U.S. patent application number 12/221498 was filed with the patent office on 2009-07-30 for methods and reagents for vaccination which generate a cd8 t cell immune response.
This patent application is currently assigned to Oxxon Therapeutics Limited. Invention is credited to Tom Blanchard, Sarah C. Gilbert, Tomas Hanke, Adrian V.S. Hill, Andrew McMichael, Magdalena Plebanski, Jorg Schneider, Geoffrey L. Smith.
Application Number | 20090191237 12/221498 |
Document ID | / |
Family ID | 10813841 |
Filed Date | 2009-07-30 |
United States Patent
Application |
20090191237 |
Kind Code |
A1 |
McMichael; Andrew ; et
al. |
July 30, 2009 |
Methods and reagents for vaccination which generate A CD8 T cell
immune response
Abstract
New methods and reagents for vaccination are described which
generate a CD8 T cell immune response against malarial and other
antigens such as viral and tumour antigens. Novel vaccination
regimes are described which employ a priming composition and a
boosting composition, the boosting composition comprising a
non-replicating or replication-impaired pox virus vector carrying
at least one CD8 T cell epitope which is also present in the
priming composition.
Inventors: |
McMichael; Andrew; (Beckley,
GB) ; Hill; Adrian V.S.; (Old Headington, GB)
; Gilbert; Sarah C.; (Headington, GB) ; Schneider;
Jorg; (Barton, GB) ; Plebanski; Magdalena;
(Melbourne, AU) ; Hanke; Tomas; (Old Marston,
GB) ; Smith; Geoffrey L.; (Oxford, GB) ;
Blanchard; Tom; (Banjul, GM) |
Correspondence
Address: |
HAMILTON, BROOK, SMITH & REYNOLDS, P.C.
530 VIRGINIA ROAD, P.O. BOX 9133
CONCORD
MA
01742-9133
US
|
Assignee: |
Oxxon Therapeutics Limited
Oxford
GB
|
Family ID: |
10813841 |
Appl. No.: |
12/221498 |
Filed: |
August 4, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10653624 |
Sep 2, 2003 |
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12221498 |
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09454204 |
Dec 9, 1999 |
6663871 |
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10653624 |
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PCT/GB98/01681 |
Jun 9, 1998 |
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09454204 |
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Current U.S.
Class: |
424/199.1 |
Current CPC
Class: |
A61K 2039/55522
20130101; C12N 2760/16122 20130101; C12N 15/86 20130101; A61P 35/00
20180101; C12N 2740/15034 20130101; A61K 39/21 20130101; A61P 31/20
20180101; A61P 31/16 20180101; A61P 37/04 20180101; Y02A 50/30
20180101; A61P 31/12 20180101; C07K 14/445 20130101; A61K 2039/5256
20130101; A61K 39/0011 20130101; A61K 39/39 20130101; A61P 31/18
20180101; C12N 2760/16134 20130101; A61K 2039/5258 20130101; A61K
39/145 20130101; A61K 2039/545 20130101; C12N 2710/10343 20130101;
A61K 39/12 20130101; A61K 2039/53 20130101; A61K 2039/57 20130101;
A61K 38/1709 20130101; A61K 2039/51 20130101; A61K 39/015 20130101;
A61K 2039/54 20130101; C12N 2710/24043 20130101; A61P 33/06
20180101; C12N 2710/24143 20130101; C12N 2740/16134 20130101; C12N
2740/16234 20130101; C07K 14/005 20130101 |
Class at
Publication: |
424/199.1 |
International
Class: |
A61K 39/00 20060101
A61K039/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 9, 1997 |
GB |
GB9711957.2 |
Claims
1. A method of boosting a primed CD8.sup.+ T cell immune response
against a tumor antigen in a mammal, comprising administering to
said mammal a source of one or more CD8.sup.+ T cell epitopes of
said tumor antigen, wherein the source of CD8.sup.+ T cell epitopes
is a non-replicating or a replication-impaired viral vector.
2. The method of claim 1, wherein the non-replicating or
replication-impaired viral vector is a recombinant poxvirus
vector.
3. The method of claim 2, wherein the recombinant poxvirus vector
is an MVA vector.
4. The method of claim 2, wherein the recombinant poxvirus vector
is an avipox virus vector.
5. The method of claim 2, wherein the recombinant poxvirus vector
is a fowlpox virus vector.
6. The method of claim 1, wherein the mammal is a primate.
7. The method of claim 1, wherein the tumor antigen comprises one
or more CD8.sup.+ T cell epitopes of melanoma.
8. The method of claim 1, wherein the tumor antigen comprises one
or more CD8.sup.+ T cell epitopes of breast cancer.
9. The method of claim 1, wherein the tumor antigen comprises one
or more CD8.sup.+ T cell epitopes of colon cancer.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 10/653,624, filed Sep. 2, 2003, which is a divisional of U.S.
application Ser. No. 09/454,204, filed on Dec. 9, 1999, which is a
continuation of, and claims priority to, International Application
No. PCT/GB98/01681, filed 9 Jun. 1998 which designates the United
States, and which is a continuing application of GB9711957.2 filed
9 Jun. 1997. The entire teachings of the above applications are
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] A general problem in vaccinology has been an inability to
generate high levels of CD8 T cells by immunization. This has
impeded the development of vaccines against several diseases
including malaria.
[0003] Plasmodium falciparum malaria causes hundreds of millions of
malaria infections each year and is responsible for 1-2 million
deaths annually. The development of an effective vaccine against
malaria is thus a major priority for global public health. A
considerable body of immunological research over the last twenty
years had led to the identification both of candidate vaccine
antigens from the parasite and immunological mechanisms on the host
that are likely to protect against infection
[0004] A major problem has been the identification of a means of
inducing a sufficiently strong immune response in vaccinated
individuals to protect against infection and disease. So, although
many malaria antigens are known that might be useful in vaccinating
against malaria the problem has been how to deliver such antigens
or fragments of them known as epitopes, which are recognized by
cells of the immune system, in a way that induces a sufficiently
strong immune response of a particular type.
[0005] It has been known for many years that it is possible to
protect individuals by immunizing them with very large doses of
irradiated malaria sporozoite given by bites from infected
mosquitoes. Although this is a wholly impractical method of mass
vaccination it has provided a model for analyzing the immune
responses that might be mediating protective immunity against
sporozoite infection (Nardin and Nussenzweig 1993).
[0006] A considerable amount of research over the last decade or
more has indicated that a major protective immune response against
the early pre-erythrocytic stage of P. falciparum malaria is
mediated by T lymphocytes of the CD8+ ve type (CD8+ T cells). Such
cells have been shown to mediate protection directly in mouse
models of malaria infection (Nardin and Nussenzweig 1993). Such T
cells have also been identified in individuals naturally exposed to
malaria and in volunteers immunized with irradiated sporozoite
(Hill et al. 1991; Aidoo et al. 1995; Wizel et al. 1995). There is
much indirect evidence that such CD8+ T cells are protective
against malaria infection and disease in humans (Lalvani et al.
1994).
[0007] CD8+ T cells may function in more than one way. The best
known function is the killing or lysis of target cells bearing
peptide antigen in the context of an MHC class I molecule. Hence
these cells are often termed cytotoxic T lymphocytes (CTL).
However, another function, perhaps of greater protective relevance
in malaria infections is the ability of CD8+ T cells to secrete
interferon gamma (IFN-.gamma.). Thus assays of lytic activity and
of IFN-.gamma. release are both of value in measuring a CD8+ T cell
immune response. In malaria these CD8+ ve cells can protect by
killing the parasite at the early intrahepatic stage of malaria
infection before any symptoms of disease are produced (Seguin et
al. 1994).
[0008] The agent of fatal human malaria, P. falciparum infects a
restricted number of host species: humans, chimpanzees and some
species of New World monkey. The best non-human model of malaria is
the chimpanzee because this species is closely related to humans
and liver-stage infection is observed consistently unlike in the
monkey hosts (Thomas et al. 1994). Because of the expense and
limited availability of chimpanzees most laboratory studies of
malaria are performed in mice, using the rodent malaria species P.
berghei or P. yoelii. These latter two models are well studied and
it has been shown in both that CD8+ ve lymphocytes play a key role
in protective immunity against sporozoite challenge.
[0009] Previous studies have assessed a large variety of means of
inducing CD8+ T cell responses against malaria. Several of these
have shown some level of CD8+ T cell response and partial
protection against malaria infection in the rodent models (e.g. Li
et al. 1993; Sedegah et al. 1994; Lanar et al. 1996). However, an
effective means of immunizing with subunit vaccines by the
induction of sufficiently high levels of CD8+ T lymphocytes to
protect effectively against malaria sporozoite infection has not
previously been demonstrated.
[0010] In recent years improved immune responses generated to
potential vaccines have been sought by varying the vectors used to
deliver the antigen. There is evidence that in some instances
antibody responses are improved by using two different vectors
administered sequentially as prime and boost. A variety of
combinations of prime and boost have been tested in different
potential vaccine regimes.
[0011] Leong et al. (Vaccines 1995, 327-331) describe immunizing
mice firstly to DNA expressing the influenza haemagglutinin (HA)
antigen and then with a recombinant fowlpox vector expressing HA.
An enhanced antibody response was obtained following boosting.
[0012] Richmond et al. (Virology 1997, 230: 265-274) describe
attempts to raise neutralizing antibodies against HIV-1 env using
DNA priming and recombinant vaccinia virus boosting. Only low
levels of antibody responses were observed with this prime boost
regime and the results were considered disappointing.
[0013] Fuller et al. (Vaccine 1997, 15:924-926 and Immunol Cell
Biol 1997, 75:389-396) describe an enhancement of antibody
responses to DNA immunization of macaques by using a booster
immunization with replicating recombinant vaccinia viruses.
However, this did not translate into enhanced protective efficacy
as a greater reduction in viral burden and attenuation of CD4 T
cell loss was seen in the DNA primed and boosted animals.
[0014] Hodge et al. (Vaccine 1997, 15: 759-768) describe the
induction of lymphoproliferative T cell responses in a mouse model
for cancer using human carcinoembryonic antigen (CEA) expressed in
a recombinant fowl pox virus (ALVAC). The authors primed an immune
response with CEA-recombinant replication competent vaccinia
viruses of the Wyeth or WR strain and boosted the response with
CEA-recombinant ALVAC. This led to an increase in T cell
proliferation but did not result in enhanced protective efficacy if
compared to three wild type recombinant immunizations (100%
protection), three recombinant ALVAC-CEA immunizations (70%
protection) or WR prime followed by two ALVAC-CEA immunizations
(63% protection).
[0015] Thus some studies of heterologous prime-boost combination
have found some enhancement of antibody and lymphoproliferative
responses but no significant effect on protective efficacy in an
animal model. CD8 T cells were not measured in these studies. The
limited enhancement of antibody response probably simply reflects
the fact that antibodies to the priming immunogen will often reduce
the immunogenicity of a second immunization with the same
immunogen, while boosting with a different carrier will in part
overcome this problem. This mechanism would not be expected to be
significantly affected by the order of immunization.
[0016] Evidence that a heterologous prime boost immunization regime
might affect CD8 T cell responses was provided by Li et al. (1993).
They described partial protective efficacy induced in mice against
malaria sporozoite challenge by administering two live viral
vectors, a recombinant replicating influenza virus followed by a
recombinant replicating vaccinia virus encoding a malaria epitope.
Reversing the order of immunization led to loss of all protective
efficacy and the authors suggested that this might be related to
infection of liver cells by vaccinia, resulting in localization of
CTLs in the liver to protect against the hepatocytic stages of
malaria parasites.
[0017] Rodrigues et al. (J. Immunol. 1994, 4636-4648) describe
immunizing mice with repeated doses of a recombinant influenza
virus expressing an immunodominant B cell epitope of the malarial
circumsporozoite (CS) protein followed by a recombinant vaccinia
virus booster. The use of a wild type vaccinia strain and an
attenuated but replication-competent vaccinia strain in the booster
yielded very similar levels of partial protection. However the
attenuated but replication competent strain was slightly less
immunogenic for priming CD8 T cells than the wild type vaccinia
strain.
[0018] Murata et al. (Cell. Immunol. 1996, 173: 96-107) reported
enhanced CD8 T cell responses after priming with replicating
recombinant influenza viruses and boosting with a replicating
strain of vaccinia virus and suggested that the partial protection
observed in the two earlier studies was attributable to this
enhanced CD8 T cell induction.
[0019] Thus these three studies together provide evidence that a
booster immunization with a replicating recombinant vaccinia virus
may enhance to some degree CD8 T cell induction following priming
with a replicating recombinant influenza virus. However, there are
two limitations to these findings in terms of their potential
usefulness. Firstly, the immunogenicity induced was only sufficient
to achieve partial protection against malaria and even this was
dependent on a highly immunogenic priming immunization with an
unusual replicating recombinant influenza virus. Secondly, because
of the potential and documented side-effects of using these
replicating viruses as immunogens these recombinant vectors are not
suitable for general human use as vaccines.
[0020] Modified vaccinia virus Ankara (MVA) is a strain of vaccinia
virus which does not replicate in most cell types, including normal
human tissues. MVA was derived by serial passage >500 times in
chick embryo fibroblasts (CEF) of material derived from a pox
lesion on a horse in Ankara, Turkey (Mayr et al. 1975). It was
shown to be replication-impaired yet able to induce protective
immunity against veterinary poxvirus infections (Mayr 1976). MVA
was used as a human vaccine in the final stages of the smallpox
eradication campaign, being administered by intracutaneous,
subcutaneous and intramuscular routes to >120,000 subjects in
southern Germany. No significant side effects were recorded,
despite the deliberate targeting of vaccination to high risk groups
such as those with eczema (Mayr et al. 1978; Stickl et al. 1974;
Mahnel et al. 1994;). The safety of MVA reflects the avirulence of
the virus in animal models, including irradiated mice and following
intracranial administration to neonatal mice. The non-replication
of MVA has been correlated with the production of proliferative
white plaques on chick chorioallantoic membrane, abortive infection
of non-avian cells, and the presence of six genomic deletions
totaling approximately 30 kb (Meyer et al. 1991). The avirulence of
MVA has been ascribed partially to deletions affecting host range
genes K1L and C7L, although limited viral replication still occurs
on human TK-143 cells and African Green Monkey CV-1 cells
(Altenburger et al. 1989). Restoration of the K1L gene only
partially restores MVA host range (Sutter et al. 1994). The host
range restriction appears to occur during viral particle
maturation, with only immature virions being observed in human HeLa
cells on electron microscopy (Sutter et al. 1992). The late block
in viral replication does not prevent efficient expression of
recombinant genes in MVA. Recombinant MVA expressing influenza
nucleoprotein, influenza haemagglutinin, and SIV proteins have
proved to be immunogenic and provide varying degrees of protection
in animal models, although this has never been ascribed to CD8+ T
lymphocytes alone (Sutter et al. 1994, Hirsch et al. 1995; Hirsch
et al. 1996). Recombinant MVA is considered a promising human
vaccine candidate because of these properties of safety and
immunogenicity (Moss et al. 1995). Recombinant MVA containing DNA
which codes for foreign antigens is described in U.S. Pat. No.
5,185,146 (Altenburger).
[0021] Poxviruses have evolved strategies for evasion of the host
immune response that include the production of secreted proteins
that function as soluble receptors for tumor necrosis factor,
IL-1.beta., interferon (IFN)-.alpha./.beta. and IFN-.gamma., which
normally have sequence similarity to the extracellular domain of
cellular cytokine receptors (Symons et al. 1995; Alcami et al.
1995; Alcami et al. 1992). The most recently described receptor of
this nature is a chemokine receptor (Graham et al. 1997). These
viral receptors generally inhibit or subvert an appropriate host
immune response, and their presence is associated with increased
pathogenicity. The Il-1.beta. receptor is an exception: its
presence diminishes the host febrile response and enhances host
survival in the face of infection (Alcami et al. 1996). We have
discovered that MVA lacks functional cytokine receptors for
interferon .gamma., interferon .alpha..beta., Tumor Necrosis Factor
and CC chemokines, but it does possess the potentially beneficial
IL-1.beta. receptor. MVA is the only known strain of vaccinia to
possess this cytokine receptor profile, which theoretically renders
it safer and more immunogenic than other poxviruses. Another
replication-impaired and safe strain of vaccinia known as NYVAC is
fully described in Tartaglia et al. (Virology 1992, 188:
217-232).
