Note: Descriptions are shown in the official language in which they were submitted.
W093/03762 PCT/AU92/0~23
15~2~
Immuno~timulation
Field of the Invention
The present invention relates to the use of
costimulator inducers to augment or to boost the immune
response in patient~ with deficiencies in T-cell function,
in particular helper T-cell function, and to novel
compositions useful in increasing responsivene~s of
T-cells, in particular helper T-cells. Nore specifically,
the invention relates to the use of the E. coli
outer-membrane proteins OmpA, OmpF or TraT to augment the
immune response to antigens in immunocompromised
individuals and to compositions including these proteins.
Background of the Invention
During the development of an immune response, a
~15 certain type of T-cell, known as the helper T-cell, which
;~bears the CD4 phenotype, is required to assist the B-cell
to differentiate into a plasma cell which in turn secretes
~oluble antibody. Before undergoing activation and
proliferation, helper T-cells, with the aid of the T-cell
receptor and other accessory molecules, must first
recognise antigens on th~ surface of antigen-presenting
cells (APC) such as macrophages, dendritic cells or
B-cells. Nore recent data are consistent with the
hypothesis that ~-cells require two signals for their
acti~ation. One signal is delivered as a result of the
binding of a peptide to a Class ~I Na~or
histocompatibility complex (MHC) molecule on the APC, and
the subsequent interaction of this NHC-peptide complex
with the T-cell receptor. Although a necessary condition,
T-cell receptor occupancy by the MHC-peptide and its
associated biochemical consequences are not sufficient to
induce T-cell activation. For most cells, a second signal
or co-stimulator molecule must be provided by the APC
(Lafferty, Prowse and Simeonovic, 1983; Mueller, Jenkins
;35 and Schwartz, 1989). Bacterial products, such as LPS and
:
WOs3/03762 PCT/AU92/OW23~
2i1~24 `
- 2 -
BCG, which have been shown to elicit co-stimulator
activity from APC are known ~s costimulatory inducers
(Janeway, 1990). It is suggested that costimulation
inducers will augment or improve the immune response of
individuals suffering from Immunodeficiency syndromes
which ~re associated, at least in p~irt, by a lack of
helper T-cells.
Acquired Immunodeficiency Syndrome (AIDS) is a
debilitating disease of man characterized by high
morbidity and mortality of infected individuals. The
disease, which is characterized by an initial infection
with a lentivirus, the Human immunodeficiency virus (HIV),
is diagnosable in its early stageæ by the presence of
antibodies in serum against the HIV and/or the presence of
the virus in the serum of asymptomatic individuals.
Almost without exception these asymptomatic individuals go
on to develop full blown AIDS with its many associated
complication~, which ultimately leads to death of the
inifected individuals.
During the course of the disease, HIV positive
~: individu~ls show a progr~ssive depletion of their helper
T-cell population tCD4-positive cells (CD4 )1 with an
increase in the numbers of CD8 cells. Accompanying the
1088 of CD4 cells, infected individuals show a
progressive loss of their ability to mount a protective
immune response to HIV, or to a number of opportunistic
pathogens which may invade the infected individuals. This
chronic depletion of helper (CD4+) T-cells, and the
resultant impairment of cell-mediated immunity, correlates
closely with disease progression towards AIDS (Fahey et
al, 1990; ~ange et al, 1989).
In an attempt to halt the s~read of AIDS amongst
at-risk individuals, and to develop a cure for the
treatment of infected individuals, many research groups
have concentrated their efforts on the development of a
~:
W093/03762 PCT/AU92/~23
_ 3 _ 2~ 24
vaccine again~t the HIV. Thus, since the initial
isolation of the HIV, some 10 years ago, many
~ophisticated virological and biotechnologic~l approache~
have b~en used in the de~ign and production of a multitude
of candidate vaccine~ (Hu et al, 1987; ~ennedy et al,
1987). Almo~t without exception the variou~ vaccine
candidate~ have failed dismally in clinical trial~. The
failure of the many vaccine candidate~ and the inexorable
progression of the di~ease in infected patients towards
full blown AIDS, has led many to the pessimistic view that
the development of an effective AIDS vaccine i8 almost
impossible. This view is not, however, shared by
Desrosiers and co-workers (1989) who have recently
reported promising results vaccinating rhesus monkeys with
`~ 15 inactivated HIV preparations.
