Note: Descriptions are shown in the official language in which they were submitted.
WO 95/21625 21 8 3 2 6 0 PCT/US95/01866
PROLACTIN AS A VACCINE ADJUVANT
Background of the Invention
The use of vaccines to prevent diseases in humans, farm
livestock, sports animals and household pets is a common
practice, and considerable effort has been, and is being, made
to extend this practice to cover a more extensive array of
diseases to which these patients are subject. For example, the
use of rabies vaccine in animals is by now commonplace, and
efforts are being made to obtain suitable vaccines to immunize
animals against other dise~ses.
One problem that frequently is encountered in the course
of active immunization is that the antigens used in the vaccine
are not sufficiently immunogenic to raise the antibody titer to
sufficient levels to provide protection against subsequent
challenge or to maintain the potential for mounting these
levels over extended time periods. Another problem is that the
vaccine may be deficient in inducing cell-mediated immunity
which is a primary immune defense against bacterial and viral
i nfection .
In order to obtain a stronger humoral and/or cellular
response, it is common to administer the vaccine in a
formulation containing an adjuvant, a material which enhances
the immune response of the patient to the vaccine. The most
commonly used adjuvants for vaccines are oil preparations and
alum. The mechanisms by which such adjuvants function are
not understood, and whether or not a particular adjuvant
preparation will be sufficiently effective in a given instance is
not predictable.
In addition, with the advent of gene therapy it has been
reported that some success has been accomplished with using
genes or "naked DNA" as vaccines. However, as with some of
the conventional vaccines, the immune response obtained was
insufficient to afford immunization.
WO 95/21625 ~ 18 ~ 2 6 0 PCT/US95/01866
Accordingly, there is a need for additional effective
adjuvant preparations which are suitable for potentiating
vaccines for animals in general, and particularly in humans.
5 Summary of the Invention `
The present invention relates to a composition for
enhancing the immune response of an animal to an infectious
disease vaccine wherein the composition comprises prolactin.
Preferably, the composition is human prolactin and the animal
1C to be vaccinated is, as well, human.
The present invention further relates to a composition
for enhancing the immune response of an animal to an
infectious disease vaccine wherein the composition comprises
prolactin cDNA. Human prolactin cDNA is preferred.
In another aspect, the invention relates to a method of
enhancing the immune response of a subject animal to an
infectious disease vaccine comprising co-administering an
effective amount of prolactin or prolactin cDNA along with a
vacci ne .
Brief Description of the Drawing
Figure 1 shows the amino acid sequence for the prolactin
protein .
Figure 2 shows the nucleic acid sequence for the
25 prolactin cDNA.
Figure 3 is a graph illustrating the Bovine serum albumin
(BSA)-specific antibody response of rats immunized with BSA
alone or BSA + prolactin.
Figure 4 is a graph illustrating a comparison of the BSA-
30 specific proliferative response of rat PBL, at 101 day timepoint, between four rats receiving BSA alone versus BSA +
prolactin .
WO 95/21625 ~ 2 6 0 PCT/US95/01866
Detailed Description of the Invention
Definitions
As used herein, "prolactin" refers to a polypeptide
5 obtained from tissue cultures or by recombinant techniques and
other techniques known to those of skill in the art, exhibiting
the spectrum of activities characterizing this protein. The
word includes not only human prolactin (hPRL), but also other
mammalian prolactin such as, e.g., mouse, rat, rabbit, primate,
0 pig and bovine prolactin. The amino acid sequence of a
recombinant hPRL is shown in Figure 1. The recombinant PRL
(r-PRL) is preferred herein.
The term "recombinant prolactin", designated as r-PRL,
preferably human prolactin, refers to prolactin having
15 comparable biological activity to native prolactin prepared by
recombinant DNA techniques known by those of skill in the art.
In general, the gene coding for prolactin is excised from its
native plasmid and inserted into a cloning vector to be cloned
and then inserted into an expression vector, which is used to
20 transform a host organism. The host organism expresses the
foreign gene to produce prolactin under expression conditions.
As used herein, the term "adjuvant" has its conventional
meaning, i.e., the ability to enhance the immune response to a
particular antigen. Such ability is manifested by a significant
25 increase in immune-mediated protection. Furthermore, the
term "genetic adjuvant" refers to prolactin cDNA which
comprises the complement to the DNA sequence encoding the
prolactin protein as defined above. The sequence for prolactin
cDNA is shown in Figure 2.
