OA12855A - Vaccinated. - Google Patents

Description

1 012855 5 THIS INVENTION relates to a therapeutic vaccine and to a prophylactic vaccine, to a method of making a therapeutic vaccine and to a method of making aprophylactic vaccine, to a method of treating a person or animal infected with a lipid-containing infectious organism, to a substance or composition for use in a method oftreatment of an infection caused by a lipid-containing infectious organism, to a substance 10 or composition for use in vaccinating a person or animal against an infection caused by alipid-containing infectious organism, to the use of a substance or composition in themanufacture of a médicament for use in the treatment of an infection caused by a lipid-containing infectious organism and to the use of a substance or composition in themanufacture of a médicament for use as a vaccine for the therapy or prophylaxis of an 15 infection caused by a lipid-containing infectious organism.

Tropical Africa has been regarded as the home of HIV/AIDS, malaria,filariasis, tuberculosis and a myriad of other chronic tropical and parasitic infections. Thequestion which arises is why are these infections chronic and why can the immune 20 Systems of infected persons not deal effectively with them.

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Infectious diseases hâve caused enormous and prolonged human sufferingand deaths throughout man's history. Certain infectious agents cause chronic infectionsfrom the start. The diseases they cause in man include for example, tuberculosis, leprosy, 012855 mycoses at various sites in the body, malaria, Onchocerca volvulosis and microfilaria that cause "river blindness". The viruses include the well-known human immune deficiencyvirus HIV, the human T-lymphocyte leukemia virus HLTV1, the hepatitis B virus, thehepatitis C virus, the herpes simplex virus, the cytomegalo virus CMV, the Epstein Barr 5 virus EBV of Burkett's Lymphoma and other viruses that cause lymphomas and othermalignant tumor disease in man.

Various animal species hâve similar or analogous microorganisms andviruses that cause chronic infections and tumors in them, for example bovine tuberculosis, 10 retroviruses such as SIV in monkeys, ’scrapies' in sheep, or malignant tumors in chicken,mice, cats and cattle. These lists are not exhaustive for humans or animais.

In order to understand the présent invention, it is necessary to understandthe basic éléments of the évolution of microorganisms and viruses, which 15 play an important part in shaping their présent structure and their behaviours in the body.This understanding has provided the insight into, and the basis for, the design of the effective vaccines of the invention.

In this spécification, the term microorganism should be understood to include20 bacteria, fungi, and parasites which affect humans and animais but to exclude viruses.

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Without being bound by theory, the Applicant proposes the following as thebasis for, and its understanding of, the invention. Most microorganisms that cause chronicinfections in man must invade the human body from the outside. Before invading the Ο 1 2856 body, they would ail hâve had a separate and independent existence and évolution thathas lasted perhaps millions of years in the earth's environment. In the process oftransmission form one person to another, these rnicroorganisms would again frequentlyhâve had to survive as independent agents for short period in the environment before 5 gaining access to a new host. They hâve ail therefore been exposed to and hâve survivedin the environment at some point in their évolution.

The earth's environment, to which these rnicroorganisms hâve beenexposed, is hostile in some ways to ail forms of life because the sun, a constant part of our I0 environment, dehydrates ail living things exposed to it. The sun is particularly hostile tornicroorganisms because the very small amount of body water in such organisms caneasily be lost by déhydration and this can cause the death of the organism.Microorganisms that hâve managed to survive in the earth's environment under the sunare therefore mutants that hâve acquired the ability to retain and conserve the water within 15 them.

Lipids and lipid-like substance are relatively résistant to déhydration andmost living surfaces exposed to the sun's rays or heat hâve a lipid material on them toreduce water losses. Microorganisms that hâve survived as independent agents in the 20 earth's environment are therefore mutants that hâve acquired a lipid covering to stop or reduce water loss from their surfaces. The amount of the lipid covering on the microorganism will clearly détermine its capacity to survive in the environment.

Microorganisms that hâve a thick lipid covering can survive better than those with a thin lipid covering and the varying capacities of most microorganisms to survive in the earth's 012855 environment as independent agents are related to the varying thicknesses of their lipidcoverings. The tubercle bacillus, for example, can survive for about a year in domesticdust and is known to hâve a very thick lipid coat. This is clearly demonstrated in the Zeihl

Nilsson stain. 5

Protective lipid coats that prevent water loss from the surface ofmicroorganisms cover over, and in some way eventually hide, the true antigens whichconfer identity, specificity, or uniqueness to the microorganism concerned. When true andrecognizable antigens from any source gain access to the body, as a first line of defense, 10 natural kil 1er cells normally produce cytokines that immediately destroy them. As a secondline of defense, the long-term immune responses of the body (by antibodies and cell-mediated immune responses) will détermine whether or not these true antigens persist inthe body. 15 When microorganism with lipid coverings invade the body in a normal infection, in the absence of the inaccessibility of their true antigens beneath the lipid coats,the immédiate response by cytokines and the long-term and effective immune responsesto the microorganism do not occur or occur very poorly. In contrast, the immuneresponses provdked by the lipid coat on the surface of the microorganism are not related 20 to the true antigens beneath and will not seriously affect them. The microorganisms therefore survive in the body as chrohic infections. By covering over their true antigens with a lipid coat, microorganisms hâve therefore been able to conserve their body water and ensure their survival first in the environment and later to escape from the immune response of the body and so persist as chronic or as repeated infections. ό 012855

The lipid coat of microorganisms can be stripped off in vitro with lipid solventssuch as chloroform or ether to expose their true antigens. The duration of action of thelipid solvent will dépend on the amount and nature of the lipid coat to be removed. Once 5 exposed, the true antigens of the microorganisms can be re-injected as a kind of vaccineinto an uninfected body. They will elicit from the body the immédiate response of naturalkiller cells with cytokines and later by long-term immune responses with antibodies. Whenconfronted by the same microorganisms in a subséquent infection, these cytokines andimmune responses will penetrate the lipid coat and destroy the true antigens beneath and 10 so the vaccinated body is protected from the infecting microorganisms.

Expérience has shown that for a préventive vaccine to achieve full immuneprotection of an uninfected body, it is necessary that: the vaccine has exposed true antigens that are related to those of theI5 microorganism concerned and that these true antigens are accessible to the immune

System, the person to be vaccinated has a competent immune System and there is no pre-existing infection or antibodies to the microorganism concerned in the sérum of the body to be vaccinated. 20

An established infection Jmay persist or be repeated because the trueantigens of the microorganism concerned hâve never been exposed to the immuneSystem and there are no antibodies to the true antigens in the person. In such a case, theexposed true antigens on the vaccine will provoke the immédiate production of cytokines 0128 7 r* rU.

U and the substances that penetrate the infection microorganism and kill it beneath its lipidcovering. The amount of cytokines produced and the numbers of microorganisms killedwill dépend on the amount of the vaccine used and the numbers of available natural killercells. In addition, the true antigens of the vaccine will provoke for the first time immune 5 responses that will reinforce and eliminate the microorganism. Such a vaccine injecteddirectly into the infected person in sufficient amounts and at suitable intervals will serve asa therapeutic vaccine.