One of the ma~or problems in the development of an
effective vaccine to combat already e~tablished HIV
nfections i8 that the HIV itself invades and insctivates
the CD4~ helper T-cells, which are the very cells which
mu~t be stimulated for an individusl to mount a protective
immune response. During the course of a normal immune
respon~e, activated CD4 helper cells produce cytokines
` such a8 Interleukin-2 (IL-2) which are known to drive the
clonal proliferation of primed T-cell~, which can
ultimately lead to the elimination of virally infected
cells. It has been shown, however, that the addition of
exogenous IL-2 to the peripheral blood mononuclear cells
(PBNC) of HIV-seropositive individuals, can restore both
antigen- and mitogen-driven blastogenesis in vitro (Bell
et al., 1990). Similar stimulation of PBMC in vivo,
leading to the production of CD4-derived IL-2, would help
to maintain strong CD8-associated antivir~l immunity. It
would appear, therefore, that there is considerable merit
in adopting therapies that lead to an increase in the
level of CD4~ T-lymphocyte~, particul~rly during the
`.
W093/03762 PCT/AU92/0~23~i~
211S~24
-- 4 --
asymptomatic stage of HIV-induced disease.
Nevertheless, IL-2 which has been recommended for
approval by the FDA ( Stone R., Science, 255, 528, 1992)
as an immunotherapeutic for the treatment of kidney
cancer, has a number of disturbing side effects
including...."circulatory problems that can be as severe
as heart attacks and strokes".... There is clearly a need
therefore for an effective immunostimulant which i5 devoid
of the undesirable side effects attributed to IL-2.
One approach to improving the prognosis of
HIV-infected individuals, would be to increase either the
absolute number, or the responsiveness of helper T-cells
in HIV-infected individuals and thereby improve the
individual~s capacity to mount an immune-attack on
HIV-infected cells and to develop effective responses to
opportunistic pathogens.
In international patent application No PCT/AU87/00107
it is disclosed that in complexes with an immunogen TraT,
OmpA and OmpF act as pvtent immunoad~uvants in
immunocompetent hosts. There is, however, no disclosure
in this reference that TraT, OmpA or OmpF have any ability
.
to increase the responsiveness of helper ~-cells.
The present inventors have made the surprising
finding that these proteins increase the responsiveness of
helper ~-cells from patients suffering a deficiency in
helper T-cell function. Further, the present inventors
have found a synergistic effect on helper T-cell
responsiveness between these proteins and other antigens.
Summary of the Present Invention
Accordingly, in a first aspect the pre~ent invention
consists in a composition comprising in admixture a
protein selected from the group consisting of TraT, OmpA,
OmpF and parts thereof, at least one other antigen and a
pharamaceutically acceptable carrier.
In a second aspect the present invention consists in
`
W093/03762 PCT/AU92/~423
211~ 42~
-- 5 --
a compo~ition comprising a protein selected from the group
consisting of TraT, OmpA, Omp~ and parts thereof, coupled
to an antigen selected from the group con~i~ting of HIV
antigens, influenza virus antigens, diphtheria antigens,
whooping cough antigens, measles antigens, tetanus
antigens, Pneumocystis antigens, Candida antigen~,
Toxoplasmosis antigens, CytomegaIovirus antigens, hepatitis
antigens, polio antigens, combinations thereof and
individual subunit proteins, peptides or polysaccharides
isolated from said antigens, and a pharmaceutically
acceptable carrier.
In a third aspect the present invention consists in a
method of increasing immune responsiveness in a patient
with an immunodeficiency, the method comprising
administering to the patient a composition comprising an
effective amount of a protein selected from the group
con~isting of TraT, OmpA, OmpF and parts thereof and a
pharmaceutically acceptable carrier.
In a fourth aspect the present invention consists in
the use of a composition comprising an effective amount of
a protein ~elected from the group con~isting of TraT, OmpA,
OmpF and parts thereof and a pharmaceutically acceptable
carrier, diluent and/or excipient in the manufacture of a
medicament for increasing immune responsiveness in a
patient with a deficiency in immune function.
In a preferred embodiment of the present invention the
responsiveness of T-cells is increased and the patient has
a deficiency in T-cell function.
In a further preferred embodiment of the present
invention the T-cells are helper T-cel~s and the patient
has a deficiency in helper T-cell functions.
In a preferred embodiment of the present invention the
pharmaceutically acceptably carrier is a hydrophobic depot
carrier. Suitable depot carriers include alhydrogel,
proteosomes and liposomes.
W093/03762 PCT/AU9V~2
211~42~ ~
-- 6 --
In a further preferred embodLment of the present
invention the at least one other antigen is seleeted from
the group consisting of HIV antiqens, influenza virus
antigens, diphtheria antigens, whooping eough antigens,
measles antigens, tetanus antigens Pneumoeystis antigens,
Candida antigens, Toxoplasmos i8 antigen~, Cytome~alovirus
antigens, hepatitis antigens, polio antigens and
combination~ thereof and individual subunit proteins,
peptides or polysaceharides isolated from said antigens.
It is presently preferred, however, that the at least one
other antigen is a HIV antigen, diphtheria toxoid or
tetanus toxoid, and most preferably a HIV antigen seleeted
from gp4118] peptide and V3 loop peptide.
In yet a further preferred embodiment of the present
invention the protein is TraT or a part thereof.
