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Thursday, May 21, 2009
The Greatest Racket in History
newswire article reporting global 25.Jul.2006 04:17
genetic engineering | health
GMO Disease Epidemics: (2) Hepatitis B Vaccine
author: Myron (Mike) Stagman, PhD e-mail:e-mail: artemesium@gmail.com
Genetic Engineering is a nightmare technology that has caused many disease epidemics, documented but unpublicized. This is the 2nd in a series revealing the epidemics. It features the horrors of GE MEDICINE. Read and take heed -- it may save your health or life.
Myron (Mike) Stagman, PhD
Concerned Citizens Information Network
www.ccin.info (environmental website)
www.MyronStagman.com
email: artemesium@gmail.com
GMO DISEASE EPIDEMICS:
(2) Hepatitis B Vaccine
I. The Face of Frankenstein's Monster
Genetic Engineering Biotechnology (GE, GM, GMOs) evades Evolution's safeguards by injecting genes of one species into a totally unrelated species, something impossible in Nature.
GM evades Evolution's safeguards, and may therefore give rise to PATHOGENS that can -- and HAVE, many times -- caused crippling and deadly DISEASE EPIDEMICS -- documented but unpublicised.
Genetic Engineering is also ideal for developing horrific biological and other military weapons --- sufficient cause to OUTLAW GM.
The Greatest Racket in History (Listen to this !): ---
GM contamination is virulent, spread by wind, bird, insect, animals and travelling human beings. Co-Existence with GM is completely impossible, only fools or liars would say differently.
GM has contaminated crops in areas of North America which dwarf the UK and even Western Europe. Planetary contamination is inevitable, if We don't stop it.
The Biotechnology corporations know oh-so-well how contamination spreads and spreads and spreads.
Now get this: Through their political influence, these Corporations have been able to acquire the obscene, sacrilegious, and indeed revolutionary PATENTS on LIFE-FORMS.
Before these revolutionary Patents on Life, a farmer could have sued -- successfully without any doubt -- the Biotechnology corporation for having contaminated, poisoned his crops.
However, given these Patents on Life (here, on the seeds), the Corporation can -- and DOES, successfully -- sue the victimised farmer for (Hold your nose) "infringement of patent".
[Our tainted, corrupted political systems allow this monstrosity.]
Thus the formula the reader might keep in mind:
GM Contamination + Patents on Life-Forms equals Corporate Ownership Rights in the crops of victimised farmers.
This formula spells the serfdom and ruin of all small family farmers on this planet. (They are the majority of the population in Asia, Africa, and Latin America).
The Chemical-Drug-Biotechnology syndicate and its Enforcers (corrupted governments in the West and Third World, corporation-dominated organisations like the WTO, World Bank, IMF) have started a WORLDWIDE AGRICULTURAL REVOLUTION.
They want to destroy small family farming, and have only gigantic Corporate GM Plantations with farmers working as serfs.
[This process is underway, especially in South America where the U.S. Military has begun to intervene on behalf of GM Plantations to crush farmer opposition.]
Already, according to the Research Foundation for Science, Technology and Ecology (RFSTE) in New Delhi, 40,000 farmers in India have committed suicide primarily as a result of the Corporate Enforcers' so-called "free trade" policies --- dumping heavily-subsidised Western farm produce onto glutted Indian markets, pushing GM crops, insisting that Patents on Life-Forms be accepted so farmers must sign contracts with GM Corporations and can no longer save seeds, etc.
The Greatest Racket in History --- GM Contamination + Patents on Life-Forms equals Corporate Proprietary Rights in the crops of victimised farmers, and spells the serdom and ruin of small family farmers everywhere on this planet.
We MUST OUTLAW GM and PATENTS on LIFE-FORMS -- or else !!
(e.g. No more organic, healthy food --- you and your children will only be able to eat unsafe GM)
See the Action Plan in Part III designed to OUTLAW GM and PATENTS on LIFE-FORMS.
But beforehand, read about the first in our series of "GM Disease Epidemics".
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II. The Horror of Genetically-Engineered Medicine and the GM Hepatitis B Vaccine Epidemic
(a) Genetically engineered medicine and vaccines are extremely dangerous and should be outlawed. GM medicine/vaccines are entirely unnecessary as well, and insult the Hippocratic Oath.
[Comment: The Drug Corporations would drop genetically-engineered drugs like a hot potato were it not for the abusive Patent laws (awarding monopolies), especially Patents on Life-Forms, and the corruption of Government which insulates the corporations from serious liability.]
- - - - - - - - - -
(b) Item: London, March 2006 --- Six healthy male volunteers were given an experimental drug manufactured by a German biotechnology company, TeGenero. The drug, supposedly intended as an "immune stimulant", wasgenetically engineered.
It came as little surprise to this observer when it was announced that all 6 men suffered multiple organ failure and nearly died, and that one of them may remain in a coma for a year or more.
One of the 6 victims said he felt like his brain was "on fire". "I thought my eyeballs were going to pop out."
Either this victim or another has been nicknamed "the Elephant Man', because his head swelled out to three times its normal size !!
It was also unsurprising that the BBC, International Herald Tribune (New York Times), etc.,apparently suppressed the fact that the drug was genetically engineered. (Could they conceivably be accused of ignorance? Surely not possible.)
["Calamitous GM Drug Trial Raises Questions About Modern Science", GM Watch, Weekly Watch 170, 6 April 2006; "A drug trial catastrophe", Prof. Joe Cummins, Ban-GEF online newsletter, 19 March 06; "Victims' agony as 'Elephant Man' drugs firm goes bust", Neil Sears, Daily Mail, 4 July 2006]
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(c) The Hepatitus B vaccine was genetically engineered. On account of this vaccine, during the 1990s there were, in the USA alone, more than 17,000 cases of hospitalisations, injuries and deaths, including the deaths of 72 children, reported to the Vaccine Adverse Event Reporting System (VAERS) of the U.S. government.
[
[Dr. Philip Incao's testimony to the Health Committee of the Ohio House of Representatives, 1 March 1999]
Note: a former FDA (U.S. government Food and Drug Administration) Commissioner wrote in 1993 in the prestigious medical journal, JAMA, that a study showed "only about 1 percent of serious events attributable to drug reactions are reported to the FDA".
USA Question: Are serious events attributable to vaccines better reported to VAERS?
UK Question: Is the MMR vaccine genetically engineered? If it is, one can readily understand why AUTISM might conceivably be linked to it.
- - - - - - - - - - -
(d)Labelling, Identification and GM Causation of Diseases
GM drugs are generally (entirely?)unlabelled. This alone is outrageous.
If you want to have a little fun, ask your doctor if the drug he/she is prescribing happens to be genetically engineered. Doctors do not like being asked important questions for which they have not the foggiest notion of an answer.
In the last two decades, any drug that has been associated with particularly nasty adverse reactions, should have been analysed (and its patent searched) for possible GMO content and GM causation of those adverse reactions.
This of course has not been done. (never? probably Never. Ordinary scientists, who are pretty uniformly on 'Their side' -- they wouldn't dare. And 'Our side' is not bright enough to have thought of it. )
Recommendation: Start doing it now.
- - - - - - - -
(e) Extremely Important Fact: "The third largest cause of death and illness in the [western] world is medical intervention [essentially, prescribed drugs]." --- Paul Flynn MP, House of Commons Health Select Committee report on "The Influence of the Pharmaceutical Industry", April 2005, vol. 2, p. 153
Only cancer and heart disease are bigger killers than prescribed drugs. As for heart disease, mark that the arthritis pain reliever, Vioxx, caused an estimated 140,000 heart attacks and up to 55,000 deaths in the USA alone.
[ Dr. David J. Graham, former associate safety director of the FDA; and see "Intimidation, Politics and Drug Industry Cripple U.S. Medicine", Ritt Goldstein, Inter Press Service, 31 December 2004 ]
Question:Is Vioxx genetically engineered? Will anyone ever do an analysis or search the patent for that information??
Indeed, in the United States (can Europe be any different?)prescribed drugs are surely the leading cause of death and illness, at least equal to cancer and heart disease combined, and quite possibly a multiple of this.
[see "Modern Health Care System is the Leading Cause of Death", Gary Null PhD, Carolyn Dean MD ND, Martin Feldman MD, et al, www.mercola.com]
- - - - - - - - - -
(f) How many drugs are now genetically engineered?
8% of total global pharmaceutical market sales are accounted for by genetically engineered drugs.
[Memorandum by the BioIndustry Association (PI 147), "The Influence of the Pharmaceutical Industry"]
Worse, one-third of all drugs in development are biologics !
[It is unclear whether the Memorandum refers to the UK only, or the Western drug corporations in general. I think the latter.]
This ought to frighten anyone who thinks he or she may ever again see a doctor or hospital.
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III. Conclusion and What To Do
Human beings and Nature, as we have known them, cannot survive in a GMO-Frankenstein world.
The nightmare technology of Genetic Engineering Biotechnology, and its incestuous Patents on Life-Forms, must be OUTLAWED.
How To Do It?
There is only one way to overcome this extremely powerful, ruthless Corporation-Government-corporate Mass Media axis.
It is to conduct grassroots Public Education Campaigns to educate and politically activate the General Public, university / college students in particular.
The General Public !! Stop forever talking to the Converted. To get around the corporate media's suppression and distortion of truth, one must talk to people and go door-to-door with leaflets and vocal messages.
And major efforts must be made to inform and activate university students.
Important Hints:
(1) Concentrate on university students.
And senior citizens.
I speak to you now, elder citizens like myself. Put moral purpose into your lives and help rescue this planet and your grandchildren.
(2) AVOID NGOs and all established organisations, especially large ones. They are not what they seem.
Their hierarchies and decision-making are corporation-influenced if not outright purchased.
Never ever join them. Never ever give them Money.
[see George Monbiot's very important article in The Guardian, 4 September 2001, "Sleeping with the Enemy: Consumer and Environmental Groups are Getting into Bed with Big Corporations]
[Note: In developing countries there are some exceptions, but very very very few in the West. If by a miracle one of them ever conducts a genuine and sensible public education campaign, then you can help out. But NEVER join or give money to them.]
The environmental field especially is riddled with wealthy, prestigious, deceptive organisations adept at dilutng and derailing environmental causes, GM in particular.
[I am thinking at this moment of one such organisation I have observed over a long period of time, in my view a very slick and dangerous FOE of environmentalism, originally created by polluting Corporations to deal with the growing environmental movement.
Don't go anywhere near this one.]
When they talk about being 'practical' and seeking 'Coexistence' with GM, let a red flag go up.
Nothing short of OUTLAWING GENETIC ENGINEERING and PATENTS ON LIFE-FORMS will save us and our planet.
Do your educational work as individuals or in small groups of people you know.
