Attenuated maturation deficient chlamydia vaccine

Abstract

The use of a novel plasmid-dependent mediated expression (DOPE) technology proves that conditional depletion of the chlamydia specific protein GrgA can lead to a great reduction in the reticulate (RB) proliferation rate and almost completely lack of the formation of the EB, thereby destroying the normal chlamydia development cycle. This conditioned GrgA-deficient Chlamydia permits the study of Chlamydia growth and development modulation and can be used as a basis for attenuated, mature deficient but immunogenic bacteria as a non-toxic vaccine against Chlamydia.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is an international application of U.S. Patent Application No. 63 / 362,120 filed on March 29, 2022. The entire contents of this application are incorporated herein by reference as if fully set forth herein.

[0003] Government funding

[0004] This invention was made with government support under Grant Nos. AI140167 and AI154305 awarded by the National Institutes of Health. The government has certain rights in the invention. Background of the Invention 1. Technical Field

[0006] The present invention relates to the fields of medicine and biology, in particular to the study of Chlamydia. Specifically, the present invention relates to a method for interrupting the developmental cycle of Chlamydia by conditional knockout of the gene grgA encoding the GrgA protein, thereby producing attenuated, maturation-defective Chlamydia suitable for use as a vaccine against Chlamydia diseases in humans and animals. 2. Background technology

[0007] Chlamydia is the most common sexually transmitted bacterial infection in humans worldwide. It can cause serious consequences, including increased discharge, burning sensations, and swelling of the testicles, as well as pelvic inflammatory disease, ectopic pregnancy, and infertility in both men and women. Symptoms can also appear in the anus, eyes (sometimes leading to blindness), throat, and lymph nodes. Many people infected with Chlamydia have no symptoms, or symptoms may not appear until after an incubation period of several weeks. Chlamydia also causes community-acquired respiratory infections in humans and can be a risk factor for cardiovascular disease and age-related neurodegenerative diseases. A vaccine would be particularly useful because infected people may not realize they are infected.

[0008] Chlamydia is also a ubiquitous pathogen in animals, including important commercial livestock, protected wild animals and other animals, including but not limited to cattle, pigs, sheep, goats, guinea pigs, birds (poultry), cats, mice, rabbits and snakes. However, there is no chlamydia vaccine for human use, and the effectiveness and safety of animal chlamydia vaccines are still uncertain. In addition, there is no antibiotic that selectively inhibits Chlamydiae at present. Therefore, there is a need for a vaccine according to the present invention in this area.

[0009] Chlamydiae are obligate intracellular bacterial parasites with a unique developmental cycle that includes two cell forms. The first is the elementary body (EB), which is infectious but does not divide and can survive temporarily in the extracellular environment. The second is the reticulated body (RB), which is proliferative but not infectious and replicates inside the host cell. The infectious cycle of the obligate intracellular bacterium Chlamydiae begins when the EB of the obligate intracellular bacterium Chlamydia enters a eukaryotic host cell. Within a vacuole ("inclusion") in the host cytoplasm, the EB differentiates into the proliferative but non-infectious RB. After repeated rounds of replication, as RB accumulates within the inclusion, the RB redifferentiates into the non-dividing EB, which then leaves the host cell to infect other host cells or spread to new hosts. See Figure 1A As shown in the figure, EB and RB are shown as small circles and large circles, respectively.

[0010] The developmental cycle of Chlamydia is regulated by transcription. After EB enters the host cell, its early genes are activated within the first few hours, allowing primary differentiation to RB. From about 8 hours after infection, the mid-cycle genes, which account for the vast majority of all Chlamydia genes, begin to be expressed, allowing RB replication. About 24 hours after infection, the late genes are activated, allowing secondary differentiation of RB back to EB.

[0011] As subunits of the RNA polymerase (RNAP) holoenzyme, sigma factors recognize and bind to specific DNA gene promoter elements, allowing RNAP to initiate transcription. Chlamydia encodes three sigma factors, called σ 66 , σ 28 and σ 54 σ 66 RNAP holoenzyme is involved in the expression of early, intermediate and late genes, while σ 28 RNAP and σ 54 RNAP is responsible for transcribing only a subset of late or mid-late genes. A few genes have tandem promoters, and their expression is regulated by multiple sigma factors.

[0012] The understanding of the mechanisms of Chlamydia development has been hampered by the lack of powerful genetic tools to knock out essential genes. Therefore, it is important to develop convenient methods to knock out these genes to identify their biological functions in Chlamydia growth and development. Summary of the invention

[0013] Thus, the information disclosed herein shows the generation of knockout Chlamydia that do not express grgA and are maturation-defective, thereby producing avirulent bacteria that can be used for research and vaccine production. Specifically, the present invention relates to a knockout Chlamydia that does not express GrgA. Preferably, the knockout Chlamydia is selected from the group consisting of: C. trachomatis, C. pneumoniae, C. psittaci, C. muridarum, C. suis, C. abortus, C. felis, C. pecorum, C. ibidis, C. avium, C. gallinacea, etc. Most preferably, the gene knockout Chlamydia is selected from the group consisting of Chlamydia trachomatis, Chlamydia pneumoniae and Chlamydia psittaci to prevent human chlamydial diseases and zoonotic chlamydial diseases.

[0014] The present invention also relates to a vaccine comprising the above-mentioned gene knockout Chlamydia and a pharmaceutically acceptable carrier. The present invention also relates to a method for stimulating an immune response to Chlamydia in a subject in need thereof, comprising: administering the above-mentioned vaccine to the subject. The present invention also relates to a method for producing neutralizing antibodies to Chlamydia in a subject in need thereof, comprising: administering the above-mentioned vaccine to the subject.

[0015] In another aspect, the invention relates to a method for determining whether a bacterial gene in a bacterial cell is essential for its growth or development, the method comprising: (a) determining the growth or development of the bacterial cell when the gene is intact and expressed; (b) disrupting the gene and determining the growth or development of the bacterial cell when the gene is ineffective; (c) introducing a plasmid containing an inducible version of the bacterial gene into the bacterial cell having the disrupted bacterial gene, and determining the growth or development of the bacterial cell when the bacterial gene is induced and when the bacterial gene is not induced. Preferably, the bacterial cell is a Chlamydia spp. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1A The developmental cycle of wild-type Chlamydia is shown.

[0017] Figure 1B The variation cycle of the grgA gene knockout Chlamydia mutant is shown.

[0018] Figure 2 The domain structure of the GrgA protein is shown.

[0019] Figure 3The underlying principle of the DOPE technique is shown, which allows physiological and mechanistic interrogation of essential genes encoded by chromosomes. Black and gray lines represent chromosome and plasmid sequences, respectively. Half arrows indicate transcription start sites. EG: essential gene; iEG: inducible EG; chr: chromosome; NA: not applicable; g: growth; d: development.

[0020] FIG. 4A to FIG. 4E It involves confirmation of the disruption of chromosomally encoded grgA by a group II intron and re-expression of GrgA from a transformed plasmid in the DOPE technique. Figure 4A A set of schematic diagrams of the grgA alleles, the locations of the intronic target sites, diagnostic primers and the sizes of PCR products obtained with different primer sets. Abbreviations: itsm: mutated intronic target site; Chr: chromosome; bp: base pair. Figure 4B is used Figure 4A Gel images of PCR products amplified by the indicated primer sets with DNA of wild-type C. trachomatis L2 (L2 / cg), L2 / cg transformed with the his-grgA-itsm expression plasmid (L2 / cg-peig), and L2 with an aadA disruption and complemented with chromosomal grgA of peig (L2 / cgad-peig). Figure 4C Sanger sequencing traces of PCR products are shown, showing the intronic targeting site in L2 / cg, the mutations around this site that confer resistance to intronic targeting in peig, and the grgA intronic junction region in the chromosome of L2 / cg-peig. Arrows and asterisks indicate wild-type bases and corresponding mutant bases, respectively. Figure 4D The Western blot shows that only chromosomally encoded GrgA was detected in L2 / cg-peig cultured in medium without ATC, and both chromosomally encoded GrgA and plasmid-encoded His-GrgA were detected in L2 / cg-peig cultured in medium containing ATC for 14 hours. Figure 4E is a Western blot showing the time-dependent loss of plasmid-expressed GrgA in L2 / cg-peig following ATC withdrawal.

