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WO2005112567A2 - Microbicide vaginal - Google Patents

Microbicide vaginal Download PDF

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Publication number
WO2005112567A2
WO2005112567A2 PCT/US2005/015212 US2005015212W WO2005112567A2 WO 2005112567 A2 WO2005112567 A2 WO 2005112567A2 US 2005015212 W US2005015212 W US 2005015212W WO 2005112567 A2 WO2005112567 A2 WO 2005112567A2
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WO
WIPO (PCT)
Prior art keywords
lactobacillus
rantes
chemokine
vaginal
strain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2005/015212
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English (en)
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WO2005112567A9 (fr
WO2005112567A3 (fr
Inventor
Anthony L. Devico
Tina Kish-Catalone
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The University of Maryland Biotechnology Institute
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The University of Maryland Biotechnology Institute
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Anticipated expiration legal-status Critical
Publication of WO2005112567A3 publication Critical patent/WO2005112567A3/fr
Publication of WO2005112567A9 publication Critical patent/WO2005112567A9/fr
Ceased legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/195—Chemokines, e.g. RANTES
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0034—Urogenital system, e.g. vagina, uterus, cervix, penis, scrotum, urethra, bladder; Personal lubricants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins

Definitions

  • the present invention relates generally to a method of delivering an antiviral agent, and more particularly, to the use of a recombinant Lactobacillus strain for delivering a highly specific and potent anti-HIV agent into a female mammal, especially at mucosal surfaces. Nucleic acid constructs for these applications are also provided.
  • a limited number of preventative options for protection against the sexual transmission of HIV-1 are available including abstinence, vaccines, barrier methods, and microbicides.
  • abstinence is not ideal for many individuals due to personal, social and cultural issues.
  • Third, the male condom has not demonstrated the desired effectiveness in preventing HIV- transmission. However, effectiveness is dependent upon the consistent and correct use of the condom, which females may not be able to negotiate with their male partners due to social and culture inequalities.
  • a new approach to preventing the transmission of HIV- 1 is the development of a microbicide, which is female-controlled.
  • microbicides designed to prevent HIV transmission can be categorized according to their mechanism of action: 1) agents that alter the vaginal environment; 2) agents that inhibit pathogen movement by creating a physical barrier; 3) agents that inhibit fusion to the target cell; 4) agents that block or prevent pathogen binding or attachment to target cells; 5) agents that directly kill or inactivates pathogen.
  • Microbicide development has focused largely on using a variety of gels, creams, and lotions as vehicles for vaginal delivery.
  • Several safety and efficacy issues are critical in the development of a vaginal microbicide.
  • Studies have indicated that inflammation and epithelial disruption, such as genital lesions, result in an increased incidence of HIV-1 transmission.
  • the present invention provides for a unique vaginal microbicide product that utilizes a natural component of the vaginal ecosystem, Lactobacillus, to deliver a highly specific and potent anti-HIV agent, RANTES or natural isoforms thereof.
  • This strategy has several distinct advantages over conventional anti-HIV preventative methods.
  • -2 RANTES a natural isoform of full-length human chemokine RANTES, selectively and strongly binds to the CCR5 receptor, which is the major HIV-1 co-receptor.
  • CCR5 receptor which is the major HIV-1 co-receptor.
  • the Lactobacillus species is part of the natural vaginal micro flora. Therefore, use of this species as a vehicle induces minimal toxicity and immunogenicity.
  • Lactobacillus exhibits many beneficial characteristics for use as a microbicide delivery method.
  • hydrogen peroxide producing Lactobacillus maintains the acidic vaginal pH, which is a natural barrier to infection by several sexually transmitted pathogens, including HIV-1.
  • lactobacillus readily colonizes the vaginal tract, allowing for adequate coverage and protection of the mucosal surfaces.
  • lactobacillus products have been accepted as safe for human use.
  • lactobacillus can be genetically engineered to produce and secrete recombinant proteins.
  • the present invention relates to a delivery vector for administering an anti HIV agent in a mammalian female, the vector comprising a lactobacillus strain engineered to express a chemokine that binds to CCR5 receptors.
  • the chemokine is RANTES or a variant thereof wherein amino acid residues from the N- terminal are removed. More preferably, the variant is a natural isoform such as active-2 RANTES.
  • Another aspect of the present invention relates to a method of delivering an anti HIV agent in a mammalian female, the method comprising contacting vaginal mucosal tissue of a mammalian female with a lactobacillus strain engineered to express a chemokine that binds to CCR5 receptors.
