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WO2005093043A1 - Vaccins immunocontraceptifs par adn et utilisations de ceux-ci - Google Patents

Vaccins immunocontraceptifs par adn et utilisations de ceux-ci Download PDF

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Publication number
WO2005093043A1
WO2005093043A1 PCT/US2004/006216 US2004006216W WO2005093043A1 WO 2005093043 A1 WO2005093043 A1 WO 2005093043A1 US 2004006216 W US2004006216 W US 2004006216W WO 2005093043 A1 WO2005093043 A1 WO 2005093043A1
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bacterial host
lactate dehydrogenase
salmonella
dna
group
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PCT/US2004/006216
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English (en)
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Barrie G. Kitto
Daniel C. Hirschhorn
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Research Development Foundation
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0006—Contraceptive vaccins; Vaccines against sex hormones
    • 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
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/542—Mucosal route oral/gastrointestinal

Definitions

  • the present invention relates generally to the field of immunocontraceptives. More specifically, the present invention relates to methods of controlling animal populations using immunocontraceptives expressed as DNA vaccines.
  • Rodent pests have infiltrated human activities and caused great economic and social impact. They can cause massive destruction of crops and foodstuffs and spread fatal diseases to people and domestic animals.
  • the so-called comensal rodents include the Norway rat (Rattus norvegicus), the roof or house rat (Rattus rattus), and the several su bspecies of mice Mus musculus (domesticus, spretus, mecedonicus, hortulanus, molossinus, and castaneous). According to estimates by the Food and Agricultural
  • a rat produces on average 40 droppings a day. At this rate, ten rats will produce 146,000 droppings a year. The same amount of rats can produce 54 liters of urine. If only a few droppings or a small amount of urine finds its way to food intended for human consumption it would most likely be rejected. This is because, among other things, waste products are carriers of disease. In addition, rodent carcasses killed by poisons can render useless entire reservoirs of grain and other crops. Rats, mice, and other rodents are capable of carrying and transmitting a variety of diseases to human and other animals. Viruses rodents transmit cause serious diseases in man with fatality rates up to
  • Rodents can transmit a variety of rickettsial diseases using vectors such as ticks, mites, fleas, or mosquitoes.
  • the principal rickettsial diseases that inflict human populations include louse-borne typhus (Rickettsia prowazeki), Rocky Mountain spotted fever, Boutonneuse fever, tustsugamushi diseases (Scrub typhus), and murine typhus (Rickettsia typh ⁇ ).
  • Rodents are also reservoirs of a great number of bacterial diseases. In fact, they are particularly known for carrying bacteria such as Salmonella, plague, Lyme disease, Borrelia, and leptospirosis.
  • Mechanical/physical rodent control includes shooting, flooding burrows with water, use of ultrasound or electromagnetic waves. Using high tech devices such as ultrasound or electromagnetic waves is not only an extremely expensive alternative but also has never been shown to produce consistent results. Trapping will produce some results under very limited conditions such as a house. However, trapping does not protect crops and it is only used in conjunction with chemical rodenticides to remove surviving animals from treated areas.
  • Biological rodent control refers to the use of predators, pathogens, and parasites that induces mortality or migration of rodents.
  • Ecological rodent control refers to changing the environment of a particular infested area such that it is not attractive to rodents anymore. This means that the environment has to be sufficiently different to make rodents uncomfortable. Ecological control has only been shown to be effective in combination with other methods of control. Genetical rodent control refers primarily to the possibility of altering the normal gene pool within a population by introducing a deleterious gene or introducing sterile males. For example, introducing genes such as the one causative of the Gruneber Lethal Syndrome that results in the death of 25% of the offspring before puberty has been explored. However, natural selection will operate to eliminate such genes from the population. Introducing sterilized individuals into the population was tested on several occasions by releasing infertile Norway rat males into the population.
  • Chemical poisons or rodenticides are the most widely used and efficient of all available methods for rodent control.
  • the introduction of these agents provided society with an extremely powerful tool in the battle against rodents.
  • certain poisons were no longer effective to control infestation at all and new chemicals have to be developed.
  • the use of chemical poisons is not an effective strategy because rodents breed rapidly and those that were killed are readily replaced.
  • Reducing fertility is a more preferable and effective method to control rodents and other mammalian pests.
  • sterilization is considered to have more potential and more impact on a population than conventional killing approaches.
  • an anti-fertility vaccine or immunocontraceptive is to induce immune response against proteins from the reproductive system, thereby leading to infertility.
  • contraceptive vaccines are for the control of rodents and other mammalian species.
  • a successful contraceptive vaccine will have to block fertilization or embryonic development, be species specific, and provoke a sustained immune response. Additionally, an effective mechanism which is cost effective to manufacture and administer for transmitting the vaccine safely throughout the target population must be found.
  • a fundamental issue in the development of immunocontraceptive agents for the control of wild species is the method of delivery.
  • the prior art is deficient in efficient methods of delivering immunocontraceptives useful in controlling animal populations such as rodents.
  • the present invention fulfills this long-standing need and desire in the art by providing immunocontraceptives expressed as DNA vaccines.
  • the present invention provides a fertility control agent that is cost-effective, humane and species specific for the control of animal populations.
  • the fertility control agent comprises a genetically engineered bacterial host that has been modified to produce the sperm-specific protein lactate dehydrogenase-C (LDH-C).
  • LDH-C lactate dehydrogenase-C
  • animals such as rodents eat these modified bacteria, their immune system produces antibodies that attack their sperms. Because the antibodies react to sperm, the instant fertility control agent affects the fertility of both males and females. Not only would the males have less viable sperms, the females would also have antibodies to the sperms entering their reproductive systems. The induced sterility is only maintained as long as the animals ingest the bacteria.
