EP0537247A1 - Variantes de virus non pathognes - Google Patents

Variantes de virus non pathognes

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Publication number
EP0537247A1
EP0537247A1 EP91912526A EP91912526A EP0537247A1 EP 0537247 A1 EP0537247 A1 EP 0537247A1 EP 91912526 A EP91912526 A EP 91912526A EP 91912526 A EP91912526 A EP 91912526A EP 0537247 A1 EP0537247 A1 EP 0537247A1
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Prior art keywords
hiv
nonpathogenic
virus
variant
strain
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EP0537247A4 (en
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George Pieczenik
Michael Scolaro
Roy Durham
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IMMUVAX
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IMMUVAX
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the field of this invention is the area of nonpathogenic variant viruses. More particular, the invention relates to spontaneously arising mutant viruses, specifically exemplified by those variants of Human Immunodeficiency Virus which are not pathogenic and which can be administered to Acquired Immune Deficiency Syndrome patients to generate an immune response and to ameliorate clinical condition as well as the course of the disease.
  • a virus is composed of genetic material, either DNA or RNA, surrounded by a protein coat. Replication of a virus requires the help of a living cell. At least the genetic material of the virus, and in some cases other components, enter a living cell which then accomplishes replication of the virus genetic material, synthesis of various viral proteins, including the proteins infected cell which, depending on the infecting virus, may be killed by the infection. The released viruses exist in an essentially vegetative state until such time as a virus encounters a susceptible cell, reinitiating the cycle.
  • RNA which, during infection becomes reverse transcribed into DNA, which integrates in the host cell chromosome. The virus becomes, in effect, a component of the host cell. Pathogenic effects occur if the virus carries a gene which is harmful to the host.
  • the pathogenicity appears to be a consequence of the cytotoxic effects of a virus infection on the particular class of cells which the virus is able to infect.
  • the paralysis which sometimes accompanies poliovirus infection, appears due to the ability of the virus to infect and kill peripheral nerve cells.
  • the virus is particularly cytotoxic to T-cells. It is the destruction of the T-cells in HIV-infected patients which leads to breakdown of the immune function in the condition known as AIDS .
  • HIV human immunodeficiency virus
  • AIDS acquired immune deficiency syndrome
  • the present invention serves to improve the current situation by providing a systematic methodology for isolating nonpathogenic variant virus strains and stabilizing them against reversion so as to provide safe, reliable vaccines.
  • a systematic theory is presented which predicts that attenuation generally results from a single base change in the absence of experimentally increased selection pressure. Consequently, attenuated strains can be found in any population of infected individuals, provided the population is large enough that the mutation will have occurred, given natural mutation frequency. Once a nonpathogenic variant strain is identified and its altered nucleotide sequence characterized, reversion to pathogenicity can be essentially prevented by introducing a second site, stabilizing mutation.
  • nonpathogenic variant HIV strains at a frequency of about 1 in 500 was predicted by the theory described herein, and at least two have now been found in a well-characterized HIV patient population.
  • the nonpathogenic variant HIV strains disclosed herein can be genetically modified to yield a live vaccine against HIV infection.
  • the nonpathogenic variant HIV strains described herein successfully compete against the pathogenic strain.
  • a patient can be treated by superinfection with a nonpathogenic variant strain.
  • Figure 1 displays a matrix which shows the decoding of the N-terminal sequence of gene V of bacteriophage f1, and the mirror-image patterns within the coding sequences.
  • An attenuated virus is a strain of virus that is capable of infecting host cells and inciting an immune response in a nonimmune infected host, but produces no symptoms or only mild symptoms in an infected host.
  • Sabin Type III poliovirus normally produces an asymptomatic infection in a non-immune human.
  • Cowpox virus normally produces only a single mild lesion at the site of vaccination. Both are examples of attenuated virus strains. Both induce an immune response in vaccinated individuals, which confers protective immunity against pathogenic strains.
  • pathogen is used to characterize an organism that causes disease in a non-immune animal or human. A virus is pathogenic if it causes disease.
  • pathogenicity and/or virulence can be assessed for different strains of a virus, depending on the severity of the clinical symptoms that accompany infection of a non-immune animal or human by the various strains. There will be a range of symptoms and variations in severity among infected individuals, animal or human. Assessment of pathogenicity and/or virulence is therefore a matter of clinical observation, skill and experience. While not strictly quantitative, an ordinarily skilled clinician can evaluate the pathogenicity and virulence of a virus strain using categories such as asymptomatic, mild, moderate, moderately severe, severe, and the like. Therefore, one ordinarily skilled in the art can identify and distinguish infections by a nonpathogenic virus strain from those by a pathogenic strain. For the purposes of this application, the terms pathogenicity and virulence are used interchangeably.
  • a nonpathogenic variant strain is a mutant of a pathogenic strain which differs from the latter in that it is nonpathogenic, or is markedly less pathogenic, than the pathogenic strain.
  • a nonpathogenic variant strain may be competitive with a pathogenic strain in an infected patient; if so, it can become the predominant strain isolated from an infected patient. This will occur especially in the case of viruses which are sufficiently able to evade the immune system so as to establish chronic infections.
