CA2085897A1 - Nonpathogenic variant virus - Google Patents

Abstract

Disclosure is made of nonpathogenic variant viruses, which may be generated through genetic engineering or which may arise spontaneously in a population of infected individuals. As specifically exemplified, the invention relates to nonpathogenic HIV variants which are competitive with pathogenic strains, and thus, nonpathogenic variant can be used to ameliorate the clinical condition of AIDS patients.

Description

WO91~19795 PCT/US91/04374 ~O ~ ~OG~N~C ~A~IA~T VI~8 This application is a Continua~ion-in-Part of U.S.
Pat~nt Application Seri~l No. 07/540,529, ~iled June l9, l990, and U.S. ~atent Application 5erial No. 07/625,958, filed Deqem~er l1, l990, both of which are incorporated by reference herein~

: TEC~N~C.a~ D OF ~ 9 ~, ;10 : The field of this in~ention is the area of onpatho~enic ~a~ian~;virU~esO Mor~ particularr the i~vention ~: ::
relates to spontaneously arising mu~ant viruses, speci~ically ~exempli-fied by thQse variants of Human Immunodeficiency Virus which are not pathogenic and which can be adminis~ered to Acquired Imfflune Deficiency Syndr~e patien~ ~o generate an immune response and;to~am~liorate clinical condition as well as~the course`of:the~diseas~.

CXGROUND OF T~

A significa~ number of inf~cti~us diseases in ~umans ~,:;20 and animals are caused by viruses. A ~irus is c~mposed of gene~ic material, eit~er DNA or ~N~, surround~d by a pro~ein coatO ~eplication of a virus re~uir~s the help of a li~ing ; cell. At l~ast ~he;gen~tic material of ~he virus, and in some cases o~her co~pon~nts, ~nter a livinq cell which then acco~plish~ replicati.on of ~he~ vir~s g@netic ~a~rial, synth@sis of variou~ ~ral proteins, inclu~ing the pro~eins infected cell which, depending on the in~cting viru5~ may b~

WO91/1979S P~T/US91/04374 killed by the infection. The released viruses exist in an essentially vegetative ~tate un~il such time as a virus encounters a susceptible cell, reinitiating the cycle.

A large variety of viruses infective to humans or animals is kIlownL Both pathogenic and nonpathogenic viruses exist, although those most studied are the pathogenic viruses.
Among the pathogenic viruse~, strains of varying degrees of virulence are commonly observedO The ~echanisms by which viruse~ exert their pathogenic eff~cts are not ~ell understood.
For one group of viruses, the retroviruses, their manner of replication appears to be responsible for their pathogenicity.
The genetic ~aterial of retroviruse~ is RN~ which, during infection becomes reverse tran~cribed into DNA, which integra~es in the ho~t cell chromoso~e. The virus becomes, in ~lS e~fect, a component of the host cell. Pathogenic effects occur if the virus carries a gene which is harm~ul to the host. For non~retroviruses, the pathogenicity appears to be a con~equence f the~ytotoxic effect-c of a YirUS inf~ction on the particular , ~ ~
; class of`cells which the virus is:able to infect. For ~xample, Z0 ~ the paralysis which sometimes accompani~s polio~irus infection, : appears du~ to:the a~ility of the viru5 to infect and kill pe~ripheral nerve ~el:Is. In the case of human imm~nodeficiency :~irus ~(HIV), the ~rirus i5 particularly ::ytotoxic to T cells.
It i~ the destruction~of the T-cells in Xnl-infected patients ~25~ which: leads to breakdown of th~ immurle function in the ~ondition Xnown as AIDS.

Strat~gies for dealing with virus disease have focused most intensely on im~unization. In the classic work of ~dward : : Jenner, immunity to sma~lpox was conf~rred on the subjects :: :
` expc)sed ~o cowpox, a relat~d Vinl5 ~hich, although pathogenic:
in s::at~le, functions as an attenuated~;train in humans. 9th~r exa~ s of vaccin~ b2lsed on a~t6!nua~ed ~l:rains are known, mos~ notab~y the Sabin ~acc:ine for polio. In ~he a~b~ nce of the for~uitous di~covery of a naturally occurrillg attenuated .

WO91/19795 P~T/U~91/04374 strain, attempts to generate an attenuated live virus have been based on repeated sexial passage of the virus through susceptible cells or tis~ues. That technique can only be employsd if susceptible cell~ or tissues are available. The approach ha also been limited by the suspicion among many researchers that mutation rate~ in vîruses a-r~ so high that, in the absence of a selection procedure, the likelihood of finding an atte~uated strain i~ extremely small. One rationale ~: for the production of attenuated strains is that serial passage somehow permitted a serie~ of mutations to accumulate, each : aontributing in~a minute way to loss of pathogenicity without co~promising viability.: Since such mutations would not be ; expected to accumulate~in ~he absence of some selective force which favors viability in the culture system, the latter is, in effect, a form of 6election pressure imposed by the investigator. For example, in the case of polio, the serial passage through thousands:of chick e~bryos could have resulted in~selecting a poli w irus strain that was better adapted to replication in c~iok e~ ryo through one or more mutations while ~2~0~ simultaneously becoming nonpathogenic to humans.

Recently,~ however, Ubertini et al. (1988) Vaccine 6~:48~1, ha~e~shown that the Sabin Type III strain of poliovirus, wi~ely-used~as an~oral vaccine, differs from the neurovirulent "Leon~' strain~only at~nucleotide position 472. The Leon strain ~25~ hos~a cytidine~(C)~where the Sabin Type III strain has uridine (U).~ The Sa~in Type~ III strain is known to revert to pathogenicity after~passage through a vaccinated individual, and has been implicated in the ~ast majority of rep~rted paralytic cases of poliomyeli~is in the U~S. and U~K. (Evans ~: et:: al. (l9 5) Nature 3l~, 548; WHO Consultive Group on Poliomyelitis Vaccines,~Geneva, 8-l0, Jul~, l98g). The single base change which result~ in 108s or gain of virulence is in a nun-transla~ed:region o~ the viral genome~ Ubertini et al.
~ ; (1988) al o reported the discovery of a stabilized variant of -~;35: Sabin Type:III which failed to revert to pathogenicity, :
.

W091/1~795 . PCT/U~91/~4374 apparently by sporting a second, compensating mutation such that a single-step reversion at nucleotide position 472 fails to rectore neurotoxicity. No mechanism sr rationale to explain these phenomen~ has been published. However, the seemingly ra~dom and unpredictable nature of the~e events suggests that systematic methodology for obtaining and stabilizing attenuated virus strains is currently unavailable. It is well established since the definitive work of Luri~ S. E. and Delbruck, M.
(1943) Genetics 28:491 that ~utation precedes selection. The attenuated virus is not created by the passage conditions, it is merely selected from a pre-existing pool of variants that have occurred by spontaneous or induced mutation. Heretofore, ~a fortuitous choice of passage conditions was the key to successfully isolati~g an attenuated virus.

~15 ~ The human immunodeficiency virus (HIV) is the causative agent of a fatal disease, the acquired immune deficiency syndrome:(AIDS). The epidemiology of th~ disease suggests that HI~ is usually transmit ed as a blood-borne dicease and also ~ by intimate ~exual~contact. Although certain groups in the ;~ 20:~ ~U~.S. are currently more severely affected by the disease then others, the entire population is at risk~ No cure is known and : no effective vaccine is currently available.

SUMMARY OF THE INVEN$ION
:
: The present invention ser~es to improve the current 25 : ~situation by providing~a systematic methodology for isolating nonp~hogenic variant virus strains a~d s~abilizing them against reversion so as to provide safe, reliable vaccines.
A systematic theory i presented which predicts that ~ attenuation generally results from a single base change in the :; 30 ab~ence sf experimentally increas~d selection pre~sure.
Consequently, attenuated strains can be found in any population of infected individuals, provided the population is large enough that ~he ~utation will have occurred, giv~n natural WO91/19795 PCT/US9l/04374 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. The existence of nonpathogenic variant H~V strains ~t a frequency of about : l in 500 was predicted ~y the theory described herein, and at : least two have now been found in a well-characterized HIV
patient population. Following ~.he teachings herein, the ~ nonpathogenic variant HIV strains disclosed herein can be genetically modi~ied to~ yield a live vaccine a~ainst HIV
infection. The nonpathogenic variant HIV strains described herein succe6sful1y:compete against the pathogenic strain. A
patient can be ~reated by superinfection with a nonpathogenic ~ariant strain.

;~15;~ BRIEF DESCRI~ION OF THE.FIGURE

Figure~1 displays a ~atrix which shows the dec~ding of the N-terminal sequence of gene V of bacteriophage fl, and the mirror image patterns within the~coding sequences. There are ::only:two possible~palindrom~ic codings:f~r such a sequence, one ~70~ of whicb contaLns internal-ter~inators.

DETAILED DES~R~PTION OF TNE_INVENTION

The ~followin~terms~ àre used herein as de~in~d: An attenuat~yi~g~ is~ a strain ~f virus that is capable of infeat~ing host:cells and~incitin~ an immune response in a non-~-~ 25: ~immune in~ected host, but produces no symptoms or only mild ;, . ~ ; ~
:; ~ymptoms in an~in~ected ho~t. For example, Sabin ~ype III
: poliovirus no ~ ally ~produces~an:a~y~pto~atic infection in a non-i~mune hu~an.~Co~pox virus normally produces only a single : mild lesion at the~si~e of vaccination. Both are examples of :
~-39 ~ a~tenuated viru8 strains. :~B~th induce an immune r~ponse in , ~: va~cinated individuals,: which confers protecti~e i~munity ~;: against pathogenio strains.

:~:

The term pathoaen is used to characterize an organism that causes disease in a non-immune animal or human. A virus is pathogenic if i~ causes disea~e There can be differing degrees of virulence associated with 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 ~10 `therefore a matter~ ~of clinical observation, skill and experience. While not strictly quantitative, an ordinarily skilled clinician~;can eYaluate the pathogenicity and virulence of a virus strain using categories such as asymptomatic, mild, moderate, moderately severe, severe, and the like. Therefore, ~15;~ one ordinarily s~illed in the art can identify and distinguish infection~ by a nonpathogenic virus strain ~rom those by a pathoyenic strain. For the purposes of this application, the terms~pathogenicity and~virulence are used interchangeably.

A non~athoaenic variant strain i~ a mutant sf a 0~ patho~enic strain which~differs from the latter in that it is nonpathogenic, or ~is markedly less pathogenic, than the pathogenic; strain.~ A~ nonpathogenic variant s~rain may be competitiue with a pathogenic strain in~ an infected patient;
if~so,~it can~become~the predominant~ strain isolated from an ~25~ ;infécted patient.;~This will occur especially in the case of viruses which are sufficiently abIe to evade the immune system ~ so as to establish ~hronic infections. ~ patient in wh~m a '~ ~ co~mpeti~ive nonpathogenic variant has aris~n or has been intr~duced wiIL eventually~fail~to yie~d detec~able pathogenic 30~ virus, since the nonpathogenic variant will have outgrown the pa ~ ogenic strain.

