U.S. patent application number 10/297611 was filed with the patent office on 2003-10-09 for immunization through oral administration of a vaccine with an edible product.
Invention is credited to Galun, Eithan, Galun, Esra, Gauss-Mueller, Verena, Gilad, Mali Ketzinel, Mitchell, Leslie.
Application Number | 20030190332 10/297611 |
Document ID | / |
Family ID | 24385828 |
Filed Date | 2003-10-09 |
United States Patent
Application |
20030190332 |
Kind Code |
A1 |
Gilad, Mali Ketzinel ; et
al. |
October 9, 2003 |
Immunization through oral administration of a vaccine with an
edible product
Abstract
A vaccine produced in edible plant and/or animal products, as
well as a method of second-generation vaccine development through
the production of at least one complete structure of a pathogen in
a transgenic plant or animal is described. Preferably, the present
invention enables the production of virus-like particles in edible
food plants, through the co-expression of a plurality of proteins
and/or of a plurality of portions of such proteins. The
co-expression of viral structural proteins should enhance the
immunogenicity of the transgenic antigen by providing all elements
necessary to induce a protective immune response. In one variation
of the method the immune response to the transgenic vaccine would
be induced by presenting the transgenic proteins as part of an
edible plant structure, utilizing this to stimulate the mucosal
immune system. Alternatively, the vaccine produced in transgenic
plant/animal products could be purified and used as an immunogen in
other vaccination strategies. A corollary to this claim is the use
of transgenic plant-derived viral vaccines as recombinant vectors
to shuttle host or therapeutic genes to the liver or other tissues
of the body utilizing the natural trophism of the virus. A second
method of vaccination, that involves the stimulation of mucosal
immunity, includes the use of conventional vaccines that are
applied to the surface of the rectum to induce an immune response
that is manifest both locally (in the intestinal mucosa) and
systemically (blood and other body tissues).
Inventors: |
Gilad, Mali Ketzinel;
(Jerusalem, IL) ; Galun, Esra; (Rehovot, IL)
; Mitchell, Leslie; (Jerusalem, IL) ; Galun,
Eithan; (Har Adar, IL) ; Gauss-Mueller, Verena;
(Lubeck, DE) |
Correspondence
Address: |
Anthony Castorina
G E Ehrlich
Suite 207
2001 Jefferson Davis Highway
Arlington
VA
22202
US
|
Family ID: |
24385828 |
Appl. No.: |
10/297611 |
Filed: |
May 16, 2003 |
PCT Filed: |
June 15, 2001 |
PCT NO: |
PCT/IL01/00550 |
Current U.S.
Class: |
424/227.1 ;
435/235.1 |
Current CPC
Class: |
A61P 1/16 20180101; C12N
7/00 20130101; A61P 31/14 20180101; A61K 2039/542 20130101; C12N
15/8242 20130101; C07K 14/005 20130101; C12N 15/8257 20130101; A61P
31/12 20180101; C12N 15/8258 20130101; C12N 2770/32422 20130101;
C12N 2770/32461 20130101 |
Class at
Publication: |
424/227.1 ;
435/235.1 |
International
Class: |
A61K 039/29; C12N
007/00; C12N 007/01 |
Claims
1. A method for administering a viral vaccine to a subject, the
viral vaccine being for a virus entering the subject through a
gastrointestinal mucosa, wherein a target organ of the virus is not
the intestine, the viral vaccine comprising an engineered viral
particle, the engineered viral particle comprising a group of
co-expressed plurality of viral proteins or portions thereof,
wherein said group of co-expressed plurality of viral proteins or
portions thereof is expressed in an edible plant material, the
method comprising: administering the edible plant material
comprising the viral vaccine to the gastrointestinal mucosa of the
subject.
2. The method of claim 1, wherein the gastrointestinal mucosa is a
rectal mucosa, such that the viral vaccine is administered to the
rectal mucosa of the subject.
3. The method of claim 2, wherein the viral vaccine is in a form of
a suppository.
4. The method of claim 2, wherein the virus is HAV (Hepatitis A
virus).
5. The method of claim 4, wherein the viral vaccine contains a
concentration of antigen in a range of from about 0.75 to about
7500 EL.U.
6. The method of claim 1, wherein the subject is a lower
mammal.
7. The method of claim 1, wherein the subject is a human.
8. The method of claim 1, wherein the viral vaccine is a
commercially available vaccine originally administered by injection
to the subject.
9. A method for delivering at least one viral encapsidated gene
through a gastrointestinal mucosa of a subject, the at least one
viral encapsidated gene being a Hepatitis A virus (HAV) gene, the
method comprising: administering the at least one viral
encapsidated gene to the gastrointestinal mucosa of the subject,
wherein the at least one viral encapsidated gene is contained in an
engineered viral particle.
10. The method of claim 9, wherein the gastrointestinal mucosa is a
rectal mucosa, such that the viral vaccine is administered to the
rectal mucosa of the subject.
11. The method of claim 10, wherein the viral vaccine is in a form
of a suppository.
12. The method of claims 9-11, wherein the subject is a lower
mammal.
13. The method of claims 9-11, wherein the subject is a human.
14. The method of claim 9-13, wherein the at least one viral
encapsidated gene is presented in a virus-like particle, present in
an edible plant material.
15. The method of any of claims 9-14, wherein said edible material
contains at least one complete viral structure as an antigen.
16. The method of claim 15, wherein said at least one complete
viral structure is a viral capsid structure.
17. The method of claim 16, wherein said viral capsid structure is
said outer capsid.
18. A method for administering a viral vaccine for Hepatitis A
virus (HAV) to a subject, wherein a target organ of HAV is the
liver, the method comprising: administering the viral vaccine for
HAV to a gastrointestinal mucosa of the subject, wherein the HAV is
contained in an engineered viral particle, said engineered viral
particle comprising a group of coexpressed plurality of viral
proteins or portions thereof, wherein said engineered viral
particle is expressed in an edible plant material and said edible
plant material is ingested by the subject.
19. The method of claim 18, wherein the viral vaccine contains at
least one viral encapsidated gene for HAV.
20. The method of claim 18, wherein the viral vaccine consists
essentially of attenuated killed virus.
21. The method of claim 18, wherein the viral vaccine comprises
attenuated killed virus without an additional adjuvant.
22. The method of claim 18, wherein the viral vaccine consists
essentially of HAV related particles.
23. The method of claim 18, wherein the gastrointestinal mucosa is
a rectal mucosa, such that the viral vaccine is administered to the
rectal mucosa of the subject.
24. A vaccine against a disease-causing pathogen for administration
to a subject, comprising: an entirety of a biologically significant
structure of the disease-causing pathogen, said entirety of said
biologically significant structure being expressed by an edible
plant material in an engineered viral particle, wherein genes for
said plurality of proteins are introduced to said edible plant
material.
25. The vaccine of claim 24, wherein said biologically significant
structure is a plurality of proteins being co-expressed by said
edible plant material.
26. The vaccine of claim 25, wherein said edible plant material is
transgenic for said genes, such that said genes are stably inserted
into a genome of said edible plant material.
27. The vaccine of claim 26, wherein the disease-causing pathogen
is HAV (Hepatitis A virus), such that said genes are HAV genes.
28. The vaccine of claim 24, wherein said edible plant material is
administered to the subject by being eaten by the subject.
29. The vaccine of claim 28, wherein the subject is a human
being.
30. A method for preparing the vaccine of claim 29, wherein said
biologically significant structure is a plurality of proteins, the
method comprising: obtaining genes corresponding to said plurality
of proteins; and inserting said genes Into said edible plant
material.
31. The method of claim 30, wherein said plurality of proteins is a
co-expressed group of viral proteins.
32. The use of a vaccine against a virus, contained in an edible
plant material, for oral administration to a subject, to protect
the subject against the virus, wherein said edible material
contains a plurality of co-expressed viral proteins or portions
thereof.
33. The use of claim 32, wherein said edible material contains at
least one complete viral structure as an antigen.
34. The use of claims 32 or 33, wherein the virus is HAV (Hepatitis
A) virus.
35. An engineered viral vaccine, comprising: a plurality of genes,
including at least one viral encapsidated gene and at least one
non-viral gene; and a carrier adapted to administration of the
vaccine to the gastrointestinal mucosa of a subject.
36. The vaccine of claim 35, wherein said viral sequences code for
a group of co-expressed plurality of viral proteins or portions
thereof.
37. An engineered viral vaccine, comprising: at least one viral
encapsidated gene; and a carrier adapted to administration of the
vaccine to the gastrointestinal mucosa of a subject; wherein
administration of the vaccine induces tolerance in said
subject.
38. A method for administering a viral vaccine for Hepatitis A
virus (HAV) to a subject, wherein a target organ of HAV is the
liver, the method comprising: administering the viral vaccine for
HAV exclusively to a gastrointestinal mucosa of the subject,
wherein an amount of viral particles in the viral vaccine is not
greater than an equivalent amount of viral particles being:
administered through intramuscular injection.
39. The method of claim 38, wherein said gastrointestinal mucosa is
a rectal mucosa.
40. The method of claim 38, wherein the viral vaccine is
administered orally.
41. The method of any of claims 38-40, wherein said amount of viral
particles is equivalent to no more than about 75 EL.U.
42. A method for administering a viral vaccine for Hepatitis A
virus (HAV) to a subject, the method comprising: administering the
viral vaccine for HAV exclusively to a gastrointestinal mucosa of
the subject, wherein said gastrointestinal mucosa comprises at
least rectal mucosa, and wherein an amount of viral particles in
the viral vaccine is in a range of from about 0.75 to about 7500
EL.U.
43. A method for administering a viral vaccine for Hepatitis A
virus (HAV) to a subject, the method comprising: administering the
viral vaccine for HAV exclusively orally to the subject, wherein an
amount of viral particles in the viral vaccine is in a range of
from about 0.75 to about 7500 EL.U.
44. A method for preparing a vaccine against a disease-causing
pathogen for administration to a subject, the method comprising:
identifying a biologically significant structure of the
disease-causing pathogen, said biologically significant structure
comprising a plurality of proteins wherein said plurality of
proteins is a co-expressed group of pathogen proteins; obtaining
genes corresponding to said plurality of proteins; and inserting
said genes into an edible plant material, said edible plant
material expressing said biologically significant structure in an
engineered viral particle.
Description
[0001] This Application is a Continuation-in-Part Application of
PCT Application No. PCT/IL01/00550, filed on Jun. 15, 2001; and of
U.S. patent application Ser. No. 09/596,060, filed on Jun. 16, 2000
(now U.S. Pat. No. 6,368,602); both of which are hereby
incorporated by reference as if fully set forth herein.
FIELD OF THE INVENTION
[0002] The present invention relates to a method of immunizing a
subject against a pathogen through administration of a vaccine with
an edible product, and in particular, to such a method in which the
vaccine is created through a genetically modified plant as a
component of the edible tissue of the plant. Preferably, the
vaccine is directed against Hepatitis A virus (HAV). The present
invention also relates to the use of conventional HAV vaccines for
mucosal immunization by application of the vaccine to the rectal or
nasal mucosal surface, optionally in the form of a suppository or
other such formulation, for the purpose of inducing an immune
response to the virus that protects against hepatitis A. The
present invention also relates to the immunotherapeutic use of
virus like particles that are composed of viral structural proteins
forming a capsid or a virus like structure.
BACKGROUND OF THE INVENTION
[0003] Infectious diseases are a worldwide cause of morbidity and
mortality. Those particularly at risk are individuals with weaker
immune systems such as very young children or those with congenital
and acquired immunodeficiencies. Despite the impact of vaccination
programs for many of the pathogens, infectious diseases are still
the primary cause of childhood mortality, worldwide (The World
Health Report 1997). Travel and other contact between populations
have increased the risk for spread of bacterial and viral
pathogens, thereby demanding a higher degree of community
protection than ever before. The most efficient and effective
strategy for preventing these diseases is by mass vaccination
(Review; Vaccine supplement, Nature Medicine 1998;4:474-534).
However, despite significant technological progress in
bioengineering and advancements in large-scale production, vaccines
remain costly and often, unavailable in sufficient quantity and/or
supply to conduct mass vaccination programs. Currently, most
vaccines are administered parenterally by injection. Further, such
vaccines must be kept refrigerated until administered. Thus, mass
vaccination programs currently require a large supply of vaccine,
maintenance of the cold chain, and supplies of sterile syringes and
needles. These limitations make population immunization campaigns
difficult to execute in some jurisdictions, particularly in Third
World countries, or in emergency response situations such as
threatened use of a pathogen in biological warfare or
bioterrorism.
[0004] HAV (Genus: Hepatovirus, Family: picornaviriade) is an
example of a pathogen for which the vaccine is currently difficult
to administer through such mass vaccination programs. As it is a
highly communicable and infectious, water or food-borne pathogen,
HAV has also been recognized by the US Centers for Disease Control
(CDC) as a pathogen with bioterrorism potential
(WHO/CDS/CSR/EDC/2000.7, Hepatitis A, World Health Organization,
Dept. of Communicable Disease Surveillance and Response document.
http//www.who.int/emc). HAV is highly contagious and transmitted
principally by the fecal-oral route (Koff R. S. Hepatitis A. Lancet
1998; 351:164349) but it may also be acquired by sexual (anal-oral)
contact and blood transfusions. The course of hepatitis A is highly
variable and age-dependent. In the very young, cases of hepatitis A
are usually asymptomatic and can only be detected by identifying
biochemical or serologic changes in the blood. However,
asymptomatic individuals still shed large amounts of virus in their
feces, and therefore, are a significant reservoir for disease
spread. The severity of disease increases with age of the patient
and symptoms range from mild/transient--to severe/prolonged--to
fulminant fatal hepatitis. The clinical course of symptomatic
hepatitis A typically displays: an asymptomatic preclinical
incubation period of 10-50 days when viral shedding is maximal; a
prodromal (preicteric) phase lasting approx. a week characterized
by loss of appetite, fatigue, fever, gastrointestinal symptoms,
dark urine and pale stools; and an icteric phase with jaundice
(bilirubin>20-40 mg/L). This latter phase usually coincides with
the onset of the immune response (at approx. 10 days after
infection) and the reduction of viremia. Nevertheless, viral
shedding into the stools may continue for several weeks.
Occasionally (more often in patients who are more than 50 years
old), fulminant hepatitis with severe liver necrosis, high fever,
hepatic encephalopathy with coma and seizures may occur, leading to
death in 70-90% of such cases. Co-infection with hepatitis C or B
complicates outcome and reduces prognosis of recovery. However, in
the majority of cases the disease is self-limiting, convalescence
long, and recovery occurs with bed rest. Often convalescent
patients experience fatigue for up to one year. Relapsing hepatitis
occurs in 3-20% of cases (usually, within 4-15 weeks) and
persistent cholesteric hepatitis with high bilirubin levels is
occasionally observed. There are no specific treatments for
hepatitis A and current methods of prevention are described
below.
[0005] There is only one known HAV serotype and a single
immunodominant conformation antigenic site (described above) to
which virus-neutralizing antibodies are directed
(WHO/CDS/CSR/EDC/2000.7, Hepatitis A. World Health Organization,
Department of Communicable Disease Surveillance and Response
document. http:/www.who.int/emc; Gauss-Muller V, Zhou M Q, von der
Helm K, Deinhardt F. Recombinant proteins VP1 and VP3 of hepatitis
A prime for neutralizing antibody response. J Med Virol
1990;31;277-83; Wang C D, Tschen S -Y, Heinricy U, et al. Immune
response to hepatitis A virus capsids after infection. J Clin
Microbiol 1996; 34:707-13). While antibodies to individual
structural and nonstructural HAV proteins may be detected, they
have no known protective function. Therefore, intact whole viral
particles are always used for immunization. In acute HAV infection
both IgM and IgG class antibodies are detected and are diagnostic.
HAV-specific IgM may persist for up to 6 months. Appearance of
specific IgG is associated with reduction of viremia and
acquisition of lifelong immunity to HAV. Liver disease symptoms,
which appear at the onset of the immune response, have been
attributed to cellular immunity rather than to immune
complex-mediated tissue damage or to direct viral cytopathogenic
effects (Viral Infections in Humans, Epidemiology and Control
(3.sup.rd Edition). Evans A S, editor. New York, Plenum Medical
Book Co., 1998; Karayiannis P, Jowett T, Enticott M, et al.
Hepatitis A virus replication in tamarins and host immune response
in relation to pathogenesis of liver damage. J Med Virol 1986;
18:261-76, Kurane I, Binn L N, Bancroft W H, Ennis F A Human
lymphocyte responses to hepatitis A virus-infected cells:
interferon production and lysis of infected cells. J Immunol 1985;
135:2140-4).
[0006] Currently, there are at least four HAV vaccines that have
received regulatory approval and are in current use (Prevention of
hepatitis A through active or passive immunization. Recommendations
of the Advisory Committee on Immunization Practices (ACIP). CDC,
MMWR 1999, 48(RR1-12):1-37.). All consist of whole
formalin-inactivated HAV that has been grown in various primate
cell lines and purified. As viral titers are inevitably low these
vaccines are in limited supply, expensive and therefore are subject
to exclusive use for controlling disease outbreaks or for
immunizing travelers to hepatitis A-endemic areas. Hence, present
HAV vaccines can only be purchased for small-scale programs and not
for mass immunization. Further, as such vaccines may be
administered only by injection, syringes, needles, and trained
staff must be available, adding to the cost and difficulty of
delivery and limiting population uptake for those who have fear of
needles. Clearly, new solutions for cheap and effective mass
vaccination are needed. Providing a vaccine through a simple method
could significantly increase vaccine uptake and population
protection against HAV. Two possible solutions are: the delivery of
HAV vaccine as an edible product (i.e., a transgenic plant-produced
HAV vaccine); or in the form of a suppository or similar
formulation that may be applied to the surface of the nasal cavity
or rectum. These methods would induce immunity to HAV via
stimulation of the mucosal immune system.
[0007] The mucosal immune system, an interconnected network of
lymphocytes and accessory cells located throughout mucosae of the
oral and nasal cavities, bronchi, gut, urinary and genital systems,
functions both nonspecifically and specifically as a first line of
defense against pathogens entering the body via these routes. It
has both inductive sites (located in lymphoid follicles of nasal
and rectal mucosae, and Peyer's patches located on the serosal
surface of the small intestine) and effector molecules (which
include secretory IgA produced by lymphocytes located throughout
the mucosae, and cytokines elaborated by CD4+ helper T-cells) as
well as cytotoxic CD8+ T-cells and intraepithelial leukocytes (IEL)
which are cellular effectors of mucosal immunity. While it is
separate from the systemic immune system (spleen, thymus, lymph
nodes and peripheral blood lymphocytes), it is interconnected as
its immune cells also circulate systemically. Vaccination studies
have shown that mucosal immunization provokes both local and
systemic immunity, while injection of antigen usually leads only to
systemic immunity. Moreover, as the mucosal immune system is
interconnected, vaccination at one mucosal site (such as the nasal
cavity) can lead to expression of immunity at a distant mucosal
site (such as the rectum or vagina), as well as systemically.
