Patent Grant
11471520
This application includes an electronically submitted sequence listing in .txt format. The .txt file contains a sequence listing entitled “15024361PC0_ST25_SequenceListing” created on May 3, 2019 and is 8 KB in size. The sequence listing contained in this .txt file is part of the specification and is hereby incorporated by reference herein in its entirety.
The present invention relates to the fields of medicine and in particular to a multi-component live attenuated Shigella vaccine which comprises additional antigens from Shigella serotypes and fimbrial colonization antigens from Enterotoxigenic Escherichia coli (ETEC). This composition produces a vaccine with enhanced and broader protection against Shigella and ETEC pathogens useful against diarrheal disease, including dysentery.
Colonization factors, including fimbriae from Enterotoxigenic E. coli (ETEC) are critical antigens and targets for vaccine antigens to induce antibodies that block colonization and disease. Isolates of ETEC express antigenically distinct colonization factor antigens. A vaccine with broad coverage against ETEC therefore should include multiple colonization factor antigens. The major colonization factor antigens previously recognized include CFA/I and CS1 through CS6.
Enterotoxigenic Escherichia coli (ETEC) cause 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.
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. 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.
Three main families of Colonization Factor Antigens (CFAs) that cause diarrheal illness or dysentery in humans are encoded by ETEC, including 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.
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.
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 (Vidal 2019). 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.
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: S. dysenteriae (15 serotypes), S. flexneri (15 serotypes), S. boydii (19 serotypes), and S. sonnei (one serotype).
S. sonnei and S. flexneri are the most important causes of disease in industrialized settings as well as in less developed regions (Livio, 2014). 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 with heterologous S. flexneri serotypes in an animal model (Noriega, 1999). 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.
Extended analysis of serotype distribution provides strategies for improvement of coverage against circulating Shigella isolates. There is a need in the art to extend direct coverage by the addition of attenuated derivatives of S. flexneri 1b and 7a. S. flexneri 1b accounted for 7.5% of all Shigella isolates in GEMS and expresses type I antigen. The inclusion of this important serotype increases direct coverage of the vaccine and provides the Type 1 antigen to increase coverage against other Type 1 strains. S. flexneri 7a (formerly named 1c), accounted for 2% of all GEMS Shigella isolates overall and has been identified as the predominant serotype in other epidemiological studies. This serotype is included in the inventive vaccine because it has a unique O-antigen structure that does not include type- and group-specific antigens found on the other serotypes and therefore is not expected to be covered by a quadravalent vaccine. In summary, the addition of S. flexneri serotypes 1b and 7 increases coverage and provides cross protection.
Therefore, the present invention provides a Shigella-ETEC vaccine with increased coverage of a broader range of ETEC and Shigella isolates, including CS14 antigens and serotypes (S. flexneri 7a, or S. flexneri 1b).
Specifically, the invention relates to a vaccine composition for prophylaxis and treatment of diarrheal disease and dysentery, comprising:
a. a live attenuated Shigella strain selected from the group consisting of Shigella flexneri serotype 7a, Shigella flexneri serotype 1b, and both Shigella flexneri serotype 7a and Shigella flexneri serotype 1b;
b. enterotoxigenic Escherischia coli coli (ETEC) surface antigen CS14, wherein the ETEC CS14 is expressed in one or both or the live attenuated Shigella strains.
Certain embodiments of the invention relate to a vaccine as described above, further comprising one or more of:
c. a live attenuated strain of Shigella sonnei;
d. a live attenuated strain of Shigella flexneri serotype 2a;
e. a live attenuated strain of Shigella flexneri serotype 3a; and
f. a live attenuated strain of Shigella flexneri serotype 6.
Certain embodiments of the invention relate to a vaccine as described above, further comprising:
c. a live attenuated strain of Shigella sonnei;
d. a live attenuated strain of Shigella flexneri serotype 2a;
e. a live attenuated strain of Shigella flexneri serotype 3a;
f. a live attenuated strain of Shigella flexneri serotype 6; and
g. optionally, an attenuated strain of Shigella dysenteriae.
Additional embodiments of the invention include a vaccine as described above, wherein one or more of the live attenuated Shigella flexneri strains is engineered to express one or more enterotoxigenic Escherichia coli (ETEC) antigens. Examples of ETEC antigens include, but are not limited to coli surface antigens (CS)1, CS2, CS3, CS4, CS5, CS6, CS14, Colonization Factor Antigen 1 (CFA/1), eltA2eltB (LTB), a tip adhesin, and Shiga toxin B (StxB).