  • a g enetically modified bacterial host that expresses an immunocontraceptive comprising an egg- or sperm-specific polypeptide.
  • the contraceptive agent is useful in controlling the size of an animal population by inducing sterility in animals that have ingested the genetically- engineered bacteria.
  • methods of using these contraceptive agents to control the population size of animals that has reached pest levels are provided.
  • Figure 2 is a map of pcDNA3.1 -LDH-C designed to express the complete sequence of lactate dehydrogenase-C.
  • Figure 3 shows PCR results of DNA extracted from a vaccinated mouse. Lane 1 , stomach DNA 2 ml; lane 2, stomach 1 ml; lane
  • FIG. 3 shows average and individual serum IgG response to DUA-Salmonella vaccination.
  • the IgG was measured by indirect ELISA on days 0, 16, 23, 34, 45, and 87. Each arrow represents the day that the vaccine was administered.
  • the serum was diluted 1 :500.
  • Figure 5 shows average and individual serum IgA response to
  • Salmonella-DNA vaccine Titrating amounts of either recombinant lactate dehydrogenase-C or heat-killed Salmonella were used in each group.
  • the legend includes: Black bar, target cells and responder cells from the pcDNA3-LDH-C-treated animals; Red bar, target cells and responder cells from the pcDNA3-GFP treated mice (control); Green bar, only responder cells from the pcDNA3-LDH-C-treated mice (control); Yellow bar, responder cells from the pcDNA3-GFP treated mice (control); Blue bar, mitomycin C- treated antigen presenting cells only (control/baseline).
  • the ELISA is measuring BrdU incorporation.
  • immunocontraceptive refers to an immunogenic composition comprising an antigen that can induce immune responses against the cells of an animal's reproductive system, thereby leading to loss of fertility in the treated animal.
  • the present invention discloses development of new methods for the control of animal populations that reach pest proportion.
  • the present invention incorporates recently d eve l o ped immunocontraception technologies.
  • the basic premise of immunocontraceptives is to orally immunize animals by using bait formulations containing proteins from the animals' own reproductive system so that immune responses that block fertilization are induced in the animals after ingestion of such baits.
  • Immunocontraception is an attractive method for reducing the population size of animals with high fecundity, and it is believed that sterilizing animals using such immunocontraceptives can reduce targeted animal populations to acceptable levels in an efficient, cost-effective, humane and, importantly, a species-specific manner.
  • Lactate Dehydrogenase C4 As An Antigen For Immunocontraception
  • lactate dehydrogenase-C All vertebrate tissue contains lactate dehydrogenases (EC 1.1.1.27). This tetrameric protein has multiple isozymes assembled primarily by two subunits, A and B, that can form 5 different molecular forms.
  • Lactate dehydrogenase-C4 (LDH-C4) is a sixth isozyme of lactate dehydrogenase that exclusively expresses in the testis of mammals and other animal species.
  • LDH-C4 differs from the other lactate dehydrogenase isozymes in amino acid composition and in its catalytic properties. Most interestingly, sera raised against LDH-C do not cross-react with the A or B subunits.
  • lactate dehydrogenase-C can be used as a contraceptive agent come from studies where antibodies were passively transferred to animals. In this experiment, pregnancy suppression occurred in mice treated with rabbit anti-lactate dehydrogenase-C serum. Active immunization of purified lactate dehydrogenase-C produced infertility in female mice, rabbits, and baboons (Lerum and Goldberg, 1974; Goldberg, 1973; Goldberg et al., 1981 ).
  • lactate dehydrogenase-C4 antigen has allowed for B-cell epitope mapping of the molecule and subsequent synthesis of species-specific immunodominant epitope peptides (Kaumaya et al., 1992; Hogrefe et al., 1987).
  • Immunodominant epitopes of lactate dehydrogenase-C4 can be identified by computer algorithms for B-cell epitope prediction (Van Regenmortel and Daney de Marcillac, 1998). The predicted epitopes can be corroborated by sequence comparison with related mammalian lactate dehydrogenase-C immunodominant epitopes.
  • lactate dehydrogenase-C4 antigenic domains include amino acids 5-17, 44-58, 61- 77, 97-110, 101-115, 180-210, 211-220, 231-243, 283-306, and 307-316.
  • a peptide vaccine consisted of a lactate dehydrogenase-C4 epitope chemically linked to a diphtheria toxoid has been tested in rabbits. The diphtheria toxoid was used with the double purpose of carrier protein and adjuvant.
  • the peptide was further adapted for baboon uses by using the human sequence, and up to 75% reversible contraception was shown in 15 female baboons (O'Hern et al., 1995).
  • a completely synthetic lactate dehydrogenase-C4 peptide vaccine consisting of just 39 amino acids coupled to a "promiscuous" T-cell epitope from tetanus toxin was capable of reducing fertility by 62% in female baboons (O'Hern et al., 1997).
  • a promiscuous T-cell epitope is a short sequence capable of providing a T- helper response needed for antibody production in a wide range of species and multiple genetic backgrounds.
  • LDH-C4 lactate dehydrogenase-C4
  • Table 1 shows a comparison of amino acids 1-15 of LDH-C4 from several species. From this table, it can be inferred that this particular sequence is divergent enough to provide some level of species specificity.
  • Table 1 Lactate Dehydrogenase-C4 Amino Acids 1-15 From Several Species Amino acid differences between mouse LDH-C and other species are underlined.
  • Fragments of the lactate dehydrogenase-C4 protein can be generated by methods known to those skilled in the art, e.g., by enzymatic digestion of naturally occurring or recombinant lactate dehydrogenase-C4 protein, by recombinant DNA techniques using an expression vector that encodes a defined fragment of lactate dehydrogenase-C4, or by chemical synthesis.
