Note: Descriptions are shown in the official language in which they were submitted.
WO 9~/12880 PCr/US90/02~6~
7 ~
Generation of Hy~rid Genes_and Proteins by
,.-. - Virus-Mediated Recombination . . .
,
-
~_ r
n'~ r ~ nls L~J~ ' .r.C- _ 5~ ) Y~ L~ . ,;3.:~; C~ ' r,
ackqround ,. ~ ,c: s~
;:~ . . _.. .
eçombinationsinvolves;tho breakage and~cr~ss-3
` ..5 wise ~euniQn~of~nucleic acid~strands;within~homolo- o~
. -gou.s~sequences.~6This*phenomeno~.is a-critical r -- ~ o,
feature-of evolution-, particu~axly in:eukaryotes;-~
. genetic~exchange-through~recombinantion works~~ ;.
't ;~ i, ., '.'` ." constal~tly~tcs~ end~-randzrearrange genom-ic-sequences~ r
10 creating genetic-diversity?by producing different ~
combinatlons of genetic alleles.. 3 ' ,._ 1 . , ., m' - .` '. . -
Intermolecular recombination is~ the~term used .~to-describe recomb.tnation between~two:dif~erent :-
nucleic acid molecules,-for example, between two ~:
15 homolo~ous chromosomes durin~.meiosis, or between
different viral genomes present in the same infected
cell. Intermolecular recombination has been docu- '~
' mented in a number of different animal viruses, :
including RNA viruses such as picornaviruses (King
20 et al., 1982. Cel1 29:921-928; Tolskaya et al.,
1983. Viroloqy 124:121-132) and influenza virus
(Fields and winter. 198Z. Cell 28:303-313) as well
as DNA viruses.
WO9~/12880 ~ PCT/~S90/02060
The phenomenon of lntermolecular recombination
between viral genomes and exogenous DNA has been
exploited for the development of certain viruses,
including adenoviruses (Morin et al. 1987. Proc.
Natl. Acad. Sci. USA 84:4626-4630~, herpesviruses
(Lowe et al. 1987. Proc. Natl. Acad. Sci. USA
84:3896-3900) and poxviruses (Panicali and Paoletti.
. 1982. Proc. Natl. Acad. Sci. USA 79:4927-4931;
Paoletti and Panicali, U.S. Patent No~ 4,603,112) as
live vectors for the cloning and expression of
foreign DNA sequences. Vaccinia Yirus, an orthopox
virus, has been most extensively characterized and
developed as an infectious eukaryotic cloning vector
(Paoletti and Panicali, U.S. Patent No. 4,603,11Z).
: 15 Heterologous genes, including those encoding anti-
gens from a ~ariety of pat~ogens, have been èx- ~
r~ .' presse.~ in~ ~ is~ector system;LoIn all cases~~the ~.
. forei~n..-.gene product.-.expressed by~the:recombinant ~__'h 2
vaccinia virus.was similar~or~Identical to^the.gene ~e
-~ 20 product.. synthesized under.native conditions. In ~
some i.nstances,-vaccination of;laboratory animals~~ ~~
ith.recombinant~acci~ia-vlruses-~has~protected- 5_2-~
these animals~against_challenge:with the correlate~
pathoc;ens (Paoletti et.al~ 1984..Proc. Natl. Acad. ~
. 25 Sci.';USA 81:193-197; Rien~-et al.~lg84. Nature '-
312:163-166; Alizon et-al. 1984..Nature 312:757-760; --
Boyle et al. 1985. Gene 35:169-177; Vilma et al. -
1988. Science 242:1058-1061).... .s.~
In addition to intermolecular recombination
(recombination between different nucleic acid - -
molecules),..intramolecular recombination (recom-. . -
bination between homologus sequences present on a
WO9(~12~ 3 ~ ~ 3~ ~ P~/US90/02060
single nucleic acid molecule) also has been doc~-
: mented in a number of animal viruses, including
picornaviruses, influenza virus, herpesvirusei~, and
poxviruses. In vaccinia vlrus, for example, it has
been shown that tandemly duplicated identical
sequences are genetically unstable ~Panicali and
Paoletti. 1582. Proc. Natl. Acad. Sci. USA,
79:4927-4931; Ball. 1987. J. Virol. 61:1788-1795;
Spyropoulos et al. 1988. J. Virol.-62:1046-1054);
intramolecular recomhination.between these identical
sequences results.in a condensation o~ the-dupli~
cated~structure.to yield a single;copy o~:the
'' previouisly duplicated sequences. -Recombinant '''~
~;' vaccinia viruses.that'contain such tandem dupli~
~ 15 cations:can be stably maintained only=when a gene ~~- -~
encoding a selectable~marker (such as'the thymidinë'
- .- kinase'gene-,.o~.æ gene encoding-drug resistance~ s~
p~aced between th~duplicated 'sequences: c:The-~ 9~
' - intramolec~la~ recombinatioi~`event tha~'resùlts inS~ -
;. . ' - 20 condensation of~the duplicated:structure also'';' ;~
results:in excisio~:of the marker gene. -Thus, ~ ~
7,~ . . selection~fo~,th~:~function`of.the marXer gene'alsoiW
selects for th.e presence of the duplication~ ' ?-'-~
~-~,Althoug~.intramolecular recombination between
tandemly.arranged,~identiCal DNA sequences has been~ ':
documented,.:there has thus far been no examination `
of in vlvo int:ramolecular recombination, between ~''
~on-identical, but related (i.e.,.partially homolo-
gous) DNA sequences
Different but related genes have been docu~
'` mented in a variety of natural systems. One example
may be found among certain pathogens that exhibit
antigenic variation. For example, the occurrance of
WO90/l28~0 P~T~'S90/02060
2 ~
antigenic and genomic variation in human immuno-
deficiency virus (HIV), the causative agent of
AIDS, has been demonstrated by a number of
researchers (Starcich et al.-1986. Cell, 45:637-648;
Hahn et al_ 1986. Science, 232:1548-1553). These
' studies have revealed that changes are found -:~' --
primarily in the viral-envelope (env) gene, which
encodes the antigens against'which`a major component
of the antibody response is directed. .~~
10 ~ The antigenic diversity exhibited by HIV and'by `'
~: other pathogenic organisms presents.:obstacles to the
.' . -5~ . - formulatio~of vaccines:effective against:these ~- ~
pathogens,-as a vaccine.effective against one~
variant or subtypë may fail to protect against.--~;
15 related strains. sIn order.to de~elop successful -- :
. ... . vaccination protocols for these pathogens, it will
"~S.-,.~.bé necessa ~_.to elicit .i~ ~ e re ~ onses capable~of~:
'.'recogni.z.ing t e many-~ariant ep topes present in-~t
'.' . -. family~of diverse but:related-proteins:expressed:by_
~ '' 20 the pathogens.--:In-order~to achieve this, it may~be `~
'.'': ............... necessary to immunize~with a-vaccine formulation in~
'''' ' - 'whichjmany; if ~not all,~of these:~riant epitopes ~2
are represented. ~
-~?~nother.example-of different but.related genes
.. .. 25 may.be--.found.among the interferons.;~The interferons
(IFN)-comprise a grouFr.of related proteins that are
encoded by three distinct gene famil.ies, designated;
IFN-alpha,'IFN-beta,.and IFN-gamma. The 14 genes'in
the IFN-alpha family share approximately 80-90% ~~
30 homolo~y. -There is also limited homology (ap- `'
proximately 30%) between genes in the IFN-alpha and
IFN-beta families (Joklik, W.~. 1985. Int~rferons.
W0~0~l288(~ P~ PCT/US90/02060
Pages 281-308 in Fields et al., eds. Virology. Raven
Press, N.Y., N.Y.) - .
All interferons possess both antiviral and
anticellular activity; all cause intPrference with .
multiplication of viruses and regulate a variety of
: cellular functions. -However,-they vary greatly in
the relative extent to which they express these
activities in various types of cells. Recently,
,recombinant DNA technologies have been used in an -:
lO effort to,produce hybrid interferons with altered
biological,and pharmacological properties; _Several~
. . 7hybrid human:IFN-alpha interferon species-have been
,- constructed (Streuli et al. l98L.:Proc..... Natl. Acad.:
Sci. USA.,78:2848-2852';.Weck et al. 1981.... Nucleic
'15 Acids Res.--9:6153-6166);_these were found to differ
;' greatly.in their:ability to.inhibit-the.multipliGa-
'' ' tio~of.yarious.YlrUse5~ different'mammalian'cells -
'''Sa,n~d,in-.,inf,e,cted:anlmals~Weck~et~al.~1982; Infect;~
~ ~;.. . , - . --
'~'; 7~ ' -' '' Immun.~35:,66,0-665t.ryThus--,int~ma~:be~possibile to' '~
..~ - . .. ...