~; Tr~T, OmpF and OmpA are outer membrane proteins of
Gram negative baeteria. The TraT protein is an outer
membrane protein of eertain strains of E.eoli whieh is
re~ponsible for the resistanee of these strains to killing
by serum. The OmpA and OmpF proteins also fall in the same
elass of proteins. These proteins may be obtained from
other Gram negative baeteria sueh as E.eoli or Salmonella
speeies. It is, however, presently preferred that the
proteins are obtsined from strains of E.eoli.
The studies presented in this invention have shown an
ability to enhanee the level and/or aetivity of helper
T-eells in individuals with deficiencies in helper T-eell
function which indicates that TraT, OmpF and OmpA of
E. eoli and parts thereof ean function as eostimulator
indueers separate from, but with similar function to, the
eostimulator ~ndueers BCG and LPS, as deseribed by
Janeway (1990).
The present inventors have shown that the eostimulstor
inducer activity of outer membrane proteins TraT and OmpA
~: :
W093/03762 PCT/AU92/0~23
~il5~2~
-- 7
of E. coli can be used to enhance the ~timulation of helper
T-cell~, derived from HIV-positive individuals, ln the
presence of antigen, and specifically, peptides derived
from the viral proteins or recall antigen~ such as
Diphtheria toxoid (DT) and Tetanus toxoid (TT).
In contra~t to BCG and LPS, TraT, OmpA and OmpF do not
produce undesirable side-effects such as endotoxic shock
and granuloma formation at the in~ection site.
TraT, OmpF and OmpA can, therefore, be u~ed as
inducerc of co~timulatory activity in antigen presenting
cell~ and thereby stimulate helper T-cells in the induction
of immune responses to, for instance, a number of
HIV-derived antigens, and thereby overcome the CD4-positive
T-cell non-responsiveness in HIV-infected individuals. ~-
The clinical outcome of increa~ed helper T-cell
numbers i8 improved immune function which in turn will
re~ult in an increased capacity of an individual to combat ~`
opportun~stic infection~.
The u~e of these molecules would greatly improve the
efficacy of candidate AIDS vaccine~ by stimulating the
~`~ production of helper T-cells. Alternatively, when used in
con~unction with other antigens to which an individual has
previously devaloped memory T-cells, these molecules will
enhance the overall level of immunity of the individual.
The ability of these molecules to restore helper
T-cell function could also be exploited to enhance helper
T-cell production in immunodeficiency conditions such as
those which may arise following certain types of cancer,
orgsn transplantation and various autoimmune conditions.
The compositions of the present invention are prepared
by mixing, preferably homogeneously mixing, TraT, OmpA or
OmpF or a part of TraT, OmpA or OmpF, which part stimulates
an antigen presenting cell to provide a co~timulator signal
for helper T-cells, with a pharmaceutically acceptable
carrier, diluent, and/or excipient using standard methods
:
w093/03762
PCT/AU92/~23
211~42'~
- 8 -
of pharmaceutical preparation.
Preferably the method additionally compri~e~ u~ing at
least one other antigen in the preparation of the
pharmaceutical composition. The antigen may be an antigen
against which it is desirable to raise an immune response
in the patient. For instance in AIDS patients HIV antigens
may be used. Other antigens which might be used include
influenza virus antigens, diphtheria antigens, whooping
cough antigens, measles antigens Pneumocystis antigens,
Candida antigens, Toxoplasmosis antigens, Cytomegalovirus
antigens, combinations thereof and individual subunit
proteins, peptides or polysaccharides isolated from said
antigens.
The TraT, OmpA and OmpF proteins which can be used in
accordance with the present invention may be purified from
publicly available standard E. coli strains which produce
these proteins.
One such strain of ~ coli is ATCC 67331 which was
deposited with the American Type Culture Collection of
12301 Parklawn Drive, Rockville MD 20852, U.S.A. on 2 Narch
1987. Purification of TraT, OmpF and OmpA from E. coli is
describQd in International Patent Application No.
PCT/AU8~/00107 (WO 87/06590).
Alternatively these proteins may be obtained from
other bacterial strains which carry recombinant DNA
molecules encoding these proteins, and purified by a method
appropriate to the site of production of the recombinant
TraT, OmpA or OmpF protein.
Where parts of these proteins, which stimulate an
antigen presenting cell to provide a co~timulatory activity
for helper T-cells are to be used, the required parts can
be identified and prepared as follows.
The intact molecule is employed to identify the
receptor which binds the molecule on the antigen presenting
~ 35 cell. The intact molecule is then digested by standard
:
W093/03762 2 11 ~ ~ 2 ~PCT/AU92/~23
_ 9 _
protein digestion teehniques and the parts genersted are
assayed for binding to the identified reeeptar. Those
parts whieh ean bind and stimulste produetion of
eostimulatory aetivity by the antigen presenting eell are
suitable for use in the eomposition~ and methods of the
pre~ent in~ention.