  • Fragment of lactate dehydrogenase-C4 have been synthesized in combination with additional elements such as diptheria toxoid or a promiscuous T-cell epitope of tetanus toxin and shown to have comparable contraceptive effectiveness to lactate dehydrogenase-C4 in several species (O'Hern et al., 1995, 1997).
  • Other carriers considered include the use of complete cholera toxin, its beta subunit, or a genetically or chemically detoxified form of the toxin. This agent has been shown to be the strongest mucosal adjuvant and can be more adequately used when oral and mucosal vaccines are considered.
  • mucosal adjuvants from bacterial toxins or other sources could be used (Piazza, 2001).
  • One of ordinary skill in the art would recognize that the present invention is equally applicable to other animal or pest populations. Human activities, while driving some species to extinction or the brink of extinction, have provided a safe haven for many other species to proliferate to levels where they can pose great danger. Some of these species have become a menace to local flora, fauna, or, paradoxically, to humans themselves. There are multiple examples in many parts of the world where mammals have reached pest densities. For example, the white-tailed dear (Odocoileus virginianus) has been a menace in North America for almost a century.
  • contraceptive vaccines for controlling vertebrate pests including deer.
  • many vertebrate pests share many characteristics with the rodents and most models developed for their immunocontraceptive control can be equally applied.
  • other animals to which the present invention is applicable include any mammalian species capable of eliciting an immune response against proteins of their own reproductive system.
  • deer elephants, water buffalo, feral horses, foxes, urban or wild dogs, urban or wild cats, rabbits, and other potentially overpopulated species causing economic damage to society could be targeted.
  • Crucial to the development of a successful contraceptive bait is the selection of a proper carrier for the immunological agent.
  • the carrier should be stable, capable of being inexpensively and efficiently produced, environmentally safe and attractive as a food to the targeted ani mals. Based on these requirements, the present invention discloses a DNA vaccine that could be orally accessible using an attenuated bacterium.
  • the contraceptive agents could be prepared into a bait formulation that could be strategically placed among the targeted animal populations. The an imals would be sterilized upon consumption of the vaccine, thus promoting population control.
  • DNA vaccines consist of a plasmid with a strong mammalian promoter (usually a viral promoter), an antigenic ge ne, a polyadenylation sequence, and a termination signal.
  • the plasmid is grown in bacteria, purified, and administered to a host by a variety of methods, usually by injection in saline or in colloidal gold using a "gene gun".
  • the cells take up the plasmid and the antigenic protein is endogenously produced. This leads to the production of antibodies and cell-mediated immunity specifically against the antigen, thus protecting the host against the antigen.
  • DNA vaccination possesses several advantages that make this strategy desirable.
  • a valuable benefit is that genetic vaccination produces antigens that are identical to proteins produced endogenously by viruses and self proteins. This is because the antigens undergo the same treatment as native products, such as post-translational modification and cellular localization. This is important because recombinant proteins and other vaccination methods do not show, in some instances, comparable immunity.
  • a second advantage is that bacterial DNA is shown to provide an immunomodulatory effect by itself. Certain sequences of bacterial origin contain the unmethylated CpG motifs known to activate host defense mechanisms. DNA vaccination could eliminate the use of adjuvants, which often exhibit high toxicity, in a number of vaccination strategies.
  • piasmids are considerably cheaper than other vaccination methods.
  • the amount of plasmid necessary to induce an immune response is generally small (on the order of micrograms).
  • the vectors can easily be modified, adapting the antigen by modifying DNA sequences.
  • multiple genes expressing an array of epitopes could be used as a single vaccine component that can specifically target a desired response such as B or T cell stimulation. Examples of chimeric genes used to enhance immunity and provide protection have been reviewed in detail by Bona and Bot (2000).
  • the idea behind the delivery of DNA vaccines by attenuated bacteria lies in the fact that the microorganism can invade or be phagocytized by host cells. Once inside the cells the bacterium escapes the phagocytic vesicle using a series of mechanisms involving various virulence factors. After the organism breaks open into the cytoplasm, it dies due to previous genetic modifications that cause its attenuation (although, the infectivity mechanisms are kept intact). This causes release of the piasmids into the cytoplasm. The plasmid then reaches the nucleus by a poorly defined mechanism. The plasmid is episomally transcribed, the mRNA is transported to the cytoplasm, and the antigen gets expressed and modified.
  • bacteria as a vehicle for plasmid vaccines should solve some of the problems of DNA vaccination and provide additional benefits.
  • One of the advantages is that it can act as a powerful natural adjuvant by two distinct mechanisms. Since the bacteria target the inductive sites in the host, the antigen gets expressed at the most effective places. Secondly, bacterial cell wall and the unmethylated CpG bacterial sequences are capable of activating innate immune response. Usually the piasmids used in DNA vaccination have high copy number and each bacterium can possess hundreds or even thousands of copies. This is particularly interesting since in the event that the bacterium is short lived and does not invade the cells, its lysis might provide the surrounding tissues with enough plasmid to elicit an immune response.
  • bacteria are convenient since their production, storage, and transportation is practical and cost effective. Lastly, bacteria can be treated with ordinary antibiotics in the case of an adverse reaction. To date, the use of bacteria as carriers of DNA have been concentrated mainly on several species of Salmonella (typhimurium, typhi, flexneri) and to a lesser extent Lysteria monogenesis, Yersinia, Shigella, and BCG.
  • Salmonella typhimurium, typhi, flexneri
  • Lysteria monogenesis mainly on several species of Salmonella (typhimurium, typhi, flexneri) and to a lesser extent Lysteria monogenesis, Yersinia, Shigella, and BCG.