General Method
Formulations containing prolactin for adjuvant purposes
are most conveniently administered by intramuscular or
subcutaneous injections or intraperitoneal although other
35 methods of administration are possible.
WO 95/21625 21 8 3 2 6 0 PCT/US95/01866
Standard formulations are either liquid injectables or
solids which can be taken up in suitable liquids as suspensions
or solutions for injection. Suitable excipients are, for example,
water, saline, dextrose, glycerol, ethanol, and so forth.
5 Nontoxic auxiliary substances, such as wetting agents, buffers,
or emulsifiers may also be added. m
Sustained and continuous release formulations are of
considerable variety and could be used i'rr~'the method of the
present invention, as is understood by those skilled in the art.
Prolactin can be administered separately from the
vaccine or in combination with the vaccine. When prolactin is
combined with the vaccine, the composition administered
contains an immunogen that is effective in eliciting a specific
response to a given pathogen or antigen, a pharmaceutically
acceptable vaccine carrier and an immunopotentiating amount
of prolactin. The vaccine will normally be administered per
manufacturer's instructions. Other adjuvants may be
administered either with the vaccine or together with the
prolacti n .
Prolactin will typically be used to enhance the protection
afforded by animal or human vaccines that are considered
"weak" (i.e., provide diminished protection in terms of level,
extent, and/or duration). Examples of such vaccines are
bacterins such as Pseudomonas Staphylococcal, Enterotoxin
Streptococci, cytomegalovirus, HIV, Bordetella bacterin,
Escherichia coli bacterins, Haemophilus bacterins,
Leptospirosis vaccines, Moraxella bovis bacterin, Pasteurella
bacterin and Vibrio fetus bacterin and attenuated live or killed
virus products such as bovine respiratory disease vaccine
(infectious bovine rhinotracheitis, parainfluenza-3,
respiratory syncytial virus), bovine virus diarrhea vaccine,
equine influenza vaccine, feline leukemia vaccine, feline
respiratory disease vaccine (rhinotracheitis-calicipneumonitis
viruses), canine parvovirus vaccine,transmissible
gastroenteritis vaccine, pseudorabies vaccine, and rabies
vaccine.
WO 95/21625 ~ 1 8 3 ~ 6 0 PCT/US95/01866
In addition, because we have demonstrated in vitro and in
vivo data that indicate that prolactin can enhance the immune
response to an immunogen and thereby function as a vaccine
adjuvant, it is believed that the exogenous administration of
5 the prolactin gene would result in the expression of prolactin
in vivo which would be available to function as an adjuvant to
any immunogen whether administered through conventional
means or via gene inoculation. The "genetic adjuvant" could be
produced by inserting prolactin cDNA into a DNA delivery
0 vehicle (e.g., plasmid vectors, liposomes, viral vectors). This
could be accomplished as described by Pellegrini 1., et al.,
Molec. Endocrinolgy, 6, 1023 (1992), Maniatis T., et al.,
Molecvlar Cloning: A Laboratory Manual, 2nd Edition, Cold
Spring Harbor Press (1989) and Felger P., et al., Proc. Natl.
Acad. Sci., 84, 7413, (1991). The "genetic adjuvant" is then
administered along with either cDNA encoding the immunogen
in an appropriate delivery vehicle or "naked" (i.e., solely the
cDNA). In addition, the "genetic adjuvant" could be
administered along with the immunogen itself. The injection
20 sequence would be optimized per immunogen, i.e., the prolactin
cDNA could be co-administered with the immunogen or
immunogen cDNA, or administered in advance or subsequent to
their administration. It is believed that the prolactin cDNA
could be inserted into the same DNA delivery vehicle. Various
25 routes of administration could be used.
EXAMPLE 1
Co-mitogenicity of recombinant human prolactin (r-hPRL)
Peripheral blood Iymphocytes (PBL) were isolated from
30 the blood of normal human volunteers by density gradient
centrifugation on Ficoll Paque (Pharmacia). Heparinized blood
was diluted 3 fold in phosphate-buffered saline (PBS) and
centrifuged at 2000 rpm for 20 minutes. The buffy coat,
located on the surface of the red blood cell pellet and
35 consisting of white blood cells, was collected and diluted with
an equal volume of PBS. The diluted buffy coat was layered on
~183~60
WO 95/21625 ~CT/US95/01866
Ficoll Paque (6 mls of buffy coat on 4 mls of Ficoll Paque in a
15 ml tube) and centrifuged for 30 minutes at 1400 rpm. The
PBL layer, found at the Ficoll-plasma interface, was collected
and the cells were washed three times in PBS. PBL were then
resuspended at 2x106/ml in serum-free AIM-V medium from
Gibco and added to the wells of round bottom 96 well
microtiter plates in a 100 ~LI volume (2x105 PBL/well).