Hence, mucosal immunization has a greater potential for inducing
effective immunity. Mucosal immunization is also relatively easy
and inexpensive to undertake as a means of mass vaccination as it
can be executed without special training or equipment. Since
currently used HAV vaccines are administered systemically (by
injection) and not locally (to the gut mucosa) they may not be
particularly effective at inducing immunity where it is initially
needed--i.e., at the gastrointestinal mucosa where the virus first
penetrates and where it possibly replicates in the early stages of
disease. In contrast, vaccines that are applied locally to the gut
are likely to be more effective at inducing T-cell and antibody
responses both locally and systemically (Mestecky J, Fultz P N,
Mucosal immune system of the human genital tract. J Infect Dis
1999; 179:S470-S474; Lehner T, Wang Y, Ping L, et al. The effect of
route of immunization on mucosal immunity and protection. J Infect
Dis 1999; 179:S489-S492).
[0008] Recently, it was demonstrated that vaccination against
bacteria, such as salmonella, or viral pathogens, such as HIV, may
possibly be enhanced through the rectal administration of
attenuated or killed bacteria/viruses (see, for example:
Nardelli-Haefliger D, Kraehenbuhl J P, Curtiss R 3rd, et al. Oral
and rectal immunization of adult female volunteers with a
recombinant attenuated Salmonella typhi vaccine strain, Infect
Immun. 1996;64:5219-24; Lehner T. Bergmeier L, Wang Y, Tao L,
Mitchell E. A rational basis for mucosal vaccination against HIV
infection. Immunol Rev. 1999 ; 170;183-96). Rectal, nasal or oral
administrations of vaccines have been shown to elicit both humoral
and cellular immune responses that protect against infection.
Results have varied according to the type of antigen, adjuvant
(materials which enhance the immune response), dose, and route
used. A highly successful oral immunogen is the Sabin polio vaccine
that has been shown to induce high levels of protective
virus-neutralizing antibodies.
[0009] Many human pathogens enter the body via mucosal routes such
as the surfaces of the gastrointestinal, oral-nasal, genitourinary
tracts. Hence, mucosal immunization, which has the potential to
stimulate both local (mucosal) and systemic immunity, is
theoretically, the preferred strategy. Unfortunately, at the
present time HAV and many other vaccines are delivered only by
injection, limiting their uptake and utility. Many of these might
be adaptable to oral, rectal and nasal immunization strategies.
Even for those vaccines that are available in an orally
administered form, however, mass vaccination programs are difficult
to perform because of storage and handling requirements for the
vaccines, as well as the cost and availability. A thermostable,
inexpensive-to-produce product which is readily produced in large
quantities would be the solution to the world's current and
emergency response vaccine needs. In an attempt to overcome some of
these problems, several groups have produced genetically engineered
plants capable of expressing bacterial or viral proteins. For
example, cholera toxin B subunit oligomers were expressed in
transgenic potato plants, although the plants were not tested for
immunogenicity (Arakawa et al., Expression of Cholera Toxin B
Subunit Oligomers in Transgenic Potato Plants. Transgenic Research,
6:403413; 1997). Similarly, the rabies virus glycoprotein, which
coats the outer surface of the virus, has also been expressed in
transgenic tomato plants, although again, the plants were not
tested for immunogenicity (McGarvey et al. Expression of the Rabies
Virus Glycoprotein in Transgenic Tomatoes. Biotechnology,
13:1484-1487, 1995). Also, U.S. Pat. No. 5,914,123 describes
vaccines that are expressed in plants, but also does not provide
any data concerning the immunogenicity of these vaccines. U.S. Pat.
Nos. 5,612,487 and 5,484,719 also describe plant-derived anti-viral
vaccines but similarly, do not provide immunogenicity data. Not all
plant-derived vaccines that have been evaluated in vaccination
studies have proven to be immunogenic. In many cases normal oral
consumption of the plant product only triggered a partially
protective immune response in animals and humans (Mason et al.
Expression of Norwalk virus capsid protein in transgenic tobacco
and potato and its oral immunogenicity in mice. PNAS, 93:5335-5340,
1996).
[0010] A partial explanation for the low immunogenicity of many of
these plant-based vaccines is that the targeted antigens have been
expressed as isolated proteins or peptides, and not in the context
of the intact pathogen. Hence these transgenic antigens cannot
assume the correct conformation for induction of neutralizing
antibodies. For example, HAV capsid proteins expressed individually
do not elicit neutralize antibodies. This is not surprising, as
earlier studies showed that other viruses and bacteria exhibit
similar behavior when only portions of the overall structure are
administered as conventional vaccines (Almond and Heeney. AIDS
vaccine development in primate models. AIDS 12(suppl A):S133-140,
1998; Mayr et al. Development of replication-defective adenovirus
serotope 5 containing the capsid and 3C protease coding regions of
Foot-and-Mouth Disease virus as a vaccine candidate. Virology,
263:496506, 1999; and Wigdorovitz et al. Induction of a protective
antibody response to Foot-and-Mouth Disease virus in mice following
oral or parenteral immunization with alfalfa transgenic plants
expressing the viral structural protein VP1. Virology, 255;347-353,
1999). However, in other cases oral immunization has proven
successful in animal models and in human volunteers (Xiang Z, Ertl
H C J. Induction of mucosal immunity with a replication-defective
adenoviral recombinant. Vaccine 1999; 17:2003-8 [a report comparing
nasal, rectal, vaginal and injection methods of immunization with a
rabies subunit vaccine produced as an adenovirus recombinant];
Kapusta J, Moedelska A, Figlerowicz, et al. A plant-derived edible
vaccine against hepatitis B virus. FASEB J 1999; 13:1796-99 [a
report concerning a HAV subunit vaccine produced in transgenic
plants and administered by feeding]. These observations indicate
that virus-like particles or some subunit antigens administered
orally are capable of inducing an immune response that may be
protective; yet these same reports indicate that different systems
of antigens generate apparently different levels of protection and
immune responsiveness. However, these reports do not concern intact
viral particles produced in transgenic plants.
[0011] Oral administration of viral antigens may also be used to
induce specific tolerance, which may be a desired outcome of
immunization. Previous studies in which viral subunits (hepatitis B
surface antigen, HbsAg) were used in oral immunization showed that
this strategy reduced inflammation caused by viral infection (see
for example; Ilan Y, Chowdhury J R. Induction of tolerance to
hepatitis B virus: can we "eat the disease" and live with the
virus? Med Hypotheses 1999;52(6):505-9 [a report concerning the
induction of tolerance to EBsAg to transform patients with severe
chronic active hepatitis B into healthy HAV carriers]; Ilan Y,
Sauter B, Chowdhury N R, et al. Oral tolerization to adenoviral
proteins permits repeated adenovirus-mediated gene therapy in rats
with pre-existing to adenoviruses. Hepatology 1998;27 (5):1368-76
[a study concerning oral administration of replication defective
recombinant adenovirus generated in mammalian tissue culture for
the purpose of inducing oral tolerance to abrogate the host immune
response to adenovirus and to prolong adenovirus mediated gene
therapy]. The results of these studies support, that tolerance to
viral antigens may be induced through oral administration of viral
subunits, or whole virus. However, none of the studies concern
virus or viral subunits that have been generated in a transgenic
plant system, nor do they demonstrate the induction of tolerance to
multiple viral antigens presented in a chimeric format. Also, none
of the reports describe the induction of tolerance with engineered
viral particles.
SUMMARY OF THE INVENTION
[0012] The background art does not teach or suggest a vaccine that
is produced by edible plant and/or animal materials, for regular
consumption (that is, through oral administration), which contains
at least one complete structure of a disease-causing pathogen. The
background art also does not teach or suggest that such a complete
structure may optionally be a plurality of proteins, or
alternatively may comprise a single molecule that mimics the
structure of pathogen. The background art also does not teach or
suggest a successfully immunogenic vaccine for viruses such as HAV.
Also, the background art does not teach or suggest a method for
producing such vaccines in transgenic plants and/or animals. The
background art does not teach that a chimeric virus like particle
containing elements of two pathogens may be used to induce specific
immunologic tolerance that may be exploited for immunotherapeutic
purposes--i.e., to reduce tissue inflammation induced by a
pathogenic protein, or to downregulate the immune response to allow
the persistence of a viral vector used to transfer new genes into
host tissue.
[0013] There is thus, a need for, and it would be useful to have, a
vaccine that is able to successfully elicit a protective immune
response to disease-causing pathogens, through the production of at
least one complete structure of the pathogen, regardless of whether
the pathogen is viral, bacterial or parasitic in nature.
[0014] There is also a need for an immunotherapeutic vaccine to
serve as a method of enhancing immunologic unresponsiveness to
viral proteins that may be involved in inducing host tissue
inflammation, or as a method of enhancing the persistence of viral
vectored genes in host tissue in gene therapy.
[0015] The present invention overcomes these problems of the
background art, and also provides a solution to a long-felt need
for producing vaccines in edible plant and/or animal products, by
providing a new developmental method for second generation
protective or immunotherapeutic vaccines or gene vectors, through
the production of at least one complete structure of a pathogen in
a transgenic plant or animal, as well as by providing the vaccines
themselves. Preferably, the present invention enables the
production of virus-like particles in edible food plants, through
the co-expression of a plurality of proteins and/or of a plurality
of portions of such proteins as recombinant peptide structures
capable of attaining sufficient immunogenic conformational
structure to give rise to an immune response that will protect
against, and/or ameliorate disease.
[0016] As a preferred example of the operation of the present
invention, a vaccine was developed for hepatitis A virus (HAV).
Previous attempts to vaccinate with isolated HAV capsid proteins
failed to elicit a protective immune response, because isolated HAV
proteins are incapable of eliciting neutralizing antibodies that
recognize specific conformational structures on the viral particle
created only after the assembly of the capsid. To overcome this
problem, preferably the present invention includes the construction
of two HAV plasmids carrying a non-infectious HAV genome lacking
the 5'UTR, for stable transformation of plants. In one plasmid
(pGPPat.OMEGA.HAV) transcription of HAV is driven from the patatin
promoter for expression in tomato fruits, and in the second plasmid
(pJDHAV) transcription is under the control of the 35SCaMV promoter
and should be expressed in the green parts of transgenic plants. To
induce an immune response to the HAV transgenes, the edible parts
of transgenic plants are ingested. Assessment of the immunogenic
potential of these vaccines is by feeding of transgenic plant
material to experimental animals.
[0017] According to preferred embodiments of the present invention,
the technology that is developed for vaccines can also optionally
and more preferably be applied for immunotherapy to block or dampen
immune-mediated inflammatory tissue responses or for gene targeting
to specific organs through the consumption of edible plant
components containing known infectious viral particles as shuttles
for the genes of interest. As described below, the present
invention is optionally able to induce tolerance, which may
preferably be used to reduce an immune response selectively, for
example for gene therapy.
[0018] Using HAV as a model pathogen, the present invention also
embodies a method for administering a regular HAV vaccine to a
subject by absorption through a mucosal tissue, particularly
through the mucosa of the rectum, but also including the nasal
mucosa. This method enables the HAV vaccine to be administered to
the subject rectally or nasally, for example as a suppository or
other dosage form, to successfully immunize the subject against
HAV. Thus, the methods of present invention overcome problems of
background art methods of administration that are associated with
current methods of vaccination against HAV--viz, availability and
supply of inactivated whole viral vaccines, expense, maintenance of
the vaccine in proper storage conditions that typically include
refrigeration, need for special equipment and expertise for vaccine
delivery. As edible or topically applied mucosal vaccines may be
self-administered, large-scale immunization programs would be
feasible.
[0019] With regard to a vaccine administered through mucosal
tissue, as described in greater detail below, rectal immunization
of mice with HAVRIX.TM., an alum adjuvanted whole inactivated HAV
vaccine induced strong HAV-specific IgA and cellular proliferative
responses both systemically and in the intestinal mucosa that were
significantly greater than those elicited by intraperitoneal
delivery of the same vaccine. A further potential advantage of
mucosal immunization is that response to natural reimmunization
through oral ingestion of pathogens may be stronger than that after
parenteral vaccination.
[0020] Hereinafter, the digestive system is defined as mouth,
esophagus, stomach, small intestine, large intestine and rectum, or
any portion thereof.
[0021] Hereinafter, the term "treatment" of a disease and/or
condition also optionally includes prevention of the disease and/or
condition thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] The foregoing and other objects, aspects and advantages will
be better understood from the following detailed description of a
preferred embodiment of the invention with reference to the
drawings, wherein;
[0023] FIG. 1A is a schematic diagram for describing the generation
of plant-derived VLP (virus-like particles). As shown, the
structural core proteins are preferably expressed in plant
material, more preferably through stable transfection and
expression of the relevant gene(s) in the plant material. These
proteins then preferably assemble to form a capsid-like structure,
or virus-like particle, which may either optionally be empty (right
side of figure) or may alternatively contain a gene (left side of
figure), for example for performing gene therapy.
[0024] FIG. 1B is a schematic diagram of the HAV-transfection
vector for use in creating transgenic plants which carries a 6.7 kb
fragment of the whole open reading frame (ORF) of the HAV, lacking
the viral 5'UTR sequence, under the control of the plant patatin
promoter and the omega (TMV) translation-enhancer box. A nos
terminator is located 3' to the HAV ORF. The vector carries the
selection gene for kanamycin resistance (shown as the far left box,
followed by the box that stands for the patatin promoter and omega
enhancer, followed by the box that stands for the HAV ORF, and
ending with the box on the far right, showing the nos
terminator).
[0025] FIG. 1C shows the presence of HAV-specific RNA sequences in
BY-2 cells transfected with pG35S.OMEGA.HAV (a plasmid containing
the entire HAV coding sequences under the control of the
fruit-specific patatin promoter and the omega enhancer sequence) as
determined by HAV specific polymerase chain reaction (PCR)
techniques, using primers from the VP1-VP3 viral structural region
of HAV: lanes 1-4 HAV-transfected cells; lane 5 negative control,
and lane P positive control.
[0026] FIG. 2 depicts a dot blot and hybridization result with a
radioactively labeled HAV-specific cDNA probe, which was used to
detect HAV-specific sequences in DNA isolated from tomato fruits of
12 transgenic plant lines. As seen in the figure all the 12 plants
contained integrated HAV DNA sequences.
[0027] FIG. 3 shows distribution of HAV-specific DNA sequences in
the leafy parts of the transgenic plants as determined by
HAV-specific PCR. M, molecular weight size marker; lanes 1-6, DNA
from tomato leaves; P, positive control.
[0028] FIG. 4 shows radiolabeled HAV-specific dotblot cDNA probe
analysis of RNA extracted from tomato fruits of 17 HAV transgenic
tomato plants. To eliminate the possibility that positive signals
might be due to residual DNA in the extracted plant tissue, the
extracts were treated with DNase I and the dot blot analysis was
repeated FIG. 4B). FIG. 4A; rows A1-A4, A6-A9, B1-B4, B6-B10, RNA
from tomato fruits; D1 & D3, negative control; D10, positive
control. FIG. 4B: A1-A9, B1B2, C1-C8, D1-D2, DNase-treated RNA
extracts from tomato fruits; B-3, 100 pg HAV plasmid treated with
DNase; B8 & D8, 100 and 200 pg, respectively of untreated HAV
plasmid.
[0029] FIG. 5 shows Western blot analysis of HAV proteins from four
different tomato lines that were earlier shown to express HAV RNA.
Total proteins were extracted from tomatoes of lines 7-1, 12-1,
31-1 and 31-2 were subjected to Western blot analysis using a
HAV-specific antibody raised to 70S HAV empty capsids. Results
showed that HAV proteins were detectable at various levels in
tomato fruits from all four transgenic plant lines.
[0030] FIG. 6 illustrates the experimental protocol used in two
sets of experiments in which mice were fed with HAV transgenic or
control tomato fruits to determine the potential immunogenicity of
the plant-derived HAV vaccine.
[0031] FIG. 7 shows results of oral immunization of mice with
transgenic tomato fruits. Balb/c mice were passively fed 5 times at
weekly intervals with fruits from 4 different transgenic tomato
lines, as indicated. Sera were tested for HAV-specific antibodies
one week after the last feeding. The mice were subsequently boosted
with 100 microliters of HAVRIX.TM. 1440 given intraperitoneally, 3
months after the first feeding with transgenic tomatoes. Sera were
tested for HAV-specific antibody levels 3, 7 and 14 days later.
[0032] FIG. 8 shows the kinetics of primary and secondary anti-HAV
antibody responses of Balb/c mice who were immunized with varying
doses of HAVRIX.TM. 1440: 100 microliters, 10 microliters, 1
microliter and 0.1 microliters, Blood was collected at 3, 7 and 14
days after vaccination and serum titers of total HAV-specific
antibodies were determined.
[0033] FIG. 9 shows the antibody response in mice after vaccination
against HAV. The same groups of mice that were fed HAV transgenic
tomatoes were immunized once with 50 microliters of
BIO-HEP-B.RTM.(a commercial vaccine containing HbsAg), given
intraperitoneally. Anti-HbsAg antibody levels in the serum were
measured 7 and 14 days later.
[0034] FIG. 10 is a graph of the comparative serum titers measured
in mice after administration of HAVRIX.TM. 1440 vaccine (containing
1440 ELISA Units (EU) of formalin-inactivated HAV adjuvanted with
alum) by various routes. Mice (6/treatment group) received 100
microliters of HAVRIX.TM. by either the intranasal (IN),
intrarectal (IR), oral (PO), intramuscular (IM), or intraperitoneal
(IP) routes, twice on Days 0 and 21. Sera were collected 35 days
after the first immunization and assessed for total HAV-specific
antibody levels using the HAVAB EIA (Abbott Laboratories).
[0035] FIG. 11 illustrates the experimental protocol used for
assessing the HAV-specific IgA response and cellular response to
rectal or oral administration of unadjuvanted inactivated whole HAV
antigen.
[0036] FIG. 12 shows the comparative frequencies of HAV-specific
IgG antibody forming cells (AFC) observed in the spleens of mice
that were immunized either intraperitoneally (IP) or intrarectally
(IR) with varying doses of HAVRIX.TM. 1440 vaccine. Data shown in
the figure represent the mean number of IgG-AFC determined by
ELISPOT assays in spleen cell populations from 5 mice in each
treatment group.