.,- ' 20 improve.:upon the func~ion o~ natural interferons:by` ;-
:, generating:hybrid interferons with-particular - ~
;æ; :~ :- applications3for-c~inical use-ln~specific 5itua-? ~
tions,:both.:in the antiviral area and the anticancer
SummarY,O* the Invention
~ . This invention pertains to methods of generat-
ingi,in vivor,hybrid-DNA structures.:from two re- :
lated, but differentr DNA sequences through a
process of genetic recombination within or between
viral genomes, and to the expression of the hybrid
Ij W~90/12X~0 PCT/US9~/02060
7 ~
~enes created by these methods in recombinant
viruses. Methods Qf this invention exploit the
phenomenon of intramolecular recombination (i.e.,
recombination between homolcgous DNA sequences ~
present within a given viral genome). The method is
based-.upon the phenomenon of ~irus-mediated re- -~~
combination to generate, in a viral genome, hybrid ~
genes from two related but nonidentical genes,
provided that these genes share regions of homology;
10 An example of related'-genes are genes encoding~
different variants of a~given polypeptide.~
dAccording to the methods of-ithis invention,'-two
related genes are introduced in tandem'into~a-viral~ ~'
genome, preferably a pox~iral~genome.'''-These tandem'
15 structures are genetically unsta~le~in:viruses. ~~
. During replication o~ the viral genome,'intra- -~ 3~ -
e~cular_reco~hination between~'homologou~-'regions-t~ - _
S ~ shared~by the~relate~-,genes resu~ts~in-'condensation~
;` - of the duplicated~structure-td~yiela~à-singlè~ m r
' 20 hybrid:sequence containing portions of-~the-two'~
original.sequences. ;Nevertheless-, it''is''possible''to
' ~' select:for ~iruses~that~main~ain~a:~tandemiduplica-~
tion in--the genome if~l) a:gene that encodes a ''~'~
selectable marker, under the control of a poxviral'
25 transcriptional promoter, is placed between the
duplicated sequences and 2) the recombinant poxvirus ~-
is propagated under conditions that select for the
function of the marker gene. Because~recombination
between the tandemly arranged genes results in the
30 excision of the marker gene, only those viruses that
retain the duplication, together with the ''
WO90~28X0 PCT~S~0/02060
3 7 ~
intervening mar.~er gene, will survive under
selective growth conditions.
A recombinant virus that contains in its genome
two tandemly arranged, related genes (separated by -
an appropriate marker gene) can serve as the pro- -
genitor of a population of progeny viruses, each of~
which contains a different hy~rid gene composed of
portions of the.two original genes. When the ~~
parental virus containing tandem DNA sequences ~--
10 separated by a.marker gene is allowed to replicate~`under _on-selective.growt~.conditions,-prDgeny
viruses generated_byjintramolecular-recombinationr~2
"' ' between homologous.sequences will.be propagated. -::;
This intramolecular rec'ombinaticln event results in '
}5 excision.of the marker gene. .Because the recom~
bination even~ can occur-between-any homologous~
t~ - .'re.$ions-shared-byjthe-two~genes r 'each-of the`proge'ny - ~- ..
' viruses wil~:contain a different~hybrid'gene~resuIt' .-
.' ~' ing from the condensation of theeoriginaI-DNA;~ - r-~
-' 20 sequences.:~-Thus~.-a.spectrum of-hybrid genes can be i-
generated from~two:related~genes:-that contain--~':---'-
. ; . -- regions~of.'DNA_sec~ence~homol'ogy;tfThe~incIusion~of3
appropriate transcriptional si'gnaIs''upstream o~'the'~
first of~the 1-wo duplicatecl DNA sec~ences will '''
25 ultimately,al:Low for the expression~of:the hybrid -
~genes created by. the intra~olecular recombination~
events.-:. .- - ; ~ .~. .. ~ - .
The methods of.this invention can be used to :-
produce a mixture of live recombinant viral vectors
capable of expressing hybrid genes~derived from
genes encoding antigenically distinct variants of a
WO90/12880 ~ 37~ PCT/~S90/02060
given polypeptide. If this polypeptide represents
an immunologically important antigen of a pathogenic
organism, particularly an organism that exhibits
antigenic and genomic variation, the viral vectors
so produced may be used as live recombinant vac- -
: cines,^these-.vaCcines may elicit immune responses to
a broad range of serological types and subtypes of
the pathogen,.as the hybrid genes produced by this '
method.may mimic the genetic changes that occur in
10 the.pathogen in nature.~..In one-embodiment of-this ~-
invention,;~.:a.recombinant:,vaccini-a virus~is,con- -~- ~
, , . structed to..contain in tandem,-~en~elope~'genes`from '~
, '' ' two d~fferent~strains.of human immunodeficiency -~
virus (HIV) L namely the'`~E:and the BHlO strains of -
.:' 15 HIV-l~ he progeny-:vaccini-a viruses generated after ~'
.intramolecular recombination between the two HI~
e3genes contain~-a.~spectrum~of'::hy~rid-envel~ope' --
;Y ,~'."~ en,e,sgderi~edofrom:-1the-arigina~ two genesiofrthe~~
..:.~ ''"- different~HIV=l.,str,ains.~ ~C ~ ~52i-e_~OO ~ C _ I ~r~ r
':.. . 20 ~ ~This-invent~on:-can also be-used to'~generate~
:~ ~ chimeric proteins-;for-other,.applications.- ~or
., - c ':.- ex3mple~-recombination~between-genes-encoding 'S5 ' -9
re,lated interferons:will~ genera~e hybrid genes.~
encoding.ch~meric:proteins, with.nove~ pharmaco- ' .'
25 logical propertie5... These:chimeric interferons may ::
exhibit improved function by comparison to'natural '~
interferons with regard to their applicability for
therapeutic use in specific situations~. :
,-~- In addition, vaccinia-mediated.recombination
30 between genes encoding related antigens can be used
to map type-specific antigenic determinants. For
, W~ 9()/1~8~0 PCr/U~90/02060
3 7 ~
example, a large number of vaccinia recombinants
expressing nov2l hy~rid antigens can be generated by
recomkination bet~een genes encoding related anti-
gens from two different serotypes of an organism.
5 The ability of ,type-specific monoclonal antibodies -
to bind individual hybrid proteins in the vaccinia
recombinants can be used to map specific epitopes.
Brief Descr~_ion of the_Drawinqs
Figure l illustrates a method of generating
10 tandem DNA sequences -~nd chimeric- genes in vaccinia
,~ using,, a single-step approach, ~e~ ?. - -~
Figure 2 illustrates a step-wise method oi~ -
generating tandem DNA sequences and chimeric genes -
in vaccinia. ~
Figure 3 ilIustrates construction of a plasmid
~'t~ `. pABT4075-containing the-HIV-l strain ,BHlO env~ gene:.. --~
~, - .-- : fused.to the E~_ coli-,lacZ gene_"~ The gene is,unde~,
, ~ - , , .
the tra~nscriptional control,of ,the-,vaccinia 7.5K. -.-:
'. . promotor._ Figure 3A shows,the intermediate step in
2~ creation of pA.l:T4075 which. is constructed of -j, - .- .
,~," , pA~LTl67_.~ Flgu,r~ 3~3 shows ,construction of the~rector
pAbT4075., ,,., .._._. ,,." ; _ .. ~,, ~,. j,. ~, . ;.,
Fi;gure 4,il] ustrates construction of..a plasmid
(pAbT8523) containlng the HIV-l.strain RF env gene_.-
25 Figure ~4A shows- construction of the plasmid ,.:
pAbT4524B containing both DraI and NotI sites. --
Figure 4E~ shows;construction, of the plasmid
pAbT4533, created ~y ligating together fragments of
the plasmids pAbT4027 and pAbT4524B. - Figure ~IC.
.
. WO90fl2880 PCT/US90/02060
3~ ~
--10--
shows construction of pAbT8523, created by ligating
sections of two plasmids, pAbT4533 and pRF3HS.
-- Figure S illustrates construction of plasmid
pAbT4082 containing the HIV-l RF env gene modified
at the 5' end of the gp41-encoding region.- Figure i
-' - 5A shows creation of the~'plasmid pAbT8526,- created-~
by.ligating fragments of pAbT4533, pAbT8523 and
pRF3HS. Figure 5B shows the creation of pAbT4082,
derived from the partial.digestion'of'the plasmid
10 pA~T8526.~ ?
s~..FIgure 6 il~ustrate's:constructlon--of'a donor ~
~ ... .. plasmid (pAbT4085) contai'ni'ng~the;vàccinial~irus -V
.. thymidine Xinase gene:-fIanked by'~wo-HIV-l?env
genes~' Figure 6A shows'creation of the plasmicT-''~~
15 pAbT4083 derived from ligation of fragments from` -
pAbT4075-:and-pAbT4'082~ igure 6B-shows créa~lon of -
~.--~ ~d~ p, 4085'~'de^ri'ec--*r'o~ 1' at n eg nts rO~
-: '; ;.~-:and~pAbT400.9l-~he~rdonOr3~plasmic~a-ls'o~contaIns~thè~'RF~ --. -- :
. .-env gene-encocling'gpl20'~un~er~:the c'on'~r~i~c~f~ 9h~
- 20 vaccinia viral promotor''a'nd'thé-lacZ'gene'fused~
... in-frame to the:BHlO~'env`ge'ne.~ c r
5.,~ ' ''' -~o~igure ~c ii~ a~schema~ic-representation of th~A~
steps involvecl in generating a vaccinia virus ''~
containing.related~ but:l'nbn-'iden'tic'ai-genes ànd
steps~in~olvecl-ln~'producing a-recombinan~ virus ~'iC)
which has'undergone':a condensation event to genërate- '-
a fused HIV-l env;gene. ~'-' :;'''' ' ' -'''' - - '''
Figure 8 illustrates construction of-plasmid -:
pAbT4105.containing one'particular:condensed env
gene derived from HIV-1 strains BH10 and RF. Figure
8A shows creation of pAbT4105 derived from ligation
W~9~ X~0 PCT/US9~/Q2060
7 ~
~11--
of plasmid pAbT2009 to a 3 Kb vaccinia virus genomic
fragment containing a condensed env yene. Figure 8B
is a restriction map of plasmid pAbT4105 including
sites which define the RF/BHlO junction.