[0022] It has long been recognized that live viruses have some
attractive features as recombinant vaccine vectors including a high
capacity for foreign antigens and fairly good immunogenicity for
cellular immune responses (Ellis 1988 new technologies for making
vaccines. In: Vaccines. Editors: Plotkin S A and Mortimer E A. W B
Saunders, Philadelphia, page 568; Woodrow G C 1977. In: New
Generation Vaccines 2.sup.nd Edition. Editors: Levine M M, Woodrow
G C, Kaper J B, Cobon G, page 33). This has led to attempts to
attenuate the virulence of such live vectors in various ways
including reducing their replication capacity (Tartaglia J et al.
1992 Virology 188: 217-232). However such a reduction in
replication reduces the amount of antigen produced by the virus and
thereby would be expected to reduce vaccine immunogenicity. Indeed
attenuation of replicating vaccinia strains has previously been
shown to lead to some substantial reductions in antibody responses
(Lee M S et al, 1992 J Virology 66: 2617-2630). Similarly the
non-replicating fowlpox vector was found to be less immunogenic for
antibody production and less protective than a replicating
wild-type vaccinia strain in a rabies study (Taylor J et al. 1991
Vaccine 9: 190-193).
SUMMARY OF THE INVENTION
[0023] It has now been discovered that non-replicating and
replication-impaired strains of poxvirus provide vectors which give
an extremely good boosting effect to a primed CTL response.
Remarkably, this effect is significantly stronger than a boosting
effect by wild type poxviruses. The effect is observed with
malarial and other antigens such as viral and tumor antigens, and
is protective as shown in mice and non-human primate challenge
experiments. Complete rather than partial protection from
sporozoite challenge has been observed with the novel immunization
regime.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1 shows the construct used to express Ty-VLP with the
malaria epitope cassette CABDHFE. CTL epitopes are from P.
falciparum STARP (sporozoite threonine- and asparagine-rich
protein) (st), LSA-1 (liver stage antigen 1) (1s), CSP
(circumsporozoite protein) (cp), TRAP (thrombospondin-related
adhesive protein) (tr), LSA-3 (liver stage antigen 3) (la) and
Exp-1 (exported protein 1) (ex). Helper epitopes are from the P.
falciparum CS protein, the M. tuberculosis 38Kd antigen and Tetanus
Toxoid. NANP is the antibody epitope from CS and AM is the adhesion
motif from P. falciparum TRAP (Muller et al 1993). The length of
the complete string is 229 amino acids.
[0025] FIG. 2 shows a schematic outline of the H, M and HM
proteins. The bar patterns on the schematic representations of the
polyepitope proteins indicate the origin of the sequences. The
positions of individual epitopes and their MHC restrictions are
depicted above and below the proteins. Pb is the only epitope
derived from the protein of P. berghei. All other epitopes in the M
protein originate from proteins of P. falciparum:
cs--circumsporozoite protein, st--STARP, Is--LSA-1 and tr--TRAP.
BCG--38 kDa protein of M. tuberculosis; TT--tetanus toxin.
[0026] FIG. 3 shows malaria CD8 T cell ELISPOT data following
different immunisation regimes. Results are shown as the number of
peptide-specific T cells per million splenocytes.
[0027] FIGS. 4A-4D show that malaria CD8 T cell ELISPOT (FIGS. 4A
and 4C) and CTL levels (FIGS. 4B and 4D) are substantially boosted
by a recombinant MVA immunisation following priming with a plasmid
DNA encoding the same antigen. The ELISPOT counts are presented on
a logarithmic scale.
[0028] FIG. 5 shows the CTL responses induced in BALB/c mice to
malaria and HIV epitopes by various immunisation regimes employing
plasmid DNA and recombinant MVA. Levels of specific lysis at
various effector to target ratios are shown.
[0029] FIG. 6 shows the results of ELISPOT assays performed to
measure the levels of specific CD8+ T cells to the malaria epitope
pb9 following different immunisation regimes. Groups of BALB/c mice
(n=3) were immunised as indicated (g.g.=gene gun). The time between
all immunisations was 14 days. ELISPOT assays were done two weeks
after the last immunisation.
[0030] FIG. 7 shows the CTL responses against influenza NP in
different mouse strains. Mice of different strains were immunised
twice two weeks apart with a DNA vaccine V1J-NP encoding for the
influenza nucleoprotein (open circles) or primed with the same DNA
vaccine and two weeks later boosted with recombinant MVA expressing
influenza virus nucleoprotein (closed circles). The CTL activity
was determined in a standard .sup.51Cr-release assay with MHC class
I-matched target cells.
[0031] FIGS. 8A-8H show CTL responses against different antigens
induced in different inbred mouse strains. Mice were immunised with
two DNA vaccine immunisations two weeks apart (open circles) or
primed with a DNA vaccine and two weeks later boosted with a
recombinant MVA expressing the same antigen (closed circles). The
strains and antigens were: FIG. 8A, C57BL/6, P. falciparum TRAP;
FIG. 8B, DBA/2, E. coli b-galactosidase; FIG. 8C, BALB/c, HM
epitope string CTL activity against malaria peptide (pb9); FIG. 8D,
DBA/2, HM epitope string CTL activity against pb9; FIG. 8E, BALB/c;
HM epitope string CTL activity against HIV peptide; FIG. 8F, DBA/2,
HM epitope string CTL activity against HIV peptide; FIG. 8G,
BALB/c, tumour epitope string CTL activity against P1A-derived
peptide; and in FIG. 8H, DBA/2, tumour epitope string CTL activity
against P1A-derived peptide. Each curve shows the data for an
individual mouse.
[0032] FIGS. 9A-9E show sporozoite-primed CTL responses are
substantially boosted by MVA. Mice were immunised with: FIG. 9A,
two low doses (50+50) of irradiated sporozoites; FIG. 9B, two high
doses (300+500) of sporozoites; FIG. 9D, low-dose sporozoite
priming followed by boosting with MVA.PbCSP; FIG. 9E, high dose
sporozoite priming followed by boosting with MVA.PbCSP. CTL
responses following immunisation with MVA.PbCSP are shown in FIG.
9C.
[0033] FIGS. 10A and 10B show CTL responses primed by plasmid DNA
or recombinant Adenovirus and boosted with MVA. Groups of BALB/c
mice (n=3) were primed with plasmid DNA (FIG. 10A) or recombinant
Adenovirus expressing .beta.-galactosidase (FIG. 10B). Plasmid DNA
was administered intramuscularly, MVA intravenously and Adenovirus
intradermally. Splenocytes were restimulated with peptide TPHPARIGL
[SEQ ID NO: 69] two weeks after the last immunisation. CTL activity
was tested with peptide-pulsed P815 cells.
[0034] FIGS. 11A-11C show CTL responses in BALB/c mice primed with
plasmid DNA followed by boosting with different recombinant
vaccinia viruses. Animals were primed with pTH.PbCSP 50 .mu.g/mouse
i.m. and two weeks later boosted with different strains of
recombinant vaccina viruses (10.sup.6 pfu per mouse i.v.)
expressing PbCSP. The different recombinant vaccinia virus strains
were: FIG. 11A, MVA; FIG. 11B, NYVAC; and WR in FIG. C. The
frequencies of peptide-specific CD8+ T cells were determined using
the ELISPOT assay.
[0035] FIG. 12 shows frequencies of peptide-specific CD8+ T cells
following different routes of MVA boosting. Results are shown as
the number of spot-forming cells (SFC) per one million splenocytes.
Each bar represents the mean number of SFCs from three mice assayed
individually.
[0036] FIG. 13 shows the survival rate of the two groups of mice.
Sixty days after challenge eight out of ten mice were alive in the
group immunised with the tumour epitopes string.
[0037] FIG. 14 shows results of an influenza virus challenge
experiment. BALB/c mice were immunised as indicated. GG=gene gun
immunisations, im=intramuscular injection, iv=intravenous
injection. Survival of the animals was monitored daily after
challenge.
[0038] FIG. 15 shows detection of SIV-specific MHC class
I-restricted CD8+ T cells using tetramers. Each bar represents the
percentage of CD8+ T cells specific for the Mamu-A*01/gag epitope
at the indicated time point. One percent of CD8 T cells corresponds
to about 5000/10.sup.6 peripheral blood lymphocytes.
[0039] FIG. 16 shows CTL induction in macaques following DNA/MVA
immunisation. PBMC from three different macaques (CYD, DI and
DORIS) were isolated at week 18, 19 and 23 and were restimulated
with peptide CTPYDINQM [SEQ ID NO: 54] in vitro. After two
restimulations with peptide CTPYDINQM [SEQ ID NO: 54] the cultures
were tested for their lytic activity on peptide-pulsed autologous
target cells.
DETAILED DESCRIPTION OF THE INVENTION
[0040] It is an aim of this invention to identify an effective
means of immunizing against malaria. It is a further aim of this
invention to identify means of immunizing against other diseases in
which CD8+ T cell responses play a protective role. Such diseases
include but are not limited to infection and disease caused by the
viruses HIV, herpes simplex, herpes zoster, hepatitis C, hepatitis
B, influenza, Epstein-Barr virus, measles, dengue and HTLV-1; by
the bacteria Mycobacterium tuberculosis and Listeria sp.; and by
the protozoan parasites Toxoplasma and Trypanosoma; and certain
forms of cancer e.g. melanoma, cancer of the breast and cancer of
the colon.
[0041] We describe here a novel method of immunizing that generated
very high levels of CD8+ T cells and was found to be capable of
inducing unprecedented complete protection against P. berghei
sporozoite challenge. The same approach was tested in higher
primates and found to be highly immunogenic in this species also,
and was found to induce partial protection against P. falciparum
challenge. Induction of protective immune responses has also been
demonstrated in two additional mouse models of viral infection and
cancer.
[0042] We show further than the novel immunization regime that is
described here is also effective in generating strong CD8+ T cell
responses against HIV epitopes. Considerable evidence indicates
that the generation of such CD8+ T cell responses can be expected
to be of value in prophylactic or therapeutic immunization against
this viral infection and disease (Gallimore et al 1995; Ada 1996).
We demonstrate that strong CD8+ T cell responses may be generated
against epitopes from both HIV and malaria using an epitope string
with sequences from both of these micro-organisms. The success in
generating enhanced immunogenicity against both HIV and malaria
epitopes, and also against influenza and tumor epitopes, indicates
that this novel immunization regime can be effective generally
against many infectious pathogens and also in non-infectious
diseases where the generation of a strong CD8+ T cell response may
be of value.
[0043] A surprising feature of the current invention is the finding
of the very high efficacy of non-replicating agents in both priming
and particularly in boosting a CD8+ T cell response. In general the
immunogenicity of CD8+ T cell induction by live replicating viral
vectors has previously been found to be higher than for
non-replicating agents or replication-impaired vectors. This is as
would be expected from the greater amount of antigen produced by
agents that can replicate in the host. Here however we find that
the greatest immunogenicity and protective efficacy is surprisingly
observed with non-replicating vectors. The latter have an added
advantage for vaccination in that they are in general safer for use
in humans than replicating vectors.
[0044] The present invention provides in one aspect a kit for
generating a protective CD8+ T cell immune response against at
least one target antigen, which kit comprises: [0045] (i) a priming
composition comprising a source of one or more CD8+ T cell epitopes
of the target antigen, together with a pharmaceutically acceptable
carrier; and [0046] (ii) a boosting composition comprising a source
of one or more CD8+ T cell epitopes of the target antigen,
including at least one CD8+ T cell epitope which is the same as a
CD8+ T cell epitope of the priming composition, wherein the source
of CD8+ T cell epitopes is a non-replicating or
replication-impaired recombinant poxvirus vector, together with a
pharmaceutically acceptable carrier; with the proviso that if the
source of epitopes in (i) is a viral vector, the viral vector in
(ii) is derived from a different virus.
[0047] In another aspect the invention provides a method for
generating a protective CD8+ T cell immune response against at
least one target antigen, which method comprises administering at
least one dose of component (i), followed by at least one dose of
component (ii) of the kit according to the invention.
[0048] Preferably, the source of CD8+ T cell epitopes in (i) in the
method according to the invention is a non-viral vector or a
non-replicating or replication-impaired viral vector, although
replicating viral vectors may be used.
[0049] Preferably, the source of CD8+ T cell epitopes in (i) is not
a poxvirus vector, so that there is minimal cross-reactivity
between the primer and the booster.
[0050] In one preferred embodiment of the invention, the source of
CD8+ T cell epitopes in the priming composition is a nucleic acid,
which may be DNA or RNA, in particular a recombinant DNA plasmid.
The DNA or RNA may be packaged, for example in a lysosome, or it
may be in free form.
[0051] In another preferred embodiment of the invention, the source
of CD8+ T cell epitopes in the priming composition is a peptide,
polypeptide, protein, polyprotein or particle comprising two or
more CD8+ T cell epitopes, present in a recombinant string of CD8+
T cell epitopes or in a target antigen. Polyproteins include two or
more proteins which may be the same, or preferably different,
linked together. Particularly preferred in this embodiment is a
recombinant proteinaceous particle such as a Ty virus-like particle
(VLP) (Burns et al. Molec. Biotechnol. 1994, 1: 137-145).
[0052] Preferably, the source of CD8+ T cell epitopes in the
boosting composition is a vaccinia virus vector such as MVA or
NYVAC. Most preferred is the vaccinia strain modified virus ankara
(MVA) or a strain derived therefrom. Alternatives to vaccinia
vectors include avipox vectors such as fowlpox or canarypox
vectors. Particularly suitable as an avipox vector is a strain of
canarypox known as ALVAC (commercially available as Kanapox), and
strains derived therefrom.
[0053] Poxvirus genomes can carry a large amount of heterologous
genetic information. Other requirements for viral vectors for use
in vaccines include good immunogenicity and safety. MVA is a
replication-impaired vaccinia strain with a good safety record. In
most cell types and normal human tissues, MVA does not replicate;
limited replication of MVA is observed in a few transformed cell
types such as BHK21 cells. It has now been shown, by the results
described herein, that recombinant MVA and other non-replicating or
replication-impaired strains are surprisingly and significantly
better than conventional recombinant vaccinia vectors at generating
a protective CD8+ T cell response, when administered in a boosting
composition following priming with a DNA plasmid, a recombinant
Ty-VLP or a recombinant adenovirus.
[0054] It will be evident that vaccinia virus strains derived from
MVA, or independently developed strains having the features of MVA
which make MVA particularly suitable for use in a vaccine, will
also be suitable for use in the invention.
[0055] MVA containing an inserted string of epitopes (MVA-HM, which
is described in the Examples) has been deposited at the European
Collection of Animal Cell Cultures, CAMR, Salisbury, Wiltshire SP4
0JG, UK under accession no. V97060511 on 5 Jun. 1997.
[0056] The term "non-replicating" or "replication-impaired" as used
herein means not capable of replication to any significant extent
in the majority of normal mammalian cells or normal human cells.
Viruses which are non-replicating or replication-impaired may have
become so naturally (i.e. they may be isolated as such from nature)
or artificially e.g. by breeding in vitro or by genetic
manipulation, for example deletion of a gene which is critical for
replication. There will generally be one or a few cell types in
which the viruses can be grown, such as CEF cells for MVA.
[0057] Replication of a virus is generally measured in two ways: 1)
DNA synthesis and 2) viral titre. More precisely, the term
"non-replicating or replication-impaired" as used herein and as it
applies to poxviruses means viruses which satisfy either or both of
the following criteria: [0058] 1) exhibit a 1 log (10 fold)
reduction in DNA synthesis compared to the Copenhagen strain of
vaccinia virus in MRC-5 cells (a human cell line); [0059] 2)
exhibit a 2 log reduction in viral titre in HELA cells (a human
cell line) compared to the Copenhagen strain of vaccinia virus.