[0037] FIG. 13 shows the comparative frequencies of HAV-specific
IgM antibody forming cells (AFC) observed in the lamina propria
cell populations harvested from mice that were immunized either
intraperitoneally (IP) or intrarectally (IR) with varying doses of
HAVRIX.TM. 1440 vaccine. Data shown in the figure represent the
mean number of IgM-AFC determined by ELISPOT assays in lamina
propria cell populations from 5 mice in each treatment group.
[0038] FIG. 14 shows the comparative frequencies of HAV-specific
IgA antibody forming cells (AFC) observed in the lamina propria
cell populations harvested from mice that were immunized either
intraperitoneally (IP) or intrarectally (IR) with varying doses of
HAVRIX.TM. 1440 vaccine. Data shown in the figure represent the
mean number of IgA-AFC determined by ELISPOT assays in lamina
propria cell populations from 5 mice in each treatment group.
[0039] FIG. 15 shows the comparative maximal HAV-specific
stimulation indices (SI) observed in the spleen cell (SPLC)
populations harvested from mice that were immunized either
intraperitoneally (IP) or intrarectally (IR) with varying doses of
HAVRIX.TM. 1440 vaccine. SPLC were stimulated in vitro with
concentrations of whole inactivated HAV varying from 20 to 2.5
EU/mL and lymphocyte proliferation was determined by uptake of
radioactive DNA precursor (tritiated thymidine) after 7 days of
culture. Data shown in the figure represent the mean of maximal
stimulation index recorded from 5 mice in each treatment group.
[0040] FIG. 16 shows the comparative maximal HAV-specific
stimulation indices (SI) observed in intestinal lamina propria
lymphocyte (LPL) populations harvested from mice that were
immunized either intraperitoneally (IP) or intrarectally (IR) with
varying doses of HAVRIX.TM. 1440 vaccine. LPL were stimulated in
vitro with concentrations of whole inactivated HAV varying from 20
to 2.5 EU/mL and lymphocyte proliferation was determined by uptake
of radioactive DNA precursor (tritiated thymidine) after 7 days of
culture. Data shown in the figure represent the mean of mammal
stimulation index recorded from 5 mice in each treatment group.
[0041] FIG. 17 shows the comparative maximal HAV-specific
stimulation indices (SI) observed in intestinal Peyer's patch cell
(PPC) populations harvested from mice that were immunized either
intraperitoneally (IP) or intrarectally (IR) with varying doses of
HAVRIX.TM. 1440 vaccine. PPC were stimulated in vitro with
concentrations of whole inactivated HAV varying from 20 to 2.5
EU/mL and lymphocyte proliferation was determined by uptake of
radioactive DNA precursor (tritiated thymidine) after 7 days of
culture. Data shown in the figure represent the mean of mammal
stimulation index recorded from 5 mice in each treatment group.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0042] The present invention is of a vaccine produced in edible
plant and/or animal products, as well as a method of producing a
second-generation HAV vaccine through expression of at least one
complete structure of a pathogen in a transgenic plant or animal,
as well as by providing the vaccines themselves. Preferably, the
present invention enables the production of virus-like particles in
edible food plants, through the co-expression of a plurality of
proteins and/or of a plurality of portions of such proteins. A
"virus-like" particle is therefore herein defined as a group of
co-expressed plurality of viral proteins and/or portions of such
proteins. The co-expression of viral structural proteins should
enhance the proper presentation of viral related antigens to the
human immune system.
[0043] As a preferred example of the operation of the present
invention, a vaccine was developed for hepatitis A virus (HAV).
Previous attempts to vaccinate with isolated capsid proteins failed
for HAV, because isolated HAV proteins were not sufficiently
immunogenic to elicit neutralizing antibodies, which recognize
specific conformational structures on the viral particle created
only after the assembly of the capsid. To overcome this problem,
preferably the present invention includes the construction of two
HAV plasmids carrying a non-infectious HAV genome lacking the
5'UTR, for stable transformation of plants. In one plasmid
(pGPPat.OMEGA.HAV) transcription of HAV is driven from the patatin
promoter for expression in tomato fruits, and in the second plasmid
(pJDHAV) transcription is under the control of the 35SCaMV promoter
and should be expressed in the green parts of transgenic plants. To
assess the immunogenicity of the protein products of the HAV
transgenes, the edible parts of transgenic plants were fed to
experimental animals and their HAV-specific immune response was
determined by laboratory tests.
[0044] According to preferred embodiments of the present invention,
the technology which is developed for vaccines can also optionally
and more preferably be applied for immunotherapeutic purposes,
including but not limited to, oral immunization with chimeric virus
like particles exhibiting specific tissue trophism to induce
specific immunologic tolerance to pathogen antigens involved in
inducing host tissue inflammation; or the use of chimeric virus
like particles to target one or more genes, such as foreign or new
genes for example, to specific organs, through the consumption of
plant material containing non-infectious viral particles as gene
vehicles.
[0045] To provide a comparison model system for oral administration
of any type of vaccine against a pathogen that does not actually
attack any portion of the digestive system, a method for orally
administering a vaccine against HAV was developed. The development
of an animal model for testing is described in Section 1. The
method was first tested by using a traditional HAV vaccine
(HAVRIX.TM.) as described in greater detail below in Section 3.
Next, transgenic plant products were fed to laboratory mice that
served as a model mammalian system, as described in Section 2.
Transgenic plants were constructed to express genes encoding
proteins from the outer capsid of HAV, which served as the model
pathogen. The HAV transgenic plants were fed to mice to determine
if the HAV transgenes were expressing HAV capsid proteins in an
immunogenic form and if these proteins were capable of eliciting
HAV-specific antibody and cellular immune responses For comparison
to conventional injection methods for vaccinating against HAV the
immune response of the mice who were fed the HAV transgenic plant
materials was compared to those elicited in other mice by a
commercial HAV vaccine consisting of inactivated whole HAV
adjuvanted with alum to enhance the immune response. The latter
vaccine was administered to the mice by injection. These
investigations are described in detail in Section 2, below.
[0046] To determine the utility of mucosal immunization via other
routes such as the rectum or nasal cavity, as an alternative
strategy to current methods of immunizing against HAV, conventional
HAV vaccines containing alum-adjuvanted whole inactivated HAV, or
whole inactivated HAV without alum, were employed for rectal,
nasal, and oral immunization of mice. These investigations are
described in detail in Section 3, below.
[0047] The present invention also optionally and preferably
provides virus-like particles, which may also optionally be termed
engineered viral particles, which are more preferably composed of a
group of co-expressed plurality of viral proteins or portions
thereof. Most preferably, the group of co-expressed plurality of
viral proteins or portions thereof is expressed in an edible plant
material. Hereinafter, the terms "engineered viral particles" and
"virus-like particles" are used interchangeably. The use of such
virus like particles, optionally and preferably in mucosal (oral,
nasal or rectal) immunization may be used to induce specific immune
tolerance to expressed viral antigens that are involved in chronic
inflammatory diseases. Alternatively, this method may optionally be
used to generate virus like particles encapsidating foreign or host
genes as a gene delivery vehicle for gene therapy, that favors
tissue specific targeting and permits prolonged tissue expression
of the encapsidated gene. The induction of immune tolerance to the
structural proteins of the delivery vehicle by oral immunization
may optionally be exploited to enhance gene expression in target
tissue.
[0048] Section 1: HAV as a Model Pathogen
[0049] The transgenic plant HAV vaccine was developed as a
comparison model system for oral immunization against a pathogen
that does not actually attack any portion of the digestive system
but which uses the intestinal mucosal as a portal of entry in its
infective process. The method of oral administration of a regular
HAV vaccine is itself novel and non-obvious, as it is the first
example of successful oral administration of such a vaccine, which
is normally administered through intramuscular injection. Thus, the
comparison model is also an inventive vaccine and method for
administration thereof and is part of the present invention.
[0050] The gastrointestinal tract is a major port of entry for many
pathogens (bacterial, viral, and parasitic) that impose a
significant health burden. HAV, a positive-stranded RNA virus, is
an example, HAV enters the human body through the gastrointestinal
system, and migrates to the liver for tissue specific replication,
thereby causing liver damage and clinical hepatitis.
[0051] To immunize the subject against the HAV disease, the
comparison model of the present invention also includes mucosal
(intrarectal) administration of conventional HAV vaccines, such as
the currently available HAV vaccine, HAVRIX.TM. (SmithKline Beecham
Biologicals).
[0052] The preferred vaccination strategy according to the present
invention involves the exposure of the intestine to HAV particles
either by oral administration or by topical application to the
rectal mucosa, thereby potentially eliciting both an antibody
response (optimally, neutralizing antibodies) and a cellular immune
response as illustrated by data from the investigations described
below.
[0053] Section 2. Construction of Transgenic Plants Expressing HAV
Antigens and Their Use in Oral Immunization of Mice
[0054] Transgenic plants capable of expressing at least one
complete, and appropriately assembled, HAV capsid structure were
genetically engineered. According to this example, transgenic
tomato plants were engineered to produce HAV viral particles that
would be non-infectious because their genome excludes about 730
nucleotides of its 5' NTR, without which, they can neither
replicate nor express their own proteins. Elimination of this part
of the viral genome does not affect the external capsid structure
of the virus that is seen by the immune system, and therefore, by
implication, the potential immunogenicity of the plant-produced HAV
vaccine. Thus, when HAV transgenic tomatoes are eaten, the
non-infectious viral particles with their immunogenic capsid
proteins would be ingested as well. The plant-generated HAV
virus-like particles should subsequently be taken up by M-cells on
the surface of the gastrointestinal tract and transferred to
antigen presenting cells and lymphocyte components of the mucosal
immune system resulting in a HAV-specific response as illustrated
by the data shown below.
[0055] FIG. 1A shows an example of the different types of
virus-like particles that could optionally be produced in plants.
On the right hand side of the figure, empty virus-like particles
are shown, which could optionally be used for immunization against
a particular virus as a vaccine for example. However, such
particles could also optionally be used to induce tolerance, in
order to actually specifically reduce the immune response to the
particles, and hence to the viral capsid for example. For example,
such tolerance could also optionally be used to reduce inflammation
by reducing the immune response. Reduction of the immune response
may optionally and preferably be desired in order to reduce the
effects of diseases that have a viral component, preferably for
inflammatory diseases that have a viral component. It should be
noted that for some diseases, only particular variants that have a
viral component may optionally be treated with the present
invention.
[0056] A number of mechanisms of mucosal immunization, such as for
rectal, nasal and targeted iliac lymph node immunization for
example, have also been shown to induce non-cognate immune
responses that reduce viral infection, for example for SIV (simian
immunodeficiency various) and for SHIV (chimeric virus of SIV and
SHIV). As the specific mechanisms involved would also influence the
recruitment of inflammatory cells to tissues where the pathogen is
established, they could also optionally be exploited to alter the
inflammatory response and disease severity and progression (e.g.,
in chronic hepatitis C infection in the liver).
[0057] However, the use of empty viral particles is preferably not
limited only to the treatment of diseases through either tolerance
or immunization. Optionally, chimeric virus-like particles, that
may for example include proteins from a plurality of viruses, may
be used to induce both tolerance and immunity simultaneously. Such
a combination is preferably determined according to the structure
of the different proteins incorporated and/or their relative
amounts. Chimeric virus-like particles may also optionally include
one or more non-viral proteins.
[0058] The left hand side of the figure shows virus-like particles
that encapsidate one or more genes, shown as a transgene. These
virus-like particles may optionally be used to induce tolerance to
the delivery vehicle, which may itself optionally be a virus
containing the gene More preferably, empty virus-like particles are
administered before the particles containing the transgene, in
order to induce tolerance. Such tolerance is preferred in order to
avoid inducing an immune response against the delivery vehicle of
the transgene, and/or the transgene itself It should be noted that
the capability to induce tolerance may be affected by a number of
factors, including but not limited to, previous patient or subject
exposure to one or more pathogens, and/or previous administration
of one or more vaccines and/or other immunizing materials to the
patient or subject. Therefore, optionally and most preferably, the
virus-like particle contains proteins that are tailored to an
individual patient and/or to a group of patients.
[0059] According to preferred embodiments of the present invention,
selective tolerance to the antigens of a vector may also optionally
be performed when the vector is a novel virus or a chimeric vector
consisting of a xenovirus into which specific proteins have been
incorporated that would enhance tissue targeting. For example, this
vector could optionally be a gene therapy vector, for administering
a transgene to a subject. Promoting tolerance to such a vector
would be useful if the vector bad to be used on more than one
occasion in the same subject, for example for repeated
administration of the vector for gene therapy, as the initial
induction of tolerance would obviate an immune response on
secondary immunization. Thus, the vector could be used more than
once without concerns of rejection.
[0060] All of these different effects are more preferably obtained
by administering the plant material containing the virus-like
particles to the subject, for example as food (oral administration)
or through other mucosal administration, such as rectal and/or
nasal immunization.
[0061] Depending upon the type of virus from which the proteins of
the virus-like particles are taken, the virus-like particle may
also optionally be targeted. For example, one or more HAV proteins,
as described below, would preferably enable the virus-like particle
to be targeted for the liver. One of ordinary skill in the art
could easily select the proper protein(s) from the proper types of
virus(es) for such targeting.
[0062] 2.1 Construction of Plasmid Vectors with the HAV Genome and
Analysis of their Expression in Transgenic (Tg) Tomato Plant
Tissues:
[0063] A 6.7 kb XbaI-PmeI HAV/7 cDNA fragment lacking the viral
5'UTR, and the additional 10 bp from the HAV translational start
site, was isolated from plasmid pHAV/7. This fragment was inserted
into the modified BSKS plasmid that contained the ten 5' coding
nucleotides of HAV/7. Further engineering inserted the 35SCaMV
promoter and the Omega (TMV) translation-enhancer box in front of
the HAV/7 coding region and a nos terminator behind this region.
The promoter-HAV-terminator combination was finally cloned into a
binary vector (for Agrobacterium tumefaciens-mediated genetic
transformation) pGPTV-kan, termed pG35S.OMEGA.HAV. This vector was
introduced into Agrobacteria with a helper plasmid and BY-2 tobacco
plant cells were transformed with pG35S.OMEGA.HAV. Kan.sup.R cell
lines with were selected and established. The presence of HAV
sequences in transfected BY-2 cells was confirmed by polymerase
chain reaction amplification (PCR) using primers from the VP1-VP3
viral structural region of the virus (FIG. 1C).
[0064] A similar vector to the pG35SHAV was constructed replacing
the 35.S CaUV promoter with the patatin promoter, which allows the
transgene to be expressed in tomato fruits. This vector was termed
pGPPATHAV (illustrated in FIG. 1B) was used to transform tomato
plants by the Agrobacterium tumefaciens transformation procedure.
It was previously assured that the patatin promoter, indeed, caused
the expression of a marker gene (GUS) in the fruits of the
transgenic plants. After genetic transformation of tomato tissue
with this HAV construct (pGPPATHAV), 17 lines of transgenic (Tg)
tomato plants were derived (46 plants in each line). These lines
were assessed for the presence of HAV sequences in their genomic
DNA. FIG. 2 depicts a dot blot and hybridization result with
radiolabeled HAV cDNA probe of DNA extracted from tomato fruits of
the Tg plant lines. As seen in the figure, all 12 lines contained
HAV-specific DNA sequences confirming integration of the transgene.
Some plant lines were also assessed by PCR for presence of
HAV-specific sequences in DNA extracted from their leaves (FIG.
3)
[0065] Overall, DNA and RNA were extracted from 72 Tg plants, for
the assessment of HAV integrated DNA sequences. In those plants
showing integrated HAV DNA sequences, the presence of HAV RNA
transcripts in nucleic acids extracted from the fruits was
investigated. Total RNA extracted from Tg and control tomato plants
was probed for HAV-specific RNA sequences by dot blot and
hybridization assays FIG. 4A). To exclude the possibility that the
positive signal seen was due to residual DNA in the RNA samples,
the RNA extracted from the tomato fruits was treated with DNase I
and the dot blot analysis was repeated (FIG. 4B). As seen in the
figure; in some of the RNA samples, the specific signal was weaker
after DNase I treatment (e.g. A1, A2, A5). However with other
samples (A3, A5, A6, A8 and B1), the signal was observed to be
higher than that of the control tomato (B2). Plants testing
positive for HAV mRNA transcripts were further examined for
expression of the inserted RNA. The predicted HAV protein products
were detected by Western blot analysis with specific anti-HAV
antibodies that included a mouse monoclonal anti-HAV (Biodesign
International, Saco, Me., cat No. C65885M); a pooled antiserum
produced from Balb/c mice immunized twice at 10-day intervals with
72 EU of HAVRIX.TM. vaccine administered intraperitoneally; and
HAV-specific antibody raised to 70S HAV empty capsids. FIG. 5 shows
a diagram of the observed expression of HAV proteins from four
different tomato lines (7-1, 12-1, 31 -1 and 31-2), which had
previously shown HAV RNA expression. Tomato tissue taken from all
four Tg lines expressed HAV capsid proteins that were detected with
the anti-HAV antibody directed to 70S empty capsids. Moreover, the
HAV capsid proteins, VP1, VP2, and VP3 that together make up the
conformational site to which virus-neutralizing antibodies are
directed were detected in all four lines. Thus, the transgenic
tomato plants not only express HAV proteins, but also more
importantly, express those HAV capsid proteins relevant to the
induction of a protective immune response.
[0066] 2.2. Evaluation of HAV Transgenic Plant Vaccines in
Mice:
[0067] Two sets of feeding experiments employing similar protocols
(shown in FIG. 6) were performed. Four groups of 4-5 week old
female BALB/c mice (n=5, each) were fed tomatoes from Tg lines 7-1,
12-2, 31-1, and 31-2. Five control mice were fed with normal wild
type tomatoes, such that they were exposed to the tomatoes
continuously and fed ad libitum. In the second experiment separate
test and control groups were fed the same tomatoes but were given
10 .mu.g of cholera toxin (CT, Sigma-Aldrich) just prior to
feeding. To ensure uptake, the mice were deprived of their standard
chow for 12 hours before in the second experiment, such that
exposure to the tomatoes was periodic and not continuous. The
tomatoes were weighed, then left in the cages for 6 h, and then
weighed again to estimate consumption. A total of 5 feedings were
offered on Days 0, 7, 14, 21, and 28. Animals were bled on Day 35
for the preparation of sera for interim antibody testing. Sera from
mice in each of the treatment groups were pooled for testing for
total HAV-specific antibodies by a commercial inhibition ELISA
(HAVAB EIA, Abbott Laboratories, Diagnostic Division, Abbott Park,
Ill.).