As will be readily understood by persons skilled in
the art homologues and analo~ues of TraT, OmpA and OmpF
eould be used in the present invention with similar
effeet. It is intended that the use of sueh homologues and
analogues are eneompassed within the seope of this
applieation.
The antigens to be used in eompositions and method of
the present invention may be any antigen against which it
is desirable to raise an immune response in an
immunoeompromised or immunosuppressed patient.
Examples inelude, for instanee, antigens of the HIV
~ueh as gp41[8~ peptide whieh may be of use to stimulate
blastogenesis of HIV-speeifie lymphoeyte~ in HIV-infected
patient~. Other antigen~ might inelude influenza virus
antigens, diphtheria antigens, whooping eough antigens,
measles antigens Pneumoeystis antigen~, Candida antigens,
oxoplasmoæis antigens, Cytome~alovirus antigens,
eombinations thereof and individual subunit proteins,
peptides or polysaceharides isolated from said antigens.
The eompositions of the invention may be prepared
using standard pharmaceutieal teehniques.
Where an antigen is to be used in the composition, it `
may be admixed with the costimulator indueer in the depot.
Alternatively, the antigen and eostimulator inducer may be
eomplexed by ehemical con~ugation using ehemieal
modifieation and/or linking groups where required. For
proteinaeeous antigens, the eostimulator indueer and
antigen eould be provided as a fusion protein, by
reeombinant DNA techniques.
wo9i/lo356~ 2 ~ PCT/AU92/0042~
-- 10 --
In eaeh case, it is to be understood that the proeess
for ~oining the antigen to the eostimulator indueer ~hould
not de~troy the desired antigenicity of the antigen or the
eo~timulator indueer aetivity of the TraT, OmpA, OmpF or
part thereof.
The eostimulator-indueer or eostimulator-inducer and
antigen ean be formulated in a depot carrier. Where both
components are to be included it is desirable to keep them
together. A depot carrier is suitable to aehieve this and
the types of depot carrier which can be used include
alhydrogel, proteosomes and liposomes. The compositions
~ are prepared by standard techniques appropriate to the
carrier being used.
Where the eostimulator-inducer is to be used without
lS antigen or where the costimulator-indueer i~ complexed or
fused to the antigen, traditional carriers other than depot
earrier~ ean also be used.
The eomposition of the present invention i8 preferably
admini~tered parenterally to the patient by standard
teehniques of parenteral administration~
Typieally 100ng-lOmg of eaeh eostimulator indueer and
antigen is used in each dose.
The preeise dose and ratio of each costimulator
indueer and antigen to be used will depend ons (i) the type
and nature (e.g. immunogenicity) of the antigen;
(ii) the genetic background of the ~ub~ect; (iii) the
immunologieal history of the sub~ect; and (iv) the type of
immune response (e.g. humoral, lymphocyte-mediated,
macrophage-mediated or granulocyte-mediated) one is seeking
to modify.
A skilled addressee will be able to determine the
appropriate ratio of costimulator inducer to antigen by
systematieally varying the relative dose and proportions of
eostimulator to antigen until the desired immune response
has ~een aehieved.
: ~ ~ ..... ... -
W093/03762 2 11 ~ 4 2 ~ PCT/AU92/00423
- 11 -
It is recognised that a number of factor will affect
the determination of an appropriate dosage for a particular
patient. Such factors include the age, weight, sex,
general health and concurrent disea~e status of the
patient. The determinatiQn of the appropriate dose level
for the particular patient is performed by standard
pharmaceuti-al techniques.
Patients for whom the use of the methods and
compositions of the invention is envisaged are patients
having a deficiency in helper T-cell function such a~
patients suffering from disease states including autoimmune
diseases, some cancers and AIDS, and patients where an
immunosuppressed state is artificially induced during
treatment of a particular disease state or condition, for
` 15 instance transplant patients and cancer patients undergoing
chemotherapy or radiotherapy.
The method of the invention might be used to raise
their h lper T-cell levels in general or the inclusion of
specific antigens can be desirable in order to raise helper
T-cell levels in order to protect the patient from specific
infections which could prove fatal in their
~;~ immunocompromised or immunosuppressed state.
` While it is understood that the primary focus of the
present invention is the treatment of human patients the
present invention is equally applicable for the treatment
of non-human animals. Accordingly, as used herein the term
"patient" is intended to cover both non-human and human
animals.
In order that the nature of the present invention may
be more clearly understood, preferred forms thereof will
now be described with reference to the following examples.
.
W093/03762 PCT/AU92/0~2~
2`~15~24 `~ ;l
- 12 -
ExAMeLE 1.
(i) TraT augm~nts the in vitro T-cell ~roliferative
re~Fonse~ elicited by immunodominant HrV-derived
~ynthetic peptides.