  • Salmonella typhimurium As A Carrier For DNA Vaccines, more is known about the role this bacterium plays in delivering DNA vaccines than any other bacterial species. After the bacterium is orally delivered, it penetrates the intestinal wall through the M cells. This enables the bacteria to infect professional antigen presenting cells located immediately below the epithelium and leads to an inflammatory response. This is followed by Sa/mot?e//a-specific antibody and cell mediated immune responses. Metabolically attenuated Salmonella strains have been developed that retain their infectivity and immunogenicity but do not lead to infection.
  • DNA vaccine technology for immunocontraception is suggested by the demonstration that significant antibody levels can be obtained in vaginal secretions following a local inoculation with a model DNA-based antigen delivered by a gene gun.
  • gene gun delivery is not a feasible approach for the control of mammals, in particular rodents. Therefore a more pragmatic solution is needed.
  • DNA-based immunocontraceptive is that the antigen produced will have native conformation and post-translational modifications in contrast to antigen produced in bacteria. This can be critical with certain auto-epitopes, especially when the production of antibodies is required.
  • antigen that is produced endogenously eliminates the need for purification, stabilization, and other requirements that protein antigens require, thus significantly reducing costs, particularly when Salmonella is used to transport DNA.
  • Model contraceptive vaccines have been primarily focused on inducing serum antibody responses and little evidence exists on mucosal immunization.
  • a D A-Salmonella hybrid vaccine would not only be able to elicit a systemic humoral response but, based on previous evidence, it could also stimulate the mucosal branch providing adequate antibody titers in the reproductive tract. This is of particular importance since past reports have not been able to detect a direct correlation between humoral antibodies and infertility.
  • the first report of a DNA vaccine for contraceptive purposes was published by Rath et al. (2002).
  • the authors used a construct containing the entire sequence of the egg antigen zona pellucida B (ZPB). Immunization of male BALB/cJ mice with ZPB DNA elicited significant antibodies that were able to bind to the native protein in a hemizona assay. (The hemizona assay is a test to determine the ability of the sperm to penetrate the egg). These preliminary results showed the feasibility of DNA vaccines to be used for immunocontraception. Accordingly, the present invention is directed to a genetically modified bacterial host that expresses an immunocontraceptive comprising an egg- or sperm-specific polypeptide or antigenic fragment thereof.
  • the sperm-specific polypeptide includes rat or murine lactate dehydrogenase-C.
  • Representative polypeptides of murine lactate dehydrogenase-C include EQLIQNLVPEDK (amino acids 5-17; SEQ ID NO. 1 ), GLADELALVDADTDK (amino acids 44-58; SEQ ID NO. 2), GEALDLDQHGSLFLSTPK (amino acids 61-77; SEQ ID NO. 3), LGVNPTSCHGWVLGEHGDSSVPIWSGVNVAGVTLK (amino acids 180-210; SEQ ID NO. 4), SLNPAIGTDK (amino acids 180-210; SEQ ID NO. 5), QVVEGGYEVLDK (amino acids 231-243; SEQ ID NO.
  • egg specific polypeptides include egg zona pellucida glycoproteins 1 , 2, and 3, e.g. PVTQSGPLRLELRLATDK (SEQ ID NO. 10), FGIHGPR (SEQ ID NO. 11) (Skinner et al., 1999; Sadler et al., 2000).
  • immunocontraceptive polypeptides include LNSSSSQFQIHGPR (SEQ ID NO. 12), CPKPDHTVTPDFYLA PPTTPEPFTPHAFALHPIPDHTLAGSGHTGLTTLYPEQSFIHPTPAPPSLGPGP A (SEQ ID NO. 13), WFLQSDNEDARIHSLYGMISC (SEQ ID NO. 14), ALNNRFQIKGVELKS (SEQ ID NO. 15), NCAYKTTQANK (SEQ ID NO. 16), CQADSGLQN RLALFTFPNISETNVTYLFGHEENSTEHAMKGVC (SEQ ID NO. 17) (Hardy et al., 2002).
  • contraceptive agents are useful in controlling the size of an animal population by inducing sterility in animals that have ingested the genetically-engineered bacteria.
  • the bacterial hosts include Salmonella typhimorium strains SL3261 or SL7202, other Salmonella species, Yersinia enterocolitica, Shigella flexner, Listeria monocytogenes, and recombinant Escherichia coli.
  • the present invention is also directed to methods of using these contraceptive agents to decrease the fertility of an animal.
  • the bacteria could be freeze-dried or be prepared through other methods as a bait formulation. Alternatively, the bacteria can be washed and administered to the animals in the wild with the purpose of spreading the bacteria from animal to animal.
  • Susceptible animals include mice, rats, deer, elephants, water buffalo, feral horses, foxes, urban or wild dogs, urban or wild cats, rabbits, and other potentially overpopulated species causing economic damage to society.
  • the following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion.
  • the present examples, along with the methods, procedures, treatments, molecules, and specific compounds described herein are presently representative of preferred embodiments.
  • One skilled in the art will appreciate readily that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those objects, ends and advantages inherent herein. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.
  • DNA contraceptive required the construction of appropriate plasmid vaccine constructs.
  • two vectors were considered: one encoding the entire cDNA sequence of mouse lactate dehydrogenase-C and one encoding a derived antigenic peptide of lactate dehydrogenase-C.
  • the vector of choice was pcDNA3.1 since it features a strong mammalian promoter from the cytomegalovirus and other features that increase gene expression in mammalian cells.
  • pcDNA3.1 -LDH-C The pcDNA3.1-LDH-C, designed to express the compete lactate dehydrogenase-C sequence, was provided by Dr. Erwin Goldberg from Northwestern University and no further modification was performed with this construct.
  • a map of the vector is shown in Figure 2.