In a little of your SPARETIME (perhaps 3 or 4 hours a week, but regularly, as part of an ethical life-style):
Educate and activate the Public by writing and telephoning and just talking to your family members, friends and acquantances;
Write letters and make telephone calls to local, regional and national media;
Write, call and especially visit your legislative representatives and other government personnel, and
Take appropriate Direct Action.
Good Luck to You.
P.S. For a detailed Action Plan on conducting education/activation campaigns (e.g. composing A4 leaflets for distribution, lobbying your legislative representative) see "Creating a Grassroots Democracy While Outlawing Genetic Engineering" on the Indymedia Biotech website. Or email me and request it.
For extensive information on the casualties and unprecedented dangers of Genetic Engineering, see "Genetic Engineering Fact-Sheet" on the website of the Concerned Citizens Information Network, www.ccin.info
homepage: homepage: http://www.ccin.info
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US Patent 5733540 - Protection from viral infection via colonization of mucosal membranes with genetically modified bacteria
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to the manipulation of the bacterial flora normally residing harmlessly on mucosal surfaces to interfere with infectious processes. Specifically, this invention provides for modification of non-pathogenic floral bacteria to confer upon them the capacity to bind to (and functionally inactivate) specific viruses. Although this disclosure describes a method of preventing infection by viruses which infect through mucosal surfaces, the skilled practitioner will recognize that the invention may potentially be applied to any pathogen which infects at a mucosal surface, including bacteria, fungi, and parasites.
2. Information Disclosure
Cytoplasmic expression of heterologous proteins by bacteria has been widely practiced for well over two decades. However, expression of heterologous proteins specifically onto the external surfaces of bacteria has been achieved only in the past few years. Surface expression systems for both gram-positive and gram-negative bacteria are known, e.g., U.S. Pat. No. 5,348,867, and WO 93/18163.
SUMMARY OF THE INVENTION
This invention provides for a method of protecting an animal from a viral infection comprising contacting a mucosal surface of the host with an amount of transformed bacteria sufficient to colonize the mucosal surface and to protect the animal from viral infection, said bacteria having been transformed with genetic material so as to confer upon the bacteria the capacity to bind the virus. More specifically, this invention provides for transformed bacteria that bind virus or other pathogens using naturally occurring receptors, domains of receptors or antiviral antibodies that are the products of the genetic material.
Preferred hosts are humans. Where the naturally occurring receptors are known, genes encoding those receptors may be used to transform the bacteria. When the specific viral/host receptors are not known, genes encoding antiviral antibodies or fragments thereof may be used to transform the bacteria. For example, for retroviruses that are covered with human leukocyte antigens �HLA DR!, antibodies against these antigens are useful. Accordingly, this invention can be used against rotavirus, papillomavirus, adenovirus, respiratory syncytia virus, corona virus, cytomegalovirus, coxsackievirus, echovirus, hepatitis A virus, rhinovirus, human immunodeficiency virus, poliovirus, Epstein-Barr virus, parainfluenza virus and herpes simplex virus using bacteria able to bind to conserved determinants on their respective capsids.
The bacteria may also be modified to express a specific carbohydrate moiety which serves as the receptor for the virus onto its normal surface proteins. For example the bacteria may be transformed with genetic material which causes the addition of sialic acid which permits the bacteria to bind to an influenza virus.
The bacteria may also be modified to cause fusion between the bacterial membrane and the viral envelope, if present. An example is the transformation of bacteria so that it can fuse with bound viral particles through a fusogenic domain engineered into the virus-binding polypeptide.
Colonization of mucosal membranes is an essential element of this invention and it is preferred that the transformed bacteria is conferred with sufficient selective advantage to permit it to compete effectively with resident bacteria to allow said transformed bacteria to successfully colonize and survive indefinitely on a selected mucosal surface. One selection advantage is an enhanced ability to adhere to a host mucosal surface through a domain in the heterologous protein which binds to a determinant on a selected mucosal surface. Selective advantage might also be conferred by the use of antibiotic resistant transformed bacteria where antibiotics are co-administered with the transformed bacteria. Other advantages include the use of products that degrade the biofilm of the mucosal membrane. Such products would include DNAses, peptidases, and hyaluronidases.
Preferred mucosal surfaces are in the following organs: nasopharynx, oropharynx, esophagus, small intestines, large intestines, rectum, vagina, and penis.
Transformed bacteria are applied to a mucosal surface through the use of a liquid solution, foam, suppository, sponge, or capsule. Where the target mucosal layer is in the vagina, the bacteria can be transformed to target sexually transmitted pathogens such as but not limited to HIV, HPV, HSV, gonorrhea, syphilis and chlamydia. Nonbacteriocidal spermicides might be co-administered with the bacteria.
The invention also embraces a means to prevent the spread of a viral pathogen from an infected individual to others with transformed bacteria by administering an amount of transformed bacteria sufficient to colonize the mucosal surfaces of the infected individual wherein said bacteria bind and inactivate infectious viral particles exiting the infected host. The modifications and targets being as stated above.
The transformation of the bacteria can be either in vitro or in vivo whereby the resident musocal bacterial flora of a host is transformed with a desired foreign genetic material by directly introducing into resident microfloral bacteria a genetic vector said vector conferring the ability of the bacteria to bind and inactivate viral pathogens of the host and thereby affording protection of the host from infection by the viral pathogen. Examples of vectors include replication defective bacteriophage.
The invention further includes inactivating infectious viral particles in suspect water supplies by the addition of engineered bacteria capable of binding and irreversibly inactivating specific viruses.
In addition to methods, this invention also embraces compositions of matter comprising a bacteria selected for its ability to colonize the mucosal membrane of a host and transformed to express a host receptor or an antibody specific for a target virus on its cell surface in an amount sufficient to bind and inactivate the target virus. The preferred compositions are as described above for the various methods.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates the ViroShield™ concept. Viruses normally gain entry into a host by binding to specific receptors expressed on the host cell surface. Expression of the same receptors on the surface of bacteria on mucosal surfaces will cause the majority of the viruses to bind to the bacteria instead, where they are functionally inactivated, thus preventing infection of the underlying host cells.
DETAILED DESCRIPTION
A. Introduction.
Most viruses infect via mucosal surfaces. A review of this process can be found in Murray, P. R., et al., Medical Microbiology, 2nd Edition, (hereinafter Murray, et al., 1994). The creation of a virus blocking bacterial flora in the mucosal surfaces by allowing colonization of bacteria transformed to bind and inactivate virus is particularly advantageous. Colonization of mucosal layers is a routine undertaking. Most mucosal layers are typically teeming with bacteria, and changes in flora attendant to pathogenic bacterial infection and administration of antibiotics is a common event. The routine nature of the floral changes on mucosal surfaces is a key advantage of the invention. The following discussion will also provide means to enhance the ability of transformed bacteria to colonize mucosal layers.
B. General methods
The techniques of amplification of genetic sequences with the polymerase chain reaction (PCR), cutting and splicing DNA into plasmids, transformation of bacteria with plasmids, and assays for antibody binding are all well known biotechnology methods and detailed descriptions of these methods can be found in a number of texts including Protocols in Molecular Biology, Molecular Biology of the Cell, and Sam brook, et al., Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 1989.
C. Viral targets.
The following is a list of viral targets. They are categorized by their respective organs of entry.
1. Upper respiratory tract (URT).
A large number of viruses infect the naso- and oropharynx, either via air droplets or direct contact. These include human rhinoviruses (HRV), adenovirus, coxsackievirus, influenza, parainfluenza, respiratory syncytia virus (RSV), Epstein-Barr Virus (EBV), and cytomegalovirus (CMV).
2. Gastrointestinal (GI) tract.
These viruses include rotavirus, Norwalk agent, hepatitis A (HAV), poliovirus and other picornaviruses.
3. Vaginal: human immunodeficiency virus (HIV), human papilloma virus (HPV), herpes simplex virus (HSV) 2, CMV, hepatitis B virus (HBV), hepatitis C virus (HCV).
D. Viral receptors.
A virus must enter a host cell to replicate. To enter a cell, viruses require surface receptors on the host cell (Murray, et al., 1994). More specifically, the virus must first bind to a molecule on the surface of the target cell. The receptors for a number of viruses have been determined in recent years. The following is a representative list and is not meant to be a limitation of the invention.
1. HRV, major group→ICAM-1, domains 1 and 2 (Lineberger, D. W., et al., Virus Research, 24(2):173-86, 1992.)
2. Influenza virus→sialic acid
3. HIV→CD4, domains 1 and 2
4. Poliovirus→PVR (poliovirus receptor, an immunoglobulin superfamily protein)
5. EBV→CD21 (complement receptor 2, the receptor for C3d)
6. HSV→heparin sulfate
7. HBV→IgA receptor
8. Adenovirus→Vitronectin receptor
E. Mucosal Surfaces are Normally Colonized by Bacterial Flora (Murray, et al., 1994)
The upper respiratory tract (URT) consists of the nasopharynx, oropharynx (oral cavity and larynx), paranasal sinuses, and the middle ear. The paranasal sinuses and the middle ear are normally sterile. However, the stratified squamous epithelium of the naso- and oropharynx are teeming with a varied microbial flora. The microflora of the nose consists mainly of coagulase-negative staphylococci, with some diphtheroids (aerobic and anaerobic), and nonhemolytic streptococci. The most prominent members of the flora of the mouth and pharynx are the alpha streptococci. Some gram-negative anaerobes (esp. Bacteriodes) and other cocci are also found.
The colon contains the largest total population of bacteria of any mucosal surface in the human body. It is estimated that >1011 bacteria/g of colonic content exists in healthy individuals, representing over 400 species. Anerobic bacteria outnumber aerobic ones by a factor of 100-1,000. Bacteroides is the predominant genus. Bifidobacterium, Enterobacteriaceae, Streptococci, and Lactobacilli are also prominent. The small intestine is populated by a similar profile of organisms as the colon, but at much lower numbers. The stomach and proximal small intestine are nearly sterile, while the distal small intestine contains approximately 1/10 of the bacterial content of the colon.
The vaginal mucosa is also colonized by a large number of bacteria. Lactobacilli are the predominant species in the normal menarchal vaginal microflora, being present in nearly 100% of normal women. Lactobacilli 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.
F. ViroShield™: Prevention of pathogen binding to host cells will prevent infection
Since viruses require binding to a receptor on the target cell surface for infection, strategies directed at inhibiting the interaction of a virus with its host receptor should be effective at preventing infection. The use of a bacterial shield against viral pathogens on mucosal surfaces is termed a ViroShield™.
The concept of the ViroShield™ type bacteria can be illustrated by the viral agents causing the common cold. The viral agents for the common cold are mainly consisting of the rhinovirus major group which bind to the ICAM-1 receptor in humans. Soluble ICAM-1 molecules expressed through recombinant DNA technology have been found to be effective in inhibiting HRV binding to susceptible cells and preventing infection (Martin, S., et al., Antimicrob Agents Chemother, 37(6):1278-84, 1993, hereinafter Martin, et al., 1993).
