Biological female contraceptive

Abstract

The present application provides a symbiont engineered to express an antibody fragment that inhibits sperm motility or fertilization, and the like. The present application provides use of the engineered symbiont or the composition containing the same as an effective contraceptive method for a female.

Description

[0001] This application is a divisional application of the same named application for a patent filed on May 4, 2011, application 201180022904.9. TECHNICAL FIELD

[0002] The present application provides a symbiont engineered to express an antibody fragment that inhibits sperm motility, fertilization or a combination thereof, and compositions containing the same. The present application provides the use of such an engineered symbiont or a composition containing the same as a contraceptive. BACKGROUND

[0003] There is a need to develop new contraceptives to provide convenient birth control to all individuals, regardless of social, financial or educational constraints.

[0004] Although there are many different contraceptives available today, none of the contraceptives is without problems. The most commonly used contraceptives to date are preparations of oral estrogens, progestins, or a combination thereof, which, although effective, have been implicated in the development of tumors in certain populations with long term use.

[0005] Barrier methods, such as diaphragms or intrauterine devices, show reduced efficacy, as well as non-compliance (for the former) and a greater risk of pelvic inflammatory disease (in the case of the latter). Spermicides are also used in this regard, but are less effective, and a higher risk of sexually transmitted diseases is associated with their use.

[0006] Thus, there is a need for a minimally invasive contraceptive that is easy to use, regardless of the above limitations. SUMMARY

[0007] In one embodiment, the present application provides a genetically engineered cell that produces an antibody fragment against a sperm antigen, and as such can be used as a contraceptive.

[0008] In one embodiment, the present application provides an engineered female reproductive tract symbiotic bacterium, wherein the bacterium is engineered to express an antibody or fragment thereof against sperm that is effective to prevent sperm motility, sperm-egg fusion or sperm penetration of the egg, or a combination thereof. In certain embodiments, the antibody fragment is a single chain Fv molecule (scFv).

[0009] In some embodiments, the antibody fragment specific to sperm antigens is human or humanized. In some embodiments, the antibody fragment specifically interacts with antigens or fragments thereof located at the acrosome or plasma membrane site of the sperm. In some embodiments, the antibody fragment specifically interacts with antigens or fragments thereof located at the sperm neck region. In some embodiments, the antibody fragment is secreted from engineered bacteria, while in other embodiments, the antibody fragment binds to or is attached to the bacterial cell wall.

[0010] In some embodiments, the engineered bacteria are *Lactobacillus*, or in some embodiments, *Lactococcus*. In some embodiments, the engineered bacteria are *Escherichia coli* Nissle 1917 strain. In some embodiments, the engineered bacteria are *Streptococcus gordonii*. In some embodiments, the engineered bacteria are *Lactobacillus jensenii* or *Lactobacillus cristata*.

[0011] In some embodiments, the scFv specifically interacts with a peptide having at least 90% identity with SEQ ID NO: 1 or 2. In some embodiments, the scFv has a sequence having at least 90% identity with SEQ ID NO: 8. In some embodiments, the scFv is encoded by a polynucleotide having a sequence having at least 90% identity with SEQ ID NO: 7.

[0012] In some embodiments, the present invention provides compositions comprising the engineered bacteria described herein. In some embodiments, the composition is in the form of a vaginal suppository, cream, or foam. In some embodiments, the contraceptive is combined with or contained in a vaginal ring.

[0013] In some embodiments, the present invention provides a method of contraception comprising the step of contacting female reproductive tract cells with an amount of engineered bacteria of the present invention sufficient to inhibit or prevent sperm motility, sperm-egg fusion, or egg penetration in the female. In some embodiments, according to this aspect, the method comprises applying an amount of a composition containing engineered bacteria sufficient to inhibit or prevent sperm motility, sperm-egg fusion, or egg penetration into the reproductive tract of a female in need.

[0014] In some embodiments, the present invention provides a method for reducing the incidence of pregnancy in a female population, the method comprising the step of contacting reproductive tract cells of a female subject with an amount of engineered bacteria of the present invention sufficient to weaken, inhibit, or prevent sperm motility, sperm-egg fusion, or egg penetration in the female subject, thereby reducing the incidence of pregnancy in the female and thus reducing the incidence of pregnancy in the female population.

[0015] In some embodiments, the present invention provides a method for reducing the fertilization rate of a female population, the method comprising the step of contacting female reproductive tract cells with engineered bacteria of the present invention in an amount sufficient to weaken, inhibit or prevent sperm motility, sperm-egg fusion or egg penetration in the female, thereby reducing the fertilization rate in the female and thus reducing the fertilization rate in the female population.

[0016] In some embodiments, the present invention provides the use of the engineered bacteria or compositions described herein in the preparation of a medicament for reducing the incidence of pregnancy in a female population. In some embodiments, the present invention provides the use of the engineered bacteria or compositions described herein in the preparation of a medicament for reducing the incidence of fertilization in a female population. Brief description of the attached diagram

[0017] Figure 1 The relative affinities of isolated and cloned phage-displayed scFvs to sperm peptides, as determined by ELISA, are graphically illustrated. Columns 1–3 show the binding of the three scFvs to a peptide derived from sperm FA-1, while columns 4–6 show the binding of the three scFvs to a peptide derived from sperm YLP(12). BSA was used as a negative binding control.

[0018] Figure 2 The relative affinity of scFvs exhibited by several isolated and cloned bacteriophages for sperm peptides used as probes is described. Clones are considered to have high affinity when the OD value associated with the peptide is higher (compared to BSA controls or samples not incubated with the peptide). In particular, clones 102, 103, 105, and 106 showed relatively high affinity for the peptide, and the in vitro and in vivo characteristics of clone 102 are further described.

[0019] Figure 3 The results of ELISA binding assays identifying a series of non-binding scFvs are graphically presented. These non-binding scFvs were subsequently used as controls for comparison with clone 102. Figure 2 As shown, clone 102 was found to have good affinity for the peptide used as a probe. In this case, a similar scFv clone, J112, is typically used. Controls A and B were also tested, representing samples without scFv phage or without both peptide and scFv, respectively. Binding was detected by measuring OD in the samples (probed using an anti-phage HRP-labeled antibody (anti-PVIII antibody)).

[0020] Figure 4A This is the plasmid map of the pSLP111.1 vector. Figure 4BThese are photographs depicting the NcoI and NotI digestion products of the plasmid. The plasmid was sheared at 37°C for three hours using NcoI- and NotI. The plasmid inserts are clearly shown in several representative clones.

[0021] Figure 5A The nucleic acid and amino acid sequences of J102 are described respectively (SEQ ID NO: 7-8). Figure 5B The various structural domains in J102scFv are described.

[0022] Figure 6 illustrates the specific binding of Lactobacillus expressing J102 to mouse sperm in vitro. Figure 6A These are light micrographs showing the significant binding of Lactobacillus expressing scFv to mouse sperm; almost every sperm cell evaluated showed significant staining. Figure 6B This is a light micrograph of Lactobacillus expressing J112 (used as a control), in which no obvious binding with mouse sperm is visible.

[0023] Figure 7 graphically illustrates the in vivo evidence of the efficacy of Lactobacillus expressing J102. Figure 7A Pregnancy rates were plotted in female mice treated with either Lactobacillus jannulatae expressing antisperm scFv J102 or control Lactobacillus jannulatae expressing unconjugated sperm scFv (J112) in two trials; in each trial, effective contraception was demonstrated in mice treated with J102 compared to mice treated with J112. Figure 7B It was drawn, as follows Figure 7A The total number of pups and the number of pups per cage in each of the two trials were described; compared with the control, the total number of pups and the number of pups per cage were reduced in the case of Lactobacillus japonicus expressing J102. Invention Details

[0024] In one embodiment, the present invention provides recombinant cells (in one embodiment, which include microorganisms) and a method of using them to produce a spermicidal compound. In one embodiment, the method and cells provide an effective method of contraception.

[0025] In one embodiment, the recombinant cells are female reproductive tract symbionts. In another embodiment, the symbiont is bacteria.

[0026] In one embodiment, the recombinant cells are non-mammalian cells that remain in the mucosa of the female reproductive tract sufficient to express compounds that interfere with sperm motility, sperm-egg fusion, or sperm penetration of the egg for a period of time. In some embodiments, when expressed in the female reproductive tract of the treated object by a symbiotic organism colonized therein, the compound results in reduced fertility in said object. In some embodiments, when expressed in the female reproductive tract of the treated object, the compound results in reduced pregnancy rates in a population of such symbiotic organisms.

[0027] In one embodiment, the present invention provides engineered symbiotic organisms expressing spermicide compounds, which are capable of colonizing the female reproductive tract or its regions.

[0028] In one implementation, cells are engineered to express nucleic acid fragments including regulatory sequences preceding (5' non-coding) and following (3' non-coding) coding sequences, and capable of being expressed as a specific protein.

[0029] Genetically engineered bacteria can be engineered to have defects in a specific gene by any method known to those skilled in the art, or engineered biotechnology can be engineered to overexpress a specific gene by methods known in the art.

[0030] In one embodiment, the construct is introduced into the bacteria of the present invention to enable selection of homologous recombination events in the bacteria for a gene knockout process. Those skilled in the art can readily design knockout constructs, including positive and negative selection genes, to effectively select transfected cells that have undergone homologous recombination events with respect to the construct.

[0031] In another embodiment, changes in gene expression, activity, and function, particularly enhancement, attenuation, and elimination of gene expression, can be achieved using a gene construct integrated into the cell genome. In yet another embodiment, changes in gene expression, activity, and function can be achieved using an extrachromosomal gene construct, and in one embodiment, this construct is maintained outside the chromosome.

[0032] In one implementation, the term construct or vector refers to a nucleic acid delivery vehicle containing a target sequence that has been subcloned into a vector.

[0033] To generate the vector of the present invention, a polynucleotide encoding the target sequence can be ligated into a suitable expression vector system suitable for transducing / transforming prokaryotic cells and directing the expression of the recombinant product within the transduced / transformed cells. It should be understood that such suitable vector systems can be readily modified using common recombination techniques to replace, duplicate, or mutate existing promoter or enhancer sequences and / or introduce any additional polynucleotide sequence, such as a sequence encoding an additional selectable marker or a sequence encoding a reporter gene.

[0034] According to another embodiment, the vector further includes a regulatory element, such as a promoter that regulates the expression of isolated nucleic acids. Such promoters are known to be cis-acting sequence elements required for transcription because they are used to bind to DNA-dependent RNA polymerases, which transcribe sequences downstream of the polymerase. In another embodiment, the vector may include an inducible promoter, or a promoter that constitutively expresses the target sequence.

[0035] The vector of the present invention may further include an origin of replication and can proliferate in more than one species of prokaryotic cells, and in some embodiments, the vector may be constructed to facilitate its integration into the genome of a selected organism. In other embodiments, as those skilled in the art will understand, the vector may be, for example, a plasmid, a baculovirus shuttle vector, a phage particle or phage, or any suitable vector.