[0021] FIG. 5A to FIG. 5E Transmission electron microscopy images (TEM) are presented, showing the lack of EB formation at different late developmental points in L2 / cgad-peig cultured in medium without ATC. Figure 5A and Figure 5B TEM images of L2 / cGad-PEIG cultured in medium without ATC and medium with 1 nM ATC at 35 hours after inoculation, respectively. Figure 5C and Figure 5DTEM images of L2 / cGad-PEIG cultured in medium without ATC and medium with 1 nM ATC at 45 hours after inoculation, respectively. Figure 5E TEM images of L2 / cGAD-PEIG cultured in medium without ATC 60 hours after inoculation.

[0022] Fig. 6A Schematic representation of the mechanism of GrgA deficiency by TetR repression in L2 / cgad-peig cultured in ATC-free medium. Figure 6B Shown are mutations identified in the tetR gene in plasmids isolated from L2 / cg-peig EBs formed in ATC-free medium. Shown are the wild-type nucleotide sequence (SEQ ID NO: 17), wild-type amino acid sequence (SEQ ID NO: 18), mutant nucleotide sequence (SEQ ID NO: 19), and mutant amino acid sequence (SEQ ID NO: 20). Figure 6C Schematic representation of the loss of grgA repression in L2 / cg-peig EBs formed with ATC-free medium. DETAILED DESCRIPTION

[0023] 1. Definition

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art are generally understood. Although various methods and materials similar or equivalent to the methods and materials described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. However, it is understood by those skilled in the art that the methods and materials used and described are examples and may not be the only methods and materials applicable to the present invention. In addition, due to the inherent changes in measurement, unless explicitly stated to the contrary, any temperature, weight, volume, time interval, pH, salinity, molar concentration or amount of substance concentration, range, concentration and any other measured value, amount or digital expression given herein are intended to be approximate, rather than accurate or critical numbers.

[0025] As used herein, the term "about" means plus or minus 20 percent of the listed value, so that, for example, "about 0.125" means 0.125±0.025, and "about 1.0" means 1.0±0.2.

[0026] As used herein, the term "Chlamydial species" refers to any species in the genus Chlamydia or Chlamydophila, including but not limited to: Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia muridarum, Chlamydia suis, Chlamydia abortus, Chlamydia felis, Chlamydia ruminants, Chlamydia ibis, Chlamydia avium, Chlamydia poultry, etc.

[0027] As used herein, the term "administration" and its synonyms refer to the introduction of a medicament into a subject, and can be performed using various methods of administration or delivery systems known to those skilled in the art for administering a medicament, pharmaceutical composition, or vaccine. The modes of administration include, but are not limited to, nasal and oral administration, or intravenous, subcutaneous, intramuscular, or intraperitoneal injection, rectal or vaginal administration by suppository or enema, or topical administration directly into or into a target tissue (such as the eye), or by any route or method of delivering a therapeutically effective amount of a drug or vaccine composition to the cells or tissues to which it is targeted.

[0028] As used herein, the term "vaccine" means any biological material preparation containing or producing an antigenic substance, which, after being administered to a subject, causes the subject to actively acquire immunity to at least the pathogenic organism from which the antigenic substance is derived. Vaccines can be used for prophylactic or therapeutic delivery.

[0029] As used herein, the terms "treat", "therapeutic" and the like refer to obtaining a desired physiological effect. "Treatment" includes: (a) preventing or reducing the likelihood of a condition or disease or its symptoms in a subject who may be susceptible to the condition or disease but has not yet been diagnosed as having the condition or disease; (b) inhibiting the condition or disease or its symptoms, such as preventing or reducing its development; and (c) alleviating, relieving or ameliorating the condition or disease or its symptoms, such as, for example, causing regression of the condition or disease or its symptoms. Therefore, treatment refers to administration for the purpose of treatment. "Therapeutically effective amount" refers to an amount that produces a physiological response that improves an infection or its symptoms or eliminates an infection or its symptoms more quickly. Therefore, the amount also includes an amount that prevents or reduces the risk of pathological changes in individuals with asymptomatic infection.

[0030] As used herein, the terms "prophylactic," "prophylactically," and the like refer to preventative treatment, which may refer to the complete or partial prevention of an infection or disease condition. Prevention is a preventative measure used to reduce the likelihood or severity of a disease or condition, such as infection with a species of the genus Chlamydia. A "prophylactically effective amount" refers to an amount that induces an immune response, as described below. Such an amount preferably results in no disease upon subsequent exposure or infection, less severe disease upon subsequent exposure or infection, more rapid remission of disease in subsequent exposure or infection, or less chance of transmission of the organism to subsequent hosts.

[0031] As used herein, the phrase "induce an immune response" and its synonyms refer to inducing a physiological response of the subject's immune system to an antigen. An immune response may include an innate immune response, an adaptive immune response, or both. A protective immune response confers memory to a subject's immune cells, the effect of which is that, when exposed to the same or similar antigen for the second time, one or more of the following characteristics characterize the secondary immune response: a shorter incubation period than that resulting from exposure to the selected antigen without prior exposure to an immune composition (vaccine); the production of antibodies (preferably neutralizing antibodies) lasts longer than the production of antibodies resulting from exposure to the selected antigen without prior exposure to an immune composition; changes in the type and quality of antibodies produced compared to the type and quality of antibodies produced after exposure to the selected antigen without prior exposure to an immune composition; an increase in the average affinity (binding constant) of antibodies to the antigen compared to the average affinity of antibodies to the antigen resulting from exposure to the selected antigen without prior exposure to an immune composition; and / or other characteristics known in the art.

[0032] As used herein, "subject" refers to any animal, including humans. This includes humans, primates, farm animals (including cattle, horses, pigs, sheep, goats, etc.), laboratory animals such as rodents (including mice, rats, guinea pigs, etc.) and rabbits, etc., birds (such as chickens, turkeys, geese, ducks and other poultry), companion animals (including dogs, cats, etc.) and reptiles (including snakes, etc.). "Subjects in need thereof" refers to any subject suffering from chlamydia infection, suspected chlamydia infection, or optionally potentially susceptible to developing chlamydia infection.

[0033] 2. Overview

[0034] The developmental cycle of the obligate intracellular bacterium Chlamydiae is initiated by the entry of its protozoa (EB) into eukaryotic host cells. Within vacuoles (inclusions) in the host cytoplasm, EBs differentiate into proliferative but non-infectious reticulum bodies (RBs). After rounds of replication, the intracellular RB population asynchronously redifferentiates into non-dividing EBs, which then exit the host cell by cell lysis or cell extrusion. Understanding the mechanisms of Chlamydiae development has been hampered by the lack of powerful genetic tools to knock out essential genes.

[0035] Therefore, the Directed Expression (DOPE) technique was developed to allow functional and mechanistic interrogation of essential Chlamydia genes. This technique demonstrated that conditional ablation of the Chlamydia-specific protein GrgA resulted in a dramatic reduction in Chlamydia growth rate and an almost complete lack of RB to EB differentiation. As GrgA is essential for RB to EB conversion, GrgA-deficient Chlamydia are completely dependent on conditional re-expression of the GrgA protein from an engineered plasmid to complete the cycle of forming infectious EBs.

