (6 / 40 total vaccinees) the plasmid was maintained in the vaccine strain and induced responses to CFA / I and LTB, providing proof of principle that humans can respond to heterologous antigens delivered via an attenuated Shigella live vector.

[0106] Notably, one volunteer in the highest dose cohort produced very high frequencies of IgA and IgG CFA / I- specific antibody secreting cells (ASC) (61 and 44 ASC / 106PBMC, respectively). Robust serum IgG and IgA ASC responses to Shigella LPS were also induced in the vaccinated subjects. ASC responses, which are a measure of mucosally primed circulating B-lymphocytes specific for the vaccine antigens, constitute a pool of cells capable of homing back to mucosal tissues where they will produce mucosal antibodies (slgA) and are regarded as a potential correlate of protection.

[0107] Example 3: Combined Sfo'ge / / a-ETEC Vaccine.

[0108] In order to optimize delivery of a vaccine that can provide additional value by protecting against two important pathogens, a combined Shigella- ETEC vaccine was developed wherein live attenuated strains of Shigella were engineered to express critical antigens from ETEC.

[0109] The expression of multiple antigens including the most clinically relevant fimbrial antigens CFA / I, CS1, CS2, CS3, CS4, CS5, and heat labile toxin (LT) antigens in 5 different attenuated Shigella strains initially was demonstrated using stabilized plasmid expression systems. These vaccine constructs induced immune responses to each heterologous antigen and the Shigella live vector following immunization of guinea pigs as single strains or in a mixed inoculum. Importantly, the immunized animals were protected against challenge with all wild type Shigella strains represented in the mix; the heterologous antigens did not diminish the immunogenicity of the Shigella live vector.

[0110] This supplied the important proof of concept that the Shigella live vector-based strategy can be successful. Therefore, a system for stable chromosomal insertion of genes encoding ETEC antigens and demonstrated induction of immune responses to all vaccine components was developed, (described below). A preferred vaccine strain according to the invention (.S'. sonnei CVD 1233-SP::CS2-CS3-V2) represents the two most preferred components of a multivalent broadly protective Shigella-ETEC vaccine. Preliminary data supports the safety, immunogenicity and protective efficacy of these vaccines.

[0111] Stabilization loci in the .S', sonnei virulence plasmid, pINV (encoding O-Ag and invasion phenotype), facilitated an engineering strategy that maximized maintenance of pINV in WT and vaccine strains of S. sonnei. Maintenance of pINV in WT challenge strains is important for induction of protective repsonses in vaccine strains. Stabilization of pINV has transformed the ability for laboratory manipulation and insertion of heterologous genes as well as large scale manufacture of consistent products.

[0112] Our basic research findings of ETEC antigen gene expression and regulation has facilitated engineering strategies to optimze expression of heterolgous antigens with native morphology in Shigella live vectors to induce maximal immune responses.

[0113] Example 4: .S'. sonnei Vaccines.

[0114] Polymorphism analysis provided an approach to stabilize 5. sonnei pINV for vaccine development, so stabilizing modifications (engineered V apBC .S’, sonnei with the sequence ofVapBC S.flexnert) were introduced in pINV in S. sonnei WT strain 53G and vaccine strain CVD 1233. The resulting stabilized strains are 53G- SP and CVD 1233-SP respectively.