[0036] Examples of such vectors are described and used in the Examples section below. However, those skilled in the art will understand that the invention is not limited to the use of any such vectors, and that it is common practice in the art to create new vectors and / or modify existing vectors for the purpose of optimizing the heterologous expression of inserted or incorporated sequences (with the vector serving as a gene delivery / engineering delivery tool for said sequences).

[0037] Some examples of suitable vectors include: M. Posno et al., Appl Environ Microbiol. 1991 June; 57(6): 1822-1828; M Shimizu-Kadota et al., Appl Environ Microbiol. 1991 November; 57(11): 3292-3300; T. Duong et al., Microbial Biotechnology Volume 4, Issue 3, pages 357-367, May 2011; VV Aleshin et al., Mikrobiologiia Volume: 69, Issue: 1, Pages: 75-80; X. Liu et al., Antimicrobial agents and chemotherapy 2006, vol. 50, no. 10, pp. 3250-3259; WO / 2005 / 112567; Luca Vangelista et al., Antimicrobial Agents and Chemotherapy, July 2010, pp. 2994-3001, Vol. 54, No. 7; US Patent 7,179,458; US Patent 7,754,467; Caren J. Chancey et al. The Journal of Immunology, 2006, 176: 5627-5636; US Patent 5,733,540 et al.

[0038] The desired nucleic acid sequence can be incorporated into cells using a variety of methods known in the art. Nucleic acid constructs can be used to stabilize or transiently transfect or transduce cells.

[0039] Many known techniques exist in the art for introducing vectors into the cells of the present invention, such as, but not limited to, direct DNA uptake techniques, and phage, plasmid, linear DNA, or liposome-mediated transduction, receptor-mediated uptake, and magneto-electroporation methods using calcium phosphate-mediated and DEAE-glucan-mediated introduction methods, electroporation, or liposome-mediated transfection (for further details, see, for example, "Methods in Enzymology," Vol. 1-317, Academic Press, Current Protocols in Molecular Biology, eds. Ausubel FM et al., Greene Publishing Associates, (1989), and Molecular Cloning: A Laboratory Manual, 2nd edition, Sambrook et al., Cold Spring Harbor Laboratory Press, (1989), or other standard laboratory manuals). The use of nucleic acid-coated particles for bombardment is also involved. It should be understood that any such method can be used to introduce the desired sequence into cells to produce the cells of the present invention and to implement the methods of the present invention.

[0040] Electroporation has been successfully used to transform a variety of cells.

[0041] Gene transfer into bacteria can also be achieved using bacterial conjugation, which relies on direct contact between donor and recipient cells. The bacterial conjugation process may involve mixing "donor" and "recipient" cells together in close contact. Conjugation occurs through the formation of cytoplasmic junctions between the donor and recipient bacteria, and newly synthesized donor DNA is directly transferred into the recipient cell. The recipient in conjugation receives DNA from the donor bacteria via horizontal transfer. The donor in conjugation transfer may possess a conjugation plasmid, a conjugation transposon, or a mobile plasmid.

[0042] In some cases, conjugation requires only a donor and a recipient. This occurs when the plasmid to be transferred is a conjugated and mobile self-propagating plasmid (i.e., carrying the tra gene and the gene encoding the Mob protein). Typically, the process involves the following steps: 1) a nick is formed in the double-stranded plasmid DNA at a specific location on oriT; 2) single-stranded DNA is released to the recipient through a pore or pili structure; 3) a DNA-relaxing enzyme cleaves the double-stranded DNA on oriT and binds to the released 5' end (forming a relaxed body as an intermediate structure); and 4) subsequently, helper protein complexes assemble on oriT to facilitate the DNA transfer process.

[0043] Transferring donor plasmids to recipients may also require "triparental" conjugation. In this type of conjugation, donor cells, recipient cells, and "helper" plasmids are involved. The donor cell carries a mobile plasmid, or conjugation transposon. The mobile vector contains oriT (a gene encoding a nickase) and a gene encoding a Mob protein; however, the Mob protein alone is insufficient for genome transfer. Therefore, unless the helper plasmid provides a suitable conjugation system (located in the donor or "helper" cell), the mobile plasmid cannot facilitate its own transfer. A conjugation plasmid is needed to form a mating pair and transfer DNA because this plasmid encodes a transfer protein (Tra) involved in pore or pili formation.

[0044] When used for, for example, cells, or nucleic acids, proteins, or vectors, the terms "recombinant" or "recombinant-modified" indicate that the cell, nucleic acid, protein, or vector has been modified by introducing a heterologous nucleic acid or protein or by altering the native nucleic acid or protein, or that the cell is derived from such a modified cell. Thus, for example, recombinant cells express genes not found in the native (non-recombinant) form of the cell, or native genes that are abnormally expressed, poorly expressed, or not expressed at all in non-recombinant cells.

[0045] When used for nucleic acids, the term "heterologous" indicates that the nucleic acid contains two or more subsequences that do not exhibit the same interactions found in the heterologous nucleic acid in their native state. For example, recombinant nucleic acids typically produce nucleic acids having two or more sequences from unrelated genes arranged to produce new functional nucleic acids, such as a promoter from one source and a coding region from another. Similarly, a heterologous protein indicates that the protein contains two or more subsequences that do not exhibit the same interactions found in the heterologous protein in its native state (e.g., fusion proteins).

[0046] The term "expression cassette" refers to a recombinant or synthetically produced nucleic acid that has a specific set of nucleic acid elements that allow a particular nucleic acid to be transcribed in a host cell. An expression cassette can be a plasmid, a virus, or part of a nucleic acid fragment. Typically, an expression vector contains the nucleic acid to be transcribed, operatively linked to a promoter.

[0047] In another embodiment, the nucleic acid used in this invention comprises RNA or DNA analogs derived from nucleotide analogs. This term includes oligonucleotides composed of naturally occurring nucleic acid bases, sugars, and covalently linked nucleotides (backbone), as well as oligonucleotides having a non-naturally occurring portion that have similar effects. Such modified or substituted oligonucleotides are often preferred over their natural forms because they possess desired properties, such as enhanced cellular uptake, enhanced affinity for nucleic acid targets, increased stability in the presence of nucleases, and / or effective gene silencing.

[0048] Nucleic acids used in this invention can be produced through any synthetic or recombination process, such as those known in the art. Nucleic acids can be further modified using techniques known in the art to alter their biophysical or biological properties. For example, nucleic acids can be modified to increase their stability against nucleases (e.g., "capping"), or to alter their lipophilicity, solubility, or binding affinity to complementary sequences.

[0049] The DNA of the present invention can also be chemically synthesized using methods known in the art. For example, DNA can be chemically synthesized from four nucleotides, either whole or in part, using methods known in the art. Such methods include those described in Caruthers (1985). DNA can also be synthesized by preparing overlapping double-stranded oligonucleotides, filling the gaps, and joining the ends together (see generally Sambrook et al. (1989) and Glover et al. (1995)). DNA containing functional homologs of expressed proteins can be prepared from wild-type DNA by site-directed mutagenesis (see, for example, Zoller et al. (1982); Zoller (1983); and Zoller (1984); McPherson (1991)). The obtained DNA can be amplified using methods known in the art. A suitable method is the polymerase chain reaction (PCR) method described in Saiki et al. (1988), Mullis et al., U.S. Patent No. 4,683,195, and Sambrook et al. (1989).

[0050] In another embodiment, in vitro transposition can be performed on genomic DNA in a cloning vector, such as a cloning vector, bacteriophage, plasmid, or BAC (bacterial artificial chromosome). Similar high-density mutagenesis can be performed in non-naturally capable organisms using cloning allele-replacement vectors (see, for example, U.S. Patent No. 6,207,384, the method of which can be used to engineer the organisms described herein).

[0051] For example, their transforming ability, ability to express heterologous proteins, and / or mucosal surface can be used to select suitable bacterial host strains. Standard techniques such as rubidium chloride or electroporation can be used to make bacterial hosts capable of transformation (see, for example, Wei et al., J. Microbiol. Methods 21: 97-109 (1995)).

[0052] Electroporation transformation of symbiotic organisms such as *Lactobacillus japonicus* can be performed by modifying, for example, the standard method described by Luchansky et al. (J. Dairy Sci. 74: 3293-3302 (1991)). In short, freshly inoculated *Lactobacillus japonicus* is cultured in MRS medium (e.g., at 37°C and 5% CO2 to an OD of 0.6–0.7).600 Bacterial cells are harvested, washed, and resuspended in a cold solution of sucrose and MgCl2. Competent cells are then mixed with DNA and electroporated. Cells are then allowed to recover and plated onto selective agar plates containing a selective agent such as an antibiotic. The engineered cells are then administered to the target organism, for example, in suppositories, creams, or foam formulations.

[0053] Optionally, before introducing the bacteria of the present invention into the vagina, antibiotic pretreatment can be used to pre-clean the mucosal surface of the native bacteria (see, for example, Freter et al., Infect. Immun., 39: 686-703 (1983)). The antibiotics can be administered orally or directly into the vagina.

[0054] In some embodiments, the present invention provides a non-limiting method for selecting bacterial strains engineered according to the method of the invention, which are capable of effectively colonizing a mucosal surface on which the bacteria are applied. This method may include repeatedly selecting rapidly colonizing bacteria on an animal or human mucosal layer. For example, wild-type bacterial strains are applied to the mucosal surface, and said bacteria are repeatedly isolated and cultured in vitro, returning to the mucosal surface at each step. In some embodiments, according to this aspect, bacteria with enhanced colonization capacity are ultimately obtained.

[0055] In another embodiment, the present invention provides a non-limiting method for engineering bacterial strains according to the method of the invention, which can effectively colonize the mucosal surface on which the bacteria are applied. This method may include expressing a fusion protein on the surface of the recombinant bacteria. The fusion protein consists of a host-binding domain linked to a target polypeptide. The host-binding domain allows the bacteria to bind with high affinity to certain determinants (proteins or carbohydrates) on the selected host mucosal surface, thereby conferring a survival advantage over the resident microflora.

[0056] Another exemplary method for engineering the bacterial strains of the present invention includes inducing the expression of heterologous proteins in native bacterial communities by introducing genes via bacteriophages. Various bacteriophage vectors have been developed for use in different bacteria. For example, bacteriophage vectors based on the temperate bacteriophage adh can be used (see, for example, Raya et al., J. Bacteriol. 174: 5584-5592 (1992) and Fremaux et al., Gene 125: 61-66 (1993)). This vector integrates site-specifically into the host chromosome at defined bacteriophage (attP) and bacterial (attB) attachment sites. Similarly, Lactobacillus-specific bacteriophages can be used to transfer vectors or other polynucleotides into the Lactobacillus chromosome. Lactobacillus-specific phages include mv4 (Auvray et al., J. Bacteriol., 179: 1837-1845 (1997)), adh (Fremaux et al., Gene 126: 61-66 (1993)), gle (Kakikawa et al., Gene 175: 157-165 (1996)), and those belonging to Bradley group A or B of vaginal lactobacillus isolates (Kilic et al., Clin. Diagn. Lab. Immunol. 8: 31-39 (2001)).