.~ ~
Immuni~y i~ the: ~tate a hieved in an individual ani~al or hu~an such that i~fection by a patho~nic viru~ result~ in WO91/1979~ PCT/US91/04374 an asymptomatic infection or an infection of reduced severity.
Immunity is usually the re-cult of stimulating production of antibodies, usually against coat protein or other external components of the virus. In order ~o provide protective i D unity, a protei~ of an attenuated virus must elicit antibodies that bind and inactivate pathogenic ~trains. The antibodies raised against the attenuated strain ~ust react with the pathogenic strains essentially as well as with the attenuated strain. The antibody reactivity can be readily ~l0 compared by known in vitro tests, which will be predictive of :~ ~a vivo effectivene~s. In the case of a genetic change o~tside : the struc~ural gene of the virus, the immunological reactions of the attenuated strain will be identical to pathogenic strains, both in vitro and in vivo.

Genotypic selection is the term used to deæcribe the - ~ ~process of selection at~the nucleotide level for efficiency and stability, by operation of the biochemical procs ses of replication, transrription, reverse transcription, ~ranslation and expression. 5election at the genot~pi~ level can lead to 20:~ ~ :convergent~solutions 5Uch that nonpathogenic mutants can be ::selected for in the absence of an a ~ ificial phenotypic sele~tion~ Gehotypic selection~ is the means whereby nonpathogenic :mutants ;arise in the absence ~f an artificial sel:ective~environment. ~The concept of genotypic selection has 25~ been~described~by~Pieczenik, G.~ (I9801 J. ~ol. Biol. 138:879-884; Pieczenik,;G. (~980) Proc. Nat. Acad. Sci. 77:3539-3543;
ànd~Crick, F.:H.~.~et:al.~ (1976~ in Or~iain ~ Life, Vol. 7, 389-~ ~ 397. Briefly,~it is proposed that there are features of : nucleotide se~uenc~s which confer greater stability, or greater ~;30 ~ efficiency of expression, than others, given that there are many :possible sequences for expressing and contr~lling ~: expression of the sa~e set o~ proteins. ~ust as i~ comput~r software or pIain Enylish, there ar~ ~any alternativQ ways to con~ey the same information, but so~e are ~ore e~icient, or ~35 ~econ~ical, than others. The basic principle of genotypic ~: :

WO~1/197g5 PCT/US91/0~374 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 populatio~ without selection.

~:: . :Evoluti~nary convergence occurs at the genotypic level, according to the theoretical discussions of Crick et al. (1976) ; origins of Life 7:389-397; Pieczenik (1980) Proc. Natl. Acad.
: Sci; USA 77:3539-3543; Pieczenik (1980~ J. Molec. Biol.
879-884. Selection acts directly on nucleic acid sequen¢e and structure via the biochemical machin~ry for replication, :transcription and translation~ Genotypic selection can result in constraints whic~ are: observed as nucleotide sequence patterns of various kinds. Patterns can be generated from a series of constraints.:If selection is sufficiently strong and ; if ~several selective~ mechanisms act on the same nucleotide 20 ~ sequence, the~ ~converg~nt ~and prQdictable solutions are :possible, ~given :that~ nucleotide sequence changes are independent of one another, and only chànges that are expressed to~give the whole organis~ or virus a reproductive advantage are~preserved.

25~ A~ example of this type of prediction was ~irst presented in Pieczenik~et:al. ~1972~ J. Mol. Biol. 9Q:191-214, which suggested that there were at l~ast two types of '~ :: nucleotide constraints, both a consequence of selection at the :
nucleotide level:. One was called: the internal-terminator ~30: ~ constraint, which held that there wa~ a selectiYe advantage in ~: having a terminator out-of-phas~in the beginning of a se~uence (Pieczenik et al. (1972) Bioch~. Biophys, ~ 152-165)~ The u~e and suppre~sion of internal-terminators for read through regulation of protein translation is now a common observation :

WO~}/19795 PCT/US91/04374 in many viral and bacterial systems, and is presently seen in HIV sequences.
",:
An interpretation of the second constraint, a true palindromic single-stranded constraint, held that "single-5strandedness" would confer an ad~antage to loop regions of ~- loop-stem structures and to the sides of double stranded structures. Random RNA Eequences can be folded into extensive : secondary structures given ~U r GC, and GU base-pairs.
.: Genotypic select$on for "single strandedness" would give these lo:~ randomly folded structures adaptive shapes.

, me fîrst DNA se~uences contained true palindromes, i.e., Mutate E.~ Tatum or the phi X 174G sequence ATG.TTT.CAG.ACT.TT;~ phi x 174F T.GCT.GGT ~nb~S.;9..:g~

The:independent existence of the internal-terminator 15~ ;constraint~(a ~syntactical constraint) and the palindromic single-strand ~:constraint (a physical constraint) implies independent~sele~tion~for these sequence constraints. However, if~ qenotyp~¢~ selection~ for ~internal-terminators and :pal~indromicity~act~ on :~the same nucleotide sequence, the

2~0~evolutionary opt:ions~ for ;such a seque~ce become extremely :li~itedO : :~

For~exampl~ ;for all sequenoes that are eighte~n nuclootides ~ ~long ~ and : contain~ ~: internal-terminators palindromic~lly~:arranged~, there are only 27 possi~le sequences, 2~:i.e~, URRURRURR/RRURRURRU, where R is A or Gr but KR ca~not be G~. There are 3 ter~inator sequences. Therefore, there are

3 x:3~=::27 ~e ~ ences that are each 9 nucleotides long.
: : Since~it is a mirror image~sequence, the oth~r 9 nucleotides ~ ,, `are: fixed and therefore there are only 27 such possib}e :3;0~sequ~nces. ::

~, :

~::

. WO91/197~S PCT/US91/04374 On the other hand, the total number of sequences that are 18 nucleotides long equals 4~ = 68,719,476,736. Imposing two such weak constraint~ on the same region of nucleotide sequence allows a reduction in evolutionary options for that sequence of Z7 . (41~) = 3.93 x lo~10. Therefore, proposing ¢oncerted genotypic selection allows for a ten billion-fold : ~ potential reduction in ~volutionary options for a nucleotide ~: sequence, independent of protein constraintsO
: :
one of these 27 predicted sequences codes for the ribosome binding-site of fl bacteriophaga's single-stranded DNA
binding protein gene V, i.e., fMET ILE-LYS-VAL-GLU-ILE-LYS
coded by AUG-A W -AAA-GU/U-GAA-AW -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.
15 ; : :This argues against~:the~Ximura-Jukes theories which would require that all synonymous codings for a particular protein have the possibility of existing egually frequently.

The average~tota~ nu~ber of synonymous codings for a protein is;61~20X, whére X is the amino acid length. Thus în 20~ a:5 amino~:acid~:sequence,~one should find 263.9 (3.05~3 or, by directly multiplying~;synonymous coding options (3x3x~x2x3x23, 288 ~possible codings.~ ;:Fig. l is~a~matrix that shows ~he decoding for fl bacteriophage~gene V protein's amino terminus and the mirror-image~patterns conta~ined within these ~odings.
~2~5~ It~shows:only two palindromic codings for such a ~equence, one of~which contains :internal-terminators.

This su~gests that neutra} amino acid substitutions and particularly that~ synonymQu~ codon replacements are evolutionarily selectable. : Therefore, an examination of 30~ : synon ~ ous codings~ ~for: identical proteins in differant organi~ms, ~r even~nucleotide:poly~orphisms, can decide whether ~ere i~ seleGtion at the ~ucleotide level which will lead to : c~vergent coding solutions.

Another example of Genotypic Selection is the selection imposed by the existence of GU base-pairs through mutation.
Unlike AU and GC base-pai~s, GU base-pair~ were originally p~s~ulated by ~rick as necessary for his wobble hypothesis.
Lat~r, Crick et al., su~ra, showed that the base-pairing interactions between tRNA and mRNA evolved ~rom a 5 base-pair interaction that employs the conserved U~ which is 5~ to the anti-codon in all tRNA molecules ~except ~Met tRNA) in potential GU base-pairs. This model all~ws ~r a flip of the ~10 anti-codo~ loop which reads the mRNA three ba e~ at a time (S' to 3') though there ~re five bases involved in each tRNA-mRNA
~:~ int~raction. A five base-pair ~NA interaction is relatively stable. The se~uence constraint that ~his type of model of translation impose on the evolutionary history of m~NA, a~ a 1~ consequence of GU ba~-pairing, is a R,N,Y (purine, any nu~leotide, pyrimidine) bias on coding seguen~es.

; ~ RNA interactions o~ the t ~ e dsscribed above, i.e., intramolecular:base-pairing in loop-ste~ struçtures and inter-mol~cular base-pairing:in tRNA-mR~A type interactions, or snRNA
:~20 ~ ~ splicing int~ractions,:rely on the structural fact that GU
: base-pair.~ occur and are~ acceptable base-pairs in these interactions. The structural stability of a GU ba~e-pair in a hairpin struc~ure is~ significantly lower than ~hat of a GC
or an AU base-pair.~ The en~rgy and:shape o~ a G~ ba~e-pair i5 5~ su:ch that:it cannot stabilize short stretches o~ ba~;pairing.
: Aæ~iv~ base-pairs:~are ind~pendently stzble, a GU base-pair flanked by two 5 base~pair stretches wo~ld ba stable. H~wever, a GU bas~-pair ~lank~d by two 3 base-pair regions would not be stable. Therefore, a 7 base pair region that mutat~ its ~:30 middl~ ba~e-pair to a GU base-pair ~ould go fxom stable to ~nstabl~, wherea~ an 11 ba~e-pair region h~ving mu~ated itæ
: middle ba~e~pair to GU would 8till be ~table.
~' ~
.

WO 91/197~5 PCI/US91~04374 This argument puts a minimum size of 11 bases on a side of a loop-stem structure for a mutation of a middle base-pair to a GU base-pair not to detectably change the stability of the structure . Because this is a loop-stem structure , i . e ., 2 2 nucleotide bases as the minimum interaction target, it is not detectably af fected 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 ., ~ three nucleotide base-pairs surrounding a GU
-~10 . base-pair) is not at all stable.

Therefore ~wo 4 to 5 GC or AU base-pairs surrounding :a GC base-pair or an AU base-pair that mutates to a GU base-:
pair is a stable, relatively non-detectable structural change in RNA-RNA interactions. This means a minimum target size of 18 to 22 nuc}eotides ~or 2Q nualeotides~ on average.

In summary, 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 th stability and shape ~ of GU base-pairs in RNA structures can give an estimate of the minimum size requixed for structural stability of RNA
interactions involved in mutating to GU base-pairs from non-: GU~base-pairs, the relative frequency of GC and AU base-pairs in these structures and in~eractions can give us mutation `~2~5 : direction.