A suboptimal dose of the T cell mitogen concanavalin A
(Con A; 0.2 ~g/ml) was added in a 50 ,ul volume together with
0 50 ~l of varying concentrations of r-hPRL (0-1000 ng/ml
final). Cultures were done in triplicate. The cells were
incubated at 37'C/5% CO2 for 72 hours and the amount of
proliferation measured by tritiated thymidine incorporation.
Tritiated thymidine (0.5~1 Ci/well) was added for the last 18
hours of incubation and cell-associated radioactivity was
measured by scintillation counting after harvesting the cells
onto glass fiber filters using a Skatron 96 well cell harvester.
Results, obtained with cells from different individuals,
shown in Table 1 below, indicated that r-hPRL was able to
enhance the proliferative response of T Iymphocytes to a
suboptimal concentration of Con A. This co-mitogenic activity
was best observed with r-hPRL concentrations of 1 -10 ng/ml,
illustrated in Figure 3.
Tab/e 1
Co-mitogenic activity of recombinant
human prolactin (cpm +/- SEM)
Con A + r-hPRL (ng/ml) Donor 1 Donor 2 Donor 3
No prolactin 22323+4585 35942+810 16549+1618
0.1 22949+2003 34040+1446 17083+1895
35882+3665 45839+2137 27590i3151
1 0 32832+1972 37658+150 22991 +2358
100 25963+4855 35009i2105 22674+1662
1000 23990 1534 35921 +1690 26646+2574
WO 95t21625 ~ 18 3 2 fi O PCT/US95/01866
EXAMPLE 2
Enhancement of antigen-specific proliferation by r-hPRL
To test the ability of r-hPRL to enhance the proliferative
5 response of human T cells to a specific antigen, PBL were
incubated with various concentrations of r-hPRL and
streptokinase, a common antigen to which most individuals are
exposed. Cultures were performed in triplicate in the wells of
96 well round bottom microtiter plates and consisted of 100
111 PBL (2x105/well), 50 ,ul streptokinase (25 ,ug/ml final) and
50 ~l of r-hPRL at varying concentrations (0-1000 ng/ml
final). Proliferation was measured by tritiated thymidine
incorporation after 6 days of culture at 37C/5%CO2.
The results, shown in Table 2 below, indicated that r-
hPRL, at a concentration of 1 ng/ml, significantly enhanced
streptokinase-induced proliferation.
Effect of recombinant human prolactin
on streptokinase-specific proliferation
Streptokinase + r-hPRL (ng/ml) Proliferation (cpm +/- SEM)
No prolactin 31807+4235
0.1 30220+5448
50964+6469
1 0 35620+11318
1 00 36713+2230
1 000 33494+7990
WO 95/21625 2 1 8 3 2 6 0 PCI~/US95/01866
EXAMPLE 3
Effect of prolactin in enhancing the immune response to an
immunogen
Twenty-four 150 gram male Sprague-Dawley rats were
divided into 4 groups. The contro~group received an
intraperitoneal injection of 10 ~-,ug BSA mixed with alum. The
other 3 groups received intraperitoneal injections of 10 ~9
BSA mixed with alum along with either 180 ~19 prolactin, 375
~9 prolactin or 750 ,ug prolactin. Tail vein bleeds were taken
weekly for 4 weeks and the serum evaluated for antibody to
BSA by a Radioimmunosorbent Assay (RIA). The animals were
boosted after the 4th bleed with 10,ug BSA mixed with alum.
Tail vein bleeds were taken over a 7 week period to obtain
serum which was evaluated for the development of antibody to
BSA by RIA.