[0060] Examples of poxviruses which fall within this definition are
MVA, NYVAC and avipox viruses, while a virus which falls outside
the definition is the attenuated vaccinia strain M7.
[0061] Alternative preferred viral vectors for use in the priming
composition according to the invention include a variety of
different viruses, genetically disabled so as to be non-replicating
or replication-impaired. Such viruses include for example
non-replicating adenoviruses such as E1 deletion mutants. Genetic
disabling of viruses to produce non-replicating or
replication-impaired vectors has been widely described in the
literature (e.g. McLean et al. 1994).
[0062] Other suitable viral vectors for use in the priming
composition are vectors based on herpes virus and Venezuelan equine
encephalitis virus (VEE) (Davies et al. 1996). Suitable bacterial
vectors for priming include recombinant BCG and recombinant
Salmonella and Salmonella transformed with plasmid DNA (Darji A et
al. 1997 Cell 91: 765-775).
[0063] Alternative suitable non-viral vectors for use in the
priming composition include lipid-tailed peptides known as
lipopeptides, peptides fused to carrier proteins such as KLH either
as fusion proteins or by chemical linkage, whole antigens with
adjuvant, and other similar systems. Adjuvants such as QS21 or
SBAS2 (Stoute J A et al. 1997 N Engl J Medicine 226: 86-91) may be
used with proteins, peptides or nucleic acids to enhance the
induction of T cell responses. These systems are sometimes referred
to as "immunogens" rather than "vectors", but they are vectors
herein in the sense that they carry the relevant CD8+ T cell
epitopes.
[0064] There is no reason why the priming and boosting compositions
should not be identical in that they may both contain the priming
source of CD8+ T cell epitopes as defined in (i) above and the
boosting source of CD8+ T cell epitopes as defined in (ii) above. A
single formulation which can be used as a primer and as a booster
will simplify administration. The important thing is that the
primer contains at least the priming source of epitopes as defined
in (i) above and the booster contains at least the boosting source
of epitopes as defined in (ii) above.
[0065] The CD8+ T cell epitopes either present in, or encoded by
the priming and boosting compositions, may be provided in a variety
of different forms, such as a recombinant string of one or two or
more epitopes, or in the context of the native target antigen, or a
combination of both of these. CD8+ T cell epitopes have been
identified and can be found in the literature, for many different
diseases. It is possible to design epitope strings to generate a
CD8+ T cell response against any chosen antigen that contains such
epitopes. Advantageously, the epitopes in a string of multiple
epitopes are linked together without intervening sequences so that
unnecessary nucleic acid and/or amino acid material is avoided. In
addition to the CD8+ T cell epitopes, it may be preferable to
include one or more epitopes recognized by T helper cells, to
augment the immune response generated by the epitope string.
Particularly suitable T helper cell epitopes are ones which are
active in individuals of different HLA types, for example T helper
epitopes from tetanus (against which most individuals will already
be primed). A useful combination of three T helper epitopes is
employed in the examples described herein. It may also be useful to
include B cell epitopes for stimulating B cell responses and
antibody production.
[0066] The priming and boosting compositions described may
advantageously comprise an adjuvant. In particular, a priming
composition comprising a DNA plasmid vector may also comprise
granulocyte macrophage-colony stimulating factor (GM-CSF), or a
plasmid encoding it, to act as an adjuvant; beneficial effects are
seen using GM-CSF in polypeptide form.
[0067] The compositions described herein may be employed as
therapeutic or prophylactic vaccines. Whether prophylactic or
therapeutic immunization is the more appropriate will usually
depend upon the nature of the disease. For example, it is
anticipated that cancer will be immunized against therapeutically
rather than before it has been diagnosed, while anti-malaria
vaccines will preferably, though not necessarily be used as a
prophylactic.
[0068] The compositions according to the invention may be
administered via a variety of different routes. Certain routes may
be favoured for certain compositions, as resulting in the
generation of a more effective response, or as being less likely to
induce side effects, or as being easier for administration. The
present invention has been shown to be effective with gene gun
delivery, either on gold beads or as a powder.
[0069] In further aspects, the invention provides: [0070] a method
for generating a protective CD8+ T cell immune response against a
pathogen or tumor, which method comprises administering at least
one dose of a recombinant DNA plasmid encoding at least one CD8+ T
cell epitope or antigen of the pathogen or cancer, followed by at
least one dose of a non-replicating or replication-impaired
recombinant pox virus encoding the same epitope or antigen; [0071]
a method for generating a protective CD8+ T cell immune response
against a pathogen or tumor, which method comprises administering
at least one dose of a recombinant protein or particle comprising
at least one epitope or antigen of the pathogen or cancer, followed
by at least one dose of a recombinant MVA vector encoding the same
epitope or antigen; [0072] the use of a recombinant non-replicating
or replication-impaired pox virus vector in the manufacture of a
medicament for boosting a CD8+ T cell immune response; [0073] the
use of an MVA vector in the manufacture of a medicament for
boosting a CD8+ T cell immune response; [0074] a medicament for
boosting a primed CD8+ T cell response against at least one target
antigen or epitope, comprising a source of one or more CD8+ T cell
epitopes of the target antigen, wherein the source of CD8+ T cell
epitopes is a non-replicating or a replication-impaired recombinant
poxvirus vector, together with a pharmaceutically acceptable
carrier; and [0075] the priming and/or boosting compositions
described herein, in particulate form suitable for delivery by a
gene gun; and methods of immunization comprising delivering the
compositions by means of a gene gun.
[0076] Also provided by the invention are: the epitope strings
described herein, including epitope strings comprising the amino
acid sequences listed in table 1 and table 2; recombinant DNA
plasmids encoding the epitope strings; recombinant Ty-VLPs
comprising the epitope strings; a recombinant DNA plasmid or
non-replicating or replication impaired recombinant pox virus
encoding the P. falciparum antigen TRAP; and a recombinant
polypeptide comprising a whole or substantially whole protein
antigen such as TRAP and a string of two or more epitopes in
sequence such as CTL epitopes from malaria.
Example Formulations and Immunization Protocols
Formulation 1
[0077] Priming Composition: DNA plasmid 1 mg/ml in PBS
Boosting Composition Recombinant MVA, 10.sup.8 ffu in PBS
[0078] Protocol: Administer two doses of 1 mg of priming
composition, i.m., at 0 and 3 weeks followed by two doses of
booster intradermally at 6 and 9 weeks.
Formulation 2
Priming Composition: Ty-VLP 500 .mu.g in PBS
Boosting Composition: MVA, 10.sup.8 ffu in PBS
[0079] Protocol: Administer two doses of priming composition, i.m.,
at 0 and 3 weeks, then 2 doses of booster at 6 and 9 weeks. For
tumor treatment, MVA is given i.v. as one of most effective
routes.
Formulation 3
[0080] Priming Composition: Protein 500 .mu.g+adjuvant (QS-21)
Boosting Composition Recombinant MVA, 10.sup.8 ffu in PBS
[0081] Protocol: Administer two doses of priming composition at 0
and 3 weeks and 2 doses of booster i.d. at 6 and 9 weeks.
Formulation 4
[0082] Priming Composition: Adenovirus vector, 10.sup.9 pfu in
PBS
Boosting Composition Recombinant MVA, 10.sup.8 ffu in PBS
[0083] Protocol: Administer one or two doses of priming composition
intradermally at 0 and 3 weeks and two doses of booster i.d. at 6
and 9 weeks. The above doses and protocols may be varied to
optimise protection. Doses may be given between for example, 1 to 8
weeks apart rather than 2 weeks apart.
[0084] The invention will now be further described in the examples
which follow.
EXAMPLES
Example 1
Materials and Methods
Generation of the Epitope Strings
[0085] The malaria epitope string was made up of a series of
cassettes each encoding three epitopes as shown in Table 1, with
restriction enzyme sites at each end of the cassette. Each cassette
was constructed from four synthetic oligonucleotides which were
annealed together, ligated into a cloning vector and then sequenced
to check that no errors had been introduced. Individual cassettes
were then joined together as required. The BamHI site at the 3' end
of cassette C was fused to the BglII site at the 5' end of cassette
A, destroying both restriction enzyme sites and encoding a two
amino acid spacer (GS) between the two cassettes. Cassettes B, D
and H were then joined to the string in the same manner. A longer
string containing CABDHFE was also constructed in the same way.
TABLE-US-00001 TABLE 1 CTL Epitopes of the Malaria (M) String Amino
acid HLA Cassette Epitope Sequence DNA sequence Type restriction A
Ls8 KPNDKSLY AAGCCGAACGACAAGTCCTTGTAT CTL B35 SEQ ID NO.: 2 SEQ ID
NO.: 1 Cp26 KPKDELDY AAACCTAAGGACGAATTGGACTAC CTL B35 SEQ ID NO.: 4
SEQ ID NO.: 3 Ls6 KPIVQYDNF AAGCCAATCGTTCAATACGACAACTTC CTL B53 SEQ
ID NO.: 6 SEQ ID NO.: 5 B Tr42/43 ASKNKEKALII
GCCTCCAAGAACAAGGAAAAGGCTTTGAT CTL B8 SEQ ID NO.: 8 ATC SEQ ID NO.:
7 Tr39 IAGGLALL GGTATCGCTGGTGGTTTGGCCTTGTTG CTL A2.1 SEQ ID NO.: 10
SEQ ID NO.:9 Cp6 MNPNDPNRNV ATGAACCCTAATGACCCAAACAGAAACGT CTL B7
SEQ ID NO.: 12 C SEQ ID NO.: 11 C St8 MINAYLDKL
ATGATCAACGCCTACTTGGACAAGTTG CTL A2.2 SEQ ID NO.: 14 SEQ ID NO.: 13
Ls50 ISKYEDEI ATCTCCAAGTACGAAGACGAAATC CTL B17 SEQ ID NO.: 16 SEQ
ID NO.: 15 Pb9 SYIPSAEKI TCCTACATCCCATCTGCCGAAAAGATC CTL mouse
H2-K.sup.d SEQ ID NO.: 18 SEQ ID NO.: 17 D Tr26 HLGNVKYLV
CACTTGGGTAACGTTAAGTACTTGGTT CTL A2.1 SEQ ID NO.: 20 SEQ ID NO.: 19
Ls53 KSLYDEHI AAGTCTTTGTACGATGAACACATC CTL B58 SEQ ID NO.: 22 SEQ
ID NO.: 21 Tr29 LLMDCSGSI TTATTGATGGACTGTTCTGGTTCTATT CTL A2.2 SEQ
ID NO.: 24 SEQ ID NO.: 23 E NANP NANPNANPNANPN
AACGCTAATCCAAACGCAAATCCGAACGC B cell ANP CAATCCTAACGCGAATCCC SEQ ID
NO.: 26 SEQ ID NO.: 25 TRAP AM DEWSPCSVTCGK
GACGAATGGTCTCCATGTTCTGTCACTTGT Heparin GTRSRKRE
GGTAAGGGTACTCGCTCTAGAAAGAGAGAA binding SEQ ID NO.: 28 SEQ ID NO.:
27 motif F Cp39 YLNKIQNSL TACTTGAACAAAATTCAAAACTCTTTG CTL A2.1 SEQ
ID NO.: 30 SEQ ID NO.: 29 La72 MEKLKELEK
ATGGAAAAGTTGAAAGAATTGGAAAAG CTL B8 SEQ ID NO.: 32 SEQ ID NO.: 31
ex23 ATSVLAGL GCTACTTCTGTCTTGGCTGGTTTG CTL B58 SEQ ID NO.: 34 SEQ
ID NO.: 33 H CSP DPNANPNVDPNANP GACCCAAACGCTAACCCAAACGTTGACCC T
helper Universal NV AAACGCCAACCCAAACGTC epitopes SEQ ID NO.: 36 SEQ
ID NO.: 35 BCG QVHFQPLPPAVV CAAGTTCACTTCCAACCATTGCCTCCGGC T helper
KL CGTTGTCAAGTTG SEQ ID NO.: 38 SEQ ID NO.: 37 TT QFIKANSKFIGIT
CAATTCATCAAGGCCAACTCTAAGTTCAT T helper E CGGTATCACCGAA SEQ ID NO.:
40 SEQ ID NO.: 39
Table 1
[0086] Sequences included in the malaria epitope string. Each
cassette consists of the epitopes shown above, in the order shown,
with no additional sequence between epitopes within a cassette. A
BglII site was added at the 5' end and a BamHI site at the 3' end,
such that between cassettes in an epitope string the BamHI/BglII
junction encodes GS. All epitopes are from P. falciparum antigens
except for pb9 (P. berghei), BCG (M. tuberculosis) and TT
(Tetanus). The amino acid and DNA sequences shown in the table have
SEQ ID NOS. 1 to 40 in the order in which they appear.
[0087] FIG. 1 shows the construct used to express Ty-VLP with the
malaria epitope cassette CABDHFE. CTL epitopes are from P.
falciparum STARP (sporozoite threonine- and asparagine-rich
protein) (st), LSA-1 (liver stage antigen 1) (1s), CSP
(circumsporozoite protein) (cp), TRAP (thrombospondin-related
adhesive protein) (tr), LSA-3 (liver stage antigen 3) (la) and
Exp-1 (exported protein 1) (ex). Helper epitopes are from the P.
falciparum CS protein, the M. tuberculosis 38Kd antigen and Tetanus
Toxoid. NANP is the antibody epitope from CS and AM is the adhesion
motif from P. falciparum TRAP (Muller et al 1993). The length of
the complete string is 229 amino acids as shown in the table 1
legend, with the amino acid sequence:--
TABLE-US-00002 [SEQ ID NO: 41]
MINAYLDKLISKYEDEISYIPSAEKIGSKPNDKSLYKPKDELDYKPIVQY
DNFGSASKNKEKALIIGIAGGLALLMNPNDPNRNVGSHLGNVKYLVKSLY
DEHILLMDCSGSIGSDPNANPNVDPNANPNVQVHFQPLPPAVVKLQFIKA
NSKFIGITEGSYLNKIQNSLMEKLKELEKATSVLAGLGSNANPNANPNAN
PNANPDEWSPCSVTCGKGTRSRKREGSGK.
[0088] The HIV epitope string was also synthesised by annealing
oligonucleotides. Finally the HIV and malaria epitope strings were
fused by joining the BamHI site at the 3' end of the HIV epitopes
to the BglII site at the 5' end of cassettes CAB to form the HM
string (Table 2).
TABLE-US-00003 TABLE 2 CTL Epitopes of the HIV/SIV Epitope String
Re- Epitope striction Origin YLKDQQLL (SEQ ID NO.: 42) A24, B8
HIV-1 gp41 ERYLKDQQL (SEQ ID NO.: 43) B14 HIV-1 gp41 EITPIGLAP (SEQ
ID NO.: 44) Mamu-B*01 SIV env PPIPVGEIY (SEQ ID NO.: 45) B35 HIV-1
p24 GEIYKRWII (SEQ ID NO.: 46) B8 HIV-1 p24 KRWIILGLNK (SEQ ID NO.:
47) B*2705 HIV-1 p24 IILGLNKIVR (SEQ ID NO.: 48) A33 HIV-1 p24
LGLNKIVRMY (SEQ ID NO.: 49) Bw62 HIV-1 p24 YNLTMKCR (SEQ ID NO.:
50) Mamu-A*02 SIV env RGPGRAFVTI (SEQ ID NO.: 51) A2, H-2Dd HIV-1
gp120 GRAFVTIGK (SEQ ID NO.: 52) B*2705 HIV-1 gp120 TPYDINQML (SEQ
ID NO.: 53) B53 HIV-2 gag CTPYDINQM (SEQ ID NO.: 54) Mamu-A*01 SIV
gag RPQVPLRPMTY (SEQ ID NO.: 55) B51 HIV-1 nef QVPLRPMTYK (SEQ ID
NO.: 56) A*0301, HIV-1 nef A11 VPLRPMTY (SEQ ID NO.: 57) B35 HIV-1
nef AVDLSHFLK (SEQ ID NO.: 58) A11 HIV-1 nef DLSHFLKEK (SEQ ID NO.:
59) A*0301 HIV-1 nef FLKEKGGL (SEQ ID NO.: 60) B8 HIV-1 nef
ILKEPVHGV (SEQ ID NO.: 61) A*0201 HIV-1 pol ILKEPVHGVY (SEQ ID NO.:
62) Bw62 HIV-1 pol HPDIVIYQY (SEQ ID NO.: 63) B35 HIV-1 pol
VIYQYMDDL (SEQ ID NO.: 64) A*0201 HIV-1 pol
Table 2
[0089] Sequences of epitopes from HIV or SIV contained in the H
epitope string and assembled as shown in FIG. 2. The amino acids in
the table have SEQ ID NOS: 42 to 64 in the order in which they
appear.