In ail chronic infections, however, demonstrable antibodies to the10 microorganism are normally présent in the infected body. The continued infection in spiteof these antibodies is proof of their irrelevance or incompétence. Such antibodies,moreover, may immobilize any vaccine related to the microorganism that is introduceddirectly into the infected body. In that case, one must carry out a form of immunotherapyand provoke new and effective immune responses that would completely destroy and 15 prevent further infections by the same microorganisms.

The invention provides a new vaccine which is prepared from themicroorganisms of a current infection by exposing their true antigens by removing theirlipid coating with lipid solvents. With a competent immune System, it is possible to 20 artificially reproduce in vitro for the infected person conditions that are similar to those for apreventative vaccination in a normal unînfected person.

According to the method of the invention, peripheral leukocytes with competent immune cells taken from the infected person are washed free of ail antibodies. 012855 8

The washed leukocytes contain competent immune cells not committed to the currentinfection and free to react to new antigens. The washed cells are suspended in vitro in amedium that contains the infected person's sérum from which the immunoglobulin orantibodies hâve been removed. The situation in vitro is now similar or analogous to that 5 seen in an uninfected person before an infection (cord sérum without antibodies to themicroorganism can replace the person's sérum). The vaccine is added to the medium andincubated with the immune competent cells and then the mixture is re-injected into theinfected person. The re-injected cells and their descendants would continue in vivo whatwas started in vitro and the new immune response provoked would progressively and 10 completely kill the microorganisms. This would, paradoxically, achieve a kind ofpreventative vaccination of the infected person similar to injecting the vaccine directly intoan uninfected person. The use of the vaccine in an established infection in this manner isimmunotherapy of the infection. A normal preventative vaccine is also a kind ofimmunotherapy that is administered to an uninfected person in advance of the infection 15 and confers immunity by killing or the immunotherapy of any microorganisms that infectthe vaccinated person.

The invention thus allows the use of a vaccine to prevent and to treat anestablished infection by any microorganism which has had its lipid coat removed in vitro to 20 expose its true antigen.

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Viruses are believed to hâve arisen from the nucleic material of pre-existingcells. Although they exist as separate entities, viruses dépend entirely on living cells fortheir vital functions and survival. They hâve not therefore been exposed to the hostile 012855 9 effects of the sun as were free-living microorganisms. Those viruses that hâve evolvedinto chronic infections in man and animais such as the HIV and other retroviruses, theHepatitis B virus, the Hepatitis C virus, the EBV, the CMW, the Herpes simplex virus, thetumor-causing viruses in man and animais etc. are ail enveloped viruses. The viral 5 envelopes hâve been acquired from their host cells in the course of their évolution toensure their survival in the body.

The DNA viruses such as the EBV, CMV and Hepatitis B viruses acquiredtheir envelopes from the nuclear membrane of the host cell as they leave the nucléus from 10 which they developed. The RNA viruses such as the retroviruses and Hepatitis C virusacquired their respective envelopes from the cell wall membrane as the mature virus finallyleaves the host cell in which it has developed. Because they carry envelopes that are ofhost cell origin, enveloped viruses are perceived as 'hybrid' viruses, combining viral coresthat are foreign to the body with envelopes that are of host origin. For that reason, 15 enveloped viruses are perceived by the host immune System as 'partly self.

An effective immune response by the host that killed the virus with itsenvelope would also kill ail those host cells from which the viral envelope was derived. Inthe case of Hepatitis B and C, the liver cells would be killed as well. In the case of HIV, ail 20 the CD4+ cells would be eliminated. In ail such situations, this would lead to serious andfatal autoimmune diseases. To avoid killing the body with the virus the immune responseof the host is therefore obliged to be ineffective. In taking its envelope from the host cellmembranes, these viruses hâve 'deliberately' taken important ’hostages' with which to'blackmail' the immune Systems of the body into ineffective responses. These ineffective 012855 10 immune responses ensure the persistence of the enveloped viruses as chronic infectionsin the body.

The viral envelope, like the cell membrane from which it is derived, contains5 phospho-lipoproteins that can be easily destroyed with lipid solvents such as chloroform orether. When the envelope has been destroyed, the rest of the viral antigens, nowconsidered a vaccine when injected into an uninfected person, will be perceived by theimmune System as non-self and completely foreign. Appropriate immune responses areprovoked that are effective in destroying only the viral cores or non-enveloped parts of the 10 virus, in keeping with the viral antigens used in the vaccine. Any subséquent infection ofthe vaccinated body by the same enveloped virus will hâve only its core destroyed leavingits envelop intact. Empty envelopes hâve no biological importance in the person. Thevaccinated person would be immune against the enveloped virus concerned. Bydestroying the envelopes of enveloped viruses, the rest of the viral antigens constitute a 15 valid and effective vaccine for the virus concerned. Administered to an uninfected person,such a vaccine will achieve a permanent préventive immunity by destroying only the viralcore of any subséquent enveloped virus of the same type that infects the body.

In an established infection by an enveloped virus, the host nature of the 20 envelope on the virus blackmails the natural killer cells and blocks any effective long-termimmune responses that could kill the virus. However, when the viral envelope is destroyedin vitro and the rest of the antigens re-injected as a vaccine into persons with theestablished infection, they are perceived as completely foreign and non-self. The vaccineprovokes immédiate cytokines that will penetrate the viral envelopes and cell membranes 01285( containing enveloped viruses and kill the viruses within. The amount of the cytokinesproduced will dépend on the amount of the vaccine used and the State and number of thenatural killer cells in the patients. When the infection has been eliminated or reduced toinsignificant levels, a long-lasting preventative vaccination or immunotherapy of the 5 patient is achieved as follows.

After verifying that the patient's immune System is competent and thatviruses hâve been eliminated or reduced significantly (in the case of HIV, a count of CD4+above 4 - 500, and a viral count below 50 copies/ml), héparine blood is withdrawn from 10 the patient. The peripheral leucocytes in the blood are washed free from ail traces ofantibodies to the virus. The immune competent cells in the washed leucocytes notcommitted to the virus are suspended with others in a medium containing the patient'ssérum from which immunoglobulins and antibodies hâve been removed. (Cord sérum withno antibodies to the virus can replace the patient's antibody-free sérum. The sérum in the 15 mixture ensures that the antigens of the vaccine are properly presented to the immuneSystem). The vaccine is added to this mixture and after incubation it is re-injected into thepatient. This is immunotherapy of the patient and is similar to the injection of a préventivevaccine into an uninfected person with a normal immune System and no infection or antibodies to the virus. 20

The re-injected mixture will continue in vivo the process that was started in vitro and the new immune responses provoked by the immunotherapy éliminâtes from the body any remnants of viruses and prevent any future re-infection by the same enveloped 12 virus. Thus a vaccine can be prepared by destroying the envelope of any given envelopedvirus and used to prevent or treat an infection by the same enveloped virus.