Approximately 60 ICAM-1 molecules can bind to a single HRV virion (Hoover-Litty, H. and Greve, J. M., J Virol, 67(1):390-7, 1993). This correlates with the fact that the HRV capsid is an icosahedral complex composed of 60 copies of each of the viral coat proteins (Smith, T. J., et al., J Virol, 67(3):1148-58, 1993). The actual receptor binding site on the HRV capsid was found to be a surface depression or "canyon" by X-ray crystallography (Oliveira, M. A., et al., Structure, 1 (1):51-68, 1993). This canyon is sufficiently small in size such that antibody molecules cannot fit, and this is one reason why humans are susceptible to repeated infections by HRV since the virus is resistant to antibody binding at this key neutralization site.
After binding to ICAM-1, a conformational change is induced in the capsid which causes the release of the viral RNA into the host cell (Martin, et al., 1993). Soluble ICAM-1 molecules are relatively ineffective at inducing capsid conformational change and thus functional inactivation of virions (Martin, et al., 1993, and Crump, C. E., et al., Antimicrob Agents Chemother, 38(6):1425-7, 1994 hereinafter Crump, et al., 1994); however, chimeric molecules combining the HRV-binding domains (1 and 2) of ICAM-1 and constant regions of immunoglobulin (Ig) molecules (IgA, IgG, or IgM) showed some effect. This effect is thought to be due to the ability of chimeric molecules to dimerize (IgA and IgG) or multimerize (IgM) via the association of the Ig domains. Multimeric receptors more closely resemble the natural state on cell surfaces, where several immobilized receptors binding to a virion may induce conformational distortion to the capsid to cause vital RNA release.
Expression of ICAM-1 on the external surface of mucosal bacteria is an extension of the multimeric-molecule strategy, with key additional benefits. Since bacteria are considerably larger than viral particles, bound HRV should be readily immobilized onto the bacterial surface. A key advantage of this invention is that only a few ICAM-1 receptors must be bound in order to effectively immobilize and neutralize a virion, whereas strategies involving soluble ICAM-1 molecules potentially must cover all 60 binding sites in order to ensure complete neutralization of a single virion. Furthermore, ICAM-1 molecules on bacterial surface are efficient in inducing capsid conformational change because simultaneous binding of several ICAM-1 molecules immobilized on a bacterial surface to several faces of a vital capsid will distort the geometry of the viral capsid and lead to conformational change and premature viral RNA release.
Viral RNA released into the bacteria are readily degraded by the abundant nucleases within bacterial cytoplasm. This leads to irreversible inactivation of viral particles, which is a key advantage of this invention because binding itself is a reversible process, and binding of soluble ICAM-1 molecules, chimeric molecules, or other drugs to HRV without functional inactivation would still leave a significant fraction of viral particles free to bind host cells at any given time. With ViroShield™ type mucosal bacteria, each bacteria is capable of irreversibly inactivating a large number of viral particles, ensuring that the majority of any vital inoculum would be eliminated before they can infect underlying host cells.
Accordingly, soluble CD4 molecules have also been shown to be effective in binding and preventing infection of HIV to target cells in vitro (Orloff, S. L., et al., J Virol, 67(3):1461-71, 1993). However, results of clinical trials with intravenously administered soluble CD4 molecules have been disappointing (Moore, J. P., et al., Aids Res Hum Retroviruses, 9(6):529-39, 1993). The reason is not due to lower binding affinity of primary vs. laboratory isolates of HIV to soluble CD4, but rather that primary isolates are less prone to inactivation after binding to soluble CD4 (Ashkenzai, A., et al., Proc Natl Acad Sci, 88:7056-7060, 1991 and Turner, S., et al., Proc Natl Acad Sci USA, 89(4):1335-9, 1992).
The expression of CD4 on bacterial surfaces should facilitate irreversible inactivation of all strains of HIV. In particular, CD4 expression on the surface of Lactobacilli on the vaginal mucosa would be effective at preventing HIV infection through vaginal intercourse. E. coli similarly transformed would be effective against HIV transmission via rectal intercourse.
An important point to keep in mind is the distinction between infection and clinical disease. For any pathogen, there is a minimum inoculating dose necessary to cause clinical symptoms from an infection. Exposure to an inoculum below this dose normally does not lead to clinical disease. Therefore, to successfully prevent disease, a strategy does not necessarily need to inactivate every particle of an inoculating dose of a virus, but rather to reduce the number of viable viral particles below the minimum infectious dose.
Since the ViroShield™ approach aims to prevent entry of a viral pathogen into a host, it not only prevents clinical disease, but should prevent infection altogether. Standard vaccines do not prevent entry of viral pathogens into a host. This may be important as certain viruses are known to trigger autoimmune processes in some hosts, regardless of whether they cause clinical infection.
Potential applications:
______________________________________ Virus Receptor Portal of Entry Suitable Bacterial Host ______________________________________ HRV ICAM-1 URT URT flora- Strept gordonii or Influenza sialic acid URT/LRT Staph xylosus Adenovirus Vitronectin URT Strept or Staph HIV CD4 vaginal mucosa Lactobacillus HSV 2 heparin sulfate vaginal Lactobacillus ______________________________________
G. Neutralization of pathogens upstream of their infection site
The only mucosal surfaces in the body relatively free of bacterial colonization are that of the stomach, upper small intestines, and lower respiratory tract. A few important viruses infect at the upper small intestines, the most significant of which are rotavirus and poliovirus (Murray, et al., 1994). Since bacterial counts in this area are low, even if all of these bacteria express receptors for the virus, it may not be possible to completely inactivate an inoculating dose of that virus. However, to reach the small intestines, viral particles must first enter the oral cavity and travel through the esophagus both are heavily colonized by bacteria. Therefore, it may be possible that bacteria on oropharyngeal/esophageal mucosal surfaces expressing viral receptors can absorb/inactivate enough viral particles to significantly decrease the infectious inoculum delivered to the small intestines.
Viruses that infect the lower respiratory tract include influenza, parainfluenza, and RSV (Murray, et al., 1994). Vital particles inhaled into respiratory tract via droplets will settle out along various portions of the respiratory mucosa depending on the physical properties of the virion, droplet, and flow. Engineered bacteria along these viruses' path through the URT may absorb/inactivate sufficient numbers of vital particles to reduce the inoculating dose reaching the lower respiratory tract below the minimum required for clinical disease.
H. Prevention of exit of pathogens to infect other uninfected hosts.
This invention also provides for a method of preventing the exit of the virus from an infected host. Preventing a pathogen from exiting an infected host would mean preventing spread of the pathogen to a number of uninfected individuals, which would be extremely important from a public health viewpoint. Rapid spread of a pathogen may wipe out entire villages in third world countries. ViroShield# should be useful even in already infected hosts by absorbing/inactivating viral particles as they exit the host. Even if ViroShield™ is unable to prevent infection of rotavirus or poliovirus for reasons discussed above (section G), engineered bacteria in the colon may still absorb/inactivate viral particles before they exit the host.
I. Use of engineered bacteria in potentially-contaminated water to inactivated virions
In third-world countries, viruses may be transmitted rapidly through inadequately treated water supplies. Fecal-orally transmitted viruses, such as rotavirus, may exist in low titers in the drinking water of a village after contamination by a single infected individual, and go on to infect a number of uninfected individuals. Non-pathogenic bacteria expressing rotavirus receptors may be added to suspect water supplies to absorb/inactivate viral particles in these settings, as long as the ingestion of the engineered bacteria is not harmful to a host. This approach should be an effective and economical means of quickly controlling orally-transmitted viruses in third-world countries.
J. Sources of genes which confer virus-binding capacity
The capacity to bind a virus may be conferred onto a bacteria in at least three ways. The first is by making the bacteria express on its surface the normal host receptor for the virus, such as ICAM-1 for HRV (major group) and CD4 for HIV. These are normal human proteins and the complete sequences of many of these genes have been determined and are stored in the database GeneBank. An advantage to this approach is that it is not readily avoided by viral mutation. If the virus mutates such that it no longer binds to the receptor expressed on bacteria, it would also lose its ability to bind to its target cell and thus no longer be infectious.
The second method is by expressing an antibody fragment (or any peptide with the capacity to bind a specific target on the surface of the virus) on the bacterial surface against a conserved determinant on the viral surface, such as VP4 on poliovirus, or gp120 on HIV. Antibody fragments (and peptides) against essentially any antigen can now be selected from a phage-display library (Marks, J. D., et al., J Biol Chem, 267(23):16007-10, (1992)).
Once appropriate clones are found, the gene coding for the antibody fragments can then be isolated and used. In addition, it was recently found that enveloped viruses, in the process of budding out of a host cell, carry along on their envelope certain host surface proteins, such as HLA DR on HIV (Arthur, et al, Science, 258(5090):1935-1938, (1992)). Thus, the human HLA DR molecule is a normal constituent of the HIV envelope. Antibody fragments directed against a conserved epitope of the HLA DR molecule may be capable of binding all isolates of HIV, and would be particularly effective in preventing male-to-female HIV spread when expressed on the surface of bacteria on the vaginal mucosa, or HIV transmission via anal intercourse when the engineered bacteria is applied to the rectum.
The third means of binding a virus by a bacteria is through the expression of certain carbohydrate moieties on the bacterial surface. A number of viruses use carbohydrate moieties as the receptor for entry into a host cell. One prominent example is the influenza virus which binds to sialic acid. Bacteria may be made to produce the enzyme sialic acid transferase in its cytoplasm which would lead to addition of sialic acid residues on normal surface proteins, thus causing influenza viruses to bind to said bacteria. The complete gene sequences of many bacterial carbohydrate transferases are known and appear in the literature.
K. Expression Systems for surface expression in bacteria
The expression of heterologous proteins on the surface of bacteria generally takes advantage of the normal surface proteins of the bacteria. It is becoming known that certain sequences within proteins direct them for export out of the bacterial cytoplasm, while others help to anchor a protein to the cell membrane. Hybrid proteins are created in which a heterologous protein sequence replaces the exposed portion of a normal surface protein, leaving the localization signal sequences intact. Several outer membrane proteins have been exploited as targeting vehicles for the localization of heterologous proteins, including the E. coli outer membrane protein maltoporin (LamB), E. coli pilin proteins K88ac and K88ad, E. coli outer membrane porins PhoE, OmpA, and OmpC, and the S. typhimurium Flagellin and TraT lipoprotein (U.S. Pat. No. 5,348,867).
A more detailed discussion of surface expression of proteins on the surface of gram-negative bacteria may be found in U.S. Pat. No. 5,348,867, and for gram-positive bacteria in PCT WO 93/18163.