[0036] Since the RB in the mutant Chlamydia cannot differentiate back to the progeny infectious EB, the conditional GrgA-deficient Chlamydia is attenuated and has very low or no infectivity. Therefore, the present invention relates to a GrgA gene knockout that can prevent the formation of the infectious form of Chlamydia and methods of using it. This bacterial mutant can be used to study the growth and development regulation of Chlamydia.

[0037] In humans and animals, GrgA-deficient Chlamydia can only infect a limited number of host cells and cannot spread to other cells in the same host or to other hosts. However, maturation-deficient RB still elicits host immune responses against Chlamydia. GrgA-deficient Chlamydia are ideal attenuated vaccine candidates for humans and animals, and thus maturation-deficient bacteria can be used as non-toxic vaccines.

[0038] GrgA is a Chlamydia-specific transcriptional regulator identified by promoter DNA pull-down that binds σ 66 and σ 28 , and activates transcription of multiple Chlamydial genes in vitro and in vivo. RNA-Seq analysis of Chlamydia trachomatis conditionally overexpressing GrgA, combined with GrgA in vitro transcription assays, has allowed the identification of two additional genes encoding transcription factors, euo and hrcA, as members of the GrgA regulon. The immediate-early genes euo and hrcA are both transcribed during early and intermediate stages. Euo is a repressor of Chlamydial late genes, while HrcA regulates the expression of multiple protein chaperones that are essential for bacterial growth. These findings suggest that GrgA regulates early Chlamydial development.

[0039] To further define the role of GrgA in Chlamydia physiology, we attempted to identify the target group II intron. TM ) insertion mutagenesis to disrupt grgA was unsuccessful. Previously, saturation chemical mutagenesis also failed to generate a grgA null mutant. TM While chemical mutagenesis has been successfully used to disrupt many non-essential Chlamydial genes, our findings suggest that grgA is an essential gene. In this work, we have demonstrated that grgA is indeed an essential gene by developing and applying a novel genetic tool that we call DOPE (Dependent on Plasmid Mediated Expression). Importantly, we show that GrgA is necessary for RB to EB differentiation later in the developmental cycle and is further required for optimal RB growth. Thus, this report suggests that a single Chlamydial regulatory factor is required for the formation of progeny EBs.

[0040] 3. Summary of results

[0041] It was shown that the Chlamydia gene grgA is essential in the developmental cycle of Chlamydia.

[0042] A conditional knockout of C. trachomatis grgA was generated.

[0043] A new genetic tool (dependent plasmid-mediated expression (DOPE) technology) was developed for studying essential genes in Chlamydia.

[0044] DOPE technology showed that conditional knockout of GrgA gene resulted in slow growth of Chlamydia and lack of RB to EB differentiation.

[0045] 4. Implementation of the Invention

[0046] A. Introduction

[0047] The developmental cycle of the obligate intracellular pathogen Chlamydia is marked by the primary differentiation of the infectious body (EB) cell type into the proliferative reticulated body (RB) and the secondary differentiation of the RB into the EB. The detailed mechanisms regulating these transitions are unclear. In this study, we developed a novel strategy called DOPE (dependent on plasmid-mediated expression) that allows the knockdown of essential genes in Chlamydia. Importantly, we demonstrated that the Chlamydia-specific transcription factor GrgA is essential for the secondary differentiation of the RB into the EB. The conditional GrgA-deficient Chlamydia we developed will be valuable for future studies of Chlamydia growth, development, and pathogenicity. Furthermore, since EB formation is absolutely required for Chlamydia to spread within infected individuals and for Chlamydia to spread to new hosts, our maturation-deficient Chlamydia system could serve as an attractive attenuated vaccine methodology for the prevention of Chlamydial disease.

[0048] Currently known experimental vaccines are ineffective against species of human Chlamydia and are not approved for use in humans. Palet al. (2020) showed that vaccination with the major outer membrane protein of Chlamydia (MOMP; a highly conserved antigenic protein exposed on the surface present in both RB and EB) did induce protection in mice. Human clinical trial data can be viewed online at pubmed.ncbi.nlm.nih.gov / 31416692 and Abraham et al., Lancet 19(10):P1091-1100,2019. Chlamydia RB alone is ineffective as a vaccine because very large amounts of RB are inefficiently produced and RB of sufficient purity cannot be obtained without contaminating infectious EB. Therefore, the field needs new vaccines for this important pathogen.

[0049] In bacteria, all RNA synthesis is catalyzed by a single RNA polymerase (RNAP). The RNAP holoenzyme (RNAPholo) consists of a catalytic core (RNAPcore) and a sigma factor that recognizes the promoter sequence. Transcription factors regulate RNA synthesis by binding to DNA or both DNA and RNAP. A transcription factor named GrgA was identified. GrgA is a Chlamydia-specific protein expressed in both EB and RB. It binds sigma in vitro. 66 (main σ factor) and σ 28 (one of two alternative sigma factors) and activates sigma in vitro and in vivo 66 Dependent genes and σ 28 GrgA overexpression leads to increased transcription of many genes, including two immediate early genes encoding transcription factors, named Euo and HrcA. Conditional knockout of GrgA leads to a significant reduction in RB growth and a complete abolition of EB formation in the offspring. Based on this, the GrgA-mediated transcriptional regulatory network (TRN) is considered to control the growth, development and pathogenicity of Chlamydia.

[0050] The developmental cycle of Chlamydia is controlled by the transcriptome, which in turn is controlled by sigma factors of RNA polymerase and transcription factors. GrgA activates transcription of multiple Chlamydial genes in vitro and in vivo. In this work, a mutant Chlamydia lacking the grgA gene was developed. It was found that this gene is essential for the conversion of RB to EB during the developmental cycle. Mutant Chlamydia form replicative RBs, but they fail to differentiate back into infectious EBs. See Figure 1B These RBs are still able to elicit an immune response against Chlamydia in the host.

[0051] We attempted to disrupt grgA by insertional mutagenesis using a suicide plasmid carrying a group II intron containing aadA, which confers spectinomycin resistance. However, diagnostic PCR analysis of spectinomycin-resistant Chlamydia by these mutagenesis attempts failed to localize the group II intron within grgA, suggesting an off-target insertion. The group II intron failed to disrupt grgA, indicating that grgA is an essential gene. We also attempted to conditionally knock down GrgA expression using CRISPR / dCas9 interference technology. However, we found that the CRISPR / dCas9 interference system was highly toxic to Chlamydia trachomatis, which is contrary to the results reported in the literature.

[0052] B.GrgA

[0053] GrgA is a chlamydial transcription factor. It is present in the chlamydial cell forms EB and RB and stimulates several σ2 transcription factors that are active at different stages. 66 Dependent promoter and σ 28GrgA can therefore stimulate transcription from several promoters that control the expression of genes that are essential for Chlamydia growth and development.

[0054] GrgA overexpression has been found to 66 - and σ 28 -dependent mechanism inhibits the growth of Chlamydia trachomatis. 66 Is the major sigma factor that is required for transcription of most Chlamydial genes throughout the developmental cycle. Figure 2 , which shows the domain structure of the wild-type GrgA protein.

[0055] C. Chlamydia Composition

[0056] Although the examples given here focus on Chlamydia trachomatis, the present invention can be used for any Chlamydia species and strains within the Chlamydia and Chlamydophila genera. For example, the present invention can be used for any strain that is infectious to humans or animals. Examples include, but are not limited to, Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia muris, Chlamydia suis, Chlamydia abortus, Chlamydia felis, Chlamydia ruminants, Chlamydia ibis, Chlamydia avium, Chlamydia poultry, and the like.

