Note: Descriptions are shown in the official language in which they were submitted.
~'` 93/14126 212 8 211 PCT/GB93/00102
A MAJOR HISTOCOMPATIBILITY COMPLEX CLASS II ANTIGEN IN A VACCINE AGAINST AN
IMMUNODEFICIENCY VIRUS
This inYention relates to vaccines against
immunodeficiency ~iruses.
There has been pessimism about the prospects for a
successful vacci~e against AIDS. The obstacles have often
appeared to be insuperable. An effec~ive vaccine must prevent
infection by a ~irus which destroys CD4' cells, which can
integrate into the host DNA and which exhibits rapid antigenic
variation. Furthermore, protection must be ef f ecti~e at
mucosal surfaçes, the primary site of entry, a~d against both
cell-free a~d cell-associated virus. The simian
immunodeficiency ~irus (SIV)-macaque model, developed in the
:~ : USA1~2, was adopted by the:~United Xingdom MRC AIDS Directed
Programme with the primary objecti~e of;establishing that
vaccination was feasible and that these obstacles to success
: could be overcome.
We have now demons~rated that a major
histocompat:ibility complex~(MHC) class II antigen can protect
animals in th~ SI~-macaque model. :Accordingly, the inv~ntion
20:~pr~vides~a class II:~antigen for use:in;a me~hod of treatment of
the:human or anîmal body ~y therapy,~in particular for use as a
:vaccine against an immunodeficiency ~irus.
The invention~also provides a pharmace~tical
composition compris~ing ~ pharma~ceutically acceptable carrier or
25~ d~iluént~and,~as:active lnyred}entj a MHC class II antigen. The
inYention ~urther providPs use of a MHC class II anti~en in the
:: ma~nùfacture~of~a~medicament for use as a vaccine against an
mmunodeficiency~viru~
: n ~ The antigen is-pref~rably a human class II antigen.
The antigen m y~therefore be a HLA-DP, HL~-DQ or ~LA~R antigen
such as the ~LA-DR4 antigen. These are known antigens and can
be:obtain~d~in puri~ied form. Th y may be prepared as
recombinant:proteins.~
Alternat:ively,- the class II antigen may be gi~en
presented by transiected cells, i.e. by cells t~ansfected with
a gene encoding the antigen and which consequently express the
: ~ ~ antigen . Transfected cells which may be administered to a
human may be transfe:cted cells of a human diploid cell line.
Such cell lines have been tested for safety for the purpose of
~: :
WO93/14126 21~ $ 21 ~ PCT/GB93/00102
-- 2
human vaccine manufacture. An appropriate cell line is the
MRC5 cell line.
Allogeneic lymphocytes whirh present a class II
antigen may be administered to a patient. The lymphocytes may
be given as li~e cells, for example as a blood transfusion.
Alternati~ely they may also be given as fixed or inactivated
cells. The lymphocytes may be ones in which the expression of
the class II antigen has been enhanced, for example by
stimulation with a mitogen or gamma-interferon~
The antigen may be used to vaccinate a host against
an immunodeficiency virus. The host may be a human or animal
but typically~it will be wished to vaccinate a human against a
human immunodeficiency virus (HIV). That virus may be HIV~l or
HI~-2. A prophylactic treatment for disease states
I5 attributable to infection by an immunodeficiency virus can
there~ore be provided. The class II antigen may in particular
act as an AIDS vaccine.
An~effective amount of the antigen is administered
to:~a~host it is wished to vaccinate. The antigen in whichever
20~ form~, can be given~parenterally, for example subcutaneously,
intramuscularly or~intravenously. The amount of antigen per
dose:depends on a variety of factors such as the age and the
condition of the~subject involved. A parenteral dose typically
; consists of from:20~g to l mg of antigen, for example from 50
2~5 ~t~o~500 ~g of ant~igen.~ A numb:ér of doses may be given, for
example from~2~;to 4;~d~oses over a period of up to six months.
: Each~:dose may:be~glven~one~or~two months apart.
An agent~f~or use as a vaccine against an
immunodeficiency virus: is~ therefore provid~d. A pharmaceutical
3Q composition also comprising~a pharmaceutically accepta~le
carrier or diluent can be formulated. The composi~ion is thus
sterile and pyrogen-free. The composition may also comprise an
adjuvant such as Al:(~OH)~3;or saponin~
Compositions for intramuscular or subcutaneous
~; 35 injections may contain together with the antlgen a
pharmaceutically accepta~le carrier, e.g. s~rile water, olive
~:~ oil, ethyl oleate, glycols e.g. propylene glycol, and if
desired, a suitable amount of lidocaine hydrochloride~ The
solutions for intr:avenous injections or infusions may contain
W093/14126 21~ ~ 21~ PCT/GB93/00102
as carrier, for example, sterile water or preferably they may
be in the form of sterile aqueous isotonic saline solutions.