1. Construction of Vectors
Plasmids 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. Plasmids have been constructed for surface expression of heterologous proteins in E. coli (e.g. pTX101 as described in U.S. Pat. No. 5,348,867), Streptococcus gordonii (e.g. pVMB20-GP232 transformation system, as described in PCT/US93/02355), and others. Both systems contain a signal sequence which directs a polypeptide to the cell surface, with an insert site for the desired heterologous gene, and an antibiotic resistance gene to help in selection of transformed bacteria. Other suitable streptococci include the lactic streptococci which have been widely transformed (De Vos, FEMS Microbiology Reviews, 46:281-295 (1987)).
Starting from the appropriate vector plasmid for each selected bacterial host, the plasmid will be digested with appropriate restriction enzymes to expose the cloning site. Then the desired heterologous gene will be ligated into the plasmid.
2. Transformation of bacterial cells
Appropriate bacterial host strains are selected for individual pathogens, heterologous protein or molecule, mucosal surface, and expression plasmid combination. The bacterial host will be rendered competent for transformation using standard techniques, such as the rubidium chloride method. Once transformed with the recombinant plasmid containing the desired heterologous gene, the bacteria will be grown in the appropriate media (e.g. LB media with 0.2% glucose). Transformed bacteria will be selected by adding the antibiotic to which the plasmid contains a resistance gene such that only transformed bacteria would survive.
3. Demonstration of expression of desired heterologous molecule on bacterial surface
Expression of the heterologous gene can be constitutive or induced by stimulating the promoter to which it is attached, such as with IPTG. Surface expression of the heterologous molecule will be demonstrated by staining the bacteria with fluorescent-labeled antibodies against the desired molecule, looking for a surface fluorescence pattern. Furthermore, binding of the target pathogen by the transformed bacteria can be demonstrated by fixing the transformed bacteria onto a slide, incubating with the target pathogen, then staining with fluorescent antibodies against the target pathogen in one color (e.g. red), and against the transformed bacteria in another color (e.g. green), showing that the target pathogens (red) are closely associated with the transformed bacteria (green).
L. Irreversible inactivation of bound viruses
To ensure inactivation of the virus after binding to the transformed bacteria, the process of binding must trigger concomitant release of viral genetic material. In this way, bacterial nucleases can degrade the viral genetic material, thus irreversibly inactivating the virus. Many viruses, such as HRV, release their genetic material after binding to immobilized receptors on the target cell surface through a conformational shift of the viral capsid (Martin, et al., 1993). This situation should be successfully mimicked by expression of the receptor on the surface of bacteria. Some viruses, such as HIV and influenza, contain fusogenic domains in their coat proteins which facilitate release of genetic material after binding (Murray, et al., 1994). Different mechanisms are engineered into bacteria to ensure release of genetic material and thus irreversible inactivation of specific viruses.
M. Successful Colonization of Engineered Bacteria
Colonization of mucosal membranes with non-recombinant bacteria is well-known. It was optimally achieved by co-administering antibiotics along with bacteria resistant to that antibiotic (Freter, R., et al., Infection and Immunity, 39(2):686-703, 1983). Under normal conditions colonization disappears within 1-2 weeks after antibiotics are discontinued, as the resident microflora recovers and reestablishes itself (Bennet, et al., 1992). To enhance colonization the following three methods are suggested.
The first method is to repetitively select for rapid colonizing bacteria on animal or human mucosal layers. For example, one would apply a wildtype bacterial strain to a mucosal surface and repetitively isolate and in vitro culture bacteria, returning at each step to the mucosal surface. Ultimately, an enhanced colonizing bacterium is obtained.
The second method is to have the recombinant bacteria express fusion proteins on their surface, which consist of a virus-binding domain and a host-binding domain. The host-binding domain will allow the bacteria to bind to certain determinants (protein or carbohydrate) on a selected host mucosal surface with high affinity, thus conferring the bacteria a slight survival advantage over the resident microflora. This has the added advantage of ensuring continued co-expression of the virus-binding domain, which would otherwise serve the bacteria no intrinsic survival benefit and therefore its expression may otherwise dwindle with time.
The third method is to induce the already resident microflora themselves to express the virus-binding protein by introducing the gene via bacteriophage. Bacteriophage has been used successfully to introduce genetic material into bacteria for some time. A number of bacteriophage vectors have been developed for different bacteria. Lactobacillus is likely the most suitable strain for vaginal mucosa and bacteriophage vectors optimized for lactobacillus are available for this invention. A bacteriophage vector has recently been developed for Lactobacillus gasseri based on the temperate bacteriophage φadh (Raya, R. R., et al., J Bacteriology, 174(17):5584-5592, 1992 and Fremaux, C., et al., Gene, 125:61-66, 1993). This vector undergoes site-specific integration into the host chromosome at defined phage (attP) and bacterial (attB) attachment sites. Optionally, the fusion gene may be placed under control of a strong promoter optimized for lactobacillus into the vector, along with a `suicide` gene under control of an inducible promoter.
Certain agents may also be added to a unit dose of the bacteria to aid in colonization. Many bacteria on mucosal surfaces secrete capsular materials which coalesce to form a biofilm which covers the entire mucosal surface. It may be beneficial to add an enzyme which digests this biofilm material to promote penetration of the engineered bacteria into the biofilm for more successful colonization. The enzymes include DNAses, peptidases, collagenases, hyaluronidases, and other carbohydrate degrading enzymes. Antibiotics (to which the engineered bacteria itself is not susceptible) may also be added to decrease the number of resident bacteria on the mucosal surface in order to make room for the engineered bacteria.
N. Persistent Expression of Heterologous Protein
As mentioned above, theoretically, expression of a foreign gene which serves a bacteria no purpose would likely dwindle over time, and the foreign gene would eventually be lost (Cardenas, L. and Clements, J. D., Vaccine, 11(2):126-135, 1993). To enhance persistent expression of the heterologous protein construct, an incentive may be created for the bacteria to express the gene. One way is the approach outlined above: create a fusion protein with virus-binding and host-binding domains so that the host-binding capability would confer a selective advantage to the bacteria to ensure the fusion proteins persistent expression. Another approach may be to create an internal requirement for the heterologous protein such that transformed bacteria that stop expressing the protein would die.
0. Vehicles for delivery/dosing regimen
Delivery of engineered bacteria to a desired mucosal surface depends on the accessibility of the area and the local conditions. Engineered bacteria may be placed in a saline solution for delivery to the naso- and oropharynx, or in a foam for delivery onto the vaginal or rectal mucosa. Mucosal surfaces less readily exposed--e.g. esophagus and trachea--may require a more viscous vehicle such as glycerin or sugar which facilitates the coating of the lining as it travels down the tract. Access to the small intestines and colon will require survival through the acid conditions of the stomach, hydrolytic enzymes secreted by the pancreas, and the antimicrobial effects of bile. Protective capsules have been suggested for protecting bacteria through the upper GI transit (Henriksson, A., et al., Appl Environ Microbiol, 57(2):499-502, 1991, hereinafter Henriksson, et al., 1991). It has been found that some strains in the colonic flora are inherently capable of surviving these conditions and therefore would be suitable for use in the GI tract.
Bacteria are self-replicating, so theoretically if an engineered bacteria 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 bacteria 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. Oral administration of 108 -1011 viable bacteria has produced transient colonization of colonic mucosa (Henriksson, et al., 1991 ). It is expected that colonization of the URT and vaginal mucosa will require less, as low as 106 viable bacteria, since these surfaces are more directly accessible and do not pose the acid and other harsh conditions of the upper GI tract.
To deliver genes directly into bacteria already resident on a mucosal surface, bacteriophage which specifically infect a selected bacteria will be used as the vector. Bacteriophage are viruses which infect bacteria. Examples include bacteriophage lambda, M13, and T7 which all infect Escherichia coli, and φadh which infects Lactobacillus gasseri. The nucleic acid of the selected bacteriophage may be manipulated such that the heterologous 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 heterologous gene(s). These replication-defective bacteriophage particles can then be introduced onto a desired mucosal surface to infect selected floral bacteria. The typical dosage would be 108 to 1012 PFU/ml applied to the mucosal surface. The proportion of solution to the treated surface should approximate 0. 1 to 1.0 ml per square centimeter of mucosal surface. The vehicle would be similar to the vehicle described above for the bacteria.
P. Situations particularly suited for this invention
1. To prevent infection from viruses for which no effective vaccine is presently available: HIV, HPV, HSV, Hepatitis A Virus, Varicella Zoster Virus (chickenpox), Rotavirus, etc.
2. Any individual who wants to minimize his/her risk of contracting viral URIs/influenza, especially those who travel frequently, work at public places (healthcare providers, school teachers, etc.), have young children, and those with important upcoming events who cannot risk being ill.
3. Immunosuppressed individuals-since ViroShield™ represents a completely additional layer of protection on the mucosal surfaces, it does not rely on normal function of the immune system, and in fact should work in conjunction with the immune system.
4. Third world countries where administration of vaccines may be difficult and unreliable; ViroShield™ against rotavirus would be particularly useful in these situations.
5. Individuals with allergic reactions to certain components in a vaccine preparation, such as eggwhite proteins in the preparation of the flu vaccine.
6. Individuals traveling to third-world countries where certain viruses are endemic, such as Hepatitis A and Poliovirus.
7. Individuals with significant risk factors for sexually-transmitted diseases.
8. Protection of livestock animals from pathogenic viral infection.
Q. Definitions
Bacteria: Minute, unicellular prokaryotic organisms that are classified as lower protists. They may occur as symbionts, parasites, or pathogens of humans and other animals, plants, and other organisms. Most of the mucosal surfaces of humans and animals are heavily colonized by a wide variety of bacteria, which serve a number of useful functions to the host.
Biofilm: A complex network of different bacteria and extracellular matrix materials secreted by the bacteria which become confluent as a film on many mucosal surfaces.
Colonize: As applied to the bacterial flora, a state in which a bacteria resides harmlessly on a host mucosal surface. The residency time may be from 2 days to permanent, but more typically 1 week to 1 month.
Conserved determinant: The portion of a protein which is common amongst many variants of the protein. This is important in viruses because there are often numerous strains of a single virus, each with slightly different variations in the viral proteins. A conserved determinant on a viral protein refers to an epitope which is common in all strains of the virus.
Disease: As applied to a viral infection, this is a state in which a host suffers harmful effects from a viral infection, either immediate (e.g. fever, chills, bodyaches, etc.) or long-term (e.g., chronic hepatitis and hepatocellular carcinoma from chronic hepatitis virus types B and C infections, and cervical cancer from chronic HPV infection).
Fusion: As used in this document, refers to the act of merging of two membranes such that the contents of the two entities combine into a single unit.
Genetic material: Generally DNA which contains at least one gene and the regulatory elements which affect the expression of that gene.
Host receptor: A molecule on the surface of the host (target) cell to which a virus attaches in order to gain entry into the host cell.
Hosts: The hosts include both animals and humans. The invention is useful for protecting livestock animals including mammals and birds.
Inactivation: The process of rendering an infectious agent no longer capable of infecting a host.