[0057] Such a composition may also optionally include a carrier such as a suitable culture medium or buffer solution, and some optional components such as a pH adjuster, salts, sugars, and the like.

[0058] D.GrgA gene knockout

[0059] The inventors found that knocking out this particular gene would produce amazing results. Finding the right gene is the key. Gene knockout is conditional gene knockout or gene disruption.

[0060] E.DOPE Technology

[0061] Conceptually, inactivation of essential genes in Chlamydia (e.g., grgA) using DOPE can be achieved in any species of the genus Chlamydia using a variety of strategies (e.g., group II intron insertion mutagenesis, homologous recombination, and CRISPR inactivation). This methodology is useful for determining whether a particular gene (any gene) is essential for the bacterial developmental cycle. Figure 3 First, we need to determine whether the gene is functional, that is, whether the cell grows and develops normally when the gene is expressed normally. Figure 3 , row A. Next, disrupt the gene and test the cells to see if there are any developmental failures when the gene is not functional. Figure 3 , row B. Then, a plasmid containing the inducible gene is produced and inserted into the cell. When the gene is induced ( Figure 3 , row C) and when the gene is not induced ( Figure 3 If disruption of a gene results in a loss of cell development that is corrected when the gene is induced, then the gene can be considered essential.

[0062] The DOPE technique can be used to study essential genes in Chlamydia or other bacterial species. DOPE has several advantages over techniques for studying essential genes that rely on downregulation using an inactive CRSPR-associated d-protein (dCas), and complementation using constitutive expression from a transforming plasmid. Because specific genes are disrupted by introns, DOPE does not have the "off-target" effects of CRISPR interference. More importantly, DOPE does not suffer from the non-specific toxicity of dCas9. With a modified, carefully calibrated, and tunable anhydrotetracycline (ATC) inducible system, DOPE can study essential genes such as GrgA that are toxic when constitutively expressed from a recombinant plasmid.

[0063] We attempted to derive CtL2 (L2 / cgad-peig) with a conditional knockout of GrgA using DOPE. We used group II intron insertion mutagenesis to disrupt the grgA gene to further investigate the role of GrgA in Chlamydia physiology. However, multiple attempts resulted in two transformants with grgA intact. These negative results, coupled with the failure of saturation chemical mutagenesis studies and transposon mutagenesis to generate grgA deletion mutants, suggested that grgA might be an essential gene. We then attempted to knock down GrgA expression by CRISPR transcriptional interference using inactive Cas9 from multiple sources, but found that the CRISPR / dCas9 system was toxic to Chlamydia in the absence of any guide RNA.

[0064] Compared with previously reported plasmid-mediated complementation techniques, DOPE allows precise expression manipulation of genes of interest and is suitable for studying genes such as GrgA that are toxic upon overexpression. Unlike CRISPR interference, DOPE does not produce off-target effects or general nonspecific toxicity.

[0065] F. Vaccine Composition

[0066] Vaccines and vaccine compositions according to the present invention include conditional GrgA knockout Chlamydia as described herein. The present invention relates to a vaccine composition comprising a Chlamydia with conditional knockout of GrgA. Preferably, the species is Chlamydia trachomatis, but any Chlamydia or Chlamydia species is suitable. For example, Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia muris, Chlamydia suis, Chlamydia abortus, Chlamydia felis, Chlamydia ruminants, Chlamydia ibis, Chlamydia avium, Chlamydia poultry, etc. may also be used.

[0067] The RB of the GrgA knockout mutant contains MOMP and other proteins that confer effective immunity to disease. Therefore, this vaccine can generate immunity to these proteins, including the highly conserved antigen MOMP, similar to the natural immunity that humans develop after exposure to species of the genus Chlamydia. This is an attenuated vaccine in which the infectivity of the vaccine is retained, but it can be safely administered because progeny will not be formed.

[0068] Vaccines or vaccine compositions generally contain some type of bacterial carrier, such as an appropriate culture medium or buffer for administration to a subject. Therefore, in some preferred embodiments, the vaccine is administered to a subject as a pharmaceutical composition. Such pharmaceutical compositions may contain salts, buffers, adjuvants, or other compounds that contribute to improved efficacy. In certain embodiments, adjuvants are used to induce or improve a specific immune response. For a description of adjuvants, see Warren et al. (Ann. Rev. Biochem., 4: 369-388, 1986), the entire disclosure of which is incorporated herein by reference. Examples of materials suitable for vaccine compositions are well known to those skilled in the art and are described in Remington's Pharmaceutical Sciences (Osol, A, Ed, Mack Publishing Co, Easton, Pa., pp. 1324-1341 (1980), the relevant disclosure of which is incorporated herein by reference.

[0069] In certain embodiments, the vaccine can be formulated into a liquid preparation (including aqueous or non-aqueous solutions, suspensions, emulsions, etc.) suitable for intravenous injection, intraarterial injection, intraperitoneal injection, etc. to achieve systemic administration. Other components such as buffers, electrolytes, preservatives, dispersants, pH regulators, osmotic pressure regulators, sugars, etc. can also be optionally added. The vaccine can be contained in a suitable container, such as a vial, a prepared and filled syringe, or any suitable container known in the art, preferably sterile.

[0070] G. Methods of vaccination

[0071] The vaccine according to the present invention is expected to be suitable for any animal susceptible to infection with one or more chlamydia, including humans. Vaccinated subjects include humans, primates, monkeys, farm animals (including cattle, horses, pigs, sheep, goats, etc.), laboratory animals such as rodents (including mice, rats, guinea pigs, etc.), rabbits, etc., birds (such as chickens, turkeys, geese, ducks and other poultry), companion animals (including dogs, cats, etc.) and reptiles (including snakes, etc.).

[0072] The subject includes any animal suffering from chlamydia infection, suspected chlamydia infection or potentially susceptible to developing chlamydia infection. Therefore, the vaccine can be used for prevention or treatment. The vaccine is preferably used for prevention to induce immunity in the subject or host, including neutralizing antibodies, thereby reducing the possibility or severity of chlamydia infection, or preferably can prevent infection. For treatment, it is preferably possible to improve the natural immunity of the infected subject and enhance its ability to clear the infection, thereby alleviating the condition more quickly.

[0073] Administration of a conjugate vaccine (or the antisera it elicits) can be for either "prophylactic" or "therapeutic" purposes. In the prophylactic case, the vaccine is administered before any symptoms of bacterial infection appear. As described above, the effect of a prophylactic administration of a vaccine is preferably to prevent or attenuate subsequent infection. In the therapeutic case, the vaccine is provided after actual symptoms of infection are detected or a positive test for infection is obtained. A therapeutic administration of a vaccine preferably has the effect of attenuating the actual infection.

[0074] H. Dosage and Administration Regimen

[0075] The specific dosage depends on the age, weight, sex and physical condition of the subject being treated and the method of administration. Those skilled in the art can easily determine the appropriate dosage based on these factors and factors known to other technicians. A single subject can be administered one or more doses.

[0076] In a preferred embodiment, the appropriate amount of a vaccine for inducing an immune response in a subject comprises administering a therapeutically effective amount or a prophylactically effective amount of the vaccine to a subject in need thereof. The amount may consist of one dose or multiple doses (such as boosters). Therefore, the number of administrations may vary. For example, administration may be one-time, or monthly, annually, or less frequently. The actual amount administered, the dosage of the medication, and the number of boosters may be determined by skilled medical personnel. This depends on the age, sex, and weight of the subject, the stage of the disease, and the severity of the treatment (including preventive treatment). Treatment prescriptions, such as dosage decisions, fall within the responsibilities of general practitioners and other physicians.