S A. Selection of the HIV-derived peptide (gp41t8]).
The ma~ority of the immunodominant sequences of the
Human Immunodeficiency virus type-l (HIV-l) are coded by
hypervariable gene sequences and these sequences are
interspersed with regions that are highly conserved
amongst HIV-l strains. Immunogenic viral proteins that
show minimal strain-to-strain variation and that
consistently elicit both humoral and cell-mediated immune
responses may be useful components for inclusion in a
subunit vaccine. In this connection, Bell and co-workers
(Bell et al, 1992) have studied HIV-seronegative sub~ects
and HIV-infected individuals classified as asymptomatic
(AS), as AIDS-related complex (ARC) or as AIDS. In
accordance with the clinical classification system of the
; CDC (Centers for Disease Control, 1986), AS HIV-infected
individuals constituted CDC Group II/III; ARC patients
were CDC Group IVA/IVC2, and AIDS were CDC Group IVCI/IV D.
They initially determined which of three short synthetic
peptides derived from the conserved sequences of the
envelope gp 120 (amino acids 262-284), gp41 (aa 579-601),
and core pl7 (aa 106-125) regions of the HTLV-IIIB
isolate, could elicit B-cell as well as T-cell responses
in HIV infected individuals. Only the gp41-derived
sequence was immunogenic at the ~- and T-cell levels. The
gp41 region was characterized further by using a series of
overlapping synthetic peptides derived from a conserved
region of the envelope gp41 (aa 572-613). The authors
subsequently identified an immunodominant dodecamer (aa
593-604; termed gp4118]) which consistently evoked both
T-blastogenic and antibody responses in asymptomatic
HIV-seropositive individuals to a lesser extent in ARC,
:
W093/03762 2 ~ 2 ~ PCT/AU92/~23
- 13 -
but not in AIDS patients.
B. The ~ynthe~i~ of HIV-l-derived peptide~.
The two peptides, gp4118] and V3 loop derived from the
gpl20 region of HIV-l, were synthesized on an Applied
S Biosystems No. 430A peptide synthesizer following the
manufacturers instructions. The peptides were purified by
chromatography on G-25 Sephadex (Pharmacia) in 10% Acetic
Acid, followed by Reverse Phase HPLC on a VYDAC C-18
column using a linear gradient of 5-60% acetonitrile in
O.1% TFA. The sequences of the peptides synthesised are
as follows:
R-S-S-op4118~: To improve the solubility of this peptide,
Arg-Ser-Ser was added to the amino terminal end of the
gp41181 sequence viz.,
Arg-Ser-Ser-Leu-Gly-Ile-Trp-Gly-Cys-Ser-Gly-Lys-Leu-Ile-Cys.
V3 loop peptide:
Asn-Thr-Arg-Lys-Ser-Ile-Arg-Ile-Gln-Arg-Gly-Pro-Gly-Arg-Ala-
Phe-Val-Thr-Ile-Gly-Lys-Ile-Gly-Asn.
C. A~se~sment of human T-cell ~roliferative response~.
Peripheral blood mononuclear cells (PBMC) were i~olated by
Ficoll-Paque (Pharmacia) gradient centrifugation, and
200,000 PBMC were cultured in 0.2 ml RPMI-160 medium
(Cytosystems Pty. Ltd., containing 10~ human AB serum, 50
mM esch of penicillin and streptomycin, 2.5 mM glutamine
and 2 mN HEPES buffer solution, pH 7.4) in 96-well
round-bottom microtitre plates in the presence of gp41~8]
or V3 loop synthetic peptides (2yN), rIL-2 ~lOu/ml),
Diphtheria toxoid (DT; Commonwealth Serum Laboratories,
Melbourne, Australia, 1570Lf units/ml; 4 and 40 ~g/ml),
Tetanus toxoid (TT; Commonwealth Serum Laboratories,
Melbourne, Australia, 100Lf units/ml; 5 and 50 yg/ml);
TraT and OmpA (purified as described by Croft et al.
1991; 40 yg/ml). After 6 days of culture at 37C
`:
W093/03762 PCT~AU92/~23~
21~L5~24
- 14 -
(with or without antigen), each culture wa8 pul8ed
overnight with 50yl of H-thymidine ~20~Ci/ml;
Amersham, U.g.), harvested on glass-fibre filter papers
and counted in a liquid scintillation spectrometer
(Beckman, U.S.A.).
Re~ult~ were expressed a~ Stimulation Indices (S.I.) which
were calculated as follows:
S.l. = mean counts per minute (c.p.m.) with antigen - mean c.p.m. without antigen
mean c.p.m. without antigen
D. Definition of TraT- and IL-2-mediated effects on
gp41~8]-. V3 peptide-. Diphtheria to~id ~pT)-.or Tetanus
to~oid (TT)- specific lymphoproliferation.