Embodiments of the invention also include vaccine compositions as described above, which further comprise a pharmaceutically acceptable carrier.
In addition, some embodiments relate a vaccine composition as described above, wherein the Shigella strains contain mutations in guaBA and sen.
An embodiment of the invention relates to a vaccine composition as described herein, comprising:
a. live attenuated S. sonnei, strain CVD 1233S, which expresses ETEC antigens CS2 and CS3;
b. live attenuated S. flexneri serotype 2a, strain CVD 1208S, which expresses ETEC antigens CFA/L and LA2 TB;
c. live attenuated S. flexneri serotype 3a, strain CVD 1213, which expresses ETEC antigens CS1 and CS5;
d. live attenuated S. flexneri serotype 6, strain CVD 1215, which expresses ETEC antigens CS4 and CS6;
e. live attenuated S. flexneri serotype 1b, strain CVD 1224, which expresses ETEC antigens CS14;
f. live attenuated S. flexneri serotype 7a, strain CVD 1242, which expresses one or more ETEC tip adhesin antigens; and
g. optionally, live attenuated S. dysenteriae, strain CVD 1254, which expresses ETEC antigens CFA/1 and LTB;
In other embodiments, the invention provides a method of vaccinating a subject in need thereof against diarrheal disease, comprising administering a vaccine composition as described above to the subject. Preferably, resides in an area where diarrheal disease is endemic and/or is a child of less than 5 years old.
Unless defined otherwise, all technical and scientific terms used 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 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.
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.
As used herein, the term “prophylaxis,” in the context of a disease, which 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 “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; 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.
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 of the 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.
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.
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 10 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.
As used herein, the terms “diarrhea,” “diarrheal disease,” or “dysentery” refer to an episode of three or more loose bowel movements or any number of loose stools containing blood in a 24-hour period, caused by bacterial infection.
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.
As used herein, the term “CF” refers to colonization factor, the most common of which include CFA/I.
As used herein, the term “CS” refers to coli surface antigen, a group of antigens from ETEC.
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.
As used herein, the term “ST” refers to heat stable enterotoxins, a group of peptide toxins produced by bacterial strains such as ETEC.
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 LT plus the B subunit.
As used herein, the term “ETEC” refers to enterotoxigenic Escherichia coli, a group of E. coli that produce special toxins that stimulate the lining of the intestine, causing them to secrete excessive fluid, leading to diarrhea.
As used herein, the term “ETEC tip adhesin antigen” refers to antigens from tip adhesins of ETEC. Adhesins are a type of virulence factor that facilitates adhesion of the pathogen, as step in pathogenesis.
As used herein, the term “StxB” refers to the E. coli toxin, Shiga toxin B subunit.
Decades of literature, including field trials and volunteer challenge studies support the protective efficacy of live attenuated vaccines, which present the entire antigenic repertoire of the pathogen and which are delivered via the most immunologically relevant, oral route. Clinical trials demonstrated that mutations in the guaBA operon, that render Shigella unable to synthesize guanine nucleotides de novo, resulted in vaccine strains that were safe and immunogenic in volunteers. Additional mutations in the set and sen genes, encoding Shigella enterotoxins, can extend the attenuation of the vaccine strain, CVD 1208S. This attenuating strategy to additional Shigella component strains is used here to form a multivalent vaccine which confers broad protection. Thus, a multivalent formulation composed of 6 live attenuated strains of Shigella expressing ETEC colonization factor antigens and antigens to induce toxin neutralizing antibodies against heat labile (LT) and heat stable (ST) toxins is being developed.
Strategies for improvement of coverage against circulating Shigella isolates were developed here in order to extend direct coverage by the addition of attenuated derivatives of S. flexneri 1b and 7a. S. flexneri 1b accounted for 7.5% of all Shigella isolates in GEMS and expresses type I antigen. The inclusion of this important serotype increases direct coverage of the vaccine. S. flexneri 7a (formerly named 1c), accounted for 2% of all GEMS Shigella isolates overall and has been identified as the predominant serotype in other epidemiological studies. This serotype was included in the inventive vaccine because it has a unique O-antigen structure that does not include type- and group-specific antigens found on the other serotypes and therefore is not expected to be covered by any previous vaccine.