Ail immune responses in the body utilize spécifie complément in one form or5 another. Even when the immune response is ineffective, as in most chronic infections bymicroorganisms and enveloped viruses, complément is used ail the same. In time,complément spécifie to the microorganism or virus is in short supply having been used up in a futile attempt to eliminate the chronic infection. 10 The immunotherapy started in vitro that provokes new and effective immune responses in the patient will also require complément. This new demand for complémentcan only aggravate the existing chronic shortage and worsen the clinical situation of thepatient. It is therefore essential that the viral load or the microorganism be substantiallyreduced in the patient by other means so that the complément produced thereafter is 15 available for completing the new and effective immune responses provoked by theimmunotherapy achieved in vitro as described above.

One method for reducing the viral load of microorganisms, as mentionedabove, is to start by first giving moderately large doses as simple direct injections into the 20 patient of the vaccine with true antigens exposed or viral envelopes removed. These do not really vaccinate the patient but provoke the natural killer cells to produce cytokines that should kill large numbers of the viruses or microorganisms. The vaccine is then similar to a drug. If repeated at suitable intervals, this killing can significantly reduce the viral load or microorganisms and their need for complément to insignificant levels. The complément 012855 13 produced or freed in the body after that is then available for the subséquentimmunotherapy started in vitro that provokes effective immune responses in the body.This latter is carried out only when there is evidence that the viral load of microorganismsin the patient do not make significant demands on complément. 5

Another quick method for ensuring that the immunotherapy started in vitro iseffective in the patient with an on-going chronic infection is to provide extra complément bythe transfusion of fresh blood to the patient. The transfusion is most useful when the newimmune responses begin to appear, usually after about 10 - 14 days from the 10 immunotherapy. The blood transfusion should be repeated until the virus ormicroorganism is completely eliminated.

Thus, according to a first aspect of the invention, there is provided a methodof making a vaccine, the method including the steps of 15 extracting a biological fluid obtained from a person or animal infected with a lipid- containing infectious organism with a lipid-containing solvent, the biological fluid containingthe lipid-containing infectious organism and the extraction producing an aqueous phase,which contains the infectious organism with the lipid substantially removed, and a lipid-containing organic phase, and 20 isolating the aqueous phase.

The lipid-extracting solvent may be chloroform. 14 012855

The biological fluid may be whole blood, plasma, pleural fluid, cerebrospinalfluid, culture fluid or other localized body fluid. The lipid-containing infectious organismmay be a virus, a bacterium, for example a tuberculosis or leprosy bacterium, a protozoa,a fungus or a mould of the type which contains lipid or lipid-like material in its cell wall. In 5 particular, it may be an enveloped virus such as an immunodeficiency virus for examplethe human immunodeficiency virus, or HIV. It may, instead, be an enveloped virus such asHepatitis B, Hepatitis C, Epstein Barr, Herpes simplex, a cytomégalovirus a human T-cellleukemia virus HTLV1, a cancer virus or any other similar enveloped virus. I0 The extraction process of the biological fluid containing the lipid-containing infectious organism may be by mixing, swirling, vortexing, rotating or by any other suitableprocess which would be familiar to the person skilled in the art. The extraction time will besufficient to allow the chloroform to solubilise ail, or substantially ail, of the lipid containedin the infectious organism and will vary with the extraction process. For example in the 15 case of vortex mixing, such as in the case of HIV, the vortexing may be carried out over aperiod of about 30 - 60 minutes and typically about 30 minutes.

The method may include the further step of concentrating the aqueous phaseto produce a concentrate containing the infectious organism or of otherwise isolating the 20 infectious organism with the lipid substantially removed, for example by lyophilisation or byany other technique which would be familiar to the person skilled in the art.

The vaccine produced in this way may be used as an auto-vaccine for theperson from whom the biological fluid was extracted. Instead, the method may include the 1.5 additional step of isolating and culturing the infectious organism from an infected pers.oninhabiting a particular geographical région. In this form, the vaccine may be used eitherprophylatically (as a true vaccine) or for the treatment of persons from that geographicalrégion. This aspect of the invention is particularly important in the case of a virus such as 5 HIV which differs in different geographical areas.

The method may include the step of diluting the biological fluid or of dilutingthe aqueous extract in order to obtain antigens from approximately 100 - 200 viralparticles per ml. 10

The ratio (volume : volume) of the chloroform to the biological fluid used inthe extraction step may be between about 3 . 1 and 5 : 1. It is preferably about 3:1.

According to a another aspect of the invention, there is provided a method of15 making a first therapeutic vaccine, the method including the steps of extracting, with a lipid-extracting solvent, a biological fluid obtained from a person oranimal infected with a lipid-containing infectious organism, the biological fluid containingthe lipid-containing infectious organism, and the extraction producing an aqueous phasewhich contains the infectious organism with the lipid substantially removed and a lipid- 20 containing phase, separating the aqueous phase'from the lipid-containing phase, isolating a leukocyte fraction from the blood of the person or animal, the isolation being conducted so that the leukocyte fraction contains substantially no plasma, free lipid- 012855 16 containing infectious .organism or free antibodies to the lipid-containing infectiousorganism, and combining at least some of the aqueous phase with at least some of the leukocytefraction to produce the vaccine. 5 It will be appreciated that a part of the leukocyte fraction will comprise infected ce Ils.

The first therapeutic vaccine prepared as described above will be used onthe patient from whom the biological fluid and the leukocyte fraction were obtained. 10 However, in a development of this embodiment of the invention, the leukocyte fractionfrom an infected person in a particular geographical area may be combined with a vaccineprepared from isolated cultured virus obtained from another infected person from thatgeographical area as described above to produce a second therapeutic vaccine. 15 Thus the invention extends to a method of making a second therapeutic vaccine, the method including the steps of isolating and culturing an infectious organism from a biological fluid obtained from atleast one person or animal infected with a lipid-containing infectious organism to produce acomposition containing the cultured lipid-containing infectious organism, 20 extracting, with a lipid-extracting solvent, an aqueous solution of the lipid-containing infectious organism, the extraction producing an aqueous phase which contains theorganism with the lipid substantially removed and a lipid-containing phase, separating the aqueous phase from the lipid-containing phase, Ûî285£ 17 isolating a leukocyte fraction from the blood of another person or animal infectedwith the lipid-containing infectious organism, the isolation being conducted so that theleukocyte fraction contains substantially no plasma, free lipid-containing infectiousorganism or free antibodies to the lipid-containing infectious organism, and 5 combining at least some of the aqueous phase with at least some of the leukocyte fraction to produce the second therapeutic vaccine.

The lipid-extracting solvent may be any suitable solvent and may be achlorinated hydrocarbon such as chloroform. It may, instead, be a hydrocarbon or an 10 ether. In particular it is noteworthy that chloroform is suitable for use in the method of theinvention even though PCT/IB01/01099 clearly indicates that chloroform dénaturés manyplasma proteins and is unsuitable for use with fluids which will subsequently be administered back to the animal or human. 15 The leukocyte fraction may be obtained by withdrawing a blood sample from the person or animal, separating the red blood cells from the plasma by sédimentation,separating the leukocytes from the plasma, for example by centrifugation, and washing theleukocytes free of residual plasma and antibodies with normal saline. 20 The second therapeutic vaccine will be used on the patient from whom the leukocyte fraction was obtained.