  • the present invention relates to a vaginal microbicide for the reduction of HIV-1 transmission, the vaginal microbicide comprising a lactobacillus strain engineered to express a chemokine that binds to CCR5 receptors forming a receptor-ligand complex.
  • the chemokine is RANTES or a variant thereof wherein amino acid residues from the N-terminal are deleted. More preferably, the variant is an active-2 RANTES wherein the first two amino acids on the N-terminal are deleted.
  • a still further aspect of the present invention relates to a method of reducing HIV replication, the method comprising delivering a vaginal microbicide comprising a lactobacillus strain engineered to express a chemokine that binds to CCR5 receptors forming a receptor-ligand complex.
  • the chemokine is RANTES or a variant thereof wherein amino acid residues from the N-terminal are deleted. More preferably, the variant is an active-2 RANTES wherein the first two amino acids on the N-terminal are deleted.
  • Another aspect of the present invention relates to a method of reducing HIV replication, the method comprising delivering a vaginal microbicide comprising a lactobacillus strain engineered to express a chemokine that binds to CCR5 forming a receptor-ligand complex and in combination with an additional antiviral agent.
  • the chemokine is RANTES or a variant thereof wherein amino acid residues from the N-terminal are deleted. More preferably, the variant is an active- 2 RANTES wherein the first two amino acids on the N-terminal are deleted.
  • the additional antiviral agent may include, but is not limited to nucleoside RT inhibitors, such as Zidovudine (ZDV, AZT), Lamivudine (3TC), Stavudine (d4T),
  • nucleoside RT inhibitors such as Zidovudine (ZDV, AZT), Lamivudine (3TC), Stavudine (d4T)
  • TDF Delaviradine
  • Efavirenz Efavirenz
  • NVP Nevirapine
  • T-20 Fuzeon
  • SQV Saquinavir
  • RTV Ritonavir
  • IDV Indinavir
  • NFV Nelfinavir
  • AMV Amprenavir
  • LV Lopinavir
  • Atazanavir Combivir
  • ZDV/3TC Kaletra
  • RTV/LPV Trizivir
  • CCR5 inhibitors/antagonists such as SCH-C, SCH-D, PRO 140, TAK 779, TAK- 220, RANTES analogs, AK602, UK-427, 857, monoclonal antibodies;
  • viral entry inhibitors such as Fuzeon (T-20), NB-2, NB-64, T-649, T-1249, SCH-C, SCH-D, PRO 140, TAK 779, TAK-220, RANTES analogs, AK602, UK-427, 857; and functional analogs or equivalents thereof.
  • HIV-1 infection occurs mainly through binding of the HIV-1 envelope glycoprotein, gpl20, to the CD4 receptor on human immune cells.
  • efficient viral fusion and subsequent entry into the host cell requires HIV binding to a chemokine co- receptor.
  • the major co-receptor for HIV infection is CCR5.
  • Inhibition of HIV-1 infection can be achieved by blocking either gpl20-CD4 binding or gpl20-CCR5 binding.
  • Developing targeted ligands that block gpl20-CD4 binding has been problematic because CD4 is essential for several critical functions, including T-cell activation.
  • the development of a targeted ligand that binds CCR5 avoids this obstacle.
  • the rationale for the development of CCR5-targeted ligands is fourfold.
  • CCR5 binding of CCR5 effectively blocks HIV infection.
  • CCR5 is well represented on mucosal epithelium and is the major co-receptor used for HIV-1 entry.
  • studies have demonstrated that loss of CCR5 is correlated with strong natural resistance to HIV-1 infection and there are no apparent health consequences associated with lack of CCR5 expression.
  • the importance of CCR5 for initial transmission of HIV-1 is highlighted by the fact that individuals lacking expression of CCR5 (the CCR5- ⁇ 32 homozygous genotype) are usually resistant to infection (Liu, et al., 1996).
  • CCR5 cell-surface density correlates with disease progression in infected individuals (Lin, et al, 2002).
  • the inhibitory effect of RANTES is proposed to act through blocking of CCR5 as well as through down- regulation of the coreceptor from the cell surface.
  • increase in the level of RANTES has been correlated with resistance to infection or a more favorable clinical prognosis, likely because of competition of the chemokines with HIV-1 for binding to CCR5.
  • Preferred proteins of the present invention are chemokines as discussed above, more preferably RANTES, natural isoforms and variants thereof.
  • the present invention also includes proteins with a homology to RANTES and characterized by having a binding affinity for receptor CCR5.
  • proteins having at least 85% amino acid sequence identity to the naturally occurring RANTES, more preferably at least 95% may be used in the present invention.
  • Such variations in the amino acid sequence are made by conservative substitutions, deletions and/or insertions into the RANTES amino acid sequence.
  • a more preferred protein is 2-RANTES, which is defined herein as a RANTES absent the two N-terminal amino acids.
  • Other amino acid deletions may include up to 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18 or 20 N- terminal amino acid deletions to RANTES.
  • Modifications and changes can be made in the structure of a polypeptide of the present invention and still obtain a molecule having RANTES peptide characteristics.