[0068] In the first experiment, HAV-specific serum antibody was not
observed in any of the treatment groups at the interim bleeding
taken on Day 35 and thereafter (FIG. 7). To determine if oral
administration had primed for a secondary response to HAV (in the
absence of a detectable primary antibody response) the mice in all
groups were subsequently boosted three months after the first
feeding (indicated by the arrow in FIG. 7) by intraperitoneal (IP)
injection with 100 .mu.L of HAVRIX.TM. 1440 vaccine (an
alum-adjuvanted commercial vaccine containing 1440 ELISA units [EU]
of inactivated whole HAV) and the antibody response was followed by
test bleedings taken on Days 95, 98, 102, and 109 as illustrated in
FIG. 7. HAV-specific antibodies were detected after Day 98 in serum
pools collected from the control, and Tg tomato groups 12-2, 31-1,
and 31-2. Two groups of mice, the control group fed wild type
tomatoes, and mice fed tomatoes from 31-1 Tg line, developed
HAV-specific antibody responses against HAV with kinetics
resembling a primary immune response. Two other groups (those fed
Tg tomatoes from lines 12-2 and 31-2) demonstrated responses with
slower kinetics. However, no antibody response was observed in mice
that were fed Tg tomatoes from Line 7-1, as performed in the first
experiment. Without wishing to be limited by a single hypothesis,
the latter observation suggested that the antigen (or quantity of
antigen) expressed by this Tg line might have induced oral
tolerance to HAV antigens, a known possible outcome of oral
immunization. The slower kinetics observed in the responses
observed in mice that were fed Tg tomatoes from lines 12-2 and 31-2
also suggested some negative influence of priming by oral
administration of HAV antigens. Hence, these results suggested that
continuous exposure of the immune system of the mice to the HAV
antigens appeared to induce immune tolerance instead of
immunization against the virus. The usefulness of the induction of
tolerance was described above, and is also addressed in greater
detail below, in Sections 3 and 4.
[0069] To establish whether oral immunization with the transgenic
HAV antigen had, indeed, induced specific immune tolerance to HAV a
series of experiments were conducted. First, the kinetics of the
primary and secondary antibody response to administration of the
HAV vaccine were characterized by immunizing Balb/c mice with
HAVRIX.TM. 1440 (FIG. 8). The commercially available HAVRIX.TM.
vaccine is also known as HAVRIX.TM. 1440 vaccine, and is a sterile
suspension containing formaldehyde-inactivated hepatitis A virus
(HM175 hepatitis A virus strain) adsorbed onto aluminium hydroxide.
The virus was propagated in MRC5 human diploid cells. Before viral
extraction the cells were extensively washed to remove culture
medium components. A virus suspension was then obtained by lysis of
the cells, followed by purification using ultrafiltration
techniques and gel chromatography. The virus was inactivated with
formalin. The vaccine contained 1440 E.U. (Elisa units) of viral
antigens, in a 1.0 ml dose volume. The inactive ingredients in the
vaccine were aluminum (from aluminium hydroxide), 0.25 mg; amino
acids for injection, 1.50 mg; disodium phosphate, 0.575 mg;
monopotassium phosphate, 0.100 mg; sodium chloride, 4.500 mg;
potassium chloride, 0.115 mg; polysorbate 20, 0.025 mg;
2-phenoxyethanol, 2.500 mg, neomycine sulfate, 10 ng; formaldehyde,
100 micrograms; water, 0.5 ml; all contained in a 1.0 ml dose
volume. The vaccine was obtained from SmithKline Beecham
Biologicals S.A. (Belgium). Each group of 5 mice received a
different dose of vaccine, as indicated. After 3, 7 and 14 days,
the mice were bled and sera were tested for anti-HAV
antibodies.
[0070] Mice receiving 100 microliters of vaccine developed a
typical primary antibody response: 7 days after the vaccination,
HAV-specific antibodies were detected at low levels and remained
unchanged for 14 days (FIG. 8). HAV-specific antibodies were not
detected in mice receiving the lower amounts of HAVRIX.TM. 1440
(10, 1 and 0.1 microliters) over the period of evaluation. To
determine if the lower doses of HAVRIX.TM. primed immunologic
memory in the absence of a detectable primary antibody response,
the mice were boosted with 100 microliters of HAVRIX.TM. 1440 one
month after the first vaccination (indicated by the arrow in FIG.
8) to determine whether a secondary antibody response could be
elicited. Presence of serum HAV-specific antibodies was checked 3,
7 and 14 days after the vaccination. As seen in FIG. 8, in the
group of mice that developed detectable primary antibody response,
a brisk secondary anti HAV specific antibody response was found,
with rapid kinetics (the antibodies were detectable as early as 3
days after boosting) and these reached a higher level than in mice
primed with the lower doses of HAVRIX.TM.. Mice in the groups that
were primed with lower doses of HAVRIX.TM., also demonstrated a
HAV-specific antibody response but with slower kinetics compared to
that of the group who received the highest priming dose of
HAVRIX.TM.. These results showed that low doses of HAVRIX.TM. given
intraperitoneally could prime for immunologic memory (in the
absence of a detectable primary antibody response) and that this
memory was sufficient to result in a detectable antibody response
upon challenge with HAVRIX.TM.. Alternatively, the response
observed in those animals that were originally immunized with the
lower doses of HAVRIX.TM., then subsequently re-immunized with 100
microliters of HAVRIX.TM. 1440, may have represented a true primary
immune response to the vaccine. In order to show that the
inhibition of the immune response observed in the initial feeding
experiment with HAV Tg tomatoes was specific to the HAV antigen,
the mice from that experiment were immunized with an irrelevant
antigen, the HbsAg of the hepatitis B virus using a commercial HAV
vaccine preparation (BIO-HEP-B.RTM., Bio-Technology Israel). Mice
were immunized once with BIO-HEP-B.RTM. and HbsAg-specific antibody
levels were measured 7 and 14 days after the administration of the
vaccine. As shown in FIG. 9, mice in all treatment groups developed
a specific HbsAg antibody response indicating that oral
administration of Tg HAV antigens had not induced a generalized
state of immunologic nonresponsiveness.
[0071] In the second experiment, again all animals proved to be HAV
seronegative after administration of Tg HAV vaccines. The second
group which were co-administered cholera toxin (CT), a mucosal
adjuvant shown to enhance the immune response to other antigens
given orally, also did not develop measurable immunity after
feeding with Tg tomatoes. All mice were subsequently given a
subimmunogenic dose (72 EU) of HAVRIX.TM., intraperitoneally (IP),
one week later. Two to three weeks later, animals were killed and
their spleens were removed for analysis of HAV-specific antibody
forming cell (AFC) numbers by ELISPOT assay and in vitro lymphocyte
proliferative responses by lymphocyte stimulation tests
(LST)(methods described in Section 3). Sera were prepared for
antibody testing. In marked contrast to the results of the first
experiment, feeding with Tg tomatoes from Line 7-1 appeared to have
stimulated a HAV-specific memory response. This resulted in
enhanced HAV-specific antibody and cellular responses to a
subsequent IP injection of a sub-immunogenic dose of HAVRIX.TM.
vaccine relative to control animals that were fed normal tomatoes
(Tables 1 and 2, below). This type of response was not observed
with the other Tg tomato lines that were evaluated.
[0072] Conclusions: Therefore, while the data from the first set of
feeding experiments with HAV transgenic tomatoes suggested the
possibility of induction of specific oral tolerance (to HAV
antigens), which without wishing to be limited to a single
hypothesis, may be due to the continuous exposure of the mice to
the transgenic tomatoes, data from the second set of experiments
revealed that the ingested transgenic HAV antigen could also prime
for HAV-specific immunologic memory allowing for a strong secondary
response upon parenteral challenge. It may also be possible that
differences in the amounts of transgenic HAV antigen ingested by
the animals between the two experiments account for these different
outcomes, again without wishing to be limited by a single
hypothesis.
1TABLE 1 Immune responses of animals passively fed with HAV-Tg
tomato line 7-1 or control (normal) tomatoes after boosting with 72
EU of HAVRIX .TM. given intraperitoneally. Max. SI Animal No. of
AFC/10.sup.6 SPLC.sup.a Attain- Total HAV- No. Treatment IgG IgM
IgA ed.sup.b Specific Ab.sup.c 7-1A 7-1 alone 0 4 0 3.5 0 7-1B 7-1
alone 6 38 0 7.5 15 7-1C 7-1 alone 10 68 0 6.0 21 7-1/6 7-1 alone
2.1 25.4 1.7 4.2 20 C37 Control 0 5 0 2.7 0 C39 Control 0 11.6 2
2.1 22 C40 Control 0 45 0 2.1 4 C42 Control 0 0.5 0 1.1 14 7-1/7
7-1 + CT 4.2 15.8 0 4.6 19 7-1/8 7-1 + CT 0 15.8 0.8 5.7 21 7-1/9
7-1 + CT 0 14.2 0.4 8.1 0 7-1/10 7-1 + CT 10.4 10.4 0 9.4 4 7-1/11
7-1 + CT 0.4 3.75 0.8 8.1 20 7-1/12 7-1 + CT 0 9.2 0 6.2 19 C38
Control + CT 0 0.4 0.4 1.8 0 C41 Control + CT 0 24.6 0.8 3.0 11 C43
Control + CT 0 4.6 0 2.7 2 C44 Control + CT 0.8 10.4 0.8 7.4 0 C45
Control + CT 1.25 12.5 0 3.4 14 .sup.aAFC (antibody forming cell)
frequencies in spleen cell (SPLC) populations as determined by
ELISPOT assays for detecting HAV-specific IgG, IgM, and IgA class
antibodies. Values shown are corrected for background observed on
wells coated with coating buffer only. .sup.bValue shown is the
maximum stimulation index (SI) obtained in SPLC proliferation
assays in which a range of 20 to 2.5 EU/mL of formalin-fixed whole
HAV was used to stimulate SPLC from treated and control animals.
.sup.cTotal HAV-specific serum antibody units as determined by a
quantitative commercial inhibition EIA (HAVAB, Abbott
Laboratories).
[0073]
2TABLE 2 Comparison of medians (calculated from data shown in TABLE
1) by treatment. No. of AFC/10.sup.6 cells Max. SI Total HAV
Treatment IgG IgM IgA Attained Specific Ab 7-1 alone 4.1 31.7 0 5.1
17.5 Control 0 11.6 0 2.1 4.0 7-1 + CT 0.2 12.3 0.2 7.2 19.0
Control + CT 0 10.4 0.4 3.0 2.0
[0074] Section 3: Rectal Immunization of Mice with Whole HAV
Vaccines
[0075] In order to test the second method of the present invention,
an animal model was developed for the assessment of the mucosal and
systemic immune responses to whole HAV following rectal
administration of formalin-inactivated whole virus either in the
form of unadjuvanted semi-purified HAV prepared from FRhK4 cells or
as the commercial vaccine, HAVRIX.TM. consisting of highly purified
whole HAV, inactivated and adsorbed with alum to enhance its
immunogenicity. A series of experiments were conducted and are
described in chronological order below.
[0076] 3.1 Preliminary Investigations of the Comparative Immune
Response to Delivery of HAVRIX.TM. via Mucosal and Conventional
Systemic Routes:
[0077] In the first experiment, female Balb/c mice, aged 4-6 weeks,
were injected with varying amounts of HAVRIX.TM. at a concentration
of 720 EU/mL, using three different routes and doses. 100
microliters (72 EU) intraperitoneally (IP); 100 microliters (72 EU)
intrarectally (IR); or 20 microliters (14.4 EU) intranasally (IN).
The vaccine was administered on Days 0 and 21. In a companion
experiment using a higher-titered HAV vaccine, HAVRIX.TM. 1440,
mice (6/treatment group) received 100 microliters (144 EU) by
either the intranasal (IN), intrarectal (IR), oral (PO),
intramuscular (A, or intraperitoneal A) routes, twice on Days 0 and
21. In both experiments sera were collected from the mice 35 days
after the first immunization and were then tested for total
specific HAV antibodies using a commercial kit (HAVAB, Abbott
Laboratories), Cellular responses to HAV were evaluated by three
methods: the lymphocyte stimulation test which evaluated lymphocyte
proliferation to in vitro stimulation with HAV virus; HAV-specific
assays for detection of cytotoxic T lymphocytes (CTLs) from their
ability to mediated in vivo killing of target cells expressing HAV
antigens; and the in vivo delayed hypersensitivity test (footpad
swelling in response to injection of HAV virus). The results of the
first experiment using the lower titered HAVRIX.TM. 720 formulation
and variable doses revealed that mice receiving the vaccine through
the IP (conventional route) or IR routes, developed anti-HAV
antibodies, whereas mice that received the vaccine intranasally,
did not (data not shown). The lower titer HAVRIX.TM. 720
formulation contains the same ingredients as described above for
the HAVRIX.TM. 1400 formulation, except that the former only
contains 720 E.U. of HAV antigens. The vaccine was obtained from
the same source as the HAVRIX.TM. 1400 formulation, as described
above. The results of the second experiment, shown in FIG. 10 were
similar, showing that all the mice that received the vaccine IP or
IM, while only about 50% of the mice that received the vaccine IR,
developed anti-HAV antibodies. The response after IP or IM
vaccination was higher than after immunization by the mucosal
routes (IR, PO, IN). Mice that were vaccinated intranasally or
orally developed barely detectable levels of anti-HAV antibodies.
Evidence of a cellular immune response in the HAV seropositive mice
was sought. Specific responses to in vitro stimulation with HAV in
proliferation assays could not be detected in any of the treatment
groups. Similarly, no CTL response was observed in cytotoxicity
assays, likely due to the presence of alum adjuvant that is known
to suppress CTL activity. In delayed type hypersensitivity
experiments, HAVRIX.TM. or PBS as control was injected to the
footpad of the vaccinated mice, and swelling was measured after 24
and 48 hours. Regardless of the immunization route, most of the
mice showed a marked reaction after HAVRIX.TM. injection but not
after PBS injection (data not shown).
[0078] 3.2 Investigations of Systemic and Mucosal Immune Responses
to Inactivated Whole HAV:
[0079] As the results of preliminary experiments with HAVRIX.TM.,
an alum-adjuvanted whole HAV vaccine suggested low immunogenicity
of the vaccine when it was administered by mucosal (IR, IN, PO)
routes in comparison with systemic (IP, IM) routes, a series of
experiments were conducted to ascertain whether the presence of
alum adjuvant, or dosage levels, influenced the responses observed
in the preliminary experiments. These experiments were also
designed to develop or implement the methodology required to assess
intestinal mucosal immune responses. In these experiments, only the
IR or PO routes of immunization were used, as these were considered
relevant to the infectivity and pathogenicity of HAV.
[0080] 3.2.1 Immunization of Mice with Whole HAV Without Alum:
[0081] The first series of experiments addressed the issue of alum
adjuvant as a potential inhibitor of the mucosal immune response to
HAV. For these experiments, a semi-purified preparation of HAV
grown on FRhK4 monkey kidney cells and inactivated with formatin
was used as a rectal, oral, or intraperitoneally delivered
immunogen. Transgenic tomato HAV antigen was also used as an oral
immunogen. The experimental protocol is illustrated in FIG. 11.
Female (non-SPF) Balb/c mice, aged 4-6 weeks at the outset of
experiments were used and kept under standard non-SPF conditions.
Specific antigens employed were: semi-purified formalin-inactivated
HAV (strain HM-175 grown in FRhK4 cells, as in HAVRIX.TM. vaccine)
purchased from Microbix Biosystems, Inc, Toronto, Canada (HAV Grade
2 antigen, cat. No EL 25-02), that according to the manufacturer,
contained 2.2 mg/mL of protein and approx. 6.0 EU/mL of HAV. This
antigen was used to immunize mice in two different presentation
formats. For intraperitoneal (IP) immunization, the antigen was
emulsified 1:1 with incomplete Freund's adjuvant (IFA). For
immunization via the intrarectal (IR) or oral (PO) routes, the
antigen was diluted 1:1 with sterile saline and delivered with a
No. 1 surgical catheter attached to a tuberculin syringe. Oral
antigen was offered passively and invasive gavage was not employed.
For all immunizations in this series of experiments dosage volume
was 100 microliters is (containing approx 110 micrograms of total
protein or 0.3 E.U. of HAV).
[0082] Transgenic plant HAV vaccines produced by two tomato strains
(2-21 and 3-12), both of which were shown to express mRNA for the
HAV insert, were also evaluated. For immunization, approx. 0.4 g of
lyophilized transgenic plant material was suspended in 4 mL of
sterile saline and sonicated on ice for 3 min using 10-second
bursts. The resultant material was centrifuged and the supernatant
used to immunize mice, giving each animal 100 microliters PO.
Sterile saline either emulsified with IFA (for i.p. injections) or
given alone (for IR or PO routes) served as a negative control.
[0083] The immunization protocol, schedule and numbers of animals
used were as follows. Animals immunized via the IP or IR routes
received two doses at 12-day intervals while animals immunized
orally received 4 doses at 5-day intervals. Three weeks after the
final immunizations approx. 0.5 mL of blood was obtained from the
tail vein of each mouse. To determine which mice were likely to
have responded to immunization sera were prepared and tested using
a commercial competitive inhibition enzyme immunoassay
(ETI-AB-HAVK-3 [anti-HAV], Sorin BioMedica, Italy) designed for
measuring total HAV-specific antibody in human serum with a
sensitivity of <20 WHO mIU/mL As the assay is a competitive
inhibition method, in which the antibody to be tested competitively
inhibits binding of HAV-specific antibody provided with the kit,
the assay was used to determine if any of the mice had produced
HAV-specific IgG capable of inhibiting the binding of the assay
reagent. While none of the animals tested was definitively positive
for HAV-specific IgG by this method, two of the mice immunized with
HAV/IFA via the IP route had borderline levels of specific IgG.
[0084] As these negative results did not preclude the possibility
that the mice may have responded in the mucosal immune compartment,
the animals were subsequently randomized among the treatments and
tested in two groups. One group was killed in subgroups of 6-8 mice
over a period spanning 7-28 days after the last immunization.
Spleens and Peyer's patches were harvested and cell populations
were prepared for stimulation in vitro with HAV or control antigens
(see below) and evaluation of the HAV-specific IgA response using
ELISPOT and EIA, and HAV-specific proliferation indices as
described in detail below. The second group of mice was allowed to
rest an additional three weeks, and then they received a single
intradermal (ID) boost of HAV antigen (27.5 .mu.g of total protein
or 0.6 EU). These mice were killed as groups of 6-8 over a period
of 7-21 days. This latter group was designated as "in vivo boosted"
in contrast to the former group whose cells were restimulated with
HAV antigen in vitro.