In another embodiment of the invention, the aqueous phase produced from the extraction of the cultured lipid-containing infectious organism can be used directly as a 012855 18 prophylactic vaccine for normal non-infected persons living in the geographical région inwhich the person or animal from which the biological fluid was obtained and in which thelipid-containing infectious organism is prévalent. 5 According to another aspect of the invention, there is provided a vaccine prepared by a method which includes the steps of extracting a biological fluid obtainedfrom a person or animal infected with a lipid-containing infectious organism withchloroform, the biological fluid containing the lipid-containing infectious organism, and theextraction producing an aqueous phase which contains the infectious organism with the 10 lipid substantially removed and a lipid-containing phase, andisolating the aqueous phase.

According to another aspect of the invention, there is provided a firsttherapeutic vaccine prepared by a method which includes the steps of 15 extracting, with a lipid-extracting solvent, a biological fluid obtained from a person or animal infected with a lipid-containing infectious organism, the biological fluid containingthe lipid-containing infectious organism, and the extraction producing an aqueous phasewhich contains the infectious organism with the lipid substantially removed and a lipid-containing phase, 20 separating the aqueous phase from the lipid-containing phase, isolating a leukocyte fraction from the blood of the person or animal, the isolation being conducted so that the leukocyte fraction is substantially free of plasma, free lipid-containing infectious organism and free antibodies to the lipid-containing infectiousorganism, and 01285f 19 combining at least some of the aqueous phase with at least some of the leukocytefraction to produce the vaccine.

According to another aspect of the invention, there is provided a second5 therapeutic vaccine prepared by a method which includes the steps of isolating and culturing an infectious organism from a biological fluid obtained from atleast one person or animal infected with a lipid-containing infectious organism to produce acomposition containing the cultured lipid-containing infectious organism, extracting, with a lipid-extracting solvent, an aqueous solution of the lipid-containing10 infectious organism, the extraction producing an aqueous phase which contains the organism with the lipid substantially removed and a lipid-containing phase,separating the aqueous phase from the lipid-containing phase,isolating a leukocyte fraction from the blood of another person or animal infected with the lipid-containing infectious organism, the isolation being conducted so that the15 leukocyte fraction contains substantially no plasma, free lipid-containing infectious organism or free antibodies to the lipid-containing infectious organism, and combining at least some of the aqueous phase with at least some of the leukocyte fraction to produce the second therapeutic vaccine. 20 According to another aspect of the invention, there is provided a method of treating a person or animal infected with a lipid-containing infectious organism, the methodincluding the steps of extracting a biological fluid obtained from a person or animal infected with a lipid-containing infectious organism with chloroform, the biological fluid containing the lipid- 012855 20 containing infectious organism, and the extraction producing an aqueous phase whichcontains the infectious organism with the lipid substantially removed and a lipid-containingorganic phase, isolating the aqueous phase, and 5 administering an effective amount of the aqueous phase to the person or animal.

The method may include the further step of concentrating the aqueous phaseto produce a concentrate or of otherwise isolating the infectious organism with the lipidsubstantially removed as hereinbefore described and administering the resulting 10 substance, optionally with a pharmaceutically acceptable adjuvant or carrier, to the person or animal.

According to another aspect of the invention, there is provided a method oftreating a person or animal infected with a lipid-containing infectious organism, the method 15 including the steps of extracting, with a lipid-extracting solvent, a biological fluid obtained from the personor animal, the biological fluid containing the lipid-containing infectious organism, and theextraction producing an aqueous phase which contains the infectious organism with thelipid substantially removed and a lipid-containing phase, 20 separating the aqueous phase from the lipid-containing phase,

J isolating a leukocyte fraction from the blood of the person or animal, the isolationbeing conducted so that the leukocyte fraction is substantially free of plasma, free lipid-containing infectious organism and free antibodies to the lipid-containing infectiousorganism, 012856 21 combining at least some of the aqueous phase with at least some of the leukocytefraction to produce a composition, and administering a therapeutically effective amount of the composition to the person or animal.

According to a another aspect of the invention, there is provided a method oftreating a person or animal infected with a lipid-containing infectious organism, the methodincluding the steps of isolating and culturing an infectious organism from a biological fluid obtained from at10 least one person or animal infected with a lipid-containing infectious organism to produce a composition containing the cultured lipid-containing infectious organism, extracting, with a lipid-extracting solvent, an aqueous solution of the lipid-containing infectious organism, the extraction producing an aqueous phase which contains theinfectious organism with the lipid substantially removed and a lipid-containing phase, 15 separating the aqueous phase from the lipid-containing phase, isolating a leukocyte fraction from the blood of another person or animal infected with the lipid-containing infectious organism, the isolation being conducted so that theleukocyte fraction contains substantially no plasma, free lipid-containing infectiousorganism or free antibodies to the lipid-containing infectious organism, 20 combining at least some of the aqueous phase with at least some of the leukocyte

fraction to produce a composition, andJ administering a therapeutically effective amount of the composition to the person or animal. 012855 22

The method may include the step of subjecting the person or animal to afresh blood transfusion to increase the complément or complement-like proteins in theblood. The complément System is composed of a group of sérum proteins that play amajor rôle in the défensive immune response (Microbiology, 2nd Ed, Prescott, Harley and 5 Klein) and the blood transfusion increases the levels of the proteins in a person or animalwith a severely compromised immune System.

According to another aspect of the invention, there is provided a thirdtherapeutic vaccine which includes leukocytes which hâve been exposed in vitro to a 10 vaccine produced by the substantial removal of the lipid portion of a lipid-containinginfectious organism, the exposure taking place substantially in the absence of the freelipid-containing infectious organism and in the presence of a medium containing sérumwhich is also free of antibodies to the lipid containing infectious organism. 15 The leukocytes will typically be exposed at about 37°C for about 4 hours.

The sérum proteins provide opsonins which enable the therapeutic vaccine toachieve permanence in the body. 20 According to another aspect of the invention there is provided a prophylactic vaccine prepared by a method which includes the steps of culturing a biological fluid obtained from a person or animal infected with alipid-containing infectious organism, 012855 23 extracting the culture containing the lipid-containing organism with a lipid-extractingsolvent, the extraction producing an aqueous phase which contains the infectiousorganism with the lipid substantially removed and a lipid-containing phase, and isolating the aqueous phase. 5

The invention extends to a substance or composition for use in a method oftreatment of an infection caused by a lipid-containing infectious organism the substance orcomposition comprising a vaccine or a first or a second or third therapeutic vaccine as hereinbefore described. I0

The invention extends further to a substance or composition for use invaccinating a person or animal against an infection caused by a lipid-containing infectiousorganism, the substance or composition comprising an isolated and cultured infectiousorganism as hereinbefore described. 15

The infectious organism may be HIV with its lipid envelope substantially removed.