[0057] Certain agents that do not irritate mucosal epithelial cells can also be added to a unit dose of bacteria to aid colonization. Numerous bacteria on the mucosal surface secrete a membrane material that coalesces to form a biofilm covering the entire mucosal surface. Adding enzymes that digest this biofilm material to promote the engineered bacteria's penetration of the biofilm (and thus more successful colonization) may be beneficial. Such enzymes include DNases, peptidases, collagenases, hyaluronidases, and other carbohydrate-degrading enzymes. Antibiotics to which the engineered bacteria are not susceptible can also be added to reduce the number of native bacteria on the mucosal surface, thereby facilitating the effective colonization of the engineered bacteria.

[0058] Expression of heterologous polynucleotides or polypeptides using P59 (van der Vossen et al., Appl. Environ. Microbiol. 58: 3142-3149 (1992)) or P23 (Elliot et al., Cell 36: 211-219 (1984)) promoters can be constitutive. Alternatively, expression can be controlled by inducible promoters. For example, Bacillus amylase (Weickert et al., J. Bacteriol. 171: 3656-66 (1989)) or xylose (Kim et al., Gene 181: 71-76 (1996)) promoters and Lactococcus lactis nisin promoters (Eichenbaum et al., Appl. Environ. Microbiol. 64: 2763-2769 (1998)) can be used to drive inducible expression. Furthermore, acid- or base-inducible promoters can be used. For example, promoters that are active under the relatively acidic conditions of the vagina (e.g., those described in U.S. Patent No. 6,242,194) can be used. Alternatively, promoters that are induced by changes in the vagina in response to semen can be used. For example, base-inducible promoters are used to induce expression in response to increased alkalinity in the vagina caused by semen introduction.

[0059] Various signaling and anchoring sequences are known to direct peptide expression to membranes, extracellular spaces, or cell walls (e.g., via covalent linkage with peptidyl dextran). Exemplary signaling sequences include the signal sequence of α-amylase from *Lactobacillus amylovorus* (Giraud & Cuny, Gene 198:149-157 (1997)) or the signal sequence of the S-layer gene (cbsA) from *Lactobacillus curvature* (e.g., MKKNLRIVSAAAAALLAVAPVAA (SEQ ID NO: 3) or MKKNLRIVSAAAAALLAVATVSA (SEQ ID NO: 4)). Signaling sequences are typically located at the amino terminus of the peptide.

[0060] Anchoring sequences are typically located at the C-terminus of the encoded protein sequence. Anchoring sequences include, for example, cell wall binding sequences; sequences LPQ(S / A / T)(G / A), where residues in parentheses indicate different options for that position; and hydrophobic sequences, and optionally, charged sequences. In some embodiments, the anchoring sequence comprises:

[0061] VTRTINVVDPITGKISTSVQTAKFTREDKNSNAGYTDPVTGKTTMNPWTPAKQGLRAVNVEQIKGYVAKVDGNVDAVVVTPDSANMVVTITYQANKPEGQNITNKKDTVPDPADGIKNKDD LPDGTKYTWKEVPDVNSVGEKTGIVTVTFPDGTSVDVKVTVYVDPVVESNRDTLSKEANTGNTNVAKAATVTSSKVESKKTLPQTGSKTEQVGILGLAIATVGSLLGLGVNRKKRQK(SEQ ID NO: 5); or,

[0062] KKAEEVKNNSNATQKEVDDATNNLKQAQNDLDGQTTDKSKLDEAIKSADDTKSTDKYNNASDDTKSKFDEALKKAEEVKNNSNATQKEVDDATKNLKQAQNDLDGQTTNKDAINDAIKD ANNAKGTDKYNNASDDTKSKFDDALKKAEDVKNDSNANQKEVDDATKNLKNTLNNLKGQPAKKANLIASKDNAKIHKQTLLPQTGTETNPLTAIGIGLMALGAGIFAKKKRKDDEA(SEQ ID NO: 6), or,

[0063] A sequence substantially identical to SEQ ID NO:5 or SEQ ID NO:6.

[0064] The correct localization and folding of peptides can be determined using standard methods. For example, cell wall enriched fractions of Lactobacillus can be obtained by suspending bacteria in a buffer solution (e.g., 25% sucrose, 1 mM EDTA, 10 mM Tris-HCl, pH 8.0), followed by treatment with cell wall degrading enzymes (e.g., lysozyme and mutalysin), and then separating the resulting protoplasts by differential centrifugation (Piard et al., J. Bacteriol. 179: 3068-3072 (1997)). Fractions can be screened by Western blotting to confirm expression in the cell wall.

[0065] Standard methods can also be used to determine the folding and biological activity of expressed peptides. For example, an ELISA assay using antibodies specific to naturally folded peptides can be used to confirm the folding and three-dimensional structure of the peptide. Of course, biological activity assays will vary depending on the activity of the peptide. For example, for peptides that bind to sperm, a standard binding assay can be used to detect the expressed peptide.

[0066] When synthesizing a gene that exhibits improved expression in host cells, it is desirable to engineer the gene so that its codon usage frequency approximates the preferred codon usage frequency in the host cells. The percentage deviation of the preferred codon usage frequency of the synthesized gene from the frequency used in the host cells can be calculated by first determining the percentage deviation of the usage frequency of individual codons from the frequency in the host cells, and then obtaining the average deviation of all codons.

[0067] The polynucleotide sequence encoding a specific polypeptide can be altered to match the codon usage of a specific host. For example, the codon usage of *Lactobacillus* can be used to derive polynucleotides encoding the polypeptide of the present invention and containing preferred *Lactobacillus* codons. The frequency of preferred codon usage displayed by the host cell can be calculated by averaging the frequencies of preferred codon usage across a large number of genes expressed by the host cell. This analysis is preferably limited to genes highly expressed by the host cell. For example, Pouwels & Leunissen (Nucleic Acids Res. 22: 929-936 (1994)) provided codon usage frequencies of highly expressed genes displayed by various *Lactobacillus* species. Codon usage tables are also available via the Internet.

[0068] In another embodiment, the vector involved in this invention further comprises the insertion of a heterologous nucleic acid sequence encoding a labeled polypeptide. The labeled polypeptide may include, for example, green fluorescent protein (GFP), DS-red (red fluorescent protein), secreted alkaline phosphatase (SEAP), β-galactosidase, luciferase, or any number of other reporter proteins known to those skilled in the art.

[0069] By modifying the amino acid sequence of naturally occurring GFP from Aequorea victoria, a variety of Aequorea-associated GFPs with useful excitation and emission spectra have been designed (Prasher et al., 1992, Gene, 111: 229-233; Heim et al., 1994, Proc. Natl. Acad. Sci. USA, 91: 12501-12504; PCT / US95 / 14692).

[0070] In one implementation, a nucleic acid having a sequence encoding a spermicide compound is transferred into a heterologous organism, resulting in expression.

[0071] Methods for detecting the expression product are well known in the art, and in one embodiment may include RNA blotting, PCR, HPLC, mass spectrometry, ELISA, RIA, or protein blotting analysis [see “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis, JE, ed. (1994); “Current Protocols in Immunology”, Volumes I-III Coligan, JE, ed. (1994); Stites et al. (eds.)].

[0072] In some embodiments, antibiotic resistance kits may be introduced into the organism described in this invention as markers confirming the expression of the introduced construct. In some embodiments, antibiotic susceptibility kits are introduced into the organism as a safety protocol for introducing constructs into the subject, and in some embodiments, to eliminate contraceptive effects and promote safe pregnancy, for example, by administering a short course of antibiotic treatment to the subject (which clears biological contraceptives).

[0073] In some embodiments, the present invention provides engineered cells expressing a spermicidal compound. In one embodiment, the spermicidal compound may include an antisperm antibody or a fragment thereof that effectively inhibits sperm motility, sperm-egg fusion, or sperm penetration of an egg, or a combination thereof.

[0074] Antibodies exist, for example, as intact immunoglobulins or as a variety of well-characterized fragments. In some embodiments, scFv fragments are considered as part of engineered organisms, compositions, kits, and for use in the methods of the present invention. In some embodiments, other fragments considered include F(ab')2, a Fab' monomer. A Fab' monomer is essentially a Fab having a portion of a hinge region (see Paul (ed.) Fundamental Immunology, 3rd ed., Raven Press, NY (1993)). Although various antibody fragments are defined in terms of the digestion of intact antibodies, those skilled in the art will understand that such fragments can be synthesized de novo by chemical methods or by using recombinant DNA methods. Therefore, the term antibody as used in the present invention also includes antibody fragments generated by modifying intact antibodies, or antibody fragments synthesized de novo using recombinant DNA methods (e.g., single-stranded Fv(scFv)).

[0075] This invention describes an exemplary method for preparing such cells; however, those skilled in the art will understand that any spermicidal compound or combination of compounds can be recombined in a symbiotic organism.

[0076] In some embodiments, antibody fragments are used that are spermicidal or otherwise inhibit sperm motility, sperm-egg fusion ability, or sperm penetration of the egg in various animal species. For example, in some embodiments, antibody fragments, such as scFv, specifically interact with highly conserved sperm-specific epitopes in animal species and humans, thereby allowing in vivo efficacy testing in animal models, while validation of the same antibody fragment, such as human scFv or its humanized form, can be performed in human clinical trials.

[0077] In some embodiments, as those skilled in the art will understand, such antibodies / antibody fragments may be generated as exemplified in this invention, or as described in Xu et al., Arch Androl. 1994 Sep-Oct; 33(2): 141-4; Clarke et al., Arch Androl. 1995 Jul-Aug; 35(1): 21-7; WO0107083A1; U.S. Patent Nos. 5,830,472, 5,753,231, 5,227,160, or by any suitable method. Engineered symbionts to express such compounds may also be accomplished by any method described in PNAS 102: 11993-11998 (2003).

[0078] Fully human antibodies / antibody fragments are particularly desirable for contraception in human women. Human antibodies / antibody fragments can be prepared using a variety of methods known in the art, including the phage display method described herein using a library of antibody / antibody fragments derived from human immunoglobulin sequences. See U.S. Patent Nos. 4,444,887 and 4,710,111; and WO98 / 46645; WO99 / 50433; WO98 / 24893; WO98 / 16654; WO96 / 34096; WO96 / 33735; and WO91 / 10741, each of which is incorporated herein by reference in its entirety and includes the examples provided below.

[0079] It should be understood that any references cited in this article are considered to be incorporated into the article in their entirety by way of citation.