[0090] FIG. 2 shows a schematic outline of the H, M and HM
proteins. The bar patterns on the schematic representations of the
polyepitope proteins indicate the origin of the sequences (see
tables 1 and 2). The positions of individual epitopes and their MHC
restrictions are depicted above and below the proteins. Pb is the
only epitope derived from the protein of P. berghei. All other
epitopes in the M protein originate from proteins of P. falciparum:
cs--circumsporozoite protein, st--STARP, Is--LSA-1 and tr--TRAP.
BCG--38 kDa protein of M. tuberculosis; TT--tetanus toxin.
[0091] For the anti-tumour vaccine an epitope string containing CTL
epitopes was generated, similar to the malaria and HIV epitope
string. In this tumour epitope string published murine CTL epitopes
were fused together to create the tumour epitope string with the
amino acid sequence:
MLPYLGWLVF-AQHPNAELL-KHYLFRNL-SPSYVYHQF-IPNPLLGLD [SEQ ID NO: 65].
CTL epitopes shown here were fused together. The first amino acid
methionine was introduced to initiate translation.
Ty Virus-Like Particles (Vlps)
[0092] The epitope string containing cassette CABDH was introduced
into a yeast expression vector to make a C-terminal in-frame fusion
to the TyA protein. When TyA or TyA fusion proteins are expressed
in yeast from this vector, the protein spontaneously forms virus
like particles which can be purified from the cytoplasm of the
yeast by sucrose gradient centrifugation. Recombinant Ty-VLPs were
prepared in this manner and dialysed against PBS to remove the
sucrose before injection (c.f. Layton et al. 1996).
[0093] Replication-defective recombinant Adenovirus with a deletion
of the E1 genes was used in this study (McGrory et al, 1988). The
Adenovirus expressed E. coli .beta.-galactosidase under the control
of a CMV IE promoter. For immunisations, 10.sup.7 pfu of virus were
administered intradermally into the ear lobe.
[0094] Peptides were purchased from Research Genetics (USA),
dissolved at 10 mg/ml in DMSO (Sigma) and further diluted in PBS to
1 mg/ml. Peptides comprising CTL epitopes that were used in the
experiments described herein are listed in table 3.
TABLE-US-00004 TABLE 3 Sequence of CTL Peptide Epitopes MHC
sequence Antigen restriction LPYLGWLVF P1A tumour L.sup.d (SEQ ID
NO.: 66) antigen SYIPSAEKI P. berghei CSP K.sup.d (SEQ ID NO.: 67)
RGPGRAFVTI HIV gag D.sup.d (SEQ ID NO.: 68) TPHPARIGL E. coil
L.sup.d (SEQ ID NO.: 69) b-galactosidase TYQRTRALV Influenza A
virus K.sup.d (SEQ ID NO.: 70) NP SDYEGRLI Influenza A virus
K.sup.k (SEQ ID NO.: 71) NP ASNENMETM Influenza A virus NP D.sup.b
(SEQ ID NO.: 72) INVAFNRFL P. falciparum TRAP K.sup.b (SEQ ID NO.:
73)
[0095] The amino acid sequences in Table 3 have SEQ ID NOS: 66 to
73, in the order in which they appear in the Table.
Plasmid DNA Constructs
[0096] A number of different vectors were used for constructing DNA
vaccines. Plasmid pTH contains the CMV IE promoter with intron A,
followed by a polylinker to allow the introduction of antigen
coding sequences and the bovine growth hormone transcription
termination sequence. The plasmid carries the ampicillin resistance
gene and is capable of replication in E. coli but not mammalian
cells. This was used to make DNA vaccines expressing each of the
following antigens: P. berghei TRAP, P. berghei CS, P. falciparum
TRAP, P. falciparum LSA-1 (278 amino acids of the C terminus only),
the epitope string containing cassettes CABDH and the HM epitope
string (HIV epitopes followed by cassettes CAB). Plasmid pSG2 is
similar to pTH except for the antibiotic resistance gene. In pSG2
the ampicillin resistance gene of pTH has been replaced by a
kanamycin resistance gene. pSG2 was used to make DNA vaccines
expressing the following antigens: P. berghei PbCSP, a mouse tumour
epitope string, the epitope string containing cassettes CABDH and
the HM epitope string. Plasmid V1J-NP expresses influenza
nucleoprotein under the control of a CMV IE promoter. Plasmids
CMV-TRAP and CMV-LSA-1 are similar to pTH.TRAP and pTH. LSA-1 but
do not contain intron A of the CMV promoter. Plasmids RSV.TRAP and
RSV.LSA-1 contain the RSV promoter, SV40 transcription termination
sequence and are tetracycline resistant. For induction of
.beta.-galactosidase-specific CTL plasmid pcDNA3/His/LacZ
(Invitrogen) was used. All DNA vaccines were prepared from E. coli
strain DH5.alpha. using Qiagen plasmid purification columns.
Generation of Recombinant Vaccinia Viruses
[0097] Recombinant MVAs were made by first cloning the antigen
sequence into a shuttle vector with a viral promoter such as the
plasmid pSC11 (Chakrabarti et al. 1985; Morrison et al. 1989). P.
berghei CS and P. falciparum TRAP, influenza nucleoprotein and the
HM and mouse tumour epitope polyepitope string were each expressed
using the P7.5 promoter (Mackett et al. 1984), and P. berghei TRAP
was expressed using the strong synthetic promoter (SSP; Carroll et
al. 1995). The shuttle vectors, pSC11 or pMCO3 were then used to
transform cells infected with wild-type MVA so that viral sequences
flanking the promoter, antigen coding sequence and marker gene
could recombine with the MVA and produce recombinants. Recombinant
viruses express the marker gene (.beta. glucuronidase or .beta.
galactosidase) allowing identification of plaques containing
recombinant virus. Recombinants were repeatedly plaque purified
before use in immunisations. The recombinant NYVAC-PbCSP vaccinia
was previously described (Lanar et al. 1996). The wild type or
Western Reserve (WR) strain of recombinant vaccinia encoding PbCSP
was described previously (Satchidanandam et al. 1991).
Cells and Culture Medium
[0098] Murine cells and Epstein-Barr virus transformed chimpanzee
and macaque B cells (BCL) were cultured in RPMI supplemented with
10% heat inactivated fetal calf serum (FCS). Splenocytes were
restimulated with the peptides indicated (final concentration 1
.mu.g/ml) in MEM medium with 10% FCS, 2 mM glutamine, 50 U/ml
penicillin, 50 .mu.M 2-mercaptoethanol and 10 mM Hepes pH7.2
(Gibco, UK).
Animals
[0099] Mice of the strains indicated, 6-8 weeks old were purchased
from Harlan Olac (Shaws Farm, Blackthorn, UK). Chimpanzees H1 and
H2 were studied at the Biomedical Primate Research Centre at
Rijswick, The Netherlands. Macaques were studied at the University
of Oxford.
Immunisations
[0100] Plasmid DNA immunisations of mice were performed by
intramuscular immunisation of the DNA into the musculus tibialis
under anaesthesia. Mouse muscle was sometimes pre-treated with 50
.mu.l of 1 mM cardiotoxin (Latoxan, France) 5-9 days prior to
immunisation as described by Davis et al (1993), but the presence
or absence of such pre-treatment was not found to have any
significant effect on immunogenicity or protective efficacy. MVA
immunisation of mice was performed by either intramuscular (i.m.),
intravenous (into the lateral tail vein) (i.v.), intradermal
(i.d.), intraperitoneal (i.p.) or subcutaneous (s.c.) immunisation.
Plasmid DNA and MVA immunisation of the chimpanzees H1 and H2 was
performed under anaesthesia by intramuscular immunisation of leg
muscles. For these chimpanzee immunisations the plasmid DNA was
co-administered with 15 micrograms of human GM-CSF as an adjuvant.
Recombinant MVA administration to the chimpanzees was by
intramuscular immunisation under veterinary supervision.
Recombinant human GM-CSF was purchased from Sandoz (Camberley, UK).
For plasmid DNA immunisations using a gene gun, DNA was
precipitated onto gold particles. For intradermal delivery, two
different types of gene guns were used, the Acell and the Oxford
Bioscience device (PowderJect Pharmaceuticals, Oxford, UK).
Elispot Assays
[0101] CD8+ T cells were quantified in the spleens of immunised
mice without in vitro restimulation using the peptide epitopes
indicated and the ELISPOT assay as described by Miyahara et al
(1993). Briefly, 96-well nitrocellulose plates (Miliscreen MAHA,
Millipore, Bedford UK) were coated with 15 .mu.g/ml of the
anti-mouse interferon-.gamma. monoclonal antibody R4 (EACC) in 50
.mu.l of phosphate-buffered saline (PBS). After overnight
incubation at 4.degree. C. the wells were washed once with PBS and
blocked for 1 hour at room temperature with 100 .mu.l RPMI with 10%
FCS. Splenocytes from immunised mice were resuspended to
1.times.10.sup.7 cells/ml and placed in duplicate in the antibody
coated wells and serially diluted. Peptide was added to each well
to a final concentration of 1 .mu.g/ml. Additional wells without
peptide were used as a control for peptide-dependence of
interferon-.gamma. secretion. After incubation at 37.degree. C. in
5% CO.sub.2 for 12-18 hours the plates were washed 6 times with PBS
and water. The wells were then incubated for 3 hours at room
temperature with a solution of 1 .mu.g/ml of biotinylated
anti-mouse interferon-.gamma. monoclonal antibody XMG1.2
(Pharmingen, Calif., USA) in PBS. After further washes with PBS, 50
.mu.l of a 1 .mu.g/ml solution of streptavidin-alkaline-phosphatase
polymer (Sigma) was added for 2 hours at room temperature. The
spots were developed by adding 50 .mu.l of an alkaline phosphatase
conjugate substrate solution (Biorad, Hercules, Calif., USA). After
the appearance of spots the reaction was stopped by washing with
water. The number of spots was determined with the aid of a
stereomicroscope.
[0102] ELISPOT assays on the chimpanzee peripheral blood
lymphocytes were performed using a very similar method employing
the assay and reagents developed to detect human CD8 T cells
(Mabtech, Stockholm).
CTL Assays
[0103] CTL assays were performed using chromium labelled target
cells as indicated and cultured mouse spleen cells as effector
cells as described by Allsopp et al. (1996). CTL assays using
chimpanzee or macaque cells were performed as described for the
detection of human CTL by Hill et al. (1992) using EBV-transformed
autologous chimpanzee chimpanzee or macaque B cell lines as target
cells.
P. Berghei Challenge
[0104] Mice were challenged with 2000 (BALB/c) or 200 (C57BL/6)
sporozoites of the P. berghei ANKA strain in 200 ml RPMI by
intravenous inoculation as described (Lanar et al. 1996). These
sporozoites were dissected from the salivary glands of Anopheles
stephensi mosquitoes maintained at 18.degree. C. for 20-25 days
after feeding on infected mice. Blood-stage malaria infection,
indicating a failure of the immunisation, was detected by observing
the appearance of ring forms of P. berghei in Giemsa-stained blood
smears taken at 5-12 days post-challenge.
P. Falciparum Challenge
[0105] The chimpanzees were challenged with 20,000 P. falciparum
sporozoites of the NF54 strain dissected from the salivary glands
of Anopheles gambiae mosquitoes, by intravenous inoculation under
anaesthesia. Blood samples from these chimpanzees were examined
daily from day 5 after challenge by microscopy and parasite
culture, in order to detect the appearance of low levels of P.
falciparum parasites in the peripheral blood.
[0106] Mice were challenged with 1.times.10.sup.5 P815 cells in 200
.mu.l of PBS by intravenous inoculation. Animals were monitored for
survival.
Influenza Virus Challenges
[0107] Mice were challenged with 100 haemagglutinating units (HA)
of influenza virus A/PR/8/34 by intranasal inoculation. Following
challenge the animals were weighed daily and monitored for
survival.
Determining Peptide Specific CTL Using Tetramers
[0108] Tetrameric complexes consisting of Mamu-A*01-heavy chain and
.beta..sub.2-microglobulin were made as described by Ogg et al
(1998). DNA coding for the leaderless extracellular portion of the
Mamu-A*01 MHC class I heavy chain was PCR-amplified from cDNA using
5'primer MamuNdeI: 5'-CCT GAC TCA GAC CAT ATG GGC TCT CAC TCC ATG
[SEQ ID NO: 74] and 3' primer: 5'-GTG ATA AGC TTA ACG ATG ATT CCA
CAC CAT TTT CTG TGC ATC CAG AAT ATG ATG CAG GGA TCC CTC CCA TCT CAG
GGT GAG GGG C [SEQ ID NO: 75]. The former primer contained a NdeI
restriction site, the latter included a HindIII site and encoded
for the bioinylation enzyme BirA substrate peptide. PCR products
were digested with NdeI and HindIII and ligated into the same sites
of the polylinker of bacterial expression vector pGMT7. The rhesus
monkey gene encoding a leaderless .beta..sub.2-microglobulin was
PCR amplifed from a cDNA clone using primers B2 MBACK: 5'-TCA GAC
CAT ATG TCT CGC TCC GTG GCC [SEQ ID NO: 76] and B2MFOR: 5'-TCA GAC
AAG CTT TTA CAT GTC TCG ATC CCA C [SEQ ID NO: 77] and likewise
cloned into the NdeI and HindIII sites of pGMT7. Both chains were
expressed in E. coli strain BL-21, purified from inclusion bodies,
refolded in the presence of peptide CTPYDINQM [SEQ ID NO: 54],
biotinylated using the BirA enzyme (Avidity) and purified with FPLC
and monoQ ion exchange columns. The amount of biotinylated refolded
MHC-peptide complexes was estimated in an ELISA assay, whereby
monomeric complexes were first captured by conformation sensitive
monoclonal antibody W6/32 and detected by alkaline phosphatase
(AP)-conjugated streptavidin (Sigma) followed by colorimetric
substrate for AP. The formation of tetrameric complexes was induced
by addition of phycoerythrin (PE)-conjugated streptavidin
(ExtrAvidin; Sigma) to the refolded biotinylated monomers at a
molar ratio of MHC-peptide:PE-streptavidin of 4:1. The complexes
were stored in the dark at 4.degree. C. These tetramers were used
to analyse the frequency of Mamu-A*01/gag-specific CD8+ T cells in
peripherial blood lymphocytes (PBL) of immunised macaques.
Example 2
[0109] Previous studies of the induction of CTL against epitopes in
the circumsporozoite (CS) protein of Plasmodium berghei and
Plasmodium yoelii have shown variable levels of CTL induction with
different delivery systems. Partial protection has been reported
with plasmid DNA (Sedegah et al. 1994), influenza virus boosted by
replicating vaccinia virus (Li et al. 1991), adenovirus (Rodrigues
et al 1997) and particle delivery systems (Schodel et al. 1994).
Immunisation of mice intramuscularly with 50 micrograms of a
plasmid encoding the CS protein produced moderate levels of CD8+
cells and CTL activity in the spleens of these mice after a single
injection (FIGS. 3, 4A-4D).