A live attenuated derivative of S. flexneri 1b was developed. The vaccine derivative of S. flexneri contains a deletion in the guaBA operon that is auxotrophic for guanine, and is defective in intracellular replication, attenuated for virulence in guinea pigs and immunogenic following immunization in guinea pigs (100% immunized animals exhibited a 2-7 fold rise in serum anti-S. flexneri 1b IgG following a single immunizing dose). In some embodiments, a deletion in set and/or sen can be added to this strain to enhance safety.
ETEC attach to the lining of the human small intestine by means of protein colonization factors (CFs), after which bacterial toxins stimulate intestinal secretion resulting in diarrhea. CFs from ETEC are critical antigens and targets for vaccine antigens to induce antibodies that block colonization and disease. Isolates of ETEC express antigenically distinct fimbrial types. A vaccine with broad coverage against ETEC therefore should include multiple fimbrial types. The major fimbrial antigens previously recognized include CFA/I and CS1 through CS6. 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. The only minor CF statistically associated with diarrhea in the GEMS study was CS14 (Vidal 2019). Addition of a CS14 antigen in a vaccine that includes the major colonization factors extends coverage of the vaccine.
In some embodiments, the invention provides a vaccine with broad protection against a high percentage of ETEC isolates by including a minor CF (CS14) to induce antibodies against CS14. In some embodiments, this is a live attenuated Shigella strain (e.g., CVD 1208S) expressing CS14. In some embodiments, CVD 1208S::CS14 is engineered by insertion of an operon encoding CS14 into the chromosome of a Shigella strain (e.g., CVD 1208S). Immunization of guinea pigs with CVD 1208S::CS14 induced antibodies that recognize CS14. In some embodiments, the vaccine produces mucosal SIgA Shigella anti-O and ETEC anti-CF and anti-LT antibodies.
Epidemiological data from the GEMS study revealed that a previously under-recognized minor fimbrial antigen from ETEC, CS14, was prevalent in isolates from children with moderate-to-severe diarrhea (MSD). Importantly, this was the only minor fimbrial antigen that was significantly associated with MSD. Inclusion of this fimbrial antigen in the instant invention vaccine would broaden the coverage of ETEC isolates.
The Shigella vaccine strategy of the present invention includes using a combination of a live attenuated strain of Shigella that represents the most prevalent species and serotypes found in clinical isolates. The vaccine includes S. sonnei (1 serotype) and S. flexneri serotypes 2a, 3a, and 6. These three S. flexneri serotypes were identified as expressing each of the type- and group-specific antigens found on the other 16 flexneri serotypes. Animal data supports cross protection between these serotypes. Two additional S. flexneri serotypes, 7a (previously referred to as serotype Ice) and 1b, were identified as among the most prevalent in the GEMS study. S. flexneri 7a has a unique O-antigen that does not express type- and group-specific antigens found on the other serotypes. Including a live attenuated derivative of S. flexneri 7a in the vaccine also broadens the overall coverage against Shigella isolates. Additionally, S. flexneri serotype 1b also would broaden protection since this serotype was found on 7.5% of Shigella isolates in the GEMS.
The genes encoding ETEC fimbrial antigen CS14 have been cloned from wild type ETEC strain into an expression plasmid. Using AttTn7 methodology developed by McKenzie and Craig for E. coli, the operon encoding CS14 was inserted in a permissive site on the chromosome of Shigella vaccine strain CVD 1208S forming CVD 1208S::CS14. Immunization of guinea pigs with CVD 1208S::CS14 induced antibodies that recognize CS14. Alternatively, the fimbrial tip adhesin of CS14, CsuD, can be expressed on the surface of Shigella live vectors to induce responses against this subunit which is responsible for host cell binding. The gene encoding the tip adhesin, csuD was engineered in tandem with the structural subunit gene, csuA2 on a plasmid. The plasmid directed expression of the CS14 tip adhesin in Shigella vaccine strain CVD 1208S. Immunization of guinea pigs with CVD 1208S (pCsuA2D) induced antibodies that recognized CS14. Purified tip proteins delivered parenterally can be used.