  • certain amino acids can be substituted for other amino acids in a sequence without appreciable loss of peptide activity. Because it is the interactive capacity and nature of a polypeptide that defines that polypeptide's biological functional activity, certain amino acid sequence substitutions can be made in a polypeptide sequence (or, of course, its underlying DNA coding sequence) and nevertheless obtain a polypeptide with like properties.
  • Amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary substitutions which take various aspects of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine, as shown below.
  • the present invention thus contemplates functional or biological equivalents of a peptide as set forth above.
  • Amino acid residues can be added to or deleted from RANTES and natural isoforms thereof through the use of standard molecular biological techniques without altering the functionality of the peptide. For example, portions of the RANTES and natural isoforms thereof can be removed to create truncated RANTES. The truncated peptide retains the properties of RANTES.
  • RANTES nucleotide and amino acid sequences for humans and other animals are publicly available in public databases.
  • Genbank Accession Nos. for RANTES is set forth below. (as obtained from http://www2.ncbi.nlm.nih.gov/genbank/ query_form.html):
  • polynucleotide means a sequence of nucleotides connected by phosphodiester linkages. Polynucleotides are presented herein in the direction from the 5' to the 3' direction.
  • a polynucleotide of the present invention can be a deoxyribonucleic acid (DNA) molecule or ribonucleic acid (RNA) molecule. Where a polynucleotide is a DNA molecule, that molecule can be a gene or a cDNA molecule.
  • Nucleotide bases are indicated herein by a single letter code: adenine (A), guanine (G), thymine (T), cytosine (C), inosine (I) and uracil (U).
  • the polynucleotide of the invention comprises SEQ ID NO 1, SEQ ID NO: 2 or variants thereof.
  • nucleic acid is introduced into a bacterial host cell.
  • the introduction may employ any available technique.
  • suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage.
  • the introduction may be followed by causing or allowing expression from the nucleic acid, e.g. by culturing host cells under conditions for expression of the gene.
  • Growing the cells in culture under conditions for expression of the RANTES or natural isoforms thereof may be employed to verify that the Lactobacillus strain contains the encoding nucleic acid and is able to produce the encoded material.
  • the present invention provides a method of delivering a biologically active dose of a polypeptide in vivo, the method comprising administering to an individual a Lactobacillus strain containing nucleic acid for expression of RANTES or a natural isoform thereof.
  • preferred bacteria include Lactobacillus and a preferred route of administration may be by application to mucosal surfaces.
  • the delivery of RANTES or a natural isoform thereof to a mammalian female for effective treatment is preferably included in the microflora of the vaginal environment.
  • the microflora of the human cervicovaginal environment provides a natural barrier against infection by incoming sexually transmitted pathogens.
  • the vaginal ecosystem is typically dominated by Lactobacillus species that are facultative anaerobes, and produce large amounts of lactic acid as the end products of sugar fermentation. This creates an acidic environment, which is not suitable for many bacterial strains.
  • approximately 70-90% of the vaginal microflora consists of Lactobacillus.
  • Lactobacillus species produce substances with antimicrobial properties including, lactacidin, acidolin, lactin 13, and hydrogen peroxide. Furthermore, several species of Lactobacillus, which normally colonize the human vaginal tract, maintain strong cell adhesion to the mucosal surface, thus providing adequate coverage of secreted bactericidal compounds. In addition, Lactobacillus has been demonstrated to possess biosurfactant proteins that physically inhibit the binding of pathogens. Thus, a Lactobacillus strain that colonizes the human vaginal tract is an effective delivery vector that can be readily engineered to express functional recombinant proteins of the present invention.
  • any Lactobacillus strain may be used that has a high affinity towards the vaginal epithelium, i.e. a high capability of adhering to the epithelial cells of the vagina, in that this property enables bacteria interaction with the vagina mucosa under both physiological and pathological conditions and thus re-establishment of microflora and optimal pH conditions.
  • the Lactobacillus strain is selected from Lactobacillus brevis, Lactobacillus salivarius subs, salicinius, Lactobacillus salivarius subs, salivarius, Lactobacillus jensenii, Lactobacillus catenaforme, Lactobacillus minutus and Lactobacillus gasseri. More preferably, the Lactobacillus strain is Lactobacillus jensenii.
  • the Lactobacillus strain expresses the RANTES or natural isoform thereof from nucleic acid contained within it.
  • the nucleic acid may comprise one or more nucleic acid constructs in which nucleic acid encoding the RANTES or natural isoform thereof is under control of appropriate regulatory sequences for expression in the Lactobacillus strain.