  • This plasmid contains the cDNA of mouse lactate dehydrogenase-C between two EcoRI sites.
  • the plasmid was transformed into XL-1 blue electrocompetent cells (Stratagene).
  • the plasmid was isolated using the Quiaprep Spin Plasmid kit (Quiagen) following the manufacturer's instructions.
  • pcDNA3.1-SPV A second construct, pcDNA3.1-SPV, was designed to express an immunodominant LDH-C epitope.
  • SPV Short Peptide Vaccine
  • SPV Short Peptide Vaccine
  • a plasmid with the intact SPV region was used as a template for PCR so that the product comprises solely of the SPV sequence.
  • the sequence of this minigene includes amino acids 5-15 of lactate dehydrogenase-C coupled via a short spacer to a promiscuous T-cell epitope from tetanus toxin. It does not contain a starting ATG codon.
  • lactate dehydrogenase-C amino acid 1 being an ATG
  • This new minigene containing amino acids 1-15 of lactate dehydrogenase-C, makes a more "natural" peptide vaccine in addition to including a start codon.
  • the PCR product was then cloned into the pCR2.1 (Invitrogen, Ca) TA vector. This vector is conveniently designed for cloning PCR products easily without purification. Small fragments such as the SPV can be conveniently cloned and the complete procedure takes 2- 3 days.
  • DNA Vaccine Vector in vitro
  • the first step in the experiment was to develop a standardized protocol for plasmid transfection. This was accomplished by using the appropriate cell lines and the appropriate vectors.
  • the cell line chosen was the COS-7 cells. This mammalian cell line has been designed to maximize protein expression in transfection experiments and has been widely described in numerous successful transformations.
  • plasmid pcDNA3.1-GFP green fluorescent protein
  • the transfection method that was used involved the use of lipid-DNA complexs; in particular, the Lipofectamine reagent from Gibco BRL. Although other methods were perfectly acceptable, the Lipofectamine protocols have been well established especially for COS-7 cells. In addition, many samples can be transfected at the same time since the preparation of the lipid-DNA complexes is extremely easy. For this experiment, large quantities of highly purified plasmid DNA were needed. Cesium chloride gradients were used for this purpose and virtually limitless ( ⁇ 10 milligrams) amounts of nucleic acids were obtained without detectable impurities. For transfecting COS-7 cells with lipid-DNA complexes, several trials were performed with various amount of plasmid and Lipofectamine reagent to obtain optimal results.
  • EXAMPLE 3 Expression of DNA Vaccine Vector In Salmonella
  • pcDNA3.1-LDH-C and pcDNA3.1-SPV were considered as the vaccine candidates to be tested.
  • the pcDNA3-GFP was used as a negative control.
  • the piasmids were transformed into Salmonella typhimorium strain SL3261.
  • the Salmonella were subsequently grown without aeration and with high salt concentrations (regular LB medium plus 1.5%). Under these conditions, the Salmonella up-regulate the expression of a series of genes in the pathogenicity islet portion of the chromosome that help the bacteria increase its infectivity. This has the effect of making better vaccine candidates, as the attenuated bacteria become more intrusive without turning more pathogenic.
  • Salmonella that has been grown under this environment will emit a strong putrid odor characteristic of infective bacteria. Under these conditions, the Salmonella take several days to achieve acceptable numbers of bacteria in the cultures. This decreases the amount of plasmid since the ampicillin concentration needed to keep the bacteria under selection pressure gradually decreases. The option of supplementing the culture with additional ampicillin is not adequate nor it is to exchange the medium several times. Therefore, an alternative procedure was devised. The Salmoenlla were grown overnight with aeration until the culture was very dense. The next day, the cells were harvested and resuspended in fresh LB supplemented with sodium chloride and the antibiotic. No aeration was provided for 4 to 5 hours. The bacteria at that point started emitting the characteristic fetid odor.
  • the cells were harvested, washed in Hanks' balanced solution, and resuspended in a minimum volume of medium. A pinch of sugar (sucrose) was added to make the formulation more flavorsome for the mice. Bacteria were counted by consecutive dilution and plating in ampicillin containing plates such that only the cells containing the plasmid were accounted for.
  • EXAMPLE 4 Vaccine Preparation A positive colony from the transformed Salmonella was grown into a 50 ml LB medium culture supplemented with 50 mg/L of ampicillin overnight at 37°C, shaken at 200 rpm. Next day a 1 L culture was started under the same conditions and was grown overnight. The cultures were centrifuged at 6000 rpm in 500 ml sterile tubes for 10 minutes, the supernatant was discarded and the pellet was resuspended in 1 L of LB medium supplemented with 1.5 % NaCI and 50 mg/L of ampicillin. The flasks were sealed with a plastic plug so that the culture would be grown under anaerobic conditions.
  • a high salt concentration and low oxygen concentration is believed to up-regulate the expression of genes that will render a more pathogenic bacterium.
  • the transformed Salmonella were incubated for 5-6 hours at 37°C with minimal agitation (50 rpm). The cells were harvested by centrifugation as above and washed twice with Hank's balanced solution, pH 7.0, with some sucrose added to sweeten the solution and make it more palatable. The pellet from the last wash was resuspended in a minimum amount of buffer and the concentration of bacteria was adjusted to give approximately 10 9 colony forming units in 300 ul.
  • a Qiaquick plasmid prep was performed on the Salmonella at this stage to check the integrity of the plasmid.
  • EXAMPLE 5 Animals And Vaccine Administration Six-week-old female Balb/c mice were purchased from Charles River Laboratories, caged in groups of 4 or 5, and housed at the University of Texas Animal Resource Center facilities. Water and food were removed from the cages 5 hours prior to vaccine administration. Normal feeding followed but each water bottle contained 1 g/L of ampicillin. This was done since, from previous experience, such Salmonella levels (even attenuated strains) made the animals very sick and this sickness was relieved by ampicillin. The vaccine was prepared the day of the administration and was given to the animals by gavage using a blunt-ended stainless steel needle.