Analysis of the distribution of Shigella serotypes associated with diarrhea from the GEMS study identified S. flexneri 1b and 7a as serotypes that would increase the coverage of our multivalent Shigella vaccine. The new live attenuated Shigella flexneri strain of serotype 1b was engineered using selected strains (103489 and 204584) from the GEMS collection with antibiotic susceptibility. Deletions into the guaBA and sen genes were introduced using lambda red recombination (
S. flexneri 1b 103849ΔguaBA
S. flexneri 1b 204584ΔguaBA
S. flexneri 1b 204584ΔguaBA
The new live attenuated Shigella flexneri strains of serotype 1b and 7a can be engineered using selected strains from the GEMS collection with antibiotic susceptibility. Deletions into the guaBA and sen genes would be introduced. Including S. flexneri 1b and 7a increases direct Shigella coverage in the combined vaccine. The entire CS14 encoding operon can be used to express full length fimbriae. In the alternative, for the tip adhesin expression system, the gene, csuD, encoding the tip subunit only, can be used. Individual strains can be lyophilized, combined into a single combined formulation into a sachet and resuspended in buffer prior to oral delivery (as for CVD 103HgR, VAXCHORA™). See Table 2, below, for a summary of the overall formulation for a selected multivalent vaccine.
Shigella Vaccine, Strain
S. sonnei
S. flexneri 2a
S. flexneri 3a
S. flexneri 6
S. flexneri 7a
S. dysenteriae
A multivalent Shigella-ETEC vaccine of the invention includes 6 live attenuated strains of Shigella (top six lines of Table 2) expressing a total of at least 8 CFs plus LTA2B, and optionally also a live attenuated S. dysenteriae strain expressing StxB. Table 2 shows an example of how the combinations of Shigella vaccine strains and ETEC antigens can be distributed according to embodiments of the invention. However, any live attenuated Shigella strain can be used to express any of the combinations of CFs shown. Any live attenuated Shigella vaccine also can be used to express a single CF.
Another approach to broaden coverage of an ETEC vaccine is based on formulating a mix of fimbrial tip adhesin proteins. Fimbrial CFs can be classified based on the amino acid sequence relatedness of their tip adhesin proteins with several important ETEC CFs falling into Class 5 fimbriae assembled by the alternate chaperone pathway. Whereas the major fimbrial subunit proteins that create the stalks of these fimbriae differ substantially from one another antigenically, their tip adhesin proteins are highly conserved into three sub-classes. Antibody against one adhesin of the subclass cross protects against attachment by other members. Thus, protection may also be broadened by this strategy. Selecting which tip adhesins to include in a multivalent vaccine requires knowing the frequency of the CFs among ETEC globally; so GEMS data inform this vaccine strategy as well. Another strategy to broaden ETEC vaccine coverage is to include non-fimbrial surface antigens, e.g., EtpA and EatA.
Analyzing the array of CFs among GEMS ETEC isolates has provided important information to guide ETEC vaccine development and future deployment. Since ST-only and LT/ST strains are strongly incriminated as the key ETEC pathogens, a fimbrial-based ETEC vaccine that included CFA/I, CS1-6 and CS14 can confer increased protection.
Vaccines
The vaccines of the invention are designed to prevent and ameliorate diarrheal disease and dysentery by immunizing a subject in need for a group of antigens associated with these diarrheal diseases. The vaccines according to this invention contain a live attenuated strain of Shigella flexneri serotype 7a; a live attenuated strain of Shigella 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 and E. coli.
Advantageously, the vaccines are at least pentavalent or hexavalent, combining a mixture of 5-6 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 1b, and S. flexneri strain 7a are mixed together. The vaccine most preferably contains at least S. flexneri strain 1b, and S. flexneri strain 7a, and also contains S. flexneri strain 2a, S. flexneri strain 3a, and S. flexneri strain 6. Preferably, the vaccines provide broad coverage for the Shigella antigens and the common serotypes that are 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 species, 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.
The Shigella strains CVD 1233S, CVD 1208S, CVD 1213, CVD 1215, CVD 1224, CVD 1242, and CVD 1245 are made and stored at the Center for Vaccine Development at the University of Maryland. See Tables 1 and 2. Other strains may be available to the person of skill in the art.