[0110] For comparison groups of BALB/c mice (n=5) were injected
intravenously with 10.sup.6 ffu/pfu of recombinant vaccinia viruses
of different strains (WR, NYVAC and MVA) all expressing P. berghei
CSP. The frequencies of peptide-specific CD8+ T cells were measured
10 days later in an ELISPOT assay. MVA.PbCSP induced 181+/-48,
NYVAC 221+/-27 and WR 94+/-19 (mean+/- standard deviation)
peptide-specific CD8+ T cells per million splenocytes. These
results show that surprisingly replication-impaired vaccinia
viruses are superior to replicating strains in priming a CD8+ T
cell response. We then attempted to boost these moderate CD8+ T
cell responses induced by priming with either plasmid DNA or MVA
using homologous or heterologous vectors. A low level of CD8+ T
cells was observed after two immunisations with CS recombinant DNA
vaccine alone, the recombinant MVA vaccine alone or the recombinant
MVA followed by recombinant DNA (FIG. 3). A very much higher level
of CD8+ T cells was observed by boosting the DNA-primed immune
response with recombinant MVA. In a second experiment using ten
mice per group the enhanced immunogenicity of the DNA/MVA sequence
was confirmed: DNA/MVA 856+/-201; MVA/DNA 168+/-72; MVA/MVA
345+/-90; DNA/DNA 92+/-46. Therefore the sequence of a first
immunisation with a recombinant plasmid encoding the CS protein
followed by a second immunisation with the recombinant MVA virus
yielded the highest levels of CD8+ T lymphocyte response after
immunisation.
[0111] FIG. 3 shows malaria CD8 T cell ELISPOT data following
different immunisation regimes. Results are shown as the number of
peptide-specific T cells per million splenocytes. Mice were
immunised either with the PbCSP-plasmid DNA or the PbCSP-MVA virus
or combinations of the two as shown on the X axis, at two week
intervals and the number of splenocytes specific for the pb9
malaria epitope assayed two weeks after the last immunisation. Each
point represents the number of spot-forming cells (SFCs) measured
in an individual mouse. The highest level of CD8+ T cells was
induced by priming with the plasmid DNA and boosting with the
recombinant MVA virus. This was more immunogenic than the reverse
order of immunisation (MVA/DNA), two DNA immunisations (DNA/DNA) or
two MVA immunisations (MVA/MVA). It was also more immunogenic than
the DNA and MVA immunisations given simultaneously (DNA+MVA 2w),
than one DNA immunisation (DNA 4w) or one MVA immunisation given at
the earlier or later time point (MVA 2w and MVA 4w).
[0112] FIGS. 4A-4D shows that malaria CD8 T cell ELISPOT and CTL
levels are substantially boosted by a recombinant MVA immunisation
following priming with a plasmid DNA encoding the same antigen. A
AND C. CD8+ T cell responses were measured in BALB/c mice using the
g interferon ELISPOT assay on fresh splenocytes incubated for 18 h
with the K.sup.d restricted peptide SYIPSAEKI [SEQ ID NO: 67] from
P. berghei CSP and the L.sup.d restricted peptide TPHPARIGL [SEQ ID
NO: 69] from E. coli .beta.-galactosidase. Note that the ELISPOT
counts are presented on a logarithmic scale. B and D. Splenocytes
from the same mice were also assayed in conventional
.sup.51Cr-release assays at an effector: target ration of 100:1
after 6 days of in vitro restimulation with the same peptides (1
.mu.g/ml).
[0113] The mice were immunised with plasmid DNA expressing either
P. berghei CSP and TRAP, PbCSP alone, the malaria epitope cassette
including the P. berghei CTL epitope (labelled pTH.M), or
.beta.-galactosidase. ELISPOT and CTL levels measured in mice 23
days after one DNA immunisation are shown in A and B respectively.
The same assays were performed with animals that received
additionally 1.times.10.sup.7 ffu of recombinant MVA expressing the
same antigen(s) two weeks after the primary immunisation. The
ELISPOT and CTL levels in these animals are shown in C and D
respectively. Each bar represents data from an individual
animal.
[0114] Studies were also undertaken of the immunogenicity of the
epitope string HM comprising both HIV and malaria epitopes in
tandem. Using this epitope string again the highest levels of CD8+
T cells and CTL were generated in the spleen when using an
immunisation with DNA vaccine followed by an immunisation with a
recombinant MVA vaccine (Table 4, FIG. 5).
TABLE-US-00005 TABLE 4 Immunogenicity of Various DNA/MVA
Combinations as Determined by Elispot Assays Immunisation 1
Immunisation 2 HIV epitope Malaria epitope DNA-HM DNA-HM 56 .+-. 26
4 .+-. 4 MVA-HM MVA-HM 786 .+-. 334 238 .+-. 106 MVA-HM DNA-HM 306
.+-. 78 58 .+-. 18 DNA-HM MVA-HM 1000 .+-. 487 748 .+-. 446 None
DNA-HM 70 .+-. 60 100 .+-. 10 None MVA-HM 422 .+-. 128 212 .+-.
94
[0115] Table 4 shows the results of ELISPOT assays performed to
measure the levels of specific CD8+ T cells to HIV and malaria
epitopes following different immunisation regimes of plasmid DNA
and MVA as indicated. The numbers are spot-forming cells per
million splenocytes. The HM epitope string is illustrated in FIG.
2. BALB/c mice were used in all cases. The malaria epitope was pb9
as in FIGS. 2 and 3. The HIV epitope was RGPGRAFVTI [SEQ ID NO:
51]. The immunisation doses were 50 .mu.g of plasmid DNA or
10.sup.7 focus-forming units (ffu) of recombinant MVA. All
immunisations were intramuscular. The interval between
immunisations 1 and 2 was from 14-21 days in all cases.
[0116] FIG. 5 shows the CTL responses induced in BALB/c mice to
malaria and HIV epitopes by various immunisation regimes employing
plasmid DNA and recombinant MVA. Mice were immunised
intramuscularly as described in the legend to table 3 and in
methods. High levels of CTL (>30% specific lysis at
effector/target ration of 25:1) were observed to both the malaria
and HIV epitopes only after priming with plasmid DNA and boosting
with the recombinant MVA. The antigen used in this experiment is
the HIV-malaria epitope string. The recombinant MVA is denoted
MVA.HM and the plasmid DNA expressing this epitope string is
denoted pTH.HM. Levels of specific lysis at various effector to
target ratios are shown. These were determined after 5 days in
vitro restimulation of splenocytes with the two peptides pb9 and
RGPGRAFVTI [SEQ ID NO: 51].
[0117] Comparison of numerous delivery systems for the induction of
CTL was reported by Allsopp et al. (1996). Recombinant Ty-virus
like particles (Ty-VLPs) and lipid-tailed malaria peptides both
gave good CTL induction but Ty-VLPs were better in that they
required only a single immunising dose for good CTL induction.
However, as shown here even two doses of Ty particles fail to
induce significant protection against sporozoite challenge (Table
7, line 1). Immunisation with a recombinant modified vaccinia
Ankara virus encoding the circumsporozoite protein of P. berghei
also generates good levels of CTL. However, a much higher level of
CD8+ T cell response is achieved by a first immunisation with the
Ty-VLP followed by a second immunisation with the MVA CS vaccine
(Table 5).
TABLE-US-00006 TABLE 5 Immunogenicity of Various Ty-VLP/MVA
Combinations as Determined by ELISPOT and CTL Assays Immunisation 1
Immunisation 2 ELISPOT No % Specific Lysis Ty-CABDH Ty-CABDH 75 15
MVA.PbCSP MVA.PbCSP 38 35 Ty-CABDH MVA.PbCSP 225 42 Ty-CABDH MVA.HM
1930 nd
Table 5 Results of ELISPOT and CTL assays performed to measure the
levels of specific CD8+ T cells to the malaria epitope pb9
following different immunisation regimes of Ty-VLPs and recombinant
MVA virus as indicated. The CTL and ELISPOT data are from different
experiments. The ELISPOT levels (spots per million splenocytes) are
measured on un-restimulated cells and the CTL activity, indicated
as specific lysis at an effector to target ratio of 40:1, on cells
restimulated with pb9 peptide in vitro for 5-7 days. Both represent
mean levels of three mice. BALB/c mice were used in all cases. The
immunisation doses were 50 .mu.g of Ty-VLP or 10.sup.7 ffu (foci
forming units) of recombinant MVA. All immunisations were
intramuscular. The interval between immunisations 1 and 2 was from
14-21 days. MVA.HM includes cassettes CAB. Priming of an Immune
Response with DNA Delivered by a Gene Gun and Boosting with
Recombinant MVA
Immunogenicity and Challenge
[0118] The use of a gene gun to deliver plasmid DNA intradermally
and thereby prime an immune response that could be boosted with
recombinant MVA was investigated. Groups of BALB/c mice were
immunised with the following regimen: [0119] I) Three gene gun
immunisations with pTH.PbCSP (4 mg per immunisation) at two week
intervals [0120] II) Two gene gun immunisations followed by MVA
i.v. two weeks later [0121] III) One intramuscular DNA immunisation
followed by MVA i.v. two weeks later.
[0122] The immunogenicity of the three immunisation regimens was
analysed using ELISPOT assays. The highest frequency of specific T
cells was observed with two gene gun immunisations followed by an
MVA i.v. boost and the intramuscular DNA injection followed an MVA
i.v. boost (FIG. 6).
[0123] FIG. 6 shows the results of ELISPOT assays performed to
measure the levels of specific CD8+ T cells to the malaria epitope
pb9 following different immunisation regimes. Groups of BALB/c mice
(n=3) were immunised as indicated (g.g.=gene gun). The time between
all immunisations was 14 days. ELISPOT assays were done two weeks
after the last immunisation.
CTL Induction to the Same Antigen in Different Mouse Strains
[0124] To address the question whether the boosting effect
described above in BALB/c mice with two CTL epitopes SYIPSAEKI [SEQ
ID NO: 67] derived from P. berghei CSP and RGPGRAFVTI [SEQ ID NO:
68] derived from HIV is a universal phenomenon, two sets of
experiments were carried out. CTL responses to the influenza
nucleoprotein were studied in five inbred mouse strains. In a first
experiment three published murine CTL epitopes derived from the
influenza nucleoprotein were studied (see Table 3). Mice of three
different H-2 haplotypes, BALB/c and DBA/2 (H-2.sup.d), C57BL/6 and
129 (H-2.sup.b); CBA/J (H-2.sup.k), were used. One set of animals
was immunised twice at two week intervals with the plasmid V1J-NP
encoding the influenza nucleoprotein. Another set of identical
animals was primed with V1J-NP and two weeks later boosted
intravenously with 10.sup.6 ffu of MVA.NP, expressing influenza
virus NP. The levels of CTL in individual mice were determined in a
.sup.51Cr-release assay with peptide re-stimulated splenocytes. As
shown in FIG. 7, the DNA priming/MVA boosting immunisation regimen
induced higher levels of lysis in all the mouse strains analysed
and is superior to two DNA injections.
[0125] FIG. 7 shows the CTL responses against influenza NP in
different mouse strains. Mice of different strains were immunised
twice two weeks apart with a DNA vaccine V1J-NP encoding for the
influenza nucleoprotein (open circles) or primed with the same DNA
vaccine and two weeks later boosted with recombinant MVA expressing
influenza virus nucleoprotein (closed circles). Two weeks after the
last immunisation splenocytes were restimulated in vitro with the
respective peptides (Table 3). The CTL activity was determined in a
standard .sup.51Cr-release assay with MHC class I-matched target
cells.
CTL Induction to Different Antigens in Different Mouse Strains
[0126] The effect of MVA boosting on plasmid DNA-primed immune
responses was further investigated using different antigens and
different inbred mouse strains. Mice of different strains were
immunised with different antigens using two DNA immunisations and
compared with DNA/MVA immunisations. The antigens used were E. coli
.beta.-galactosidase, the malaria/HIV epitope string, a murine
tumour epitope string and P. falciparum TRAP. Compared with two DNA
immunisations the DNA-priming/MVA-boosting regimen induced higher
levels of CTL in all the different mouse strains and antigen
combinations tested (FIGS. 8A-8H).
[0127] FIGS. 8A-8H show CTL responses against different antigens
induced in different inbred mouse strains. Mice were immunised with
two DNA vaccine immunisations two weeks apart (open circles) or
primed with a DNA vaccine and two weeks later boosted with a
recombinant MVA expressing the same antigen (closed circles). The
strains and antigens were: C57BL/6; P. falciparum TRAP in A. DBA/2;
E. coli b-galactosidase in B. BALB/c; HM epitope string CTL
activity against malaria peptide (pb9) in C. DBA/2; HM epitope
string CTL activity against pb9 in D. BALB/c; HM epitope string CTL
activity against HIV peptide in E. DBA/2; HM epitope string CTL
activity against HIV peptide in F. BALB/c; tumour epitope string
CTL activity against P1A-derived peptide in G. DBA/2; tumour
epitope string CTL activity against P1A-derived peptide in H.
Sequences of peptide epitopes are shown in table 3. Each curve
shows the data for an individual mouse.
Sporozoites can Efficiently Prime an Immune Response that is
Boostable by MVA
[0128] Humans living in malaria endemic areas are continuously
exposed to sporozoite inoculations. Malaria-specific CTL are found
in these naturally exposed individuals at low levels. To address
the question whether low levels of sporozoite induced CTL responses
can be boosted by MVA, BALB/c mice were immunised with irradiated
(to prevent malaria infection) P. berghei sporozoites and boosted
with MVA. Two weeks after the last immunisation splenocytes were
re-stimulated and tested for lytic activity. Two injections with 50
or 300+500 sporozoites induced very low or undetectable levels of
lysis. Boosting with MVA induced high levels of peptide specific
CTL. MVA alone induced only moderate levels of lysis (FIGS.
9A-9E).
[0129] FIGS. 9A-9E show sporozoite-primed CTL responses are
substantially boosted by MVA. Mice were immunised with two low
doses (50+50) of irradiated sporozoites in FIG. 9A; two high doses
(300+500) of sporozoites in FIG. 9B; mice were boosted with
MVA.PbCSP following low-dose sporozoite priming in FIG. 9D; high
dose sporozoite priming in FIG. 9E. CTL responses following
immunisation with MVA.PbCSP are shown in FIG. 9C.
Recombinant Adenoviruses as Priming Agent
[0130] The prime-boost immunisation regimen has been exemplified
using plasmid DNA and recombinant Ty-VLP as priming agent. Here an
example using non-replicating adenoviruses as the priming agent is
provided. Replication-deficient recombinant Adenovirus expressing
E. coli .beta.-galactosidase (Adeno-GAL) was used. Groups of BALB/c
mice were immunised with plasmid DNA followed by MVA or with
Adenovirus followed by MVA. All antigen delivery systems used
encoded E. coli .beta.-galactosidase. Priming a CTL response with
plasmid DNA or Adenovirus and boosting with MVA induces similar
levels of CTL (FIGS. 10A-10B).
[0131] FIGS. 10A-10B show CTL responses primed by plasmid DNA or
recombinant Adenovirus and boosted with MVA. Groups of BALB/c mice
(n=3) were primed with plasmid DNA (FIG. 10A); or recombinant
Adenovirus expressing .beta.-galactosidase (FIG. 10B). Plasmid DNA
was administered intramuscularly, MVA intravenously and Adenovirus
intradermally. Splenocytes were restimulated with peptide TPHPARIGL
[SEQ ID NO: 69] two weeks after the last immunisation. CTL activity
was tested with peptide-pulsed P815 cells.
Immunogenicity of the DNA Prime Vaccinia Boost Regimen Depends on
the Replication Competence of the Strain of Vaccinia Virus Used
[0132] The prime boosting strategy was tested using different
strains of recombinant vaccina viruses to determine whether the
different strains with strains differing in their replication
competence may differ in their ability to boost a DNA-primed CTL
response. Boosting with replication-defective recombinant vaccinia
viruses such as MVA and NYVAC resulted in the induction of stronger
CTL responses compared to CTL responses following boosting with the
same dose of replication competent WR vaccinia virus (FIGS.