[0077] The Chlamydia used in the vaccine according to the present invention is produced by inducing GrgA expression using ATC in the culture medium. ATC has been removed from the vaccine preparation and is not present in the subject, so when the vaccine is administered to the subject, the expression of GrgA has been turned off. This prevents continued infection and results in increased levels of released immunogenic but non-infectious RB.

[0078] I. Conclusion

[0079] Since the first publication demonstrating reproducible transformation of Chlamydia with shuttle vectors 12 years ago, the Chlamydia research community has utilized reverse genetics tools to study gene function through ectopic gene overexpression, gene knockdown, and other approaches. Even so, the study of Chlamydia and other biological systems has been hampered by the lack of effective strategies for perturbing or depleting truly essential genes. In this report, we developed a tightly regulated inducible expression system, termed DOPE, that can knock down essential genes in Chlamydia. The DOPE system is a convenient and versatile tool not only to establish the essential properties of genes, but also to determine their underlying mechanisms. Unlike CRISPR interference, DOPE has no off-target effects or general nonspecific toxicity.

[0080] Our inability to generate grgA null mutants using gene-targeted mutagenesis and random mutagenesis by the Valdivia laboratory strongly suggests that grgA is essential for the growth and survival of Chlamydia. By knocking down GrgA expression using the DOPE strategy, we found that GrgA plays a key role in maintaining RB replication efficiency and is absolutely required for RB differentiation to EB. Surprisingly, although previous studies have shown that two immediate early transcription factors, euo and hrcA, are readily upregulated after induction of GrgA overexpression, the genome replication kinetics data presented here indicate that the absence of ATC in culture does not affect primary EB differentiation to RB. However, these data do not exclude the possibility that GrgA plays a role in primary differentiation, as the amount of GrgA prepackaged into EBs could be sufficient to support primary differentiation of EBs to RBs. In line with this view, large amounts of GrgA were detected in C. trachomatis EBs. Our ongoing transcriptome analysis will elucidate the mechanisms by which GrgA regulates RB growth and RB differentiation to EBs.

[0081] EB formation is absolutely required for the spread of Chlamydial infection within the infected host and to new hosts. Because RB and EB share most of the immunodominant antigens (e.g., major outer membrane proteins), conditionally GrgA-deficient, “maturation”-defective Chlamydia are potential candidates for attenuated Chlamydial vaccines, provided that strategies are developed to adequately prevent EB from escaping the gene expression control system in the DOPE plasmid. In addition, maturation-defective Chlamydia are also a useful system for studying the role of RB in anti-Chlamydial immunity.

[0082] In summary, the data presented here strongly suggest that GrgA plays an important role in the growth of Chlamydia and is essential for the differentiation of RB to EB. The very low level of EB formation detected from cultures without ATCs is likely due to leaky GrgA expression in very few cells. Conditional GrgA-deficient Chlamydia are a valuable model to study RB replication and RB to EB differentiation. For example, Chlamydia with maturation defects due to the lack of GrgA could be used as a non-toxic vaccine to prevent Chlamydial disease.

[0083] 5. Examples

[0084] The present invention is not limited to the specific processes, compositions or methods described, as such processes, compositions or methods may vary. The terminology used in the description is intended only to describe a particular version or embodiment and is not intended to limit the scope of the invention, which is 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, preferred methods, devices and materials are now described. All publications mentioned herein are incorporated by reference in their entirety as references; nothing herein shall be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention.

[0085] Example 1: General Materials and Methods

[0086] A. Primers and other sequences

[0087] The following primers were used here.

[0088] Table 1. Primers

[0089]

[0090]

[0091] The sequence of pTargetron-aadA-gagA67 is as follows:

[0092]

[0093]

[0094]

[0095]

[0096] The sequence of pTRL2-peig is as follows:

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103] B. Carrier

[0104] pTRL2-grgA-67m (carrying a grgA allele resistant to intronic insertions between nucleotides 67 and 68) was constructed by assembling three DNA fragments using the NEBuilder HiFi DNA Assembly Kit (New England Biolabs). All three fragments were polymerized using Q5 DNA polymerase (New England Biolabs). TM ) was amplified from pTRL2-His-GrgA. Fragment 1 was generated using primers pgp3-pgp4-F and His-RBS-R (Table 1 above). Fragment 2 was generated using primers RBS-His-F and GrgA-67-R (Table 1 above). Fragment 3 was generated using primers GrgA-67-F and pgp4-pgp3-R (Table 1 above).

[0105] pDFTT3(aadA) is a Targetron vector that uses group II intron mutagenesis to disrupt chlamydial genes and was provided by Dr. Derek Fisher (Southern Illinois University, IL). To construct pDFTT3(aadA)-GrgA-67, which is designed to disrupt the open reading frame of grgA, two PCR fragments were first generated using pDFTT3 as a template. Fragment 1 was obtained using primers GrgA67_IBS1 / 2 and University primers (Table 1 above), while fragment 2 was obtained using primers GrgA67_EBS2 and GrgA67_EBS1 / δ (Table 1 above). The two fragments were combined and PCR amplified. The resulting full-length intron-targeted fragment was digested with HindIII and BsrGI and ligated with HindIII- and BsrGI-digested pDFTT3(aadA). The ligation product was transformed into E. coli DH5α and plated on LB agar plates containing 500 μg / ml spectinomycin and 25 μg chloramphenicol. The authenticity of the insert in pDFTT3(aadA)-grgA-67m was confirmed using Sanger sequencing.

[0106] C. Host Cells and Culture Conditions

[0107] Mouse fibroblast L929 cells were used as host cells for Chlamydia trachomatis transformation and EB preparation. Unless otherwise stated, human vaginal carcinoma HeLa cells were used for experiments to determine the effects of GrgA depletion on Chlamydia growth and development. Both L929 and HeLa cell lines were grown in Dulbecco's modified Eagle's medium (DMEM) (Sigma Millipore) containing 5% and 10% fetal bovine serum (vol / vol), respectively. TM ) for monolayer culture. Gentamycin (final concentration: 20 μg / mL) was used to maintain uninfected cells and was replaced with penicillin (10 units / mL) and / or spectinomycin (500 μg / mL) as detailed below. Uninfected and infected cells were cultured in an incubator at 37°C, 5% CO2.

[0108] D. Chlamydia

[0109] Wild-type Chlamydia trachomatis L2 434 / BU (L2) was purchased from ATCC. This strain was selected because: 1) it is the best model organism for research; 2) its genome is almost identical to those serotypes that are tropistic for genital epithelial cells; 3) it is easy to grow in cell culture; 4) almost all genetic tools are developed using this organism. Chlamydia strains also contemplated for use in the present invention include any Chlamydia or Chlamydophila species.

[0110] L2 / cg-peig was obtained by transforming L2 EBs with pTRL2-grgA-67m using calcium phosphate, as described in the previous art. The transformants were plated on L929 monolayers and selected with penicillin. L2 / cgad-peig was obtained by transforming L2 / cg-peig in the same manner with pDFTT3(aadA)-grgA-67m. ATC was added to the culture immediately after transformation to induce the expression of GrgA from pDFTT3(aadA)-grgA-67m. Twelve hours later, spectinomycin D (final concentration: 500 μg / ml) was added to the culture medium to initiate selection. L2 / cgad-peig EBs were expanded using L929 cells and purified by ultracentrifugation on MD76 density gradients. The purified EBs were resuspended in sucrose-phosphate-glutamate (SPG) buffer; a small aliquot was then taken and stored at -80°C. Unless otherwise stated, all Chlamydia cultures were supplemented with cycloheximide (final cycloheximide concentration in the culture medium was 1 nM) to optimize Chlamydia growth.