[0085] Preparation of Serum, Spleen Cell and Peyer's Patch Cell
Populations:
[0086] Animals were killed by an overdose of chloral hydrate. Blood
was collected by cardiac puncture, allowed to clot overnight at 4
C. Serum was prepared and kept frozen at -20.degree. C. until
tested. The spleen and Peyer's patches (approx. 4-8 per animal)
were removed into sterile RPMI-1640 medium (GIBCO/BRL, Biological
Industries, Beit Haemek, Israel) containing antibiotics
(penicillin, 100 U/mL and streptomycin, 100 micrograms per mL,
GIBCO/BRL) and transported to the laboratory for further
processing. The organs were washed once in sterile RPMI. Spleen
cell (SPLC) suspensions were prepared by teasing apart the spleen
in 5 mL of RPMI+antibiotics using sterile forceps. The suspension
was transferred into a sterile conical bottomed 15 mL tube and the
tissue debris allowed to settle. The supernatant containing
suspended SPLC was transferred into a fresh tube and the cells were
pelleted by centrifugation (1500 rpm, 7 min) at room temperature.
The cells were washed twice by centrifugation (1500 rpm, 5 min)
through RPMI+antibiotics then viable cell numbers were determined
by Trypan Blue exclusion using a Neubauer hemocytometer. The SPLC
were then resuspended in 10 mL of the same medium containing 10%
fetal calf serum (FCS, HyClone Laboratories, Tarom Applied
Technologies, Petah Tikvah, Israel) at a concentration of
1.times.10.sup.6 cells/mL for use in subsequent assays. Similarly,
Peyer's patches were teased apart in 5 mL of RPMI+antibiotics
containing 1.5 mg/mL of Dispase (Sigma-Aldrich, Milwaukee, Wis.).
The Peyer's patch cell (PPC) suspension was transferred into a
conical-bottomed 15 mL tube and incubated with shaking for 45-60
min at 37.degree. C. to dissociate lymphoid cells from the tissue
stroma. The PPC were then pelleted by centrifugation and washed
twice through RPMI+antibiotics and numbers of viable cells were
determined as described above. As PPC numbers were usually limited,
the final suspensions in 4.5 mL of RPMI containing antibiotics and
10% FCS, whose densities ranged from 1.times.10.sup.4 to
5.times.10.sup.5 cells/mL, were used in assays without further
adjustment.
[0087] Determination of Capacity of SPLC and PPC to Synthesize and
Secrete HAV-Specific IgA Antibodies in vitro and Determination of
Proliferative Response to HAV Antigens:
[0088] After determination of cell densities, SPLC and PPC
preparations from each mouse were pipetted in 1-mL aliquots into 5
wells of flat-bottomed sterile 24-well tissue culture plates. For
each cell population, separate wells received: medium only
(unstimulated negative control); pokeweed mitogen (Sigma) at a
final concentration of 10 micrograms per mL and HAV antigen
(Microbix) diluted to give final concentrations per well of 11,
5.5, 2.8, and 1.4 micrograms per mL. The plates were incubated for
5 days at 37.degree. C., 5% CO.sub.2 in air. At the end of this
culture period, 0.7 mL of supernatant was removed from each well
and stored at -20.degree. C. for determination of HAV-specific IgA
by enzyme immunoassay (EIA, see below). For assays in which
HAV-specific IgA forming cells were evaluated after in vitro
stimulation the cells were treated as follows. Cells from each well
were harvested into conical-bottomed tubes, washed once by
centrifugation through RPMI+antibiotics and resuspended in 1 ml of
RPMI containing antibiotics and 10% FCS. Viable cell numbers after
in vitro stimulation were determined by Trypan Blue exclusion and
related to cell densities in the initial cell population to give a
stimulation index (SI). SI's.gtoreq.2.0 were considered a positive
response to stimulation.
[0089] ELISPOT Assays to Determine Frequencies of IgA Antibody
Forming Cells (AFC) in Systemic (SPLC) and Mucosal (PPC) Immune
Compartments:
[0090] To detect cells producing specific IgA antibodies in both
the spleen and Peyer's patches, an ELISPOT assay was adapted from a
standard protocol Lewis D J M & Hayward C M M, Stimulation of
Mucosal Immunity. In: Vaccine Protocols; A. Robinson, G H Farrar, C
N Wiblin, Eds., Humana Press, Totowa, N.J., 1996, pp 187-195). The
principle of this assay is that cells forming specific IgA will
secrete it onto the surface of antigen-coated microplate wells,
leaving an "imprint" which may be detected by conventional
enzyme-linked immunosorbent assay (ELISA) techniques. As
nonspecific background reactivity is inherent in the ELISPOT
method, all assays contained controls for reactivity with FRhK4
antigens (present in the original immunogen) and nonspecific
binding to other assay constituents (coating buffer control). Wells
of 96-well polystyrene tissue culture plates (Costar 3596, Corning
Inc., Corning, N.Y.) were coated overnight with HAV antigen
(Microbix), or FRhK4 lysate, (prepared by freeze thawing uninfected
cells and clarifying the supernatant by centrifugation at 15,000
rpm for 20 min), diluted to 20 micrograms per mL of protein in
standard ELISA coating buffer (HCO.sub.2/CO.sub.3.sup.2-, pH 9.6).
Negative control wells received coating buffer only. Coating volume
was 100 microliters per well and duplicate wells were coated for
each antigen or control.
[0091] The following day the antigens were discarded and the wells
were washed 4 times with sterile phosphate buffered saline (PBS, pH
7.4). The wells were subsequently blocked for 2-4 h at room
temperature with 5% FCS in sterile PBS (250 microliters per well).
The blocking solution was discarded and SPLC or PPC suspensions
were added to the wells and the plates were incubated for 24 h at
37.degree. C. in 5% CO.sub.2 in air, taking care to keep the plates
level and undisturbed during this period. SPLC were incubated at a
density of 100,000 cells/well in a volume of 100 microliters per
well while PPC were used at densities of 1000-50,000 cells/well
(100 microliters per well) according to their yields. At the end of
the incubation period the cells were discarded and the microplate
wells were washed 5 times in PBS containing 0.05% Tween 20 (PBST).
To detect spots where antigen-specific IgA-forming cells (IgA-AFC)
had secreted their antibodies into the antigen-coated microplate
wells, goat-anti-mouse IgA (affinity purified from Kirkegaard &
Perry Labs [KPL], Gaithersburg, Md.) diluted 1:500 in PBST
containing 5% normal rabbit serum (NRS, Biological Industries, Beit
Haemek, Israel), 100 microliters per well was added and the
microplates were incubated overnight at 4.degree. C. The antibody
was discarded and the wells were washed 5 times with PBST. Next,
alkaline phosphatase-conjugated rabbit anti-goat IgG
(affinity-purified, KPL) diluted to 1:1000 in PBST containing 5%
NRS, 100 microliters per well, was added and the microplates were
incubated for 2 h at room temperature. The antibody solution was
discarded and the microplates were washed 5 times with PBST. To
detect spots representing single AFC, alkaline phosphatase
substrate (BCIP/NBT SigmaFast, Sigma) prepared according to the
manufacturer's directions, was added (100 microliters per well),
and the enzymatic reaction was allowed to proceed at room
temperature in the dark for 45 min before stopping it by rinsing
the plates 3 times in distilled water. The plates were covered with
aluminum foil and stored at 4.degree. C. until AFC counting. Spots
were enumerated using an inverted microscope with 400.times.
magnification. Spots in duplicate wells for HAV and control
antigens were counted and averaged. To calculate the net number of
HAV-specific AFCs, the number of spots in counted the control wells
were averaged then this average was subtracted from the averaged
number of spots calculated in wells coated with HAV antigen. The
net HAV-specific spot (AFC) number was reported (Tables 3-8,
below).
[0092] EIA for Detection of HAV-Specific Antibodies in Mouse Serum
and Tissue Culture Supernatants:
[0093] During the course of these experiments, preliminary
development of an enzyme immunoassay (EIA) for detecting
HAV-specific IgA antibodies in mouse serum or in tissue culture
supernatants was undertaken. Wells of polystyrene EIA microplates
(Nunc Immunopolysorp, Fisher Scientific, Israel) were coated
overnight at 4.degree. C. with HAV antigen (Microbix) or FRhK4
antigen, diluted to 20 micrograms per mL of protein in
HCO.sub.2.sup.-/CO.sub.3.sup.2-, pH 9.6 coating buffer as described
above (100 microliters per well, in duplicates). Negative control
wells received coating buffer only. The antigen or control
solutions were discarded and the wells were washed three times in
PBS containing 0.05% Tween 20 (PBST). Wells were subsequently
blocked for 1 h at room temperature with 250 microliters of
blocking buffer (5% NRS in PBST). The blocking solution was
discarded and the wells were washed 5 times with PBST. Tissue
culture fluids to be tested for in vitro production of HAV-specific
IgA or dilutions of mouse serum from immunized animals (1:20 and
1:200 in blocking buffer) were subsequently added (100 microliters
per well, single determinations for each antigen and negative
control) and the plates were incubated overnight at 4.degree. C.
These solutions were discarded and the wells washed 5 times in PBST
as before. Next, goat anti-mouse IgA (KPL) diluted to 1:500 in
blocking buffer (100 microliters per well) was added and the plates
were incubated for 2 h at room temperature. The wells were emptied
and washed 5 times with PBST. Alkaline phosphatase (AP)-conjugated
goat anti-rabbit IgG (KPL, 1:1000 dilution in blocking buffer, 100
microliters per well) was added and the plates were incubated for a
further 2 h at room temperature. After discarding the AP conjugate
and washing the wells 5 times with PBST, AP substrate (SigmaFast
NBT substrate, Sigma, prepared according to the manufacturer's
directions was added and the plate was incubated for 1 h at room
temperature. Absorbance at 405 nm (A.sub.405) determined using an
EIA microplate reader (Tecan Spectra Rainbow). For each animal and
treatment, results were expressed as a signal-to-noise (S/N) ratio
obtained by dividing the A.sub.405 determined with antigen (either
HAV or FRhK4) by the A.sub.405 recorded in wells receiving coating
buffer only. S/N ratios.gtoreq.2.0 were considered positive.
[0094] Lymphocyte Stimulation Assays:
[0095] To assess lymphocyte recognition of HAV antigens in both the
systemic (represented by SPLC) and gut mucosal (represented by PPC)
immune compartments, in vitro lymphocyte proliferation assays were
performed. Proliferation assays were performed as follows for
experiments where the response to in vivo boosting with HAV was
evaluated. SPLC and PPC (when in sufficient quantity) were adjusted
to 1.times.10.sup.6 cells/mL in RPMI containing antibiotics and 10%
FCS. Proliferation assays were performed in flat-bottomed 96-well
tissue culture plates. Each well received 100 microliters of cell
suspension, 100 microliters of medium (as above), and 10
microliters of one of the following: medium only (unstimulated
control); phytohemagglutinin (Sigma) prepared to give a final well
concentration of 10 micrograms per mL; or HAV antigen (Microbix)
diluted to give final well concentrations of 11, 5.5, 2.8, and 1.4
micrograms per mL. All concentrations are expressed as micrograms
of protein/mL. Duplicate wells were set up for each stimulant. The
plates were incubated for 5 days at 37.degree. C. in 5% CO.sub.2 in
air. To determine the extent of cellular proliferation, 2 .mu.Ci of
.sup.3H thymidine (Amersham, Israel) was added to each well during
the last 18-24 hours of culture. The cells were harvested by
hypotonic lysis and their DNA collected onto glass fiber filters
for determination of incorporated radioactivity by liquid
scintillation counting. Stimulation indices (SI) were determined by
dividing the averaged (over duplicates) cpm obtained for the
stimulated wells by the averaged cpm obtained for the negative
control (medium only) wells. SI's.gtoreq.2.0 were considered
positive.
3TABLE 3 Results of Bulk Culture Experiments: Mice immunized with
HAV intraperitoneally (IP). LST EIA ELISPOT Immunization
Stimulation Specific IgA No.IgA-AFC Protocol/ Index (SI).sup.b
(S/N).sup.c (Mean.sup.d) Stimulant.sup.a SPLC PPC SPLC PPC SPLC PPC
HAV/IFA IP #1: None -- -- 1.7 2.1 0 0 Mitogen (10 .mu.g/mL).sup.e
0.5 1.9 1.7 1.8 1.5 0 HAV (11 .mu.g/mL) 0.5 2.3 2.3 2.0 10.1 1.5
HAV (5.5 .mu.g/mL) 0.5 0.8 1.7 1.5 17 0 HAV (2.8 .mu.g/mL) 0.7 2.6
1.8 1.6 0 0 HAV (1.4 .mu.g/mL) ND ND ND ND ND ND HAV/IFA IP #2:
None -- -- 1.5 1.4 5 0 Mitogen (10 .mu.g/mL).sup.e 1.3 1.6 1.5 1.0
0 0 HAV (11 .mu.g/mL) 2.3 1.3 1.6 1.6 25.5 0.5 HAV (5.5 .mu.g/mL)
1.5 2.2 1.5 1.3 11.2 0 HAV (2.8 .mu.g/mL) 1.0 1.9 2.0 1.6 21.7 0
HAV (1.4 .mu.g/mL) ND ND ND ND ND ND Footnotes to Table 3:
.sup.aImmunization protocol (for animal) and stimulant used (final
concentration) in bulk culture for 5-6 days before testing.
.sup.bStimulation index (SI) determined from number of viable
cells/mL (as determined by Trypan Blue exclusion hemocytometer
counts) in stimulated wells divided by the number of viable
cells/mL in unstimulated (medium only) wells. SI .gtoreq. 2.0
considered positive. .sup.cHAV-specific IgA S/N (signal;noise
ratio) as determined using EIA by dividing A405 nm measured in
wells coated with HAV antigen by A405 nm measured in wells
receiving coating buffer only. S/N .gtoreq. 2.0 considered
positive. .sup.dNo. HAV-specific IgA-AFC: average no.spots counted
in wells coated with HAV above averaged background of spots counted
in wells coated with FRhK4 antigen or coating buffer only. .sup.eIn
bulk culture experiments mitogen was pokeweed mitogen (PWM); in
proliferation assays involving mice boosted in vivo by an
additional ID injection of HAV, the mitogen was phytohemagglutinin
(PHA). SPLC = spleen cells PPC = Peyer's patch cells
[0096]
4TABLE 4 Results of Bulk Culture Experiments: Mucosally-immunized
mice. LST EIA ELISPOT Immunization Stimulation Specific IgA
No.IgA-AFC Protocol/ Index (SI).sup.b (S/N).sup.c (Mean.sup.d)
Stimulant.sup.a SPLC PPC SPLC PPC SPLC PPC HAV IR #1: None -- --
1.4 1.1 0 0 Mitogen (10 .mu.g/mL).sup.e 1.1 0.3 1.3 2.3 0 0 HAV (11
.mu.g/mL) 1.2 0.6 1.6 1.8 43 0 HAV (5.5 .mu.g/mL) 1.0 1.2 1.4 1.7
31.3 0 HAV (2.8 .mu.g/mL) 2.2 1.3 1.4 1.5 16.5 0 HAY (1.4 .mu.g/mL)
ND ND ND ND ND ND HAV IR #2: None -- -- 1.3 1.5 65.7 0 Mitogen (10
.mu.g/mL).sup.e 0.1 3.0 1.1 1.6 0 0 HAV (11 .mu.g/mL) 1.3 8.0 1.4
1.5 20.2 0 HAV (5.5 .mu.g/mL) 1.0 5.4 1.5 1.7 39 0 HAV (2.8
.mu.g/mL) 1.0 6.1 1.4 2.2 55.2 0 HAV (1.4 .mu.g/mL) ND ND ND ND ND
ND HAV IR #3: None -- -- 1.0 1.1 0 0 Mitogen (10 .mu.g/mL).sup.e
0.1 2.0 0.9 1.9 0 0 HAV (11 .mu.g/mL) 1.1 5.0 0.9 1.3 0 0 HAV (5.5
.mu.g/mL) 1.2 3.0 2.0 1.6 0 0 HAV (2.8 .mu.g/mL) 0.4 3.9 1.1 1.7 17
0 HAV (1.4 .mu.g/mL) ND ND ND ND ND ND HAV IR #4: None -- -- 1.0
1.0 1.5 0 Mitogen (10 .mu.g/mL).sup.e 1.1 1.7 1.3 1.4 0 0 HAV (11
.mu.g/mL) ND ND ND ND ND ND HAV (5.5 .mu.g/mL) 1.2 2.4 1.4 1.4 2 0
HAV (2.8 .mu.g/mL) ND ND ND ND ND ND HAV (1.4 .mu.g/mL) ND ND ND ND
ND ND HAV IR #5: None -- -- 1.4 1.5 0.5 0 Mitogen (10
.mu.g/mL).sup.e 0.8 1.4 1.3 1.4 0 0 HAV (11 .mu.g/mL) ND ND ND ND
ND ND HAV (5.5 .mu.g/mL) 1.1 1.6 1.4 1.4 0 0 HAV (2.8 .mu.g/mL) ND
ND ND ND ND ND HAV (1.4 .mu.g/mL) ND ND ND ND ND ND HAV PO: None --
-- 1.4 1.5 24 0 Mitogen (10 .mu.g/mL).sup.e 0.4 4.2 1.6 0.9 0 0 HAV
(11 .mu.g/mL) ND ND ND ND ND ND HAV (5.5 .mu.g/mL) 1.2 1.7 1.5 1.4
17 0 HAV (2.8 .mu.g/mL) ND ND ND ND ND ND HAV (1.4 .mu.g/mL) ND ND
ND ND ND ND .sup.aImmunization protocol (for animal) and stimulant
used (final concentration) in bulk culture for 5-6 days before
testing. .sup.bStimulation index (SI) determined from no. of viable
cells/mL (as determined by Trypan Blue exclusion hemocytometer
counts) in stimulated wells divided by the number of viable
cells/mL in unstimulated (medium only) wells. SI .gtoreq. 2.0
considered positive. .sup.cHAV-specific IgA S/N (signal:noise
ratio) as determined using EIA by dividing A405 nm measured in
wells coated with HAV antigen by A405 nm measured in wells
receiving coating buffer only. S/N .gtoreq. 2.0 considered
positive. .sup.dNo.HAV-specific IgA-AFC: average no.spots counted
in wells coated with HAV above averaged background of spots counted
in wells coated with FRhK4 antigen or coating buffer only. .sup.eIn
bulk culture experiments mitogen was pokeweed mitogen (PWM); in
proliferation assays involving mice boosted in vivo by an
additional ID injection of HAV, the mitogen was phytohemagglutinin
(PHA).