  • EXAMPLE 7 Blood Collection And Vaginal Washes To measure antibody responses in serum and vaginal secretions, the animals were bled and vaginally washed on selected days. To prepare the serum, the mice were first restrained, their tail cut at the tip, and a few drops (7-10) of blood were collected in a tube for each animal. The blood was incubated for 20 minutes at 37°C and then at 4°C overnight to complete the clotting process. The blood was spun down in a microcentrifuge at 10000 rpm for 10 minutes. The serum was carefully removed, transferred into a fresh tube, and was either immediately used or kept frozen at -80°C.
  • Vaginal washes were performed by inserting a blunt pipette tip containing 50 ml of phosphate buffer saltine into the vagina and moving the liquid up and down 10 times. The wash was transferred into a microtube and was kept immediately at 4°C and stored under the same conditions overnight where the large particles were allowed to sediment to the bottom of the tube.
  • the plates were washed 4 times with PBST (phosphate buffer saltine pH 7.4, 0.5% Tween 20 (Sigma)), incubated at room temperature in blocking buffer (PBST with 1 % protease-free BSA (Roche Biochemicals)) for 30 minutes and washed again once. Serum or vaginal washes were added to the wells up to 50 ml in blocking buffer and the plates were incubated for 1 hour at room temperature. When IgA was tested both serum and vaginal washes were diluted 1 :10. When IgG was tested serum was diluted 1 :500 and vaginal washes were again diluted 1 :10.
  • the plates were washed 3 times as described above and then a secondary affinity purified goat anti- either mouse IgG or IgA coupled to a horseradish peroxidase was added. Both antibodies were obtained from Kirkegaard & Perry Laboratories (Gaithersburg, MD). For IgG assessment, the antibodies were used at 1 :10,000 dilution in blocking buffer. For IgA, a 1 :1000 dilution was used. After one hour incubation at room temperature the plates were washed 4 times and 100 ml of hydrogen peroxide-2,2'-Azino-bis-(3- ethylbenzthiazoline-6-sulfonic acid (ABTS) from Moss Inc.
  • ABTS hydrogen peroxide-2,2'-Azino-bis-(3- ethylbenzthiazoline-6-sulfonic acid
  • T helper cell (Th cell) proliferation assays were performed in order to determine the extent of activation that LDH-C and Salmonella confers and the efficacy of the vaccine to stimulate T cells.
  • the assays were performed by a modification of the protocols by the Corradin et al. (1977) and Rosenwasser and Rosenthal (1998) references. Mice were sacrificed by carbon dioxide asphyxiation and were immediately moved to sterile conditions.
  • Spleens were removed and placed in 10 ml of RPMI 1640 (Invitrogen) supplemented with 2 mM L- glutamine, 1X antibiotic-antimycotic solution (Invitrogen), 50 mM b-mercaptoethanol, and 10 % fetal calf serum (Invitrogen).
  • the spleens were cut in small pieces and were then moved to a sterilel O ml syringe.
  • the syringe was connected to a 25 mm Easy Pressure Syringe Filter Holder (Gelman Laboratory, Ann Arbor, Ml) and the cells were collected in a sterile 15 ml tube. From this point on, the cells were kept on ice.
  • Splenocytes were centrifuged at 1250 rpm for 10 minutes and the supernatant was removed.
  • the pellet was resuspended in 5 ml sterile lysing buffer (150 mM NH 4 CI, 1.0 mM KHCO 3 , 0.1 mM EDTA, pH 7.4) and incubated at room temperature for 5 minutes with occasional shaking.
  • RPMI medium was added to fill the tube to 15 ml and the samples were centrifuged at 1250 rpm for 10 minutes. The supernatant was discarded and the cells were washed with media once more.
  • the pellet was resuspended in 5 ml and splenocyte recovery was determined by counting the cells using a hemocytometer. The cell concentration was adjusted to 1X10 6 cells/ml. This Th cell assay needs cells that process the antigen but do not proliferate themselves causing unspecific proliferation. These antigen- presenting cells were prepared as splenocytes from untreated mice using the above protocol and then were treated with mitomycin-c that prevents cellular proliferation without killing the cells by intercalating in the chromosomal DNA. Once the splenocytes were obtained, they were centrifuged at 1250 rpm for 10 minutes and the pellet was resuspended in 2 ml of PBS.
  • a fresh solution of mitomycin-c (Sigma) was prepared in PBS at 0.5 mg/ml and was kept in the dark at all times (the substance is extremely light sensitive).
  • One hundred ml of the mitomycin-c solution was added to each ml of splenocytes and the cells were incubated at 37°C for 20 minutes.
  • the tubes were then filled to 15 ml with complete RPMI medium and centrifuged at 1250 rpm for 10 minutes and the supernatant was discarded. The wash was repeated two additional times. The pellet was then adjusted to 1X10 6 cells/ml in complete RPMI.
  • Purified splenocytes (1X10 5 cells) from vaccinated animals were plated in flat-bottom microwell sterile plates with or without 1X10 5 mitomycin-c-treated antigen presenting cells.
  • Recombinant lactate dehydrogenase-C 100, 10, or 1 mg/ml
  • heat-killed Salmonella 50, 10, or 1 ml
  • no antigen as negative controls were also added to the wells.
  • the recombinant lactate dehydrogenase-C was brought to 1 mg/ml with PBS, and was filtered-sterilized using a Millex-GV syringe driven filter unit with a 0.22 mm PDVF membrane (Millipore Co., Cork Ireland).