Bovine serum albumin (BSA)-specific proliferation of
peripheral blood Iymphocytes from rats immunized with BSA
+/- r-hPRL
To measure the effect of r-hPRL on the cellular response
of rats immunized with BSA, blood was collected from
individual animals sacrificed 101 days after boosting. To
isolate peripheral blood Iymphocytes (PBL), blood samples
were diluted 4 fold in the phosphate-buffered saline (PBS) and
centrifuged at 2000 rpm for 20 minutes. The buffy coat was
collected and contaminating red blood cells were removed by
the addition of Tris-ammonium chloride Iysis buffer followed
by a 10 minute incubation at 37C. PBL were then washed
twice in PBS and resuspended at 5x106/ml in RPMI-1640
medium supplemented with 100 u/ml penicillin, 100 ,ug/ml
streptomycin, 20 mM Hepes buffer, 2 mM L-glutamine, 5x10-5
M 2-mercaptoethanol and 5% heat-inactivated fetal calf serum.
PBL were added to the wells of flat bottom 96 well microtiter
plates in a 100 ,ul volume (5x105 cells/well) and cultured in
the presence of medium alone (background control) or 1000
!lg/ml BSA added in a 100 ,ul volume. Cultures were done in
WO 95/21625 21 8 3 2 6 0 PCT/US95/01866
triplicate. Proliferation was measured by tritiated thymidine
incorporation after 5 days of culture at 37C/5% CO2.
The results indicated that, overall, PBL rats immunized
with BSA + 180 ,ug rhPRL displayed higher levels of BSA-
5 specific proliferation than PBL from rats immunized withantigen alone. This observation suggests that r-hPRL may act
to enhance the cellular component of the immune response to
an immunizing antigen. Results are compiled in Table 3 below
and are illustrated in Figures 5 and 6.
Table 3
BSA-specific proliferation of rat PBL (cpm +/- SEM)
101 days after boosting
Group Background BSA-specific
response
BSA alone
Rat 1 918 + 35 1236 + 100
Rat 2 559 + 169 1392 + 185
Rat 3 614 + 51 930 + 265
Rat 4 242 + 21 2122 + 257
BSA + 180
~9 PRL
Rat 1 426 + 99 2552 + 30
Rat 2 269 + 18 756 i 37
Rat 3 723 + 185 4328 + 77
Rat 4 676 + 29 2023 + 397
2183~60
WO 95/21625 PCT/US95/01866
SEQUENCE LISTING
(l) GENERAL INFORMATION~
5 (i) APPLICANT: Richards,Susan
Kaplan, Johanne
Moscicki, Richard
(ii) TITLE OF INVENTION: PROLACTIN AS ADJUVANT
(iii) NUMBER OF SEQUENCES: 2
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: William G. Gosz
(B) STREET: One Kendall Square
(C) CITY: Ca",bridge
(D) STATE: MA
(E) COUNTRY: U.S.A.
(F) ZIP: 02139
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOSIMS-DOS
2 5 (D) SOFTWARE: Patentln Release #l .0, Version #l .2
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
3 0 (C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Gosz, William G
(B) REGISTRATION NUMBER: 27,787
(C) REFERENCE/DOCKET NUMBER: GEN 4-2.0
-10-
WO 95121625 ~ 18 3 2 6 0 PCTIUS95/01866
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 5088722583
(B) TELEFAX: 6173747225
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 351 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
2 0 (Vi) ORIGINAL SOURCE:
(A) ORGANISM: human prolactin
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Thr lle Gly Phe His Met Pro Arg Leu Cys His Glu Cys Lys Phe Arg