c~ Figure 9 illustrates the construction and
ser~uencin~ of a phage (pAbT4106~~-containing the
junction between the HIV-l RF and BHlO env sequences
in the hybrid gene from vAbTl68-l. ~Figure 9A'shows
creation of pAbT4106 by ligation of a fragment from
plasmid pAbT4lO5,with phage ml3mpl9.~~Figure 9B
shows the partial~nuclectide~sequences of a portion~
o~pAbT4106.containing the'fused-`en~~gene,''as well-
- as regions of,the,parental RF and BHlO:env genes ';~
from..pAbT4085. , ~ 3 ~ '~
Figure,-lO:illustrates~'the'restriction map ~
(Figure:lOA~ and.nucleotide sequence (Figure lOB~ of
.,r. ..`.- .~:' another~condensed'env~gene--f rOm the-~recombinant~S~ ;L .~
- - - , -;vi~us~vA~T168--2 . - i~J ~ :r269 ~' ~0 'J~ "; .L~ J' ,.~_' _C~
~ Figure,11^shows~^the`'approach~for^~generation of'-
20 c~i;meric'HIV:env genes in~vaccinia'virus;~
.,Figure 12.~shows-the str~cture ofdhybrid ~
genes in intermediate recombinant5r~ 3~ 2
~,~;Figure-l3-shows.thelstrUCtUre of-hybrid env
genes,in.condensed recombinants.'-:~ .t~
Detailed Description o~~the..Inven ion -'-- ' '?-'
~ --,The~ above and variaus other'objects and ad-
vantages of.,the-present invention-are achieved by
methods-for.the-generation, ln vivo,'~lof hybrid DNA-;
structures from t,wo related but different DNA - -
se~uences through various genetic recombination ~
,, WOgO/12880 PCT/U~90/02~6~
2 ~ 7 ~
events within or between viral genomes, and the
expression of the hybrid genes created by these
methods in recombinant viruses.
1. D~A Sequences Useful for Generation of Hybrids
DNA sequences suitable for.use~in methods-of 3
this inventicn can be any two or more DNA sequences
that share regions of homology-(i.e., regions of '-
similar DNA sequence). ,They can include DNA se- -
quences encoding structural or f.~nctional.components
of,~various pathogens.~eThe sequence homology that -'
'.. ,. ,, , . directs,,recomblnatlon b,etween related:DNA sequences
may be locallzed ,to.short regions of. nucleotide-~ ~-
identity. For example, the extent of nucleotide--~:
sequence.identity,necessary for intramolecular' ';
,,:,., ~"";,~, 15 recombination-in yeast can.be~as little as 10~.,` ^ ,
'nucleot~des,-,~Mazza,ra ,a d-~r.een,~1983,~2.~ bstract:~I6' . .
' -- The Molecular Biolo~y of,Yeast, Cold.Spring-Harbor,''v' , -"~
' New York)-.9~The min,,mum-degree_of..homology~.required .
for intramoleçular,recombination.between related DNA '~-
sequences can.be,determined experimentally-~or each
'''' ;''~:' ' set of re:lated sequences~ g~ 5,.~r C ~ -9~-`e
.As a particular;.example?-:it is known that the
env genes encoding the.envelope~glycoproteins-o~'~'"
;, human immunodeficiency virus (HIV), the causative
""" 25 agents of AIDS, vary widely among di~ferent,is~lates '~
of HIVr and that each envelope glycoprotein variant
elicits primaxily type-specific neutraliæîng anti-
bodies (i.e.., antibodies capable of neutralizing ~-'-
only the cognate strain;~ Ho, D.D..et al., 1988. '~'-
30 Science, 239:1021-1023~. Although the nucleotide :'-
W~90/12~80 P~T/US90/(12060
-13-
sequences of these HIV envelope genes are ~1idely
divergent, they nevertheless contain regions of DNA
sequence homology suitable for the generation of
hybrid genes by the methods of this invention.
.. In a specific embodi~ent, hybrid genes are
- qenerated from the env gene of~the R~ strain of '
HIV-L and the env gene of the BHlO:~IV-l strain.
2;`9 ^Host Virus
-
r;-.Hybrid DNA se~uences can be gPnerated in vivo :'
in.-any virus (e.g_, poxvirus) in.which intramolecu-
~ar.:recombination can occur between duplicated ' -':
se~uences. ~The.term "intramolecular recombination"-'
refers to reco~bination within a single DNA molecule
as opposed to recombination between DNA molecules.~ ~
~ ' 15' In~.particul2r,-.this term-is used herein to mean~
", ,t ~',Z, .`~ recombination'withi~ th~ genomic DN~ of a-~irus'.~
' ;3~ ~h~ pre~errad virus u~5ed in the methods-of'this
''~"'~'"'`~~ ':...inven~io~-~s the;pox..virus~,.~accinia'virus.'' The'`' -~.
' . ' virus;has.been developed as an~infectious''eukaryotic
cloning-vector.(Paoletti''and' PanicaIi,'United~States
. ..' ' -~ Patent No_-4,60~ an*~recombinant~'vaccinia virus
has bee~ used successfully as a vaccine in.several -
experimental systems . The virus is considered
non-oncogenic~.has a wel~-characterized genome, and.
can ca~ry--large amounts of foreign DNA without loss
of infectivity (Mackett, M. and G.L. Smith. l986. J.
Gen. Virol.,. 67:206.7-2082). : -
_
W090/l2X~0 3 ~ ~ PCr/VS90/02060
-14-
3. Generation of Recombi~ant Viruses that
- Contain Tandemlv Arran~ed, Related DNA
Sequences Inserted in the Genome ~
. _ _ .
The generation of recombinant v~ruses (e.g.,
vaccinia viruses):that contain tandemly-arranged an~
related DNA elements inserted in the genome is based
on the following methods. Briefly, to produce the
recombinant viruses, donor plasmids are constructed `
that.,contain elements *o ~e inserted.into ~he viral
.
. 10 genome,.~,These elements are flanke~ by DNA~sequences-
- homologous to DNA sequences-present.~ the.~genome of
the,,host virus;. these sequences'will-serve.to~direct
ins",ertion of the elements.to a particular region of~
the,,-,vi.ral,genome:by in vivo recor~ination.~-Cells -5
are infected with-:virus..(e.g~-r-vaccinia Yirus~ and :-
~ . . ~ . . . .
u~2,.the ir~ .cted cell-s~rare-..a~s~ transfec~ed~with~-the~^-3
-,,-:,~ ;.. -:- ~onor-~lasmids.-. ~Homologous recombination~-between
. ;,. .-.plasmi~ DNA;,and ~irus~ A results.~n--.the ~ormat'ion~~:_
~f recombinant.viruses that incorporate...the desired '
elements~f.rom the_donor plasmid~.~ ~arious ~irus -:r-
~l,e~tion,._schemes,5~.e.g.~ see~rSpyropoulos.et-al_,3
1988~ J. Viro].:,62:1046-1054,.. Falkner-:and Moss -~
lg88. J. Viro].,,62:1849-1854,. ~rank~ et.al~,~ 1985.:'~
Mol~ Cell Bio],.-:5:1918 1924). can be incorporated--:o.-:
int~ this,general approach,:-in.order t~ permi~ thei- ~,r
identification and isolation of recombinant,viruses
Specific details of two approaches ~sed in.this
invention are described below.
WO90/1~80 ~Cr/US90/02060
~'3~7~
-15-
a. Donor 21asmids for in vivo recombination with
. _
_st virus
In the first approach (Figure 1), tandemly
arranged related genes are inserted into the genome-
-5 of;the virus (e.g vaccinia virus):in a single _
;.recom~-ination event by means of a--specially designed
donor vector. .. - - '
A preferred donor vector contains (in a 5' to `
3'_orientation)~
10 t~ . i)'._ a transcriptional promoter; :- ~
ii).~a first gene or gene fragment, encoding a i
,_ _'L'/ polypeptide o~ interest, under'the'c^ontroI
- of the transcriptional promoter; '~
.~2iii)~a gene encodi~g a selectable marker, also:
.' 15 ~ under the control of'a transcriptional
fi ~nC J' '- promoter; -~
- `'.y'iy~ second gene.or gene fragment`containing~
7,~ ';' region(s) of~DNA,~sequence~hombl'ogoùs.to _d5
'' :.' 5r.~ th~,~irst.gene.,~-encoding~'a' second--poly-
'~' ' 20 ~ peptide:of'interest. ;'
The~preferred clonor vector:also contains.viral DNA~
.-m. se.~ences~labeled L.and~ in ~:igure l)~-fIankïn~:~hè
construct of elements.i-iv, the flanking segments '~;
being,homologous~to a:region of:the viral genome. ...''l
into which the tandem genes.are to be inserted. .~ ~ r ~S
The.,transcriptional promoters in'i and iii are
req~ired for the expression of the adjacent genes in~
vivo,_~For,vaccinia virus, for example, these may
include any of the well-characterized and isolated ;~
transcriptional promoters,-such as the 7.5K promoter
(Venkatesan et al. 1981 Cell 25:805-813); the BamF
promoter (Panicali et al. 1983 Proc. Natl. Acad.