[0097]
5TABLE 5 Results of Bulk Culture Experiments: Negative Controls LST
EIA ELISPOT Immunization Stimulation Specific IgA No.IgA-AFC
Protocol/ Index (SI).sup.b (S/N).sup.c (Mean.sup.d) Stimulant.sup.a
SPLC PPC SPLC PPC SPLC PPC Saline/IFA IP: None -- -- 1.8 1.5 0 0
Mitogen (10 .mu.g/mL).sup.e 0.3 1.0 1.7 1.5 0 0 HAV (11 .mu.g/mL)
1.6 1.1 1.7 1.5 4.2 0 HAV (5.5 .mu.g/mL) 0.6 2.0 1.6 1.4 1.8 0 HAV
(2.8 .mu.g/mL) 0.8 1.2 1.5 1.4 0 0 HAV (1.4 .mu.g/mL) ND ND ND ND
ND ND Saline PO: None -- -- 1.4 1.5 0 0 Mitogen (10 .mu.g/mL).sup.e
1.0 2.3 1.6 0.9 0 0 HAV (11 .mu.g/mL) ND ND ND ND ND ND HAV (5.5
.mu.g/mL) 0.9 1.3 1.5 1.4 0 0 HAV (2.8 .mu.g/mL) ND ND ND ND ND ND
HAV (1.4 .mu.g/mL) ND ND ND ND ND ND .sup.aImmunization protocol
(for animal) and stimulant used (final concentration) in bulk
culture for 5-6 days before testing. .sup.bStimulation index (SI)
determined from no. of viable cells/mL (as determined by Trypan
Blue exclusion hemocytometer counts) in stimulated wells divided by
the no viable cells/mL in unstimulated (medium only) wells. SI
.gtoreq. 2.0 considered positive. .sup.cHAV-specific IgA S/N
(signal:noise ratio) as determined using EIA by dividing A 405 nm
measured in wells coated with HAV antigen by A405 nm measured in
wells receiving coating buffer only. S/N .gtoreq. 2.0 considered
positive. .sup.dNo. HAV-specific IgA-AFC: average no. spots counted
in wells coated with HAV above averaged background of spots counted
in wells coated with FRhK4 antigen or coating buffer only. .sup.eIn
bulk culture experiments mitogen was pokeweed mitogen (PWM); in
proliferation assays involving mice boosted in vivo by an
additional ID injection of HAV, the mitogen was phytohemagglutinin
(PHA).
[0098]
6TABLE 6 Results of Experiments Following Boosting of Immunity to
HAV in mice primed via the IP route. LST EIA ELISPOT Immunization
Stimulation Specific IgA No.IgA-AFC Protocol/ Index (SI).sup.b
(S/N).sup.c (Mean.sup.d) Stimulant.sup.a SPLC PPC SPLC PPC SPLC PPC
HAV/IFA IP #1: None -- -- 4.4 ND 12.3 2.5 Mitogen (10 .mu.g/mL) 2.6
1.4 3.0 ND ND ND HAV (11 .mu.g/mL) 2.0 0.8 2.5 ND ND ND HAV (5.5
.mu.g/mL) 0.7 1.2 2.5 ND ND ND HAV (2.8 .mu.g/mL) 0.9 1.1 3.5 ND ND
ND HAV (1.4 .mu.g/mL) 1.2 1.8 2.2 ND ND ND HAV/IFA IP #2: None --
-- 3.2 ND 0 7.5 Mitogen (10 .mu.g/mL).sup.e 1.3 2.8 2.2 ND ND ND
HAV (11 .mu.g/mL) 0.9 2.4 1.7 ND ND ND HAV (5.5 .mu.g/mL) 1.4 3.0
1.7 ND ND ND HAV (2.8 .mu.g/mL) 1.1 2.7 1 6 ND ND ND HAV (1.4
.mu.g/mL) 1.2 4.2 1.6 ND ND ND .sup.aImmunization protocol by
animal. Animals were boosted ID with 27.5 micrograms of HAV protein
(Microbix HAV antigen) 10-28 days before testing. .sup.bStimulation
index (SI) determined from .sup.3H-TdR incorporation experiments:
no.cpm incorporated into cells in stimulated wells divided by the
no.cpm incorporated into cells in unstimulated (medium only) wells.
Mitogen used was PHA. SI .gtoreq. 2.0 considered positive.
.sup.cHAV-specific IgA S/N (signal:noise ratio) as determined using
EIA by dividing A405 mn measured in wells coated with HAV antigen
by A405 nm measured in wells receiving coating buffer only. Mitogen
used was PWM. S/N .gtoreq. 2.0 considered positive. .sup.dNo.
HAV-specific IgA-AFC: average no.spots counted in wells coated with
HAV above averaged background of spots counted in wells coated with
FRhK4 antigen or coating buffer only.
[0099]
7TABLE 7 Results of Experiments Following Boosting of Immunity to
HAV: Mucosally-Immunized Mice. LST EIA ELISPOT Immunization
Stimulation Specific IgA No.IgA-AFC Protocol/ Index (SI).sup.b
(S/N).sup.c (Mean.sup.d) Stimulant.sup.a SPLC PPC SPLC PPC SPLC PPC
HAV IR #1: None -- -- 2.0 ND 2.0 1.5 Mitogen (10 .mu.g/mL) 0.9 4.5
1.7 ND ND ND HAV (11 .mu.g/mL) 0.6 2.1 1.1 ND ND ND HAV (5.5
.mu.g/mL) 0.8 3.0 1.3 ND ND ND HAV (2.8 .mu.g/mL) 1.1 3.9 1.1 ND ND
ND HAV (1.4 .mu.g/mL) 0.6 4.1 1.2 ND ND ND HAV IR #2.: None -- --
1.1 ND 0 0.3 Mitogen (10 .mu.g/mL).sup.c 1.1 4.1 1.3 ND ND ND HAV
(11 .mu.g/mL) 1.0 0.8 1.2 ND ND ND HAV (5.5 .mu.g/mL) 0.9 1.2 1.3
ND ND ND HAV (2.8 .mu.g/mL) 0.7 0.6 1.0 ND ND ND HAV (1.4 .mu.g/mL)
0.7 1.0 1.5 ND ND ND HAV IR #3.: None -- -- 1.5 ND 4.8 0 Mitogen
(10 .mu.g/mL).sup.e 2.1 0.5 1.5 ND ND ND HAV (11 .mu.g/mL) 2.0 0.5
1.5 ND ND ND HAV (5.5 .mu.g/mL) 2.3 0.7 1.3 ND ND ND HAV (28
.mu.g/mL) 8.0 1.1 1.3 ND ND ND HAV (1.4 .mu.g/mL) 2.2 1.3 1.3 ND ND
ND HAV IR #4.: None -- -- 1.2 ND 1.0 2 Mitogen (10 .mu.g/mL).sup.e
1.7 2.4 1.3 ND ND ND HAV (11 .mu.g/mL) 2.4 1.0 1.3 ND ND ND HAV
(5.5 .mu.g/mL) 1.7 1.2 1.3 ND ND ND HAV (2.8 .mu.g/mL) 8.0 1.1 1.3
ND ND ND HAV (1.4 .mu.g/mL) 1.7 1.4 1.2 ND ND ND HAV IR #5.: None
-- -- 1.0 ND 0 0 Mitogen (10 .mu.g/mL).sup.e 1.8 0.7 1.1 ND ND ND
HAV (11 .mu.g/mL) 1.2 0.5 1.8 ND ND ND HAV (5.5 .mu.g/mL) 1.1 0.8
1.5 ND ND ND HAV (2.8 .mu.g/mL) 1.1 0.8 1.8 ND ND ND HAV (1.4
.mu.g/mL) 0.9 0.8 1.3 ND ND ND HAV IR #6: None -- -- 1.3 ND 0 0
Mitogen (10 .mu.g/mL) 0.6 0.7 1.0 ND ND ND HAV (11 .mu.g/mL) 0.6
0.8 1.3 ND ND ND HAV (5.5 .mu.g/mL) 1.0 0.7 1.4 ND ND ND HAV (2.8
.mu.g/mL) 0.7 0.9 1.4 ND ND ND HAV (1.4 .mu.g/mL) 0.7 0.7 1.2 ND ND
ND HAV IR #7: None -- -- 2.7 ND 5.8 0.5 Mitogen (10 .mu.g/mL) 2.0
0.8 2.6 ND ND ND HAV (11 .mu.g/mL) 1.6 1.1 2.7 ND ND ND HAV (5.5
.mu.g/mL) 2.3 0.9 2.9 ND ND ND HAV (2.8 .mu.g/mL) 1.6 0.9 2.7 ND ND
ND HAV (1.4 .mu.g/mL) 1.0 1.1 2.6 ND ND ND HAV IR #8: None -- --
2.4 ND 2.5 7.5 Mitogen (10 .mu.g/mL) 1.2 1.8 2.8 ND ND ND HAV (11
.mu.g/mL) 1.2 1.9 2.4 ND ND ND HAV (5.5 .mu.g/mL) 1.5 1.7 2.7 ND ND
ND HAV (2.8 .mu.g/mL) 1.0 3.1 2.8 ND ND ND HAV (1.4 .mu.g/mL) 1.3
2.8 2.7 ND ND ND HAV IR #9: None -- -- 2.8 ND 1.3 2.0 Mitogen (10
.mu.g/mL) 0.8 3.3 2.8 ND ND ND HAV (11 .mu.g/mL) 0.8 3.1 2.8 ND ND
ND HAV (5.5 .mu.g/mL) 0.8 2.7 2.9 ND ND ND HAV (2.8 .mu.g/mL) 0.8
3.1 3.6 ND ND ND HAV (1.4 .mu.g/mL) 0.5 2.8 3.0 ND ND ND HAV IR
#10: None -- -- 2.9 ND 0.5 4.5 Mitogen (10 .mu.g/mL) 1.3 1.7 2.6 ND
ND ND HAV (11 .mu.g/mL) 1.2 1.1 2.8 ND ND ND HAV (5.5 .mu.g/mL) 1.4
1.8 2.5 ND ND ND HAV (2.8 .mu.g/mL) 1.2 1.5 2.9 ND ND ND HAV (1.4
.mu.g/mL) 1.3 1.5 2.9 ND ND ND HAV PO#1: None -- -- 3.6 ND 1.5 7.8
Mitogen (10 .mu.g/mL) 1.2 2.9 2.9 ND ND ND HAV (11 .mu.g/mL) 0.9
11.6 2.6 ND ND ND HAV (5.5 .mu.g/mL) 1.1 1.1 2.6 ND ND ND HAV (2.8
.mu.g/mL) 0.7 0.9 2.5 ND ND ND HAV (1.4 .mu.g/mL) 1.2 1.4 3.1 ND ND
ND HAV PO#2: None -- -- 2.8 ND 2.3 11.8 Mitogen (10 .mu.g/mL) 1.5
4.1 3.3 ND ND ND HAV (11 .mu.g/mL) 1.2 1.7 2.7 ND ND ND HAV (5.5
.mu.g/mL) 1.8 1.9 3.2 ND ND ND HAV (2.8 .mu.g/mL) 0.9 2.0 2.7 ND ND
ND HAV (1.4 .mu.g/mL) 1.1 2.2 3.0 ND ND ND T12-2 PO #1.: None -- --
2.6 ND 5.0 6.0 Mitogen (10 .mu.g/mL) 0.1 1.6 3.2 ND ND ND HAV (11
.mu.g/mL) 2.4 1.5 3.2 ND ND ND HAV (5.5 .mu.g/mL) 2.5 1.0 3.0 ND ND
ND HAV (2.8 .mu.g/mL) 1.5 1.3 3.1 ND ND ND HAV (1.4 .mu.g/mL) 2.0
1.9 3.1 ND ND ND T12-2 PO #2.: None -- -- 2.8 ND 5.0 3.0 Mitogen
(10 .mu.g/mL) 1.1 0.9 0.7 ND ND ND HAV (11 .mu.g/mL) 1.0 0.8 2.5 ND
ND ND HAV (5.5 .mu.g/mL) 1.3 0.8 3.1 ND ND ND HAV (2.8 .mu.g/mL)
1.0 1.1 3.0 ND ND ND HAV (1.4 .mu.g/mL) 2.1 1.2 3.2 ND ND ND T12-2
PO #3.: None -- -- 2.3 ND 4.5 1.0 Mitogen (10 .mu.g/mL) 1.1 2.3 2.3
ND ND ND HAV (11 .mu.g/mL) 1.1 1.2 2.3 ND ND ND HAV (5.5 .mu.g/mL)
1.0 1.6 3.9 ND ND ND HAV (2.8 .mu.g/mL) 0.6 1.0 7.2 ND ND ND HAV
(1.4 .mu.g/mL) 0.9 2.3 2.1 ND ND ND T12-3 PO #1: None -- -- 2.6 ND
2.0 0 Mitogen (10 .mu.g/mL) 1.5 1.6 2.4 ND ND ND HAV (11 .mu.g/mL)
0.9 1.8 2.6 ND ND ND HAV (5.5 .mu.g/mL) 1.1 1.2 2.0 ND ND ND HAV
(2.8 .mu.g/mL) 0.5 1.0 1.9 ND ND ND HAV (1.4 .mu.g/mL) 0.5 1.3 1.8
ND ND ND T12-3 PO #2. None -- -- 0.5 ND 4.3 2.8 Mitogen (10
.mu.g/mL) 0.8 2.2 2.1 ND ND ND HAV (11 .mu.g/mL) 2.2 2.1 1.6 ND ND
ND HAV (5.5 .mu.g/mL) 2.1 2.2 2.2 ND ND ND HAV (2.8 .mu.g/mL) 0.5
2.7 2.3 ND ND ND HAV (1.4 .mu.g/mL) 0.7 2.0 1.8 ND ND ND T12-3 PO
#3: None -- -- 2.0 ND 2.0 0.8 Mitogen (10 .mu.g/mL) 1.1 1.6 1.5 ND
ND ND HAV (11 .mu.g/mL) 1.6 1.6 2.1 ND ND ND HAV (5.5 .mu.g/mL) 1.7
1.1 2.0 ND ND ND HAV (2.8 .mu.g/mL) 1.2 1.2 2.1 ND ND ND HAV (1.4
.mu.g/mL) 1.0 1.3 2.1 ND ND ND .sup.aImmunization protocol by
animal. Animals were also boosted ID with 27.5 micrograms of HAV
protein (Microbix HAV antigen) 10-28 days before testing.
.sup.bStimulation index (SI) determined from .sup.3H-TdR
incorporation experiments: no. of cpm incorporated into cells in
stimulated wells divided by the no. of cpm incorporated into cells
in unstimulated (medium only) wells. Mitogen used was PHA. SI
.gtoreq. 2.0 considered positive. .sup.cHAV-specific IgA S/N
(signal:noise ratio) as determined using EIA by dividing A405 nm
measured in wells coated with HAV antigen by A405 nm measured in
wells receiving coating buffer only. Mitogen used was PWM. S/N
.gtoreq. 2.0 considered positive. .sup.dNo. of HAV-specific
IgA-AFC: average no.spots counted in wells coated with HAV above
averaged background of spots counted in wells coated with FRhK4
antigen or coating buffer only.
[0100]
8TABLE 8 Results: Negative Controls (not boosted). LST EIA ELISPOT
Immunization Stimulation Specific IgA No.IgA-AFC Protocol/ Index
(SI).sup.b (S/N).sup.c (Mean.sup.d) Stimulant.sup.a SPLC PPC SPLC
PPC SPLC PPC Saline PO: None -- -- 1.6 ND Mitogen (10 .mu.g/mL) 1.1
2.4 2.6 ND ND ND HAV (11 .mu.g/mL) 2.0 1.9 1.9 ND ND ND HAV (5.5
.mu.g/mL) 1.1 1.5 2.2 ND ND ND HAV (2.8 .mu.g/mL) 0.3 0.9 1.9 ND ND
ND HAV (1.4 .mu.g/mL) 0.9 1.8 1.0 ND ND ND Saline IR#1: None -- --
2.0 ND Mitogen (10 .mu.g/mL) 1.3 1.5 2.4 ND ND ND HAV (11 .mu.g/mL)
1.4 1.0 2.3 ND ND ND HAV (5.5 .mu.g/mL) 1.7 1.1 3.0 ND ND ND HAV
(2.8 .mu.g/mL) 1.1 1.1 3.6 ND ND ND HAV (1.4 .mu.g/mL) 0.8 1.4 2.1
ND ND ND Saline IR #2: None -- -- 2.2 ND Mitogen (10 .mu.g/mL) 0.9
1.0 2.7 ND ND ND HAV (11 .mu.g/mL) 1.4 0.8 2.1 ND ND ND HAV (5.5
.mu.g/mL) 1.2 1.1 2.1 ND ND ND HAV (2.8 .mu.g/mL) 0.9 1.0 2.0 ND ND
ND HAV (1.4 .mu.g/mL) 1.1 1.1 1.8 ND ND ND .sup.a-dSee footnotes to
Table 7. Animals were not boosted with HAV.
[0101] Summary and Conclusions:
[0102] These table results are summarized as follows. The hepatitis
A viral (HAV) antigen (purchased from Microbix as a semi-purified
preparation grown in FRhK4 monkey kidney cells) is immunogenic in
mice when delivered by the intraperitoneal (IP) route emulsified
with incomplete Freund's adjuvant (IFA); intrarectally (IR) by
direct application or orally (PO) by feeding. HAV antigen given by
IP, IR, or PO routes generates HAV-responsive (memory) lymphocytes
in both the spleen cell (SPLC) and Peyer's patch cell (PPC)
populations that can be detected by in vitro proliferation assays
or after in vivo boosting with HAV. In mice immunized via the
mucosal (IR or PO) routes the proliferative response appears to be
more prevalent and more vigorous in the PPC population than in the
SPLC population, indicating that mucosal routes of immunization are
effective at eliciting local (intestinal) immune responses. HAV
antigen given by the IP, IR or PO routes also gives rise to
HAV-specific IgA forming cells in both the SPLC and PPC populations
which can be detected by ELISPOT assays either directly (after
immunization) or indirectly (after in vitro boosting in bulk
culture--data not shown). However, cells forming IgA that was
reactive with FRhK4 antigens as well as other assay constituents
are also detectable by ELISPOT assays. Therefore, results have been
expressed as the net number of positive (HAV-specific) spots after
subtraction of this background reactivity. HAV-specific cells
present in the SPLC and PPC populations can be induced to
synthesize and secrete HAV-specific IgA detectable by direct enzyme
immunoassay (EIA) after in vitro stimulation in bulk culture with
pokeweed mitogen (PWM or HAV antigen at various concentrations. Two
transgenic tomato vaccines given orally also appeared to be
immunogenic in that they provoked immunologic memory lymphocytes in
the spleen and Peyer's patches that were detectable by
proliferation assays.