  • Suitable vectors comprising nucleic acid for introduction into the Lactobacillus strain can be chosen or constructed and containing appropriate regulatory sequences, including promoter sequences, terminator fragments, enhancer sequences, marker genes and other sequences, as appropriate.
  • Vectors for delivering the nucleotide sequences into the Lactobacillus may be plasmids, viral e.g. "phage, or phagemid.
  • phage a virus
  • Any expression vector capable of expression in Lactobacillus is suitable for the instant invention, including secretion expression vectors (de Vos, W., Current Opinion in Microbiol, Vol. 2, pp.
  • Plasmids may be used as a vector and are circularized DNA molecules commonly found in bacteria. They replicate independently from the bacterial host genome via an origin of replication (ori) site. Genes inserted into a plasmid are readily transcribed if placed downstream of appropriate promoter sequences. Certain promoter sequences exist which are regulated by external factors such as the molecule IPTG. A number of plasmids have been optimized for individual bacterial host strains, most notably E. coli.
  • the plasmid will be digested with appropriate restriction enzymes to expose the cloning site. Then the desired nucleotide sequence for RANTES can be ligated into the plasmid and the plasmid introduced in the bacterial host. Once transformed with the recombinant plasmid containing the desired nucleotide sequence, the bacteria is grown in the appropriate media (e.g. LB media with 0.2% glucose). Transformed bacteria will be selected by adding an antibiotic to which the plasmid contains a resistance gene such that only transformed bacteria would survive. Expression of the nucleotide sequence can be constitutive or induced by stimulating the promoter to which it is attached, such as with IPTG. Expression of RANTES or a natural isoform thereof can be demonstrated by staining with fluorescent-labeled antibodies against the desired molecule.
  • appropriate media e.g. LB media with 0.2% glucose
  • the coding sequence for RANTES or natural isoforms thereof is part of a nucleic acid vector and under the regulatory control of a promoter.
  • a promoter employed in accordance with the present invention is preferably expressed constitutively in the bacterium.
  • Use of a constitutive promoter avoids the need to supply an inducer or other regulatory signal for expression to take place.
  • the promoter directs expression at a level at which the bacterial host cell remains viable, i.e. retains metabolic activity and growth is maintained.
  • the promoter may be homologous to the bacterium employed, i.e. one found in that bacterium in nature.
  • a lactobacillal promoter may be used in a Lactobacillus.
  • the nucleic acid construct may comprise a secretory signal sequence.
  • the nucleic acid encoding the RANTES or natural isoform thereof may provide for secretion of the RANTES (by appropriately coupling a nucleic acid sequence encoding a single sequence to the nucleic acid sequence encoding the polypeptide).
  • Ability of a bacterium harboring the nucleic acid to secrete the polypeptide may be tested in vitro in culture conditions that maintain viability of the organism.
  • Suitable secretory signal sequences include any of those with activity in Gram positive organisms including Lactobacillus. Such sequences may include the ⁇ - amylase secretion leader of Bacillus amyloliquefaciens or the secretion leader of the Staphylokinase enzyme secreted by some strains of Staphylococcus, which is known to function in both Gram-positive and Gram-negative hosts.
  • a bacteriophage may be used as the vector.
  • Bacteriophages are viruses that infect bacteria.
  • An example includes bacteriophage phi.adh, which infects Lactobacillus gasseri.
  • the nucleic acid of the selected bacteriophage may be manipulated such that the RANTES gene(s) replaces the genes coding for bacteriophage coat proteins, rendering the bacteriophage replication-defective. Adding these recombinant DNA molecules into cell lysates containing functional bacteriophage proteins will lead to assembly of functional bacteriophage particles carrying the RANTES gene(s).
  • These replication-defective bacteriophage particles can then be introduced onto the vaginal mucosal surface to infect selected floral Lactobacillus.. The typical dosage would be 10 to 10 PFU/ml applied to the mucosal surface.
  • Lactobacillus are self-replicating, so theoretically if an engineered Lactobacillus successfully colonizes a mucosal surface, it should persist indefinitely. However, numerous factors may limit the indefinite survival of an engineered bacterial population on a given mucosal surface, the most significant factor being the fierce competition for space by a number of different bacteria on any mucosal surface. Therefore, it is envisioned that applications of engineered Lactobacillus to a mucosal surface will need to be repeated on a regular basis; optimal dosing intervals are routine to determine, but will vary with different mucosal environments and bacterial strain. The dosing intervals can vary from once daily to once every 2-4 weeks. It is expected that colonization of vaginal mucosa will require less, as low as 10 6 viable Lactobacillus bacteria, since these surfaces are more directly accessible and include an environment for continued colonization.