WO90/t2880 PCT/US90/02
20~4379
-16-
Sci. USA 80:5364-5368); the 40K promoter (Rosel et
al. 1986. J Virol. 60:436-449); the 30K promoter '
(Perkus et al. 1985 Science 229:981-984); the tk
- promoter,(Hruby et,al. 1983 Proc. Natl. Acad. Sci.'
USA 80-.3411-3415) and the llK promoter (Bertholet'et
al__l985- Proc.: Natl_,Acad.-Sci.. USA.82:2096-2100).~ :
- The gene encoding the selectable marker ~ele- ::
.... ~. . , ment iii) is,required for.the maintenance of the
tandemly duplicated structure during viral replica- :
:-: 10 tion. Several types of marker.genes can be used. A
.'.'.': . .~... ..' pa~rticularly preferred marker gene is;the gene
~ enc~oding *,h,ymidine kinase,~ tk)-. ~:~'iruses-that
,',,.','~,,:'.. .:' contain this,gene can.grow in:.theipresenc'e of the
.,,;,., '.,,; ,, chemical.methotreXate,.-while~.viruses lacking this
.. 15 gene can,noti_(,C,omplone-Piccardo et al.. ,- 1919. J.
;.'.. '. '. : .^ ' Virol. 31:28i-287). Thus, recombinants that contain
.qene canibe~se}-cted;o~:the.basis:of their
abilit~,to gro,w,~ met otrexate.~ ~a)~..g~, '
~,~l',;',',,.~ -, ~ ''' ,' ". ~ Ot er-lselectable:.markers.include theivaccinia
~"'",""' " .- " ' - 20 virus host range (29K) gene-:(Gill'ard et~'al;, Proc.
.;,.: . '.. ' Natl.~Acad.. Sci.. USA. 83:5S73-5S77-1986), or genes'~
, .,",, ~"""s ! ~ - '' ' t.,at ~,oner-ch mica~.o tib'iot-' 'esistance'^suc~-?2
.. . . as the~E~coli.Neo .or,qPt~genes:~Franke et al'~
Mol. Cell~Biol. 5:1918-1924~1985;~Falkner and~Moss?
.: ' . 2S 1988. J~Virol. 62:.1849-1854~1988) ;~ 3 ~ ~s ~ J `~
. . "'.-'' '. ; -. '. ' . 3_ In.,,,the,second approach (Figure'2)',~tandemly
arranged related genes are inserted into the viral''^-~
. , genome,via.two separate.and sequential recombination.
events. -.~ first gene, under. the control of~a ` ~ r
transcriptional promoter,-is:inserted into the viral~
genome using standard techniques.of in ~ivo:recom-' '
bination. The second related gene, together with a'.
. . . ~ : .
- - .;
WOgO/l2~80 PCT/US9~/02~60
2~37~
gene encodins a selectable marker te.~., thymidine
kinase) is then inserted into the virus containing
the first gene. This insertion occurs via recom-
bination between the first gene, contained on the
viral genome,~and the second gene r -contained on a --
donor plasmid., This second recombination event ?--
generates a virus that contains two hybrid genes in
tandem; these genes are separated by a gene encoding
the selectable, marker. 3 . :;:~ ~~ ; ' ; ' - '
lQ ~ In,this,-second-,approachr the.-donor plasmid - :.
. vector,for,.~inserting a second, related-gene into a-'''
,.' ~irus~that,already;.contains.a.first:~related:gene has
the following ,elements~
~ ,i) , a, gene or-gene:fragment-containing one or'
~r_ " .;, more regions homologous.to a first gene ~:
already present in:the-genome of a~recom-
, ` binant virus; and3~s-? :~JDL~s. ~i~s j5
-. ii) a gene encoding a selectable marker, under ..,. -
r .~
~ .. the control~of~a-'trans'cri~ional~rpromoteri~ `
~, , ~ , ; _ _ . . _ _ ..................... ...... . _
;' 20 ~,pr,~ef,erred marker-.gene is the.. tk~gene" as~described
pr,e,v~iouslyt~ s s ~ 5 ~ r .-
` y~,~tgE:l~r~J ~0 ~ d~ ~r:~9n~ 3~ ?-'~ 9,5
b. Inteqration of foreiqn DNA.sequences into the:-i
viral qenome-and-isolation of'recombinants~ ' ' '~''
As descri.bed briefly above,.:homologous-recom- -'
25 binati.on.~etween donor plasmid DNA and virus DNA'in~
an-infected cell results in the formation of recom-'
binant-viruses that have-incorporated the desired ':
elements. , Appropriate.host cells for ln vivo`-- ~
recombination are generally eukaryotic cells that 3
r 30 can be infected by the virus (e.g., vaccinia virus)--
and transfected by the plasmid vector. Examples of
~; WO90/12880 ~ 3 7 ~ RCT~ISsO/O~o~
-18-
such cells are chic~ embryo fibroblasts, HuTK143
(human) cells, and CV-l and BSC-40 (both monkey
kidney~ cells. Infection of cells with vaccinia
virus and transfection o~ these cells with plasmid
vectors is accomplished by techniques standard in
the art~such:as Panicali and Paolett'i, Uni~ed'States
Patent No. 4,603,112, incorporated.by reference
.... .: herein.
In both of the approaches described above,'
selection for function of-the marker gene (e.g., the
tkjgene~ following in vivo^.recombination permits:--
:.... .. ,-;~,. ~so,lat,ion.,of~recombinants that contai~:tandemly~
arranged and related genes (i.e ;:~genes~'that share~-
regions of DNA homoloqy~ Both approaches (Figures
1 and.~).will yield recombinant.-viruses~i~ which the -:
,,,marker~gene,is.positioned between,the two tandemly
arranged and related genes_6 ;~
~3~r.~ 3x-~s.- 9id_09 r~ n~ 9 9~- ? ~ i ' ' -
4~e~cl-ntr-amolecular-RecombInatiQn~o- sr._
~9d,-Methods of~t~is-invention-for~generating^hy~rid '~`
' 20 genes from two related genes in a recombinant'-virus~-
. ' `~;'''' are based upon the genetic instability of tandemly
arranged non-identical DNA.sequencPs,that'.share . '..
regions of homolo~y..? Intramolecular recomb'ination'`~:
, between,regions:o.homology,shared'.by,~,he two'~
"''''"''"'" '25 related genes, which occurs during the~course of-;~
normal viral. replication, results in the formation :
of a~,-single hybri~ gene co~posed o~ portions of the :
two original genes. Because-the recombination'' '~ ''~
events~can occur rando~ly between any homologous ~'
sequences contained on the-two genes, these-events -`
will generate a spectrum of hybrid genes.
WO90/1~X80 PCT/US90/02060
7 ~
--19--
In viruses generated by approach l or approach
2 (Figures l and 2, respe~tively), a gene encoding a
selectable marker resides b~tween the two tande~ly
arranged.,related genes. This arrangement permits -
isolation and propa~ation.:of viruses containing the
., genetically unstable:tandem gene arrangement.
Viruses are propagated under conditions that permit
growth-of only those recombinants that contain the'''
marker gene. ~
Intramolecular recombination between homologous `:
sequences,on,the;.two tandemly arranged; related '~
genes results,,in,-.the excisio~ of:the marker'gene.'-!~
Viruses that have~undergone intramolecular'recom-~~''
blnation,to generate.a~iybrid gene can be iden~
..15 tifiedr, and_in some cases,'selected for,'by virtue'- ;,;
'. o the lack of,the,function encoded:by-the~now-' - ''
~t ~ . absent ma~Xer gene~e For example-,~ when-'the~tk gen 3`'
-`- ''~';.'~i~Ls`'used as the selectable marker, those-progeny~
'~.','-.'-viruses that have undergone intramolecular recom-
''20 bination, with the:concomitant~excision'of~the;tk '
. . ~
gene,-ca~n_be,~selected,-in a,medium`containing-5
. .. bromo-,2,~=deoxyuridine~(BUdR~ or trifluorothymidine.