[0115] Plasmid stability was first confirmed by performing plasmid loss assays in vitro. Single pINV+ colonies were grown in broth overnight and then plated on congo red agar (for visual distinction of pINV+ red colonies and pINV- white colonies) to quantitate the emergence of pINV- colonies. After about 25 generations of growth, no pINV- colonies were detected in CVD 1233-SP, while approximately 40% of pINV-bacteria emerged from the non-stabilized vaccine strain. See FIG. 5. The invasion, intracellular replication and plasmid loss of these strains were tested in HT-29 cells and human enteroids using gentamicin protection assays. In both models, there was no difference in initial (30- min time point) total numbers of intracellular WT, or vaccine strains. See FIG. 6. At 4 hours post-infection, the number of intracellular WT bacteria increased about 10-fold. In contrast there were fewer intracellular numbers of both vaccine strains at 4 hours as expected due to the attenuating guaBA mutation that restricts intracellular growth. When the intracellular bacteria were quantitated as pINV+ or pINV-, a high proportion of colonies of unstabilized WT 53G and unstabilized vaccine strain CVD 1233 were pINV-. About 20-50% of total bacteria lost pINV. See FIG. 6, white bars. In contrast no pINV- colonies were detected following infection with CVD 1233-SP (FIG. 6). Stabilization of pINV in the attenuated strain significantly reduced spontaneous loss of pINV during enteroid infection, demonstrating a major advancement for live attenuated S. sonnei vaccine candidates.

[0116] FIG. 5 shows stable maintenance of pINV in CVD 1233-SP following growth for -25 generations in vitro (****p<0.0001; t-test). For the data presented in FIG. 6, which shows invasion, intracellular growth and plasmid loss in WT versus stabilized strains in human enteroids, enteroids were infected with WT 53G, or vaccine strains. Bacteria were recovered from 9 independent infections following 30-minute or 4-hour gentamicin treatment. Colonies were tested for the presence (red bars) or loss (white bars) of pINV at each time point (*p<0.01; **p<0.001, one-way ANOVA with Dunn’s multiple comparisons test).

[0117] Vaccine strains CVD 1233 and CVD 1233-SP were tested in the guinea pig model for immunogenicity and protection. Both strains induced similarly robust anti-S. sonnei anti-LPS serum IgG and mucosal IgA responses and provided efficacy rates of 86% (CVD 1233) and 100% (CVD 1233-SP) against WT challenge. Given the importance of S. sonnei in the U.S., its propensity to develop MDR, and increasing prevalence in developed countries, maximized pINVstabilization will be highly advantageous to the advancement and translation of live attenuated .S'. sonnei vaccines as well as other S. sonnei vaccine modalities to manufactured products.

[0118] Example 5: Optimization of CS2 and CS3 Expression in CVD 1233-SP.

[0119] The results have supported the use of CVD 1233-SP as a live vector for the expression of 2 key ETEC colonization factors, CS2 and CS3. Using the methods described above, the operons encoding these antigens were engineered in tandem, driven by the constitutive mLpp promoter, into the chromosome of CVD 1233-SP downstream of the glmS gene to create strain CVD 1233-SP::CS2-CS3-V1. This strain was used to immunize guinea pigs where it was demonstrated to be immunogenic and induced robust responses to the .S'. sonnei live vector as well to CS2 and CS3. Additional sequence analysis of upstream regulatory regions of the CS2- encoding operon supported further genetic modifications consisting of optimization of the promoter region upstream of CS2 that resulted in higher level expression of CS2 and CS3 in CVD 1233-SP::CS2-CS3 version 2 (V2). See FIG. 2, which shows expression of CS2 and CS3 in CVD 1233-SP::CS2-CS3-V2. Cell lysates of Shigella live vectors and CS-expressing ETEC controls were probed with anti-CS2 or anti-CS3, as indicated.

[0120] Immunization with 1233-SP: :CS2-CS3-V2 induced even higher serum and mucosal antibody responses to CS2 and CS3 in guinea pigs than VI. See FIG. 3, which presents antibody responses following immunization with CVD 1233-SP::CS2-CS3-V-1 (open circle) or V-2 (open triangle). Serum (FIG. 3A through FIG. 3C) and tears (FIG. 3D through FIG. 3F) from guinea pigs before immunization (BL1 and Tl) or post one (BL2, T2) or two (BL3, T3) doses of VI or V2 were tested by ELISA for IgG and IgA against the S. sonnei live vector, CS2 and CS3. Each point represents an individual animal. **p<0.01 two-tailed Mann-Whitney U test.