Mucosal surface: The epithelial membranes which line the inner interface of the body with the environment, including the respiratory tract, gastrointestinal tract, and genitourinary tract.
Receptors: As applied to viral receptors include the native protein and the functional domains that provide the specific binding characteristics that define these proteins as receptors of virus binding.
Selected for its ability to colonize the mucosal layer: As applied to bacteria refers to bacteria which have been chosen by either selective pressure or by deliberate genetic transformation to enhance ability to colonize mucosal surfaces. The ability whether in terms of absolute numbers or in residency time is defined as at least double the wildtype's ability to colonize.
Selective advantage: Certain features which when conferred upon a bacteria cause the bacteria to be better adapted to survive in a specific environment such that it will have a greater chance than other bacteria in the same environment to survive and flourish in that environment.
Transform: As applied to bacteria, the introduction of foreign genetic material into a bacteria for the purpose of causing said bacteria to express the foreign gene(s).
Viral infection: The introduction of a virus into a host or a host cell. This does not necessarily suggest harmful effects suffered by the host and needs to be distinguished from clinical disease. This is an important concept since ViroShield™ represents a way to prevent infection altogether, while standard vaccines do not actually prevent infection but may prevent disease.
Virus: An infectious agent that consists of proteins and genetic material, either DNA or RNA, both of which are arranged in an ordered array and are sometimes surrounded by an envelope. A virus is generally smaller than a bacterium and is an obligate intracellular parasite at the genetic level; it uses the cell machinery to produce viral products specified by the viral nucleic acid. They are classified into 5 classes based on the type of nucleic acid (ssDNA, dsDNA, dsRNA, +strand RNA, -strand RNA), and a sixth class which is capable of reverse-transcribing +RNA into DNA (retroviruses, e.g. HIV).
All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.
EXAMPLES
The following examples are provided by way of illustration only and not by way of limitation. Those of skill will readily recognize a variety of noncritical parameters which could be changed or modified to yield essentially similar results.
A. The following examples teach the expression of the receptor for HRV, major group, ICAM-1, on the surface of Esherichia coli.
1. Expressing ICAM-1 domain on the surface of E. coli using plasmid pTX101
ICAM-1 domains 1 and 2 (the minimal receptor for HRV, major group) are expressed on the surface of E. coli through the creation of a fusion protein with aa 1-9 of normal surface protein Lpp and aa 46-159 of OmpA using a system described in U.S. Pat. No. 5,348,867. The DNA segment coding for domains 1 and 2 of ICAM-1 will be amplified using polymerase chain reaction (PCR) with primers designed to introduce in-frame EcoRI restriction sites flanking aa 1-168 of ICAM-1.
Plasmid TX101, as described in U.S. Pat. No. 5,348,867, contains the β-lactamase gene spliced into an EcoRI site, which is removed by EcoRI digestion followed by separation of the linearized plasmid and the β-lactamase gene with agarose electrophoresis. The PCR amplified ICAM-1 gene segment is ligated to the purified plasmid. E. coli strain JM109 is rendered competent by the rubidium chloride method and transformed with the pTX101-ICAM construct using electroporation.
The Lpp-OmpA-ICAM construct is under the control of the strong Ipp promoter, which is inducible by IPTG (isopropyl thiogalactoside). Thus, IPTG stimulation will lead to high-level expression of the fusion protein. Transformed bacteria will be grown at 24° C. as this expression system works best (highest surface expression) at that temperature.
2. Ascertaining surface expression of ICAM-1 and demonstrating HRV binding.
Immunofiuorescence is used to confirm proper ICAM-1 expression on the bacterial surface. Transformed bacteria are applied to a glass slide and fixed with methanol. Slides will be treated with murine mAbs against ICAM-1, washed extensively, then reacted with goat-anti-mouse IgG conjugated with rhodamine.
Fluorescence is observed under a Confocal Fluorescence Imaging System MRC-500 Bio-Rad microscope. To demonstrate HRV binding to the transformed bacteria, slides with fixed bacteria are incubated with HRV, washed extensively, then reacted with murine mAbs against HRV coat protein. After washing, the slides will be treated with goat-anti-mouse IgG conjugated with rhodamine and visualized as described above.
3. Neutralization of HRV infection of HeLa cells by transformed bacteria in in vitro assay.
Early infection of HeLa cells in vitro by HRV will be monitored by detecting HRV mRNA inside infected HeLa cells by Northern blot analysis. A semipermeable membrane with pores of sufficient size to allow passage of HRV but not bacteria or HeLa cells is placed on top of a monolayer of HeLa cells in a tissue culture flask. Transformed or unmodified bacteria are layered onto the semipermeable membrane, then HRV is added on top of the bacteria and allowed to infect the underlying HeLa cells.
After an appropriate amount of time for infection, (2-6 hrs), the bacteria and semipermeable membrane are removed, and the HeLa cells washed extensively. The cells are then lysed, and their total RNA isolated for Northern analysis which is a standard method useful for detecting HRV mRNA which is an indication of infection.
4. Methods of formulating transformed bacteria in an appropriate vehicle (foam, DNAse, etc.) for use in animal and human hosts:
Transformed bacteria are formulated in a number of vehicles for animal application, For use in the nasopharynx, transformed bacteria may be mixed in saline and applied as a nasal spray. The bacteria are added to the saline at concentration of 106 to 108 cells/ml and applied twice daily in a directed spray of 0.1 ml solution/cm2 area of nasal mucosa.
B. The expression of a viral receptor on the surface of Streptococcus gordonii.
The following examples teach the expression of ICAM-1 on the surface of a gram-positive bacteria, since gram-positive bacteria are prominent members of the nasopharyngeal flora. The expression system is based on the one described in patent PCT/US93/02355. The examples provided herein use Streptococcus gordonii, but are readily adaptable to other gram-positive bacteria due to a common motif, LPXTGX, which allows the anchoring of proteins on the surface of essentially all gram-positive bacteria.
1. Expression of ICAM-1 on the surface of Streptococcus gordonii.
S. gordonii, strain GP232 described by Fischetti et al. in WO 93/18163 is used. In this strain, the gene which encodes for the M6 surface protein of S. pyogenes (contains the LPXTGX motif), emm-6.1, and an ermC gene (erythromycin resistance) disrupted by a cat (chloramphenicol acetyltransferase) gene have been inserted into the chromosome of GP232 downstream of a strong chromosomal promoter. GP232 expresses M6 on its surface, and is susceptible to erythromycin. Integration vector pVMB20 constructed by Fischetti et al. allows for the insertion of heterologous DNA sequences into emm-6.1. pVMB20 contains a functional (undisrupted) ermC gene, and is a 6.3-kb E. coli plasmid which does not replicate in S. gordonii. pVMB20 is digested with KpnI and HindIII to release a 538-bp KpnI/HindIII segment within emm-6.1, but leaving the LPXTGX motif intact. The ICAM-1 gene is PCR amplified using amplification primers specially designed to obtain an in frame KpnI/HindIII insert containing domains 1 and 2 of ICAM-1. The insert is then ligated into the digested vector.
Nucleotide sequence analysis of the pVMB20:ICAM-1 construct confirms the proper (in frame) insertion. The plasmid will be linearized and used to transform GP232 by standard methods. Homologous recombination between the 5' end of the emm-6.1 gene and the 3' end of the ermC gene, present on both the GP232 chromosome and the plasmid, allows for the integration of the ICAM-1 gene and the functional ermC gene into the GP232 chromosome.
Transformants are selected by screening for erythromycin-resistance, in media containing 5 μg/ml erythromycin.
2. Ascertaining surface expression of ICAM-1 and demonstrating HRV binding.
As in example A2, surface expression of ICAM-1 is verified by immunofluorescence using antibodies specific for ICAM-1. HRV binding will also be demonstrated as in example A2.
3. Neutralization of HRV infection of HeLa cells by transformed bacteria in in vitro assay.
Neutralization of HRV infection of HeLa by transformed GP232 will be demonstrated as in example A3.
C. Antibody expression on a E. coli.
The following examples teach the expression of an antibody fragment against a conserved determinant on the HLA DR molecule on the surface of E. coli. Since the HIV envelope is found to contain approximately 375 copies of the HLA DR molecules from its host (Arthur, L. O., et al., Science, 258(5090):1935-8, 1992.) an antibody fragment against a conserved determinant on HLA DR will bind to all isolates of HIV.
A phage display system exists which allows for the rapid selection of antibody fragments against essentially any target (Marks, J. D., et al., J Biol Chem, 267(23):16007-10, 1992.) is utilized to select for an antibody fragment with high affinity against a conserved determinant on HLA DR.
1. Selection for an antibody fragment with high affinity against a conserved determinant on HLA DR.
A phage library consisting of approximately 1014 bacteriophage each displaying a unique antibody fragment (scFv) on its surface is used (E.g., G. Winters, MRC, Cambridge, UK). Phage binding to HLA DR is selected by taking advantage of the fact that activated T cells express HLA DR, while resting T cells do not. All phage that bind activated T cells will be selected, then of this population, phage that bind resting T cells are removed. This process effectively isolates the subpopulation of phage that bind to HLA DR, and a few T cell activation markers. B cells express HLA DR constitutively. Subjecting this subpopulation of phage to B cells allows for selection of anti-HLA DR phage only, because B cells do not express T cell activation markers.
Of the phage that bind HLA DR, the ones that bind to conserved determinants are selected by screening the subpopulation against B cells of a variety of HLA DR specificities, and selecting only the clones that bind to every B cell specificity. If more than one clone is identified, the one with the highest binding affinity is used. Binding affinities in excess of 10-8 to 10-12 are preferred.
2. Method of expressing an antibody fragment against a conserved determinant on HLA DR on surface of E. coli using plasmid pTX101.
The VH and VL domains of the selected scFv are cloned using suitable primers designed to introduce in-frame EcoRI restriction sites at the N-terminus of the VH and the C-terminus of the VL. The PCR amplified gene segment is ligated into the EcoRI site of pTX101. JM109 bacteria are transformed with the plasmid, and surface expression of the fusion protein will be induced with IPTG at 20° C. as described in example 1.
3. Method of ascertaining surface expression of antibody fragment and demonstrating HIV binding.
Immunofluorescence is performed to confirm proper anti-DR scFv expression on the bacterial surface. Transformed bacteria are applied to a glass slide and fixed with methanol. Slides are treated with soluble human HLA DR molecules, washed, murine mAbs against HLA DR, washed, then reacted with goat-anti-mouse IgG conjugated with rhodamine. Fluorescence will be observed under a Confocal Fluorescence Imaging System MRC-500 Bio-Rad microscope. To demonstrate HIV binding to the transformed bacteria, slides with fixed bacteria are incubated with HIV, washed extensively, then reacted with murine mAbs against the HIV coat protein gp120. After washing, the slides are treated with goat-anti-mouse IgG conjugated with rhodamine and visualized as described above.