  • a patient in whom a competitive nonpathogenic variant has arisen or has been introduced will eventually fail to yield detectable pathogenic virus, since the nonpathogenic variant will have outgrown the pathogenic strain.
  • Immunity is the state achieved in an individual animal or human such that infection by a pathogenic virus results in an asymptomatic infection or an infection of reduced severity. Immunity is usually the result of stimulating production of antibodies, usually against coat protein or other external components of the virus.
  • a protein of an attenuated virus In order to provide protective immunity, a protein of an attenuated virus must elicit antibodies that bind and inactivate pathogenic strains. The antibodies raised against the attenuated strain must react with the pathogenic strains essentially as well as with the attenuated strain. The antibody reactivity can be readily compared by known .in vitro tests, which will be predictive of in vivo effectiveness.
  • Genotypic selection is the term used to describe the process of selection at the nucleotide level for efficiency and stability, by operation of the biochemical processes of replication, transcription, reverse transcription, translation and expression. Selection at the genotypic level can lead to convergent solutions such that nonpathogenic mutants can be selected for in the absence of an artificial phenotypic selection. Genotypic selection is the means whereby nonpathogenic mutants arise in the absence of an artificial selective environment. The concept of genotypic selection has been described by Pieczenik, G. (1980) J. Mol. Biol.
  • genotypic selection is that those nucleotide changes which lead to more efficient expression, or to greater stability of the genome, will have an adaptive advantage independent of the environment. Therefore, random mutations that lead to such adaptive advantage will be selected for and will propagate through the population of organisms. Similarly, synonymous nucleotide changes do not fix randomly in the population without selection.
  • the first DNA sequences contained true palindromes, i.e.. Mutate E. Tatum or the phi X 174 G sequence ATG.TTT.CAG.ACT.TT: phi X 174 F T.GCT.GGT.CAG.ATT.GGT.CGT.
  • One of these 27 predicted sequences codes for the ribosome binding-site of f1 bacteriophage's single-stranded DNA binding protein gene V, i.e., fMET-ILE-LYS-VAL-GLU-ILE-LYS coded by AUG-AUU-AAA-GU/U-GAA-AUU-AA. This is the first time a nucleotide sequence was predicted to exist a priori and such predictions are strong evidence for direct genotypic selection. This argues against the Kimura-Jukes theories which would require that all synonymous codings for a particular protein have the possibility of existing equally frequently.
  • Fig. 1 is a matrix that shows the decoding for fl bacteriophage gene V protein's amino terminus and the mirror-image patterns contained within these codings. It shows only two palindromic codings for such a sequence, one of which contains internal-terminators.
  • Genotypic Selection is the selection imposed by the existence of GU base-pairs through mutation. Unlike AU and GC base-pairs, GU base-pairs were originally postulated by Crick as necessary for his wobble hypothesis. Later, Crick et al., supra.
  • sequence constraint that this type of model of translation imposes on the evolutionary history of mRNA, as a consequence of GU base-pairing, is a R,N,Y (purine, any nucleotide, pyrimidine) bias on coding sequences.
  • RNA interactions of the type described above i.e., intramolecular base-pairing in loop-stem structures and intermolecular base-pairing in tRNA-mRNA type interactions, or snRNA splicing interactions, rely on the structural fact that GU base-pairs occur and are acceptable base-pairs in these interactions.
  • the structural stability of a GU base-pair in a hairpin structure is significantly lower than that of a GC or an AU base-pair.
  • the energy and shape of a GU base-pair is such that it cannot stabilize short stretches of base-pairing. As five base-pairs are independently stable, a GU base-pair flanked by two 5 base-pair stretches would be stable.
  • this is a loop-stem structure, i.e., 22 nucleotide bases as the minimum interaction target, it is not detectably affected by a GC or AU to GU base-pair mutation.
  • This target size could possibly go down to 18 (nine on each side) and still be stable, but 14 nucleotides (seven on each side, i.e., 2 three nucleotide base-pairs surrounding a GU base-pair) is not at all stable.
  • any RNA interactions that are necessary to preserve contiguous structure must have a minimum of 20 base-pairs so as not to be disrupted by the mutation of a GC or an AU base-pair to a GU base-pair. While the stability and shape of GU base-pairs in RNA structures can give an estimate of the minimum size required for structural stability of RNA interactions involved in mutating to GU base-pairs from nonGU base-pairs, the relative frequency of GC and AU base-pairs in these structures and interactions can give us mutation direction.
  • GC and AU base-pairs occur in RNA interactions and RNA structures at a higher frequency than GU base-pairs. Therefore, there is an asymmetry in mutation direction of AU base-pairs and GC base-pairs to GU base-pairs. This appears at a higher frequency of C to U, as opposed to U to C transitions and also, a higher A to G transition frequency than G to A. This gives a direction to the evolution of RNA structures and interactions; that is, C to U and A to G, as follows:
  • the present invention is based on four fundamental insights: 1) that a loss of pathogenicity is not necessarily maladaptive for a virus, 2) that a single base change can confer loss of pathogenicity, 3) that such a base change can occur by genotypic selection, and 4) that such a change, once identified, can be stabilized by an introduced second site mutation that effectively presents reversion to pathogenicity.