[0111] E. Immunofluorescence staining

[0112] L2 / cgad-peig was inoculated at an MOI of 0.3 inclusion forming units onto subconfluent HeLa monolayers grown on 6-well plates. After centrifugation at 900 g for 20 minutes, the cells were cultured at 37°C in medium containing 0 or 1 nM ATC for 30 hours. The infected cells were then fixed with cold methanol, blocked with 10% fetal bovine serum prepared in phosphate-buffered saline (PBS), and stained sequentially with monoclonal L21-5 anti-major outer membrane protein antibody and FITC-conjugated rabbit anti-mouse antibody. The immunostained cells were finally counterstained with 0.01% Evan blue (in PBS) and then analyzed on Olympus TM Images were taken on an Olympus IX51 fluorescence microscope using a constant exposure time for each channel. TM Red and green fluorescence images were collected on an IX51 fluorescence microscope. Using PictureFrame TM Then use the Java-based ImageJ TM Software to process images.

[0113] F. IFU assay

[0114] L2 / cgad-peig EB stocks or frozen harvests of L2 / cgad-peig cultured with or without ATC were thawed, diluted 1 to 10, and then inoculated onto L929 monolayers on 96-w plates in medium containing 1 nM ATC and 1 μg / mL cycloheximide. After centrifugation at 900 g for 20 minutes, cells were cultured at 37°C for 30 hours. Cell fixation and antibody reactions were performed as described above. Immunostained inclusions were counted under a fluorescence microscope without Evans blue counterstaining.

[0115] G. Diagnostic PCR and DNA sequencing

[0116] To confirm and sequence the grgA alleles in the chromosome and plasmid, the Quick-gDNA MiniPrep TM Kit (Sigma Millipore TM ) Total RNA was extracted from about 1000 infected cells. The obtained DNA was used for PCR amplification using Taq DNA polymerase. DNA fragments separated by electrophoresis using 1.2% agarose gel were extracted using a gel extraction kit (Qiagen TM ) were manipulated and purified and were subjected to Psomagen TM Sanger sequencing services provided by the Service Center (New York).

[0117] H. Quantification of genome copy number

[0118] To quantify genome copy number in culture, a Cell Lifter (Corning TM ) Remove the infected cells from the plastic plate in 6-well, 12-well or 24-well plates. Collect the cells and culture medium into an Eppendorf TM The tubes were centrifuged at 20,000 g at 4°C. The supernatant was carefully aspirated. One hundred microliters of alkaline lysis buffer (100 mM NaOH and 0.2 mM EDTA) was added to each tube to dissolve the cell pellet. The tubes were heated at 95°C for 15 minutes and then placed on ice. Four hundred microliters of 50 mM Tri-HCl (7.0) were added to each tube. The neutralized extracts were used directly for qPCR analysis (1 μL / reaction) or diluted with H2O before use.

[0119] I. Western Blotting

[0120] Carry out the detection of MOMP and GrgA as described in the art.Infect the L929 cells grown on 6-well plates with L2 / cgad-peig, and cultivate with the culture medium containing 1nM ATC.After inoculation 15 hours, 15 hours 20 minutes and 15 hours 40 minutes, make the cells in the selected hole go to the culture medium that does not contain ATC after 3 washings.After infection 16 hours (hpi), in the 1X SDS-PAGE sample buffer of 200 μ L, collect the cells in each hole, heat at 95 ℃ for 5 minutes, then with 35% amplitude ultrasonic treatment 1 minute (5 seconds on / 5 seconds off).Protein is separated in 10% SDS-PAGE gel, then transferred to PVDF membrane.Probe detection is carried out to membrane with monoclonal mouse anti-MOMP MC22 antibody, stripping and use polyclonal mouse anti-GrgA antibody to probe membrane again.

[0121] J. Transmission Electron Microscopy

[0122] To visualize intracellular Chlamydia up to 36 hpi, L929 cell monolayers grown on 6-well plates were infected as described above and cultured with medium containing or not containing 1 nM ATC. For cultures up to 36 h, cells were removed from the plastic surface using trypsin, collected in PBS containing 10% fetal bovine serum, and centrifuged at 500 g for 10 min. The pelleted cells were resuspended in EM fixation buffer (2.5% glutaraldehyde, 4% paraformaldehyde, 0.1 M cacodylate buffer) at RT, incubated for 2 h, and stored overnight at 4°C. To visualize intracellular Chlamydia at 45 and 60 hpi, the above steps would result in lysis of infected cells and inclusions. To overcome this problem, cells grown on glass coverslips were infected with trypsinization and fixed without trypsinization. In preparation for imaging, cells were first rinsed with 0.1 M cacodylate buffer, dehydrated in a graded series of ethanol, and then embedded in Eponate 812 resin at 68 °C overnight. TM 90 nm thin sections were cut on a UC6 microtome and picked up on copper grids. The grids were stained first with uranyl acetate and then with lead citrate. TIFF images were acquired using an AMT XR111 digital camera at 80 kV Philips TM The images were collected on a CM12 electron microscope. ImageJ TM The software measures the RB diameter.

[0123] Example 2: MOMP immunostaining, qPCR analysis and detection of progeny EBs. MOMP immunostaining was performed as described in Example 1. The results showed that at 34 hpi, inclusions in cultures without ATC were significantly smaller compared to cultures with 1.0 nM ATC.

[0124] Example 3: Dope-dependent plasmid expression (DOPE) gene technology.

[0125] The DOPE technology was developed to study the biological functions and intrinsic mechanisms of genes essential for Chlamydia growth and / or development.

[0126] 1. DOPE for Targetron (Group II intron) interference

[0127] 1.1. Construction of inducible GrgA expression vector (GrgADOPE plasmid)

[0128] 1.1a. Plasmids of Chlamydia species targeted using DOPE.

[0129] 1.1b. Synonymically mutate the intronic target site to render the essential gene of the plasmid carrying it resistant to the intronic targeting vector. This step increases the efficiency of selecting Chlamydiae that have essential genes disrupted by chromosomal introns, but which may not be essential genes. Group II intronic target sites can be determined by the Targetron algorithm.

[0130] 1.1c. Use an appropriate inducible expression system. The inducible system can be controlled by anhydrotetracycline or one of its derivatives, isopropyl β-d-1-thiogalactopyranoside (IPTG), theophylline, etc.

[0131] 1.2. Transformation of Chlamydia with inducible expression vectors

[0132] 1.2a. Mix the GrgA expression vector with Chlamydia EBs and calcium phosphate prepared in HEPES buffer.

[0133] 1.2b. Inoculate the above mixture onto host cells.

[0134] 1.2c. Select transformants using appropriate antibiotics.

[0135] 1.3. Construction of Targetron mutagenesis vector

[0136] 1.3a. Use of Antibiotic Resistance Genes for GrgA Expression Vectors Different antibiotic resistance genes.

[0137] 1.3b. Use the Targetron algorithm to facilitate building.

[0138] 1.4. Transformation of Chlamydia Transformed with DOPE Plasmid Using GrgA Targetron Vector

[0139] 1.4a. Perform transformation and infection according to steps 1.2a and 1.2b.

[0140] 1.4b. Transformants were selected using medium containing antibiotics that select for group II intron inserts and ATC to induce expression of GrgA from the DOPE plasmid.

[0141] 1.4c. The disruption of chromosomal grgA and the integrity of plasmid grgA were confirmed by PCR analysis and DNA sequencing.