The effects of TraT and Il-2 on proliferative responses
(expressed as Stimulation Indices) to the various
HIV-derived and recall (DT and TT) antigens have been
defined by using a modified version of a documented
formula (Denz et al., 1985):
~; An additive effect was defined as:
(AB)
(A) ~ (B)
A Synergistic effect was defined as 5
(AB~ ~ 1
(A) + (B)
and an Inhibitory effect was defined as:
(AB) ~ 1
(A) + (B)
where (A) and (B) signify the proliferative responses to
the individual agents (e.g., gp41 ~8] and TraT),
; respectively, and (AB) is the lymphoproliferative response
seen when both agents are combined and added to the same
cultures.
W093/03762 211~ 12 4 PCT/AU92/0~23
The addition of TraT to eultures of PBNC from eleven
asymptomatie (AS) individuals, thirteen patients with
AIDS-re~ated complex (ARC) and one AIDS-lymphoma patient,
S augmented the T-eell proliferative response to the gp41[8]
peptide to levels whieh were higher than that achieved by
the addition of IL-2 to eultures eontaining the gp41t8]
peptide (Table 1). In every ea~e, with the exeeption of
patient $110, a synergistic effeet was seen in eultures
that had been stimulated with a mixture of TraT and
gp4l[8] and this effeet was maximal when 40 ~g of TraT
was used in the eell eultures (Table 2). By contrast, a
synergistic effect was observed in only three from
twenty-five cases, when IL-2 was eombined with gp4118]
(Table 1). The effeet of another outer-membrane protein,
OmpA, was alæo tested on PBMC eultures of one
::~asymptomatie, and one symptomatie individual, and in both
eases a synergiætie effeet was ~een when OmpA was
eo-eultured with gp41(8) (Table 1).
~: 20 In view of the impressive results obtained with gp4118] in
the presenee of TraT, it was important to determine
whether TraT would augment the T-eell response to another
~: HIV-derived peptide. The V3 loop peptide was eonsidered a
suitable eandidate, as this peptide (La Rosa et al.,
Seienee 2~9: 932, 1990), the prineipal neutralising
determinant of HIV-l, is eurrently in elinieal trials
(Serip, No. 1703, 26, 1992). The results in Table 3 show
that in all eight cases tested TraT augmented the
proliferative responses to the V3 peptideO However, an
inhibitory effeet was observed when PBNC from the eight
individuals were eultured in the presenee of IL-2 and V3
peptide (Table 3). In summary, TraT was far more
effeetive than IL-2, a lymphokine whieh haæ been trialled
as an Immunotherapeutie (Rosenberg, Lotze and Mul , 1988),
in augmenting the T-eell responses to the HIV-derived
~ .
W093/03762
PCT/AU92/0~2~;~
211542'1
- 16 -
peptides gp41t8] and the V3 loop.
(ii) TraT augments the in vitro T-cell prolif rative
~ onses to recall anti~ens such as Dightheria
S toxoid (DT) ~nd Tetanus tQsoid (~T).
It is well esta~lished that lymphoproliferative responses
to recall antigens are impaired even during the
asymptomatic disease period when CD4-positive T-cell
numbers are often only slightly reduced (Lane et al.,
198S). Since TraT has been shown to augment HIV-specific
lymphoproliferation in both symptomatic and asymptomatic
individuals, it was reasonable to expect that it would
have a similar effect in enhancing the response to recall
antigens. The ability of TraT to enhance the response to
recall antigens will be of clinical importance in boosting
immunity and thereby enabling immunocompromised
individuals to combat opportunistic infections.
In all six patients studied (#123 to $128), TraT
~ignificantly augmented DT - and T T- specific
proliferative re~ponses (Table 4). This finding would
make TraT an attractive immunomodulator molecule for
rastoring defective T-cell responses, not only to
HIV-derived antigens, but also to a range of recall
antigens. From a clinical viewpoint, a molecule with
co-stimulator inducer-like properties such as TraT would
be an attractive immunotherapeutic for boosting general
immunity in immunocompromised individuals.
`
W093/03762 ~ 4 2 ~1 PCT/AU92/~23
~XAMPL~ 2
ElQ~Lçy$Q~çtriç-anslyuluLof T-cell subset
di~tribution indicates th~t TraT preferentially
~tiDNlate~ CD4-positive helper T-cell~.
The T-cell ~ubset distribution of Peripheral blood
mononuclear cells (PBMC), that had been stimulated with
TraT, Interleukin-2 (IL-2) or with gp41t8], after a 6-day
incubation, were analysed using immunofluorescence and
f}ow cytometry.
The phenotypes of the T-cells in proliferating cultures of
PBMC were compared with those from unstimulated cell
cultures~
T-cell phenotype analysi~.