[0080] Human antibodies or antibody fragments, such as scFv, can also be produced using transgenic mice that do not express functional endogenous immunoglobulins but can express human immunoglobulin genes. For an overview of this technique for producing human antibodies, see Lonberg and Huszar, 1995. For a detailed discussion of the techniques for producing human antibodies and human monoclonal antibodies, as well as the protocols for producing such antibodies and their fragments, see, for example, WO 98 / 24893; WO92 / 01047; WO96 / 34096; WO96 / 33735; EP 0598877; U.S. Patent Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; 5,939,598, which are incorporated herein by reference in their entirety. In addition, companies such as Abgenix, Inc. (Freemont, Calif.), Kirin, Inc. (Japan), Medarex (NJ), and Genpharm (San Jose, Calif.) can use techniques similar to those described above to provide human antibodies against selected antigens.

[0081] The antibodies of the present invention can also be modified using the methods and coupling agents described by Davis et al. (US Patent No. 4,179,337) to produce antibody fragments that substantially do not induce an immunogenic response.

[0082] In some embodiments, the present invention provides a composition comprising the engineered bacteria described herein.

[0083] In some embodiments, the composition may comprise engineered bacteria as described in this invention, wherein the composition comprises bacteria expressing different heterologous antisperm agents, the cells being contained in the same composition as part of a unit dose administered. In some embodiments, such bacteria expressing different heterologous antisperm agents may vary according to specificity to a particular antigen or epitope, or according to the antigen or epitope to which the fragment binds, or according to affinity for the epitope, or according to the isotype from which they are derived, or according to a combination of these.

[0084] In some embodiments, such compositions may also include different bacterial strains expressing the same or different heterologous antisperm agents, said strains also included in the same composition as part of the unit dose administered.

[0085] In some embodiments, the composition is a vaginal suppository, liquid, spray, foam, cream, mousse, or any carrier suitable for vaginal delivery.

[0086] In some embodiments, engineered bacteria or compositions containing them are applied to or contained in a vaginal ring, which is then applied to a female object. In some embodiments, the present invention relates to a condom containing engineered bacteria or compositions containing said bacteria applied to and retained on the outer surface of the condom, so that the wearer of the condom can transfer the engineered bacteria to their female partner during intercourse.

[0087] In some embodiments, the term "comprising" or its grammatical form means containing the indicated active agent, such as the engineered symbiotic of the present invention or the bacteriophage described in the present invention, as well as other active agents and pharmaceutically acceptable carriers, excipients, lubricants, stabilizers, etc., known in the pharmaceutical industry. In some embodiments, the term "consistently composed of" means a composition whose only active ingredient is the indicated active ingredient, i.e., the engineered symbiotic of the present invention, but may also include other agents / components / compounds used for stabilizing, preserving, etc., but not directly involved in the contraceptive effect. In some embodiments, the term "consistently composed of" may refer to a component that exerts a contraceptive effect through a mechanism different from that of the engineered symbiotic, which enhances or prolongs the contraceptive effect, or has both. In some embodiments, the term "consistently composed of" may refer to a component that promotes the release of the engineered symbiotic (e.g., by enhancing the release of the engineered symbiotic from suppository formulations or other formulations), promotes effective colonization, promotes uniform colonization, and other desired effects, which promotes the activity of the engineered symbiotic, but is not a component of the engineered symbiotic. In some embodiments, such agents may include agents that induce the expression of an antisperm agent encoded in the form of an active ingredient, or in some embodiments, agents that enhance the expression of an antisperm agent encoded in the form of an active ingredient. In some embodiments, the term "composition" refers to a composition containing an active ingredient and a pharmaceutically acceptable carrier or excipient.

[0088] The delivery of engineered bacteria to a desired mucosal surface depends on the accessibility and local conditions of the area. For example, engineered bacteria can be placed in saline solutions, creams, suppositories, or foams for delivery to the vaginal mucosa. Foams may include one or more hydrophobically modified polysaccharides, such as cellulose and chitosan. Cellulose includes, for example, hydroxyethyl cellulose, hydroxypropyl cellulose, methylcellulose, hydroxypropyl methylcellulose, hydroxyethyl methylcellulose, etc. Chitosan includes, for example, the following chitosan salts: chitosan lactate, chitosan salicylate, chitosan pyrrolidone carboxylate, chitosan itaconic acid, chitosan nicotinate, chitosan formate, chitosan acetate, chitosan gallate, chitosan glutamate, chitosan maleate, chitosan aspartate, chitosan glycolate, and quaternary ammonium-substituted chitosans and their salts. Foams may also include other components such as water, ethanol, isopropanol, glycerol, glycerol, propylene glycol, and sorbitol. Optionally, the spermicide may be included in the bacterial composition. Other examples of foams and foam delivery carriers are described, for example, in U.S. Patent Nos. 5,595,980 and 4,922,928.

[0089] In some embodiments, the bacteria can be delivered as suppositories or pessaries, see, for example, U.S. Patent No. 4,322,399. In some embodiments, the bacteria of the present invention are prepared in a preservation matrix, for example, as described in U.S. Patent No. 6,468,526, and delivered in a soluble element made of a soluble polymeric material and / or a complex carbohydrate material selected for its solubility properties, so that it remains substantially solid before use and dissolves upon use due to human body temperature and humidity, releasing the pharmaceutical material at the desired timed release and dosage. See, for example, U.S. Patent No. 5,529,782. The bacteria can also be delivered in a sponge delivery carrier, as described in U.S. Patent No. 4,693,705.

[0090] In some embodiments, the engineered bacteria need to be applied to the mucosal surface periodically; the optimized dosing interval can be routinely determined but will vary depending on the mucosal environment and bacterial strain. In some embodiments, the dosing interval can vary from once daily to once every 2-4 weeks or longer. In some embodiments, administration is based on the female menstrual cycle so that administration is performed after menstruation has stopped or nearly stopped, thereby optimizing colonization before ovulation in the treated female.

[0091] In some embodiments of introducing bacteriophages to transform native lactobacilli, the nucleic acid of the selected bacteriophage can be manipulated so that a heterologous gene replaces the gene encoding the bacteriophage capsid protein, thereby making the bacteriophage replication-deficient. Adding these recombinant DNA molecules to cell lysis buffer containing functional bacteriophage proteins will result in the assembly of functional bacteriophage particles carrying the heterologous gene. These replication-deficient bacteriophage particles can then be introduced onto the desired mucosal surface to infect selected bacterial populations. A typical dosage is 10 ppm applied to the mucosal surface. 8 -10 12 PFU / ml. The solution-to-surface ratio should be approximately 0.1 = 1.0 ml per square centimeter of mucosal surface. The transport vector is similar to the bacterial transport vector described above.

[0092] In some embodiments, the present invention provides a method of contraception comprising the step of contacting female reproductive tract cells with engineered bacteria described in the present invention. In some embodiments, according to this aspect, the method comprises applying a composition containing engineered bacteria (in some embodiments, including the composition described in the present invention) to the reproductive tract of a female subject in need of this. In some embodiments, the present invention also provides contacting the female reproductive tract with a bacteriophage described in the present invention, which in turn leads to in-situ engineering of a symbiotic flora to obtain the engineered bacteria described in the present invention.

[0093] In some implementations, the terms “contact” or “application” refer to direct and indirect exposure to the material in question.

[0094] In some embodiments, the dose administered into the female reproductive tract may be 10. 5 -10 9 One recombinant bacterium. In some embodiments, the dosage is optimized for a specific host or population. In some embodiments, such optimized dosage can be 10 7 -10 9 One recombinant bacterium, or 10 6 -10 8 One recombinant bacterium, or 10 8 -10 9 A recombinant bacterium.

[0095] Various methods for proving effective contraception are known in the art, and all of these methods are considered to be applicable to the present invention, some of which are exemplified herein.

[0096] In one embodiment, the contraceptive efficacy of the biological contraceptive of the present invention is determined by evaluating the number of offspring produced per reproductive cycle in female animals treated with the contraceptive of the present invention. For example, female mice are placed in cages with male mice 1 to 3 days after administration of the biological contraceptive. The male mice are then removed between 1 and 7 days after mating. The birth of pups in the female mice is then observed daily. The contraceptive efficacy of the administered contraceptive is determined by the reduction in pregnancy and / or the reduction in litter size after administration. For example, for animal species that produce an average of 3 or more offspring per litter (e.g., as illustrated below, when using a distantly related hybrid strain, mice produce an average of 11 or more offspring per litter), the contraceptive protein can be considered effective if the pregnancy rate or litter size is reduced by 10%-100%, 30%-100%, or 50%-100%, or 60%-100%, 70%-100%, or 80%-100%, or 90%-100%, or 95%-100%, or more. As exemplified in this invention, when evaluating the contraceptive efficacy of Lactobacillus strains expressing J102, at least 50% contraceptive efficacy was observed. Moreover, since the colonization studies conducted herein showed that Lactobacillus colonized well in approximately 50% of mice, it is expected that other animals known to be more receptive to Lactobacillus colonization will have even higher contraceptive efficacy.

[0097] If the number of offspring per litter is reduced by at least 50%, the contraceptive is considered effective. Therefore, Figure 7 shows the emergence of an effective biological female contraceptive, which includes engineered symbionts expressing antisperm agents.

[0098] In some embodiments, the effectiveness of the contraceptive of the present invention can be determined by measuring the level of antibodies expressed in mucosal secretions. Alternatively, the effectiveness of the contraceptive can be evaluated, for example, by observing antibodies binding to sperm using immunohistochemistry in secretions collected after intercourse.

[0099] As illustrated in this article, when Lactobacillus janigeri expressing antisperm scFv was colonized into mice, a decrease in pregnancy rate between 50% and 58% was observed, and the number of offspring per cage and thus per mouse was also significantly reduced.

[0100] In one embodiment, the symbiotic organism expressing the antisperm agent can be further engineered to express an agent that is effective against infection by organisms causing sexually transmitted diseases in the treated female. In one embodiment, as those skilled in the art will understand, the organism causing sexually transmitted diseases is chlamydia, HIV, HPV, etc.

[0101] In one embodiment, such an agent, effective against infection in treated females by organisms causing sexually transmitted diseases, can interfere with the infection of said organisms, or in another embodiment, such an agent can reduce the probability of infection or the infection load. In some embodiments, such an agent can interfere with the pathogenesis of the infection in the organism. It should be understood that further incorporation of any agent effective against infection in treated females by organisms causing sexually transmitted diseases is contemplated in this respect and is considered part of the invention.

[0102] Accordingly, in some embodiments, the contraceptive of the present invention can be considered as a combination of contraceptive-anti-STD therapy.

[0103] While it is anticipated that the engineered symbiotic organisms of the present invention can be further engineered, for example, to express anti-chlamydia scFv, or anti-HPV or anti-HIV scFv, or other anti-HIV agents, such as cyanovirin, it should be understood that the presence of wild-type lactobacillus colonization in the female reproductive tract provides protection against STD infections. It is also anticipated that the engineered organisms of the present invention expressing antisperm agents will be as effective, or in some embodiments, more effective than wild-type lactobacillus alone, in reducing the incidence of sexually transmitted diseases in female populations using such organisms described herein.

[0104] It should be understood that any embodiment described herein that applies to any or all methods of the present invention is considered part of the invention. All publications and patent applications referenced in this specification are incorporated herein by reference as if each individual publication or patent application were expressly and individually indicated to be incorporated herein by reference.