The invention extends further to a substance or composition for use in the20 prophylaxis of an infection caused by a lipid-containing infectious organism the substance or composition comprising a prophylactic vaccine as hereinbefore described.

The invention extends, further, to the use of substance or composition in the manufacture of a médicament for use in the treatment of an infection caused by a lipid-

24 containing infectious organism, the substance or composition comprising a vaccine or atherapeutic vaccine as hereinbefore described.

The invention extends further to the use of a substance or composition in the5 manufacture of a médicament for use as a vaccine for the prophylaxis of an infectioncaused by a lipid-containing infectious organism, the substance or composition comprising an isolated and cultured infectious organism as hereinbefore described.

The invention extends further to the use of a substance or composition in the10 manufacture of a médicament for use in the prophylaxis of an infection caused by a lipid-containing infectious organism, the substance or composition comprising a prophylactic vaccine as hereinbefore described.

The invention is now described, by way of example, with reference to the15 following Examples, the Table and the Annexures. EXAMPLE 1

The vaccine 20

Fresh fasting blood (5ml) from an HÎV sero-positive person (containing 10000 to 20000viral particles per ml) was withdrawn into a syringe with 500 units of héparine and allowedto stand at room température for 30 minutes. This plasma (1ml) was combined withchloroform (5ml) in a stérile screw-capped glass tube. The mixture was mixed using a 25 012855 vortex mixer for 5 minutes and then allowed to stand at room température for another 25minutes. Normal saline (9ml) was added and the mixture was mixed using the vortexmixer for 5 minutes and allowed to stand at room température for 10 minutes. Theresulting mixture was transferred to a stérile screw-capped glass centrifuge tube and 5 centrifuged for 15 minutes at 4000rpm. The aqueous supernatant was transferred to astérile glass Pétri dish with a base diameter of 10cm and allowed to stand under anextraction hood for 30 minutes. The resulting 10ml of vaccine constitutes 10 doses. Itcontained antigens from 1000 - 2000 viral particles per ml of vaccine. The first vaccinewas stored at minus - 20°C as a single dose. 10 EXAMPLE 2

The vaccine

In another embodiment of the invention, fresh fasting blood (5ml) from a patient withI5 100,000 - 200,000 viral particles per ml was drawn into a syringe with 500 units ofhéparine. This plasma (1ml) was combined with chloroform (5ml) in a stérile screw-capped glass tube and thoroughly mixed with a vortex mixer for 10 - 15 minutes. Themixture was allowed to stand at room température (20 - 25°C) for 45 minutes. Normalsaline (9ml) was added to the mixture and vortexed for 5 minutes and allowed to stand for 20 10 minutes at room température. The mixture was transferred into stérile screw-capped centrifuged tubes and centrifuged at 4000rpm for 15 minutes. The supernatant wastransferred to a stérile glass Pétri dish with a base diameter of 10cm and allowed to standunder an extraction hood for 30 minutes. The supernatant (1ml) was diluted with normal 012855 26 saline (9ml) to give a dilution of 1/wo- This final dilution (1ml) contained antigens fromabout 1000 - 2000 viral particles per ml and was stored in single doses at minus -20°C. EXAMPLE 3 5

The effect of auto-vaccine on viral counts (see Table 1, Annex 1 )

Auto-vaccines were prepared according to the method of Example 1, and containedantigens from 1/100 the viral count in the patient are concerned. The auto-vaccine (1ml) I0 was injected directly into the patient. This produced significant faits of viral counts in thepatient concerned except in patient no 7. In patients 1 and 2, the auto-vaccine wasinjected on 2 separate occasions 4 days apart and the absolute fall in viral count was 91 %and 75% respectively. 15 In another patient (Annex 1 (b)), 1ml of auto-vaccine was administered as 5 simple s/cinjections on 17/4/2001, 21/04/2001, 21/06/2001, 21/08/2001 and 21/10/2001. The viralcount fell from 29,566 (4.5 log10) on 16/04/2001 to below 50 (1.5log10) on 02/07/2002 15months later and the HIV serology was indeterminate. However on 17/12/2002 the viralcount had risen to 65 (1.8logio). This suggests that the auto-vaccine administered as a 20 direct injection caused the viral count to fall to the point where the HIV serology wasindeterminate but without further auto-vaccines, the viral réplication restarted. EXAMPLE 4 27 1285

The effect of auto-vaccine with washed peripheral leukocytes (see Annex 2)

The auto-vaccine was prepared according to the method of Example 1 and diluted 1/ioo. A5 blood sample (20ml) was withdrawn into a syringe with 1000 units of héparine. Thesample was transferred to a long stérile screw-capped tube with a base diameter of 1cmand allowed to stand upright for 30 minutes at -37°C. The leukocyte rich plasma abovethe red cell sédiment was aspirated off and centrifuged at 2000rpm for 10 minutes in abench centrifuge (Jouan C312). The supernatant plasma was discarded and normal 10 saline (5ml) was added to the leukocyte deposit and after vortexing for 1 minute themixture, again centrifuged at 2000 rpm for 10 minutes.

The supernatant was discarded and fresh normal saline (5ml) was added to the deposit.This process of washing the leukocytes was repeated 2 more times to remove ail traces ofantibodies from the cell deposit. The auto-vaccine (1ml) was then added to the washed 15 leukocytes and the mixture injected immediately subcutaneously into the patientconcerned. The procedure was repeated on the dates shown in Table 2 below. Therewas a marked increase of CD4 in patients and significant falls of viral counts in ail patientsand was below 50 particles in 9 of 34 patients. 20 EXAMPLE 5

The therapeutic vaccine and immunotherapy

The washed peripheral leukocytes were prepared as in Example 4. The auto-vaccine(1ml), prepared according to the method of Example 1, was added to the cell deposit 012855 28 followed by culture medium of RPMI (GIBCO) (5ml) with 10% human cord sérum free fromantibodies to HIV and other viruses in place of the patient's sérum from whichimmunolglobins hâve been removed. The mixture was incubated at 37°C for 4 hours. Thetherapeutic vaccine was re-injected directly subcutaneously into the forearm of the person 5 from whom the blood sample had been withdrawn and auto-vaccine prepared. The wholeprocedure was repeated on day 30 as a booster. A mild immune réaction in the form of mild tiredness, body aches and a mild fever lasting afew day occurred in a few patients about 10-14 days from the first immunotherapy. 10 These symptoms were controlled by anti-pyretics and anti-inflammatory drugs such asaspirin, and dexamethazone.

When these symptoms persist, they were usually stopped by a transfusion of fresh bloodwhich provided additional complément for the immune reaction provoked by the 15 therapeutic vaccine and immunotherapy. The CD4+ and viral counts were repeated every3-4 months until the viral counts were below détection levels. Monitoring was continuedannually for 3 years. In a few cases, the sero-positive status of the patient converted toHIV sero-negative after varying periods.