  • these insights it is now possible to state that with a statistical frequency of about 1 in 500 virus-infected individuals, a strain of nonpathogenic virus arises. and by screening a population of virus-infected individuals, nonpathogenic strains are found which serve as nonpathogenic variant virus strains for vaccine production.
  • nonpathogenic variant virus isolatable from an infected individual is competitive with pathogenic virus, since the variant strain is able to multiply in sufficient numbers to be detectable in the individual, and since the individual is initially identified by lacking symptoms of pathogenic infection.
  • a population of about 1500 HIV-positive patients has been followed clinically for over 10 years.
  • a few individuals were identified who had been HIV-positive for over 10 years but had remained clinically asymptomatic, with normal T-cell counts. HIV could be grown from the blood samples of some of these patients.
  • the HIV strains grew more slowly than normal and did not appear to produce the cytopathic effects in culture characteristic of pathogenic HIV.
  • these strains can be further modified by specific second site mutations, genetically engineered to stabilize the attenuated strains against reversion to pathogenicity.
  • the course of infection can be altered by superinfecting patients with a competitive nonpathogenic variant strain.
  • genotypic selection it is necessary to explain how a single nucleotide change arising by genotypic selection can result in loss of pathogenicity.
  • the genomes of many viruses are single-stranded RNA or DNA. All viruses make one or more m-RNA's which are single-stranded. Genotypic selection can act to optimize stability of these single-stranded DNA's or RNA's.
  • single-stranded nucleic acids can form a secondary structure, using base pairing interactions between nearby sequences.
  • two segments that happen to be complementary with each other can form a "stem- loop" structure, the two segments forming a locally base-paired stem, while the segment between them forms the loop.
  • Such structures are frequently observed; their stability increases as the number of base pairs forming the stem is increased.
  • RNA molecules can interact with one another in a similar manner through the interaction of segments of locally complementary sequence. Since single stranded nucleic acids are flexible molecules under physiological conditions, stemloop structures can tolerate some unpaired bases on either side of the stem, since these can simply exist puckered out of the stem structure.
  • At least five base pairs are required to stabilize a stem-loop structure. Longer stems, having more base pairs, have greater stability. A mutation in the stem which interrupts base pairing can destabilize a short stem, but can be tolerated by a longer stem. Mutations that destabilize existing stem-loop structures are less adaptive.
  • One type of mutation that can be tolerated in a stem-loop structure is the type that results in forming a G:U pair, either by an A to G mutation in an A:U pair or by a C to U change in a G:C pair. G:U pairs can form a single hydrogen bond which contributes somewhat to stem-loop stability, provided the G:U pair is flanked by 4 or 5 A:U or G:C pairs.
  • a stem-loop structure of about 20 nucleotides total length will therefore be long enough to tolerate the appearance of a G:U pair by mutation without being selected against by genotypic selection (Nussinov et al. (1984) J. Theor. Biol. 106:245-259; Ibid, pp. 261-273).
  • the mutation may nevertheless have a phenotypic consequence, as by affecting pathogenicity.
  • a mutation of this sort can persist in a population and the resultant strain can be nonpathogenic. It can also revert, by a reversal of the original mutation to restore the pathogenic sequence.
  • Once the change is identified one skilled in the art can introduce a corresponding base change in the previously unchanged member of the pair, to maintain the stem-loop integrity but virtually eliminate the probability of reversion to pathogenicity. For example, if the initial (pathogenic) sequence was G:C and a C to U mutation resulted in a nonpathogenic strain, the resulting G:U pair could be further modified by a second site mutation of the G to an A, giving an A:U pair.
  • the second mutation maintains stem-loop stability together with the nonpathogenic phenotype. Reversion of the U to C would restore pathogenicity but would destabilize the stem-loop, resulting in genotypic selection against such a revertant, and failure to propagate in the population of viruses. Other corresponding mutations, such as deletions, can also prevent viable reversions.
  • the foregoing discussion has been couched in terms of the example of a stem-loop structure. Other more complex structures are known to exist as the result of localized base-pairing interactions in single-stranded nucleic acids. These also function as potential loci for genotypic selection. The majority of the base pairs in such regions of secondary structure are A:U and G:C pairs.
  • Pathogenicity itself is generally considered maladaptive for a parasite.
  • a parasite limits the extent to which it can replicate within the host. Therefore mutations that prolong host viability, i.e., reduce pathogenicity, tend to be favored in evolution.
  • the combination of genotypic and phenotypic selection pressures leads to the existence of nonpathogenic variant strains in replicating virus populations.
  • Genotypic selection operates without regard to phenotype. It will therefore be understood that genotypically selected variants will arise that have no apparent effect on phenotype. Also, the phenotype may be affected by a mutation but the new phenotype may not confer any increase in fitness, except for that conferred by genotypic selection itself. Therefore, the improvement in fitness of a competitive nonpathogenic variant does not depend on the phenotype of nonpathogenicity, although that phenotype may further contribute to fitness.