[0105] Those skilled in the art will understand that various changes in form and detail may be made therein without departing from the spirit and scope of the invention as set forth in the appended claims. By means of no more than conventional experimentation, those skilled in the art will recognize or be able to identify many equivalent forms of the specific embodiments of the invention described herein. Such equivalent forms are intended to be included within the scope of the claims.

[0106] It should be understood that, unless otherwise indicated or clearly apparent from the context, words such as “a,” “an,” and “the” referring to articles herein mean one or more. Unless otherwise indicated or clearly apparent from the context, a claim or description including “or” or “and / or” among the members of the group is considered satisfied if one, more than one, or all of the group members are present, used for, or otherwise relate to a given product or method. The invention includes embodiments in which exactly one group member is present, used for, or otherwise relates to a given product or method. The invention also includes embodiments in which more than one or all of the group members are present, used for, or otherwise relate to a given product or method. Furthermore, it should be understood that, unless otherwise indicated or unless it clearly causes contradiction or inconsistency to those skilled in the art, the invention provides for all variations, combinations, and arrangements in various embodiments, wherein one or more definitions, elements, clauses, descriptive terms, etc., from one or more of the enumerated claims are incorporated into another claim deriving from the same basic claim. When elements exist in a list (such as in Markush form, etc.), it should be understood that each subgroup of elements is also disclosed, and any element can be removed from the group. It should be understood that, generally, when an invention or aspect of an invention is referred to as containing a specific element, feature, etc., certain embodiments of the invention or certain aspects of the invention consist of or are substantially composed of such elements, features, etc. For simplicity, not every instance of these embodiments is explicitly stated in the language of this invention. For convenience, some claims exist in the form of dependent claims, but the applicant reserves the right to rewrite any dependent claim in the form of an independent claim (to include elements or limitations of the independent claim to which it is to be subordinated and any other claim), and such rewritten claim is considered equivalent in all respects to the dependent claim, regardless of its form (modified or unmodified) before being rewritten in the form of an independent claim.

[0107] The following materials, methods, and embodiments are intended to be provided for illustrative purposes (as ways of implementing / performing the invention) and are not intended to be limiting. Example

[0108] Generally, the terminology used herein and the experimental methods used in this invention include molecular, biochemical, microbiological, and recombinant DNA techniques. These techniques have been explained in detail in the literature. See, for example, "Molecular Cloning: A Laboratory Manual," Sambrook et al., (1989); "Current Protocols in Molecular Biology," Volumes I-III, Ausubel, RM, ed., (1994); Ausubel et al., "Current Protocols in Molecular Biology," John Wiley and Sons, Baltimore, Maryland, (1989); Perbal, "A Practical Guide to Molecular Cloning," John Wiley & Sons, New York, (1988); Watson et al., "Recombinant DNA," Scientific American Books, New York; Birren et al. (eds.), "Genome Analysis: A Laboratory Manual Series," Vols. 1-4, Cold Spring Harbor Laboratory Press, New York. Methods described in York (1998); U.S. Patent Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III, Cellis, J.E., ed. (1994); "Current Protocols in Immunology", Volumes I-III, Coligan, J.E., ed. (1994); Stites et al. (eds.), "Basic and Clinical Immunology" (8th edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds.), "Selected Methods in Cellular Immunology", WH Freeman and Co.New York (1980); Available immunoassays are extensively described in patents and scientific literature, see, for example, U.S. Patent Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis," Gait, MJ, ed. (1984); "Nucleic Acid Hybridization," Hames, BD, and Higgins. SJ, ed. (1985); "Transcription and Translation" Hames, BD and Higgins SJ, eds. (1984); "Animal Cell Culture" Freshney, RI, ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984); and "Methods in Enzymology" Vol. 1317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein purification and Characterization - A Laboratory Course Manual" CSHL Press (1996), all of which are incorporated herein by reference as if fully described herein. Other general references are provided in this document. The methods contained herein are considered well known in the art and are provided for the convenience of the reader. All information contained herein is incorporated by reference.

[0109] Example 1

[0110] Preparation of anti-human sperm antibodies

[0111] Monoclonal antibodies against human sperm antigens were prepared by fusing P3-X63-Ag8-653 mouse myeloma cells with lymphocytes from Balb / c mice (immunized with human epididymal sperm dissolved in Tergitol NP-40 detergent). The antibodies were acrosome-positive for methanol-fixed sperm and plasma membrane-positive for unfixed sperm, as determined by indirect immunofluorescence assays obtained from ascites fluid of mice injected with these hybridomas. Cross-reactivity of the antibodies with mouse and rabbit sperm was verified.

[0112] As described in (Naz, RK et al., Proc Natl Acad Sci USA (1986) 83(15): 5713-7), the fertilization antigen FA-1 was purified from mouse testes dissolved in deoxycholic acid or lithium diiodosalicylate using immunoaffinity chromatography with the monoclonal antibody MA-24. Additional anti-FA-1 monoclonal antibodies were prepared as described above, and their cross-reactivity with human and rabbit sperm was also verified.

[0113] Example 2

[0114] Cloning of anti-human sperm antibodies

[0115] RNA preparation:

[0116] Hybridoma cells secreting antibodies that kill sperm or reduce sperm motility were selected. Cells were cultured and counted to treat 10 million cells using a FastTrack 2.0 kit (Invitrogen) to collect RNA. Total RNA was isolated directly from the cells using detergent lysis and protein degradation buffer. Poly(A)+ RNA was then isolated using a modified Aviv and Leder protocol where mRNA was bound to oligo dT resin. The resin was then washed with low-salt buffer to remove excess total RNA, and Poly(A)+ RNA was eluted from the resin. Spectroscopic analysis at 260 nm and 280 nm indicated the final concentration of Poly(A)+ RNA.

[0117] Reverse transcription and amplification of Poly(A)+ RNA

[0118] The epitope recognition region and complementarity-determining region (CDR) of the antibody were identified and designed as probes to detect oligonucleotides of the Ig heavy chain and light (κ) chain subunits.

[0119] Heavy chain oligonucleotides were obtained from the Ig-Prime kit (Novagen) and diluted with dH2O to a final concentration of 1.0 μg / μl.

[0120] Reverse transcription and polymerase chain reaction (PCR) amplification of each hybridoma Poly(A)+ RNA was performed in a single reaction using the Promega Access RT-PCR system. In short, 5 μg of hybridoma Poly(A)+ RNA, 1 μg of each suitable primer (primers 1 and 2 for the heavy chain, and primers 3 and 4 for the light chain), 1 μl of 10 mM dNTP mixture, 10 μl of 5×AMV reverse transcription buffer, 2 μl of 25 mM MgSO4, 1 μl of AMV reverse transcriptase, 1 μl of Tfl polymerase, and 30 μl of nuclease-free dH2O were mixed in a 0.5 mL microcentrifuge tube. PCR reactions were performed according to standard protocols, with optimized cycling temperature and time. The amplified products were analyzed on an agarose gel and purified.

[0121] Antibody cloning and sequencing

[0122] The gel-purified product was subcloned into a suitable vector that produced a high yield of the product. The vector, buffer, cDNA, and T4 DNA ligase were incubated at room temperature for 1 hour, followed by heating at 65°C for 10 minutes. Using supercompetent *E. coli* cells, DNA was added to the cells and incubated on ice, followed by heating at 42°C and adding SOC medium. The cells were then incubated in a 37°C shaking water bath for 1 hour. These cells were then plated on LB Peelere's plates containing the selection compound and incubated overnight at 37°C. Positive clones were selected and cultured to purify the plasmid.

[0123] Purify the plasmid using a Qiagen-tip 20 column, following the manufacturer's instructions.

[0124] To confirm the presence of the cDNA insert in the purified plasmid, each clone was digested with a suitable restriction endonuclease, and the digestion products were electrophoresed on an agarose gel. Clones containing the insert were sequenced.

[0125] Sequence analysis of heavy and light chain clones

[0126] Positive clones were sequenced using a Fidelity kit (Oncor) following the manufacturer's instructions. The sequencing reaction consisted of plasmid DNA, primers (such as the T3 primer located upstream of the 5' cloning site in the pCR-Script vector), and dH2O. It was heated to 95°C for 5 minutes, annealing buffer was added, and the reaction was then incubated at 37°C for 15 minutes. The reaction was continued by adding reaction buffer. 33PαATP, T4 DNA polymerase, and dH2O were used to label the reactants, and the mixture was incubated at 400°C for 15 minutes. A, C, G, or T was then added to terminate the mixture, and the mixture was incubated at 40°C for 5 minutes. The reaction was terminated by adding proteinase K solution, and the gel was heated to 95°C before loading onto an acrylamide sequencing gel. The gel was then electrophoresed at 2000 volts for 2 hours, dried under vacuum on Whatman 3mm filter paper, and exposed to X-ray film overnight at room temperature. After film development, the gel was read out in a lightbox.

[0127] Sequencing can also be performed using an automated DNA sequencer (such as the ABI Prism 377). For each clone, cDNA and primers are mixed with dideoxynucleotides labeled with four dyes and AmpliTaq polymerase FS. The entire reaction mixture is loaded into a single lane and electrophoresed on a 36 cm 5.0% acrylamide gel for easy reading. Real-time detection of individual electrophoretic fragments is achieved using laser scanning and CCD camera imaging.

[0128] Construction of expression carrier

[0129] Expression vectors, or variants thereof, as described in McCracken A. et al. (2000) Arch. Microbial. 173: 383-389; Perez-Casal, J. et al. (2003) Mol. Microbiol. 8: 809-819; Kruger C. et al. (2002) Nature Biotechnology 20: 702-706; Rao, S. et al. (2005) PNAS 102: 11993-11998; Liu X. et al. (2006) Antimicrobial Agents and Chemotherapy 50: 3250-3259; del Rio B. et al. (2008) Clinical and Vaccine Immunology 15: 1429-1435; U.S. Patent No. 7,312,076, or European Patent No. 1,011,721 B1, may be prepared by methods known in the art.

[0130] Alternatively, the carrier can be constructed as follows:

[0131] A shuttle vector was created by subcloning the *E. coli* origin of replication from pBluescript into a backbone vector (Fons, M., et al. (1997) Plasmid 37, 199-203), followed by removal of the full-length M6 coding region (PstI). This plasmid was digested with SmaI, partially digested with NdeI, filled with DNA polymerase I (Klenow fragment), and self-ligated. The resulting plasmid contained a *Lactobacillus*-compatible origin of replication (repA) and positive selection markers, such as antibiotic resistance genes, and has been used to express heterologous proteins in various *Lactobacillus* species.

[0132] The amplified antibody fragment was cloned into the vector.

[0133] The amplification products corresponding to the heavy and light chains were purified by gel purification and resuspended in dH2O. Antibodies can be recoded by assembling PCR (Stemmer WP et al., (1995) Gene 164:49-53) to more closely conform to the optimal Lactobacillus codons used.