  • the Lactobacillus bacteria may be applied in a nutrient medium, i.e. medium containing a substance or substances that sustain metabolic activity in the bacterium. Such substances may sustain viability if not growth of the bacterium. Such substances may include an energy source such as glucose and amino acids.
  • the individual to which the bacterium is administered may be human or animal, i.e. a non-human mammal.
  • a preferred Lactobacillus strain is Lactobacillus jensenii and a preferred route of administration may be by application to vaginal mucosal surfaces.
  • Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing, in addition to the preferred Lactobacillus strain of the present invention, such carriers as are known in the art to be appropriate.
  • administration is preferably in a "therapeutically effective amount," which is sufficient to show benefit to a subject.
  • Such benefit may be at least amelioration of at least one symptom.
  • the amount may be sufficient to reduce the deleterious effect on the subject of a subsequent pathogenic challenge, for instance by enhancing the immune response or reducing HIV replication or transmission.
  • the actual amount administered, and rate and time-course of administration will depend on the aim of the administration, e.g. the biological effect sought in view of the nature and severity of the challenge, and is the subject of routine optimization. Prescription of treatment, including prophylactic vaccination, for example decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors.
  • compositions according to the present invention may comprise, in addition to the preferred recombinant Lactobacillus strain, a pharmaceutically acceptable carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may depend on the route of administration. Those of relevant skill in the art are well able to prepare suitable solutions.
  • a pharmaceutical composition comprising a preferred Lactobacillus strain for administration in accordance with the present invention may comprise one or more nutrient substances, e.g. an energy source such as glucose and amino acids.
  • a composition comprising a preferred Lactobacillus strain may be administered in accordance with the present invention alone or in combination with other HIV antiviral agents, either simultaneously or sequentially, including a therapeutically effective amount of at least one antiviral agent, such as nucleoside RT inhibitors, CCR5 inhibitors/antagonists, viral entry inhibitors and their functional analogs.
  • at least one antiviral agent such as nucleoside RT inhibitors, CCR5 inhibitors/antagonists, viral entry inhibitors and their functional analogs.
  • the antiviral agent comprise nucleoside RT inhibitors, such as Zidovudine (ZDV, AZT), Lamivudine (3TC), Stavudine (d4T), Didanosine (ddl), Zalcitabine (ddC), Abacavir (ABC), Emirivine (FTC), Tenofovir (TDF), Delaviradine (DLV), Efavirenz (EFV), Nevirapine (NVP), Fuzeon (T-20), Saquinavir (SQV), Ritonavir (RTV), Indinavir (IDV), Nelfinavir (NFV), Amprenavir (APV), Lopinavir (LPV), Atazanavir, Combivir (ZDV/3TC), Kaletra (RTV/LPV), Trizivir (ZDV/3TC/ABC);
  • ZDV Zidovudine
  • AZT Lamivudine
  • 3TC Lamivudine
  • Stavudine d4T
  • CCR5 inhibitors/antagonists such as SCH-C, SCH-D, PRO 140, TAK 779, TAK- 220, RANTES analogs, AK602, UK-427, 857, monoclonal antibodies;
  • compositions of the invention are administered in substantially non-toxic dosage concentrations sufficient to ensure the release of a sufficient dosage unit of the present combination into the subject to provide the desired inhibition of the HIV virus.
  • the actual dosage administered of the RANTES or natural isoform thereof will be determined by physical and physiological factors such as age, body weight, severity of condition, and/or clinical history of the subject and preferably in a concentration of about 1 wM-25 uM, more preferably about 2-20 wM, and most preferably about 5-10 wM. It will be understood, however, that dosage levels that deviate from the ranges provided may also be suitable in the treatment of a given viral infection.
  • Lactobacillus jensenii was engineered to express -2 RANTES. This bacterial species was selected because it is one of the predominant strains of the human vaginal microflora and, as such, is an efficient colonizer of the vaginal mucosa.
  • 2 RANTES was cloned in a vector provided by Oscel, Inc. and the transformed Lactobacillus demonstrated expression of -2 RANTES by ELISA and SELDI assays. The ELISA assay is specific for the detection and quantitation of RANTES chemokine. 15 ng/mL of -2 RANTES was detected from a 5 mL culture of lactobacillus, wherein the growth conditions were optimized to increase the concentration of -2 RANTES expression.
  • Mass spectrometry analysis was performed using the Surface Enhanced Laser Desorption and Ionization (SELDI) protein chip technology. This assay can determine the exact mass of a protein that is captured using a specific human RANTES antibody. The results demonstrated that there is a peak in the range where -2 RANTES should fall.
  • SELDI Surface Enhanced Laser Desorption and Ionization