The ETEC antigens can be expressed in one or more of the Shigella strains in the multi-valent vaccine or in all of them. The ETEC antigens useful for the vaccine compositions 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/1), eltB (LTB), a tip adhesin, and Shiga toxin B (StxB). Any, or preferably all, of these antigens are expressed in at least one of the Shigella strains in the vaccine composition. In particular, CS14 is present in the vaccine, and CFA/I, CS1, CS2, CS3, CD5 and CD6 also 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 and can be found, for example in Wu et al., Infect. Immun. 79(12):4912-4922, 2011 PMC3232646.
The vaccines are live attenuated strains of Shigella, and therefore preferably have been modified so that virulence is reduced 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. 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 strains 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.
A certain embodiment of the vaccine compositions of the invention comprises live attenuated S. sonnei, strain CVD 1233S, which expresses ETEC antigens CS2 and CS3; live attenuated S. flexneri serotype 2a, strain CVD 1208S, which expresses ETEC antigens CFA/l 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 1215, which expresses ETEC antigens CS4 and CS6; live attenuated S. flexneri serotype 1b, strain CVD 1224, which expresses ETEC antigens 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.
Vaccine Compositions
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. A pharmaceutically acceptable carrier refers to any convenient compound or group of compounds 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 carriers 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.
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 vaccines of the invention preferably are administered or delivered by the oral route. Alternatively, the strains can be inactivated, using formalin, heat, or some other agent.
In some embodiments, an inactivated multivalent Shigella-ETEC vaccine would be administered by the oral route or intradermal, subcutaneous, sublingual, nasal, or intramuscular routes. Other routes of administration can include, but are not limited to local or parenteral, including: oral, intravenous, intra-arterial, intrathecal, subcutaneous, intradermal, intraperitoneal, intramuscular, or local injection; rectal or vaginal suppository, topical, nasal, buccal, transdermal, sublingual, inhalation, transmucosal, wound covering, and the like. The administration can be given by transfusion or infusion, and can be administered by an implant, an implanted pump, or an external pump, or any device known in the art. Preferably, the vaccines of the present invention are administered orally, by intramuscular injection or by nasal administration. Oral administration of the vaccine is an advantageous route of administration because it is non-invasive, needing no needle and syringe, or special equipment.
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.
Any pharmaceutically acceptable carrier and/or excipient known in the art is contemplated for use with the invention. Carriers can include, for example, solvents, diluents, other carriers, and the like to contain the active ingredient(s) in solid, liquid, or gas form. Common carriers in solid form include starch (e.g., corn 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 of semi-liquid form include gels, lipids (e.g., lipid vesicles or nanoparticles), oils, polyethylene glycols, glycerine, 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.
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 (e.g., benzyl alcohol, methyl parabens, and the like), antioxidants (e.g., ascorbic acid, sodium bisulfite, and the like), chelating agents (e.g., EDTA and the like), glidants (e.g., colloidal silicon dioxide),
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.
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.
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.
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 buffer solution. An additional embodiment includes a multi-dose package of individual dosage sachets, such as a 5-dose or a 10-dose presentation.
Doses and Regimens
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 give 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. A dose of vaccine contains about 108 colony forming units (CFU) to about 5×1010 CFU dry attenuated bacteria, or as recommended by medical personnel. Doses may vary from about 107 CFU to about 1011 CFU, preferably about 108 CFU to about 5×1010 CFU, or about 5×108 CFU to about 1010 CFU or about 108 CFU to about 109 CFU, or any dose suitable as determined by the practitioner, depending on the size, exposure, general health, age, or other factors.
Subjects
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 serotypes and 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 are 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.
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.
This invention is not limited to the particular processes, compositions, or methodologies 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. 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. 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.
Study Design and Population.
The rationale, assumptions, clinical, epidemiological and microbiological methods of GEMS, a 3-year case-control study undertaken among children less than 5 years of age in Gambia (Basse), Mali (Bamako), Mozambique (Manhiga) and Kenya (Siaya County) in sub-Saharan Africa and India (Kolkata), Bangladesh (Mirzapur) and Pakistan (Karachi-Bin Qasim Town) in South Asia, have been described. MSD was defined as an acute episode of diarrhea (3 or more loose stools during a 24-hour period) that started within the previous 7 days, was separated from another episode by 7 or more days, and was accompanied by either signs of dehydration (sunken eyes, slow recoil after an abdominal “skin pinch” or administration of intravenous fluids), dysentery or admission to hospital based on clinical concern of diarrheal disease severity.