  • Salmonella was grown in a 5 ml LB culture at 37°C with constant agitation overnight started from a glycerol stock. The bacterium was heat killed by placing the culture in 65°C water bath for 2 hours. The culture was cooled down to 4°C and was diluted 1 :1 0 in iced-cold sterile PBS before it was used in the assay. Each experimental group was set up in triplicates. The plates were covered and incubated at 37°C, 5% CO 2 , and 90% humidity for 3 days. Proliferation was quantified by a colorimetric cell proliferation ELISA, the BrdU colorimetric kit (Roche).
  • the lymphocytes were incubated with the nucleotide analog 5-bromo-2'-deoxyuridine BrdU.
  • the cells were harvest and fixed to the plates 20 hours latter.
  • the unfixed material was washed and the microwells were probed with a monoclonal antibody against BrdU coupled to horseradish peroxidase.
  • the solution was washed and then incubated with substrate solution for 20 minutes.
  • the color development was stopped with 25 ml of 1M H 2 SO 4 .
  • the plates were read at 450 nm in a Spectramax 190 microplate reader (Molecular Devices, Sunnyvale CA).
  • 10 for pcDNA3-LDH-C 10 for pcDNA3-LPV
  • 5 for pcDNA3-GFP 5 for pcDNA3-GFP.
  • Four weekly doses and one boost at day 82 of 10 8 colony- forming units were administered directly into the back of their throats forcing the formulation into their stomachs without causing extreme discomfort but ensuring systematic dispense.
  • Fasting animals not only will have empty stomachs that can easily accommodate the bacteria but will be free from the normally hostile milieu needed for digestion. The process of vaccine administration was proven to be a difficult one and lead to several unintended casualties.
  • the plasmid could be detected in the stomach but not in the spleen, which suggests that Salmonella never reached (at least at this stage) the spleen but was able to establish in the stomach. This could lead to the conclusion that, since the plasmid was present in the digestive system but not in the spleen, a more localized mucosal response would be elicited.
  • the immune system was constantly monitored by testing the antibody response in serum and vaginal washes. The antibody response was tested by indirect ELISA using recombinant lactate dehydrogenase-C to capture the specific antibodies. In particular, serum IgG and IgA and vaginal IgA were assessed.
  • Figure 4 shows that the pcDNA3-LDH-C and pcDNA3-SPV was able to elicit a modest but specific immune response compared to the control pcDNA3-GFP.
  • a high background could be seen in some GFP bleeds and it is believed that this is due to bacterial lipopolysacharides which are highly immunogenic and can cause an allergic-like inflammation response and even death.
  • Similar graphs illustrate serum and vaginal IgA are depicted in Figures 5 and 6, respectively.
  • EXAMPLE 11 Second Vaccine Trial Results from the above vaccination trial show that mice are able to make an antibody-based immune response towards lactate dehydrogenase-C when Sa/ o ⁇ e//a-DNA vaccine constructs were fed.
  • the purpose of the present vaccine trial was primarily focused on testing the effects on fertility and to assess T cell proliferative responses. An ideal contraceptive vaccine for overpopulation control would require minimum number of doses. This is true for any vaccination strategy. This trial was designed to account for this and ultimately minimize the number of doses. For this experiment, 3 doses of 10 9 colony-forming units (300 ml) were administered to Balb/c female mice on days 0, 7, and 47.
  • the DNA vaccine constructs used were the same as in the previous vaccine trial.
  • mice Eight mice were used for pcDNA3-LDH-C, six for pcDNA3-SPV, and six for the control pcDNA3-GFP. Keeping a small manageable population was needed to effectively perform adequate mating strategies.
  • the Salmonella carrying the piasmids was grown, as before, in LB supplemented with 1.5% sodium chloride without aeration for a few hours.
  • a sample of about 10 8 bacteria from each sample was used for plasmid preparation using a Qiaquick Minipreps (Qiagen, Germany). The samples were run on agarose gel to confirms that approximately equivalent amounts of plasmid were given in each dose and the integrity of the plasmid was not compromised by the sample preparation used.
  • the animals did not produce quantifiable amounts of the vaginal specific antibody until the third administration of the vaccine (which can be considered a boost). Fertility Assessment On day 65, after an adequate antibody response was detected, the animals were mated by introducing either two or three females into cages containing a single male. The males were previously tested for fertility and all the animals proved fertile. Once a female was impregnated, she was returned to her original cage. A female was considered impregnated only if she exhibited a clear post-coital vaginal plug. Under these mating conditions, all females were impregnated in 8 days, which includes at least 2 estrous cycles.
  • Table 2 shows that pcDNA3-LDH-C was able to reduce fertility in female mice from an average of 5.75 of the pcDNA3-GFP to 2. This corresponds to a reduction of about 65%.
  • the recorded average first litter size for the Balb/c genotype is 5 pups per animal (Technical Bulletin #1 , Charles Rivers Laboratory, Spring 1999). This is consistent with the average litter size found in mice treated with the peptide or control piasmids.
  • T Cell Proliferative Response Antibody production is an extraordinarly complex mechanism requiring the interaction of a variety of immune cells and signals originally stimulated by an antigen.
  • activated helper T-cells are responsible for stimulating antibody-secreting cells.
  • the ability of a vaccine to stimulate Th-cells is essential for this particular vaccination strategy since there are many factors that can contribute to the activation of lymphocytes.
  • lactate dehydrogenase-C or the Salmonella would be the primary stimulators of T- cell response.
  • To determine the key players involved in stimulating a cellular immune response the ability of both the Salmonella and the lactate dehydrogenase-C was tested in vitro using splenocytes from the vaccinated or control animals.