[0142] 1.5. Determination of GrgA dependency

[0143] 1.5a. Preparation of EBs from cultures containing ATCs. Remove ATCs from the stock solution by washing with a buffer (eg, sucrose-phosphate-glutamate solution).

[0144] 1.5b. Inoculate cells with EBs without ATC prepared in the previous step.

[0145] 1.5c. Cultivate infected cells in medium without ATC and in medium containing ATC.

[0146] 1.5d. Dependence was assessed by different methods, including but not limited to analysis of inclusion size, genome duplication and EB yield.

[0147] 2. DOPE for gene knockout using homologous recombination

[0148] 2.1. Construction of inducible GrgA expression vector (GrgADOPE plasmid)

[0149] Construct the DOPE plasmid as described in 1.1a and 1.1c. Disregard point 1.1b.

[0150] 2.2. Transformation of Chlamydia with the inducible GrgA expression vector

[0151] Transformation and selection were performed as described in 1.2.

[0152] 2.3. Construction of homologous recombination vector

[0153] A plasmid is constructed that carries the sequence flanked by the grgA coding region. All or part of the open reading frame of grgA is replaced with an antibiotic selection marker that must be different from the marker in the DOPE plasmid.

[0154] 2.4. Transformation of DOPE-transformed Chlamydia with GrgA targeting vector

[0155] Transformation, selection and confirmation were performed as described in 1.4. The Targetron vector (step 1.3) was replaced with the Targetron vector produced in step 2.3.

[0156] 2.5. Determination of GrgA dependency

[0157] Follow the steps under 1.5 for preparation of ATC-free EB stocks, infection, culture, and characterization.

[0158] 3. DOPE for gene knockout using CRISPR

[0159] 3.1. Construction of inducible GrgA expression vector (GrgADOPE plasmid)

[0160] Construct the DOPE plasmid as described in 2.1.

[0161] 3.2. Transformation of Chlamydia with the inducible GrgA expression vector

[0162] Transformation and selection were performed as described in 1.2.

[0163] 3.3. Construction of homologous recombination vector

[0164] A grgA-targeted CRISPR knockout vector was constructed, which contained a CRISPR-associated (CAS) gene, a grgA-targeted guide RNA, and an antibiotic resistance gene flanked by CRISPR target site sequences. The antibiotic selection marker in the CRISPR-targeted vector must be different from the marker in the DOPE plasmid.

[0165] 3.4. Transformation of DOPE-transformed Chlamydia with GrgA targeting vector

[0166] Transformation, selection and confirmation were performed as described in 1.4. The Targetron vector (step 1.3) was replaced with the Targetron vector produced in step 3.3.

[0167] 3.5. Determination of GrgA dependency

[0168] Follow the steps under 1.5 for preparation of ATC-free EB stocks, infection, culture, and characterization.

[0169] In summary, the grgA gene in the Chlamydia chromosome was disrupted using a group II intron carrying aadA, which confers resistance to spectinomycin. This disruption was only achieved in Chlamydia transformed with a plasmid carrying grgA, in which the intronic target site was mutated synonymously. Therefore, this gene targeting strategy is called DOPE (dependent on plasmid-mediated expression). The ATC inducible expression system was redesigned in the DOPE plasmid to significantly reduce the level of GrgA expression, which makes it possible to effectively complement the chromosomal grgA disruption without excessive growth inhibition mediated by GrgA overexpression.

[0170] Targetron TM Targetron is a group II intron-based insertional mutagenesis technique that has been successfully used to disrupt a large number of Chlamydia chromosomal genes. Therefore, in order to knock out GrgA expression in Chlamydia and study its physiological role, Targetron TM The vector contains a group II intron with a spectinomycin resistance gene specific for multiple grgA insertion sites. Despite several attempts, we were unable to generate any grgA null mutants. Spectinomycin-resistant Chlamydia were obtained from only two transformed cultures, but diagnostic PCR analysis failed to demonstrate insertion of grgA into the group II intron, suggesting nonspecific targeting. This, coupled with the failure to obtain grgA null mutants, did not allow the use of Targetron TMInsertional mutagenesis established a stable grgA null mutant, which indicated to us that grgA is essential for the growth and development of Chlamydia. As a new alternative approach to study the biological functions and potential mechanisms of grgA and other genes essential for the growth and / or development of Chlamydia, we developed the Dependent Plasmid Mediated Expression (DOPE) tool (see Figure 3 Since essential genes are required for normal growth or development of Chlamydia, disruption of these genes in wild-type Chlamydia would be lethal ( Figure 3 A and Figure 3 B) However, transformation of wild-type Chlamydia with a recombinant plasmid carrying an essential chromosomal gene downstream of an inducible promoter can disrupt the chromosomal allele in the presence of an inducer in the culture medium ( Figure 3 C) In the resulting essential gene disruption strain, withdrawal of the inducer leads to depletion of the gene product produced by the recombinant plasmid, allowing functional and mechanistic analysis of the essential gene ( Figure 3 D).

[0171] Example 4: Mechanistic interrogation of chromosomally encoded essential genes.

[0172] To apply DOPE to the study of grgA, the plasmid pTRL2-peig was constructed, which encodes an anhydrotetracycline (ATC)-inducible grgA allele (i.e., peig). See the sequence provided above. Compared to the native chromosomal grgA allele that contains a group II intronic target site between nucleotides 67 and 68, the grgA allele in peig carries a His-tag sequence and four synonymous point mutations surrounding the group II intronic target site. See Figure 4A We transformed wild-type C. trachomatis with an intact chromosome grgA (L2 / cg) with peig to obtain L2 / cg-peig.

[0173] Next, we used the targetron carrying the group II intron containing aadA TM Plasmid transformation L2 / cg-peig, this intron has an insertion site between nucleotides 67 and 68 in grgA. TM The target site has been mutated, so the vector can only be inserted into the chromosomal grgA allele ( Figure 4A ). Using Targetron TM After plasmid transformation of L2 / cg-peig, we supplemented the culture medium with ATC and spectinomycin to induce the expression of GrgA from peig and simultaneously select for chromosomal mutants carrying intron-disrupted grgA.

[0174] PCR analysis confirmed the chlamydial genotypes L2 / cg, L2 / cg-peig, and L2 / cgad-peig, which was a chromosomal grgA disruption and complemented by a plasmid. Figure 4A and Figure 4B . Figure 4C Sanger sequence tracing confirmed the nucleotide sequences surrounding the intronic target sites in L2 / cg and L2 / cg-peig and the intronic junction region of grgA in L2 / cgad-peig. As expected, Western blotting detected ATC-independent expression of chromosomally encoded GrgA and ATC-dependent expression of plasmid-encoded His-GrgA in L2 / cg-peig. Figure 4D It is worth noting that Figure 4E As shown, Western blotting also detected a time-dependent loss of GrgA in L2 / cgad-peig after ATC withdrawal. His-GrgA was almost undetectable 2 hours after ATC withdrawal. FIG. 4B to FIG. 4E The data presented in demonstrate that wild-type chromosomal grgA is successfully disrupted in L2 / cgad-peig and that GrgA expression and function are dependent on ATC-induced GrgA expression from the peig plasmid.