In. this selective:.medium, only:those viruses'which-
lack tk gene:function.can.survive.~
_.In.,vir,use.s generated by approach I';-intra~ t
molecular,~recombination between tandemIy arranged,~~
related genes.will yield a final hybrid gene that ~'
contains a single,fusion junction, i.e.', the 5' ~
portion of the hybrid gene will~ be derived from the~
5' region of one of the two original genes, while
WOgO/1~80 PCT/US90/02060
3 ~ ~
-20-
the 3' portion of the hybrid gene will be derived
from the 3' region of the second of the two original
genes. Each individual hybrid gene will c~ntain a
different fusion junction, generated by random
: 5 recombination between the homologous regions shared
by the two original genes-(Figure l~
~ In viruses-generated by approach 2, each of the
.. tandemly arranged genes is itself a hybrid gene, -`
formed by recombination between a first gene~located
10 in^the:viral-!qenome and a second, related gene ::
.. , contained:on the~donor plasmid: :Intramolecular -- -
, recombinat-ionibetWen thiS pair3cf hybrid~genes :~-~'~''
`' results~in the formation of-a.single'hy~rid~'gene-~''
. that contains two separate.fusion'junctions.~~-Thusj:
~:' '' 15 the. final hybrid gene.will~contain-5' and 3' seg~
.' . ments.from one-of:the:two origlnal genes flanking a~
'' cen,te~ seg~ent~derived~rom thë second of the two 'G_
'~.~'','', . '''',` ~`.t-',~ ' original~gene5-~ igUre' 2~._ g' ~5~
_.r~c ~ ;;~~ ;5rt~ 9~0~ L' 9-v'51~ , 51-- I 232' ~
. , 5. ,Recombinant Virus--as::~accines~ n~
::' 20 The~methods of.this-:inverttion-car.t~be-~expIoite~,:
.: . , ~. . ~ .
'.''''''' '`` togprodllce--a-~accine in the~'form(of~live~recom-S'~
binant,,,viral-vectors.capable of.'..expressing'a unique-
' . chimeric protein, or,a popu~ation.of related chi-'-' L
meric_p:roteins or,:protein segmentsrrfrom~la-patho- ~r
' ' '" ' 25 genic o:rganism.,.that exhibits antigenic ~ariation.--A
recombinant virus (e.g.~ paxvirus) that contains
tandemly arranged genes encoding.antigenically ''''':
distinct forms of a.chosen polypeptide can'serve~as~
the progenitor,of a population of progen~''viruses, ' ~;
WOgO/1~88~) 2 ~ PCT/U590/02060
each of which contains a different hybrid gene
composed of portions of the original tandemly
arranged genes.. These hybrid genes, created by
; intramolecular recombination-events, can be ex- :'
pressed by the recombinant poxviruses as chimeric
'~ po~ypeptides. .~
If the chimeric polypeptides represent an
immunoloyically.important-antigen of a pathogenic
organism (e.g., the HIV env antigen), particularly -
an organism that exhibits.antigenic and genomic - -
variation, the population of~viral~vectors-that ':'-`''
. express these polypeptides may be used as Iive~
' recombinant vaccines;.these vaccines may elicit
'. immune responses to a broad range of-serological -~:
15 types and-subtypes of the:pathogen~
As an:alternative.to the use of-the whole -` ~- -
,'.*~ pop~latio.~ of-progeny viruses~contaI~ing hybrid ~~
genes~.it-is .also..possible ta~isolate indi~iduaI '~
.- '' .' members of-lthe populationi each-of which expresses.a
- 20 different~.and unique.hybrid gene.-: The individual,' ? '~-
plaque purified.recombinants~~can be examined-for:--
': t-~ their potentia:l~to elicit i~mune responses-to:the~
several variants of the cognate pathogen; appro~
priate recombinants can .then be used, individually :
or in combinat.ion,-to formulate a.live~r'ecombinant~- '
vaccine. . .~ - i,J_' . '--1- ~'_._.-"... '. ' ..'. '.''
- An.example of the use-of the methods of this ';
invention ,i.5 the development of'a vaccine effective-'
against multiple strains of the human immuno~
deficiency viruses,- HIV-l and HIV-2, the causative''
agents of AIDS. The genetic and antigenic variation
W090/128Xt) 2 ~ '3 ~ PCT/VS90/0~060
-22-
among strains of HIV, which resides primarily in the
genes encoding the viral envelope proteins, presents
a major obstacle to the development of an HIV
vaccine, as a vaccine effective against one variant
may fail to protect against related-strains. -To 'i
-- generate a vaccine with potential for eliciting'.an:
immune response against multiple HIV strains, a
parental,poxvirus that contains, tandemly arranged -
in the genome,. envelope genes from two widely - :''''
di.vergent.HIV-~ strains ~P.~ the R~'and BHl0
strains),~can be constructed.~:Intramolecular re~
., combination.between.homologoUs-'regions! of:the two'~
HIV envelope genes in.the parental virus wiIl give~':
rise to,a-population~of progeny viruses,~:each of~
15 which contains a unique hybrid enveIope-gene~~ '3
' generated:from-the random.recombination'between
~ '-~s~ ,homologous,iregions-shared,:by the:-tw,o~original-gé~'ë's~
-"~,-,-,: , .'.~'~The c~imeric;:envelope:prote;ns-encoded--by^th'ese~ 9
--hy~r,id_genes.wi~l contain both,,nove~:c,combinations~o~
'"~ 20 epitopes as well:~as new-discontinuous-epitopes.~ 3- ~ C'
Either individual-members of this-poputation,~ a'-?j-i
i mixtu,~e;of-.se:~.ected members-of:.this~-~opulat'i'on,~-or
the entire population.of-progen~ viruses:can be''used
as ~_vaccine,to~immunize persons susceptibIe'toi-HIV '
inf,ection.,_-During. replication Or the progeny~
viruses in an immunized host, the chimeric antigens''
will,be expressed along with the normal complement
of,.poxviral genes~ These antigens will stimulate an
immunological response directed against the variety ;
of epitopes represented'in the~population; this"- -- ~
WO90/128~0 PCT/US90/02060
2 ~ 3 ~ ~
-23-
immune rPsponse may be effective against a spectrum
of HIV stains.
-. A number o other pathogens, in addition to
., HIV,-exhibit genetic and anti~enic variation which--
allows them to escape host immune surveillance.~
`S' These include various parasites, such as trypano-
somes and malarial plasmodia; and viruses, such as ~
.. .. ..
influenza virus (an orthomyxovirus), certain~:
picornaviruses, and retroviruses. The methods of
t,his invention,may be applied.to~the development of
a~vaccine for any of these pathogen~
X. O~ Y ;r ~ J 29~ ? L ~ ,'~ L'." '~
6 ~ The Generation-of-Chimeric Proteins for- ~
~ gPharmaceutical Uses --~ .. :.. .. -.~-. ^,-.--~ -~ -:
'- . ~L.~L_Although;the methods of.,this invention are -~
J~.'7~ t'-'' '1s-.part~cular}~ suitable~for developing~vaccine5. ~h'~'V ci - -
. ;rt .
. 'agains~._pathogens which.~exhibit-antigenic diversityr
;.' they can also be.us~e~,to~genërate:hybrid-'genes ~ 3~1 !
encoding chimeric proteins for:other~applications.
For example,;: 2L 'recombinant VIrUS that^contains,-~' ~~
''~ ' ' 20 tandemly:arrarlged:in-the-vira~ genome,~genes en-,-sx9 clr
coding,relatecl,interferons tor.~any.two related'genes
encoding pharmacologically important:polypeptides)'
will,,.--upon int:ramolecular recombination, give rise''
to progeny vi~ses.that contain hybrid genes; '~
25 _,-Individual progeny viruses generated by the re- -' -'"
combination.events could be isolated and the chi- -'~
meric proteins synthesized by these viruses could be
purified and examined for the desired novel bio- ':
logical properties. Alternatively, the novel hybrid
W~90~12~0 ~ ~ Q~ P~T/US90/~2~60
-24-
genes contained in the individual progeny viruses
could be isolated by standard recombinant DNA
methodologies and expressed in alternate recombinant
organisms, including E. coli,-yeast, baculovirus, or
eukaryotic cells.
7..i The Generatifan o~ Chimeric Proteins for E~itope
Mapp in~
~- -Hybrid glycoprotein_genes have~been used to map
.. ~ type-specific antigenic~determinants~ For example,~
. lO a large number of hybrid genes containing portions
of the glycoprotein genes ~rom vesicular stomatitis`
virus serotypes Indiana and New Jersey were made by
'- ~ standard techniques of recombinant DN~. These
hybrid genes were:individually~expressed in vaccinia
.i5~ v1rus and~wer~used~to-:~map antigen~crdeterminants~ 2
~ ~ `; -recol~;fnized by~ type~-speclfic;monoc tonal-~ant ~bodIes '~
.~ - ~ - (Keil and-~agner,,~1989.hf~rology~~0:392-40~
.The methods:of-thi~-invention can also be used'
-. to map-~type-specific-antigenic determinants.~-For
~ 20 example, 2giYen ~ two~related~roteins~and a~collecti'on '~S
of,~type-specific antibodies:which:recognize only one
(or.-the other):o~.the.two proteinsr the epitope~
recognized by-each~antibody--ca~:be mapped by con- -;
structing.a.recomkinant virus-containing the two : ~~
related genes.: The resulting intramoleuclar recombin- -
ation events between the two.related genes will -
generate a.population of viruses containing a- - -~
spectrum cf hybrid genes.- Antibody binding to -- -.
individual hybrid prote~ns can then-be assayed (for
example, by black plaque assay on the viral
WQ90/1~880 ' 2 ~ ~ ~ 3 7 ~ ~T/US90~U20~0
population). The genes encoding individual ~ybrids
of interest can then be ma~ped (for example, using
the pol~merase chain reaction technique to amplify
the hybrid gene in the viral genome, followed by
restriction mapping or sequence analysis) to
loca~ize-the epitope.~~ :~ ~'5 ; ~
:- The invention is illustrated further by the
following Exemplification.'''^' -
EXEMPLIFICATION
: 10 Cells and Virus o~~
E~ coli strains JMl01:stMessing et~al.,-Nucl.