GC and ~U base-pairs occur in RNA interac:tions and RNA
structures at a higher f requency than GU base-pairs .
Therefore, there is an asy~etry in mutation directiorl of ~U
base-pairs and GC base-pairs to GU base-pairs . This appears 3 0 at a higher f requency of C to U, as opposed to U to C
transitions and also, a hi~her A to G transition frequ~ncy than G to ~. Thi~3 giv~s a direc~ion to the evolution of RNA

WO 91/19795 PCr/US91/04374 structures and interactions; that is, `C to U and A to G, as 113ws:
G--C ~ G-U C ~U
A--U ~ G-U A~G

This ms~del of evolutionary direction ~nd stability of structure$ sug~ests that bacteriophage f 2 i~ convergently evolvillg into MS2 which i8 convergently evolving into R17. In addition, ~iers noted that durin~ the course of sequencing ~S2 coat protein, a c~A(s;L2~lo9) codon was ~;een to mutate (and fix in the pOpUlZltiCh oiE ~;equenced phage and molecules) to a CAG
synonymous c:odon -- again an A ~o G transition (Fiers, W.
'tChemical StruGture and Biological Ac~ivity of Bacteriophage MS2 RNA'I, in Zinder (ed.) ~1975) RNA Phaqes, Cold Spring Harbor PresQ, Cold Sprirl~ Harbor, N.Y. ~ p. 3~6. Thi~ cal~ be explained :15 as ~n ~ ~a~e-pair mutating to a GU bas~-pair~
: ~
This is the f irst time one can put a logic and : ~ direction to synonymol}s mutation~;, s;howing eviderlce of ~: con~ergenc~ at the n ucleotide level. Since t~e changes obs~rved were synonymou arld did not af~ect protein sequeTlc~, ~20 ~ this cannot~ be a consequence of proteirl functi on. It demongtrates ~ competltion and f ixa~ion between nucleotide s~ences and se~nce structures, independ~nt of sequence expr~ssion.

The pr2sent i~vention is based on four furldamental : inæ~ght~;: 1) that a 105s o:f pathogenicity is not n~cessarily maladaptive for a ~irus, 2) that a single base change can confer lo~s of pathos~enicity, 3) that such a base chang~ ~n oc:cur b~ g~otypit: sel~c:tion, and 4) th~t such a change, ~ace identified, can be ~tabilized by an introduc:ed ~;econd site 3û mu~atio~ that: ef~ectiv~ly presents re~er~;ion to pathogenicity.
~s~ a c:onsequenc:2 o~ these insights, it is now pos~ible tc7 stat~
that wit.h a ~;:t~ti~;tical fr~quenc:y of about 1 in 500 virus~
infected individuals, a ~:train of nonpathogenic virus arises, :

W091/19795 P~T/US91/04374 and by scr~ening a population of virus-infected individuals, nonpathogenic strains are found which serve as nonpathogenic variant virus strains for vaccine production. A corollary of this finding is that the 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 indlvidual is initially identified by lacking symptoms of pathogenic infection. As described in detail 10 ~ herein, a population of a~out 1500 ~IV-positive patients has : been followed clinically for over lO years. A few individuals were identified~who h~d been HIV-positive for over lO years but had remained olinically asymptomatic, with normal T-cell counts. HIV c~uld be grown from the blood samples of some of these patients. Howaver, in these cases, the HIV strains grew more slowly than ~normal and did not appear to produce the c~ opathic effects in culture characteristic of pathogenic HIV.
We :have identified a~t~:least ~wo nonpathogenic variant HIV
strains. ~:Foll~wing~the teachings herein, these ~trains can be 20~ further modi~ied:by;spec~ific second site~mutations, genetically engineered:: to stabilize the attenuated strains against ;reversion~to pathogenicity. ~ In addition, the course of infection can~:be àltered~ by superinfecting patients with a competitive nonpathogénic variant strain.

2:5 ~ In order~to~teach how and where to make the second site mutation, 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'~ which are :~ ~ single-stranded.~ Genotypic selection can act to optimize sta~ility of these single-stranded DNA's or RNA's.

It is well-known that ~ingle-strand~d n~cleic acids can form a secondary structure, using base pairing interaction~
; between nearby se~uences~ For example, two egments that W~91/197g5 PCT/US91/~ Y~

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 fre~uently ob~erved; their s~a~ility increases as the number of base pairs forming the stem i5 increased.
Two 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, stem-~10 loop structures can tolerate so~e unpaired bases on either side :of the stem, sinc~ 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, ~15~ : have greater stability. X mutstion in the stem which interrup~s base pairing can destabili~e a short ste~, but can be tolerated by a longer stem. Mutation~ that de~tabilize existing stem-loo~ structures are less adaptive. One type of ;mutation that can be tolerated in a stem-loo~ structure is the ~20~ typ~:that results in forming a;G:U pair, ~ither 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 ca~n form a:~sin~le hydrogen bond which contributes somèwhat to~stem-loop ~stability, provided the G:U pair is ;:fl;anked~by 4 or 5~A:U or G:C:pairs. A stem-loop structure of ~25~ about 20 nucleotides~total length will th~re~ore be lDng enough :` : to~t~lerate~the~appearance of a G:U pair by muta~ion without being selected against~by genotypic selection (Nussinov et al.
: ~(1984) J. Theor. Biol. ~Q~:245-259; Ibid. pp. 261-273~.

- The ~utation~ mày nevQrtheless have a phen~typîc ~30 : ~onsequence, as ~y a~ffecting pathogenici~y. A mutation of this sort can persist in;a population and th~ resultan~ strain can : be nonpathogenic~ ;It can al80 revert, by a re~ersal of the original ~uta~ion to restore the pathogen~c sequence. Once the change is identified, howe~er, one skilled in the art can ,~ .

WO91/19795 PCT~US91/04374 introduce a corresponding base change in the previously unchanged member of the pair, to maintain the stem-loop integrity but virtually elimin~te 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, givi~g 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.

;~15~ The forégoing 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-~tranded nucleic acids. These a~lso funation as potential loci for genotypic selection. The ~20~ ~mjjority of the~ base pairs in such regions of secondary structure are A:U and G:C~pairs. Because mutations to G:U can be tolerated~within~such structures, a bias will be observed in~ favor~ of A to G~and C to U mutations in replicating RNA
; virus~populations.~Where several cycles of replication have ~;~25 ~ occurred,~such that a~nonpathogenic variant has appeared, genotypic selection;~allows~that ~v~ariant to persist and even overgrow the original~pathogenic strain.

Pathogenicity ~ itself is gener~lly co~idered maladaptive~for~a parasite. By killing a host, or es~ential ~30 cells within a host, a parasite limits the extent to which it can replicate within the host. Therefore mutations that prolong host Yiability,~i.e., reduce pathogenicity, tend to be favored in evolution. The combin~tion of genot~pic and :
, WO91/1979~ PCT/US91/04374 phenotypic selection pressures leads to the existence of nonpathogenic variant strains in replicating virus populations.

Genot~pic selection operates without regard to phenotype. It will therefore be understood that genotypically select~d variants will arise that have no apparent e*fect on phenotype D ~lso, the phenotype may be af fected by a mutation but the n~w phenotype may not s::on~er any increa~3e in fitnes$, except for that conferred by genotypic selection i~self~
There~ore, the improvelaent in f itness of a competitive :10 nonpathogenic variant doe-~: not depend on the phenotypa of nonpathogenicity, although that phenotyF e may further s::ontribute t.o f itness .

The ~Erequency with which nonpathogenic ~traills call be îound in infected individuals can be ~stimated ~rom the minimum :15 target size o~ a~out 20 nuclectides, as discussed su~ra, which carl ac:cept~ a G:CT pair without disrupting Recondary st~uc:ture:
on~ simply divides 2 0 by the total length of ~h~ viral genome in nuc~eotides. ~For poliovirus, the calculated frequenay is 2Q/7431 = . on27, t~e fraction of an infected population ~20~ ~ ~ :carrying~ a~ T~onpathogenic s~rain of polio during an epidemic of th~ disease. Thè ~calculation is based on the assumptions that all sites on ~e v~irus are equally likely to mutate and hat only mutations away from the original se~uence in th~ 20 ::~ucleo~id~ target~region associated ~ith pathogenicity will ~25: ~di~rupt whatever interaction has b~en lethal to the ho~t cel}.
As has been h~n wi~h poli~virus, a C to U change can disrupt such a pathogenic interaction. Without further identifying or o~f~ring any hy~othesis as to what ~he natuæe of the interaction is,;it follows that any nuc}~otide change in the ~30 target region wQuld be e~fective to inhibit the interaction and remov~ the pathogenic:phenotype.

It is a1so understood that nonpathogenic variant~ may re ult ~ro~ other ~utational events, including other base . WO9~/19795 PC~/US91/0437 substitutions, insertions and deletions. Deletion mutations o~fer the advantage th~t reversion is extremely rare.

The frequency of nonpathogenic variants of HIV can be calculated based on the sa~e assumptions. The target size of 20 nucleotid2s divided by the genomic size of 8213 for HIV
yi~lds .0022~ Approximately one pati~nt out of 500 carries a nonpathogenic fo~m of the viru8 according to the calculation.
This analysis has been born~ out by clinical data.

~ Analysis of clinical data for HIV-in~ected patients has been complicated by the long and variable latent period between infection, (an event which cannot always be aecurately dated~
and onset of symp~oms, which are initially equivocal. The best indicator of in ection is the presence of virus, detected immunologically, or of anti~odi~s ~o the virus in a patien~'s : blood. Indicators of pathogenic effects, other than full-blo~n A~DS, include significantly lowered T-cell count, lowered ~:: percen age of T4 lymphocytes, and clinical picture of lymphadenopathy and~ other:symptom~ associated with onset of immune defici~ncyO Systématic studies are further confounded 20~ by i~sues~ of confidentiality, compliance and longitudinal tracking in ths ~ajor ~t-risk population sub-groups. The ; present study includes a :Glosely followed group of ~bout 1500 patien~s, many of whom have been monitoxed ~or more than lO
:- years. A few individuals fro~ this group were identifi~d as 25~ ~having~tested HIV-positive for at least B years and who wer~
clinichlly asymptomatie, i.e., had ~ormal T-eell eounts.and otherwi~e normal blood ehemi~try. N~verthel~s, the preeene~
of HIV in the blood of these patients could be demonstrated by "We tern'~ blot gels showing presenee of a eharaeteristîe ~IV~
~:~30~ coded prot0in, and ~y polymerase chain rea¢tion (P~R) to amp}if y and identify the presene~ of NIV DNA.