[0103] 3.2.2. Further Examination of the Mucosal Immune Response to
HAVRIX.TM. and the Effect of Dose on the Response to Rectal
Immunization:
[0104] To determine the effect of HAVRIX.TM. dose on the intestinal
mucosal and systemic immune responses to HAV, and to determine if
rectal immunization induces immunologic memory to HAV, the
following experiment was performed. In this experiment the
methodology to assess the mucosal immune response was further
refined to increase the sensitivity of detection of the immune
response. This experiment compared the immune responses in both
systemic and mucosal compartments to intrarectal (IR) immunization
and immunization via conventional intraperitoneal (IP) routes.
Immune responses were evaluated in systemic (spleen cell=SPLC) and
gut mucosal (Peyer's patch cell=PPC, lamina propria lymphocyte=LPL)
cell populations by three methods that were modified from the
procedures described above:
[0105] 1. HAV-specific ELISPOT assays measuring the production of
IgG, IgM, and IgA class specific antibody forming cells (AFC).
[0106] 2. Class-specific EIAs which measure in HAV-specific IgG,
IgM, and IgA antibodies in both serum and fecal extracts
(coproantibodies).
[0107] 3. HAV-specific lymphocyte stimulation assays that measured
the in vitro proliferative response of lymphocytes. This assay is
considered a surrogate measurement of the immune responsiveness of
CD4+ T-cell helper cells in lymphoid populations.
[0108] The measured responses of SPLC and in the sera were
considered indicative of the systemic immune response while that of
the PPCs and LPLs was considered representative, respectively, of
the inductive and effector compartments of the intestinal mucosal
immune system
[0109] Immunization of Mice and Collection of Tissues:
[0110] Four doses of HAVRIX.TM. 1440 were compared: 144, 72, 36,
and 18 EU. BALB/c female mice (Harlan Laboratories, Rehovot,
Israel), 4-6 weeks at the outset of the experiment, in groups of 5,
received three doses of HAVRIX.TM. (either IP or IR) at weekly
intervals after which, they were allowed to rest for a period of
1-3 months (Table 9). Control animals received sterile saline via
the IP or IR routes according to the same schedule. Subsequently,
three animals in each group (including the saline controls) were
boosted with 72 EU of HAVRIX.TM. given by the IP route (regardless
of the route used for the priming immunization). This strategy was
designed to mimic the situation in which the vaccine would be
delivered parenterally (by injection), in an immunization campaign
during an outbreak situation to previously immunized individuals.
Two animals in each group were not immunized further. In the saline
control groups, the immune responses of the three animals immunized
with 72 EU of HAVRIX.TM. IP, were considered to be representative
of a primary immune response to this dose/route of HAVRIX.TM.,
while the responses measured in the remaining saline controls
represented nonspecific background in the assays used. Blood and
fecal specimens (for the preparation of serum and
coproantibodies--see below) were collected prior to, and 1 week
after each immunization. Two weeks after delivery of the last
(booster) dose of HAVRIX.TM., all animals in each group were
killed. Blood was removed for preparation of sera, and the spleen
and small intestine (from the pylorus to ileocaecal junction) were
removed for analysis of systemic (spleen cell=SPLC) and gut mucosal
(PPC, LPL) lymphocyte responses. The measured response of SPLC and
serum antibody determinations were considered indicative of the
systemic immune response while those of the PPCs, LPLs, and
coproantibody determinations were considered representative of the
inductive (PPC) and effector (LPL, coproantibodies) arms of the
mucosal immune system. In contrast to the earlier experiments,
animals were kept under SPF conditions and provided with autoclaved
standard rodent chow and water ad libitum.
9TABLE 9 Treatment Groups. Number of Mice Route Priming Dose
(EU).sup.a Total Boosted.sup.b Not Boosted IP 144 5 3 2 72 5 3 2 36
5 3 2 18 5 3 2 0 5 3 2 IR 144 5 3 2 72 5 3 2 36 5 3 2 18 5 3 2 0 5
3 2 .sup.aMice were immunized three times at weekly intervals with
the dose of HAVRIX .TM. vaccine shown, and by the route indicated.
.sup.bAnimals 1-3 in each treatment group received an additional
booster immunization of 72 EU of HAVRIX .TM. administered
intraperitoneally (IP) at 1-3 months after the last priming dose.
Animals 4 and 5 in each treatment group were not re-immunized ("not
boosted").
[0111] Serum and Coproantibody Preparation:
[0112] Mice were bled via the tail vein and serum was prepared and
stored at -20.degree. C. until testing. While the mice were being
bled, fecal pellets were collected and coproantibodies were
extracted as soon as possible according to the procedure described
by DeVos, et al. DeVos T, Dick T A. A rapid method to determine the
isotope and specificity of coproantibodies in mice infected with
Trichinella or fed cholera toxin. J Immunol Meth 1991;141
285-288.).
[0113] Cell Preparations:
[0114] SPLC, PPC and LPL suspensions were prepared from individual
mice, as follows. SPLC: Spleens were removed and placed into 60 mm
petri dishes with 3-5 mL of sterile RPMI-1640 medium (GIBCO/BRL,
Biological Industries, Beit Haemek, Israel) containing antibiotics
penicillin, 100 U/mL and streptomycin, 100 .mu.g/mL, GIBCO/BRL),
placed on ice, and transported to the laboratory for further
processing. The organs were washed once in sterile RPMI or
Dulbecco's phosphate buffered saline (PBS), and then teased apart
in 5 mL of RPMI with antibiotics. The suspended SPLC were collected
by centrifugation, washed twice through RPMI with antibiotics, then
resuspended in 10 mL of the same medium containing 2%
heat-inactivated (56 C, 30 min) FBS (Biological Industries). Viable
lymphoid cells were enumerated by Trypan Blue exclusion using a
Neubauer hemocytometer. For ELISPOT and lymphocyte proliferation
assays, SPLC were adjusted to 1.times.10.sup.6 cells/mL in RPMI
medium containing antibiotics and 10% FBLS. PPC: Peyer's patches
(approx. 4-8 per animal) were removed from the serosal surface of
the gut into sterile 60-mm petri dishes containing 5 mL of sterile
RPMI-1640 medium with antibiotics. The organs were washed once in
sterile RPMI or sterile PBS then teased apart in 5 mL of the same
medium containing 1.5 mg/mL of Dispase (Sigma-Aldrich, Milwaukee,
Wis.). The cell suspension was transferred into a conical-bottomed
10-15 mL tube and incubated with shaking for 45-60 min at
37.degree. C. to dissociate lymphoid cells from the rest of the
tissue. PPCs were collected by centrifugation and washed twice
through RPMI with antibiotics. Numbers of viable lymphoid cells
were determined by Trypan Blue exclusion. As PPC numbers were
limited, the final pellet was resuspended in 2.0-2.5 mL of RPMI
with antibiotics and 10% FBS for subsequent use in ELISPOT and
lymphoproliferation assays. Cell densities (cells/mL) for each PPC
preparation were recorded for later normalization of assay results
to the estimated response had the initial cell density been
1.times.10.sup.6 cells/mL, to allow for data comparisons.
[0115] LPLs: A modification of the method of Jackson, et al.
(Jackson R J, Fujihashi I, Xu-Amano H, Kiyono H, Elson C O, McGhee
J R. Optimizing Oral Vaccines: Induction of Systemic and Mucosal
B-cell and Antibody Responses to Tetanus Toxoid by Use of Cholera
Toxin as an Adjuvant. Infect Immun 1993;61(10):4272-79) was used to
extract LPLs from the small intestine wall. The entire small
intestine was opened longitudinally and washed thoroughly with
RPMI-1640 medium or sterile PBS, then cut into segments 2-3 cm in
length. To remove intraepithelial lymphocytes (IELs) and other
lymphoid cells (principally, CD9+ T cells) located in intestinal
mucosal crypts, tissues from individual animals were placed in 10
mL of in RPMI with 2% FBS in 50-mL centrifuge tubes and gently
agitated on a mechanical shaker for 30 min at 37.degree. C. At the
end of this period, the tubes were shaken vigorously for 15 sec.
Tissues were removed with the aid of a fine mesh strainer, then
placed in fresh medium and shaken again for 15 sec, strained
through the mesh, and transferred into fresh 50-mL tubes for
further extraction in 10 mL of RPMI with antibiotics containing 1.5
mg/mL of Dispase. Enzymatic digests were carried out in three
cycles 30 min at 37.degree. C. on a mechanical shaker. At the end
of each incubation period, the LPLs present in the supernatant were
collected by centrifugation, resuspended in RPMI with antibiotics
and 2% FBS and placed on ice until the third digestion was
complete. The LPLs were pooled and collected by centrifugation.
Numbers of viable lymphoid cells were determined by Trypan Blue
exclusion hemocytometer counts, cells were resuspended in 2-2.5 mL
of RPMI with antibiotics and 10% FBS and used at their available
densities for ELISPOT and lymphocyte proliferation assays.
Individual densities were recorded and the data were normalized to
the estimated response for an initial cell density of
1.times.10.sup.6 cells/mL for subsequent data comparisons.
[0116] Immunoassays:
[0117] HAV-specific systemic and mucosal immune responses were
evaluated by three methods i) ELISPOT assays to determine the
frequencies of IgG, IgM, and IgA class antibody forming cells
(AFC); ii) enzyme immunoassays (EIAs) which measured
antigen-specific IgG, IgM, and IgG antibodies in both serum and
fecal extracts (coproantibodies); iii) lymphocyte stimulation
assays that measured the in vitro dose-related proliferative
response of lymphocytes to HAV antigen.
[0118] Antigen: The HAV antigen used for these immunoassays was
highly purified alum-free (to avoid possible interference of this
adjuvant with the antigenicity of the virus in immunoassays)
formalin-fixed HAV purchased from a vaccine manufacturer
(Aventis-Pasteur, Lyon, France). The concentration of HAV in the
undiluted preparation was 1064 EU/mL or 170 .mu.g of
protein/mL.
[0119] ELISPOT Assays:
[0120] Numbers of HAV-specific IgG, IgM, or IgA AFC in SPLC, PPC,
and LPL cell populations were determined by the method of Jackson,
et al. (Jackson R J, Fujihashi I, Xu-Amano H, Kiyono H, Elson C O,
McGhee J R. Optimizing Oral Vaccines: Induction of Systemic and
Mucosal B-cell and Antibody Responses to Tetanus Toxoid by Use of
Cholera Toxin as an Adjuvant. Infect Immun 1993;61(10):4272-79)
Briefly, wells of sterile 96-well nitrocellulose-bottomed
microplates (MAHAN S4510, Multiscreen HA plate Millipore, Bedford,
Mass.) were coated for 24 h at 4.degree. C. with 100 .mu.L of HAV
antigen diluted to a concentration of 5 .mu.g/mL in sterile
bicarbonate-carbonate coating buffer (pH 9.6). The antigen was
discarded and the wells were washed three times in sterile
Dulbecco's phosphate buffered saline (PBS, pH 7.4) then blocked for
a minimum of 1 h at room temperature with sterile 1% bovine serum
albumin (BSA) in PBS. Just before adding the cells to be tested,
the blocking solution was discarded with no further washing of the
microplate wells. All cell populations were added such that the
well volume was 100 .mu.L regardless of the cell densities of the
SPLC, PPC, or LPL populations. The numbers of SPLC added per well
were 10,000 for all preparations, while the numbers of PPC and LPL
varied according to the yield for individual preparations.
Regardless of the cell numbers, data were normalized to an estimate
of the response that would be obtained if 100,000 cells were added
to each well for each PPC or LPL preparation. Control wells
containing reagent only were included on each plate to distinguish
antigen-specific from nonspecific spots. Each cell preparation was
tested in duplicate. After adding the cells, the plates were
transferred carefully to a 37 C, 5% CO.sub.2 incubator and left
overnight. The following day, the cells were discarded and the
wells were washed with three changes of sterile PBS, then three
times with PBS containing 0.05% Tween 20 (PBST). To detect
HAV-specific antibodies of the IgG, IgM, and IgA classes,
horseradish peroxidase (HSP)--conjugated goat anti-mouse
class-specific antibodies (Southern Biotechnology Associates [SBA],
Birmingham, Ala.) diluted to 1:1000, each, in PBST containing 5%
normal goat serum (NGS) were added (100 .mu.L/well) and is
incubated for 18 hours at 4 C. These antibodies were discarded and
the wells were washed 4 times with PBST. To visualize ELISPOTs
indicative of positions of HAV-specific AFC,
3-aminoethyl-9-carbazole substrate (AEC, Sigma-Aldrich) diluted in
0.1 M acetate buffer containing 0.015% H.sub.2O.sub.2(100
.mu.L/well) was added. The plates were covered with aluminium foil
and incubated at room temperature for 20 min. The enzymatic
reaction was stopped, by washing the wells 4 times with distilled
water. ELISPOTs were enumerated using a top-illuminated dissecting
microscope. Characteristic ELISPOTs were not observed in control
wells. Results were expressed as the averaged value of duplicate
wells normalized to the estimated number of spots per 10.sup.6
cells and corrected for background (i.e., the averaged number of
ELISPOTs observed in non-immunized saline controls were subtracted
to provide a net value).
[0121] EIAs: HAV-specific antibodies of the IgG, IgM, and IgA
classes were determined by a direct EIA. Briefly, wells of 96-well,
flat-bottomed polystyrene microplates (Nunc ImmunoPolysorp, Danyel
Biotech, Rehovot, Israel) were coated with HAV antigen diluted to a
concentration of 10 .mu.g/mL in bicarbonate-carbonate buffer, pH
9.6 (100 .mu.L/well) overnight at 4 C. The antigen was discarded
and the plates were blocked for 1 h with 3% nonfat milk in PBS (pH
7.4) (200 .mu.L/well). The blocking solution was discarded and the
wells were washed three times with PBST. For serum antibody
determinations serial 2-fold dilutions (in blocking buffer) ranging
from 1:50 to 1:6400 were prepared directly in the wells such that
the final well volume was 100 .mu.L. A preimmunization BALB/c mouse
serum pool diluted the same, was included as a control in addition
to a row of wells that received dilution buffer only. Fecal
extracts for coproantibody determinations were added undiluted (100
.mu.L/well). Negative controls for coproantibody determinations
included Hank's Balanced Salt solution (HBSS), the extraction
buffer used in preparing coproantibodies; fecal extracts from
non-immunized saline control mice; and extracts of feces collected
from 30 of the mice before immunization. All determinations on each
plate were single. However, replicate assays were performed.
[0122] The plates were incubated for 2 h at room temperature or
overnight at 4.degree. C. Following this, the weft contents were
discarded and wells were washed 4 times with PBST. To detect
class-specific antibodies, BRP-goat anti-mouse IgG, IgM, or IgA
(from SBA), diluted 1:1000 in 5% NGS in PBST were added to each
well (100 .mu.L/well) and the plates were incubated for 2 h at room
temperature. The well contents were discarded, and the plates were
washed 4 times with PBST. HRP substrate (ortho-phenylenediamine in
0.1 citrate-phosphate buffer (pH 5) containing 0.015%
H.sub.2O.sub.2 (100 .mu.L/well) was added and the plates were
incubated at room temperature for 60-90 min. A.sub.450 was
determined for each well using a Spectra Rainbow microplate reader.
Endpoint titers for serum determinations were expressed as the last
serum dilution showing an absorbance value equal to or greater than
the mean plus two standard deviations of the averaged absorbance
values calculated for both sets of control wells. Results for
coproantibody determinations were expressed qualitatively as
positive or negative based on a signal-to-noise (S/N) calculated
from the absorbance measured in wells containing fecal extracts
divided by the averaged absorbance measured in all the negative
control wells S/N ratios>2 were taken to be positive for IgG and
IgM determinations, while S/N ratios>3 were taken to be positive
for IgA determinations.
[0123] Lymphocyte Proliferation Assays:
[0124] The in vitro response of SPLC, PPC, and LPLs to stimulation
with formalin-inactivated HAV was determined using standard
lymphocyte proliferation assays performed in U-bottomed 96-well
microculture plates. HAV antigen was diluted to give final well
concentrations of 20, 10, 5, and 2.5 EU/mL. Positive control wells
received PHA (Sigma-Aldrich) adjusted to a final concentration of
10 .mu.g/mL. Negative control wells received medium only. Cells
were added in a fixed volume of 100 .mu.L regardless of the density
in the original preparation. Thus, SPLC were added at an initial
density of 100,000 cells per well while PPC and LPL initial
densities varied according to individual yields. Results were
normalized to the response estimated if the initial well density
had been 100,000 cells/well. Well volumes were brought up to 0.25
mL with RPMI containing antibiotics and 10% FCS (as described
above) and the cells were incubated at 37.degree. C. in 5% CO.sub.2
in air for 7 days. During the last 18 hours of culture, the cells
were pulsed with 1 .mu.Ci/well of .sup.3H-thymidine (Amersham,
Piscataway, N.J.). The cells were harvested by hypotonic lysis and
the DNA collected onto glass fiber filters for counting (Wallac
Microbeta Harvestor and Counter, Turku, Finland). Results were
expressed as the stimulation index (SI) calculated from the
averaged cpm per well containing stimulant divided by the averaged
cpm of negative control (medium only) wells and corrected for
averaged background values observed in non-immunized saline
controls.
[0125] Statistical Methods:
[0126] Frequencies of AFCs and serum endpoint titers (taken as
absolute values) were compared using 1-tailed, 2-sample T-tests for
equal or unequal variance. F-tests were used to determine equal or
unequal variance.
[0127] Results:
[0128] Determination of HAV-Specific Antibodies by EIA and ELISPOT
Assays:
[0129] HAV-specific antibodies of IgG, IgM, and IgA classes were
measured in both sera and fecal extracts (coproantibodies) by EIA
to evaluate the specific antibody response in the systemic and
intestinal mucosal immune compartments, respectively. Similarly,
systemic (SPLC) and mucosal (PPC, LPL) AFC were enumerated by
ELISPOT assays. Results comparing the response of intraperitoneally
(IP) and intrarectally (IR) immunized animals are summarized for
each HAVRIX.TM. immunizing dose, successively, in Tables 10-14.
ELISPOT values shown in the Tables have been corrected for
background levels of activity observed in saline control mice that
received saline only, throughout the course of the experiment.
Coproantibodies are expressed qualitatively as either present (+)
or absent (-).