Acids Res_~,~9:309 (198I)')-~and MC1061 ~(Casadaban and
Cohen,-J. Mol. Biol., 1 :179 '(1980)) can be used as
the host for the growth of all plasmids. The monkey'
kidney cell iine:BSC-40-is'used for vaccinia virus '';
i "' ~-'- infections~. r ~accinia v-irusJ'-tk-~~utant~WR 417i-~
J~ ' ', ', ,', tSpyropoulos et al., J. Virol., 62:1046-1054` (19~8))`
", .. . . .
-- is used as the parental virus for in vivo recombina
~9~ , ;rc
"'~'~, 20 EnzYmes.'o~ 'b J~ o~'e~ 3~ t~ J '
rlc Restriction'enzymes'are'obtainedl from New
England BioLabs or'Boehringer-Mannheim. The large
fragment of'DNA polymerase (Klenow) is obtained from
United'States Bi'ochemical Corp. calf intestinal ~'
phosphatase (CIP)' and T4 DMA ligase is-obtained from
Boehringer-Mannheim.
_ " ~ '- ?--' - ' ' - -
. _ . . .: ,: . ,
W090/128X() PCT/US90/0~0~0
2~37~
_lecular Cloninq Procedures
Restrictior. enzyme digestions, purification of
DNA fragments and plasmids, treatment of DNA with
CIP, Klenow, ligase or linkers and transformation of
E. coli are performed_essentially as described
:- ~aniatis et al., Molecular Clonin~ A Laborator~
Manual,.Cold Spring Harbor,Laboratory, Cold Spring
Harbor, N.Y., 1982), incorporated herein by refer- _:
ence.
., 10 Preparatlon of Vaccinia Virus-Recombinants .~
Viral ~infection,,~transfections~ plaqu~ purifi-
~'~ - ' cation and virus ampl,ification-are performed essen- g
tlally~as-described (Spyropoulos,et al., J. Virol.,-:~
~.,c,-,~ 62~1046-1054 (1988),),. Infection and ~ransfection i_
,r~5.~; 15 ~ere per~ormed in the presence of~5uM meth~trexat~ : -'
's,~;`,(MTX); ~l~a~ues were,-plated,Land,purif~ed~in the ~os~n~
~,~a sence o,f;,~ X_~o~ lO ~ .L- ~g 2C~O~ ~,r2j ;- ~ ~-
.~ r~ ,.C::i g f O ': ~ V 1~ r _ o t 2 ~ i 5~ 3'1 r ~ ~1~_ 2 5 ~ 3 Z J 2 r
~. Vacclnia Virus Genomic_Anal~sis ,,-o~
.~ DNA is extracted from vaccinia virus-infected
'- 20 cells as described (Esposito et al , J. Virol~ m~ '7 OS
Methods,j2:1~5 ~1981)).~and,analyzed,by~restriction
enzy,me digestions~ and Southern,hybri,dization,as 5 ~-7
desc,rib,ed (Maniatis,et~al., Molecular,Cloning: A ?!;-
Laboratory Manual, Cold,-Spring H,~arbor Labo~atory,- ~J
Cold Spring.Harborr`N.Y., 1982)- Vaccinia virus-,~
recombinant DNA is subcloned into phage,ml3mpl8,~New.
'' England Biolabs) and sequenced by the ch~in-tennina-
tion method (Sanger _t al., Proc. National Acad.
WO'~0/12880 P~T/US9~/02~60
7 9
-27-
Sci. USA, 74:5463 (1977)). Polymerase chain
reaction was performed using a kit (Perkin Elmer
Cetus) according to manufacturer's instructions. - -
, Products and compositions described'herein are
5 hybrid proteins derived ~rom the expression of ~ c
condensed genPs contained:within viruses that have'~
undergone intramolecular recombination- according to
the,methods o~ the present invention..-~:These pro- -
teins can have antigenic properties and, when . ~ -
suitably_expre5sed,.-may serve a.variety,of purposes.' '~
~, One-suc~,use can.be proteins that elicit ~i~mune'.
.,,,~, ,~ responses to a broad ranqe.of.HIV~isolates, ~3 _i ~`0
Particular compositions of this invention are
chimeric env genes cor.taining combined sequences of
15 the RF and BH10 env genes of HIV-l.
-'rhis invention.will now ~e.more specifica~ly~
~ 'character~zed-.throug~ the .use.. of the~o~lowing~
---'' ''''''-examples, wh~ ch ar~,not construed_to.be-_~imitin~ 3
.c,~ *j. n~ L ~ ~, r !_ . 6
~ hi ~ 9J3~,; ~ 3~,~ .. ~ EXAMPL~.... ~ :. h ~ f` _ . . '_ J f . ~
"~-~. 20 ; ConstT~ction o~.~;~lasmid'containinq the~HIV'stralnl~ d~`
BH10 env-gene-:Lused to the-E. coli lacZ;cJene, under
thF control of the ~accinia ?.5K Promoter (~iqure 3)
~ nTheoHIV-1 5train BH10 env gene~is~locat'ed,on :'~
plasmid pSV-He:~v,~obtained.fro~.Blair Ferguson ~E.I.'
25 DuPont~deNemours and Co.) ~` pSV-Henv was digested `'~
with XbaI and XhoI, treated with Klenow, and the ..~r
2700 bp fragment containing the env-encoding'se-
quence was'gel-purified. pAbT4007-(See U.S. patent-
application Serial'No.~.910,501, filed September 23,~'-
W090/l~B0 PCT/US90/02060
-2~
198~) was digested with S~aI, treated with CIP, and
ligated to the 2700 bp fra~ment to create pAbT167,
as shown in Figure 3A.
c. pAbT167 was partially digested with HindIII,
and a 9500 kp partial digestion product, lacking
both the 3.' end.:o~.the.env gene and the Bam~ pra~
moter, was gel-purified and self-ligated to form
pAbT4075 as shown in-Figure 3B_ pAbT4075 contains
the BHlO env gene fused in-frame to-lac~ at the
HindIII:site.at position-Z000 in-the-BHlO env gene, - -
encoding..all o~ env protein qpl20 and-approximately -`
. ~ 300 bp of en~.protei~~gp4.1 ~;~s ;~s~-. , . z~es~
-~^ 7'~ 0~ 'e ~ - EXAMPLE 2~
; Construction-o~-a ~lasmid-containinq the'~IV:strain
:.R-F-env~qen~ mo~ifI~d'at ~he-5--~ end-of:-the~qp41~ 5
encodin~.portion:of:th~ ~en~ ~ ures ~~,~5~ rç~
. pAG3 (U.S. patent application Serial No.
.910,501, supra) was dige'sted with NdeI, treated with
Klenow, and ligated to NotI linkers (dGCGGCCGC; New!:.'`'`~ . 20 En~land-BioLab~s.).~.~The resulting:plasmid~pAbT4524~ o'~
was..digested with N'otI, treated with Klenow and ` ;
ligated to a_s~ynthetic oligonucleotlde (dGCTrTAAAGC;
Biolog~ Departmentr-.Brandeis'University)-,~containing
DraI site and restoring NotI sites flanking the i':
DraI:site in plasmid pAbT4524}3, as shown in Figure iS
-pAbT4~27~.(U.S~ patent application Serial No. ' :
910,50}, su~ra)- was digested with DraI and a 5530 bp-
frag-ment was geI-purified. pA~4524B was partially -
WQ90/1~80 ~T/US90~020~0
7 ~
-29
digested with DraI. A 2260 bp fragment was gel~
purified, treated with CIP and ligated to the 5530
bp fragment to create pAbT4533, as shown in F.Lgure
4B. ~
The HIV-l strain RF env gene was obtained on --
plasmid pRF3HS from Blair Ferguson (E . I . DuPont
deNemours and Co.). pRF3HS.was digested with AvaII,
treated with Xlenow, digested with SpeI and the
resulting`500 bp fragment-.containing the 5' end of
the RF-env:gene'was gel-purified.. .pRE3HS was ..
digested with'_EcoRI,'treated'with-Klenow;-digestèd
w~th~SpeI.and_the resulting_2lO0 b ~ ~ragment con~
' taining the 3' end of the RF env gene was gel- ... `
purified. ~pAbT4533:was digested with:SmaI', treated
15 with ? CIP and'ligated'to:the-500-`bp:and-2lOO~bp
fragment~-~to create pAbT8523,-as shown'~in-Figure 4C.`;
ipAbT8523~was digested with'EcoRI~Ztreated with~
. Klenow~, L ligated-:to an~~Xba~ 1inker?'~New-~ngland' ~~
~'' BioLabs~, digested with-?EincII,sand ^a 900 bp frag- '
20 ment containing the 5' end of RF env was gel-puri- -
fied.-~pRF3HS was~digested with XhoI,-treated-with
Klenow~;~ligated to an-Sph~-lïn~èr ~New(~ngland ~ OS
BioLabs~ dige:;ted'With HincII and a 1700 bp'frag-
ment contàining the 3' end of RF en~ was gel- ' -
2S purified. -pAbT4533 was di~ested with XbaI and SphI ;
and:ligated to the 900 and 1100 bp fragments to form
pAbT8526, as shown in Figure 5A.