~: Th~ eriteria for identifying indiYiduals earrying a ;~ nonpathogenie HIV variant inelude: l) po~i~ive reaetion for WO91/19795 PCT~US91/04374 HIV using Western blot or PCR assay, or both, 2) lack of clinical AIDS symptoms with relatively norm~l T~/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 inf~ction. 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 ~10 long-term heal~hy, HIV-positi~e individual with clinical ~ symptoms of AIDS shows stabilization of T4 cell counts and :~ : percentages and stabilization o~ his condition (See Example 3 ;: for further discussion).
The growth in culture of HIV from three of the ~;15 anomalously asymp~omatic patients was tested. The characteristic~ ~o be expected of an attenuated HIV strain are:
non-lethal to T-cells in culture; 2) independent of cells from~ a particular host (can grow on cells other ~han the donor's)~; 3) growth rate in culture slower than that of the 20 ~ pathogenic strain, possibly not observable without intervention of an inducer; 4)~ at least a one base (or base pair, if applicable) ~alteration in RNA seguence compar~d to wild-type pathogen. Two of~the three~ patients' virus isolates were found~to~::grow ~a;vitro,: however:growth was slower than wild-~2:5~ iype~:;HIY. In~:the~ thi:rd: case, no growth in culture was observed, although the~pre~ance of HIV in the patient's blood was:~confirmed,~

; Identi~ying the base changes responsible for nonpathogenicity is accomplished by sequence analysis of the ~30:~ RNA of each isolaté.:~HIY is known to be somewhat Yariable in sequence, eYen as between pathogenic isolates. On the average, the number of ~equence::variants which ~an be isolated fro~ a si~gle pati~nt iB about nine (Ratner at al. (DA~E?) Nature 13:277: Pis~her et al. Science (lg86) ~ 655). ~he base ~35~ change r~sponsible for 108s of pathogenicity can be identi~ied :

.

by several s~rategies. F~r 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~ By sequencing several o~ the nonpathogenic strains isolated from one patient and comparing those nucleotide sequences~ the region where mutations to : nonpathogenicity occur can be identified unequivocally.
Hemophiliac populations can be tested, since thes~ patients are well-studied, high risk group for HIV infection. Given the ~10 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 viru~ in question, :
as described æu~ra, per infected individual. Since asymptomatic individuals are not readily identified as infected,~the search~is preferably conducted among those most at risk, i~e~, members of a patient's household, school or ~20 ~place of :work. For screening for avirulent HIV, t~le populations of ~sexual contact~ and/or population~ sharing needles ~or injected~drug use can also be used. The choice o~
at-risk group is~based on knowledge in the art of the mode of transmission of the virus and well-understood epidemiological ~ principles. The frequenay of finding asymptomatic carriers is : less than l/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 i~ desired.

Aft~r identifying the locus of a base change yielding nonpathogenicity, one skilled in the art can introduce an appropriate s~cond ~sit~ mutation by known techniques, for example by site-specific mutagenesi~ or by poly~era~e chai~
rea~tion (PCR). Smith gl985) Ann._Rev. Gen. l9:423, PCR

Protocols, (M. A. Innis et al. eds.) (l990) Academic Press.
The appropriate mutation is one which stabilizes the original mutation by eliminating ~he possibility of base-pairing as a result of reversion at the locus of the ~irst mutation, as taught, supra. Although a second s~te mutation of the type described could arise naturally, it would only be observed with a frequency comparable to that which generated the original mutant~ Such a variant would arise only about once in every 500 individuals bearing a nonpathogenic strain. Therefore, it would be unrealistic and unacceptable to await discovery of such a variant. Theréfore the use of the techniques ~f the ; present invention to generate such strains (~ermed a Highly Adapt~e Nonpathogenic (HANP) strain herein3 are of great benefit to providing a live virus vaccine. It would app~r that the non-reverting Sabin strain described by Ubertini et al. is an example of a HANP strain of poliovirus.

By transforming appropriate host c~lls in culture with ; nucleic acid of the HANP strain, HANP virus can be generated and replicated to any~desired extent.

~20 ~ ; Use~of a H~NP strain to produce immunity can be carried out e~ither~with~live or~inactivated virus. ~ive virus can be administered~by conventional means, and can provide protective immunity~at~an acceptable ris~ level. In many instances, the degree of immunity~can be monitored, if desired, by measuring ~25~ the~recipient'~s ant;ibody ~itre at intervals after the v~ccine is~administered.~In~certain instance~, for example, with HIV, an additional safety margin can ~e provided by administering i ~
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 eries of trials with ~est sub~ects. Larger amount~ oP killed ~irus are needed to confer immunity aompared to live nonpathog~nic 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 lconservatively estimated at 1 double 5revertant per 101l to 10l2 virus) and killed virus (1 active virus per 109 to 101 inactivated particles) a~ount to an estimated l in lO20 to 1 i~ l~22 pathogens per administered virus. An adminîstered dose of approxima~ely lg of virus is only about lo~ olS particles, so only about 1 in 108 _ lol~
:10vaccine doses would contain a single pathogenic virus. The . :
foregoing estimates are approximate only and do not take into account ~iological faotors which might bias the observed values :~ : in either direction~; however, they indica~e that a high degree of safety is inherent in the use of inactivated HANP vaccine.

15~ A partiaular advantage of a live HANP vaccine is provided by the~fact~that such a virus can replicate in host : cells without destroylng them, whereas:the pathogenic strain dsstroys the host~cells.~ Therefore, a HANP strain can replicate and ultimately stimulate a protective im~une response ~20~::even in~a patient already infected~with a pathogenic ~train.
: This advantage~an 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. In the case ~f HIV, the ad~antage can be observed in early and middle infection ~tages, 25~but will not::be observed~if the p~tient is too sev~rely i~mune-compromised to~form~antibodies. Further, în the case of HIV
: and~ other: viruses :which evade the host's imm~ne response, ~:either the~nonpathogenic variant or an HANP strain can compete with and replace the p~athogenic viru5 in a host infect~d with ~30 ~; pathogenic viru~. ~:Consequently, a patient who i~ experiencing symptoms of the~disease can be superinfected with a : nonpathogenic ~ariant~strain and thereby be protected against urther ra~age~:of the d~sea~ as:~he nonpathogenic variant becomes the predominant, or only ~train in the patient. It 3~5~will be understood that the out~ome of such superinfe~tion will be influenced by the extent of permanent damage suffered by the patient at the time of superinfection.

The following examples serve to fuxther document the techni ~ es and data under1ying ~he present i~ven~ion. Samp1ing of blood, and ana1y~es of blood ample~ and isolation of T
cells were carried out by standard clinical methods. The presenae of HIV in patient blood samp1e~ was v~rified by both immunoa~say and by identifying HIV DN~ by polymerase chain reaction (PCR). A11 methods of virus cu1ture were standard ~10 :methode, used in accordance wi~h published protoco1s.

A group of about 1500 individuals testing HIV-positive has ~een traaked over a period of more than ten year~.
A1though~ 1On~itudina1 studies of EIV-infected patients are ~ ometimes ~igficu~t in prac~ic~ to carry out, ~hey are not ;15 ~ impossible~or uniqu~ Cer~ain grou~ of at-ri~k individua1 , ; for~ exa~ple hem~philiacs, are relati~ely easy to study longitudina1ly; inde~d severa1 such groups ar~ currently under tudy~. Other at-risk groups, such a~ intra~enous drug users and~homosexualc, preoent a~reater cha11enge to study over the ~20 ~ lo~g t~rm due t~ higher mobility~ and the di~ficulty of establishing mutual trust between patien~ and investigator.
The~present in~es~igation invo1ved a group of patisnt~ who were not preselected as to risk category, but rather were initia11y identi~ied as ~ positive,and subs~quent1y monitored for the ~2~ appearanoe of:clinical symptoms in order:to commence early and appropria~e trea~m~nt. While the thrus~ of the tudy was for therapeu~ic purposes, records of ~tandard tests per~or~ed o~r ~the year~ were kep~ to develop a ~ore comprehensiYe pictur~ of the di~ease during the~course of infection.
~: ' ~3~0 Three individua1s identified as t~sting poæitive for IV vv~r a p~riod of ten years or more without ha~i~g c1inica1 sy~pto~ of immune defici~ncy were ~@lecked for the ~ ~it~o ~tudie~. The~e individua1s displayed normal T-cell counts.

WO91/J9795 PCT/US9lJ04374 While the T-cell count varies over a wide range of normal ~alues, 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. ~rom two of these individuals, nonpathogenic HI~ 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 increas~d production of 10 ; antibodies specific for HIY components and increased cellular : immunity functions.~ :The best positive correlation of improved clinical outcome :with:a parameter measured in this study was for the production~of antibody specific for ~IV ccre protein pl5.
, ~ ~15 ~ : ~ : Further s~udy indicates that the donor's serum and thesera~from the four te5st pat~ients with improved health æhows : strong binding to~all~HIV pro eins, and in particular to I5kDa (pl5) in t~e Ro~he~ assay and 17kDa doublet (pl7) in certain ~ other~Western blot systems. Generally sera from test patients -20 ~ ;who exhibit mixed~response or~a d cline in health do not bind to~ the pl5 or~ pl7 ::~doublet~ on: Western blots. Severely ; ` immuno~ompr~mised:HIV-positive individuals with AIDS no longer éxh:ibit~antibodies specific for pl5 and pl7 by the Western blot as6ay~ Thus~,~the~producti~on of antibody(ies) speci~ic to pl5 ~`2~5~ (cr to~ the:~pl7: doublet) is positively correlated with the presence of~the putati~e competitive, nonpathogenic HIV variant o*~ the~present i~Yenti~on, and is a predictor, albeit not absolute, of improved clinical condition. It is understood in `the art that apparent:molecular weight~ may vary, according ~o ~30~ the:~ exact conditions used :~or polyacrylamide gel electrophoresis. ~:~

Other clinically u~eful ~;trains snay have other phenotypes with r æpect to ~he antibodieæ generated. t will be understood by ~the skilled artisan that plS- (or pl7 :

doublet-) specific antibody production is not an absolute predictor of clinical utility. However, the disclosed nonpathogenic variant virus from the donor ~corresponding to IMM-l) has had a 40% success rate in the test patient population as determined by improved health. Improved health in tho~e test patients is corre~ated with increased response in delayed-type hypersensitivity skin tests, improved sense of well-being and return to a normal life style.

: : Epid~miological studies have identified fifty additional potential donors, including twenty from the 1978 San : Francisco Hepatitis B ~ohorts Study. From independent serological studies on potential donors, five potential donors whose ~era bind to the pl5 and/or pl7 doublet on Western blots of HIV protains w~re identified.

:
For reasons of confidentiality all patients de~cribed herein are identified by patient numbers.