The results of the treatment in Example 5 on five patients are set out in Table 1 below.

Patient No. 1 2 3 4 5 CD4 before treatment (cells/ml) 342 210 214 350 846 CD4 after treatment (cells/ml) 424 443 587 1537 910 Viral load 23200 31460 42300 17300 207,743 29 012855 before treatment Viral load aftertreatment 505 4005 3218 Less than 50 Less than 50 Serology + + + -ve -ve Time (months) 5 4 7 3 4

The time refers to the number of months after the treatment before the CD4 and viral load were measured again. Viral particles could not be counted below 50. 5 A sixth patient seen and treated in October 1992 according to Example 5, and for whomCD4+ and viral loads were not measured, reported sero-negative 3 years later. The séro-négative results in case 4 and 5 above and in the 6th patient confirm that the HIV hadbeen completely eliminated from the patients. 10

The Applicant believes that the reason why HIV is not eliminated from thebody by the immune System is because it is an enveloped virus with the envelope taken from the CD4+ membranes as the mature virus leaves the infected cell. A full discussion ofthis hypothesis was published by the Applicant in Medical Hypothèses (1994) 42, 81-88 15 and Medical Hypothèses (1997) 48, 517-521. Because the envelope of HIV is derivedfrom the CD4+ membrane, the immune System perceives the virus to be “partly self. Aneffective immune response, which ignores the presence of the envelope, and kills the viruswith the envelope, would also kill ail the CD4+ cells from which the viral envelope isderived. The HIV has effectively taken a "hostage" in the form of the lipid envelope with 20 which it "blackmails" the host immune System into an ineffective response.

An effective préventive vaccine is a prophylactic immunotherapy and must meet three requirements. These are, firstly, that the vaccine must provoke an immune 30 response that kills the virus, secondly, that the immune System of the person must becompetent and thirdly that there should be no pre-existing antibodies to the virus in theperson to be vaccinated. To be affective, a therapeutic vaccine used as immunotherapy totreat HIV infection after it has occurred must also produce a prophylactic immunotherapy 5 in the HIV infected person. In other words, the HIV vaccine should provoke an immuneresponse that kills the virus in the infected person, the person should hâve immunecompétence and antibodies to HIV should be absent from that part of the immune Systemof the infected person which is to be vaccinated. At the beginning of HIV infection, and fora considérable time thereafter, the person has a competent immune System with several 10 immune units in it. These deal successfully with different infections or antigens that invadethe body at any given time. Evidence of this is that infected agents which invade the bodyin the first few years after HIV infection are successfully controlled by the immune System.

If in vitro one of the immune units not committed to the HIV présent in the 15 peripheral blood leukocytes were washed free of HIV antibodies and infectious agent andcombined with an effective HIV vaccine, the immune unit would perceive the vaccine as a"non-self and foreign antigen because of the absence of the viral envelope. An effectiveimmune response appropriate to a "non-self foreign antigen would start in vitro. When themixture is re-injected into the infected person, the vaccinated immune cells and their 20 descendents should continue the process in vivo and eventually kill onlv the core of theHIV in the person. This should eventually eliminate the virus from the body provided thereis complément available, as would hâve happened in a vaccination of a normal uninfectedperson. An essential feature of the invention is that a part of a person’s immune System 012855

contained in his peripheral leukocytes can be vaccinated in vitro in a medium free of HIV antibodies and lipid-containing infectious agent before being re-injected into the person.

Claims (26)

Ο 1 285e 32 CLAIMS

1. A method of making a vaccine, the method including the steps ofextracting a biological fluid obtained from a person or animal infected with a lipid- 5 containing infectious organism with lipid-extracting solvent, the biological fluid containingthe lipid-containing infectious organism and the extraction producing an aqueous phase,which contains the infectious organism with the lipid substantially removed, and a lipid-containing organic phase, and isolating the aqueous phase. I0

2. A method as claimed in Claim 1, in which the biological fluid is selected fromwhole blood, plasma, pleural fluid, cerebrospinal fluid, culture fluid and localized body fluid.

3. A method as claimed in Claim 1 or Claim 2, in which the lipid-containing 15 infectious organism is selected from viruses. bacteria, protozoa, fungi and moulds of the type which contain lipid or lipid-like material in their cell walls.

4. A method as claimed in Claim 3, in which the infectious organism is an enveloped virus. 20

5. A method as claimed in Claim 4, in which the infectious organism is anenveloped virus selected from human immunodeficiency virus, Hepatitis B virus, HepatitisC virus, Epstein Barr virus, Herpes Simplex virus, Cytomégalovirus, human T-cell leukemia virus and cancer virus. 012855 33

6. A method as claimed in Claim 3, in which the infectious organism is abacterium selected from tuberculosis bacteria and leprosy bacteria.

7. A method as claimed in any one of the preceding daims, in which the5 extraction process is selected from mixing, swirling, vortexing and rotating.

8. A method as claimed in any one of the preceding daims which includes thefurther step of concentrating the aqueous phase to produce a concentrate containing theinfectious organism with the lipid substantially removed. 10

9. A method as claimed in any one of Clairns 1 to 7 inclusive, which includesthe further step of concentrating the aqueous phase by lyophilisation.

10. A method as claimed in any one of the preceding daims, which includes a15 step selected from diluting the biological fluid and diluting the aqueous extract in order to obtain antigens from approximately 100 - 200 viral particles per ml.

11. A method as claimed in any one of the preceding daims, in which the lipid-extracting solvent is chloroform and in which the ratio (volume : volume) of the chloroform 20 to the biological fluid used in the extraction step is between about 3 : 1 and 5:1.

12. A method as claimed in Claim 11, in which the ratio is about 3:1. 0128Β5 '-/Ί 34

13. A method of making a first therapeutic vaccine, the method including thesteps of extracting, with a Iipid-extracting solvent, a biological fluid obtained from a person oranimal infected with a lipid-containing infectious organism, the biological fluid containingthe lipid-containing infectious organism, and the extraction producing an aqueous phasewhich contains the infectious organism with the lipid substantially removed and a lipid-containing phase, separating the aqueous phase from the lipid-containing phase,isolating a leukocyte fraction from the blood of the person or animal, the isolation I0 being conducted so that the leukocyte fraction contains substantially no plasma, free lipid-containing infectious organism or free antibodies to the lipid-containing infectiousorganism, and combining at least some of the aqueous phase with at least some of the leukocytefraction to produce the first therapeutic vaccine. 15

14. A method of making a second therapeutic vaccine, the method including thesteps of isolating and culturing an infectious organism from a biological fluid obtained from atleast one person or animal infected with a lipid-containing infectious organism to produce a 20 composition containing the cultured lipid-containing infectious organism, extracting, with a lipid-extracting" solvent, an aqueous solution of the lipid-containing infectious organism, the extraction producing an aqueous phase which contains theorganism with the lipid substantially removed and a lipid-containing phase, separating the aqueous phase from the lipid-containing phase, 35 012855 isolating a leukocyte fraction from the blood of another person or animal infectedwith the lipid-containing infectious organism, the isolation being conducted so that theleukocyte fraction contains substantially no plasma, free lipid-containing infectiousorganism or free antibodies to the lipid-containing infectious organism, and 5 combining at least some of the aqueous phase with at least some of the leukocyte fraction to produce the second therapeutic vaccine.