[0121] Serum antibodies also were able to inhibit binding of WT ETEC expressing CS2 and CS3 to HT-29 cells. See FIG. 4. Finally, all (5 / 5) immunized animals were protected against WT .S'. sonnei challenge in a Sereny test. For the data in FIG. 4, sera from CVD 1233-SP: :CS2- CS3-V2-vaccinated guinea pigs (Vacc Sera) inhibited ETEC adherence. CS2- (FIG. 4A) or CS3- (FIG. 4B) expressing ETEC were incubated with sera as indicated for 1 hour then inoculated onto HT-29 monolayers for 1.5 hours. Cells were washed vigorously and lysed for bacterial counts. *p<0.05; ****p<0.001, one-way ANOVA with Bonferroni’s multiple comparisons test.

[0122] The improved antigen expression and higher immune responses with maintenance of robust responses to the Shigella live vector, supported the advancement of this prototype product. An RCB was prepared and tested for purity, identity and potency, and further confirmed as mycoplasma negative. The RCB was used in process development wherein conditions for high level growth of CVD 1233- SP::CS2-CS3-V2 were established at the IL fermentation level. Characterization of material produced under these conditions revealed <0.01% plasmid loss.

[0123] Example 6: cGMP MCB, WCB and pilot vaccine lot production of S. sonnei live vaccine strain CVD 1233-SP::CS2-CS3-V2.

[0124] S. sonnei is responsible for a significant burden of disease and is also recognized as the predominant Shigella pathogen in industrialized settings. Furthermore, the expansion of .S'. sonnei in countries as they become more industrialized underscores the importance of this strain as a critical component in a broadly protective vaccine. The enhanced features of the vaccines described here, including stabilized pINV and optimized ETEC CF expression, support increased promise for success through the cGMP production process and as an immunogenic and efficacious vaccine in humans.

[0125] The production of cGMP MCB, WCB and vaccine lots occur in a step- wise manner as described above. Characterization of all materials is carried out as they are produced, with CVD1233-SP::CS2-CS3-V2 vaccine- specific tests. CVD1233-SP::CS2-CS3-V2 has been shown to grow to high cell counts (>109CFU / ml) with a remarkable <0.001% pINV loss, when grown at large scale (up to IL fermenter). Material from these fermentation runs maintained all required characteristics including expression of CS2 and CS3. Parameters were defined for optimal vaccine viability.

[0126] References.

[0127] All publications listed below and throughout the specification arc hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.1. Barry EM, Levine MM. A tale of two bacterial enteropathogens and one multivalent vaccine.Cell Microbiol 2019; 21(11); el3067.2. Wu T, Grassel C, Levine MM, Barry EM. Live attenuated Shigella dysenteriae type 1 vaccine strains overexpressing shiga toxin B subunit. Infect Immun 2011; 79(12): 4912-22.3. Pilla G, Wu T, Grassel C, et al. Evaluation of a Live Attenuated S. sonnei Vaccine Strain in the Human Enteroid Model. Pathogens 2021; 10(9).4. MacLennan CA, Talaat KR, Kaminski RW, Cohen D, Riddle MS, Giersing BK. Critical Needs in Advancing Shigella Vaccines for Global Health. J Infect Dis 2022; 225(9): 1500-3.5. Hausdorff WP, Scheele S, Giersing BK. What Drives the Value of a Shigella Vaccine? 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Claims

Claims1. A multivalent vaccine composition comprising one or more live attenuated Shigella bacteria which expresses an enterotoxigenic Escherichia coli antigen.

2. The multivalent vaccine composition of claim 1, wherein the Shigella bacteria are selected from one or more of S. sonnei, S. flexneri, and S. dysenteriae .

3. The multivalent vaccine composition of claim 2, wherein the Shigella bacteria are selected from live attenuated 5. sonnei, live attenuated S.flexneri serotype 2a, live attenuated S. flexneri serotype 3a, live attenuated 5. flexneri serotype 6, live attenuated 5. flexneri serotype lb, live attenuated S.flexneri serotype 7a, and any combination thereof.