  • the frequency with which nonpathogenic strains can be found in infected individuals can be estimated from the minimum target size of about 20 nucleotides, as discussed supra. which can accept a G:U pair without disrupting secondary structure: one simply divides 20 by the total length of the viral genome in nucleotides.
  • the calculation is based on the assumptions that all sites on the virus are equally likely to mutate and that only mutations away from the original sequence in the 20 nucleotide target region associated with pathogenicity will disrupt whatever interaction has been lethal to the host cell.
  • nonpathogenic variants may result from other mutational events, including other base substitutions, insertions and deletions.
  • Deletion mutations offer the advantage that reversion is extremely rare.
  • the frequency of nonpathogenic variants of HIV can be calculated based on the same assumptions.
  • the target size of 20 nucleotides divided by the genomic size of 8213 for HIV yields .0022.
  • Approximately one patient out of 500 carries a nonpathogenic form of the virus according to the calculation. This analysis has been borne out by clinical data.
  • the present study includes a closely-followed group of about 1500 patients, many of whom have been monitored for more than 10 years. A few individuals from this group were identified as having tested HIV-positive for at least 8 years and who were clinically asymptomatic, i.e., had normal T-cell counts and otherwise normal blood chemistry. Nevertheless, the presence of HIV in the blood of these patients could be demonstrated by "Western" blot gels showing presence of a characteristic HIV- coded protein, and by polymerase chain reaction (PCR) to amplify and identify the presence of HIV DNA.
  • PCR polymerase chain reaction
  • the criteria for identifying individuals carrying a nonpathogenic HIV variant include: 1) positive reaction for HIV using Western blot or PCR assay, or both, 2) lack of clinical AIDS symptoms with relatively normal T4/T8 ratios and normal number and percent of T4 cells, 3) production of HIV virus in vitro, and 4) good health for a long time after infection.
  • a further criterion can be the identification of those individuals meeting the above criteria plus the evaluation of the partners of those individuals who also have stable or stabilized T4 cell counts. The last criterion is particularly applicable when an anal receptive partner of a long-term healthy, HIV-positive individual with clinical symptoms of AIDS shows stabilization of T4 cell counts and percentages and stabilization of his condition (See Example 3 for further discussion).
  • the growth in culture of HIV from three of the anomalously asymptomatic patients was tested.
  • the characteristics to be expected of an attenuated HIV strain are: 1) non-lethal to T-cells in culture; 2) independent of cells from a particular host (can grow on cells other than the donor's); 3) growth rate in culture slower than that of the pathogenic strain, possibly not observable without intervention of an inducer; 4) at least a one base (or base pair, if applicable) alteration in RNA sequence compared to wild-type pathogen.
  • Two of the three patients' virus isolates were found to grow in vitro, however growth was slower than wild-type HIV. In the third case, no growth in culture was observed, although the presence of HIV in the patient's blood was confirmed.
  • Identifying the base changes responsible for nonpathogenicity is accomplished by sequence analysis of the RNA of each isolate. HIV is known to be somewhat variable in sequence, even as between pathogenic isolates. On the average, the number of sequence variants which can be isolated from a single patient is about nine (Ratner et al. (DATE?) Nature 313:277; Fischer et al. Science (1986) 233:655).
  • the base change responsible for loss of pathogenicity can be identified by several strategies. For example, the number of possible candidates can be drastically reduced by eliminating those not located in stem-loop or other secondary structures and those within coding regions.
  • the region where mutations to nonpathogenicity occur can be identified unequivocally. Hemophiliac populations can be tested, since these patients are a well-studied, high risk group for HIV infection. Given the number of known infected individuals, there are an estimated 2000 asymptomatic carriers of nonpathogenic HIV in the U.S. alone. For other viruses, the search for asymptomatic carriers is preferably concentrated in areas and at times of an epidemic. The frequency of occurrence of such individuals is about 20 divided by the genome size of the virus in question, as described supra. per infected individual.
  • the search is preferably conducted among those most at risk, i.e., members of a patient's household, school or place of work.
  • the populations of sexual contacts and/or populations sharing needles for injected drug use can also be used.
  • the choice of at-risk group is based on knowledge in the art of the mode of transmission of the virus and well-understood epidemiological principles.
  • the frequency of finding asymptomatic carriers is less than 1/500, since any screening procedure is likely to include uninfected individuals. The actual frequency will depend in part on the state of knowledge in the art with respect to defining an at-risk population for each virus for which an attenuated strain is desired.
  • HANP virus By transforming appropriate host cells in culture with nucleic acid of the HANP strain, HANP virus can be generated and replicated to any desired extent.
  • Use of a HANP strain to produce immunity can be carried out either with live or inactivated virus.
  • Live virus can be administered by conventional means, and can provide protective immunity at an acceptable risk level.
  • the degree of immunity can be monitored, if desired, by measuring the recipient's antibody titre at intervals after the vaccine is administered.