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Abstract

La présente invention se réfère à un microbicide vaginal comprenant une Lactobacille exprimant une chimiokines qui se lie aux récepteurs CCR5 en formant ainsi un complexe récepteur-ligand et de préférence, la chimiokines est RANTES ou une variante. Il est en outre décrit des procédés d'utilisation de lactobacille modifiée pour éviter la transmission sexuelle de HIV, dans lesquels la Lactobacille colonise le tractus vaginal et secrété la chimiokines fournissant ainsi la protection de sa surface de muqueuse.
PCT/US2005/015212 2004-05-03 2005-05-03 Microbicide vaginal Ceased WO2005112567A2 (fr)

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US60/567,643 2004-05-03

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WO2005112567A3 WO2005112567A3 (fr) 2009-04-16
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011138776A2 (fr) 2010-05-06 2011-11-10 Hervana Ltd. Contraceptifs biologiques féminins
US8686111B2 (en) 2006-07-25 2014-04-01 Mintaka Foundation For Medical Research Cytokine derivatives
US11166968B2 (en) 2015-09-29 2021-11-09 Kimberly-Clark Worldwide, Inc. Synergistic composition for maintenance of healthy balance of microflora
US12029748B2 (en) 2017-02-28 2024-07-09 Kimberly-Clark Worldwide, Inc. Synergistic composition for maintenance of healthy balance of microflora

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040116341A1 (en) * 2001-10-23 2004-06-17 Holtzman Michael J. Methods for ameliorating childhood infections

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8686111B2 (en) 2006-07-25 2014-04-01 Mintaka Foundation For Medical Research Cytokine derivatives
WO2011138776A2 (fr) 2010-05-06 2011-11-10 Hervana Ltd. Contraceptifs biologiques féminins
US11166968B2 (en) 2015-09-29 2021-11-09 Kimberly-Clark Worldwide, Inc. Synergistic composition for maintenance of healthy balance of microflora
US12419898B2 (en) 2015-09-29 2025-09-23 Kimberly-Clark Worldwide, Inc. Synergistic composition for maintenance of healthy balance of microflora
US12029748B2 (en) 2017-02-28 2024-07-09 Kimberly-Clark Worldwide, Inc. Synergistic composition for maintenance of healthy balance of microflora

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