11A-11C).
[0133] FIGS. 11A-11C show CTL responses in BALB/c mice primed with
plasmid DNA followed by boosting with different recombinant
vaccinia viruses. Animals were primed with pTH.PbCSP 50 .mu.g/mouse
i.m. and two weeks later boosted with different strains of
recombinant vaccina viruses (10.sup.6 pfu per mouse i.v.)
expressing PbCSP. The different recombinant vaccinia virus strains
were MVA in FIG. 11A; NYVAC in FIG. 11B and WR in FIG. 11C. The
superiority of replication-impaired vaccinia strains over
replicating strains was found in a further experiment. Groups of
BALB/c mice (n=6) were primed with 50 .mu.g/animal of pSG2.PbCSP
(i.m.) and 10 days later boosted i.v. with 10.sup.6 ffu/pfu of
recombinant MVA, NYVAC and WR expressing PbCSP. The frequencies of
peptide-specific CD8+ T cells were determined using the ELISPOT
assay. The frequencies were: MVA 1103+/-438, NYVAC 826+/-249 and WR
468+/-135. Thus using both CTL assays and ELISPOT assays as a
measure of CD8 T cell immunogenicity a surprising substantially
greater immunogenicity of the replication-impaired vaccinia strains
was observed compared to the replication competent strain.
The Use of Recombinant Canary or Fowl Pox Viruses for Boosting Cd8+
T Cell Responses
[0134] Recombinant canary pox virus (rCPV) or fowl pox virus (rFVP)
are made using shuttle vectors described previously (Taylor et al.
Virology 1992, 187: 321-328 and Taylor et al. Vaccine 1988, 6:
504-508). The strategy for these shuttle vectors is to insert the
gene encoding the protein of interest preceded by a
vaccinia-specific promoter between two flanking regions comprised
of sequences derived from the CPV or FPV genome. These flanking
sequences are chosen to avoid insertion into essential viral genes.
Recombinant CPV or FPV are generated by in vivo recombination in
permissive avian cell lines i.e. primary chicken embryo
fibroblasts. Any protein sequence of antigens or epitope strings
can be expressed using fowl pox or canary pox virus. Recombinant
CPV or FPV is characterised for expression of the protein of
interest using antigen-specific antibodies or including an antibody
epitope into the recombinant gene. Recombinant viruses are grown on
primary CEF. An immune response is primed using plasmid DNA as
described in Materials and Methods. This plasmid DNA primed immune
response is boosted using 10.sup.7 ffu/pfu of rCPV or rFPV
inoculated intravenously, intradermally or intramuscularly. CD8+ T
cell responses are monitored and challenges are performed as
described herein.
Example 3
Malaria Challenge Studies in Mice
[0135] To assess the protective efficacy of the induced levels of
CD8+ T cell response immunised BALB/c or C57BL/6 mice were
challenged by intravenous injection with 2000 or 200 P. berghei
sporozoites. This leads to infection of liver cells by the
sporozoites. However, in the presence of a sufficiently strong T
lymphocyte response against the intrahepatic parasite no viable
parasite will leave the liver and no blood-stage parasites will be
detectable. Blood films from challenged mice were therefore
assessed for parasites by microscopy 5-12 days following
challenge.
[0136] BALB/c mice immunised twice with a mixture of two plasmid
DNAs encoding the CS protein and the TRAP antigen, respectively, of
P. berghei were not protected against sporozoite challenge. Mice
immunised twice with a mixture of recombinant MVA viruses encoding
the same two antigens were not protected against sporozoite
challenge. Mice immunised first with the two recombinant MVAs and
secondly with the two recombinant plasmids were also not protected
against sporozoite challenge. However, all 15 mice immunised first
with the two plasmid DNAs and secondly with the two recombinant MVA
viruses were completely resistant to sporozoite challenge (Table 6
A and B).
[0137] To assess whether the observed protection was due to an
immune response to the CS antigen or to TRAP or to both, groups of
mice were then immunised with each antigen separately (Table 6 B).
All 10 mice immunised first with the CS plasmid DNA and secondly
with the CS MVA virus were completely protected against sporozoite
challenge. Fourteen out of 16 mice immunised first with the TRAP
plasmid DNA vaccine and secondly with the TRAP MVA virus were
protected against sporozoite challenge. Therefore the CS antigen
alone is fully protective when the above immunisation regime is
employed and the TRAP antigen is substantially protective with the
same regime.
[0138] The good correlation between the induced level of CD8+ T
lymphocyte response and the degree of protection observed strongly
suggests that the CD8+ response is responsible for the observed
protection. In previous adoptive transfer experiments it has been
demonstrated that CD8+ T lymphocyte clones against the major CD8+ T
cell epitope in the P. berghei CS protein can protect against
sporozoite challenge. To determine whether the induced protection
was indeed mediated by CD8+ T cells to this epitope we then
employed a plasmid DNA and a recombinant MVA encoding only this
nine amino acid sequence from P. berghei as a part of a string of
epitopes (Table 6 B). (All the other epitopes were from
micro-organisms other than P. berghei). Immunisation of 10 mice
first with a plasmid encoding such an epitope string and secondly
with a recombinant MVA also encoding an epitope string with the P.
berghei CTL epitope led to complete protection from sporozoite
challenge (Table 6 B). Hence the induced protective immune response
must be the CTL response that targets this nonamer peptide
sequence.
TABLE-US-00007 TABLE 6 Results of Mouse Challenge Experiments Using
Different Combinations of DNA and MVA Vaccine No. Infected/
Immunisation 1 Immunisation 2 No. challenged % Protection A.
Antigens used: PbCSP + PbTRAP DNA DNA 5/5 0% MVA MVA 9/10 10% DNA
MVA 0/5 100% MVA DNA 5/5 0% Control mice immunised with -
galactosidase DNA DNA 5/5 0% MVA MVA 5/5 0% DNA MVA 5/5 0% MVA DNA
5/5 0% B. DNA (CSP + TRAP) MVA (CSP + TRAP) 0/10 100% DNA (CSP) MVA
(CSP) 0/10 100% DNA (TRAP) MVA (TRAP) 2/16 88% DNA (epitope) MVA
(epitope) 0/11 100% DNA (beta-gal) MVA (beta-gal) 6/7 14% none none
9/10 10%
[0139] Table 6 Results of Two Challenge Experiments (A and B) Using
Different
[0140] Immunisation regimes of plasmid DNA and MVA as indicated.
BALB/c mice were used in all cases. The immunisation doses were 50
.mu.g of plasmid DNA or 10.sup.6 ffu of recombinant MVA. The
interval between immunisations 1 and 2 was from 14-21 days in all
cases. Challenges were performed at 18-29 days after the last
immunisation by i.v. injection of 2000 P. berghei sporozoites and
blood films assessed at 5, 8 and 10 days post challenge. CSP and
TRAP indicate the entire P. berghei antigen and `epitope` indicates
the cassettes of epitopes shown in table 1 containing only a single
P. berghei K.sup.d-restricted nonamer CTL epitope. Note that in
experiment B immunisation with the epitope string alone yields 100%
protection.
[0141] Mice immunised twice with recombinant Ty-VLPs encoding pb9
were fully susceptible to infection. Similarly mice immunised twice
with the recombinant MVA encoding the full CS protein were fully
susceptible to infection. However, the mice immunised once with the
Ty-VLP and subsequently once with the recombinant MVA showed an 85%
reduction in malaria incidence when boosted with MVA expressing the
full length CS protein, and 95% when MVA expressing the HM epitope
string which includes pb9 was used to boost (Table 7).
TABLE-US-00008 TABLE 7 Results of Challenge Experiments Using
Different Immunisation Regimes of Ty-VLPs and MVA No. Infected/
Immunisation 1 Immunisation 2 No. challenged % Protection
Ty-CABDHFE Ty-CABDHFE 7/8 13% Ty-CABDH MVA.PbCSP 2/13 85%
Ty-CABDHFE MVA-NP 5/5 0% MVA.PbCSP MVA.PbCSP 6/6 0% MVA.HM
Ty-CABDHFE 14/14 0% Ty-CABDHFE MVA.HM 1/21 95% none MVA.HM 8/8 0%
none none 11/12 9%
Table 7 Results of Challenge Experiments Using Different
Immunisation Regimes of Ty-VLPs and MVA as Indicated. BALb/c Mice
Were Used in All Cases. Immunisations were of 50 .mu.g of Ty-VLP or
10.sup.7 ffu of recombinant MVA administered intravenously. The
interval between immunisations 1 and 2 was from 14-21 days in all
cases. Challenges were performed at 18-29 days after the last
immunisation by i.v. injection of 2000 P. berghei sporozoites and
blood films assessed at 5, 8 and 10 days post challenge. CSP
indicates the entire P. berghei antigen. Ty-VLPs carried epitope
cassettes CABDH or CABDHFE as described in table 1. MVA.HM includes
cassettes CAB.
[0142] To determine whether the enhanced immunogenicity and
protective efficacy observed by boosting with a recombinant MVA is
unique to this particular vaccinia virus strain or is shared by
other recombinant vaccinias the following experiment was performed.
Mice were immunised with the DNA vaccine encoding P. berghei CS
protein and boosted with either (i) recombinant MVA encoding this
antigen; (ii) recombinant wild-type vaccinia virus (Western Reserve
strain) encoding the same antigen (Satchidanandam et al. 1991), or
(iii) recombinant NYVAC(COPAK) virus (Lanar et al. 1996) encoding
the same malaria antigen. The highest degree of protection was
observed with boosting by the MVA recombinant, 80% (Table 8). A
very low level of protection (10%) was observed by boosting with
the wild-type recombinant vaccinia virus and a significant level of
protection, 60%, by boosting with the NYVAC recombinant. Hence the
prime-boost regime we describe induces protective efficacy with any
non-replicating vaccinia virus strain. Both the MVA recombinant and
NYVAC were significantly (P<0.05 for each) better than the WR
strain recombinant.
TABLE-US-00009 TABLE 8 Challenge Data Results for DNA Boosted with
Various Vaccinia Strain Recombinants. No. Infected/ Immunisation 1
Immunisation 2 No. challenged % Protection DNA-beta gal. MVA.NP 8/8
0% DNA-CSP MVA-CSP 2/10 80% DNA-CSP WR-CSP 9/10 10% DNA-CSP
NYVAC-CSP 4/10 60%
Table 8 Results of a challenge experiment using different
immunisation regimes of plasmid DNA and various vaccinia
recombinants as indicated. BALB/c mice were used in all cases. The
immunisation doses were 50 .mu.g of plasmid DNA or 10.sup.6 ffu/pfu
of recombinant MVA or 10.sup.4 ffu/pfu of recombinant wild type
(WR) vaccinia or 10.sup.6 ffu/pfu of recombinant NYVAC. Because the
WR strain will replicate in the host and the other strains will
not, in this experiment a lower dose of WR was used. The interval
between immunisations 1 and 2 was 23 days. Challenges were
performed at 28 days after the last immunisation by i.v. injection
of 2000 P. berghei sporozoites and blood films assessed at 7, 9 and
11 days post challenge. pbCSP indicates the entire P. berghei
antigen and NP the nucleoprotein antigen of influenza virus (used
as a control antigen). The first immunisation of group A mice was
with the plasmid DNA vector expressing beta galactosidase but no
malaria antigen.
[0143] In a further experiment shown in Table 8, mice were
immunised with the DNA vaccine encoding P. berghei CS protein and
boosted with either (i) recombinant MVA encoding this antigen; (ii)
recombinant WR vaccinia virus encoding the same antigen or (iii)
recombinant NYVAC(COPAK) virus encoding the same malaria antigen,
all at 10.sup.6 ffu/pfu. A high and statistically significant
degree of protection was observed with boosting with recombinant
NYVAC (80%) or recombinant MVA (66%). A low and non-significant
level of protection (26%) was observed by boosting with the WR
recombinant vaccinia virus (Table 9). MVA and NYVAC boosting each
gave significantly more protection than WR boosting (P=0.03 and
P=0.001 respectively). These data reemphasise that non-replicating
pox virus strains are better boosting agents for inducing high
levels of protection.
TABLE-US-00010 TABLE 9 Influence of Different Recombinant Vaccinia
Strains on Protection. Immunisation 1 No. inf./ DNA Immunisation 2
No. chall. % protection CSP MVA.PbCSP 5/15 66 CSP NYVAC.PbCSP 2/15
80 CSP WR.PbCSP 11/15 26 .beta.- galactosidase MVA.NP 8/8 0
Table 9 Results of challenge experiments using different
immunisation regimes of plasmid DNA and replication incompetent
vaccinia recombinants as boosting immunisation. BALB/c mice were
used in all cases. The immunisation doses were 50 .mu.g of plasmid
DNA or 10.sup.6 ffu/pfu of recombinant MVA or recombinant wild type
(WR) vaccinia or recombinant NYVAC. The interval between
immunisations 1 and 2 was 23 days. Challenges were performed at 28
days after the last immunisation by i.v. injection of 2000 P.
berghei sporozoites and blood films assessed at 7, 9 and 11 days
post challenge. PbCSP indicates the entire P. berghei antigen and
NP the nucleoprotein antigen of influenza virus (used as a control
antigen). The control immunisation was with a plasmid DNA vector
expressing .beta.-galactosidase followed by MVA.NP. Alternative
Routes for Boosting Immune Responses with Recombinant MVA
[0144] Intravenous injection of recombinant MVA is not a preferred
route for immunising humans and not feasible in mass immunisations.
Therefore different routes of MVA boosting were tested for their
immunogenicity and protective efficacy.
[0145] Mice were primed with plasmid DNA i.m. Two weeks later they
were boosted with MVA administered via the following routes:
intravenous (i.v.), subcutaneous (s.c.), intraperitoneal (i.p.),
intramuscular (i.p.) and intradermal (i.d.). Two weeks after this
boost peptide-specific CD8+ T cells were determined in an ELISPOT
assay. The most effective route which induced the highest levels
were i.v. and i.d inoculation of MVA. The other routes gave
moderate to poor responses (FIG. 12).
[0146] FIG. 12 shows frequencies of peptide-specific CD8+ T cells
following different routes of MVA boosting. Results are shown as
the number of spot-forming cells (SFC) per one million splenocytes.
Mice were primed with plasmid DNA and two weeks later boosted with
MVA via the indicated routes. The number of splenocytes specific
for the SYIPSAEKI [SEQ ID NO: 67] peptide was determined in
INF-.gamma. ELISPOT assays two weeks after the last immunisation.
Each bar represents the mean number of SFCs from three mice assayed
individually.
[0147] Boosting via the i.v. route was compared with the i.d. and
i.m route in a challenge experiment. The i.d route gave high levels
of protection (80% protection). In the group of animals that were
boosted via the i.m. route, 50% of the animals were protected.
Complete protection was achieved with MVA boost administered i.v.
(Table 10)
TABLE-US-00011 TABLE 10 Influence of the Route of MVA
Administration on Protective Efficacy Immunisation 1 Immunisation 2
No. infected/ DNA MVA No. challenged % protection CSP CSP i.v.
*0/20 100 CSP CSP i.d 2/10 80 CSP CSP i.m. 5/10 50 Epitope epitope
i.v. 1/10 90 NP NP i.v. 10/10 0 *culminative data from two
independent experiments
Table 10 Results from challenge experiments using different routes
of MVA boosting immunisation. Animals were primed by intramuscular
plasmid DNA injection and two weeks later boosted with the
indicated recombinant MVA (10.sup.6 ffu/mouse) administered via the
routes indicated. The mice were challenged 16 days after the last
immunisation with 2000 P. berghei sporozoites and screened for
blood stage parasitemia at day 8 and 10 post challenge. Epitope
indicates the polypeptide string HM.
Alternative Routes of DNA Priming: the Use of a Gene Gun to Prime
Peptide Specific Cd8+ T Cells
[0148] Gene gun delivery is described in detail in for example in
Eisenbraun et al. DNA Cell Biol. 1993, 12: 791-797 and Degano et
al. Vaccine 1998, 16: 394-398.