  • an additional safety margin can be provided by administering HANP virus as an inactivated or killed virus.
  • the amounts of killed virus to be administered, sufficient to provide immunity, can be determined without undue experimentation by monitoring antibody titres obtained after a series of trials with test subjects.
  • killed virus Larger amounts of killed virus are needed to confer immunity compared to live nonpathogenic virus because the killed virus is unable to replicate in the host. Any technique known in the art can be employed for killing or inactivating a virus without destroying antigenicity of the coat protein or other surface proteins.
  • the combined safety factors for HANP virus (conservatively estimated at 1 double revertant per 10 11 to 10 12 virus) and killed virus (1 active virus per 10 9 to 10 10 inactivated particles) amount to an estimated 1 in 10 20 to 1 in 10 22 pathogens per administered virus.
  • An administered dose of approximately lg of virus is only about 10 11 - 10 12 particles, so only about 1 in 10 8 - 10 11 vaccine doses would contain a single pathogenic virus.
  • a particular advantage of a live HANP vaccine is provided by the fact that such a virus can replicate in host cells without destroying them, whereas the pathogenic strain destroys the host cells. Therefore, a HANP strain can replicate and ultimately stimulate a protective immune response even in a patient already infected with a pathogenic strain. This advantage can be observed only in the case of a viral disease that is slow to develop as compared with the time necessary to mount an immune response.
  • the advantage can be observed in early and middle infection stages, but will not be observed if the patient is too severely immunecompromised to form antibodies.
  • either the nonpathogenic variant or an HANP strain can compete with and replace the pathogenic virus in a host infected with pathogenic virus. Consequently, a patient who is experiencing symptoms of the disease can be superinfected with a nonpathogenic variant strain and thereby be protected against further ravages of the disease as the nonpathogenic variant becomes the predominant, or only strain in the patient. It will be understood that the outcome of such superinfection will be influenced by the extent of permanent damage suffered by the patient at the time of superinfection.
  • a group of about 1500 individuals testing HIV-positive has been tracked over a period of more than ten years. Although longitudinal studies of HIV-infected patients are sometimes difficult in practice to carry out, they are not impossible or unique. Certain groups of at-risk individuals, for example hemophiliacs, are relatively easy to study longitudinally; indeed several such groups are currently under study. Other at-risk groups, such as intravenous drug users and homosexuals, present a greater challenge to study over the long term due to higher mobility, and the difficulty of establishing mutual trust between patient and investigator. The present investigation involved a group of patients who were not preselected as to risk category, but rather were initially identified as HIV-positive and subsequently monitored for the appearance of clinical symptoms in order to commence early and appropriate treatment. While the thrust of the study was for therapeutic purposes, records of standard tests performed over the years were kept to develop a more comprehensive picture of the disease during the course of infection. Three individuals identified as testing positive for
  • HIV over a period of ten years or more without having clinical symptoms of immune deficiency were selected for the in vitro studies. These individuals displayed normal T-cell counts. While the T-cell count varies over a wide range of normal values, a reduction in T-cell count of more than 50% below an individual's baseline count (e.g., below 530) or a dramatic increase in the T8/T4 cell count ratio over the normal ratio is a strong indicator of onset of immune deficiency. From two of these individuals, nonpathogenic HIV variant viruses were cultured. One nonpathogenic HIV variant was used to inoculate a set of test patients, with mixed clinical results. In all test patients, however, there was seen increased production of antibodies specific for HIV components and increased cellular immunity functions.
  • the production of antibody(ies) specific to p15 is positively correlated with the presence of the putative competitive, nonpathogenic HIV variant of the present invention, and is a predictor, albeit not absolute, of improved clinical condition. It is understood in the art that apparent molecular weights may vary, according to the exact conditions used for polyacrylamide gel electrophoresis.
  • strains may have other phenotypes with respect to the antibodies generated. It will be understood by the skilled artisan that p15- (or p17 doublet-) specific antibody production is not an absolute predictor of clinical utility. However, the disclosed nonpathogenic variant virus from the donor (corresponding to IMM-1) has had a 40% success rate in the test patient population as determined by improved health. Improved health in those test patients is correlated with increased response in delayed-type hypersensitivity skin tests, improved sense of well-being and return to a normal life style.
  • IMM-1 Immuvax 1
  • IMM-29 and IMM-41 are from two other potential donor individuals (IMM29 and IMM-41, respectively) who have tested as HIV-positive and have remained free of ARC or AIDS symptoms for at least eight years.
  • the blood from IMM-1 (Patient #1) has yielded HIV variant virus which does not kill infected cells in cell culture, as described in Example 1. While in vitro attempts to culture nonpathogenic variant HIV from samples IMM-29 and IMM-41 have not yet been made, these blood cell samples are candidate sources of competitive nonpathogenic HIV variant strains. Example 1. HIV replication in vitro.