5 10 15
Met Thr Thr Arg Ala Asn Ser Leu Ala Thr Glu Phe His Met Pro Arg
3 0 20 25 30
Leu Ser Glu Gln Cys His Glu Cys Lys Phe Arg Met Thr Gly Glu Asn
3 5 Glu Arg Ala Thr Glu Asp Ser Tyr Met Asx Leu Ser Thr His Met Pro
~1~ 3 ~ ~ O PCT/US95/01866
WO 95/21625
Arg Leu Leu Cys Ser His Met Pro Arg Leu Asx Pro Met Arg Asn Ala
5 Glu Asn Thr Glu Arg Glu Asp Asp Glu Phe lle Asn lle Thr lle Asn
95'
:;
His Met Ala Asn Pro Arg Glu Pro Arg Leu Ala Cys Thr lle Asn Pro
100 105 110
Arg Leu Met Arg Asn Ala Ala Cys Cys Glu Ser Ser lle Asn His Met
115 120 125
Pro Arg Leu Pro Glu Pro Leu Glu Asn Gly Thr His Leu Tyr Cys His
130 135 140
Glu Cys Lys His Met Pro Arg Leu Leu Pro lle Cys Pro Gly Gly Ala
145 150 155 160
2 0 Ala Arg Cys Gln Val Thr Leu Arg Asp Leu Phe Asp Arg Ala Val Val
165 170 175
Leu Ser His Tyr lle His Asn Leu Ser Ser Glu Met Phe Ser Glu Phe
180 185 190
Asp Lys Arg Tyr Thr His Gly Arg Gly Phe lle Thr Lys Ala lle Asn
195 200 205
Ser Cys His Thr Ser Ser Leu Ala Thr Pro Glu Asp Lys Glu Gln Ala
210 215 220
Gln Gln Met Asn Gln Lys Asp Phe Leu Ser Leu lle Val Ser lle Leu
225 230 235 240
3 5 Arg Ser Trp Asn Glu Pro Leu Tyr His Leu Val Thr Glu Val Arg Gly
245 250 255
-12 -
WO 95/21625 ~ 1 8 3 2 6 ~ PCI/US95/01866
Met Gln Glu Ala Pro Glu Ala lle Leu Ser Lys Ala Val Glu lle Glu
260 265 270
Glu Gln Thr Lys Arg Leu Leu Glu Gly Met Glu Leu lle Val Ser Gln
275 280 285
Val His Pro Glu Thr Lys Glu Asn Glu lle Tyr Pro Val Trp Ser Gly
290 295 300
Leu Pro Ser Leu Gln Met Ala Asp Glu Glu Ser Arg Leu Ser Ala Tyr
305 310 315 320
Tyr Asn Leu Leu His Cys Leu Arg Arg Asp Ser His Lys lle Asp Asn
325 330 335
Tyr Leu Lys Leu Leu Lys Cys Arg lle lle His Asn Asn Asn Cys
340 345 350
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1100 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
WO 95/21625 2 1 8 3 2 6 0 PCI`/US95/01866
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
TGCCTCATTA ACTMCCACT CACATTMAA GMMTATMC ATATATATTA MMTMTCA 60
TATCCTATM TMTTMCTC ATCTMMTA CMCCTACTG TACCATATAC TMCTGMTA 120
AGACTAGCAT TATTATTCAG GATMCTMG TCCATMGAT ATGTACCATA TTATACACAT 180
TTATAGCACG GATATTACTT ACTGGATATA CTTTGATCTA TCTTGATATT TATTATTCAA 240
MTACTACGT GATATATCGC ATGTCCCMM CATGMCATC MMGGATCGC CATGGMMGG 300
GTCCCTCCTG CTGCTGCTGG TGTCMMCCT GCTGCTGTGC CAGAGCGTGG CCCCCTTGCC 360
15 CATCTGTCCC GGCGGGGCTG CCCGATGCCA GGTGACCCTT CGAGACCTGT TTGACCGCGC 420
CGTCGTCCTG TCCCACTACA TCCATMCCT CTCCTCAGM ATGTTCAGCG MTTCGATM 480
ACGGTATACC CATGGCCGGG GGTTCATTAC CMGGCCATC MCAGCTGCC ACACTTCTTC 540
CCTTGCCACC CCCGMGACA AGGAGCMGC CCMCAGATG MTCMAMG ACTTTCTGAG 600
CCTGATAGTC AGCATATTGC GATCCTGGM TGAGCCTCTG TATCATCTGG TCACGGMGT 660
ACGTGGTATG CMGMGCCC CGGAGGCTAT CCTATCCMA GCTGTAGAGA TTGAGGAGCA 720
MCCMMCGG CTTCTAGAGG GCATGGAGCT GATAGTCAGC CAGGTTCATC CTGMMCCM 780
AGMMTGAG ATCTACCCTG TCTGGTCGGG ACTTCCATCC CTGCAGATGG CTGATGMGA 840
GTCTCGCCTT TCTGCTTATT ATMCCTGCT CCACTGCCTA CGCAGGGATT CACATMMT 900
CGACMTTAT CTCMGCTCC TGMGTGCCG MTCATCCAC MCMCMCT GCTMGCCCA 960
CATCCAmC ATCTATTTCT GAGAAGGTCC TTAATGATCC GTTCCATTGC MGCTTCTTT 1020
-14 -
wo 95/21625 2 1 ~ 3 2 6 0 PCT/USg5/01866
TAGTTGTATC TCTmGMT CCATGCTTGG GTGTMCAGG TCTCCTCTTA MMMTMAA 1080
ACTGACTCGT TAGAGACATC 1100