[0134] Expression cassettes were constructed by PCR amplification and subcloned into suitable sites on the vector. Each cassette contained four components: a lactobacillus-compatible promoter element, an antibody fragment, a signal sequence for secretion, or a cell wall anchoring domain. Unique restriction sites were placed between each component from the 5' to 3' ends. PCR amplification of each component was performed using Pfu DNA polymerase. The P23 promoter from *Lactococcus lactis* (van der Vossen, JM, et al. (1987) Appl. Environ. Microbiol. 53, 2452-2457) was generated by amplification using primers 5'-GTG GAG CTC CCC GAA AAG CCC TGA CAA CCC-3' and 5'-GGAAAC ACG CTA GCA CTA ACT TCA TT-3'. To guide antibody secretion, primers were designed to amplify the *Lactobacillus curvatureii* S-layer gene (cbsA) sequence, which has unique sites at the 5' and 3' ends, from the putative ribosome binding site to the signal peptidase cleavage site. Subsequently, the amplified S-layer signal nucleotide sequence (CbsAss) corresponding to MKKNLRIVSAAAAALLAVAPVAA was digested and used to clone it into an expression cassette.

[0135] The product was ligated into a vector, and a TAA stop codon was inserted into the N-terminus of the anchor motif to ensure secretion. Sequence verification was performed before transformation into Lactobacillus strains.

[0136] Lactobacillus transformation

[0137] Bacterial strains and culture. Naturally occurring human vaginal isolates of *Lactobacillus curvatureii*, *Lactobacillus gasseri*, and *Lactobacillus janseri*, etc., can be obtained from vaginal swabs of healthy volunteers, or alternatively, commercially available strains can be used and cultured at 37°C (5% CO2 / 95% air) on de Man, Rogosa and Sharpe (MRS) or Rogosa SL (Difco) medium. Medium 199 (Invitrogen) was also used for protein expression analysis. The plasmid was introduced into *Escherichia coli* DH12S (Invitrogen) by electroporation. To preserve the plasmid, the transformed *E. coli* DH12S were cultured at 37°C in LB medium (Difco) supplemented with erythromycin (300 μg / ml). Basically, as described for *Lactobacillus gasseri* (Luchansky, JB, Tennant, MC & Klaenhammer, TR (1991) J. Dairy Sci. 74, 3293-3302), the plasmid was transformed into *Lactobacillus japonicus* by electroporation. The transformed *Lactobacillus japonicus* was routinely propagated in liquid medium containing 20 μg / ml erythromycin.

[0138] The antisperm activity of engineered lactobacilli was detected as described below.

[0139] Example 3

[0140] The production of a series of antisperm scFvs

[0141] Library Construction

[0142] Human scFv filamentous phage display library was used to isolate the tethered variable domain of human VH-VL antisperm protein. Primers designed based on sequences obtained from V Base (http: / / vbase.mrccpe.cam.ac.uk) and in accordance with the guidelines of Barbas & Lerner (Barbas, Bain et al. 1992 Proc Natl Acad Sci USA 89(10): 4457-61; Gram, Marconi et al. 1992 Proc Natl Acad Sci USA 89(8): 3576-80; Zebedee, Barbas et al. 1992 Proc Natl Acad Sci USA 89(8): 3175-9; Barbas, Amberg et al. 1993 Gene 137(1): 57-62), Winter (Hawkins, Russell et al. 1992 J Mol Biol 226(3): 889-96; Hawkins and Winter 1992 Eur J Immunol 22(3): 867-70; Hoogenboom, Marks et al. 1992 Immunol Rev 130: 41-68; Hoogenboom and Winter 1992 Immunol Rev 130: 41-68; Marks, Griffiths et al. 1992 Biotechnology (NY) 10(7): 779-83; Marks, Hoogenboom et al. 1992 J BiolChem 267(23):16007-10; Marks and Winter 1992 Behring Inst Mitt(91):6-12; Orlandi, Gussow et al. 1992 Biotechnology 24:527-31; Tomlinson, Walter et al. 1992 J Mol Biol The techniques previously described in 227(3): 776-98) and the Benhar laboratory (Azriel-Rosenfeld, Valensi et al. 2004 J Mol Biol 335(1): 177-92) were used to prepare human scFv libraries. Human spleen and peripheral blood lymphocyte cDNA were used as templates for PCR amplification of antibody genes. In this library, libraries of human heavy and light chain variable domains were linked together in a combinatorial manner to generate all possible combinations of VH-VL artificial binding molecules, which were fused to the m13 filamentous phage p3 gene, and the fused protein was subsequently displayed on the phage surface (typically a single copy per phage on average).

[0143] Sperm antigen

[0144] Peptides were designed from sperm antigens FA-1 and YLP(12), respectively. Peptide 1 below is a 35-residue peptide designed from antigen FA-1, while peptide 2 below is a YLP(12) peptide. The peptides were biotinylated to facilitate binding to magnetic beads as described below. Due to the relatively short peptide sequences, a linker was added between the biotin and the peptide in peptide 2. Phage display scFv libraries were screened for the two peptides as described below.

[0145] Antibody screening

[0146] The library stock was cultured at 37°C in LB + Amp (100 μg / ml) + 1% glucose. Helper phage M13KO7 was added, and the culture was incubated overnight at 30°C with the helper phage in the presence of 100 μg / ml Amp + 30 μg / ml Kan. The culture was rotated at 4000 RPM for 10 min, and the supernatant was filtered through a 0.45 μm filter. 1 / 5 volume of PEG / NaCl was added, and the filtrate was placed on ice for at least 1 hour, followed by rotation at 4000 RPM for 30 min. The phage-containing precipitate was then resuspended in PBS. The phage was blocked with 4% BSA for at least 30 min, and antibody selection based on magnetic beads was performed according to the protocol described in R. Kontermann and S. Dubel (eds.), Antibody Engineering Vol. 1, Springer-Verlag Berlin Heidelberg 2010, pages 267-287. Blocking was performed with 2% BSA for at least 30 min. The sealed beads were added to the sealed phage, and then the beads were removed, thereby extracting all the phages bound to streptavidin. The sealed phages were then incubated with biotin for 30 minutes, followed by incubation with the beads for 30 minutes, after which the beads were discarded.

[0147] Incubate the bacteriophage with the following peptides for 1 hour:

[0148] ACGVSRPVIACSVTIKEGSQLKQQIQSIQQSIERL (SEQ ID NO: 1) – Biotin; and YLPVGGLRRIGG (SEQ ID NO: 2) – Ahx – were then incubated with the beads for 30 minutes, followed by washing and elution. The neutralized eluent was then mixed with dh5αF+ cells at 37°C for 60 minutes and cultured overnight on LB agar plates containing 100 μg / ml Ampicillin and 1% glucose. The panning process was repeated multiple times.

[0149] Antibody fragment specificity

[0150] Peptides were used as probes for ELISA assays, and the relative affinity of fragments was assessed by OD.

[0151] E. coli TG-1 infected with the selected phage (as described above) was plated to generate individual colonies. These colonies were picked into 100 μl of YTAG medium in the wells of a sterile 96-well plate and incubated at 30°C with shaking at 150 RPM. 10 μl of the phage was then transferred to a plate containing 90 μl of YTAG + 2.5 μl / ml (5 x 10⁻⁶) 8 CFU)M13KO7 helper phage was incubated in fresh 96-well plates at 37°C without shaking for 30 minutes, followed by shaking at 150 RPM for 30 minutes. The plates were centrifuged at 4000 RPM at 14°C for 5 minutes, and the supernatant was discarded. 200 μl / well of YTAK (containing kanamycin) was added, and the plates were incubated overnight at 30°C at 150 RPM with shaking. Subsequently, the bacterial plates were centrifuged at 4000 RPM at 4°C for 5 minutes, and then 100 μl of the supernatant mixed with 100 μl of PBST was used for ELISA.

[0152] 100 μl / well of antigen and control antigen were plated onto ELISA plates and incubated overnight at 4°C to coat the plates. The ELISA plates were washed once with washing buffer (300 μl / well of PBST) and blocked with 3% milk / PBS at room temperature (RT) for 1 hour. The plates were then washed once with washing buffer. Phage was then added to the plates and incubated at room temperature for 1 hour, followed by three washes with washing buffer. 50–100 μl / well of HRP-conjugated antiphage and diluted anti-M13 phage antibody were added and incubated at room temperature for 1 hour. After three washes with washing buffer, 100 μl / well of OPD substrate solution was added and incubated at room temperature for 20 minutes. The reaction was then terminated with 50 mL of 1 M HCl solution, and the plates were read at 450 nM. Detection was performed using horseradish peroxidase (HRP)-labeled antiphage antibody, and the color obtained from the substrate-enzyme reaction was read using an automated ELISA reader.

[0153] Another ELISA protocol involves coating plates with 2 μg / ml BSA-biotin (in PBS) at room temperature for 2 hours. The plates are washed with PBST and then coated with 2 μg / ml streptavidin (in PBS) at room temperature for 2 hours, followed by another wash with PBST. The plates are then coated overnight with 2 μg / ml peptide in PBS at 4°C, washed once with PBST, and blocked with 3% milk / PBS solution at 37°C for 1 hour. The remainder of this protocol is the same as described above, except that the plates are developed with 50 μl / well of TMB (tetramethylbenzidine) stop reagent diluted 4-fold in SDDW, and stopped with 50 μl / well of 1M H2SO4 after 5 minutes.

[0154] result

[0155] Figure 1 The relative affinities of the isolated and cloned phage-displayed scFvs to sperm peptides used as probes are graphically depicted. Columns 1-3 show the binding of the three scFvs to peptides derived from FA-1, while columns 4-6 show the binding of the three scFvs to peptides derived from YLP(12). BSA was used as a negative binding control. The scFvs showed significant affinity for the indicated peptides compared to the controls. Figure 2 The relative affinity of scFv for sperm peptides used as probes by certain isolated and cloned bacteriophages is described. Figure 3 Results of an ELISA binding assay identifying a series of non-binding scFvs were presented, which were subsequently used as controls. In this paper, one such scFv clone, J112, is typically used. Clone J102 showed greater affinity than other clones, as well as controls A and B, which were samples containing neither the scFv phage nor the peptide and scFv, respectively. Binding was detected by measuring the OD in the samples probed with an anti-phage HRP-labeled antibody (anti-PVIII antibody). Some of these scFvs were subsequently used in the examples below, as appropriate, as controls and contraceptives.