  • T-cell [HIV] preparations from each sample were used to inoculate in vitro cultures of T-cells from four pooled normal (HIV-negative) healthy donors. A total of 2 x 10 7 patient's cells were added to 2 x 10 7 pooled cells from buffy coats of four healthy donors (purchased from American Red Cross and stored at -80"C. until use) . Pooled T-cell preparations were used to eliminate variations in growth possibly caused by an unknown bias in individual virus isolates for individual T-cell types. The same preparation of pooled cells was used as in all HIV growth studies. All reagents and media were obtained from commercial sources. Abbreviations used herein are standard in the art. The culture medium contained 80%(v/v) RPMI 1640,
  • FCS Fetal Calf Serum
  • MLR mixed lymphocyte reaction
  • PHA phytohaemagglutinin
  • sPHA-T cultures contained 25%(v/v) of a supernatant of irradiated and 24h PHA-stimulated human T cells.
  • sPWM-T cultures contained 25%(v/v) of a supernatant of irradiated and 24h pokeweed mitogen (PWM) -stimulated human T cells.
  • HIVAG-1 kits Abbott Laboratories
  • HIV-1 p24 antigen quantitation panel Abbott Laboratories
  • Patient #9- a 38 year old male; date of infection, 1981. This patient began having minor lymphadenopathy early in 1987 and had an episode of seborrheic dermatitis in November, 1987. He is currently asymptomatic. The patient currently displays a positive PCR test for HIV DNA, and has antibodies to gp120, gp41 and p24 (+++) as shown by Western blot analysis. The test for presence of p24 antigen was negative. The patient's 1986 T4 lymphocyte count was 304, which was elevated somewhat to 410 (25%) in early 1987, and further increased to 650 (26%) currently. His T8 lymphocyte counts were 960 (58%) in early 1987, and 1500 (60%) currently. Total lymphocyte count increased from 1650 to 2500 over the same time period. Other parameters were normal. This patient has received AZT treatment since 1988.
  • virus protein was detectable in culture. Virus protein was detectable from culture of patients #1 and #9, after a lag period. For comparison, a blood sample from a patient infected with pathogenic virus (#11) yielded significant levels of virus protein as soon as 5 days after inoculation.
  • Example 3 Symptom control by superinfection with a nonpathogenic variant HIV.
  • Patient #15 is a homosexual male who has been asymptomatic for 10 years, despite a date of infection estimated at 1979 or early 1980.
  • Patient #16 has been infected since 1978.
  • Patients #15 and #16 reported that they have been lovers since 1978, with multiple contacts outside the relationship until 1984. Since 1984 to the present, their relationship with one another has been exclusive (monogamous). Their pre-1978 lovers are now dead.
  • Both men have continuously tested positive for antibodies to HIV proteins by Western blot and negative for p24 antigen. Both men have continuously tested positive for HIV DNA by the PCR test.
  • patient #15 has the masculine "inoculator" role, patient #16 is passive (anal receptive).
  • patient #16 developed symptoms of lymphadenopathy and severe fatigue.
  • the donor of the whole blood containing the putative nonpathogenic HIV variant was Patient #1 as described in
  • Example 1 The donor was further tested to insure that neither syphilis nor hepatitis or any other active infection would be transmitted to members of the test population.
  • test population had already declined to a state of severe immunosuppression and included individuals who either could not tolerate AZT or DDI or in whom these drugs had been ineffective, and had shown progressive T4 cell loss despite standard therapies (mean T4 cell count 66, range 5132). Each had marked depletion of circulating anti-HIV antibodies as well as severely impaired cell-mediated immunity (as measured by delayed-type hypersensitivity skin test reaction to eight test antigens).
  • the test population ranged in age from 26-44, and was composed of ten men and one woman. All patients provided informed written consent prior to entering the study.
  • test patient The immunological state of each test patient was determined prior to inoculation with donor blood to determine baseline measurements. Each test patient was then monitored weekly for 12-16 weeks after inoculation. Each evaluation included clinical history, physical examination and a laboratory profile including complete blood count (CBC), platelet counts, sedimentation rate, biochemistry and electrolyte profiles, Beta-2 microglobulin, p24 antigen, lymphocyte subset panels (including NK cell and CD8 subsets). Each test patient was also skin-tested (to measure DTH) at the onset of the study against eight antigens (Merieux CMI plus mumps) and this was repeated twelve to sixteen weeks after inoculation. Circulating antibody production against nine HIV-1 antigens was measured at baseline and at weekly intervals after inoculation. Tables 2-6 display the results of this testing.
  • CBC complete blood count
  • platelet counts sedimentation rate
  • biochemistry and electrolyte profiles Beta-2 microglobulin
  • p24 antigen lymphocyte subset panels
  • lymphocyte subset panels including NK cell and
  • test patients Six of the eleven test patients remained clinically stable or improved, developing no opportunistic disease (either as recurrence or as new onset), experiencing improved appetite, weight gain, decreased fatigue and increased stamina, allowing a return to normal exercise and daily routine. Each claimed an increasded sense of well-being.
  • nine of the test patients remain in the study.
  • Four patients have improved in their conditions as compared with their status at the start of the study.
  • IMM #24 had experienced severe migrating arthralgias of two years' duration. These symptoms disappeared within twelve weeks after inoculation, allowing him to discontinue his previous high dosages of non-steroidal antiinflammatory agents.