Procedures
Stools or rectal swabs from cases and controls were cultured onto MacConkey and xylose/lysine/deoxycholate agar and three E. coli colonies per subject were identified and pooled for extraction of DNA. The DNA then was tested by a multiplex PCR containing primers to amplify eltB (LTB subunit) and est (ST). ETEC strains were shipped to the University of Chile and confirmed by PCR to detect LT and ST variants (STh and STp). Confirmed ETEC isolates were further tested by monoplex or multiplex PCRs using primers that detect target genes encoding the major CFs (CFA/I, CFA/II [CS1, CS2, CS3], CFA/IV [CS4, CS5, CS6]), and various minor CFs (CS7, CS12, CS13, CS14, CS17, CS18, CS19, CS20, CS21 and CS30) (Table 3, below).
Reference strains served as positive controls for specific CFs. Crude bacterial lysate was obtained by boiling 5 pooled colonies of each ETEC isolate in 0.1% TRITON™ X-100 (detergent) for 10 minutes, followed by centrifugation at 8000×g for 5 minutes to separate the template DNA in the supernatant from the cellular debris. PCR was performed with total bacterial DNA in a 25-μL reaction, containing 10 mmol/L deoxyribonucleotide triphosphate (dNTP) mix, 30 mmol/L MgCl2, x reaction buffer (10 mmol/L Tris-HCl, 50 mmol/L KCl), 1 U of Taq polymerase (GoTaq; PROMEGA™, Madison, Wis.), and 1 μL of template DNA. Primers were used at concentrations shown in Table 3, below. To prevent nonspecific amplification, the “hot start” technique was used. This includes preheating the reaction mixture to 94° C. for 5 minutes before adding Taq DNA polymerase. Samples were amplified for 35 cycles, with each cycle comprising 90 seconds at 94° C. for denaturation, 30 seconds at specific primers annealing temperatures, 60 seconds at 68° C. for strand elongation, and a final extension at 72° C. for 5 minutes. PCR products were electrophoresed in 2.0% agarose, stained with ethidium bromide, and amplicons identified based on expected size of the amplified product and compared with amplicons of reference strains.
Isolates were agglutinated with specific anti-O antisera. A subset of isolates were tested at the University of Gothenburg, Sweden to assess phenotypic expression of CFs using monoclonal antibodies.
Data Analysis
Analyses were restricted to ETEC cases that had a single ETEC toxin/CF genotype pattern. Prevalence of ETEC CFs was expressed as percentages in a stratified analysis by ETEC toxin profile, site and region. Strength of association between ETEC toxin and CF genotypes and MSD was examined using conditional logistic regression models in which the outcome was case-control status (MSD) and the independent variable (covariate) was whether the child's ETEC had the specific CFA (no or yes), while applying Firth's penalized likelihood approach. Matched odds ratios (ORs) and corresponding 95% confidence intervals (CIs) were obtained from these models. Pooled and site-specific analyses were conducted. Heterogeneity in ORs across the sites was examined using Chi square test for heterogeneity. A p value ≤0.05 was considered significant. Data were analyzed using SPSS version 23 (IBM™, Inc.) and SAS statistical software version 9.4 (SAS INSTITUTE™ Inc. Cary, N.C., USA).
Table 4, below, summarizes the proportion of ETEC strains that carry the major CF antigens including CFA/I and CS1-CS6, with data presented by country, continent and toxin genotype. Overall, 363 (66.2%) of 548 ST-only and LT/ST strains encoded a major CF antigen including 20.4% encoding CFA/I, 14.1% encoding CFA/II (i.e., CS3 alone or with CS1 or CS2) and 31.8% encoding CFA/IV (i.e., CS6 alone or with CS4 or CS5). The only major CF commonly observed among LT-only isolates was CS6-only, which was recorded in 44 of 258 LT-only strains (17.1%). Only 3 of 258 LT-only strains (1.2%) encoded CFA/I or CFA/II.