  • Salmonella did not seem to be as critical as suspected. This is because the signal did not increased in proportion to the amount of bacterium added to the assay. The noise (non-specific proliferation of responder cells without a target), however, increased as more antigen was added but the signal just reached a plateau. A large non-specific proliferation was expected since the Th-cells were not purified from splenocytes.
  • the splenocytes include a number of antigen presenting cells (e.g. macrophages, monocytes, etc.) that can proliferate non-specifically in the presence of any known antigen.
  • these results not only showed the importance of LDH-C in producing a helper response but also confirmed that Salmonella is an extremely efficient carrier of plasmid-based vaccines.
  • Salmonella-DHA Vaccine As Immunocontraceptive
  • the data presented above indicate that administration of Salmonella-DNA vaccine in female mice resulted in ⁇ 65% reduction in fertility. Such infertility has never before been achieved in mice with lactate dehydrogenase-C. This level of infertility (although only tested in a few mice) is by itself acceptable as a rodent control. Future experimentation will determine the efficacy of the vaccines in males and the effect the DNA- Salmonella hybrid will have when both sexes are treated with the vaccine. If 65% infertility could be achieved in females, it most likely would show much higher levels if both sexes are treated; perhaps complete sterility.
  • Salmonella-DUA vaccine can induce such a high level of infertility because this vaccine was capable of inducing a high level of IgA (see Figures 8 and 9).
  • IgA evolved to be an integral part of mucosal secretions and is more stable in the vaginal area, but its polymeric nature may be more effective in neutralizing sperms in vivo.
  • the fact that splenocytes were able to produce a robust T- helper response against lactate dehydrogenase-C but a only modest one against the Salmonella means that the antigen reached the spleen either intact or processed by professional antigen presenting cells.
  • the main antibody isotype was IgA and the expression of lactate dehydrogenase-C was detected in intestine extracts. Based on these experimental evidences the following mechanism is suggested.
  • the Salmonella reaches the epithelium and gets transported to the lymphoid tissue underneath, where it gains entry into the cells. The bacteria dies and the plasmid is shuttled to the nucleus where lactate dehydrogenase-C is eventually expressed. Lactate dehydrogenase-C is secreted in large enough quantities to stimulate the epithelial B-cells to produce IgA antibodies.
  • professional antigen presenting cells distribute the antigen to multiple lymph nodes where a T-cell response is stimulated. The validity of this mechanism could be corroborated with future experimentation.
  • One such set of experiments is to test the invasiveness of the Salmonella. This can be accomplished by orally immunizing the animals and then extracting DNA from different tissues. The extracted DNA can be subjected to PCR u sing primers directed against either the plasmid or the bacterium's chromosome. The kinetics for the establishment of Salmonella and the production of antigen should also be tested. This can be accomplished by either PCR, RT-PCR, Western blots, or immunostaining for lactate dehydrogenase-C using different tissues at different times after administration. The importance of recognizing the mechanism goes beyond a pure academic exercise.
  • Salmonella It is essential to recognize key players and the role they play in this vaccination strategy not only to make more effective vaccines but also to minimize (if possible) any components that may provide toxic effects to the target or non-target species.
  • Salmonella could be replaced by other organisms such as E. coli or even nonreplicating agents such as plasmid carriers.
  • pathogens such as Salmonella are common dwellers in rodent colonies and rats and mice are carriers of several bacterial species.
  • the introduced bacterium should be highly attenuated and cannot propagate beyond its host.
  • systems that carry the plasmid without antibiotic resistance should be pursued if any form of DNA shall be used as a contraceptive vaccine.
  • Some systems of antibiotic resistance-free piasmids are available. The following references were cited herein:

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Abstract

La présente invention concerne un vaccin immunocontraceptif comprenant un hôte bactérien de recombinaison qui a été modifié pour produire un polypeptide spécifique à l'oeuf ou au sperme, tel qu'une lactate-déshydrogénase-C de protéine spécifique au sperme (LDH-C). Lorsque des animaux tels que des rongeurs mangent ces bactéries, leurs systèmes immunitaires produisent des anticorps qui attaquent leurs spermes. Non seulement les mâles présentent moins de sperme viable, mais les femelles présentent également des anticorps au sperme entrant dans leurs systèmes reproducteurs. L'immunocontraception constitue une méthode intéressante pour réduire la taille d'une population d'animaux présentant une fécondité élevée et la stérilisation d'animaux au moyen de tels immunocontraceptifs peut réduire des populations d'animaux cibles à des niveaux acceptables de manière efficace, rentable, humaine et surtout spécifique à l'espèce.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014101367A1 (fr) * 2012-12-25 2014-07-03 成都医学院 Bactéries synthétiques pour l'expression à haute efficacité de l'acide lactique déshydrogénase c4 et son procédé d'expression

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4290944A (en) * 1980-07-31 1981-09-22 Northwestern University Antigenic peptide compound
US5480799A (en) * 1993-12-10 1996-01-02 The University Of North Carolina At Chapel Hill Sperm antigen corresponding to a sperm zona binding protein autoantigenic epitope
US5656488A (en) * 1990-11-21 1997-08-12 Washington University Recombinant avirulent salmonella antifertility vaccines

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4290944A (en) * 1980-07-31 1981-09-22 Northwestern University Antigenic peptide compound
US5656488A (en) * 1990-11-21 1997-08-12 Washington University Recombinant avirulent salmonella antifertility vaccines
US5480799A (en) * 1993-12-10 1996-01-02 The University Of North Carolina At Chapel Hill Sperm antigen corresponding to a sperm zona binding protein autoantigenic epitope

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014101367A1 (fr) * 2012-12-25 2014-07-03 成都医学院 Bactéries synthétiques pour l'expression à haute efficacité de l'acide lactique déshydrogénase c4 et son procédé d'expression

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