The MHC class II antigens can be ~afely used by
virtue of their negligible toxicity.
S The following xamples illustrate the invention.
Example l
Inacti~ate~ Vac-ines
In initial experiments relatively crude, inacti~ated
` vaccines were deliberately used (Table l). The virus infected
C8166 ceIls ~Virology, l~, 51-64, 1983 in which the cells are
called C63/CRll-2 cells) or partially purified virus,
inactivated ~ither by aldehydes or ~-propiolactone, were giYen
to gr~ups of three or four cynomolgus macaques. Four doses of
vaccine were administered with a rest period of at least six
months between the thi~d and final doses. Three different
adjuvants were used, either Quil-a (a purified saponin), SAF-l
' (Syntex emulsion containing threonyl muramyl di-peptide) or,
Freund's adjuvant.~Each group of vaccinated animals, together
with~a group of~unvaccinated controls, ~was challenged
20 ~intravenously with~10MIDsO~of the 3ZH isolate of SIVmac25l, two
weeXs~after the~fina1~dose~of vaccine.~ All control animals
became~infected. ~Virus was repeatedly isolated and proviral
DNA~detected in~peripheral blood~1ymphocytes after
amp1ification~by~polymerase chain~reactio~. Furthermore
; 25~signiflcant anti~ody responses to SIV were detected. In
contrast~there~was'~no evidence of virus infection to any of the
vaccinated~anima~1~s~by any~of these criteria. These experiments
",'~ with~,inactivated~;virus~vaccines have~been~ extended to show that
the' immunization~s~chedule can be;reduced to three doses given
at~monthly in~ervals. ~The duration o~ protecti~n was asses ed
by re-challenging animal~ four to six months after the final
dose~of ~accine~ Five~'of eight macagues were protected. Thes~
. ; ~,
resul~s together with~other published data2~3~4~5 demonstrate
that inactivated~vaocines induce a powerful protection against
35 ~SIV infection~in macaques and~that this protection is still
detectable at least S months~after the completion of
vaGcination . ;~
:
Cross-Protection
The ~readth of protection induced by SIV vaccines
was investigated by vaccinating eight rhesus and eight
cynomolgus macaques with formalin inactivated SIV using SAF-l
as adjuvant. Two weeks after the fourth dose of vaccine, four
rhesus and four cynomolgus monkeys were challenged with the
homologous virus, All eight animals were completely protected
against infection. The four remaining rhesus monkeys were
cha}len~ed with lO~ID50 of SIV~It~ (kindly supplied by Dr. M.
Murphy-Corb). These animals also resisted infection. The
remaining four cynomo~gus macaques were challenged with lOMIDso
of HIV-2SBL~9 ~kindly supplied by Drs. P. Putkonen and G~
~Biberfield). These:animals all became infected. Analysis Qf
the viruses involved in these cross-protection experiments
revealed that SIVmao251~and~SIV~lta share 83% identity in the
amino acid sequences:of their:~envelope proteins. In contrast,
SIVmac251 and HIV-2sB~are only 73% identical in the envelope
protein. The antigenic~diversity of these viruses was
established using~a:panel~of 30 monoclonal antibodies made
against the envelope:~protein of SIVmac251. Although al} of
these antibodies reacted with the vaccine virus in an ELISA
~assay, 11 failed to~reaot with~SIV~lta and 20 failed to reach
with:HIV-2sBL. These~results indicate that inactivated vaccine
~prepared fr~om SIVmac~completely~protects animals against
~chal:lenge with the antigenically distinct strain of SIV~Ita, but
~that~this ~cross-protection does not extend to the more
~distantly related~HIV-2 virus.: Thus, the antigenic variability
:of immunodeficie~cy:~viruses may not be as big an obstacle to
~successful vaccination:as~was originally feared. However, this
~conclusion~may~require~;reinterpretation in the light of anti-
cell responses:~discussed below.
Mucosal Immunity~ ~
The~problem of inducing protection at a mucosa}
~surface was investigated using the intrarectal route of
challenge. ~The~standard~challenge virus poo~ of the 32H
isolate of SIVmac251, ~ which had been used in all the previous
intraven~ous challenges,~was first titrated in rhesus macaques
using the intrarectal route. One thousand times more ~iru es
was required to infect monkeys by this route, but ~he
~subsequent course of infection was essentially
indistingulshable from that following intravenous inoculation.