Deposits have:been made with th~ American Type Culture Collection, 1230I:Par lawn Drive, Rockvil~e~ M.D. 20B52.
: Those deposits were~ of ~fresh frozen peripheral blood ~2~0:~ mononuclear~cèlls containing nonpathogenic HIV variants. The d posi~ identified~as I~muvax 1 (IMM-l) corresponds to Patient #l~, as described in Example 2. Deposits identified as IMM-29 and IM~-41 are from~two other potential donor individu~ls (IMM-29~a~nd IMM-41, respectively~ who have test~d as H~V-positiv~
~25 : : and have remained free of ARC or AIDS sympto~s for at least eight years. The blood from IMM-l (Patient #1~ has yielded HI~
varian~ virus which does not kill in~ected c~lls in cell culture, as described in ~xample l. While }a itro attempts - to~culture nonpathogenic variant HIV fr~m ~ample~ IMM-29 and IM~-41 ha~e not yet been made, these blood cell amples are candid~te source~ of competitive nonpathogenic ~IV variant ætrains.
~:
:

. W091/19795 PCT/US91/0437 Example l. HIV replication in vitro.

Blood samples of- patients l, 2 and 9 and control patients were obtained with con~ent. T-cell ~HIV~ preparations from each sample were used to inoculate ~ vitro cultures of T-cells from four pooled normal (HIV negative) healthy donors.
A total of 2 x 107 patient's cellæ wsre added to 2 x 107 pooled cells ~rom 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 10~ : possibly caused by:an unknown bias in individual virus isolates for individual~ T-cel} types. The same preparation of pooled : cells was used:as~in~all;HIV growth studies. All reagents and ~:~ media were obtained from commercial sources. Abbr-viatlons used herein are~standard in the art. I

5~ The culture~ med~um contained ~0%(v/v) RPMI 1640, 2~0%(v/v)~l99 Earle':s~sal~s~lQ%(v/v)`Fetal CaLf Serum ~FCS) inactivated ~by~ incubating :30 min ~:at 56C, 1%(w/v) human a:lbumin, :lOO IU/ml penicillin,~ lOO~g/ml streptomycin, 0.05 mg~ml~gen~amic~n.~Pooled cells were~suspended in RPMI 1640 at ~20~ ~ ml~and;incubated;with~l.25mM L-leucine methylester (Leu-0-Me)~:Sig~a Chemical ~Co.~ St. Louis, M0, for lO mln at room temperatur~ Cells~were~then wash~d:~three times in RPMI 1640 plus:2%(v} v)~FCS and~then:co-cultured with patient's:cells.

Three~types~of~cuIture were;tested, varied according ~:25~ to ~khe;~type of~;stimulated T~cel~l:s employed. :MLR ("mixed lymphocyte reactionn) cultures contained pooled cells that were ~: pre-stimulated with phytohaemagglutinin (PHA) for three days before they :were~: co-~ult~red~ with :patient's cells.
Additionally the;culture medium contained 50 U interleukin-2 30 :~ (}L-2). sPHA-T:~culturés~ oontained 25%(v/~) o~ a supernatant of irradiated and 24h PHA-stimulated human T ~ells. sPWM-T
cultures contained 25%(v/v) of a supernatant o~ irra~iated and 24h pokeweed ~itogen:~PWM)-stimulated human T cel~s. Every 5 WO 91/19795 PCr/US91/04374 days, lml culture medium plus PBL was replaced with 5 x lO6 pool cells in lml medium. For MLR cultures, pooled cells were pre~;timulated with PHA f or 3 days . For the other cultures pool cells were Leu-0-Me ~reated and the lml contained 25%
sPHA-T or sPWM-T, re~;p~c:tiYQly.

Grow~h o? HIV in culture was ~easured by the quantity of p24 antigen, usinç~ HIVAS;-l kit~i tAbbott ~aboratories~ for p24 detaction and the ~IV-l p24 antigen quantitation panel ~Abbott Laboratories) to generate a p24 standard curve.

: lO Example 2 .

Over many years of clinical exps~rience of a patient group nu~e~i~g more than 1500 individuals, a small number of patients p~r senting an: anomalous clinical picture were observed. The~;e patients had been infected with HIV for more 5~:; than eight years but rg~mained essen~ially asymptomatic and had mainl:ained nor~aal T-cell counts. From an initial scre~n of ap~aren~ly anomalous cases, the folls~wing were ~elected for fu~ther analysis:

Patient #l-, a 34 year old male; date of infection, ~20 ~ ~ l979. The patient has never sxperienced any sy~ptoms of immune ; deficiency. ~rrent tests for HIV DNA by PCR were positiYé.
~rrent Weotern blot~; ~tested positive (++~) ~or anti~odies ~o 0, gp41 and p24. ~ests for p24 antigen were negative. In ::~ 19847 th~ patient's T4: lymphocyte count sras 900: in A~S~ust, l989~ the coun~ was ll90 (35% of total T c~lls), arld T8 lymphocyte count was 1496 (44% ) . Currently, the T4 count is :~ lO85 (35%) and the q~8 count ~s 1364 (J,4%). OthQr indicators, including s~dimentation rate and lymphocy~e counts, were normal .
:: :
Patient #2- a: 41 year old ~ale; date of infection, 19790 The patien~ has had no clinical symptr~ms attributable W091~19795 PCT/US91/~4374 to immune deficiency except for a mild thrush episode in l985.
A PCR test for ~IV D~A 6 month~ ago (ll/89) was positive.
Western blot analysis for antibodies to gp l20, gp41 and p24, conducted at approxima~:ely the ~ame time, was positive, although higher levels were obsenr~d when the test was repeated thirteen ~onths later. ~ test for p24 antigen was positive (75pg/ml) in Septamber 1988, but negative thirteen months later . In lgB7, the pati~nt ' s T4 lymphocyte count was 57 6 (36%)D but had increased to 902 ~41~6) by Qctober, 1~89. His corresponding T8 lymphocy~e counts were 480 (30%) and ~80 (40%), respecti~relyD Other parameters were normal.
, :
~?atient #9- a 3~ year old mal~; date of inf~3ction, 1981. Thi~: patient b~gan having minor lymphadenopathy early in 1987 and had an epi~ode o~ seborrheic dermatitis in ~15~ No~rember, 19870 He i8: currently asymptomatic. ~he patient currently displays a positive PCR te~; for HIV DNA, æld has ~; ; antibodieæ to gpl20, gp41 and p24 ~+++) as shown by Western blot analy~;is,. The test for presence of p~4 antigen was negative. The patient ' s 1986 T4 lymphocyte c:ourlt wa~ 304, 20: ~ which was elevated ;omewhat to 410 (25%) in early 1987, and further increased to 650 (26%) currently. His T8 lymphocyte ~: ~ coun~s were 96~ (589~) :in early 1987, and 1500 ~60%) currently.
Total lymphocyte count ~iracr2ased from 1650 to 2500 oYer the ~ same time period. Other parameters w~re normal. This patient :~ 25 ~ ha~; receiv~d P~Z~ tre:atment since 1988.

Blood sam~les ~rom patients #l, #2 and #9 were cultured in vit~ro , as described in Example 1, to dete~mine whether ': virus could be cultured from thPSe patients and to test the g~owth characteristics. The results are shown in Table l.
Shown ~or compari~on is the data o~ patient #ll, who has a low (40-lO0) T4 count and lymphadenopathy symptoms.

WO9t/19795 . PCT/US91/04374 Table 1 p24 (pg/ml) ~ ç~ .Da~ 5 Day 10 ~Y_l~ Day ~Q
1 2,743 56,897 23,68822,~64 :: 2 -0- -o- -o- -0-~:~; 9 233 5,277 12,9531~,553 :~ 11 20,500 124,213 51,~2~100,~00 ~ In the cas~ of: patient #2, no virus protein was ~; detectable in culture. ~irus protein was detectable from culture~ of; patientB ~#1 and ;#g, after a lag period. For comp~rison, a :blood :s~ample ~rom a patient infected with ~ pathogenic vi:~ ~ (Y11)~ yielded ~significant levels of virus -~5~ protein as soon as 5 days after inocu1ation~

E~3~eI~ SYm~om ~contro1 by superil~fection _with a onpathQ~enic variant ~IV.

Patient~:#;15~is ~:a homosexual: male who has been ;asy~ptomatic for:~ lO:;yea~s, despite~ a date of infection ~20~ es~imated at~1979~or:;early 1980. Patient #16 has bee~ infected since~:1978O Patients~#15 and #16 reported ~hat they have been `lovers~:s:ince lg78,: with :multiple ~::::contacts ou~si~e tha re1atio~ship until~:~198~. ~Since~1984~to: the ~resent, their relationship with one another has been exclu~ive (monogamous).25 ~ ~ ~Their~pre-1978 lo~ers~are:~ow dead~ :Both men have con~inuously tested~positive for~ant~ibodies to HIV:proteins by Western blot : : and negati~e for p24~ antigen. Both~men haYe continuously tested positive for HIV DNA by:the PCR:t~æt~ Tn th~ir sexual ~ acti~ity, pat~nt #15:~s the;masculine "inocula~or~ role, :~30 patient #16 is passive ~anal receptive).

:: :

WO 91/19795 PCI`/IJS91/04374 In ~982 patient #16 developed symptoms of lymphadenopathy and ~evere fatigue. However, eight years later, his di~;ease has not progressed, he is stable, and shows clinical signs of improvement. His lates~ T4 helper cell count was 382 (20~) ~ somewhat below normal; however, the count has been stable for at least a year and a half. The latest ~4 lymphocyte count ~Eor patient #15 was 576 (24%), slightly b~low normal, but consis~ent with the pa~ient ' s good health.

The findings support the conclusion that patient ~ 5 : ~10 currently carries a nonpathogenic variant o~ ~IV, that he has transmitted the variant virus to patient #16 and that the superinfec:tion o~ pati~nt #16 with nonpathogenic variant HIV
has prev~nted the further deterioration o~ his inunune ~;yst~m and has resulted in his curxently impro~ing condition.. To the ~xtent that any pa~hogenic virus o~ palti~nt # l~ has been transmitted to patient # 15, the latter is not su~;ceptible because he is already infect~d with the more c~mpetitive, nonpathogenic variant:.

:: The patient identified in the present application as #16 was 20~ identified in ~U.S. ~ Paltsnt: Application 07/540,529, filed June 16, l990, as Patient #24, The identif ication num~er was ch~nged in: th~:present ~case to make it consistent with patient rec:ords: in the c:linical study.
, Example 4. linical Improvement ~ter Superinfection Wit}
` ~: 25 _ npa~thoqenic HIV Vari~t.

; El~ven severely immuno-csmpromised patient~ were each inoculated with a single injection of whole ~lood ~rom an HIV-~ positive don~r who ha been a~ympt~matic over a long period of :~ time (at l~ast ll years). Patients were m~nitored we~kly for 1~-16 we~s after inoculation. All ~I~ anti-vixal drugs w~xe discontinued prior to and during the study except for one : ~ :

patient who received brief treatment with interferon-a2b for Karposi's sarcoma. No adverse affects were observed after inoculation although one patient regressed and died 98 da~-s later. Five patients impro~ed clinically, one remained ~ta~e 5and the remaining four experienced a mixed response. In addition to monitoring the apparent state of health in the patients, estimates of capacity for cell-mediated immunity and the level of humoral respon~e ~o HIV was al~o monitored, thus providing objective measurement of effect of the inoculation lOwith the putative nonpathogenic HIV variant.