15. A method as claimed in Claim 13 or Claim 14, in which the lipid-extractingsolvent is selected from chlorinated hydrocarbons, hydrocarbons and ethers. 10

16. A method as claimed in Claim 15, in which the lipid-extracting solvent is chloroform.

17. A method as claimed in any one of Claims 13 to 16 inclusive, in which the15 leukocyte fraction is obtained by withdrawing a blood sample from the person or animal, separating the red blood cells from the plasma, separating the leukocytes from the plasmaand washing the leukocytes free of residual plasma and antibodies.

18. A vaccine prepared by a method which includes the steps of extracting a20 biological fluid obtained from a person or animal infected with a lipid-containing infectious organism with chloroform, the biological fluid containing the lipid-containing infectiousorganism, and the extraction producing an aqueous phase which contains the infectiousorganism with the lipid substantially removed and a lipid-containing phase, and isolating the aqueous phase. 36 0^285-

19. A first therapeutic vaccine prepared by a method which incfudes the steps of extracting, with a lipid-extracting solvent, a biological fluid obtained from a person oranimal infected with a lipid-containing infectious organism, the biological fluid containingthe lipid-containing infectious organism, and the extraction producing an aqueous phase 5 which contains the infectious organism with the lipid substantially removed and a lipid-containing phase, separating the aqueous phase from the lipid-containing phase, isolating a leukocyte fraction from the blood of the person or animal, the isolation being conducted so that the leukocyte fraction is substantially free of plasma, free lipid- 10 containing infectious organism and free antibodies to the lipid-containing infectiousorganism, and combining at least some of the aqueous phase with at least some of the leukocytefraction to produce the vaccine. 15

20. A second therapeutic vaccine prepared by a method which includes the steps of isolating and culturing an infectious organism from a biological fluid obtained from atleast one person or animal infected with a lipid-containing infectious organism to produce acomposition containing the cultured lipid-containing infectious organism, 20 extracting, with a lipid-extracting solvent, an aqueous solution of the lipid-containing infectious organism, the extraction Jproducing an aqueous phase which contains theorganism with the lipid substantially removed and a lipid-containing phase, separating the aqueous phase from the lipid-containing phase, 37 072855 isolating a leukocyte fraction from the blood of another person or animal infectedwith the lipid-containing infectious organism, the isolation being conducted so that theleukocyte fraction contains substantially no plasma, free lipid-containing infectiousorganism or free antibodies to the lipid-containing infectious organism, and 5 combining at least some of the aqueous phase with at least some of the leukocyte fraction to produce the second therapeutic vaccine.

21. A third therapeutic vaccine which includes leukocytes which hâve beenexposed in vitro to a vaccine produced by the substantial removal of the lipid portion of a 10 lipid-containing infectious organism, the exposure taking place substantially in the absenceof the free lipid-containing infectious organism and in the presence of a medium containingsérum which is also free of antibodies to the lipid containing infectious organism.

22. A prophylactic vaccine prepared by a method which includes the steps of 15 culturing a biological fluid obtained from a person or animal infected with a lipid- containing infectious organism, extracting the culture containing the lipid-containing organism with a lipid-extractingsolvent, the extraction producing an aqueous phase which contains the infectiousorganism with the lipid substantially removed and a lipid-containing phase, and 20 isolating the aqueous phase. J

23. A substance or composition for use in a method of treatment of an infectioncaused by a lipid-containing infectious organism the substance or composition comprising 012855 38 a vaccine, or a therapeutic vaccine prepared by a method as claimed in any one of daims 1-17 inclusive.

24. A substance or composition for use in the prophylaxis of an infection caused5 by a lipid-containing infectious organism the substance or composition comprising a prophylactic vaccine as claimed in claim 22.

25. Use of substance or composition in the manufacture of a médicament for usein the treatment of an infection caused by a lipid-containing infectious organism, the 10 substance or composition comprising a vaccine or a therapeutic vaccine as claimed in any one of daims 18 to 21 inclusive.

26. Use of substance or composition in the manufacture of a médicament for use in theprophylaxis of an infection caused by a lipid-containing infectious organism, the substance 15 or composition comprising a prophylactic vaccine as claimed claim 22. CHANGE IN VIRAL COUNT 3-4 WEEKS AFTER ONE DOSE OFAUTO-VACCINE Column 6 | PERCENTAGEFALL IN VIRAL LOAD | | 91% 75% 36% ί e £ m ©\ Ci so Os Ό 1 86.2% %9 66.93% 41.4% CM -5· ai S sé 4 W « ·({ « FALLNT AI URW O 0Λ O /-“S ÛD 3 Z—·\ « ô2 o ôi O © ôx O © Wl O 0 oc 0 0 ôû O © .OC O © un S Uï? P O fc- 1 w 1 10 (5.1 19 (5.1 87 (4.9 22 (4.3 >1 (4.0 6 (3.71 3 (3.61 9 (3.91 3 (3.11 1 (3.1 1( c Ό - Μη 83 en Γ" CM en CM so en r- V) 3 73 £ pri r~ CM ^Ti Qa 00 in O X Os O *3 R Μί cm CM 00 F·* V) TT 00 w O < » P* O w (6 © < g .—s z*-\ z«“\ z«—». Z—*' o O O O 0 P- £3 ε u ôû O 00 O □û logi ÛÛ O ôû O ÛÛ O ûû 0 ÛÛ O 0Û _o tT g^ U > g TC sO rc -»✓ 3 (5.2 (4.5 so en ✓ (3.01 (4.8 (3.61 (3.3 CM en S d ® “δ CM > (*- rr Γ- 00 Os r- 0 en c LT) un O Os TT os CM 0 SO 2 S H I—H 0 0 O un 00 un O P P u> O CM e-< c- TT 00 Tf un — U > <! U Tf CM so O (a. ω INT BCINE o cû ,Ο e\ £ Ûû ,ο O 00 -2 O ÛÛ Λ z—\ 0 00 0 logio) ÛÛ 0 z-"\ 0 § logio) 0 ÙÛ JO en 8; J > ml 2 (5.1 CM Un λ·** 0(5.0 (4.7 1« 00 c- (4.8 i s un en «Φ ô 5 s \0 sO SO 00 'T CM SO en T Γ*Ί 00 un CM Os un r- ’S- o tt en un O O r* H 3 40 66 20 Sü en so O\ en en CM U > - < U — CM nr sO CM M-) O O\ 0 Ot T un c- O O un O en r- so CM ’T 1—< 2® en Os O en en Os *T 00 00 1—« J É-< CM «—H «—M CM IA Λ A H b *-? b r* 00 en un O r» Γ" ’er Os TT 2 <5 O en O Os O 00 SO en un P* •A •^r un en <o en en c- un P g < 3 P 0 INI < ω s s Q a g 0 a U 2 X Ci X 00 C0 u Z X < 0 F-» CM m -*T un SO ç- 00 Os M O Ό cm S <ü