~93/14126 ~ 1 2 ~ PC~ 3/00102
Four rhesus macaques were then vaccinated subcu~aneously with
formalin-inactivated SIV using SAF-l as adjuvant. Two weeks
after the fifth dose of vaccine the animals were challenged
in~rarectally with lOMID50 based ~n the intrarectal titration.
Four unvaccinated control animals challenged at the same time
all became infected. In contrast, all four of the vaccinated
anima}s have remained uninfected over a peri~d of at least six
months. This experiment indicates that immunity can be induced
against challenge via a mucosal surface.
ChalIenqe with Cell-Associa~ed Virus
A cell associated challeng~ virus stock was prepared
from ~he spleen of a cynomolgus macaque J82 which had been
infected with the 32H isolate of SIVmac251 ten weeks
pre~iously. Aliquots of the cells were cryopreserved and then
titrated in vi ro by co-cultivation with C8166 cells (Table 2~.
The infectivity titres of the cells and their supernatant fluid
were log10 4.5 and 2.5~respectively. Thus 99% o~ the
infectivity was cell-associated and one I3so was ~qui~alent to
72 viable c211 Subs qu2nt titration of the spleen c~lls in
:20 viv~o in monkeys gave an end-point of log10 3 . 0 with on~ ID50
being equivalent to 2,300 cells. Having ~repared a~d titrated
intravenously an appropriate cell~associated YirUS challenge,
four cynomolus macaques were selected which had previously been
vaccinated subcutaneously wit~ inactivated SIV and shown to be
:25 protected against~intraveno~s cell-free virus challenge. These
animal~ which had remained free o~ virus for twelve months
following lnitial challenge were revaccinated and two weeks
later challenged intravenously with cell-associated virus
~ (Table 3). The four vaccinated animals, together with four
:~ 30 unvaccinated contr~ls~, all became:infected~ Virus and proviral
DNA were detected repeat~dly in the peripheral blood
lymphocytes. T~us a vaccine which had protected again~t
intravenous~challenge~with cell-free ~i~us grown in a human T-
:cell line failed to pro~ect against SIV infected simian spleen
: 35 cells
.
Rec~mbinant Vaccînes
The specific compounds within the inacti~ated
~; vaccine which were responsible for the pr~tection were next
sought by immunization with a variety of recombinant proteins
- 6 - PCT/GB93/00102
derived from SIV genes~ Groups of four monkeys were immunised
either with p27 expressed on yeast ~irus-like particles and
combined with aluminium hydroxide, or with purified gpl60
derived from a recombinant vaccinia virus, or gpl30 expressed
in CH0 cells, or gpl40 expressed by baculo~irus. Each of the
envelope proteins was administered with the Syntex adjuvant
formulation. ~accines were given in four doses and the animals
were challenged With lOM~Dso of SIV two weeks after t~e final
dose, to~ether with groups of four unvaccinated control
animals. ~11 of these monkeys became infected except one which
was vaccinated with the~baculovirus derived gpl40. Thus
although recom~inant proteins were able to induce high titres
; of antibody against SIV envelope, they were not able to protect
anLmals ag~inst intra~enous challenge.
~5 Immune Correlates of Protection
The immune responses which correlated with
~; protection were analysed by measuring antibo~y titres in sera
taken on the day of challenge from 55 vaccinated macaques used
`in~:thes:e studie-~. Forty three animalæ had received inactivated
vaçcines and 12 a recombinant envelope protein (Table 4).
Neutralis:ing antibodies were measured against SIVmac251 grown
as a persistent:infection in HUT-7~ cells. The mean titre of
~: neutrali;zing antibody in the~group of 32 macaques which
received :inactivated~vaccine and wère protected was log10 2.0 +
0.5i~: The same mean v~alue was found in the group of 11 animals
which~were unprotected.~ Furthermore the 11 animals vaccinated
wi~th re:co ~ inant~envelope proteins and unprotected, had a
higher mean~titre~of log1~0 2.9 + 0.~. Thus there was no clear
correlation;be~ween~ titres of neutralising antibodies and
protection in these animals. Titration o~ these sera against
: recombinant en~el:ope gp~40 by ELISA also failed to show any
:correlation with:protection. Similarly, although these
: vaccines in~uc d strong T-helper cel~l proliferation responses
to~5IV,~ and in some cases MHC class-II restricted cytotoxic
35~ cells, there was no obYious correlation between the cellular
responses to SIV and protection. Our failure to find any
~: correlation between the powerful pro~ection we have. observed
- following vaccination and any of the immune responses which we
had measured was disturbing. Howsver, it is possible that the
l h ~
~93/14126 PCT/GB93/00102
-- 7
immunological assays we used were inappropriate.