` `:
T~o-millilitre volumes of both stimulated and unstimulated
PB~C (5 x 106 cells/ml) were cultured for 6 days under
the same conditions as described in Example 1. After 6
days of culture, pelleted cells were resuspended, and the
cell suspensions layered onto 1 ml Ficoll-Hyp~que
gradients (Pharmacia), and centrifuged at 800g for 10
min. Viable (as judged by Trypan blue exclusion)
lymphoblastoid cells were collected from the interface and
washed in Hank~s Balanced Salt Solution (HBSS;
Cytosystems, Pty. Ltd~) pH 7.4. Viable cells that had
been isolated from unsorted cultures of PBMC were
phenotyped using dual combinations of fluorescent
monoclonal antibodies ~ i . Q ., CD 3/4 and CD 3/8; Coulter
Electronics Inc., Hialeah FL, V.S.A.). Two-thousand
viable cells from each PBMC culture were assayed for
surface-bound fluorescence. Using both the viable PBMC
count (i.e., after 6 days of culture with and without
~- stimulation), and the relative percentages of the
~, ~
~'
WO 93/03762 PCI`/AU92/00423~.s~
2115424 ` `
_ 18 -
respective T-cell subsets gated in each bitmap of 2,000
cells, the absolute numbers of T-cells from the
CD4-positive and CD8-positive T-cell subset~ were
calculated. The formula used for the calculation of
S subset numbers was: T-cell subset % (per 2,000 ~gated~
cells) x viable cell number (as determined by Trypan blue
exclusion). Bitmaps were continually ad~usted to encircle
the ma~ority of either stimulated or unstimulated
lymphocytes. After 6 days of culture of purified PBMC, >
90% of viable cells were consistently found to be
CD3-positive, indicating that they were predominantly
T-cells.
There was a 23 to 48% increase in absolute CD4-positive
T-cell numbers (based on absolute cell numbers in the
un~timulated cultures) when PBMC were cultured in the
presence of TraT, gp4118]), or IL-2. However, when TraT
was combined with gp4118], CD4-positive T-cells increased
by 63% (#123) and 96% ($124) respectively (Table 5). By
contrast, there was a somewhat lower (0 to 42%) increase
in CD8-positive T-cell numbers when PaNC were incubated in
the presence of any of the three stimulants, and this
increased to 10% (#123) and 79% (#124) when PBMC were
exposed to a m~xture of TraT and gp41t8] in a 6-day
; culture. Another notable feature of these results is that
after a 6-day incubation with the various stimulants,
there was a significant increase in the CD4/CD8 ratio
compared with the CD4/CD8 ratio measured immediately after
isolation of the PBMC.
The synergistic effect between TraT and gp41[8~ wa~ even
more pronounced when these stimulants were tested on the
PBMC from patients $125 and #126 (Table 6). In these two
ARC patients, the percentage increases in CD4 cell numbers
were: TraT, 58% ($125), 36% (#126); gp41t8~, 0% (#125),
18% (#126); IL-2, 3~8% (#125), 216% ($126). However,
there was a dramatic increase in CD4 cell numbers when
~ ~ '
W093/03762 2115 ~ 2 4 PcT/Au92/o~23
`; !
-- 19 --
TraT was combined with gp41181: 737% (#125) and 1,763~
(#126) respectively. The percentage increase~ in CD4 were
significantly higher than the corresponding increases for
the CD8-positive population, i.e., 74% ($125) and 185%
(#126) (Tsble 5). The preferential increase in
CD4-positive helper T-cells in cultures incubated with
TraT, or with a combination of gp41t8~ and TraT, suggests
that TraT will boost helper T-cell numbers in vivo and
~; thereby enable HIV-infected individuals to combat
opportunistic infections. Further improvement in
HIV-positive individuals would be obtained by combining
TraT with anti-retroviral sgents such as zidovudine.
Industrial Application
The current invention is applicable to the preparation of `~
vaccines designed to combat immunodeficiency disorders
such as AIDS and to the treatment of patients ~uffering a
deficiency in helper T-cell function in general.
It will be appreciated by persons skilled in the art
~;~ that numerous variations and/or modifications may be made
to the invention as shown in the ~pec~fic embodiments
without departing from the spirit or scope of the
invention as broadly described. The present embodiments
ara, therefore, to be considered in all respects as
illustrative and not restrictive.
~: ~
WO 93/03762 PCl /AU92/00423~.~
2~1~424
-- 20 --
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WO 93/03762 211 ~ 4 2 ~ PCl`/AU92tO0423
: ! .