4. The multivalent vaccine composition of claim 3 which further comprises live attenuated S. dysenteriae.

5. The multivalent vaccine composition of claim 3 which comprises live attenuated S. sonnei, strain CVD 1233SP; live attenuated S.flexneri serotype 2a, strain CVD 1208S; live attenuated S. flexneri serotype 3a, strain CVD 1213; live attenuated S. flexneri serotype 6, strain CVD 1216; live attenuated S.flexneri serotype lb, strain CVD 1224; live attenuated S.flexneri serotype 7a, strain CVD 1242; and optionally, live attenuated 5. dysenteriae 1, strain CVD 1254.

6. The multivalent vaccine composition of claim 2, wherein the one or more of the Shigella bacteria express one or more enterotoxigenic E. coli (ETEC) antigens selected from the group consisting of CFA / I, CS1, CS2, CS3; CS4, CS5, CS6, CS14, one or more ETEC tip adhesin antigens; LThA2B; or any combination thereof.

7. The multivalent vaccine composition of claim 5, wherein the one or more of the Shigella bacteria express one or more enterotoxigenic E. coli (ETEC) antigens selected from the group consisting of CAF / I, CS1, CS2, CS3; CS4, CS5, CS6, CS14, one or more ETEC tip adhesin antigens; LThA2B; or any combination thereof.

8. The multivalent vaccine composition of claim 2, which comprises live attenuated S. sonnei, strain CVD 1233SP, which expresses ETEC antigens CS2 and CS3; live attenuated S. flexneri serotype 2a, strain CVD 1208S, which expresses ETEC antigens CFA / 1 and LTB; live attenuated S. flexneri serotype 3a, strain CVD 1213, which expresses ETEC antigens CS1 and CSS; live attenuated S. flexneri serotype 6, strain CVD 121, which expresses ETEC antigens CS4 and CS6; live attenuated S. flexneri serotype lb, strain CVD 1224, which expresses ETEC antigen CS14; live attenuated S. flexneri serotype 7a, strain CVD 1242, which expresses one or more ETEC tip adhesin antigens; and optionally, live attenuated S. dysenteriae, strain CVD 1254, which expresses ETEC antigens CFA / 1 and LTB.

9. The multivalent vaccine composition of claim 1, comprising CVD 1233-SP::CS2-CS3-V2 (SEQ ID NO:2).

10. A pharmaceutical composition comprising the multivalent vaccine composition of claim 1, claim 5, claim 8, or claim 9.

11. A method of vaccination against diarrheal disease in a subject in need thereof, comprising administering to the subject the multivalent vaccine composition of claim 8 or claim 9.

12. A method of vaccine production, comprising integrating one or more ETEC antigens into the chromosome of a Shigella live vector to produce high levels of expression of the ETEC antigens.

13. A method of claim 12 wherein the Shigella bacteria are selected from one or more of 5. sonnei, S. flexneri, and 5. dysenteriae.

14. The method of claim 12 wherein the operons encoding the one or more ETEC antigens are engineered into the chromosome of S. sonnei attenuated strain CVD 1233-SP to form CVD 1233-SP::CS2-CS3-V2.

15. The method of claim 13 wherein the ETEC antigens are selected from CFA / I, CS1, CS4,CS5, CS6, an ETEC tip adhesin, LThA2B. CS2, and CS3.

16. The method of claim 15, wherein the ETEC antigens are CS2 and CS3.

17. A vaccine composition comprising a live attenuated Shigella sonnei, S.flexneri, or 5. dysenteriae bacterial strain that expresses an operon encoding the enterotoxigenic Escherichia coli antigen CS2 and an operon encoding the enterotoxigenic E. coli antigen CS3, wherein the operons encoding CS2 and CS3 are cloned in tandem behind the constitutive mLpp promoter (PmLpp) and include a deletion of 161 base pairs of DNA upstream from the CS2 ATG start codon and the CS2 ATG start codon is engineered about 9bp downstream from PmLpp ribosome binding site.

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