Among a subset of 123 ETEC isolates from MSD cases shown by PCR to encode major CFs, 96 phenotypically expressed the CF surface antigens detected by dot blot immunoassays. These included 24/26 CFA/I (92.3%), 4/7 CSI (57.1%), 10/11 CS2 (90.9%), 13/17 CS3 (76.5%), 19/23 CS5 (82.6%) and 26/39 CS6 (66.7%) isolates; no isolates encoding CS4 were tested.
aCFA/II strains are defined as encoding CS3 either alone or in combination with either CS1 or CS2 but never both CS1 and CS2. Very rarely isolates that encode CS1 without CS3 have been reported.19 The rare CFs of this nature recovered in GEMS are not included in this table.
bCFA/IV strains are defined as encoding CS6 either alone or in combination with either CS4 or CS5, but never both CS4 and CS5. Very rarely isolates that encode CS5 without CS6 have been reported. The few such isolates recovered in GEMS are not included in this table
Recognizing that 33.8% of ST-only and LT/ST strains and about 75.2% of LT-only strains do not encode a major CF, the proportion of those isolates that encoded solely one of the following characterized minor CF antigens: CS7, CS12, CS13, CS14, CS17, CS18, CS19, CS20, CS21 or CS30 was investigated. The proportion of ETEC MSD cases that had isolates encoding one of these minor CFs in the absence of a major CF and that accounted for ≥5.0% of the overall case isolates of that toxin genotype (Table 5) was determined. Among MSD cases with ST-only ETEC, only CS14, identified in 58 ST-only cases (19.9%), reached a prevalence of ≥5% (Table 5); seven of 81 cases with LT/ST isolates lacking major CFs (2.7%) also encoded CS14 alone. When encoded as the sole CS, the other minor CS antigens were uncommon (<5%) among ST-only and LT/ST isolates. See Table 5, below.
a1Percent of 502 ST-only plus LT/ST strains;
bPercent of all 258 LT-only strains
cPercent of 252 LT-only strains (6 strains were not recoverable from −70° C. storage for testing);
c1Percent of 225 LT-only strains;
dPercent of all 291 ST-only strains;
ePercent of 290 ST-only strains (1 strain was not recoverable from −70° C. storage for testing);
e1Percent of 266 ST-only strains;
fPercent of all 257 LT/ST strains;
gPercent of 255 LT/ST strains (2 strains were not recoverable from −70° C. storage for testing);
g1Percent of 236 LT/ST strains;
hPercent of all 548 ST-only plus LT/ST strains;
iPercent of 545 ST-only plus LT/ST strains (3 strains were not recoverable from −70° C. storage for testing).
Among ST-only and LT/ST ETEC strains encoding a single minor CF but no major CFs, only CS14 was significantly associated with MSD (Table 6).
aThere was no significant heterogeneity across GEMS sites (by chi square test for heterogeneity);
b1ST-CS3 only was not detected in Bangladesh.
b2ST-CS4 & SC6 was not found Gambia, Mozambique and Bangladesh;
b3ST-CS7 only was restricted to Gambia and India;
b4ST-CS12 only was not found in Mozambique and LT only-CS12 was not found in Gambia;
b5ST-CS13 only was not found in Mali and Pakistan;
b6LT only-CS14 only was not found in Mali and Bangladesh;
b7ST-CS17 only was found only in Mozambique and Bangladesh;
b8ST-SC18 only was not found in Pakistan and LT only-CS18 only was found only in Mali, Kenya and Pakistan;
b9ST-CS19 only was detected in Mali, India and Bangladesh, and LT-only-CS19 was found only in Gambia and India;
b10LT-only CS21 only was detected only in Kenya and Pakistan;
cExcludes strains that co-encoded a major colonization factor such as CFA/I, CFA/II, CFA/IV, and all other minor colonization factors.