[0156] Example 4

[0157] Engineered lactobacilli expressing scFv

[0158] Antibody cloning and sequencing

[0159] Following the established protocol, scFv was released from the phage genome by digestion with NcoI and NotI enzymes to display the complete scFv fragment in the phage clone. The gel-purified product was subcloned into the pSLP111.1 vector provided by Dr. Jos Seegers, which yielded a high yield of the product. Figure 4AA map of the vector is provided. Subcloning was performed by PCR amplification of scFv using unique primers containing an NcoI restriction site on the 5' primer and an AscI site on the 3' primer (5'-GCG CCA TGG CCG AGG TGC AGC TGT TG, 3'-GCG GGC GCG CCC CAG CAC AGT GAGTTT GGT CCC). The amplified DNA was purified by gel extraction, and the restriction sites were activated by digestion with NcoI and AscI. Subsequently, the restriction-digested DNA fragments were purified again by gel extraction and ligated (using T4 ligase) into the PSLP111.1 vector, which had previously been digested with NcoI and AscI and purified by gel extraction. The ligation compound was used to transform *E. coli* DH5α, which was prepared as competent cells using the Maniatis technique. Transformed bacteria were plated on LB agar chloramphenicol plates (10 μg / mL), incubated overnight, and colonies were picked. Plasmid DNA was prepared from 24 colonies. Plasmid DNA was prepared from these colonies using the Qiagenminiprep plasmid kit and digested with NcoI and AscI to identify colonies containing appropriately sized DNA inserts. Positive clones were then transferred into *Lactobacillus japonicus* as described below.

[0160] Purify the plasmid using a Qiagen-tip 20 column, following the manufacturer's instructions.

[0161] To confirm the presence of the cDNA insert in the purified plasmid, each clone was digested with NcoI-AscI, and the digestion products were electrophoresed on an agarose gel. Clones containing the insert were sequenced.

[0162] Sequence analysis of heavy and light chain clones

[0163] Positive clones were sequenced using the sequencing services of the Weizman Institute, employing an ABI automated sequencer with all conditions and reagents as per the manufacturer's instructions. The initial sequencing primers used were:

[0164] Forward CCATGATTACGCCAAGCTTGGGAGCC (SEQ ID NO: 9)

[0165] Reverse GAATTCAACCTTCAAATTGCC (SEQ ID NO: 10)

[0166] The amplified antibody fragment was cloned into the vector.

[0167] The gel-purified amplification product is subcloned into a suitable site on the vector. The expression cassette contains four components, including a lactobacillus-compatible promoter element, an antibody fragment, a signal sequence for secretion, or a cell wall anchoring domain.

[0168] Sequence verification was performed before transformation into Lactobacillus strains.

[0169] Lactobacillus transformation

[0170] The human vaginal isolate of *Lactobacillus japonicus* strain ATCC (strain number 25258) was used and cultured at 37°C (5% CO2 / 95% air) on de Man, Rogosa and Sharpe (MRS) medium (Difco). pSLP111.1 containing the insert fragment was introduced into *Lactobacillus japonicus* using the calcium chloride technique. The transformed *Lactobacillus japonicus* was routinely propagated in liquid medium containing 10 μg / ml erythromycin.

[0171] Sperm binding and motility assay

[0172] Mouse sperm were isolated according to existing protocols [Liu Z et al., Journal of Biological Chemistry (2010) 285, 2758-2770]. 10 6 Sperm were suspended in a conical tube in sperm buffer (Ham's F-10, supplemented with 21 mM HEPES; 4 mM sodium bicarbonate; 0.6% human serum albumin; 3.6 ml sodium lactate (60% stock solution) and gentamicin: 10 μg / ml) to preserve their ability to swim to the top of the tube, where motile sperm were collected by withdrawing small volumes (hundreds of microliters) of buffer. Sperm counts were verified by a hemocytometer, and sperm were incubated at a sperm:lactobacterial (CFU) ratio of 1:1 or 1:2.

[0173] Mouse sperm were isolated, and the binding of scFv to sperm was assessed using a phage display scFv binding assay. The motility assay described above was used to select motile sperm, which were isolated and placed in a chamber slide, air-dried, and then phage containing the target scFv (10 μL) was applied to the sperm. 8 The sample was applied to the chamber and incubated for 1 hour. After washing, sperm were detected using an anti-cp8 antibody (ENCO, Israel) linked to HRP and diluted 1:200. The chamber was then washed and visualized with DAB and peroxide.

[0174] result

[0175] Figure 4B The NcoI and NotI digestion products of the plasmid are shown. The plasmid was digested with NcoI- and NotI at 37°C for 3 hours.

[0176] A novel scFv, J102, was discovered to possess a high binding affinity for a highly conserved sperm antigen. Sequencing of this clone yielded... Figures 5A-5B The complete DNA and protein sequences shown are (SEQ ID NO: 7-8).

[0177] The binding of scFv J102 to human and mouse sperm was detected. Although scFv J102 is a fragment derived from humans, the high species conservation of the FA-1 antigen allows J102 to bind to both mouse and human sperm, as determined by in vitro assays. Figure 6A The study showed significant binding of *Lactobacillus* expressing scFv to mouse sperm, with significant staining observed in almost every sperm cell evaluated. In contrast, control *Lactobacillus* expressing scFv against non-sperm antigens did not bind significantly to mouse sperm. Figure 6B The results of human sperm binding studies are consistent with those in mice (data not shown).

[0178] As described in the Methods section above, motility studies were conducted using human sperm. While lactobacilli expressing unrelated controls showed a moderate effect on sperm motility in vitro, a significant effect of impairing sperm motility was observed when using lactobacilli expressing sperm-binding scFv. This included a 50% reduction in motility compared to the control in assays detecting the effect of scFv J102, demonstrating the potential of lactobacilli expressing sperm-binding scFv to interfere with sperm function.

[0179] Example 5

[0180] Antisperm activity of engineered Lactobacillus japonicus

[0181] Inhibition of fertilization

[0182] 10 of each species 5 One sperm cell was cold-incubated overnight in a solution of the relevant and control antibodies (diluted 1:50, 1:5, or 1:1), or in a solution of the construct that produced the control and relevant antibodies. Oocytes were then added, allowing sperm-oocyte binding to occur for 1 hour. The oocytes were subsequently washed, and the number of sperm binding per oocyte was determined and expressed as a percentage of the number of sperm binding to the control sample. The construct with the highest percentage of inhibition was further evaluated.

[0183] In vivo fertilization inhibition study

[0184] Purified relevant antibody fragments, purified control antibody fragments, engineered Lactobacillus expressing the relevant antibody fragments, and engineered Lactobacillus expressing the control antibody fragments were injected into the vaginas of females (mice, rabbits). One male was confined with 2–4 females at multiple points after the various reagents were administered into the females' vaginas. Twenty-four hours after grouping, the presence of vaginal plugs in the females was visually examined daily. Females were marked to distinguish them (e.g., by successive ear punchings), and optionally, two weeks after the onset of mating, the females were moved to separate cages. After three weeks, pregnant females and offspring were counted. The construct with the greatest contraceptive activity was then selected for further evaluation / optimization.

[0185] Fusion inhibition assays were performed as follows. Young female mice (8-10 weeks old) were injected intraperitoneally with 5 units of pregnant mare serum (PMS) in 0.9% NaCl. Forty-eight hours later, the mice were injected intraperitoneally with 5 units of hCG (human chorionic gonadotropin) in 0.9% NaCl to trigger superovulation. Fourteen-six hours after hCG injection, the released oocytes were collected and treated with hyaluronidase to remove cumulus cells. The zona pellucida was removed with a protease mixture. The oocytes without zona pellucida were incubated in culture medium with a specified concentration of peptide for 30 minutes [Hogan, B. et al., Manipulating The Mouse Embryo, 91-101, (1986)]. Sperm collected from the epididymis of male mice was matured by incubation and subjected to an acrosome reaction as described by Fleming and Yanagimachi [Gamete Res. 4, 253-273 (1981)]. The sperm was then added to oocytes in the presence of control and related antibodies, and constructs expressing the control and related antibodies, and incubated for 15 minutes. The oocytes were subsequently transferred to a sperm-free medium and incubated for an additional 1 hour and 45 minutes. The oocytes were then fixed and stained as described by Primakoff et al. [J. Cell. Biol. 104, 141 (1987)]. The total number of swollen sperm heads was then counted. Swollen sperm heads are a marker of sperm-egg fusion.

[0186] Based on these observations, several indices were calculated. The fertilization index (FI) was determined by dividing the total number of swollen heads by the total number of oocytes. The fertilization rate (FR) was the percentage of fertilized oocytes. The inhibition percentage was determined by dividing the fertilization index of the experimental peptide by the fertilization index of the control peptide. The construct with the greatest activity was selected.

[0187] Example 6

[0188] Engineered Lactobacillus japonicus used as an effective contraceptive

[0189] Following the method described in Example 4, other animal species were tested. Rabbits, rats, guinea pigs, miniature pigs, monkeys, and other species were evaluated. In this case, several constructs were evaluated, and differences in animal species were noted. The percentage of effectiveness for each construct was determined within each animal strain.

[0190] In addition, autoimmune insemination (AIF) was performed on each species using semen obtained from males with verified fertility, where sperm count and motility were assessed. Lactobacilli expressing relevant and control antibody fragments were administered intravaginally, and AIF was performed at different time points after administration. During artificial insemination, ovulation was induced by injection of, for example, 100 IU hCG. Following ovulation and artificial insemination, females were allowed to complete the pregnancy process to assess its potential teratogenic effects.

[0191] To evaluate the persistence of control lactobacilli and lactobacilli expressing relevant antibody fragments in the reproductive tract when administered to females.

[0192] For this purpose, vaginal secretions were collected by repeatedly blowing sterile PBS into the vaginal opening. The presence of scFv in the supernatant was determined by briefly rotating the liquid and mucus in a microcentrifuge. The titers of antisperm antibody fragments in serum and vaginal washes were determined by ELISA.

[0193] A time-course study was conducted. Females treated with lactobacillus were repeatedly paired with males, and pregnancy rate / contraceptive effectiveness was assessed as a function of time after treatment.

[0194] Example 7

[0195] Lactobacillus japonicus expressing sperm-binding scFv is an effective contraceptive in mice.

[0196] Mouse administration and mating studies

[0197] Female ICR mice were anesthetized with ketamine solution (100 mg / ml) at a concentration of 0.1 ml / gm body weight, 0.5 ml of xylazine (20 mg / ml), and 8.5 ml of 0.9% saline. The anesthetized mice were then administered 10 ml of xylazine solution intravaginally. 8 Freshly cultured, mid-index (determined by OD) engineered Lactobacillus japonicus strain J102 or control J112. (N = 12 mice per group). After administration, females were held supine with pelvis tilted upwards for 30–60 minutes. Females were administered Lactobacillus daily for 3 days, followed by mating with age-matched ICR males. Males and females were kept together for 7 days, rotating the males every other day, and then removed. The presence of plugs in females was then visually assessed, and the number of pregnant mice and the number of offspring produced per cage were recorded.

[0198] Mouse colonization study

[0199] Between 12 and 24 hours after the first intravaginal application, females were swab-swabled or doucheed, and the recovered sample / liquid was spread on chloramphenicol-containing MRS plates and incubated at 37°C for 24 hours. The presence of bacterial flora served as an indicator of vaginal colonization.