Res~onses to Cell Components
At this point results of a further vaccine
experiment began to emerge which offered explanation for our
observations (Table 5). This experiment was originally
designed to examine if the two doses of vaccine were sufficient
to protect against intravenous challenge with cell-fee virus.
; Four cynomol~us macaques were vaccinated with SIV-infected
C8166 cells using Quil-A as adjuvan~ at weeks 0 and 4. A
l0: control group of four animals were similarly vaccinated but
with unin~ cted Cal66 cells. Both groups were c~allenged with
~: l0MIDso of virus two weeks after the second dose of vaccine.
; ~ One of the four animals~vaccinated with SIV-infected cells
: became infected but,~:surprisingly, only two of the four;~ 15 vaccinated with uninfected~cells became i~fected. In order to
confirm these surprising results the protected animals were
; further vaccinated~at week 26 and re-challenged two weeks later
toge*her with foux~naive control macaques. Partial protection
was again observed in:the animals immunised with uninfected
C8166 cells, whereas: al1 four unvaccinated co~rol animals
became infected. ; Antibodies to the cellular component of these
vaccines were measured~by ELISA using a detergent lysate of
::C~16~6:cells as antigen~(Table 6). The mean titre of antibody
n the eight protected animals~was~log10 3~5 and in the five
:25:~ unprotected animals~ Og10 2.~4. The difference between these
two groups was high1y~significant.~ Analysis of ~nti-cell
antibody :levels~in:;all the animals which had received
inactivated vaccines~howed a similar difference between
protected~and unprotected animals. Thus there was a
~statistical correlation between the titre of antibody to C8166
cells and prsteçtion in thes~ animals.
Conc1usions
These:studies demonstrate that at leact 3 differe~t
inactivated Yaccines protect against homologous cell-free SIV.
~-~ 35 The protection induced is potent since neither virus nor
proviral DNA can be detected in the ~accinated animals over
~ prolonged perlods~fol1Owing challenge. ~ive di~ferent
: ~adjuvants and a:variety of immu~ization procedures are
effective. The inactivated vaccines pro~ect against
WO93/14126 PCT/GB93/00102
82~ ~ - 8 -
heterologous challenge with SI~delta but not against HIV-2. The
immunity against challenge is reduced but still detectable at
four and eight months post-vaccination. Parenteral vacci~ation
with inacti~ated virus protects against intrarectal challenge
with cell fr2e virus, but not against intravenous challenge
wikh SIV-infQcted ~imian spleen cells. Three different
preparations of SIV envelope protein were shown to be hig~ly
immunogenic, but failed to protect against live intravenous
challenge. The protection observed failed ta correlate with
any of the immune reactions to SIV which were measured.
Howe~er there was a correlation between protection and levels
of antibody to C8166 cells. These results suggest that the
protection observed may be mediated at least in part, by immune
res~onses to cellular components present within the inacti~at~d
vaccines.
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-- 15 --
Example 2
To ~onfirm the protection influenced by uninfected
human T cells, a second experiment was initiated (Table 6).
Groups of 4 cynomolgus macaques were vaccinated with either
C8l66 cells ~a human T cell line) or RK-13 cells (rabbit kidney
fibroblasts). A third group acted as naive controls. The
cells were gent}y fixed with 0.075% glutaraidehyde and combined
with Quil A ~a purified saponin) as adjuvant. Each d~se
comprised 2 x 108 cells and lO~g of ~uil A. The vaccines were
administered subcuta~eously at 0, 4, 8 and 16 weeks. Two weeks
; after the final dose of vaccine all 12 macaques were challenged
with lO monkey infectious doses (MID50) of simian
immunodeficiency virus (SIVmac32H) which had been grown in
C8166 cells. Virus and pro~iral DNA:was detected in all the
control animals~and those vaccinated with RK-13 cells but in
. only two of the four given C8166 cells (Table 2.l).
To:confirm:and extend this obser~ation the two
protected animals were given another dose of C8166 cells at 30
weeks. Two weeks later they, and four naive ~ontrols, w~re
;20:~:cha11enged with lO::MIDso of an antigenically distinct virus,
; SIVs~3~which had been grown in human peripheral blood
mononuc1ear cells (PB~C):from at least~two donors. The
controls were all infected but the two vaccinates remained
: protected. ~
2~5 ~ ~ :: Finally, the protected animals were vaccinated again
at~:44~ weeks and:challenged:, together with four controls, with
0:MIDso of SIVmac25~1:grown in simian PBMC. All the animals
became:infected~
This experim`ent confirms that uninfected human T
30~ cells protect against at least two antigenically distinct
strains of SIV grown in human T cells which need not be
identical with the cell;s used as the vaccine. This protection
did not extend to SIV grown in simian cells.