[0149] The mouse malaria challenge experiments described so far
using plasmid DNA to prime an immune response used intramuscular
injection of plasmid DNA.
[0150] Intradermal delivery of plasmid DNA using a biolistic device
is another route to prime specific CTL responses. Plasmid DNA is
coated onto gold particles and delivered intradermally with a gene
gun. Groups of mice (n=10) were immunised three times at two weeks
intervals with the gene gun alone (4 .mu.g/immunisation), immunised
two times with the gene gun followed by an intravenous MVA.PbCSP
boost or immunised intramuscularly with 50 .mu.g of pTH.PbCSP and
two weeks later boosted with MVA.PbCSP intravenously. Two weeks
after the last immunisation the animals were challenged with 2000
sporozoites to assess protective efficacy of each immunisation
regimen. In the group that received the intravenous MVA boost
following two gene gun immunisations one out of ten animals
developed blood stage parasitemia (90% protection). Complete
protection was observed with intramuscular DNA priming followed by
MVA i.v boosting. Seven out of 10 animals that were immunised three
times with the gene gun were infected. (30% protection) (Table
11).
TABLE-US-00012 Immunisation 1 No. inf./ % DNA Immunisation 2
Immunisation 3 No. chall. protection gene gun DNA gene gun DNA gene
gun DNA 7/10 30 gene gun DNA gene gun DNA MVA.PbCSP 1/10 90 -- DNA
i.m MVA.PbCSP 0/10 100 Naive 10/10 0
Table 11 Results of challenge experiments comparing different
routes of DNA priming (intradermally by gene gun versus
intramuscular needle injection). Groups of BALB/c mice (n=10) were
immunised as indicated. Each gene gun immunisation delivered 4
.mu.g of plasmid DNA intraepidermally. For i.m. immunisations 50 mg
of plasmid DNA were injected. Twenty days after the last
immunisation mice were challenged as described previously.
Highly Susceptible C57BL/6 Mice are Protected
[0151] C57BL/6 mice are very susceptible to P. berghei sporozoite
challenge. C57BL/6 mice were immunised using the DNA-MVA prime
boost regime with both pre-erythrocytic antigens PbCSP and PbTRAP,
and challenged with either 200 or 1000 infectious sporozoites per
mouse. (Two hundred sporozoites corresponds to more than twice the
dose required to induce infection in this strain). All ten mice
challenged with 200 sporozoites showed sterile immunity. Even the
group challenged with 1000 sporozoites, 60% of the mice were
protected (Table 12). All the naive C57BL/6 mice were infected
after challenge.
TABLE-US-00013 TABLE 12 Protection of C57BL/6 Mice from Sporozoite
Challenge No. animals inf./ % No. challenged protection 1000
sporozoites DNA followed by MVA 4/10 60 Naive 5/5 0 200 sporozoites
Naive 5/5 0
Table 12 Results of a challenge experiment using C57BL/6 mice.
Animals were immunised with PbCSP and PbTRAP using the DNA followed
by MVA prime boost regime. Fourteen days later the mice were
challenged with P. berghei sporozoites as indicated.
Example 4
Protective Efficacy of the DNA-Priming/MVA-Boosting Regimen in Two
Further Disease Models in Mice
[0152] Following immunogenicity studies, the protective efficacy of
the DNA-priming MVA-boosting regimen was tested in two additional
murine challenge models. The two challenge models were the P815
tumour model and the influenza A virus challenge model. In both
model systems CTL have been shown to mediate protection.
P815 Tumour Challenges:
[0153] Groups (n=10) of DBA/2 mice were immunised with a
combination of DNA followed by MVA expressing a tumour epitope
string or the HM epitope string. Two weeks after the last
immunisation the mice were challenged intravenously with 10.sup.5
P815 cells. Following this challenge the mice were monitored
regularly for the development of tumour-related signs and
survival.
[0154] FIG. 13 shows the survival rate of the two groups of mice.
Sixty days after challenge eight out of ten mice were alive in the
group immunised with the tumour epitopes string. In the group
immunised with the HM epitope string only 2 animals survived. This
result is statistically significant: 2/10 vs 8/10 chi-squared=7.2.
P=0.007. The onset of death in the groups of animals immunised with
the tumour epitope string is delayed compared to the groups
immunised with the HM epitope string.
Influenza Virus Challenges:
[0155] Groups of BALB/c mice were immunised with three gene gun
immunisations with plasmid DNA, two intramuscular plasmid DNA
injections, one i.m. DNA injection followed by one MVA.NP boost
i.v. or two gene gun immunisations followed by one MVA.NP boost
i.v. Plasmid DNA and recombinant MVA expressed the influenza virus
nucleoprotein. Two weeks after the last immunisation the mice were
challenged intranasally with 100 HA of influenza A/PR/8/34 virus.
The animals were monitored for survival daily after challenge.
[0156] Complete protection was observed in the following groups of
animals: [0157] two DNA gene gun immunisations followed by one
MVA.NP boost i.v.; [0158] one i.m. DNA injection followed by one
MVA.NP boost i.v.; and [0159] two i.m. DNA injections.
[0160] In the group of animals immunised three times with the gene
gun 71% of the animals survived (5/7) and this difference from the
control group was not significant statistically (P>0.05). In the
naive group 25% of the animals survived (FIG. 14) and this group
differed significantly (P<0.05) for the two completely protected
groups.
[0161] FIG. 14 shows results of an influenza virus challenge
experiment. BALB/c mice were immunised as indicated. GG=gene gun
immunisations, im=intramuscular injection, iv=intravenous
injection. Survival of the animals was monitored daily after
challenge. In a second experiment groups of 10 BALB/c mice were
immunised with MVA.NP i.v. alone, three times with the gene gun,
two times with the gene gun followed by one MVA.NP boost i.v. and
two i.m injections of V1J-NP followed by one MVA.NP boost. Two
weeks after the last immunisation the mice were challenged with 100
HA units of influenza A/PR/8/34 virus.
[0162] Complete and statistically significant protection was
observed in the following groups of animals:
[0163] two gene gun immunisations followed by one MVA.NP boost;
and
[0164] two i.m injections of V1J-NP followed by one MVA.NP
boost.
In the group receiving one MVA.NP i.v., 30% (3 out of 10) of
animals survived. In the group immunised with a DNA vaccine
delivered by the gene gun three times, 70% of the animals were
protected but this protection was not significantly different from
the naive controls. In this challenge experiment 40% (4 out of 10)
of the naive animals survived the challenge.
Example 5
Immunogenicity Studies in Non-Human Primates
Immunogenicity and Protective Efficacy of the Prime Boost Regimen
in Non-Human Primates.
[0165] In order to show that the strong immunogenicity of the DNA
priming/MVA boosting regime observed in mice translates into strong
immunogenicity in primates, the regimen was tested in macaques. The
vaccine consisted of a string of CTL epitopes derived from HIV and
SIV sequences (FIG. 2), in plasmid DNA or MVA, denoted DNA.H and
MVA.H respectively. The use of defined CTL epitopes in a
polyepitope string allows testing for SIV specific CTL in macaques.
Due to the MHC class I restriction of the antigenic peptides,
macaques were screened for their MHC class I haplotype and
Mamu-A*01-positive animals were selected for the experiments
described.
[0166] Three animals (CYD, DI and DORIS) were immunised following
this immunisation regimen:
TABLE-US-00014 week 0 DNA (8 .mu.g, i.d., gene gun) week 8 DNA (8
.mu.g, i.d., gene gun) week 17 MVA (5 .times. 10.sup.8 pfu, i.d.)
week 22 MVA (5 .times. 10.sup.8 pfu, i.d.)
[0167] Blood from each animal was drawn at weeks 0, 2, 5, 8, 10,
11, 17, 18, 19, 21, 22, 23, 24 and 25 of the experiment. The
animals were monitored for induction of CTL using two different
methods. PBMC isolated from each bleed were re-stimulated in vitro
with a peptide encoded in the epitope string and tested for their
ability to recognise autologous peptide-loaded target cells in a
chromium release cytotoxicity assay. Additionally, freshly isolated
PBMC were stained for antigen specific CD8+ T cells using
tetramers.
[0168] Following two gene gun immunisations very low levels of CTL
were detected using tetramer staining (FIG. 15). Two weeks after
the first MVA boosting, all three animals developed peptide
specific CTL as detected by tetramer staining (FIG. 15). This was
reflected by the detection of moderate CTL responses following in
vitro restimulation (FIG. 16, week 19). The second boost with MVA.H
induced very high levels of CD8+, antigen specific T cells (FIG.
15) and also very high levels of peptide specific cytotoxic T cells
(FIG. 16, week 23).
[0169] FIG. 15 shows detection of SIV-specific MHC class
I-restricted CD8+ T cells using tetramers. Three
Mamu-A*A01-positive macaques were immunised with plasmid DNA (gene
gun) followed by MVA boosting as indicated. Frequencies of
Mamu-A*A01/CD8 double-positive T cells were identified following
FACS analysis. Each bar represents the percentage of CD8+ T cells
specific for the Mamu-A*01/gag epitope at the indicated time point.
One percent of CD8 T cells corresponds to about 5000/10.sup.6
peripheral blood lymphocytes. Thus the levels of epitope-specific
CD8 T cells in the peripheral blood of these macaques are at least
as high as the levels observed in the spleens of immunised and
protected mice in the malaria studies.
[0170] FIG. 16 shows CTL induction in macaques following DNA/MVA
immunisation. PBMC from three different macaques (CYD, DI and
DORIS) were isolated at week 18, 19 and 23 and were restimulated
with peptide CTPYDINQM [SEQ ID NO: 54] in vitro. After two
restimulations with peptide CTPYDINQM [SEQ ID NO: 54] the cultures
were tested for their lytic activity on peptide-pulsed autologous
target cells. Strong CTL activity was observed.
Example 6
Immunogenicity and Challenge Studies in Chimpanzees
[0171] To show that a similar regime of initial immunisation with
plasmid DNA and subsequent immunisation with recombinant MVA can be
effective against Plasmodium falciparum malaria in higher primates
an immunisation and challenge study was performed with two
chimpanzees. Chimp H1 received an initial immunisation with 500
.mu.g of a plasmid expressing Plasmodium falciparum TRAP from the
CMV promoter without intron A, CMV-TRAP. Chimp H2 received the same
dose of CMV-LSA-1, which expresses the C-terminal portion of the
LSA-1 gene of P. falciparum. Both chimps received three more
immunisations over the next 2 months, but with three plasmids at
each immunisation. H1 received CMV-TRAP as before, plus pTH-TRAP,
which expresses TRAP using the CMV promoter with intron A, leading
to a higher expression level. H1 also received RSV-LSA-1, which
expresses the C-terminal portion of LSA-1 from the RSV promoter. H2
received CMV-LSA-1, pTH-LSA-1 and RSV-TRAP at the second, third and
fourth immunisations. The dose was always 500 .mu.g of each
plasmid.
[0172] It was subsequently discovered that the RSV plasmids did not
express the antigens contained within them, so H1 was only
immunised with plasmids expressing TRAP, and H2 with plasmids
expressing LSA-1.
[0173] Between and following these DNA immunisations assays of
cellular immune responses were performed at several time points,
the last assay being performed at three months following the fourth
DNA immunisation, but no malaria-specific T cells were detectable
in either ELISPOT assays or CTL assays for CD8+ T cells.
[0174] Both animals were subsequently immunised with three doses of
10.sup.8 ffu of a recombinant MVA virus encoding the P. falciparum
TRAP antigen over a 6 week period. Just before and also following
the third recombinant MVA immunisation T cell responses to the TRAP
antigen were detectable in both chimpanzees using an ELISPOT assay
to whole TRAP protein bound to latex beads. This assay detects both
CD4+ and CD8+ T cell responses. Specific CD8+ T responses were
searched for with a series of short 8-11 amino acid peptides in
both immunised chimpanzees. Such analysis for CD8+ T cell responses
indicated that CD8+ T cells were detectable only in the chimpanzee
H1. The target epitope of these CD8+ T lymphocytes was an 11 amino
acid peptide from TRAP, tr57, of sequence KTASCGVWDEW [SEQ ID NO:
78]. These CD8+ T cells from H1 had lytic activity against
autologous target cells pulsed with the tr57 peptide and against
autologous target cells infected with the recombinant PfTRAP-MVA
virus. A high precursor frequency of these specific CD8+ T cells of
about 1 per 500 lymphocytes was detected in the peripheral blood of
this chimpanzee H1 using an ELISPOT assay two months following the
final MVA immunisation. No specific CD8+ T cell response was
clearly detected in the chimpanzee H2, which was not primed with a
plasmid DNA expressing TRAP.
[0175] Two months after the third PfTRAP-MVA immunisation challenge
of H1 and H2 was performed with 20,000 sporozoites, a number that
has previously been found to yield reliably detectable blood stage
infection in chimpanzees 7 days after challenge (Thomas et al. 1994
and unpublished data). The challenge was performed with the NF54
strain of Plasmodium falciparum. This is of importance because the
TRAP sequence in the plasmid DNA and in the recombinant MVA is from
the T9/96 strain of P. falciparum which has numerous amino acid
differences to the NF54 TRAP allele (Robson et al. 1990). Thus,
this sporozoite challenge was performed with a heterologous rather
than homologous strain of parasite. In the chimpanzee H2 parasites
were detectable in peripheral blood as expected 7 days after
sporozoite challenge using in vitro parasite culture detection.
However, in H1 the appearance of blood stage parasites in culture
from the day 7 blood samples was delayed by three days consistent
with some immune protective effect against the liver-stage
infection. In studies of previous candidate malaria vaccines in
humans a delay in the appearance of parasites in the peripheral
blood has been estimated to correspond to a substantial reduction
in parasite density in the liver (Davis et al. 1989). Thus the
chimpanzee H1, immunised first with P. falciparum TRAP plasmid DNA
and subsequently with the same antigen expressed by a recombinant
MVA virus showed a strong CD8+ T lymphocyte response and evidence
of some protection from heterologous sporozoite challenge.
DISCUSSION
[0176] These examples demonstrate a novel regime for immunisation
against malaria which induces high levels of protective CD8+ T
cells in rodent models of human malaria infection. Also
demonstrated is an unprecedented complete protection against
sporozoite challenge using subunit vaccines (36 out of 36 mice
protected in Table 6 using DNA priming and MVA boosting with the CS
epitope containing vaccines). Induction of protective immune
responses using the DNA priming/MVA boosting regimen was
demonstrated in two additional mouse models of viral infection
influenza A model and cancer (P815 tumour model). More importantly
for vaccines for use in humans this immunisation regimen is also
highly immunogenic for CD8+ T cells in primates. Strong
SIV-gag-specific CTL were induced in 3 out of 3 macaques with
plasmid DNA and MVA expressing epitope strings. The levels induced
are comparable to those found in SIV-infected animals. The data
from the chimpanzee studies indicate that the same immunisation
regime can induce a strong CD8+ T lymphocyte response against P.
falciparum in higher primates with some evidence of protection
against P. falciparum sporozoite challenge.
[0177] Ty-VLPs have previously been reported to induce good levels
of CD8+ T cell responses against the P. berghei rodent malaria
(Allsopp et al. 1995) but alone this construct is not protective.
It has now been found that subsequent immunisation with recombinant
MVA boosts the CD8+ T cell response very substantially and
generates a high level of protection (Table 7).
[0178] Recombinant MVA viruses have not been assessed for efficacy
as malaria vaccines previously. Recombinant MVA alone was not
significantly protective, nor was priming with recombinant MVA
followed by a second immunisation with recombinant plasmid DNA.
However, a second immunisation with the recombinant MVA following
an initial immunisation with either Ty-VLPs or plasmid DNA yielded
impressive levels of protection. Non-recombinant MVA virus has been
safely used to vaccinate thousands of human against smallpox and
appears to have an excellent safety profile. The molecular basis of
the increased safety and immunogenicity of this strain of vaccinia
virus is being elucidated by detailed molecular studies (Meyer et
al. 1991; Sutter at al. 1994).