pAbT8526 was partially digested with StyI,
treated with Klenow, and a lO,600 bp fragment was
30 gel-purified and ligated to a BamHI linker '-
(dCGGATCCG; New'England'BioLabs) to create pAbT4082 -
WO 90/t2880 PCI/VS90/02060
20~37~
--30--
as shown in Figure 5B. pAbT4082 contains the HIV-l
RF env gene with a BamHI site inserted at the StyI
site at position 1600, at,the 5', end of the gp41-
encoding region, 30 bp 3' to the gpl20-gp41 junc-
5 tion.~ _ 3
--, .-. . . - EXAMPLE 3,
Construction of an IVR vector-containinq the--vac- .-
cinia virus thvmidine kinase-~ene flanked bY the,
~ qpl2Øl~ortion of~the HIV-l~strain RF~env~qene and~? ~
'~',, ,10,~ the HlV-l-strain BHlO~env qene-fused,to lacZ-~Flqure.j - ,,
,~59~ pAbT4082"was_ digested, w,ith_XbaI-and;,BamHI and,
~''the 1700_bpr fragment, co,n,taining;the-RE,gpl20 gene _ .~
t,,' , was;,gel~E?urified~ pAbT4075-was,,digested wit2~ BamHI,,-
."'.;; ~:15-~.parti.,al~.~dlgest,e,d~w~th,,,XbaI~andi~t~eated~.witL~ P, - ~
; 95,00,bp-f~ag~nent,i,w,as gel,-pu~i~ied;and ligated_.i ' '.
~i~.,' ~ .:..'''~',..~.to ~he~1700 bpo f ragment_to c,rea,te,,pAbT4083~ as.:shown;~
r" :- ,.. , in Fi~re 6A. s ~ - ",~ ~,r~ ;rg ' 2 ~.iJ ~:~Lr~ -^S . .. '' n?.'~
pAbT4~00,~-~U.S.~p~a~te-ntJapplication~seria~-No~
.y.;.-~i,,."p 2o:- 910,501~ su~ra)~,w,as dig~ested~1~with~sau31~Jand the~gooi
...... _ . :., . . .. , , -
bp Sragment containing the ~acc~nia~rirus",thymidine:
'~ kinase (tk) gen,e was gel-pur,ified.j~ pAbT4083 was ,~.
. --~.
digested~with_B~"~HI, tre,ated:with CI~"and ligated',toc, S
the~,,900 bEl ~ragment to, createjpAbT4085, as,,,shown in ~
25 Figure 6B. .~ ,r::; .. ..: ..-.
pAbT4085 contains the RF,gpl20,_unde~,the~_
control Gf,the ? ..SK promoter,- and the BH10- gpl60 ,-,
genes separated by the tk gene.,; ,,In addition, the ?
BH10 env gene is fused in-frame to the lacZ gene.--.
WO90/t~0 -PCT/US90/02~60
2~ 3~
-31-
EXAMPLE 4
.. . .
Insertion of tandem HIV-l env ~ene s~uences into
vaccinia virus and subseouent condensation of the-
. _ . .
tandem array to vield sinqle-copv hybrid env qenes
(Figure-7), ~
, DNA sequences contained within pAbT4085 contain
the tk gene flanked by two diverse env genes from
HIV-1 strains B~lO.and RF-- These env genes differ
.
b~.21% i~ the,amino acid sequence-of their exterior -
envelope proteins~(Starcich et al;r~lg86~:-cell J~
45:637) ,~Th.is~vector is.used.to.insert the~desired
'' structure into tk vaccinii .by.. in vivo~recombination.
Methotrexate is used.to'obtain and puri~y tk~'recom-
binants away ~rom,,the parenta~.tk, virusr~which '---~ :
cannot grow in the presence o.methotrexate.~ A- - '
recombinant~..,virus isi_t~ere~or~.generated whic~ q .
'' - contains both env genes as well as the tk ge~e;~.L~ ~La
~ ''(Pigure 7). ''
'- Because recombination between conserved regions
., _ . _, . .
. 20 of the tandemly arrayed e~nv genes results in dele-
;,i- ' , .. ,tio~_o~ th~,ii~tervening-sequences;:including the'.tk'
, . ~ . _ ~ _ _ _ . _ , ~ _ . _ _ . . . . ., . .. _
gene, vlrus which have undergone,condensation~,events
can be selecte~d using 5'-bromodeoxyuridine (BUdR)
which permits growth,,,o~ tk ,but not tk r virus~: As '
a ~number,of con~erved.regions spanning the env;~
sequence are available-for recombination,'a vaccinia
recombinan~ containin~:two different env genes in' :
tandem will~generate ~ diverse set of chimeric env '
genes. The E. coll lacZ.gene s included as a
marker; plaques resulting from the growth of virus`:~
WO 90/t2880 PCr/US90/02060
20~4379
--32--
containing both env genes will be colorless in the
presence of Xgal because there is no transcriptional
promoter to express lacZ, whereas virus containing a
hybrid env gene fused in-frame to lacZ will give -..:
5 rise-to blue plaques. .~
_, _ _, ~ __,,, _,,, __ _, , _ .. ~_ .. ._ _ . _ .. ,, _ .. ... . _. . . . . .. _ _ . .. . .. _ . . .
Plasmid pAbT4085 was transfected into BSC--40 .'-.
cells which.had previously -been infected with the
vaccinia -tk .strain WR 417 ~tSpyropoulos et aI r J- -
Virol.~62:1046-1054 .(1988 )) in the presence of'`
10 methotrexate_ :_When the progeny virus population was
- plated;in-the presence,-o.,f methotrexat'e 'a'nd-~overlaid 3
wi~h_X,gal~onl~_col-orless~(lacZ S:'p~aques-'were
,,, ' ' obsçrved~r::when the same::popu~:ation was` platëd in ~-
,' th,e_absence o~-methotrexate~and overlaid 'w'ith` Xgal,''
~-,i 15 blue plaques.:were- observed. Two blue recoDlbinants,--
;,~......... designated vAb'r168,-.l and~ vA~T168-2, were plaque-;; 5~
purifie.d.~i~ the~sence~~.ol~'meth~otrexa~i:;ànd2~ 9:
; 7~ t~ plified~9e ;~ g~iJ zf; L~sw 25 Z9f~9e V1~9 ri._o
z-~10-?~ 9v:~32nco .~Q~I,E.s.n~,~"~
-~Ls3 f2~ 2~ 29_ ~ ro 9'~'fi--S ~ ~.2e s L~
- 20 . Anatlvs~s-o~ ~ie~r~tructures~^of '-,two;~ acc~r~ia~-qenerated~
hvbrid env c{enes fused~-to lacz ?~Fi~ures 7, 8,- 9, 101-
~:~accin:~a~ ~cirus genomic: DNA Was~ prepalrèd from ~~~
.. th,q~, recombinar~t,~vAbT16~-1 d,eriv'ed' ~rom` the 'previous ~
Example. .) The~ DNA.was digested: with" HindIII and the '
25 r,esulting fragments separated by agarose gel elec-''-
trophoresis. r~rrhe restriction pattern' observed was '-
consistent with that expected Sor`'a recombinant ~
virus which had undergone a condensation event to'''
generate a hybrid env gene, as shown' in Figure''7.' A
,: , . .
~ . j , . -;
WO90/12880 - PCT/US90/02060
2~37~
3 kb fragment containing the putative chimeric env
gene was gel-purified and ligated to pAbT2009 (U.S.
patent application Serial No. 910,501, supra),
previously digested with HindIII and treated with
CIP,-to yield the plasmid pAbT4105, as shown in ~-
-; i Figure 8A.
j~The hybrid env gene in pAbT4105 was analyzed by
restriction enzyme mapping; a restriction map is
shown in Figure 8B. The-results~indicate that the
5' end~.of the-.hybrid env gene is derived from the RF ~;
-: env gene:and:the 3'~end:from thé BH10 env gene.~-The
~' ......... restriction analysis localized-:the:RF/BHlo junction-
' to a 118 bp region-between a StyI and HgaI site; as''
~' indicated in.Figure 8B'.
o~.To further.define the RF/BH10 junctionj - '
pAbT4105 was digested with 8glII~and'HgaI, treated` -
wIth.~l-enow and-a~2.00~bp-fragmenticon~ain'ing the~7~
hybrid junction-was-gël-purified'.-6Phagë ml3mpl8 DNA ' ..
.' . (New England BioLabs) was digested with ~incII,
'' : 20 treated with CIP and ligated.to the 200 bp fragment
-' to create pAbT4106, as shown in Figure 9A.
` - Single-stranded preparations were made of
pabT4106 and the 200 bp~insert sequenced'by..the'~
chain-termination-..method.'-'Sanger,'`Nicklen~and
Coulson,-~Proc; Natl. Acad..Sci; ~SA,~-74,~5463-~
_ _ _
~l9?7~ Tlhe corresponding regions:o~ the parental `'
RF and BH10 env genes from pAbT4085 were also se-
quenced.- The relevant.sequences of the RF, BH10 and --
vAbT168-1 hybrid env genes are shown in Figure 9B. `
Thus the RF/BH10 condensation event occurred within
a conserved 22.-bp region common to both the RF and
-34-
BH10 env genes, located 21-43 bp from the 3' end of
the RF env fragment in pAbT4085 (Figure 9B).