. ,
:: `
::
WO93Jl4126 ~ PCT/GB93/00102 -
~ 16 -
Table 6: Uninfected Cell vaccines
.... _._ _ _ . , ~ . -- _ _ I
tlACC:~NE DOSES OUTCO~ ~E OF CH ~LL.ENGE
1st 2nd 3rd l
, . . . . I
Uninfected C8166 cells + 0,4,8,16 2/4
~: Quil A 30 0/2 2/2 _
Uninfected RR-13 cells + O,4,8,16 4/4 _
~ . , . .
None _ _ 4/4
:: ~ None ; : _ 4/4
: 10 None 4/4
~ ~ ; C~ ~ ~ ~
No. monkeys infectedjNo. monkeys challenged
Exam~le 3 ~ :
~: 15 The major antigens~ present on the surface of
al~logeneic or xenogeneic~T cells are the major
hiætocompatibility:~antigens (MHC) class I and class II. To
determine i~f thes~ w~ere~responsible for the protection observed
groups of four cynomolgus macaques were immunised with either
0 ~a)~normal:mouse fibroblasts (L cells), b) L cells (8024 line)
trans:fected with~the human genes for ~ C class I tHLA B7 + ~2
mi~roglobulin)::or~:~c)~L~cells (8115 line) transfected with the
human~genes~for MHC~class II (HLA-DR4). By fluorescent
an~ibody~staining:,:over 90% of 8024 and 8115 cells were
:25~ expressing class I:or class II~antigen respectiveiy. The cells
; ::were gently fixed~in~0.075% glutaraldehyde and combined with
~ , . . .
10~: of Quil A~as:;adjuvant ~Table 7). Animals w~re given 2 x
cells subcutaneously on four~occasions at 0,4,8 and 16
weeks. Two weeks after the last do~e, all twelve animals w~re
~i ~ 30 ~challenged intràvenously with 10 MID50 f SIVmac32H grown in
C8166 cells. Al:l~the animals in groups a) and b) ~ecame
infected but only two of;four given:cells expressing class II.
; This result demonstrates that human MHC class II,
~: : : namely HLA-DR4, can protect animals against SIV grown in human
~ : 35 T cells.
: ~ : :
~1Vf~ 93/14126 ~ 1 2 ~ 21 i~ PCI`/C;B93/00102
-- 17 --
Table 7: MHC Class I or Class II Vaccines
~ . = = = l
VACCINEDOSES ~WEEKS ) OU'rCOME
a) Normal L ceIls 0,4,8,16 4/4
b) L cells (8024) 0,4,8,16 4/4
expressing class I
c) L cells (8115) 0,4,8,16 2/4
expressing class II
. "..... _ ~_
.:
No. monkeys infected~No. monkeys challenged with SIVmac32H.
~ 10
; References
1. Desrosiers RC, Ringler DJ. Use of simian
immunodeficiency viruses for AIDS research. Int
Virol 30, 301-312 (1989).
`15 2. Desrosiers RC,~Wyand MS, Kodama T et ~l. Vaccine
protection against SIV infection. Proc Natl Acad
Sci,~ New York,~86, 63s3-s? (1989).
3.~ Murphy-Corb M~, Martin LN, Davison-Fairburn B et ~l.
A formalin-inactivated whole simian immunodeficiency
2~0 ~ virus va~ccine~confers protection in macaques.
Science~246, 1293-97~(1989).
4~ Stott E3;,~Chan WL, Mills KHG et al. Preliminary
Report~ Protection of cynomolgus macaques against
simian~;1mmunodeficiency virus by fixed infected cell
~ vaccine.~ Lancet 336, 1538-41 ~l990).
5~ Carlson JR,~ McGraw TP, Keddie E et ~l. Vaccine
protect~ioni~o~f~;rhesus macaques against simian
immùnodeficiency~virus infection. AIDS Res Hum
Retro~6~ 23~9-~46~(1990).
30~ 6.~ Stott~EJ~ Kitchin PA,~Page M et al. Anti-cell
~` ; antibody~in~macaques. Nature 353, 393 (1991).
: ~
~: :`: :: ~ :
~:::; :: : :