The donor of the whole blood containing the putative nonpathogenic ~IV variant was ~atie~t #l as described in Exa~pl~ l. The donor was further ~ested to insure that neither syphilis nor hepatiti~ or any other active infection would be ;:15~transmitted to members of the test population.

~embers o~ th~ kest population had already declined to a state of evere im~unosuppression and included individ~als : who either could not tolerate AZT or DDI or in whom these drugs ~: had b en ineffective, and had shown progressive T4 cell loss :~2`0:despite standard therapies (mean T~ cell count 66, range 5 132~ ach had marked depletion of cir~ulating anti-HIV
;antibodie~:~s well as s~verely lmpaired cell-mediat~d immunity (as measured by delayed-~ype hypersensitivity skin t~st :r~action to eight test antigens). The test population ranged ~25:: ` in age from 26-44, and was composed of ten men and one w~man.
:All patients provided informed written consent prior to entering the studyO

~:The immunological s~ate of each test patient was ::: d~ermined prior to inoculation wi~h donor blood to d~termine 3ûbas~line measurements. Each ~est patient was tlhen laoni.tor~d weekly for 12-16 w~eks aftsr inoculation. Eaa~ evaluation included cliniaal his~o~y, physical exa~inatlon and a labora~ory profil~ in~luding complete blood count ~C~C), .

platelet counts, sedimentation ra~e, 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-l antigens was measured at baseline and at weekly intervals : : after inoculation. Tables 2-6 display the results of this lO . testing.
:: :
Unrelated to clinical outcome, all laboratory values ~: including T4 c:ell counts, remained stsble, except for an increase in Beta-2-microglobulin in all eleven test patients.
This suggests that~a rebound in im~une system function can be seen before actual cell numbers increase.

; within the~ f~rst seven weeks after inoculation, a marked increase~:in ~total HIV non-core antibody production : (against gp160, gp120,~p64, p53 and p31) wa~ noted in lO of ll patients. ~evel~s ~of;~antibodies specific for combined core ~20~ proteins ~p55:, p2:4 and ~pl5) also increased markedly in all :eleven:test patients.~ Five of the lO patients developed high le:v-els of antibodies~against pl5.: Four~o~ those five patients remained strongly:positive~f Dr antibodies directed against pl5, and th:is aspect ~of the ~response::to ~inoculation correlated ~25 ; ~: positively~with marked clinical improvement.

~, :
Six of the eleven tes- patients remained clinically stable or improved, developing no opporkunistic disea~e ~either : as recurrence or as new onset), experiencing improYed appetite~
weight gain, decreased~fatigue and increased stamina, allowing 30 ~ ~ a return to normal~exercise and daily routine. Each claimed an in~reased:~ense of well-being. :At about elev~n ~onths a~ter - the initial inoculation with blood containing the nonpathogenio ~: variant HI~, 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.

For example, IMM #24 had experienced severe migrating arthralgias of two years' duration. These symptoms disappeared within twelve weeks after inoculation, allowin~ him to discontinue his previous high dosages of non-steroidal anti-:inflammatory agents.
:;
Patient #37 entered the study with a single Karposils sarcoma ~KS) lesion of the skin. During the post-inoculation ~:10~ : monitoring this~lesion was observed to wax and wane in size and shade, but remained essentially stable.

Four of the eleven patients di~played mixed responses, improving with re~ard~to some clinical pro~lems, in well-being and in stamina,~ but ~continuing to experience major disease.

5:~Patient~#31;had been previously~treated for XS tmuco-cutaneous)~ with Interferon-alpha-2b from 1987-1989 with complete::remission~:~for~:one~year. KS lesions did not recur after~interferon ~reatment was:discontinùed. For several years prior,~he::had~experienced:~mild peripheral neuropathy and had ~20~suffered multiple~ episodes of:severe acute pancreatitis ssociated with:hypertriglyceridemia. During the sixteen weeks ;of~:~post-inocula~ion~ monitoring, he ~experienced improved app~tite, stamina:~:and~regression of~ the neuropathy. ~e was hospital~ized;~twice,~ however, for:brief and mild episodes of ~25~pancréatitis. He was~abl~e to resume normal activities after ~each episode including extensive travel. At nearly one year into~the study, he is~leading:a normal:life.

: Patient #25 ~had developed K~ ~of the cervical lymph nodes, tonsil,~ hard ~palate and ~kin ona month prior to ~-~30~;inoculation. ~e~experienced increased stamina and with gain during tha co~rse~o~ the study, ~ut XS lesion ize and number W091/19795 . PCT/US91/04374 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 a~ter 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 wor~activities. Hé has since regressed.

Patient #30:experienced increased stamina and marked diminution in previously debilitating arthralgias and myalgias, ~15~ but developed symptoms~:oonsistent with~Sjogren's Syndromer 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 ~esion a_ ~:20~ follow-up. ~

: :Patient #:32~had previously suffered multiple episodes of:~ ~pneumothorax,~ Pneumocystis carinii pneumonia and cytom~galovirus~(~MV)~retinitis with ~he loss of viæion in one eye.~ During the~course:of ~he study, he experienced an episode ~2~5~ of:~sspticemia, ~assoc~ated wi~ :an infected central venous catheter,~:with;~a;~brief~;epiæode of weight~loss, impaired stamina :and depréssion. :He recovered from:the~e problems, but the ~MV
~:~ retinopathy continued to prsgress despite gancyclovir therapy.
Surprisingly, howevèr,~almost all CMV activity (90%) resolved ~30~ : l6 weeks after ino~ulation. During the period after the second inoculation, this:~patient experienced~ a regression in AIDS
symptomology, and:ultimately ended his life.

, :

W~9l/1~795 PCT/US91/04374 Patient #23, a pre-terminal female patientt aged 29, regressed and died. She had suffered an episode of critical pancreatitis prîor to the entry into the study group, following which she develope~ CMV retinopathy, and magnetic resonance S imaging (MRI) results and central spinal ~luid (CSF) protein aonsistent with HIV or ~MV. She experienced an unusual and dramatic improvement in mental statu thr~e day6 after inoculation, which lasted for 8iX weeks. She was subsequently hospitalized with severe ane~ia, erosive reflex esophagitis, b}eeding ulcers and progres~ive encephalopathy. She died 14 weeks aft~r inoculation~

:~ Table 2 summarizes the clinical outcome results for the test population, along with the changes in non-core and core antibody levels. Increase in pl5-sp~cific an~ibody correlates ~15 with clinical improv~ment. A lack of a p55-specific antibody response correlates with r~gression or ~ix~d clinical response.
A ~arked incr~ase in p~4-speciflc antibody occurred in the two ati~ntst one with min~mal clinical improvement and one with s~abilized cond!tion, during the post-inoculation study period.
2:~ ~ Tab~es 2 and 3 chronicle the antibody responses to particular HIV proteins be~ore 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 week~ ~fter inoculation.
:25: Tabl~ 5 compar~s~:natural killer, cytotoxic killar, CD8 and CD8 suppressor cell~ in the AIDS patients inocul~ed with the nonpathogenic :HIV with normal ranges. Over the 16 week monitoring period following inoculation, there was no signif~cant effe~t on cell numbers, as reported in Table 5.

30; It ~hould b~ noted that finding a patient who is a~y~ptomatic for AIDS~and whose blood plates out productive ~IV
~ generaking a ~rong (3~antibody r~spons~ for all HIV pro~ein : ~pitopes ~nd ~æpecially core protein epitope~, does not ~er se ~ prove that th~ strain is nonpathog~nic. Criteria for a nonpathogenic viral strain include l) the viral strain does not kill T4 lymphocytes n vitro, 2) the viral strain propagates in culture relatively slowly, 3) it elicits an antibody ~ response to H~V, particularly with antibodies specific for viral core proteins and 4) after inoculation into an immuno-compromised AIDS patient, antibody levels inarease, 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 lO, 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 improv¢ment in patients inoculated with such a :
, nonpathogenic viral strain shows that it is also competitive ~15 with resident pathogenic viral strains.

The findings~described above describing neurological improveme~t,~arthritic relief and partial restoration of immune function,may be explained by viral infection of more than one cell~ type: `i.e., macrophage, T cells, B cells and cells 20~ i m olYed ~in the~complement pathway or~there may be indirect effe~ts of NIV or~of~superinfecting nonpathogenic HIV variants.
The return of~B~cell-mediated immunity, particularly with the production of~antibodies specific to core epitopes (p55, p24 'and pl~5), suggests~that infection and colonization by the ~,~25~ inoculated,~virus occurred. This i~ supported by the fact that few~of~the~treated~patients had antib~dies to pl5 prior to inoculation~ and~almost~al,l generated~some anti-pl5 antibody ; during the evaluation period following inoculation. In one patient (#26~ pl5 antibody increased six weeks after 30 , 'inoculation just as p24 antigen was decreasing, suggesting that one or ~ore pl5 peptide epitopes were neutralizing. This also , suggests that the~pl5 peptide epitope(s~ (for exa~ple, as a synthetic peptide s~upled to a carri~r prot~in or as a recombinant protein) could be an immunogen ~or inducing neutralizing antibodies to HIV, and thusr antibodies protective , ~

WO91/19795 . PCT/VS9l/04374 against AIDS. In fact, Tabl~s 4 and 7-8 show that the parameter most closely associa~ed 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 pl5 and/or pl7 doublet. Furthermore, in principle, a nonpathogenic variant HIV virus described herein could be used as a live virus ~accine since all eleven test patients showed increased antibody production, at least initially, to both viral core and :10 ~ envelope proteins.

The correlation of pl5-specific and/or pl7-specific : :antibody with clinical well-being suggest strongly that the viral strains which induces the production of anti-15 ~nd/or ~nti-17 antibody ~to HIV are not clinically harmful and, , ~15~ : therefore, can be considered nonpathogenic in a clinical sense.
It~is nonpathogenic in:a cellular sense ( vitro) in that it does~ not kill T4:cells in culture.

Applicants believe that:this is the first instance where:the~return of the delayed-t~ype hypersensitivity skin test 20~ :~ reaction in ~IDS patients was observed; in this case it occurred ~after ~inoculation with blood containing a no~pathogenic HIV variant. This is~a strong indicator that cell-mediated im~unity is returning and that the cells responsible for~this~im~unity are both regen~rating and~or are ` 25~ :proeected~from destruction by wild-type HIV.