[0175] In Figure 4, black and gray lines represent chromosome and plasmid sequences, respectively. Half arrows represent transcription. EG: essential gene; iEG: inducible EG; chr: chromosome; NA: not applicable; g: growth; d: development. The figure shows the disruption of chromosomally encoded grgA by a group II intron and the confirmation of the re-expression of GrgA in the transformation plasmid in the DOPE system. Figure 4A Schematic diagram showing the grgA alleles, the location of the intronic target site, diagnostic primers and the size of PCR products obtained with different primer sets. Abbreviations: itsm, mutated intronic target site; Chr, chromosome. Figure 4B Provides the use of Figure 4A Gel images of PCR products amplified by the indicated primer sets with DNA of wild-type Chlamydia trachomatis L2 (L2 / cg), L2 / cg transformed with his-grgA-itsm expression plasmid (L2 / cg-peig), and L2 complemented with chromosomal grgA disrupted with aadA with peig (L2 / cgad-peig). Figure 4C Sanger sequencing traces of PCR products are shown, showing the intronic target site in L2 / cg, the mutations around this site that confer resistance to intronic targeting in peig, and the grgA intronic junction region in the chromosome of L2 / cg-peig. Arrows and asterisks indicate wild-type bases and corresponding mutant bases, respectively. Figure 4DWestern blots showing that only chromosomally encoded GrgA was detected in L2 / cg-peig cultured in medium without ATC, and both chromosomally encoded GrgA and plasmid-encoded His-GrgA were detected in L2 / cg-peig cultured in medium containing ATC for 14 hours. Figure 4E Western blots showing time-dependent loss of His-GrgA in L2 / cgad-peig following ATC withdrawal are shown. Membranes were first probed with an anti-major outer membrane protein antibody, stripped, and then reprobed with an anti-GrgA antibody.

[0176] Compared to the native chromosomal grgA allele, which contains a group II intron target site between bases 67 and 68, the grgA allele in peig carries the His-tag coding sequence and four synonymous point mutations surrounding the group II intron target site. Figure 4A The wild-type Chlamydia trachomatis (i.e., L2 / cg) was transformed with the plasmid pTRL2-peig to obtain L2 / cg-peig. L2 / cg-peig was then transformed with the suicide plasmid pTargetron-aadA-grgA67, which carries a group II intron containing aadA that targets bases 67 and 68 of grgA (see Figure 4A Since the target site in peig is mutated, the intron can only be inserted into the cg allele (see Figure 4A ).

[0177] After transformation of L2 / cg-peig with the intronic plasmid, the culture medium was supplemented with 1 nM ATC to induce expression of GrgA from peig and 500 μg / mL spectinomycin to select for Chlamydia with grgA disrupted with the intron (aadA). Figure 4A PCR analysis with the indicated primers (see Figure 4B ) confirmed the genotypes L2 / cg, L2 / cg-peig, and plasmid-complemented chromosomal grgA-disrupted L2 / cgad-peig ( Figure 4B ). Figure 4C Sanger sequence tracing confirmed the nucleotide sequence surrounding the intronic target site in L2 / cg and L2 / cg-peig and the intronic junction region of grgA in L2 / cgad-peig. As expected, Western blotting detected ATC-independent expression of chromosomally encoded GrgA and ATC-dependent expression of plasmid-encoded His-GrgA in L2 / cg-peig. Figure 4D. Notably, Western blotting also detected a time-dependent loss of GrgA in L2 / cgad-peig after ATC withdrawal. The membranes were first probed with anti-major outer membrane protein (MOMP), stripped, and then reprobed with anti-GrgA antibody. See Example 1 for the method. By 2 hours after ATC withdrawal, His-GrgA was almost undetectable. See Figure 4E .In summary, FIG. 4B to FIG. 4E The data presented in show that grgA was successfully disrupted in the chromosome of L2 / cgad-peig, where the expression and function of GrgA depended on the presence of ATC in the culture medium.

[0178] Example 5: tetR mutation allows GrgA expression and EB escape in the absence of ATC.

[0179] Consistent with EB quantification, ultrathin section transmission electron microscopy revealed that EBs were readily detectable at 36 hours in cultures containing ATC and predominated at 45 hours. See Figure 5. In contrast, few EBs were found in cultures without ATC at 35, 45, or 60 hours. See Figure 5. Taken together, these findings suggest that GrgA deficiency plays an important role in optimal growth of RB and is essential for the generation of infectious EBs from RB. Electron microscopy revealed a lack of EBs in L2 / cgad-peig cultured in ATC-free medium at 35, 45, and 60 hours. The one nanomolar ATC cultures were not processed for EM at 60 hours because most inclusions had ruptured by this time. Note that EBs are a cell morphology approximately 400 nm in diameter and highly electron dense in cultures containing ATC. The irregularly shaped dark small particles are glycogen granules in both cultures containing ATC and without ATC.

[0180] Example 6: EB escape in ATC-free cultures of L2 / cgad-peig.

[0181] Although GrgA-null Chlamydiae exhibited a severe defect in EB formation, we were able to detect very low traces of EB background in Chlamydiae cultures lacking the ATC. Since mutations in the tetR gene and / or mutations in tetO (TetR operon) might abrogate the ability of TetR to repress grgA in L2 / cgad-peig, allowing EBs to form in the absence of the ATC, we recovered plasmids from EBs formed in ATC-free cultures and amplified them in E. coli. Notably, DNA sequencing revealed that single nucleotide polymorphisms (SNPs) in the tetR gene occurred in all 10 plasmids. Three different SNPs were detected; their locations and effects on the 208-aa TetR protein are shown in Figure 6. Two of the SNPs resulted in premature termination at codons 16 or 158, while the third SNP resulted in a frameshift at codon 64. These findings suggest that the absence of full-length TetR or a TetR defective in synthesis results in leaky expression of wild-type GrgA in the absence of the ATC.

[0182] for Fig. 6A , in L2 / cgad-peig, the plasmid allele of grgA is repressed in the presence of ATC. Figure 6B In Figure 1, tetR mutations found in plasmids isolated from L2 / cg-peig EBs formed in the absence of ATC lead to premature translation termination or frameshifts. Codon positions are indicated. Wild-type DNA and amino acids are shown in black, while the mutated or frameshifted nucleotides and the resulting translational effects are shown in red. Figure 6C , Figure 6B The mutation in grgA results in the loss of grgA repression, which enables peig to express functional TetR and subsequently form EBs.

[0183] The data presented here conclude that GrgA is required for EB formation.

[0184] References

[0185] All references listed below and the entire specification are incorporated by reference in their entirety.

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Claims

1. A gene knockout Chlamydia that does not express GrgA.

2. The gene knockout Chlamydia according to claim 1, wherein The chlamydia is selected from the group consisting of: Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci, Chlamydophila muridarum, Chlamydophila suis, Chlamydophila abortus, Chlamydophila felis, Chlamydophila pecorum, Chlamydophila niger, Chlamydophila truncatus ... ibidis), avian Chlamydia (C.avium), poultry Chlamydia (C.gallinacea), etc.

3. The gene knockout Chlamydia according to claim 2, wherein The Chlamydia is selected from the group consisting of Chlamydia trachomatis, Chlamydia pneumoniae and Chlamydia psittaci.

4. A vaccine comprising the gene-knockout Chlamydia according to claim 1 and a pharmaceutically acceptable carrier.

5. A method of stimulating an immune response to Chlamydia in a subject in need thereof, the method comprising: The vaccine according to claim 4 is administered to the subject.

6. A method for producing neutralizing antibodies against Chlamydia in a subject in need thereof, comprising: The vaccine according to claim 4 is administered to the subject.

7. A method for determining whether a bacterial gene in a bacterial cell is essential for its growth or development, the method comprising: (a) determining the growth or development of said bacterial cell when said gene is intact and expressed; (b) disrupting the gene and determining the growth or development of the bacterial cell when the gene is inactivated; (c) introducing a plasmid containing an inducible version of the bacterial gene into the bacterial cell having the disrupted bacterial gene, and determining the growth or development of the bacterial cell when the bacterial gene is induced and when the bacterial gene is not induced.

8. The method according to claim 7, wherein: The bacterial cell is a Chlamydia species.

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