-- 21 --
TABLE 2
Do~e-Re~ponse effect_of TraT
on gp4118] - ~pecific T-cell proliferation
Patient No. and Diagno~is
Treatment~ 111 112 113 114
nild
ARC AS AS AS
StLmulation Indices
`~:
Tra$ - lOyg 3.0 4.3 7.8 16.5
TraT - 20yg 5.2 7.8 18.1 35.2
TraT - 40yg 8.0 9.0 29.0 56.4
TraT - 60yg 9.0 7.7 23.2 42.5
~:: TraT - 80yg 5.4 6.8 14.7 25.7
;~ gp41t8] 10.5 5 9 7.9 19.9
TraT (lOyg) + gp4118]13.3 25.9 20.2 38.6
TraT (20yg) + gp41~8]17.2 25.3 35.5 75.8
TRAT (40yg) + gp41~8]21.7 39.7 48.0 99.3
TRAT (60yg) + gp41~8]24.8 18.8 30.8 70.0
TRAT (80yg) + gp41~8]19.3 17.8 24.5 48.2
:
WO 93/03762 PCI'~AU92/0042~
; ;
~15~2~
-- 22 --
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W O 93/03762 PC~r/AU92J00423
~ 2 il542~ ~:
- 23 -
TABLE 4
Th- au~-ent~tlon bv TrsT of T-cell prol~fer~t~vc re6pon~e~ to
Diphtherla to~o~d lDT) ant Tetsnu~ to~ud tTT)
Pat~ent No ~nd Dia~nos~
123 124 125 126 127 128
Rapos~'6 AS ~RC** ARC AS ~ild ARC
Tre~tmQnt6 S~rco~a
tSt$mul~t~on Indic-s)
TraTt40~g 10.4 5.7 7.2 12.2 12.1 11.2
DT(40~g) 6.5 5.3 3.3 5.1 6.2 6.1
DT(4yg~ 4.0 2.7 1.4 1.41 3.31 3.9
TT(SOyg) 4.5 5.4 2.8 3.7 5.5 3.0
TT(Syg) 3.0 2.8 1.6 2.7 3.1 2.7
TrsT+DT(40yg)37.6(S)* 27.0(S) 20.9(S) 24.0tS) 30.9(S) 30.2(S)
TraS~DT(4~g)21.6~S) 18.7tS) 11.5(S) 21.6(S) 23.8(S) l9.0(S)
TraT~TT (50y8) 34.0(S) 27.5(S)16.0tS) 29.5(S) 26.5(S) 21.1(S)
TraTITT(5~g)21.3(S) 13.4(S) 11.2(S) l9.5(S) 21.1(S) 19.6(S)
_
* S~nergistic effect
AS - Asymptomat~c; ** ARC - AIDS-related comple~
WO 93/03762 PCl`/AU92/00423r. ~
21i542~
- 24 -
TABLE 5
Flo~ cvto~etr~c analvsi6 of T-c-ll sub~t~ EL__t~on of
prol~eratln~ and unsti~ulated cell6 afto~ a 6-d~y ~ncubatlon
Pat~ent No. and D~a~no~6
~123 - Kapo~ '8 Sarco~a* ~124 - ~ild ARC*
Ab601ute T-cell numbers Absolute T-cell numbers
Treat~ent CD4 CD8 CD4 CD4 CD8 CD4
NIL 3.04xlO 2.7xlO 1.1 2.5xlO 2.4xlO 1.0
TraT 3.7xlO 2.28s106 1.6 3.0xlO . 2. 5Y10 1 . 2
gp41[8] 3.ax106 2.40~106 1.6 3.7x106 2.9x106 1.3
IL-2 3.9x106 2.5xlO 1.6 3.6x106 3.4x106 1.1
8]*TraT 4.9x106 2.9x106 1.7 4.9xlO~ 4~3ylO6 1.1
8]~IL-2 4.1~106 2.4x106 1.7 3.6~106 ~ 3.8x106 0.9
* CD4/CDa at day 0 was ~123-0.23; ~124-0.52
~: :
W O 93/03762 211 5 4 2 -4 PC~r~AU92tO0423
- 25 -
TABL~ 6
~low cvtometric analvsi6 of T-cell sub~et dl~tribut~Qn
of proliferating a~d unst~mulated c~118 after
~ 6-da~ i~cub6tio~
Patlent No. and Diaenos~
#125 - ARC ~126 - ARC
Absolute T-cell numbers Absolute T-cell numbers
Treatment CD4 CD8 CD4 CD4 CD8 CD4
CD8 CD8
NIL 92,0008B0,000 0.10 44,000780,000 0.06
TraT 145,0001,150,000 0.13 60,000l,S00,000 0.04
gp4118~54,000980,~00 0.06 S~,000550,000 0.09
IL-2 440,0001,160,000 0.38 139,0001,130,000 0.12
8]+TraT 770,000 1,530,000 0.50 820,000 2,220,000 0.37
[8~IL-229,0001,280,000 0.03 68,0001,310,000 O.OS
* CD41CD8 at da~ 0 vas ~125 = 0.125; tl26 - 0.09
WO 93/03762 PCI/AU92/0042~....~
211~2~ 26-
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~: -
: ~