: The pattern of stability of 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 3~0 ~ do not correlate with chan~es in clinical s~atus during a brief p iod of evaluation.~In fact, Patient #23 (who died) had the largest T4 increase~ ~from I20 at baseline to 208 in week 3).
The decrease in~ T8 cells, in the absence of con~omitant WO91/19795 PCT/U~91/04374 -3~-reduction in number of percentage of T4 cells, is considered a yood sign in that it more closely resembles a normal T4/T8 ratio~ This may be correlated with the reduced arthralgias ssen in this study~

~bout three months after the initial injection of donor blood containing the nonpathogenic variant HIV, the ten surviving test group members received second injections of the IMM-l d~nor b~ood. At about five and el~ven months into the : : studyJ the delayed-type hypersensitivity (DTH) skin test reactions were again examined. Tables 7 and 8 present test : results tak~n at about five and eleven mon~hs into the study, respectively. In all patients, the DTH response was increased over that at the initiation of the study.

Entering the twelfth month into the study, four lS ~ ~patients:are~prove~d in their general hea~th: #33, #27, #40 and ~31. The four remaining patients exhibited a mixed :: :
:response or had regressed. At 13 14 week~ into the study, Patient #23:~died of CMY-related bleeding esophagitis. Patient #3~Z regressed, and committed suicide 44 weeks into ~he study.
;2Q`~ ;:Patient::#25 left~ the study after about six months when he relocated ~o another~city. Patient #:25 has regressed after ear1i~èr~improv ent,~and~was returning~to anti~iral therapy.

The reported general clinical well-being comes not only from ~he clinician':s e~alu~tion bu~ also from positive comments ~25~ by the test patients themselves. While this may reflect a placebo af~ct, ~objec~ive measures such as dec:r~aased analgesic do~#~ges, weight gain / increased ex~rcise tolerance, improved .

WOsl/1~795 P~T/US91~04374 u stamina and restoration of humoral and mediated cell immunity as measured by ~TH support the clinical impression o~ improved health.

~ When one consider~ the b~neficial effects of Karpas' passive immunization treatments of AIDS patients, one might consider the possibility that the positive results noted herein could be explaîned by trans f erring neutralizing antibodi~s~
Kaxpas ~t alO (19~8~Proc. Natl. Acad. Sci~ USA 85:9234-9237 ;showed that at least~500 ml of antibody-containing serum was 10 ~ needed for positive results, and that those positive results do DO~ gen~rally la~t re than ;one ~onth. Monthly tran6~usions would ~hus u~ually be needed to continu2 the ;positive "Rarpa~ eff-ct."

It is~ proposed~ herein that serial scre~ning ~or ~15~ olinical and cellula~nonpathogenicity a~s well as selectio~ for c~mpet~iti~eness~against pathogenic strains will result in the id~ntification of a~more highly~co~petitive pheno~ype. The ;patient~who beco~s~cl~inicaliy "wellH then carri~s the most competi~ive strain~of~;~a nonpathogenic variant. The recipient then becomes the new donor. ~ One can continu~ to moni~or the evolution o~ more competitive strains in this manner and s~lect for ~he least pathogenic ~and most ~competitive variant by observing and screening for the most rapid return of ~lini~al w~ being. The id~ntificat~on of a ~ore ~o~pet~ e strain involves a selection process while the identification of a nonpathogenic strain involves a screening process, The competitiveness of an ~IV variant as compared with ~he pathogenic HIV strain can ~e as essed using, for example9 mix d and individual~infections of cells ~a y~ro. If variant and pathogenic strains are equal~y competiti~e, then a mixed inection with variant and pathogen at a l:l ratio should : r~ult in a 50% reduction in cell ki~ling. If, h~wever, the variant i5 mor~ c~mpetitive than pathogen, then fewer cells should be productively infected by pathogen than with the variant, and th~re should ~e greater than a 50% reduction in cell killing. ~he con~erse should be t~e in case~ wh~re the pathogenic virus i~ ~ore competitiYe than the nonpathogenic variant.

~ One explanation for~the varied~clinical results in this ::
study may be that the healthiest patient~ rQspond~d best and the sicXest patients responded the least~ ~olecular competition appears:~to have occurred in all patients by virtue of tha r~newed production of core pl5 -specif ic 2~ntibody . The inability to maintain core pl5-~peci~ic antibody production may refle t the degree o~ immunologic deterioration at end stage disea~e.

In this ~tlldy, approxi~ately SO% of t;he test patients experienced a su~tained restoration o~ antibody production :

W~91/19795 PCT/~S91/04374 after one inocul~tion with a nonpathogenic ~IV variant. From a public health perspec~ive, this resul~ uggests a method for mimicking and accelerating the natural termi~ation of an epidemic. Therefore, one can speculate that the introduction of this strain into the population can reYerse the course of this epidemic, decreasing the financial and social burden, extending the effective lat~ncy period and giving the research com~unity more time to dev~lop completely nonpathogenic, non-reverting hi~hly competitive strains.

The present invention is applicable to a wide range of viruses of eitper humans or animals. The strategies for seeking nonpathogenic variants must be modified to ta~2 accoùnt o~ ~aeh virus' mode of infection a~d clinical cour~e. Such adaptations and modifications will be e ~ edients which tho~e :
15~ skilled in the~art will understand how and why to apply in each circumstance. The use of nonpathogenic variant YirUS as eith~r ~:
: liv~ or heat-killed ~vaccines will also vary in detai~, depending on the ~irus and the animal or human to be immunized, The techniques ~f superinfection can also be varied, based on ~;~2~0~ knowl~dge of no ~ al rou~e$ of infection for ach type of virusr as thos~ skilIed in the art will readily appreciate. In the case of ~IV, method of treatment ~y superinfection with a nonpathogeni¢ variant strain provides a low risk~ inexpensive means to control the course of the disease in those who are 25~ af~licted. Bcth subjec~ive obs2rva~ions and ob~ective clinical data indicate that inoculation with nonpathoge~ic EIV variant leads to an improvement in the sta~e of a symptomatic AIDS
patient's immune function.

~NDUSTRIAL APPLICABILI~.

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 patient , will be use~ul in the treatment of AIDS.

, .

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: ' WO 91~19795 PCl'/US91/~4374 Table 3. ~ral Antibc~y Response to AIDS Viral Antigens CP160GP120 P64 P55 P53GP41 P31 P24 P15T~>t~ll IMM 25 6/2~ 3 3 2 1 2 3 3 3 0 20 (7110-lc~ 7/10 3 3 1 0 5 ~ 2 22 2 0 14 ` 818 2 2 1 1 1 2 3 2 0 14 ~: 8/15 3 3 2 1 2 2 3 3 0 19 8/22 3 3 i 0.5 1 2 3 2 0.5 16 8~29 3 2 ~ 0.5 1 3 3 3 0.5 1 8 9/5 3 3 1: 0.5 0.5 3 3 3 0.5 17.5 9/12 ~ 3 3 I 0.5 0.5 ` 2 3 3 ~.5 16.5 3 3 1 0 0.5 2 3 3 0.5 16 9n6 3 2 1 0.5 1 3 3 3 0.5 17 lOt3 3 3 1 1 1 2 3 3 1 18 lOn2 3 1 0.5 0.5 0.5 3 3 3 0.5 15 Mcan: 17 1Cc#~7~10 3 ~ 2 ; ~ O.S 3_ OS O.S 0.5 12 ` 71~26 : 3 3 I 1 1 3 1 0.5 0 13.5 ~` ~ 8/1 3: 3 I ~1 1 3 1 0.5 0 13.5 8/8 3:: 3 ::: :1: 2 1 3 0.5 a.s 0 14 8/15 ~ 3 ~ 3 2 2 :1 3~ 1 0.5 0 IS.S
8~22; ` 3 3 1 1 1 3 0.5 0.5 0 13 8/29 3 ~ 3 1 ~ 2 0.5 3 1 1 0.5 15 9/26~ 3 : ~ ~3: ~ 2 0.5 3 0.5 0.5 0 13.5 1013:. :` 3~ :2 : 1~ 2 0.5 3 0.5 1 0.5 13.5 10~ 4 ~ 3 2 ~ ;` O.S 2 0.5 3 0.5 2 0 5 14 6/12 ~ 3 ~:: 3 ~ :2 : 0 2 :2 0.5 0.5 2 15 ; (?/~Icc#l) ~7//18 3 -- -~;3 - - ~ 3 - - 2 _2 0-55 1 13.5 n6 3 3 2 0 2 3 0.5 0.5 1 1~
~; 8/1 3 3 2 0 2 2 OS 0.5 1 14 ~/8 3 3 : : I O ~ 2 0 0 1 11 ` 8/15 3 3~:: I O 1 2 0.5 0,. 1 11.5 '' 8n2 3 : 3 ~ 2 O 2 2 05 0.5 1 14 `: 8129 3 2 2 0 2 3 0.5 1~ O1 2.5 /12 3 2: 3 0 3 3 0.5 0 ~ 14.5 9/19 : 3 ` 3 ~ 3 o 3 3 2 0.5(~.5 18 9/~6 3 3 ~ 3 0 3 3 1 0.5 0.5 17 A~can: IS
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WO 91/19795 . PCI/US91/~4374 Table 3. (Page 2) GP160 GP120 P64 P55 P53GP41 P31 P24Pl~Tl>~l ~7110-l~c #1) 7/10 3_ 3 1 1 2 23 2 0 0 IS

/1 3 3 2 2 2 3 3 0.5 0 18.5 : 8/~ 3 3 1 2 0 0 t48/lS 3 3 2 2 2 3 3 0.5 0 18.5 8/22 3 3 2 2 2 2 ~ 0.5 0 16.S
: 812~ 3 2 2 2 3 3 1 3 21 9119 3 2 2 2 1 3 3 0.5 3 195 9126 3 3 2 2 1 3 2 O.S 3 l9.S
1013 3 1 ~: 2 2 2 3 3 0.5 3 19.5 10/~4 3 3: 2 ~2 1 3 3 1 3 21 Mcan: I

2J -~sn9 ~ 3 3 :: 2 2 2 3 2 2 0 l9 ~1cc~ 3 3 ; ~;2 ~ 1 1 3 3 Z _ 018 5~ 7118 : 3 : 3 2 :I 1 3 813~ ~ 3 ~; 2~ ~ ;2~ 32 3 20 18 8/lS : ~ 3 3:; ~ 3 2 :2 ~ ~ 33 2 021 8/22: ~ 3 ~3 ~:~ 2~ 2 33 2 019 8129 ~ 33 ~33 ~; ;3 0~ 1 332 32 33 22o 9112: :: 3 3: ;~ 3~ ~: 0 1 ~ 32 2 320 9/1~ ~ ~ 3 3 ; 3 ~ 33 2 321 9/26 3 ~ 3 : :~:; 3 ~ 0.5 2~ 33 3 323.5 10/~ 3~: 3 ;~: 3 ~ 05 :1 ~ 3~ 1 218.S
10124~ 3~ ~: 3 ~ 3 ~0.5 2 ~ ~: 3 2 2 321S