Vaccinia virus genomic DNA was also prepared
from recombinant vAbT168-2 and was analyzed by
Southern hypridization using HIV and DNA as a probe
The restriction map of the bybrid env gene from
vAbT168-2 is shown in Figure 10A. The map indicates
360bp sequence between a KpnI and a BsmI site in the
central region of gp120, between hypervariable
regions V2 and V3. This region of vAbT168-2 was
sequenced and is shown in Figure 10B. In vAbT168-2,
the RF/BH10 condensation event occurred within a
conserved 23 bp seuence located between nucleotides
758 and 782. vAbT168-1 and vAbT168-2 are thus two
recombinants containing different hybrid env-lacZ
genes derived by vaccinia virus-mediated recombin-
ation between adjacent RF and BH10 env genes.
EXAMPLE 6
Generation of hybrid env genes (Figures 11, 12, 13)
Having demonstrated the feasibility of using
vaccinia virus-mediated recombination to derive
hybrid env-lacZ genes, the generation of populations
of recombinant vaccinia containing full-length,
chimeric env genes was undertaken. Approach 2
(Figure 11) was used in this set of experiments.
First, the HIV-1 BH10 env gene (including I1 nucleo-
tides of 5'-proximal and 105 nucleotides of 3'
proximal untranslated sequences) was inserted, under
WO 90/1~0 2 ~ ~ ~ 3 ~ ~ PCT/US90/02060
-35-
the control of the vaccinia 40X promoter (designated
H6 in Rosel et al./ 1986 J. Virol. 60:436-449), into
....
the HindIII M region of the vaccinia genome using -
host-range selection ~See Smith,.International-- -~
patent application PCT/US89/02486, filed June 7, -~~
198g; this recombinant virus was designated vAbT271;
Next, a plasmid vectort designated pAbT4120, was
constxucted containing the 5'.portion of the HIV-l
RF env gene fused at nucleotide-1449 (i~mediately ~::
following hyper~aria~le region V~ in gpl20) to the :~ ;
lacZ gene~ .This vector_also~contained the ~ gene;
~a~kner~and Moss,:1988r ~ ~irol.~62:1849-1854J,~im
under,the_control of-the.vaccinia Dl promoter - ~~~~-
(Lee-Chen-et al.p~l988,'^Virology 163:64-79):,-for~
selection of.recombinantsr and a bacterial-replicon'' -
~and ampici~lin-resistance gene for:growth and'~
.'. 'se~ection,-~ E~--col;i,~`cCells were ~nfected-wit~
'.'.~',`~.; ': .. 'vAbT2~1~and3then Were.. transfeCted-with pAbT4120.~ A' .-~
.:. -single recombination event between.:rho~oIogbus~ ;r~;
20 . regions of the BHlO en~ gene in vAbT271 and the RF
env gene in pAbT4120 would result in the genomic'...-:- 0
- -,,-. structure_sho~n in-~E~gure~ ^containin~stwo:hvbrid
en~ genes.-,~Nine different recombinants,''designated'
vAb~373I~-were isolated-and purified'(using ~E~ and'
lacZ selection) to characterize these hybrid genes.
T~e presence o~ a bacterial replicon and ampr gene
facilitated the subcloning of the vaccinia genomic~
reg,ion containing the.5'-proximal hybrid gene.
Genomic DNA preparations-were digested with HindIII,
self-ligated..and used.to transform F,, coli to ''~
ampicillin-resistance Restriction mapping'revealed
a number.,of different hybrid junctions in these
intermediate recombinants (Figure 12). In four of
W090/12~0 PCT/US90/02~0
2~54379
-36-
these recombinants (recombinants 1, 3, 5 and 6 as
shown in Figure 12), qpt-selective conditions were
removed to allow the propagation of condensed virus
'' containing a:single, hybrid, full-length env gene.
5 The lacZ.gene served as~a marker-to distinguish ! - ~ ~
condensed-(colorless plaques) from uncondensed (blue
plaques) virus. After--four serial passages of virus
-. ,- under non-selective conditions, approximately 90%~of
the progeny plaques from each of the four popula-
: 10 tions were,colorless, indicating that'nearly'~all the
.' viral genomes had.condensed:to^a~single-env--gene. .-_
i'~'''" ~., : .;..,..: The,two~junction reg_ons in each-of-thë hybrid~genes
.~ ' from eight.individual recombinants (two~from'ëach~ ~-
,. population~, designated.vAbT373C'were mapped by~
, . .', 15 r,e,~striction,mapping of genomic DNA amplified by~the''
. . . , , ~
. polymerase chain reaction_ ,Restriction mapping~
"5~ s~ tFigures~3~-showed that eac~of.the_eight_vAbT3~3C~5 -.:-
`,.~"' ~' '`~'''''~' '-'^t`,~'~;' ''' r ses~,contain,;a:;uni ue~-hybrid-: :gene - o't o''-~''
:'.~..' '"~'''.~-. ''- ' were identica,~".chimeras~ 3~ r.c~ o^J~ -~L^r.
''" 20 De~osit,~.~o 9~ ; ~ *;~;? ~
~_~The.,plasmi~pABT4085~was p~aced on-deposi~~at~Z
the American Type,,Culture Collection in RockviIIe~ ~,
Ma,r,yland on october~6~i,1988:and.assigned Accessio'~
No,.~67~818., d~ 3 .- 31~.- 9 S ~ G'~ r .0 ~ Z"3 . . '~ r
9 n ? r? ~ ~ ~J -j ~ 9 ~
Equivalents -, _,- ,, ,, ~ , ~ .~,... .
Those,skilled in.the art will recognizer or be
able to ascertain using no more than routine ex~
perimentation,~. many equivalents to the specific ' ~'
embodiments of the invention described herein.:-:Such
equivalents are intended to be encompassed by the~ '
following claims.
W O ~0/12~80 37 20~ PCT/US90/02060
PCl' ApDlicant's Gu7d- - Vr~lunc r - Annei~ ~3
A~`''EX 1!3
.
ImerTnt~loll~tl A~tpll~011 hlo PCTI
. MICROORGAN IS MS
_ - ~ ID~ c~ns)~ o~ ~t~t~t~
. . _ .. . . .. _ ... . .
rt~ tl_t I~oa e r t naotme~l ~ntl t O' __
l~o el o~e~ ~ntrrm~ '
. ' _ . . ~ ,, . _ , -
American Type Culture Collection
. .... . _ _
~ t~ I v~ r~ ~n nnctw no ~o~t~t Ce t- ~t 60 m~ -
12301 Parklawn Drlve
Roc~rille, ~aryland Z0852
United-States of America -
. . _ _ . _._
D-t~t ol r~t~t ~eet~7n ~t~rn~ r -
,- ~ 06 0CTOBE~1988 i-3cir~iJ ~ 1' ~
. rnri lo 88) 67818
. _ . . ... _ _, ... _ _ . .. _ .
1- AOOmOI~ DIC~T1011~ 014tlt d no~ D~ ltl-l Tt~ ~nlo~t n ~D~on~o qn ~ r~ rr en~ u~ J
~ _.. _~
In respect of those designations in which a European Patentis sought,
the Applicant hëreby informs the European Patent Office under
European Rule 28(4) that,-until the publication of the mention of ch
- grant of the European Patent or until the date on which the European
" ~ Application has bee~ refused or is withdrawn or is deemed to be
~ ;~; - ~~ Xc withdrawn~ the aYailability of~the-biological materi~l deposited with
- the America Type Culture Collection under Accession ~o.67818
shall be effected only by the issue of a sampletoanexpertnomina~ed
: by the requesl:er in accordance ~ith European 'Aule 28(5).
C O~lOl~tAT~D ~ ro~ t~tlCK I~DlCAnO~t--~t~ lilt~DII ' (11 tn- ~ntl~cstlr~n~ U- n ~or ~11 Ct~on~
. . -
.__ ~
:~., ? ~ `' ~ -, 't
n. ~P~A~ ru~nl~ o 0~ lc~no~ (b~ ~n~1~not~D~DI-~
_ _ ,, _ _ . . .. . _
~b~ no~ollo~ r~t o~ uornm-t lo 1~ Int-rn~no~tl 1~ utVr~sWCnr tn~ ~I n~n ol t~ In~Uon~
~cenc ~on ~t~no~ ol D-t~o~ul )
. , _... .
.. ...... ....
_;~ ,
~. ~
, ..
_ . _ . _ . _ - _ - _ _
T~ n~t ~ I D . ln ~n- ~nl-~n-l~on-~ ~DD~ on ~rn~n O~o llo ~J cn4~t Itr ln~ no otncn
(~I~tno D 01~ __
, T~ o~ -e- D~ om ~n ~ DDIIC-n~ DT ~n- Inl-~n~ on-~ no~ ~ Crn
_~ _ __ _ . _.. __._____________._.__ . ~_
lnorl~ om~." c
! ~