4. Neutralization of HIV infection of T cells by transformed bacteria in in vitro assay
Early infection of CEM cells (a laboratory T cell line) in vitro by HIV is monitored by detecting reverse transcriptase activity within infected cells. A semipermeable membrane with pores of sufficient size to allow passage of HIV but not bacteria or CEM cells is placed on top of CEM cells in a tissue culture flask. Transformed or unmodified bacteria are layered onto the semipermeable membrane, then infective HIV is added on top of the bacteria and allowed to infect the underlying CEM cells. After an appropriate of time for infection, (i.e. 2-6 hrs), the bacteria and semipermeable membrane are be removed, and the CEM cells washed extensively. These cells are lysed, and their cytosolic contents assayed for reverse transcriptase activity as an indication of early HIV infection.
5. Methods of formulating transforming bacteria in appropriate vehicle (foam, DNAse, etc.) for use in animal or human hosts.
For the GI tract, transformed E. coli bacteria are cultured and added to a mixture of various fatty acids conventionally used for rectal administrations such as: hydrogenated cocoa nut oil, glycerin, hydrogenated palm kernel oil, or other suitable material for rectal administration. The bacteria is added to the excipients at a concentration of 106 to 108 cells per mg of excipient. Each suppository is between 3-8 grams.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.
* * * * *
Other References
* Marshall, Science, 269: 1050-1055, 1995
* Int.J.Tiss REACXIII(2) 115-122 (1991), "Lactobacilli in Relation to Human Ecology and Antimicrobial Therapy," 1991 Bioscience Ediprint, Inc
* Gastroenterology 1992;102:875-878, "Fecal Recovery in Humans of Viable Bifidobacterium sp Ingested in Fermented Milk," 1992 by the American Gastroenterological Association
* Am J Clin Nutr 1992;55:78-80, "Survival bifidobacteria ingested via fermented milk during their passage through the human small intestine: an in vivo study using intestinal perfusion 1-4," 1992 American Society for Clinical Nutrition
* Human Health: The Contribution of Microorganisms Formulation, Production and Marketing of Probiotic Products, "Commercial Aspects of Formulation, Production and Marketing of Probiotic Products," Chapter 10, S. Laulan
Inventor
* Lee, Peter Poon-Hang
Application
No. 401070 filed on 03/08/1995
US Classes:
424/93.1, WHOLE LIVE MICRO-ORGANISM, CELL, OR VIRUS CONTAINING424/93.2, Genetically modified micro-organism, cell, or virus (e.g., transformed, fused, hybrid, etc.)424/93.4, Bacteria or actinomycetales424/93.45Lactobacillus or Pediococcus or Leuconostoc
Field of Search
435/173.1, TREATMENT OF MICRO-ORGANISMS OR ENZYMES WITH ELECTRICAL OR WAVE ENERGY (E.G., MAGNETISM, SONIC WAVES, ETC.)435/173.8, Metabolism of micro-organism enhanced (e.g., growth enhancement or increased production of microbial product)435/252.3, Transformants (e.g., recombinant DNA or vector or foreign or exogenous gene containing, fused bacteria, etc.)435/244, Chemical stimulation of growth or activity by addition of chemical compound which is not an essential growth factor; stimulation of growth by removal of a chemical compound435/252.1, Bacteria or actinomycetales; media therefor424/93.1, WHOLE LIVE MICRO-ORGANISM, CELL, OR VIRUS CONTAINING424/93.2, Genetically modified micro-organism, cell, or virus (e.g., transformed, fused, hybrid, etc.)424/93.4, Bacteria or actinomycetales424/93.45Lactobacillus or Pediococcus or Leuconostoc
Examiners
Primary: Ziska, Suzanne E.
Attorney, Agent or Firm
* Townsend and Townsend and Crew LLP
US Patent References
5348867, Expression of proteins on bacterial surface
Issued on: 09/20/1994
Inventor: Georgiou, et al.5531988Bacteria and immunoglobulin-containing composition for human gastrointestinal health
Issued on: 07/02/1996
Inventor: Paul
Foreign Patent References
* WO 93/18161 WO. 09/19/1993
International Class
A01N 063/00
US Patent Application 20060283080 - Method of creating and preserving the identity of non-genetically modified seeds and grains
192.168.1.100
http://www.patentstorm.us/applications/20060283080/fulltext.html
1. A method of ensuring the exclusion of genetically modified material from food products comprising: selecting seeds from certified sources that are known to contain only non-genetically modified and non-genetically-engineered varieties (non-GMO); planting the selected seeds to produce a non-GMO crop; inspecting a grower operation and machinery to verify that the operation is free of contaminates and conforms to processing and cleanliness criteria prior to harvest; harvesting the non-GMO crop; inspecting processing facility to verify that the operation is free of contaminants and conforms to processing and cleanliness criteria prior to processing; tracking containers holding the non-GMO crop each time the crop is moved into and out of a storage container, wherein the tracking includes monitoring the field in which the non-GMO crop was grown, monitoring each of the storage containers used to hold the non-GMO crop, and recording the date of all crop
transfers; and processing the non-GMO crops into a food product, wherein said food product is placed in containers for shipment, wherein the containers possess lot-based tracking information which permits the food product to be tracked back to the field and to containers used to produce or ship the food product; and shipping the food product for use by a food processor.
2. The method of claim 1, further comprising obtaining genetic test results indicative that the crop substantially excludes genetically modified crop material.
3. The method of claim 2, further comprising certifying that the crop excludes genetically modified crop material.
4. The method of claim 3, wherein certifying that the crop excludes genetically modified crop material comprises testing for application susceptibility.
5. The method of claim 3, wherein the certifying is sufficient to indicate that the crop contains 0.01% or less genetically modified material.
6. The method of claim 3, wherein the act of certifying comprises making a non-visual verification that the crop substantially excludes genetically modified crop material.
7. The method of claim 1, further comprising obtaining genetic test results indicative that the food product substantially excludes genetically modified crop material.
8. The method of claim 7, further comprising certifying that the food product substantially excludes genetically modified crop material.
9. The method of claim 8, wherein the certifying is sufficient to indicate that the food product contains 0.01% or less genetically modified material.
10. The method of claim 7, wherein the lot-based tracking information comprises a lot identification number established when the crop is harvested.
11. The method of claim 1, wherein the lot-based tracking information comprises a lot identification number established when the crop is harvested.
12. A method of ensuring the exclusion of genetically modified material from food products comprising: planting seeds selected from sources known to contain only non-genetically modified and non-genetically-engineered varieties to produce a non-GMO crop; harvesting the non-GMO crop; and processing the non-GMO crop into shipping containers identified with non-varietal lot-based tracking information.
13. The method of claim 12, further comprising obtaining genetic test results indicative that the crop substantially excludes genetically modified crop material.
14. The method of claim 13, further comprising certifying that the processed crop excludes genetically modified crop material.
15. The method of claim 14, wherein the certifying is sufficient to indicate that the processed crop contains 0.01% or less genetically modified material.
16. The method of claim 14, wherein the act of certifying comprises making a non-visual verification that the crop substantially excludes genetically modified crop material.
17. The method of claim 12, further comprising obtaining genetic test results indicative that the unprocessed crop substantially excludes genetically modified crop material.
18. The method of claim 17, further comprising certifying that the food product substantially excludes genetically modified crop material.
19. The method of claim 17, wherein the non-varietal lot-based tracking information comprises a lot identification number established when the crop is harvested.
20. The method of claim 12, wherein the non-varietal lot-based tracking information comprises a lot identification number established when the crop is harvested.
21. A method of ensuring the exclusion of genetically modified material from food products comprising: obtaining crop products grown under controlled conditions to exclude genetically modified material; tracking the crop products using lot-based tracking information; genetically testing the crop products to determine whether genetically modified material is present; and excluding the crop products if the genetic testing indicates that genetically modified material is present at an excess level.
22. The method of claim 21, further comprising associating information obtained at multiple stages of the development of the crop products with the lot-based tracking information in a single document.
23. The method of claim 21, wherein the genetic testing is variety-independent.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
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Genetic Murder Patent
NEW DISCOVERY !! Like alcohol ( WITHOUT THE SIDE EFFECTS ) our "LIGHT" water produces great blood flow and HIGH BLOOD OXYGEN THAT CAN "CURE" ANYTHING (WASHINGTON POST, sidebar)!! Pathogens can't survive in a high blood oxygen environment while producing such amazing results blood flow can be measured, in a non-invasive way, using doppler ultrasonography! UCLA M.D. that taught at the medical school: "WE HAVE TESTED EVERY WATER PRODUCT AVAILABLE AND NOTHING COMES EVEN CLOSE TO YOURS IN GETTING BLOOD (94% WATER) TO THE EXTREMITIES!" Products for pain ("PUT IT WHERE IT HURTS") were allowed by regulators because the published results are determined on the skin i.e. like the doppler effect where the ability of blood to go through a membrane into the cells can be measured on the skin. Take a cloth, soak it in "Light" water and place it anywhere (the eyes *below) or as a typical example: "I am Diabetic and my foot turned black, I soaked my foot in your water while I watched TV. I was a week away from surgery to have my foot amputated. The skin turned rosy pink! Thank GOD for your discovery!" M.D. (After a man regrew the tip of his finger): "IF I HADN'T SEEN THIS, I WOULDN'T BELIEVE IT!" WASHINGTON POST ARTICLE(sidebar): "SAID TO CURE ANYTHING!" THIS WORKS! When I was down in Exuma, Bahamas recently somebody said: "How old do you think that man is?" They agreed between 38 and 48. I AM 79. If you buy an E5 machine, you are invited to a party at the Shohola property where you can meet people with similar success stories! Also, when you buy a water machine from us, most of the profits go to this mountain property where our camping facilities support the Boy Scouts, Girl Scouts and disadvantaged youngsters. Politics aside, if you read the Washington Post article you would wonder why one sector of society that our water helps, would make an effort to stop this support! It has nothing to do with our water machines because after 36 years in business and over 200,000 users, the results are well documented! Check out: WATER SCAMS EXPOSED! (sidebar)
ORDER BOTTLED WATER 570 296-0214 johnelliswater.com <>ORDER A MACHINE: CALL 845 754-8696 OR (CLICK ON "ORDER" AT THE TOP OF THIS PAGE) MILLIONS OF HOME OWNERS HAVE DEBILITATING DISEASES CAUSED BY TOXIC MOLD! UNFORTUNATELY, THE WATER THEY ARE DRINKING FROM THEIR DISTILLERS AND PURIFIERS DOESN'T KILL MOLD SPORES, OUR WATER DOES (DOLE BANANA, 9 MONTHS OF TESTING)!! THEY CAN'T EVEN KILL BACTERIA (ORGANA TEST, THIS WEBSITE)! INSTEAD, THEY ARE TEARING OUT THE SHEETROCK WALLBOARD IN THEIR HOMES AND TEARING DOWN THEIR HOMES! IN MANY CASES, THEY CAN NO LONGER WORK BECAUSE OF THE DEBILITATING EFFECTS THAT ARE CAUSED WHEN THE MOLD SPORES ARE INHALED AND END UP THE BLOOD STREAM (94% WATER)! OUR PATENTS SHOW WE HAVE THE ONLY WATER IN THE WORLD THAT KILLS MOLD SPORES AND THEY ARE SO DEADLY, TERRORISTS WANT TO FLY PLANES OVER THE UNITED STATES AND PUT MOLD SPORES IN THE AIR, KILLING MILLIONS OF AMERICANS (CONGRESSIONAL REPORT AND THE HISTORY CHANNEL). THE ANTHRAX SCARE IS ONLY THE BEGINNING!