[0200] result

[0201] Colonization studies in mice showed that up to 50% of the irrigation fluid produced bacterial colonies, indicating that colonization occurred in up to 50% of mice after a single intravaginal application. In animals where colonization was demonstrated by wiping, the intravaginal persistence of the applied lactobacillus was found to be high, lasting up to 5 days post-application.

[0202] Two separate experiments were conducted, in which females were treated with either Lactobacillus japonicus expressing antisperm scFv or a control strain, and in each experiment, effective contraception was demonstrated. Figure 7A Although 92% of control females became pregnant in each experiment, females treated with Lactobacillus jans expressing antisperm scFv showed a significantly lower pregnancy rate (58% or 50%).

[0203] As a result of administering *Lactobacillus japonicus* expressing antisperm scFv, the total number of progeny and the number of progeny per cage were also reduced compared to the control. Figure 7B Based on these findings, the number of offspring per mouse was also reduced in the treated females compared to the control group.

[0204] Example 8

[0205] Safety of biological contraceptives

[0206] To evaluate the effects of engineered lactobacilli on vaginal stress response. Animals, such as mice, were treated vaginally with lactobacilli once daily for 10–30 days. Animals were then sacrificed, and the reproductive tract was examined visually and microscopically.

[0207] Examining vaginal tissue for epithelial ulceration, edema, leukocyte infiltration, and vascular congestion as functions of introduced lactobacilli and engineered lactobacilli expressing controls and related antibodies. Animals treated with engineered lactobacilli were compared with animals treated with natural isolates and untreated animals. Improvements in reproductive tract histology, if present, were noted.

[0208] Example 9

[0209] Reversibility of birth control pills

[0210] Female animals that failed to become pregnant despite repeated mating (due to the administration of lactobacillus) were given antibiotics and then mated. Pregnancy was assessed as a measure of the reversibility of the engineered lactobacillus. Similarly, the persistence of lactobacillus in the animal reproductive tract was assessed. After an indication of a decrease or disappearance of the engineered lactobacillus population, the animals were mated, and their gravidity was determined.

[0211] Once safety and efficacy studies have been conducted in suitable animal models (e.g., as described above), human clinical trials are then considered.

[0212] This instruction manual also includes the following:

[0213] 1. Genetically engineered symbiotic bacteria of the female reproductive tract, wherein the genetically engineered bacteria are engineered to express an antisperm agent.

[0214] 2. The engineered bacteria of Embodiment 1, wherein the antisperm agent is an antisperm scFv antibody fragment.

[0215] 3. The engineered bacteria of embodiment 2, wherein the antisperm scFv antibody fragment is human or humanized.

[0216] 4. The engineered bacteria of Embodiment 2, wherein the scFv antibody fragment interacts specifically with the acrosome or plasma membrane.

[0217] 5. The engineered bacteria of Embodiment 2, wherein the scFv antibody fragment specifically interacts with the sperm neck region.

[0218] 6. The engineered bacteria of Embodiment 2, wherein the scFv antibody fragment specifically interacts with sperm FA-1 antigen or a fragment thereof.

[0219] 7. The engineered bacteria of embodiment 2, wherein the scFv antibody fragment interacts specifically with a peptide having at least 90% identity with SEQ ID NO: 1 or 2.

[0220] 8. The engineered bacteria of embodiment 2, wherein the scFv antibody fragment has a sequence having at least 90% identity with SEQ ID NO: 8.

[0221] 9. The engineered bacteria of Embodiment 2, wherein the scFv antibody fragment is encoded by a polynucleotide having a sequence having at least 90% identity with SEQ ID NO: 7.

[0222] 10. The engineered bacteria of Embodiment 1, wherein the symbiotic bacteria is Lactobacillus.

[0223] 11. The engineered bacteria of embodiment 8, wherein the symbiotic bacteria is Lactobacillus janniae, Lactobacillus curvatureii, or Lactobacillus casei.

[0224] 12. The engineered bacteria of Embodiment 1, wherein the symbiotic bacteria is Lactococcus.

[0225] 13. The engineered bacteria of embodiment 1, wherein the symbiotic bacteria is Escherichia coli Nissle 1917 strain.

[0226] 14. The engineered bacteria of Embodiment 1, wherein the symbiotic bacteria is Streptococcus Gordonii.

[0227] 15. A composition comprising the engineered bacteria of embodiment 1.

[0228] 16. The composition of embodiment 15, wherein the composition is in the form of a vaginal suppository, sponge, cream or foam.

[0229] 17. Use of the composition of Embodiment 15 for female contraceptive methods.

[0230] 18. Use of the composition of embodiment 15 in the preparation of a medicament for reducing the incidence of pregnancy in a female population.

[0231] 19. Use of the composition of Embodiment 15 in the preparation of a medicament for reducing the incidence of fertilization in female populations.

[0232] 20. Use of the engineered bacteria of Embodiment 1 in the preparation of a medicament for reducing the incidence of pregnancy in female populations.

[0233] 21. Use of the engineered bacteria of Embodiment 1 in the preparation of a drug for reducing the incidence of female population fertilization.

[0234] 22. An apparatus for insertion into the vagina, comprising engineered bacteria of embodiment 1.

[0235] 23. The device for insertion into the vagina according to embodiment 22, wherein the device is a ring inserted into the vagina.

[0236] 24. A method of contraception, the method comprising the step of contacting female reproductive tract cells with an amount of engineered bacteria of Embodiment 1 in the female subject that can effectively inhibit or prevent sperm motility, sperm-egg fusion, or egg penetration.

[0237] 25. The contraceptive method of embodiment 24, the method comprising the step of contacting female reproductive tract cells with an amount of the composition of embodiment 15 in which sperm motility, sperm-egg fusion, or egg penetration can be effectively inhibited or prevented in the female.

Claims

1. Genetically engineered symbiotic bacteria for use in the female reproductive tract. The genetically engineered bacteria mentioned above are contraceptive lactobacilli engineered to express antisperm agents in the female reproductive tract, and The antisperm agent is an antisperm antibody or its antigen-binding fragment, and the antisperm antibody or its antigen-binding fragment comprises: Heavy chain variable region, which includes: HCDR1, which consists of the amino acid sequence of DHDMH, HCDR2, which consists of the amino acid sequence GISWKSDSMAYRDSVKG, and HCDR3, which consists of the amino acid sequence of DQEHFDFDY; and The λ-type light chain variable region includes: LCDR1, which consists of the amino acid sequence SGSSSNLGSNTVN, LCDR2, which consists of the amino acid sequence of DNNQRPS, and LCDR3 is composed of the amino acid sequence AAWDDSLSGLV.

2. The engineered bacteria of claim 1, wherein the antisperm antibody or its antigen-binding fragment comprises: The heavy chain variable region consists of the following amino acid sequence: EVQLLESGGG LVQPGGSLRLSCAASGFTFSDHDMHWVRQA PGKGLEWVSG ISWKSDSMAY RDSVKGRFTI SRDNSKNTLY LQMNSLRAEDTAVYYCARDQ EHFDFDYWGQ GTLVTVSS; and The λ-type light chain variable region is composed of the following amino acid sequence: DIVLTQPPSA SGTPGQRVTI SCSGSSSNLGSNTVNWYQQL PGKAPKLLIY DNNQRPSGVP DRFSGSKSGT SASLAISGLR SEDEADYYCA AWDDSLSGLVFGTGTKLTVL.

3. The engineered bacteria of claim 1 or 2, wherein the antisperm agent is an antisperm scFv antibody fragment.

4. The engineered bacteria of claim 3, wherein the antisperm scFv antibody fragment is human or humanized.

5. The engineered bacteria of claim 3, wherein the scFv antibody fragment interacts specifically with the acrosome or plasma membrane.

6. The engineered bacteria of claim 3, wherein the scFv antibody fragment specifically interacts with the sperm neck region.

7. The engineered bacteria of claim 3, wherein the scFv antibody fragment specifically interacts with the sperm FA-1 antigen or a fragment thereof.

8. The engineered bacteria of claim 3, wherein the scFv antibody fragment specifically interacts with the peptide shown in SEQ ID NO: 1 or 2.

9. The engineered bacteria of claim 3, wherein the scFv antibody fragment consists of the amino acid sequence shown in SEQ ID NO:

8.

10. The engineered bacteria of claim 3, wherein the scFv antibody fragment is encoded by a polynucleotide consisting of the nucleic acid sequence shown in SEQ ID NO:

7.

11. The engineered bacteria of claim 1, wherein the symbiotic bacteria is Lactobacillus jensenii, Lactobacillus crispatus, or Lactobacillus casei.

12. A composition comprising the engineered bacteria of any one of claims 1 to 11.

13. The composition of claim 12, wherein the composition is in the form of a vaginal suppository, sponge, cream, or foam.

14. An apparatus for insertion into the vagina, comprising engineered bacteria according to any one of claims 1 to 11.

15. The device for insertion into the vagina according to claim 14, wherein the device is a ring for insertion into the vagina.

16. Use of the engineered bacteria of any one of claims 1 to 11 in the manufacture of a contraceptive kit.

17. Use of the composition of claim 12 in the manufacture of a contraceptive kit.

18. An antisperm agent, comprising an antisperm antibody or an antigen-binding fragment thereof, said antisperm antibody or antigen-binding fragment comprising: Heavy chain variable region, which includes: HCDR1, which consists of the amino acid sequence of DHDMH, HCDR2, which consists of the amino acid sequence GISWKSDSMAYRDSVKG, and HCDR3, which consists of the amino acid sequence of DQEHFDFDY; and The λ-type light chain variable region includes: LCDR1, which consists of the amino acid sequence SGSSSNLGSNTVN, LCDR2, which consists of the amino acid sequence of DNNQRPS, and LCDR3 is composed of the amino acid sequence AAWDDSLSGLV.

19. The antisperm agent of claim 18, wherein the antisperm antibody or its antigen-binding fragment comprises: The heavy chain variable region consists of the following amino acid sequence: EVQLLESGGG LVQPGGSLRLSCAASGFTFSDHDMHWVRQA PGKGLEWVSG ISWKSDSMAY RDSVKGRFTI SRDNSKNTLY LQMNSLRAEDTAVYYCARDQ EHFDFDYWGQ GTLVTVSS; and The λ-type light chain variable region is composed of the following amino acid sequence: DIVLTQPPSA SGTPGQRVTI SCSGSSSNLGSNTVNWYQQL PGKAPKLLIY DNNQRPSGVP DRFSGSKSGT SASLAISGLR SEDEADYYCA AWDDSLSGLVFGTGTKLTVL.

20. The antisperm agent of claim 18 or 19, wherein the antisperm agent is an antisperm scFv antibody fragment.

21. The antisperm agent of claim 20, wherein the scFv antibody fragment specifically interacts with the peptide shown in SEQ ID NO: 1 or 2.

22. The antisperm agent of claim 20, wherein the scFv antibody fragment consists of the amino acid sequence shown in SEQ ID NO:

8.

23. The antisperm agent of claim 20, wherein the scFv antibody fragment is encoded by a polynucleotide consisting of the nucleic acid sequence shown in SEQ ID NO: 7.

Images