  • Patient #37 entered the study with a single Karposi's sarcoma (KS) lesion of the skin. During the post-inoculation monitoring this lesion was observed to wax and wane in size and shade, but remained essentially stable.
  • KS Karposi's sarcoma
  • Patient #25 had developed KS of the cervical lymph nodes, tonsil, hard palate and skin one month prior to inoculation. He experienced increased stamina and with gain during the course of the study, but KS lesion size and number continued to increase and as a result, at six weeks after inoculation, he was treated with Interferon-alpha-2b (50 MU I.V. daily for 14 days) with moderate regression in lesion size for skin and hard palate and a complete regression of the tonsilar lesion. Interferon was discontinued after 14 days because of extreme fatigue. Four weeks later (at twelve weeks post-inoculation), a second course of interferon therapy was instituted (25 MU I.V. three times per week for 14 days), resulting in moderate regression of all lesions. The patient continued to experience weight gain and increased stamina, as well as return of nocturnal erections; he was able to engage in all normal work activities. He has since regressed.
  • Interferon-alpha-2b 50 MU I.V.
  • Patient #30 experienced increased stamina and marked diminution in previously debilitating arthralgias and myalgias, but developed symptoms consistent with Sj ⁇ gren's Syndrome, a single small KS lesion of the gingival mucosa at fourteen weeks after inoculation and a small CMV lesion of the retina. He was treated only with high dose gamma globulin for two weeks, resulting in complete regression of his retinal lesion at follow-up.
  • Patient #23 a pre-terminal female patient, aged 29, regressed and died. She had suffered an episode of critical pancreatitis prior to the entry into the study group, following which she developed CMV retinopathy, and magnetic resonance imaging (MRI) results and central spinal fluid (CSF) protein consistent with HIV or CMV. She experienced an unusual and dramatic improvement in mental status three days after inoculation, which lasted for six weeks. She was subsequently hospitalized with severe anemia, erosive reflex esophagitis, bleeding ulcers and progressive encephalopathy. She died 14 weeks after inoculation.
  • CMV retinopathy and magnetic resonance imaging (MRI) results and central spinal fluid (CSF) protein consistent with HIV or CMV.
  • MRI magnetic resonance imaging
  • CSF central spinal fluid
  • Table 2 summarizes the clinical outcome results for the test population, along with the changes in non-core and core antibody levels. Increase in p15-specific antibody correlates with clinical improvement. A lack of a p55-specific antibody response correlates with regression or mixed clinical response. A marked increase in p24-specific antibody occurred in the two patients, one with minimal clinical improvement and one with stabilized condition, during the post-inoculation study period. Tables 2 and 3 chronicle the antibody responses to particular HIV proteins before inoculation and during a 16 week post inoculation period. Table 4 illustrates the rebound of the cellular immune response, as measured by delayed-type hypersensitivity skin testing sixteen weeks after inoculation.
  • Table 5 compares natural killer, cytotoxic killer, CD8 and CD8 suppressor cells in the AIDS patients inoculated with the nonpathogenic HIV with normal ranges. Over the 16 week monitoring period following inoculation, there was no significant effect on cell numbers, as reported in Table 5. It should be noted that finding a patient who is asymptomatic for AIDS and whose blood plates out productive HIV generating a strong (3+) antibody response for all HIV protein epitopes and especially core protein epitopes, does not per se prove that the strain is nonpathogenic.
  • Criteria for a nonpathogenic viral strain include 1) the viral strain does not kill T4 lymphocytes in vitro, 2) the viral strain propagates in culture relatively slowly, 3) it elicits an antibody response to HIV, particularly with antibodies specific for viral core proteins and 4) after inoculation into an immunocompromised AIDS patient, antibody levels increase, clinical well-being returns and cell-mediated immunity returns. These are all indications that the variant strain is either less virulent than the wild-type virus or is avirulent.
  • a further criterion, but not an absolute criterion, for choosing a potential donor is the ability of serum to bind to 15 and/or 17kDa doublet proteins on Western blots of HIV proteins. The clinical improvement in patients inoculated with such a nonpathogenic viral strain shows that it is also competitive with resident pathogenic viral strains.
  • p15 antibody increased six weeks after inoculation just as p24 antigen was decreasing, suggesting that one or more p15 peptide epitopes were neutralizing.
  • the p15 peptide epitope(s) could be an immunogen for inducing neutralizing antibodies to HIV, and thus, antibodies protective against AIDS.
  • Tables 4 and 7-8 show that the parameter most closely associated with improved clinical condition was the positive response in the skin test.
  • Another parameter which was positively, but not absolutely, correlated was increased levels of antibody specific for p15 and/or p17 doublet.
  • a nonpathogenic variant HIV virus described herein could be used as a live virus vaccine since all eleven test patients showed increased antibody production, at least initially, to both viral core and envelope proteins.
  • T4 cell numbers over twelve to sixteen weeks in the test population suggests that the expected increase in the number of T4 cells will take much longer than three months, and that T4 number and T4/T8 ratio do not correlate with changes in clinical status during a brief period of evaluation.