READ ABOUT THE WATER SCAMS (below) while keeping in mind that I am a Graduate Engineer and as a result of my top secret Government Security Clearance, I was awarded military contracts with information that could have saved billions of dollars for the Federal Government including a few hundred lives lost in nuclear submarines that sank!! However, I knew that even with great expense on my part, they probably wouldn't listen then just as they won't listen today concerning the devastation caused by the Water Industry! With Government contracts I had to go with my lawyer to a Arlington, VA tribunal to prove that they had made a serious mistake on their blueprints. I PROVED I was correct and completed the contract saving thousands olf lives! Now, in the water business, millions of people are developing life threatening health problems because they won't listen (scroll down to the very bottom of this page)!
WATER SCAMS: Watch out for "professors" or ANY website that asks for "donations" regardless of how legitmate they appear!! They are involved in plagarism like the man that was fired from the University of Colorado at Boulder for plagarism!! How about his comment about the victims at 9/11: "They deserved it!"..innocent victims trying to make a living!! Our literature is copyrighted including registered trademarks. If they use it to obtain "donations" (they are criminals) ask them to explain: 1) Why do we have the best water tests in the industry by lowering the steam velocity, starting and stopping the boiling (York Labs on this website)? i.e. (a clue) When we started in business, the largest selling home distiller made water 9 times purer than the tap water. By starting and stopping the boiling (ask them if they have a degree that includes Steam Plant Design like John Ellis) the water was 827 times purer than the tap water!! Ask them why this is possible! DAH!These tests were done for the U.S. Patent Office. Also, patents give as little information as possible without giving away proprietary secrets! DAH! They don't know that? Ask them how these test results are possible or why Dole Banana, a multibillion dollar company, produced water that destroys mold spores that can kill you! Ask why? Are their chemists stupid? Also, the Washington Post article that describes our patents! Are 10,000 people/day crazy?? How about an M.D. that tested our water and taught at the UCLA Medical School for blood flow (no other water is even close in getting blood to the extremities)? Is he and his associates crazy or how about an M.D. with Degrees that include Quantum Physics at a major Hospital? Is he crazy also? How about the Washington Times with a $600 million investment in Washington D.C.: "We have had at least 50 phone calls, ranging from Los Alamos to Johns Hopkins. They all have your machines!". Are they crazy also? How can people be so dishonest in an attempt to obtain your "donations"? If you suffer from cancer because of their comments (while selling courses or a product) and suffered harm because you believed them, we have the medical reports from the largest cancer research center in the world! YOU CAN EASILY COLLECT!! Our project manager is one of eight people in a multibillion dollar research project with the largest cancer research center in the world (3 out of 4 people will get cancer)!! WE CAN PROVIDE THE INFORMATION YOU NEED TO WIN! It's like winning the Lottery for almost nothing! Look at the "Organa Test"! This is another way to win big time if anybody in the water industry or their shills has lied to you and you suffered from illnesses as a result of their duplicity. We won property worth millions, so can you!! You can protect mankind from these dishonest people while helping to save millions of lives worldwide!! Call us, we will help you live a great life beyond your wildest dreams!!
At 79 years of age, I wonder why I don't retire (I have made enough money for 10 lifetimes). However, I love this business and our customers! My son and daughter including my grandchildren can run the business for generations. Here's what makes my day: I had a man call me and say that when he was 80 he had so many problems including cancer, he wrote his will. He said he bought one of our older machines and that is why he is still alive: "I am 103 and I want the best machine you manufacture!" I said: "It took you 20 years to figure that out?" Again, we love our customers! It's not like buying a product and then, after the sale, it's over! Many of our customers, that tell us about the results they attained on any subject, have been entertained at our gorgeous Shohola, PA property. Join us!!
They have already built a bridge to land below our mountain where they are building 160 homes. These are million dollar homes and each home is on 5 acres. I mention this because the value of the mountain, will skyrocket and I was able to secure the value of this property in a low cost lawsuit!! Appraisers are having trouble putting a value on the mountain because in addition, there are miles of "Parker Glen Bluestone", a Registered Trademark, already quarried there in the 1800's. You can WIN BIG in a similar action against the water industry that is lying to you!!
Make some big money!! They can use an opinion without a problem unless they are using a Registered Trade Mark in a deprecating way! If they are, ANYONE is open to a major lawsuit especially if they are asking for donations (fraud) while selling a product! It's easy to file a lawsuit and as stated, we collected property worth millions!! In many cases, we haven't filed because these people HELP our sales i.e. chem 101 professor: A "Smoking is good for you!" university while selling plagarized chemistry courses with "Jesus Dressup", making fun of Jesus on the Cross and "Scary Bible Quotes" making fun of 90% of Americans that believe in God and linking to other people that ask for money while using four letter words that drives customers to us!! Call us for information that will amaze you (some of this information is on this website)!!
ATTENTION: HOME BUILDERS AN D WALLBOARD MANUFACTURERS THAT ARE BEING SUED!! WE CAN HELP YOU! Mold spores are found in the soil and they will grow anywhere including wallboard. However, they can't blame the manufacturer of the wallboard or the contractors for something that is natural in the environment! All these people had to do was buy one of our machines and we have a 7 page contract from Dole Banana to help you win the case. This is a multi-billion dollar company that acted responsibly to protect consumers even if consumers don't want to protect themselves. Our machines produce water that kills mold spores and they also take in the room air and destroy mold spores while they are operating. In similar fashion, they tried to hold the air conditioner manufacturers responsible for health problems caused by bacteria in the air condition systems! The Organa Test, which has been done 100's of times, can handle that one too! After being in business for 36 years and spending $700,000/year in advertising, these home owners must have heard about us. They caused their own problems by inaction!
Years ago, after watching a documentary where a man and his family developed so many health problems he lost his job and a beautiful home, I decided to do something about it!
ZOMBIE NATION? (DRUGS ARE IN ALL DRINKING WATER, Associated Press 3/10/08) Philadelphia: 57 drugs,41 million people affected. Why become a walking drug cocktail or worse as predicted by Otto von Bolschwing (declassified/CIA)?? He was already working with the CIA for 6 years because of his contacts when he came to the United States in 1954 and he was closely connected to the drug industry including Warner Lambert. Obviously, with his world wide connections to the pharmaceutical industry, they wanted to sell more drugs (not less) and as he predicted, these drugs are now in our drinking water!! As a result of the Washington Post article (we stopped counting at 100,000 machines), so many people asked for our bottled water that the largest independent food distributor in the country ($20 billion/year sales) spent a year and over one million dollars checking us out! However, they require alot more than we can currently produce! Accordingly, we are tooling up to meet the demand.
Although you will be as shocked as we were to discover his true identity, Otto von Bolschwing gave me enough information, from many of the world's top scientists, to invent a way to change water properties so that small amounts can treat vast quantities of water for the benefit of mankind!! The Washington Post article (sidebar), that describes our worldwide patents, proves this and there are plenty more benefits that we are allowed to quote from a major newspaper. Today, all water has drugs that people flush down their toilets including the markers for all the other diseases that they are trying to treat! Don't be fooled. The markers for other peoples diseases are also in the water (nice thought)!! The CIA declassification PROVES that running water through ordinary distillers, filters, reverse osmosis and "alkaline" water machines ONE TIME (even 10 times) WON'T WORK! The Third Reich figured that out during WW 2 while using drugs to control the prison population!! Drugs alter water supplies so that the above products are as useless to the population then as they are today! After all, after taking over one country after another in their quest for world domination, they obviously wanted the population to be unable to "purify" their water!! That's why our machines recycle water 100's of times/gallon (not once!) while changing the properties of water to produce "light" water (you will notice the water goes down easier)! Since your blood is 94% water, "lighter" water gives you better blood flow (above). Also, if you heat and cool water long enough in a proprietary way (100's of times/gallon), it will finally break down the hydrogen bonds so that the molecules compress or expand more easily. It's not "putting oxygen in water" because that oxygen never gets into the blood stream. When the molecules aren't under compression, the molecules are larger and the blood can hold more oxygen coming into the lungs. Men with accute emphysema (VA Hospitals) don't have to be on oxygen 24 hours a day and there are other major benefits also!! You can call us!
* If you look at the "Put It Where It Hurts" product (above) and the fact that regulators allowed the product because the results can be measured on the skin, in a non-invasive way, using doppler ultrasound (getting blood to the extremities), you will enjoy this one!! In the same vein as THE WASHINGTON POST article on this website that describes our worldwide patents, one of our customers lost his eyesight from the effects of diabetes. After buying a bottle of water, HIS EYESIGHT CAME BACK RIGHT AWAY....USING ONLY ONE BOTTLE OF WATER (improved blood flow)!! Although the response time can vary depending on the problem, it shows the results are the same as soaking your foot in our water to increase blood flow!! A boon for diabetics in saving their limbs! We have thousands of "testimonials" like this. However, some of them we aren't allowed to use. In any case, the results our customers attain after reading THE WASHINGTON POST article where 10,000 people/day travelled to obtain our water should make the results obvious! The fact that the article describes our patents: "The curative power results from the "movements" of water between two metal tanks.", should make it even more obvious!! Our machine has a stainless steel tank 8" in diameter and only 5 1/2" high with a stainless steel coil in the tank. Attached to that tank is another tank (a little boiler) with a tube that connects the two tanks so that the water can go from boiling to cooling (between the two tanks), in a proprietary way, about 3 times/minute, 100's of times/gallon! THIS CHANGES THE PROPERTIES OF ORDINARY WATER TO OBTAIN THESE FANTASTIC RESULTS!!
The "Organa Test" shows that today people have been lulled into a false sense of security! How do you control a population? WATER! Most importantly, "The Organa Test" (on this website) PROVES that these products also produce water that DOESN'T STOP DISEASE and the Water Industry knows it...hiding this from the public! These products DON'T WORK and the test has been done 100's of times! If naysayers did this test and consumed the product, you would never hear from them again!! It proves their duplicity because our water is readily available! They can do the same tests done by Organa, the UCLA Medical School (above) and Dole Banana for deadly mold spores!! Don't let the water industry fool you including their shills (that are paid to lie to you)! Make sure you read "Water Scams Exposed" and the CIA information. Otto's dossier includes many of his worldwide contacts that the United States Government needed after WW2! Click: "About the Inventor" (sidebar)...it can save your life!! Call us if you have any questions!!