U- 1ΛO UjÎ *ë3 > 012855 ANNEX1B P. B. Μ / Male 51 Years 19/4/01- SerologyHIV 16/4/0] - VC 29,566 (4.5 logio) 17/4/01-T3 1181 - T4 877 - T8 344 - T4/T8 2.2 (CPC N° 13,502) lml plasma/leucocytes @ 40°C 24hrs chloroform ll/2hrs + 9ml mlN/S 15minscentrifuged. Supematant ’/ι<χ> = Vaccine 17/4/01 - 1ml vaccine s/c. 21/4/01 - lml vaccine s/c. 21/6/01 - 4 doses of vaccine given every 2 months. 21/8/01 21/10/01. 13/12/01 -T3 1496 - T4 1007 - T8 453 - T4/T8 2.4 - VC <50 (1.7 logio) Serology indeterminate 2/7/02 - T3 1492 - T4 1028 - T8 434 - T4/T8 2.4 - VC <50 (1.7 logio) - Serology indeterminate (CPC 22,443) 17/12/02 - T3 1664 - T4 1091 - T8 539 - T4/T8 2.0 - VC 65 (1.8 log,o) -> j 072855 AUTO-VACCINATION WITH WASHED PERIPHERAL LEUKOCYTES % fall inViral load r- σ\ 97% Γ- σ\ 97% 97% O\ 97% Viral loadAfter vaccine/Date <50 (4/12/03) <50 (4/12/03) <50 (30/10/03) <50 (4/12/03) <50 (4/12/03) <50 28/10/03 <50 (13/08/02) <50 (10/09/03) Viral loadbefore vaccine/Date 2314 (7/6/03) 786 (16/01/03) 546 (11/2/03) 20641 (20/8/03) 16877 (17/6/03) 2314 (7/8/03) 3778 (27/5/03) 272,902 (6/9/01) 71 (3/6/03) CD4 afterVaccine/Date 357 19/12/03) 567 (17/4/03) 625 (21/8/03) 694 (30/10/03) 963 (28/10/03) 233 (13/08/02) 790 (7/10/03) CD4 beforeVaccine/Date 384 (10/6/03) 1021 (16/1/03 713 (01/2/03) 694 225 (10/3/03) 785 (7/8/03) 771 (27/5/03) 200 (12/7/03) 761 (03/06/03) h' <ü CZ3 où < £ U. TT O $ m CS tu 'g >Λ T CS 32yrs ! 30yrs/F ce >> 1/1 CS 29yrs/F Folder No 474 K) TJ* j 999 1102 633 Γ- ’er S 03' 1667 □ Z CS ΓΛ xr Ό r- 00 012855 οχ r- Os 97% 98% 79% 97% tZ'. 92% 71% s® ΟΛ r- 00 78% <50 (2/09/03) 13668(27/11/03) 371 (5/12/03) 6197(21/11/03) 341 (18/11/03) 28646(27/11/03) 13668 6/11/03 1380 ! (9/10/03) 3886 (23/10/03) 1369 (25/9/03) 129 (14/11/02) 172652 (1/4/03) 30430 (12/12/02) 30486 (30/5/03) 15402 (10/7/03) 59602 (9/9/03) 172652 (1/4/03) 4882 (2/7/03) 30086 (22/10/02) 6355 (1/4/03) 912 (2/9/03) 300 (6/11/03) 499 (27/11/03) 836 (18/11/03) 758 (27/11/03) 496 (6/11/03) 1116 (9/10/03) 852 (27/11/03) 456 (25/9/03) 839 (14/11/02) 279 (23/7/03) 238 (8/11/03) 424 ! (27/5/03) 1 I 939 (10/7/03) 687 (24/7/03) 867 (20/3/03) 941 (15/7/03) 742 (22/10/2) 362 (11/6/03) 35yrs/F tu "ς/5 u. >, Os en rr % k O CS 27yrs On O en 38ys/F uï Sn O es § U. O TT 04 VT) 417 822 400 Tt 00 ι/Ί j ;-O Ξ ri 1036 1 387 989 1577 o O CS en TT ΜΊ O 00 012855 sp θ'· CM Os 94% 93% 93% r OC %08 88% 10,283 (28/10/03) 157 24/11/03 116 4/12/03 2,890 27/11/03 9,541 2/12/03 6,332 18/11/03 1,032 (17/10/02) 480 (2/9/03) 125,358 (27/3/03) 2,878 17/07/03 1731 25/03/03 41,930 10/12/02 94,228 : 9/1/03 32,941 27/02/03 4,143 (7/03/02) 435 (28/10/03) 980 27/11/03 C“l O es o CM 349 27/11/03 I 607 2/12/03 1 271 18/11/03 492 (5/7/02) 579 (14/10/02) 613 (18/9/03) 267 (20/3/03) 1016 17/07/03 693 25/03/03 241 10/12/02 en rr 5 O ~ 263 ! 27/02/03 455[ 7/03/02 33yrs/F 00 36/F 1 r- CM 29/M 19ZF 1049 1936 0070 / 0670 0803 0720 1747 2005 Ch 20 04 22 O·. CM 24 un Γ4 012855 s? s® £ sp è? Os TT Os r* un ΦΟ Os Os OS Os Os Os en en VO id m p <s ITi m o en O r* en en m O oo oo en S? *fr © § es o es m es »“* un Λ es Γ** Cb es ©O CS Λ r- 'δ Ch —1 OS «n 00 r~- TT n- CS xf •—H MO un Ch en O V) es un 3,086 4/3/03 1 [7,195 CS O en es CM Ch Λ en <S en Ç en es 1 28,00 23/7/02 O »"·Ή o CS en»—1 1—4 MO OO i—H 6/3/03 OO <Z) en f-H 7/1/03 en en en en en m O en ”3· O ττ O O O es es O ch δ r-4 Ç \o es O es ,—i CS un Ch en CS >r> » Os en on KO en m un CN OC so o> CM un 'O sO t—»4 »—4 O es es en O en es P O O o en en o en 1 CM es ^t* 00 r— CO r- r—1 ç Γ~- O CM TT O\ en n* © CM en τΤ r-« un ^4 en - CS tr, es m un SQ Os r- % £ £ % U-. >> U 5n >-. >1 k h4 Sn >. 1—4 «n -çr es O 00 es en «n un un en en en 00 Os O r* 'T es O O r*· es en 00 en en en en ^-4 <5 F— en O r* 00 Os O es CS es es es en en en

\O O\ 94% 92,041 13/10/03 2,888 22/1/04 11,133 4/11/03 3,112 5/2/04 35,801 20/2/03 55,665 18/2/03 1024 13/10/03 ’T O O ~ £ MO (S 446 28/10/03 343 5/2/04 898 20/02/03 381 18/2/03 35yrs/F I 00 CS 1010 851 33 34 T