[0179] Plasmid DNA has previously been tested as a malaria vaccine
for the P. yoelii rodent malaria. High levels of, but not complete,
protection is seen in some strains but in other strains of mice
little or no protection was observed even after multiple
immunisations (Doolan et al. 1996). Although plasmid DNA has been
proposed as a method of immunisation against P. falciparum, success
has not previously been achieved. The evidence provided here is the
first evidence to show that plasmid DNA may be used in an
immunisation regime to induce protective immunity against the human
malaria parasite P. falciparum.
[0180] A similar regime of immunisation to the regime demonstrated
herein can be expected to induce useful protective immunity against
P. falciparum in humans. It should be noted that five of the
vaccine constructs employed in these studies to induce protective
immunity in rodents or chimpanzees contain P. falciparum sequences
and could therefore be used for human immunisation against P.
falciparum. These are: 1. The P. falciparum TRAP plasmid DNA
vaccine. 2. The P. falciparum TRAP recombinant MVA virus. 3. The
Ty-VLP encoding an epitope string of numerous P falciparum
epitopes, as well as the single P. berghei CTL epitope. 4. The
plasmid DNA encoding the same epitope string as 3. 5. The
recombinant MVA encoding the longer HM epitope string including
many of the malaria epitopes in 3 and 4. Similarly the plasmid DNAs
and MVA encoding HIV epitopes for human class I molecules could be
used in either prophylactic or therapeutic immunisation against HIV
infection.
[0181] These studies have provided clear evidence that a novel
sequential immunisation regime employing a non-replicating or
replication-impaired pox virus as a boost is capable of inducing a
strong protective CD8+ T cell response against the malaria
parasite. The examples demonstrate clearly a surprising and
substantial enhancement of CD8+ T cell responses and protection
compared to replicating strains of pox viruses. Because there is no
reason to believe that the immunogenicity of CD8+ T cell epitopes
from the malaria parasite should differ substantially from CD8+ T
cell epitopes in other antigens it is expected that the
immunisation regime described herein will prove effective at
generating CD8+ T cell responses of value against other diseases.
The critical step in this immunisation regimen is the use of
non-replicating or replication-impaired recombinant poxviruses to
boost a pre-existing CTL response. We have shown that CTL responses
can be primed using different antigen delivery systems such as a
DNA vaccine i.d. and i.m, a recombinant Ty-VLP, a recombinant
adenovirus and irradiated sporozoites. This is supported by the
data presented on the generation of a CD8+ T cell response against
HIV, influenza virus and tumours. Amongst several known examples of
other diseases against which a CD8+ T cell immune response is
important are the following: infection and disease caused by the
viruses HIV, herpes simplex, herpes zoster, hepatitis C, hepatitis
B, influenza, Epstein-Barr virus, measles, dengue and HTLV-1; by
the bacteria Mycobacterium tuberculosis and Listeria sp.; and by
the protozoan parasites Toxoplasma and Trypanosoma. Induction of
protective CTL responses against influenza A virus has been
demonstrated in FIG. 14. Furthermore, the immunisation regime
described herein is expected to be of value in immunising against
forms of cancer where CD8+ T cell responses plays a protective
role. The induction of protective CTL responses using the DNA prime
MVA boost regime against tumours is shown in FIG. 13. Specific
examples in humans include melanoma, cancer of the breast and
cancer of the colon.
[0182] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
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352-4.
Sequence CWU 1
1
78124DNAUnknownCTL Epitope of the Malaria String 1aagccgaacg
acaagtcctt gtat 2428PRTUnknownCTL Epitope of the Malaria String
2Lys Pro Asn Asp Lys Ser Leu Tyr1 5324DNAUnknownCTL Epitope of the
Malaria String 3aaacctaagg acgaattgga ctac 2448PRTUnknownCTL
Epitope of the Malaria String 4Lys Pro Lys Asp Glu Leu Asp Tyr1
5527DNAUnknownCTL Epitope of the Malaria String 5aagccaatcg
ttcaatacga caacttc 2769PRTUnknownCTL Epitope of the Malaria String
6Lys Pro Ile Val Gln Tyr Asp Asn Phe1 5733DNAUnknownCTL Epitope of
the Malaria String 7gcctccaaga acaaggaaaa ggctttgatc atc
33811PRTUnknownCTL Epitope of the Malaria String 8Ala Ser Lys Asn
Lys Glu Lys Ala Leu Ile Ile1 5 10927DNAUnknownCTL Epitope of the
Malaria String 9ggtatcgctg gtggtttggc cttgttg 27109PRTUnknownCTL
Epitope of the Malaria String 10Gly Ile Ala Gly Gly Leu Ala Leu
Leu1 51130DNAUnknownCTL Epitope of the Malaria String 11atgaacccta
atgacccaaa cagaaacgtc 301210PRTUnknownCTL Epitope of the Malaria
String 12Met Asn Pro Asn Asp Pro Asn Arg Asn Val1 5
101327DNAUnknownCTL Epitope of the Malaria String 13atgatcaacg
cctacttgga caagttg 27149PRTUnknownCTL Epitope of the Malaria String
14Met Ile Asn Ala Tyr Leu Asp Lys Leu1 51524DNAUnknownCTL Epitope
of the Malaria String 15atctccaagt acgaagacga aatc
24168PRTUnknownCTL Epitope of the Malaria String 16Ile Ser Lys Tyr
Glu Asp Glu Ile1 51727DNAUnknownCTL Epitope of the Malaria String
17tcctacatcc catctgccga aaagatc 27189PRTUnknownCTL Epitope of the
Malaria String 18Ser Tyr Ile Pro Ser Ala Glu Lys Ile1
51927DNAUnknownCTL Epitope of the Malaria String 19cacttgggta
acgttaagta cttggtt 27209PRTUnknownCTL Epitope of the Malaria String
20His Leu Gly Asn Val Lys Tyr Leu Val1 52124DNAUnknownCTL Epitope
of the Malaria String 21aagtctttgt acgatgaaca catc
24228PRTUnknownCTL Epitope of the Malaria String 22Lys Ser Leu Tyr
Asp Glu His Ile1 52327DNAUnknownCTL Epitope of the Malaria String
23ttattgatgg actgttctgg ttctatt 27249PRTUnknownCTL Epitope of the
Malaria String 24Leu Leu Met Asp Cys Ser Gly Ser Ile1
52548DNAUnknownCTL Epitope of the Malaria String 25aacgctaatc
caaacgcaaa tccgaacgcc aatcctaacg cgaatccc 482616PRTUnknownCTL
Epitope of the Malaria String 26Asn Ala Asn Pro Asn Ala Asn Pro Asn
Ala Asn Pro Asn Ala Asn Pro1 5 10 152760DNAUnknownCTL Epitope of
the Malaria String 27gacgaatggt ctccatgttc tgtcacttgt ggtaagggta
ctcgctctag aaagagagaa 602820PRTUnknownCTL Epitope of the Malaria
String 28Asp Glu Trp Ser Pro Cys Ser Val Thr Cys Gly Lys Gly Thr
Arg Ser1 5 10 15Arg Lys Arg Glu202927DNAUnknownCTL Epitope of the
Malaria String 29tacttgaaca aaattcaaaa ctctttg 27309PRTUnknownCTL
Epitope of the Malaria String 30Tyr Leu Asn Lys Ile Gln Asn Ser
Leu1 53127DNAUnknownCTL Epitope of the Malaria String 31atggaaaagt
tgaaagaatt ggaaaag 27329PRTUnknownCTL Epitope of the Malaria String
32Met Glu Lys Leu Lys Glu Leu Glu Lys1 53324DNAUnknownCTL Epitope
of the Malaria String 33gctacttctg tcttggctgg tttg
24348PRTUnknownCTL Epitope of the Malaria String 34Ala Thr Ser Val
Leu Ala Gly Leu1 53548DNAUnknownCTL Epitope of the Malaria String
35gacccaaacg ctaacccaaa cgttgaccca aacgccaacc caaacgtc
483616PRTUnknownCTL Epitope of the Malaria String 36Asp Pro Asn Ala
Asn Pro Asn Val Asp Pro Asn Ala Asn Pro Asn Val1 5 10
153742DNAUnknownCTL Epitope of the Malaria String 37caagttcact
tccaaccatt gcctccggcc gttgtcaagt tg 423814PRTUnknownCTL Epitope of
the Malaria String 38Gln Val His Phe Gln Pro Leu Pro Pro Ala Val
Val Lys Leu1 5 103942DNAUnknownCTL Epitope of the Malaria String
39caattcatca aggccaactc taagttcatc ggtatcaccg aa
424014PRTUnknownCTL Epitope of the Malaria String 40Gln Phe Ile Lys
Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu1 5 1041229PRTUnknownCTL
Epitope of the Malaria String 41Met Ile Asn Ala Tyr Leu Asp Lys Leu
Ile Ser Lys Tyr Glu Asp Glu1 5 10 15Ile Ser Tyr Ile Pro Ser Ala Glu
Lys Ile Gly Ser Lys Pro Asn Asp20 25 30Lys Ser Leu Tyr Lys Pro Lys
Asp Glu Leu Asp Tyr Lys Pro Ile Val35 40 45Gln Tyr Asp Asn Phe Gly
Ser Ala Ser Lys Asn Lys Glu Lys Ala Leu50 55 60Ile Ile Gly Ile Ala
Gly Gly Leu Ala Leu Leu Met Asn Pro Asn Asp65 70 75 80Pro Asn Arg
Asn Val Gly Ser His Leu Gly Asn Val Lys Tyr Leu Val85 90 95Lys Ser
Leu Tyr Asp Glu His Ile Leu Leu Met Asp Cys Ser Gly Ser100 105
110Ile Gly Ser Asp Pro Asn Ala Asn Pro Asn Val Asp Pro Asn Ala
Asn115 120 125Pro Asn Val Gln Val His Phe Gln Pro Leu Pro Pro Ala
Val Val Lys130 135 140Leu Gln Phe Ile Lys Ala Asn Ser Lys Phe Ile
Gly Ile Thr Glu Gly145 150 155 160Ser Tyr Leu Asn Lys Ile Gln Asn
Ser Leu Met Glu Lys Leu Lys Glu165 170 175Leu Glu Lys Ala Thr Ser
Val Leu Ala Gly Leu Gly Ser Asn Ala Asn180 185 190Pro Asn Ala Asn
Pro Asn Ala Asn Pro Asn Ala Asn Pro Asp Glu Trp195 200 205Ser Pro
Cys Ser Val Thr Cys Gly Lys Gly Thr Arg Ser Arg Lys Arg210 215
220Glu Gly Ser Gly Lys225428PRTUnknownCTL Epitope of HIV-1 gp41
42Tyr Leu Lys Asp Gln Gln Leu Leu1 5439PRTUnknownCTL Epitope of
HIV-1 gp41 43Glu Arg Tyr Leu Lys Asp Gln Gln Leu1 5449PRTUnknownCTL
Epitope of SIV env 44Glu Ile Thr Pro Ile Gly Leu Ala Pro1
5459PRTUnknownCTL Epitope of HIV-1 p24 45Pro Pro Ile Pro Val Gly
Glu Ile Tyr1 5469PRTUnknownCTL Epitope of HIV-1 p24 46Gly Glu Ile
Tyr Lys Arg Trp Ile Ile1 54710PRTUnknownCTL Epitope of HIV-1 P24
47Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys1 5 104810PRTUnknownCTL
Epitope of HIV-1 P24 48Ile Ile Leu Gly Leu Asn Lys Ile Val Arg1 5
104910PRTUnknownCTL Epitope of HIV-1 P24 49Leu Gly Leu Asn Lys Ile
Val Arg Met Tyr1 5 10508PRTUnknownCTL Epitope of SIV env 50Tyr Asn
Leu Thr Met Lys Cys Arg1 55110PRTUnknownCTL Epitope of HIV-1 gp120
51Arg Gly Pro Gly Arg Ala Phe Val Thr Ile1 5 10529PRTUnknownCTL
Epitope of HIV-1 gp120 52Gly Arg Ala Phe Val Thr Ile Gly Lys1
5539PRTUnknownCTL Epitope of HIV-1 gag 53Thr Pro Tyr Asp Ile Asn
Gln Met Leu1 5549PRTUnknownCTL Epitope of SIV gag 54Cys Thr Pro Tyr
Asp Ile Asn Gln Met1 55511PRTUnknownCTL Epitope of HIV-1 nef 55Arg
Pro Gln Val Pro Leu Arg Pro Met Thr Tyr1 5 105610PRTUnknownCTL
Epitope of HIV-1 nef 56Gln Val Pro Leu Arg Pro Met Thr Tyr Lys1 5
10578PRTUnknownCTL Epitope of HIV-1 nef 57Val Pro Leu Arg Pro Met
Thr Tyr1 5589PRTUnknownCTL Epitope of HIV-1 nef 58Ala Val Asp Leu
Ser His Phe Leu Lys1 5599PRTUnknownCTL Epitope of HIV-1 nef 59Asp
Leu Ser His Phe Leu Lys Glu Lys1 5608PRTUnknownCTL Epitope of HIV-1
nef 60Phe Leu Lys Glu Lys Gly Gly Leu1 5619PRTUnknownCTL Epitope of
HIV-1 pol 61Ile Leu Lys Glu Pro Val His Gly Val1 56210PRTUnknownCTL
Epitope of HIV-1 pol 62Ile Leu Lys Glu Pro Val His Gly Val Tyr1 5
10639PRTUnknownCTL Epitope of HIV-1 pol 63His Pro Asp Ile Val Ile
Tyr Gln Tyr1 5649PRTUnknownCTL Epitope of HIV-1 pol 64Val Ile Tyr
Gln Tyr Met Asp Asp Leu1 56545PRTUnknownTumor Epitope String 65Met
Leu Pro Tyr Leu Gly Trp Leu Val Phe Ala Gln His Pro Asn Ala1 5 10
15Glu Leu Leu Lys His Tyr Leu Phe Arg Asn Leu Ser Pro Ser Tyr Val20
25 30Tyr His Gln Phe Ile Pro Asn Pro Leu Leu Gly Leu Asp35 40
45669PRTUnknownCTL Peptide Epitope of P1 Tumor Antigen 66Leu Pro
Tyr Leu Gly Trp Leu Val Phe1 5679PRTUnknownCTL Peptide Epitope of
P. berghei CSP 67Ser Tyr Ile Pro Ser Ala Glu Lys Ile1
56810PRTUnknownCTL Peptide Epitope of HIV gag 68Arg Gly Pro Gly Arg
Ala Phe Val Thr Ile1 5 10699PRTUnknownCTL Peptide Epitope of E.
coli b-galactosidase 69Thr Pro His Pro Ala Arg Ile Gly Leu1
5709PRTUnknownCTL Peptide Epitope of Influenza A Virus NP 70Thr Tyr
Gln Arg Thr Arg Ala Leu Val1 5718PRTUnknownCTL Peptide Epitope of
Influenza A Virus NP 71Ser Asp Tyr Glu Gly Arg Leu Ile1
5729PRTUnknownCTL Peptide Epitope of Influenza A Virus NP 72Ala Ser
Asn Glu Asn Met Glu Thr Met1 5739PRTUnknownCTL Peptide Epitope of
P. falciparum TRAP 73Ile Asn Val Ala Phe Asn Arg Phe Leu1
57433DNAUnknown5' Primer MamuNdeI 74cctgactcag accatatggg
ctctcactcc atg 337585DNAUnknown3' Primer MamuNdeI 75gtgataagct
taacgatgat tccacaccat tttctgtgca tccagaatat gatgcaggga 60tccctcccat
ctcagggtga ggggc 857627DNAUnknownPrimer B2MBACK 76tcagaccata
tgtctcgctc cgtggcc 277731DNAUnknownPrimer B2MFOR 77tcagacaagc
ttttacatgt ctcgatccca c 317811PRTUnknowntr57 Target Epitope from
TRAP 78Lys Thr Ala Ser Cys Gly Val Trp Asp Glu Trp1 5 10
* * * * *