?/19-Xc#l~7~726 ~ ~ 2 005 -~ ~ 05 - 2 ~
/1 3 ` 3 0.5 0 0.5 2 I 1 2l3 8/~ 3 3: ~ 1 0.5 I : 3 29.5 317 U~5 3 ~3 ~ ~ 0.5 0 0.5 3 2 1,. 3 l6 8/22 3 3 ~ 0.5 0 0.5 3 2 1 215 /29 3 3 ~ O.S 31 0.5' 0.512.S
.5 o o 3~.5 0,5' O IO.S
/12 : 3 2 0.5 O O 30.5 O.S0.5 10 9~9 3 3 : 0.5 o 0 20.5 0.5 95 : 9125 3 2 0.5 0 0 3O.~i0.50.5 10 0/3 3 2 : ~0.5: 0 0 : 3O.S ~-50.5 10 10/10 3 3 1 o 0.5 31 10.5 13 Mc~n: 12 ' WO 9~/19795 PCI/US91/04374 Table 3 . ( Page 3 ) GP160 GP120 P64 P55 PS3GP41 P31 P24 P15 Tot~l (7~19-.Scc#l) 7/9 3 3 _I 0 I 1 2 0 _1l : 7/26 3 3 3 0.5 3 3 3 1 0.5 20 B/l 3 3 3 0 3 3 3 0 0.5 18.5 8/8 3 3 : 3 3 3 3 1 0 5 1 9.5 8n2 : 3 3 2 0 2 3 3 1 0 17 sns 3 : 3 3 0 2 3 3 O.S I 18.5 9~5 3 3 3 0 3 3 3 0.5 1 19.5 9112 3 2 3 0 2 3 3 O.S I 17.S
sno 3 ~ 3 0 3 3 3 0.5 2 18.5 9/26 3 2 3 : 0 2 3 3 0.5 0.5 17 :: io/lo :3 2 ; ~ 3: Q5 2 3 3. O.S O.S 17.5 M~n: 18 : IMM~30 uts 3 3 ~ ~ : 3 ; 05 3 3 3 1 1 20.5 .scc#l)_ ?/10 ~ ` _3 3 3 o _ 3 0 18 7~ :3 : 3: ~ : 3 : 3 3 3 Q.S 0 185 811~ 3~ ~ 3 ~:3~ 0 3 3 3 1 QS 195 8/8 ~ :3~ : : 3 ~3 ~ 3 8/15 3 ~ 3 : ~3 ~:0 3 3 3 1 0.5 Ig.5 8/22 3 ~ 3 ~ ~ 3 ~) 3 ~ 3 3 1 05 I~.S
8n9: 3 3 : : 3 0 3 ~: 3 3 1 0.5 19.5 : 9/5 ~3~ 3: ~: ~ : 3 0 3 ~ 3 3~ 1 O.S 19.5 9112 ~3~ : 3~ ~ ~3 ~ 0 3 3 3 1 O.S 19.5 5119 ~ ~3~ :~: 2: ~ ~ 3: 02 ~ 3 3 0.5 0 ~6.5 sn6 ~ 3 ~: 3~ : 3 :~ ~) 3 : 3 3 O.S 0 ig.5 103 :~~3 ~ ~ 2 ~: 3 ~ 0 3 : ~ 3 3 0.5 0.5 18 on~ 3 ~ ~:2 ~ 3 0 3 ~: 3 3~ 0.5 0 17.5 M~n: ~9 33~ 6/4~ ::;3 :: : ~ 3 :~: 3 :1 3: ::: 3 3 3 2 24 7/~6 ~ 3 ~ 3~ ~ 2~ 2~ _ 23__ 2__ 2 OS 1~5 3 3 `~ ~2 ~ I ~ 2 : ~: 3 3: 2 1 20 8/8 : 3 3: 2 : 1 2 3 3 2 2 21 8It S ~ 3 3 ~ 3 ~ 2 3 3 3 2 2 2 8/2~: 3 3: ~ 2 1 3 ~ 3 3 2 1 21 8n9- 3 : 3 3 ~ 0 3: : 3 3 3 3 24 g/5 :3 ~ 3~ ~ 3 : o 3~ 3 3 3 ^ 3 24 9112 3 3 : ~ 3 0 : ~ 3 3 3 3 , 3~4 9/19 3 2 : 3 O.S 3 3 3 3 . 323.5 9/~6 3 2 3 05 3 3 3 3 323.5 1013 3 3 3 0.5 3 3 2! 3 323.5 10/24 3 ~ 2 ~ 3 05 3 2 2 3 321.5 Mcan 23 , , :: ~ :
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WO 91/19795 P~/~lS91/Oq374 Table 3 . ( Page 4 ) GP160 CP120 P64 rss P53 GP41 P31 P24 P15 To~l IMM 37 6n ( n Icc#l)__8/16 _ 3 _ 3~ 2-- 2 ~ '_0.5 ~65 8/8 3 3 3 0.5 2 2 10.5 0IS
8/l S 3 3 3 0 2 2 10.5 014.5 8n2 3 3 3 ~.5 2 2 1 1 0lS.S
o~ ~29 3 2 : 3 0 2 3 0.51 0.5 15 9/5 3 3 3 0.5 3 3 0.51 0.5 17.S
9/12 3 2 : 3 o.s 3 2 0.51 a.s 15.S
9jl9 3 3 3: 0.5 3 2 0.51 0.5 }S.S
: g~26 3 3 3 0.5 3 2 0.51 0516.5 10/3 3 2 3 0.5 2 3 0.51 05lS.S101~4 ; 3 2 3 ~ ~ O.S Mcan 16 22: 3 3 3 0 3 3 3 ~ 3 ~ 3 3 3 32 326 9/5 3 ~ 3 ~ i :0 3 3 32 323 g/12 :~3 3 : ~ 3 ~ 3 2 2 2 3 21 9/26 3 3: ~ ~: 3 0 : 3 ~ 3 3 2 2 n 10/3 : 3 3 ~ 3 ~ O 3: ; 3 2 1 2 21) 10/10 :: ~ : 3 3 ~ ~ 3 0-5 3 3 32 2 22.5 10/24 ~ ~: :3~ 3 ~ ~: 3 0.5 3 3 32 2 22.3 an: 22 ;: ~' :
::
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Claims (25)

1. A method for isolating a nonpathogenic variant virus strain of a pathogenic virus comprising screening a population of individuals infected or at risk of being infected by the virus to identify those who are virus-positive and symptom-free for a period longer than the normal time for appearance of symptoms of disease caused by the virus, culturing the virus from said symptom-free, virus-positive individuals, then screening the cultured virus for characteristics of nonpathogenic variant virus and cloning any virus having characteristics of nonpathogenic variant virus.

2. A method for isolating a nonpathogenic variant HIV
strain comprising screening an HIV-positive population of individuals to identify those who have been HIV-positive and symptom-free for at least 8 years, culturing HIV from the blood cells of said symptom-free HIV-positive individuals, testing the growth rate of individual HIV cultures and cloning slow-growing virus from said cultures whereby the nonpathogenic variant HIV strain is isolated.

3. A nonpathogenic variant HIV strain isolated by the method of claim 2.

4. The nonpathogenic variant HIV strain of claim 3, wherein said strain is cultured from the blood cells of IMM-1.

5. The nonpathogenic variant HIV strain of claim 3, wherein said strain is cultured from the blood cells of one of IMM-29 and IMM-41.

6. A live HIV virus vaccine comprising a nonpathogenic variant HIV strain isolated by the method of claim 2 and a physiologically acceptable diluent,

7. A method of immunizing a human against AIDS comprising administering to said human an immunizing amount of a nonpathogenic variant HIV strain isolated by the method of claim 2.

8. An inactivated HIV virus vaccine comprising an inactivated, nonpathogenic HIV strain isolated by the method of claim 2 and a physiologically acceptable diluent.

9. A method of treating an HIV-infected patient to stabilize and/or improve the patient's clinical condition comprising superinfecting the patient with an infecting amount of a competitive nonpathogenic variant HIV strain.

10. A method of treating an HIV-infected patient according to claim 9, wherein the competitive nonpathogenic HIV
strain is cultured from blood cells of an individual who has been both HIV-positive and symptom-free for at least 8 years.

11. The method of treating an HIV-infected patient according to claim 10, wherein said individual is IMM-1.

12. The method of treating an HIV-infected patient according to claim 10, wherein said individual is one of IMM-29 and IMM-41.

13. A method of treating an HIV-infected patient to elicit an immune response against HIV core proteins comprising superinfecting the patient with an infecting amount of a competitive nonpathogenic variant HIV strain.

14. The method according to claim 13 wherein said variant HIV strain is cultured from blood cells of IMM-1.

15. The method according to claim 13 wherein said variant HIV strain is cultured from blood cells from one of IMM-29 and IMM-41.

16. A method of treating an HIV-infected patient to increase cell-mediated immunity comprising superinfecting the patient with an infecting amount of a competitive nonpathogenic variant HIV strain.

17. The method of claim 16 wherein said variant HIV strain is cultured from blood cells of IMM-1.

18. The method of claim 16 wherein said variant HIV strain is cultured from blood cells from one of IMM-29 and IMM-41.

19. A method of making a highly adaptive nonpathogenic virus strain of a nonpathogenic variant virus comprising the steps of:

a) identifying a first locus in the nucleotide sequence of the nonpathogenic variant whereat a base change correlates with a change in phenotype from pathogenicity to nonpathogenicity;

b) identifying a corresponding second locus in the nucleotide sequence of the nonpathogenic variant where base-pairing between the first locus and second locus participates in forming secondary structure of DNA or RNA of the virus;

c) making a second site mutation at the second locus such that the resulting interaction between the first and second locus preserves the secondary structure whereby a reversion to a pathogenic sequence at the first locus fails to base pair at the second locus, and;

d) transforming a host cell with DNA or RNA of the virus knowing the second site mutation, thereby producing a highly adaptive nonpathogenic virus.

20. A highly adaptive nonpathogenic HIV made by the method of claim 19.

21. A method of treating an HIV-infected patient to stabilize the patient's clinical condition comprising superinfecting the patient with an infecting amount of a highly adaptive nonpathogenic HIV made by the method of claim 19.

22. A method of immunizing a human against AIDS comprising administering to said human an immunizing amount of a highly adaptive nonpathogenic HIV strain made by the method of claim 19.

23. A method of treating cytomegalovirus infections and lesions in an HIV-infected patient comprising superinfecting the patient with an infecting amount of a competitive nonpathogenic variant HIV strain.

24. The method of treating cytomegalovirus infections and lesions in an HIV-infected patient according to claim 23, wherein said variant HIV strain is cultured from blood cells of IMM-1.

25. The method of treating cytomegalovirus infections and lesions in an HIV-infected patient according to claim 23, wherein said variant HIV strain is cultured from blood cells of one of IMM-29 and IMM-41.