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CLICK ON THE VARIOUS SIDEBAR SUBJECTS AND IF YOU WISH, SCROLL DOWN FOR FURTHER INFORMATION. OUR WATER, WHEN ADDED TO VOLUMES OF OTHER WATER, CHANGES THE PROPERTIES AND PRODUCES WATER THAT GOES THROUGH A MEMBRANE INTO THE CELLS EASIER THAN ANY OTHER WATER!! IT DOESN'T MATTER IF IT'S HUMAN (above), ANIMAL OR PLANT! NEWSPAPER PICTURES: 22' CORN, EARS OVER 2' LONG! DOLE BANANA:SOLVED THEIR MOLD/ROT PROBLEM, WHILE INCREASING GROWTH EXPONENTIALLY, JUST MIXING IT WITH ORDINARY WATER! ELIMINATING MOLD SPORES, FOUND IN ALL SOIL, INCREASES PLANT GROWTH AND SOLVES WORLD HUNGER PROBLEMS! Thousands of M.D.'s and Phd's at Government Think Tanks that use our machines state: "Textbook science can't explain these incredible results!" Unless someone has a devious agenda (since our water is readily available), they can do the "Organa Lab Test" under Scientific Data (sidebar) that has been done 100's of times!! The Lab Test is under the Letter (scroll down). If you look at the results, you will realize that if you used ANY other water in this test and consumed the product, it would probably kill you!! This should make people wake up to the coming pandemic and make them aware of the fact that "pure water" products produce water that can't stop disease...ours does!! The water industry is hiding this information from the public (below)!
BEWARE!! THE WATER INDUSTRY IS BREAKING STATE AND FEDERAL LAWS! It is illegal to hide material facts from buyers! Read the FTC Policy Statement on Deception: 10/14/83 (below). Also, they are breaking International Copyright Laws if they use our literature, images, and Registered Trademarks without permission! 55 years ago my Chemistry Professor at Lafayette College said: "You can't change the hydrogen bond angle in water!" I did (for better blood flow) and he gave me the only 100 in 30 years of teaching! (Testimonials, sidebar). Although even my patents don't disclose "light" water technology, in those days, scientists didn't realize why people going to Lourdes and Nordenau aren't "healed" unless they are drinking enough water to replace the water they are eliminating! That's why we manufacture machines!
ALKALINE WATER
TIME WARNER MUST HAVE SOLD A MILLION COPIES OF THE "PH MIRACLE" AND "SICK AND TIRED" BOOKS. SINCE THESE BOOKS EXTOLL THE VIRTUES OF OUR MACHINES WE INCREASED THE SALES OF THE BOOKS BY MENTIONING THEM IN OUR FULL PAGE ADVERTISING. OUR MACHINES NOT ONLY MAINTAIN THE PROPER PH FOR FOOD DIGESTION, THEY ARE ALSO THE ONLY PRODUCT THAT CAN PRODUCE "PURE" WATER("LESS THAN 0.1 PPM POLLUTANTS, YORK LABS) AND PRODUCES WATER THAT STOPS DISEASE WHILE, MOST IMPORTANTLY, PRODUCING GREAT BLOOD FLOW! UCLA M.D.: "NOTHING COMES EVEN CLOSE IN GETTING BLOOD TO THE EXTREMITIES!" (ABOVE). THE "ALKALINE" WATER MACHINES CAN'T DO ANY OF THIS!!
ONE OF THE EDITORS OF THESE BOOKS, PETER TOCCI, IS ONE OF OUR GREAT CUSTOMERS. HOWEVER, AFTER THE BOOKS WERE WRITTEN, ACID-ALKALINE MACHINES THAT DON'T AUTOMATICALLY PRODUCE AN ALKALINE REACTION LIKE OUR MACHINES, CAUSED SUCH HORRIFIC HEALTH PROBLEMS THE AUTHOR HAD TO LEAVE THE STATE AND SELL THEM FROM A FOREIGN COUNTRY! WHY WOULD SOMEBODY BUY ONE ANYWAY?? IF YOU WANT TO MAKE WATER MORE ALKALINE, ADD A PINCH OF BI-CARBONATE OF SODA TO THE WATER! THESE MACHINES DON'T GIVE YOU GOOD BLOOD FLOW OR ANYTHING ELSE! TELL THEM TO DO SOME OF THE TESTS ON THIS WEBSITE! OBVIOUSLY, A MULTI-BILLION DOLLAR COMPANY LIKE DOLE BANANA WOULD USE THEM FOR A MOLD-ROT PROBLEM IF THEY WORKED! THIS IS COMMON SENSE!!
MOST OF THE SOLID FOOD YOU CONSUME SHOULD BE ON THE ALKALINE SIDE TO OFFSET MEATS THAT ARE ACIDIC. HOWEVER, WHEN IT COMES TO LIQUIDS, IT'S A DIFFERENT MATTER BECAUSE THE STOMACH HAS TO MAINTAIN AN ACIDITY OF 1-1.5 PH FOR PROPER FOOD DIGESTION. APPLE CIDER VINEGAR (I PUT IT ON MY SALAD EVERY NIGHT AND IT'S USED FOR DIGESTIVE PROBLEMS), GRAPEFRUIT JUICE AND LEMON JUICE HAVE AN ACIDIC PH OF 4 AND YET THEY ALL HAVE AN ALKALINE REACTION ON THE BODY JUST LIKE OUR WATER DOES BECAUSE THE PANCREAS MAINTAINS THE PROPER PH!
THE JAPANESE GOVERNMENT RECEIVED THOUSANDS OF HEALTH COMPLAINTS ABOUT ACID-ALKALINE WATER MACHINES AND AS A RESULT, THEY CLOSED DOWN ABOUT A DOZEN COMPANIES DURING THE 70'S. DIDN'T THE BOOK AUTHOR KNOW THIS?? HERE'S WHY THEY CAUSE SERIOUS HEALTH PROBLEMS: IF YOU DRANK A GLASS OF ALKALINE WATER RIGHT NOW, YOUR STOMACH WOULD BE MORE ALKALINE FOR ABOUT TWO MINUTES, UNTIL THE PANCREAS BROUGHT IT BACK TO 1-1.5 PH FOR PROPER FOOD DIGESTION. LET'S SAY YOU DRANK WATER FROM AN "ALKALINE WATER MACHINE" ALL THE TIME: AT FIRST, IT WOULD HAVE A STIMULATING EFFECT BECAUSE THE PANCREAS IS WORKING OVERTIME TO BRING THE DIGESTIVE JUICES BACK TO 1-1.5 PH. HOWEVER, IF YOU CONTINUE, YOU ARE COURTING DISASTER!!
WE RECEIVE 100'S OF PHONE CALLS FROM PEOPLE WITH DIGESTIVE PROBLEMS BECAUSE THE PANCREAS CAN'T MAINTAIN THE PROPER PH. MANY DEVELOP IRREVERSABLE CROHN'S DISEASE AND IN THE WORST CASE SCENARIO, THE PANCREAS SUDDENLY SHUTS DOWN AND THEY DROP DEAD!! ONE MAN CALLED US AND TOLD US THAT TWO OF HIS FRIENDS DROPPED DEAD WITHOUT WARNING AND HE QUESTIONED WHY THEY WERE ALLOWED ON THE MARKET!
DEALING WITH WATER SCAMS, THE FEDERAL GOVERNMENT AND MY MILITARY CONTRACTS
When I was 25 years old, I was a Design Engineer for Douglas Aircraft at El Segundo, CA and after that a Honeywell Engineer. At the age of 30, I started my own company, hired a team of Engineers and developed a scientific product that hasn't been duplicated to this day, even if they try to copy it!! That's why it doesn't need a patent!! As a result, many of my customers are Scientists and Engineers. One man, with 7 aerospace patents that he assigned to United Aircraft including the "swept wing design" used in all aircraft, graduated from The Choate School where I graduated with the Sikorski brothers. I mention them because many famous people graduated from Choate and their father invented the helicopter. This encouraged me to become an Engineer! Jack Kennedy graduated from Choate. It was more difficult than most colleges and my friends told me college was "easy" compared to Choate. I mention that because they get a kick out of the inane comments by so-called "professors" that can't even explain how distillation works and the importance of Steam Velocity! Also, in ordinary distillation the hydrogen bond angle goes from 104 degrees in ordinary water to 101 degrees in ordinary distilled water. In addition: "You can 't change the hydrogen bond angle in water". What do they think happens when you put a pot of water on a stove or why does the solubility change as the water warms up?
HERE'S IS HOW I COULD HAVE SAVED THE FEDERAL GOVERNMENT BILLIONS OF DOLLARS AND A FEW HUNDRED LIVES:
In the early 1960's, I had Government Inspectors at my plant almost every day and they would join us for lunch, so we would got to know them as friends. One day, an inspector asked me what I thought about the material another company was using for the torpedo launch tubes in a nuclear submarine contract. I told him: "They will peel like a banana when they are fired during sea trials!" The owner of the company, we both knew, made alot of money as a contractor on similar submarine contracts and as you know, the submarines sank to the bottom of the Atlantic causing the death of at least 200 fine young men! At the time, I hoped that the Inspector would do something about it. I couldn't because I had just come back from a military court because of my problem with a mistake on the blueprints of a military contract! We told them repeatedly, that there was a serious mistake that would cost lives but they wouldn't listen!! It was like "throwing sand against the tide"! That why I finally had to hire a lawyer and PROVE them wrong in a military court (at the top of the page)!! The SAME THING is happening today in the water industry, only with much greater health implications for MILLIONS of people!! Example: the "100% Pure" water! Pure water scams revealed! $10 value" ads placed by a man in Florida. An investigation shows the man has no degree in this field! Other people that stoop for "donations" to support themselves may have a degree in another field but reveal their stupidity in this field! The Florida man was caught selling Government Recalled water distillers even running water into toxic water collection jugs (Associated Press 3/10/08) and what he does is even WORSE than the AP article with terrible health implications especially for young children! The man obviously doesn't care! We mentioned the ads to a "Back to Nature" magazine where they accept these ads but they told us, in effect: "They don't care about the health of their readers!" We have run ads in the magazine for 30 years and the former owners keep in contact with us. They wrote articles about our older machines in their magazine! I could probably stop all this deceit but as noted with my government contract problems, it may be like "throwing sand against the tide"!
This information is for educational purpose only. It is not intended to diagnose, treat, cure, or prevent disease. Statements contained herein have not be evaluated by the Food & Drug Administration, as in all health situations, as was done in this report, qualified professionals should be consulted. PROTECTED BY FOREIGN and UNITED STATES PATENTS 6,612,090 (15 claims) - METHOD PATENT 5,203,970 & 6,409,888 and patents pending.