Genomic DNA isolated from wild type (WT) S. flexneri 1b strain 103849 or 204584 was used as a template with primers Wu048 (GTGAAGGTGAAGCCCGTGAAGT; SEQ ID NO:41) and Wu049 (TGCAGCAGCATTGCGGTTACG; SEQ ID NO:42) to amplify the intact guaBA locus as a 1.8 kb band. Amplification with genomic DNA from the ΔguaBA mutant derivatives (2, 7, 9, 14, 2, 8, 11, 12, and 13) result in an 891 bp band reflecting the 900 bp deletion. NTC: no template controls. See
To confirm guanine auxotrophy in the S. flexneri 1b ΔguaBA strains, wild type or mutant derivative strains were grown on minimal M9 media plus 0.5% casamino acids with (panels B, D, and F of
To confirm intracellular replication defect in S. flexneri 1b ΔguaBA vaccine strains, wild type or mutant derivative strains were applied to monolayers of HT-29 cells for 90 minutes, washed with gentamicin, and incubated for an additional 30 minutes (0 hour) or 2 hours. Cells were lysed and intracellular bacteria quantitated. Mean CFU/mL from triplicate wells is shown for each strain in
The lambda red system described in Datsenko and Wanner, Proc. Natl. Acad. Sci. 97(12):6640-6645, 2000 was used to introduce 3 deletion mutations into two different S. flexneri 1b strains (this can be done in any S. flexneri 1b strain). Using this deletion system, fragments of DNA flanking the region to be deleted were amplified by PCR and fused to an antibiotic resistance encoding gene which is flanked by FRT sequences. (The accessory plasmids were obtained from Datsenko and Wanner). This fragment is electroporated into S. flexneri 1b which contains a helper plasmid that catalyzes recombination between the flanking regions on the fragment with genomic copies. The result is S. flexneri with FRT-antibiotic resistance gene-FRT in the genome in place of the sequences targeted for deletion (e.g., guaBA, or sen or set). The antibiotic resistance marker then is deleted by introducing a second helper plasmid that catalyzes recombination between FRT sites. The result is S. flexneri 1b with a deletion in guaBA, sen and set (performed in 3 separate steps) with no antibiotic resistance markers.
Live attenuated Shigella vaccines (S. flexneri serotypes 1b) was engineered to contain a deletion in the guaBA operon. The deletion of the enterotoxin encoding gene sen or set can be added to Shigella strains. Live attenuated S. flexneri 7a vaccine strain also can be engineered with deletions in guaBA and sen. Deletions can be introduced into wild type strains using allelic exchange or using lambda red recombination.
The operon encoding ETEC colonization factor CS14 was amplified using PCR from a wild type ETEC strain and cloned into a commercial cloning plasmid (pBAD). A modification of the Tn7 system is described by McKenzie and Craig (McKenzie 2006). The operon was subsequently cloned from pBAD using restriction digestion, into the plasmid pGRG25-PmLpp. Plasmid pGRG25-PmLpp is a modification of the pGRG25 plasmid into which the mLpp promoter has been inserted. The mLpp promoter drives high level constitutive expression of cloned downstream genes.
Introduction of pGRG25-PmLpp-CS14 into S. flexneri live attenuated strain CVD 1208S was performed to facilitate transposition of the CS14-encdong operon into the Shigella chromosome at the attTn7 site downstream from glmS. CVD 1208S::CS14 was confirmed using PCR to amplify the chromosomal region and by sequencing the chromosomal insertion. CS14 also can be inserted into the chromosome of any of the live attenuated Shigella vaccine strains by the same methods.
In a second strategy for inducing antibodies to block binding of ETEC expressing CS14, the gene encoding the structural subunit, csuA, was cloned in tandem with the gene encoding the tip adhesin, csuD, downstream. The csuA and csuD genes are not fused; each contains an independent promoter and they are transcribed and translated as individual proteins. The goal was to stabilize tip adhesin expression with the presence of the structural subunit. The two genes were cloned into the expression plasmid pBAD in which gene expression is induced with the addition of arabinose. The plasmid pBAD-CsuA2D, was introduced into Shigella vaccine strain CVD 1208S by electroporation.
Currently there are no licensed vaccines against Shigella or ETEC. Other single pathogen vaccine candidates are in various levels of clinical trials but none supply the broad coverage that will be provided by this vaccine. Five putative major vaccine markets are contemplated for vaccines according to the invention:
All references listed below and throughout the specification are hereby incorporated by reference in their entirety.
This application is a 371 national stage application of PCT Application No. PCT/US19/25602, filed Apr. 3, 2019, and claims the benefit of U.S. provisional application Ser. No. 62/651,973, filed 3 Apr. 2018. The entire contents of this application is hereby incorporated by reference as if fully set forth herein.
This invention was made with government support under grant nos. AI 109776 and AI 142725, awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
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| PCT/US2019/025602 | 4/3/2019 | WO |
| Publishing Document | Publishing Date | Country | Kind |
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| WO2019/195437 | 10/10/2019 | WO | A |
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| 20150026693 | Savarino | Sep 2015 | A1 |
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| WO2016202872 | Dec 2016 | WO |
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| 20210121553 A1 | Apr 2021 | US |
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| 62651973 | Apr 2018 | US |