  • Patient #23 who died had the largest T4 increases (from 120 at baseline to 208 in week 3).
  • the decrease in T8 cells, in the absence of concomitant reduction in number of percentage of T4 cells, is considered a good sign in that it more closely resembles a normal T4/T8 ratio. This may be correlated with the reduced arthralgias seen in this study.
  • DTH delayed-type hypersensitivity
  • the competitiveness of an HIV variant as compared with the pathogenic HIV strain can be assessed using, for example, mixed and individual infections of cells in vitro. If variant and pathogenic strains are equally competitive, then a mixed infection with variant and pathogen at a 1:1 ratio should result in a 50% reduction in cell killing. If, however, the variant is more competitive than pathogen, then fewer cells should be productively infected by pathogen than with the variant, and there should be greater than a 50% reduction in cell killing. The converse should be true in cases where the pathogenic virus is more competitive than the nonpathogenic variant.
  • One explanation for the varied clinical results in this study may be that the healthiest patients responded best and the sickest patients responded the least. Molecular competition appears to have occurred in all patients by virtue of the renewed production of core p15-specific antibody. The inability to maintain core p15-specific antibody production may reflect the degree of immunologic deterioration at end stage disease.
  • nonpathogenic variant virus as either live or heat-killed vaccines will also vary in detail, depending on the virus and the animal or human to be immunized.
  • the techniques of superinfection can also be varied, based on knowledge of normal routes of infection for each type of virus, as those skilled in the art will readily appreciate.
  • method of treatment by superinfection with a nonpathogenic variant strain provides a low risk, inexpensive means to control the course of the disease in those who are afflicted. Both subjective observations and objective clinical data indicate that inoculation with nonpathogenic HIV variants leads to an improvement in the state of a symptomatic AIDS patient's immune function.
  • Nonpathogenic variant viruses such as those disclosed herein, will be useful in the prophylaxis and treatment of disease.
  • Specifically exemplified nonpathogenic variant HIV strains which are more competitive than the pathogenic strains of AIDS patients, will be useful in the treatment of AIDS.
  • Cytotoxic Killer 1.0-7.8 Suppressor: 4-27% (40-630)

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Abstract

On décrit des variantes de virus non pathogènes, qui peuvent être générées par génie génétique ou qui peuvent apparaître spontanément dans une population d'individus infectés. Comme démontré par des exemples spécifiques, la présente invention a trait à des variantes du VIH non pathogènes qui peuvent lutter contre des souches pathogènes; il s'ensuit qu'une variante non pathogène peut être utilisée pour améliorer l'état clinique de patients atteints du SIDA.
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AU699175B2 (en) * 1994-02-14 1998-11-26 Australian Red Cross Society (Nsw Division) Non-pathogenic strains of HIV-1
JPH10500281A (ja) * 1994-02-14 1998-01-13 ザ マクファーレーン バーネット センター フォー メディカル リサーチ リミテッド 非病原性ｈｉｖ−１種
US6015661A (en) * 1994-02-14 2000-01-18 The Macfarlane Burnet Centre For Medical Research Limited Methods for the detection of non-pathogenic HIV-1 strains containing deletions in the Nef coding region and U3 region of the LTR
TWI239847B (en) 1997-12-02 2005-09-21 Elan Pharm Inc N-terminal fragment of Abeta peptide and an adjuvant for preventing and treating amyloidogenic disease
US20080050367A1 (en) 1998-04-07 2008-02-28 Guriq Basi Humanized antibodies that recognize beta amyloid peptide
US7964192B1 (en) 1997-12-02 2011-06-21 Janssen Alzheimer Immunotherapy Prevention and treatment of amyloidgenic disease
MY139983A (en) 2002-03-12 2009-11-30 Janssen Alzheimer Immunotherap Humanized antibodies that recognize beta amyloid peptide
WO2006066089A1 (fr) 2004-12-15 2006-06-22 Neuralab Limited Anticorps du peptide beta-amyloide humanises utilises dans l'amelioration de la cognition
US8784810B2 (en) 2006-04-18 2014-07-22 Janssen Alzheimer Immunotherapy Treatment of amyloidogenic diseases
WO2009017467A1 (fr) 2007-07-27 2009-02-05 Elan Pharma International Limited Traitement de maladies amyloïdogéniques
US8003097B2 (en) 2007-04-18 2011-08-23 Janssen Alzheimer Immunotherapy Treatment of cerebral amyloid angiopathy
JO3076B1 (ar) 2007-10-17 2017-03-15 Janssen Alzheimer Immunotherap نظم العلاج المناعي المعتمد على حالة apoe
US9067981B1 (en) 2008-10-30 2015-06-30 Janssen Sciences Ireland Uc Hybrid amyloid-beta antibodies

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EP0242216A1 (fr) * 1986-04-16 1987-10-21 THE UNITED STATES OF AMERICA as represented by the Secretary United States Department of Commerce Clone non cytopathique de virus lymphotropique de T-cellule humaine type III
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