[0130] Response to 144 EU of HAVRIX.TM. (Table 10):
[0131] While the mean numbers of IgG AFCs found in SPLC of mice
immunized via the IP route appeared to be higher relative to those
observed in IR-primed animals this difference was not statistically
significant (p=0.132). HAV-specific IgG AFC were not observed in
PPC and LPL populations in either group. Large numbers of IgM AFC
were found in the spleens of both groups. However, no significant
group differences were observed. IgM AFC were also found in PPC and
LPL populations of some rectally immunized mice. IgA AFCs were not
observed in SPLC of any of the animals. Nor were they found in the
PPCs of IP-immunized mice. However, they were found in abundance in
both the PPC and LPL populations of rectally immunized mice and at
a significantly higher (p=0.043) frequency in the PPC population
(LPLs, not compared). Correspondent with observed higher IgG AFC
frequencies, titers of HAV-specific IgG while appearing higher in
IP-immunized mice, were not found to be significantly different
from those of the IR group (p=0.094). Titers of specific IgM
antibodies were higher in the sera of IR-immunized mice (p=0.009)
in keeping with the consistently large numbers of IgM AFC seen in
the spleens of this group. While titers of HAV-specific IgA serum
antibodies also appeared higher in the IP-immunized mice, they were
not significantly different from the IR group (p=0.091) IgG and
IgA-class HAV-specific coproantibodies were detected in fecal
extracts from 1/5, and 2/5 rectally immunized mice, respectively,
but not in any animals from the IP group. IgM class coproantibodies
were not observed in any of the mice. The additional dose of 72 EU
of HAVRIX.TM. (given IP to Animals 1-3 in each group) two weeks
before killing the mice did not appear to enhance the frequencies
of AFC, in comparison to those found in Animals 4 and 5 in each
group who received only three doses of HAVRIX.TM..
10TABLE 10 Comparative levels of HAV-specific IgG, IgM, and IgA
class antibodies determined in mice primed intraperitoneally (IP)
or intrarectally (IR) with 144 EU of HAVRIX .TM.. Animal IP Route
IR Route Number AFC/10.sup.6 cells Titer.sup.-1 Copro- AFC/10.sup.6
cells Titer.sup.-1 Copro- (Treatment) SPLC PPC LPLs (serum) Ab SPLC
PPC LPLs (serum) Ab IgG Antibodies: 1 (B) 35 0 ND 800 - 0 0 0
<50 - 2 (B) 5 0 ND .gtoreq.6400 - 0 0 0 .gtoreq.100 + 3 (B) 5 0
ND .gtoreq.800 - 10 0 0 <50 - 4 (NB) 5 0 ND .gtoreq.200 - 0 0 0
<50 + 5 (NB) 0 0 ND .gtoreq.400 - 0 0 0 .gtoreq.50 - IgM
Antibodies: 1 (B) 290 0 ND .gtoreq.50 - 57.5 0 0 .gtoreq.200 - 2
(B) 0 0 ND .gtoreq.100 - 0 0 6 .gtoreq.200 - 3 (B) 15 0 ND
.gtoreq.100 - 62.5 8.5 0 .gtoreq.200 - 4 (NB) 15 0 ND 50 - 22.5 0 0
.gtoreq.100 - 5 (NB) 0 0 ND 50 - 67.5 0 0 .gtoreq.100 - IgA
Antibodies: 1 (B) 0 0 ND <50 - 0 0 0 <50 - 2 (B) 0 0 ND 1600
- 0 28 0 <50 - 3 (B) 0 0 ND .gtoreq.800 - 0 20 0 <50 - 4 (NB)
0 0 ND .gtoreq.1600 - 0 0 14 <50 ND 5 (NB) 0 0 ND 800 - 0 38 146
.gtoreq.1600 +
[0132] Response to 72 EU of HAVRIX.TM. (Table 11):
[0133] Again, HAV-specific IgG and IgM AFC frequencies in SPLC were
higher in IP-immunized animals than in rectally immunized mice.
This was of borderline statistical significance (IgG: p=0.053; IgM:
p=0.057). Fewer IgA AFC were observed in SPLC of both treatment
groups (no difference). In PPC populations, one animal in the
IP-immunized group had a large number of IgM AFC (no significant
group differences overall). Too few LPLs were isolated from the
IP-primed animals, hence, group comparisons for AFC were not
possible. However, IgA AFC were observed in PPC and LPL
preparations from two rectally immunized mice. Serum titers of
HAV-specific IgG (p=0.014) and IgA (p=0.038) were higher in mice
immunized via the IP route compared to the IR group. No group
differences were observed for specific IgM levels. Again, IgG, IgM,
and IgA class HAV-specific coproantibodies were observed only in
rectally immunized mice. The extra dose of 72 EU of HAVRIX.TM.
(given IP to Animals 1-3 in each group) did not appear to enhance
the frequencies of AFC or antibody levels in either IP- or
IR-immunized mice.
11TABLE 11 Comparative levels of HAV-specific IgG, IgM, and IgA
class antibodies determined in mice primed intraperitoneally (IP)
or intrarectally (IR) with 72 EU of HAVRIX .TM.. Animal IP Route IR
Route Number AFC/10.sup.6 cells Titer.sup.-1 Copro- AFC/10.sup.6
cells Titer.sup.-1 Copro- (Treatment) SPLC PPC LPLs (serum) Ab SPLC
PPC LPLs (serum) Ab IgG Antibodies: 1 (B) 8 0 ND 800 - 0 0 0 100 -
2 (B) 35 0 ND .gtoreq.3200 - 15 0 0 .gtoreq.100 - 3 (B) 5 0 ND 6400
- 5 0 16 800 + 4 (NB) 18 0 ND .gtoreq.1600 - 5 9 0 <50 - 5 (NB)
20 0 ND 3200 - 5 0 0 <50 - IgM Antibodies: 1 (B) 70 1.5 ND
.gtoreq.100 - 0 0 0 .gtoreq.100 - 2 (B) 170 4.5 ND .gtoreq.100 -
47.5 0 0 400 - 3 (B) 5 148.5 ND .gtoreq.100 - 67.5 0 0 .gtoreq.100
- 4 (NB) 100 0 ND 100 - 42.5 6.5 0 200 + 5 (NB) 105 2.5 ND 50 -
17.5 0 0 <50 - IgA Antibodies: 1 (B) 2.5 0 ND .gtoreq.200 - 0 18
0 <50 - 2 (B) 0 0 ND .gtoreq.400 - 0 28 0 <50 - 3 (B) 0 0 ND
.gtoreq.200 - 0 0 59 .gtoreq.400 + 4 (NB) 0 0 ND 1600 - 5 0 53
<50 - 5 (NB) 0 0 ND 800 - 5 11 7 <50 -
[0134] Response to 36 EU of HAVRIX.TM. (Table 12):
[0135] The frequencies of specific IgG AFC in spleens of IP-primed
animals were higher (p=0.048) than that of their IR counterparts.
Specific IgG AFC were also observed in PPC populations of one
IP-primed mouse and two IP-immunized mice and while higher in the
latter group, were not significantly different. Frequencies of
specific IgM AFC were high in both groups (not significantly
different). Specific IgA AFCs were also observed in all cell
populations in both groups (no significant differences). However, 3
of the PPC, and 4 of the LPL preparations obtained from rectally
immunized mice had strikingly high frequencies of specific IgA AFC.
Serum titers of specific IgG antibodies were also found to be
significantly higher (p=0.035) in the IP group compared to rectally
immunized mice. Titers of specific IgM antibodies were also higher
(p=0.033) in the IP group. In contrast, serum titers of
HAV-specific IgA antibodies were significantly higher (p=0.032) in
rectally immunized mice. IgA coproantibodies were found in fecal
extracts of 3/5 rectally immunized mice but only one in the IP
group.
12TABLE 12 Comparative levels of HAV-specific IgG, IgM, and IgA
class antibodies determined in mice primed intraperitoneally (IP)
or intrarectally (IR) with 36 EU of HAVRIX .TM.. Animal IP Route IR
Route Number AFC/10.sup.6 cells Titer.sup.-1 Copro- AFC/10.sup.6
cells Titer.sup.-1 Copro- (Treatment) SPLC PPC LPLs (serum) Ab SPLC
PPC LPLs (serum) Ab IgG Antibodies: 1 (B) 0 0 ND .gtoreq.400 - 0 0
0 .gtoreq.100 - 2 (B) 10 0 ND 3200 - 5 0 0 <50 - 3 (B) 0 6 ND
.gtoreq.6400 - 0 24 0 <50 - 4 (NB) 10 0 ND 1600 - 0 0 0 100 - 5
(NB) 10 0 ND .gtoreq.400 - 0 10 0 <50 - IgM Antibodies: 1 (B) 0
0 ND 200 - 32.5 0 0 50 - 2 (B) 5 0 ND 400 - 32.5 0 0 .gtoreq.50 - 3
(B) 75 0 ND 1600 - 32.5 12.5 25 .gtoreq.100 + 4 (NB) 30 5.5 ND
.gtoreq.400 - 0 0 12 50 - 5 (NB) 60 13.5 ND 400 - 27.5 0 39 50 -
IgA Antibodies: 1 (B) 0 0 ND <50 + 0 10 124 .gtoreq.400 + 2 (B)
0 0 ND <50 - 5 0 506 .gtoreq.400 + 3 (B) 2.5 0 ND <50 - 0 60
6 .gtoreq.400 - 4 (NB) 7.5 80.5 ND 200 - 0 0 53 <50 - 5 (NB) 0 0
ND <50 - 0 31 144 .gtoreq.100 +
[0136] Response to 18 EU of HAVRIX.TM. (Table 13):
[0137] IgG AFCs were not found in SPLC of any mice immunized with
18 EU of HAVRIX.TM.. Only a few IgG AFCs were observed in mucosal
cell populations. IgM AFC frequencies appeared higher (but were not
significantly so) in PPC populations from IP-immunized mice,
whereas IgM AFC frequencies were significantly higher (p=0.003) in
the spleens of the IR group. Only two animals in the i.p.-primed
group demonstrated HAV-specific IgA AFC, and only in their LPLs,
However, compared to the IP group, significantly higher (p=0.021)
frequencies of IgA AFC in LPL populations were found in 4/5
rectally immunized mice, consistent with was observed in mice
immunized with 36 EU of HAVRIX.TM. via the IR route. No significant
group differences were observed in serum titers of HAV-specific IgG
(p=0.282), IgM (p=0.116), or IgA (p=0.075) class antibodies.
Coproantibodies of the IgA class were detected in fecal extracts
from two rectally immunized mice. IgM coproantibodies were found in
the fecal sample of one rectally immunized animal while IgG
coproantibodies were detected in the feces of one of the IP
group.
13TABLE 13 Comparative levels of HAV-specific IgG, IgM, and IgA
class antibodies determined in mice primed intraperitoneally (IP)
or intrarectally (IR) with 18 EU of HAVRIX .TM.. Animal IP Route IR
Route Number AFC/10.sup.6 cells Titer.sup.-1 Copro- AFC/10.sup.6
cells Titer.sup.-1 Copro- (Treatment) SPLC PPC LPLs (serum) Ab SPLC
PPC LPLs (serum) Ab IgG Antibodies: 1 (B) 0 0 ND .gtoreq.400 - 0 0
0 400 - 2 (B) 0 0 0 .gtoreq.1600 - 0 0 0 .gtoreq.1600 - 3 (B) 0 0 0
1600 - 0 0 0 .gtoreq.3200 - 4 (NB) 0 0 0 .gtoreq.800 + 0 5 11
.gtoreq.200 - 5 (NB) 0 0 13 .gtoreq.800 - 0 0 0 1600 - IgM
Antibodies: 1 (B) 0 0 0 100 - 32.5 0 0 50 - 2 (B) 5 11.5 0 50 -
92.5 0.5 0 400 + 3 (B) 10 6.5 0 100 - 42.5 0 0 100 - 4 (NB) 0 20.5
23 <50 - 82.5 8.5 21 .gtoreq.50 - 5 (NB) 20 104.5 0 <50 -
67.5 0 11 100 - IgA Antibodies: 1 (B) 0 0 5 .gtoreq.800 - 0 16 68
<50 - 2 (B) 0 0 0 .gtoreq.1600 - 0 0 174 400 + 3 (B) 0 0 0
<50 - 0 0 0 .gtoreq.400 - 4 (NB) 0 0 0 6400 - 0 0 44 <50 - 5
(NB) 0 0 26 .gtoreq.3200 - 0 0 107 .gtoreq.800 +
[0138] Explanatory Notes (Tables 10-13):
[0139] SPLC: spleen cells; PPC: Peyer's Patch Cells; LPLs: lamina
propria lymphocytes. Values shown are estimated frequencies of
antibody-forming cells (AFC) per million cells in the three
different cell population as determined by ELISPOT assays at the
time of sacrificing the animals (see Methods).
[0140] Treatment: B=animals received 72 EU of HAVRIX.TM., i.p. two
weeks before sacrificing. NB=animals were not immunized further
after the initial 3-dose immunization schedule with HAVRIX.TM..
[0141] Serum titers: represent the reciprocal value of the endpoint
titer measured in EIAs with serum dilutions ranging from 1:50 to
1:6400 (see Methods). Reciprocal titers<50 represent a negative
value in the assay.
[0142] Coproantibodies: HAV-specific antibodies secreted into, and
extracted from, feces (see Methods). Fecal samples were not
obtained from all animals. Antibody levels which were measured by
EIA are expressed qualitatively based on a calculated signal:noise
(S/N) ratio. S/N>2 was considered representative of a positive
response in IgG and IgM determinations; a S/N ratio of >3 was
considered positive in assays for class-specific IgA antibodies
(see Methods) Values shown represent those measured in fecal
samples 1 week after boosting (Animals 1-3) and in nonboosted
control animals at the same interval. Specific antibodies were not
measurable in samples collected at earlier intervals.
[0143] ND: not determined.
[0144] FIGS. 12-14 summarize these observations of the antibody
response to various immunizing doses of HAVRIX.TM. showing that at
the highest dose (144 EU) IgG-AFC frequencies in SPLC populations
were observed to be higher overall in mice immunized parenterally
(via the IP route) in comparison to rectally immunized mice (FIG.
12), although these differences were not statistically significant.
However, a lower immunizing doses of HAVRIX.TM. (36 and 18 EU), a
strong reverse trend in frequencies of IgM- and IgA-AFC was
observed, particularly in the LPL populations (FIGS. 13 and 14,
respectively) which were observed to be significantly higher in
rectally immunized mice in comparison to those who were immunized
intraperitoneally.
[0145] Cellular Recognition of HAV Antigens in Immunized Mice as
Determined by Lymphocyte Proliferation Assays
[0146] The in vitro dose-related responses of lymphocytes present
in SPLC, PPC, and LPL populations of mice immunized with the
varying doses of HAVRIX.TM. by the IP and IR routes were compared
using conventional proliferation assays. As cell numbers in the PPC
and LPL populations were limited (on the average, permitting an
initial well density of 50,000 cells) incubations were carried out
for 7 days. SPLC that were not limited in number, were plated at an
initial density of 100,000 cells per well and results from wells
containing PPC and LPLs were normalized to estimate the response at
the same initial density. Insufficient cell numbers in some of the
LPL populations and technical limitations reduced the numbers of
comparisons using stimulation indices (SI) as the parameter.
Individual data, summarized by immunizing dose and route, are shown
in Table 14. In common practice, SI's>2.0 are considered to be
indicative of a positive response, although some interpretations
require higher cutoffs. All SI's shown in Table 14 have been
corrected for assay background (i.e., SI's determined for the same
cell populations obtained from non-immunized saline control mice
have been subtracted) and therefore, this interpretation is
considered valid here. In order to present the data comparatively,
only the maximum SI's attained in the dose response curve for each
cell preparation and for each animal, are shown in Table 14 and are
summarized for all immunizing doses of HAVRIX.TM. in FIGS. 15-17.
At higher immunizing doses of HAVRIX.TM. (144 and 72 EU) SI's were
observed to be low or negative (with a few exceptions), in all cell
populations. There were no discernible differences in the response
related to the immunizing dose or route of vaccine delivery. At
lower immunizing doses (36 and 18 EU) the SI's measured in all cell
populations were observed to be 3-->100-fold higher in animals
who were immunized via the IR route than those receiving HAVRIX.TM.
via the IP route. The strength of the in vitro response of SPLC,
PPC, and LPLs was also dose-related to the amount of stimulating
HAV added to the cultures (data not shown). The highest SI's were
observed in animals immunized with three intrarectal doses of 36 EU
of HAVRIX.TM. (Table 14). While, differences in SI's measured in
animals primed with 18 EU of HAVRIX.TM. via the IP and IR routes
were not as striking as those observed with an immunizing dose of
36 EU, they were still at least 10-fold higher in the IR group,
than in the IP groups. There were no discernible differences
between the response of animals who received only three
immunizations and those who received an additional booster dose of
HAVRIX.TM..
14TABLE 14 Comparative in vitro response to HAV antigens of
lymphocytes from mice immunized intraperitoneally (IP) or
intrarectally (IR) with varying doses of HAVRIX .TM. vaccine.
HAVRIX .TM. Maximal Stimulation Index (SI) Observed.sup.a Dose/Cell
IP Route IR Route Population 1 2 3 4 5 1 2 3 4 5 HAVRIX .TM. 144
EU: SPLC ND ND 1.9 ND 23.6 10 0 0 0.3 ND PPC 0 ND 2.4 0 0.5 4.7 ND
2.7 0 ND LPLs ND ND ND ND ND ND ND 1.8 0 0 HAVRIX .TM. 72 EU: SPLC
ND ND 5.6 ND 0.5 0 ND 0 0 ND PPC 14.7 0.1 0 0 0.5 2.4 ND 0 0 ND
LPLs ND ND ND ND ND 0 ND 26.5 ND 0 HAVRIX .TM. 36 EU: SPLC 0.9 1.6
0.9 1.0 1.0 29.7 ND 12.5 166.7 16.8 PPC 4.6 1.5 2.9 2.8 7.4 12.3 ND
1.0 133.9 182.9 LPLs ND ND 2.4 ND ND ND 23.0 80.8 14.2 53.1 HAVRIX
.TM. 18 EU: SPLC 11.7 1.4 3.6 6.3 4.0 60.0 ND 55.1 17.6 1.6 PPC 6.4
0.8 1.9 9.8 3.4 23.0 ND 0 22.8 11.7 LPLs 5.4 1.6 2.7 3.8 2.5 2.8
62.2 44.5 ND 3.1
[0147] Footnotes (Table 14):
[0148] .sup.a Value shown is the maximum stimulation index (SI)
observed in the dose response to in vitro stimulation of lymphoid
cells (see Methods) in the cell populations indicated in the first
column of the table, compared for each treatment group (IP vs. IR)
for each animal (1-5) in the treatment groups.
[0149] ND: not determined due to insufficient cell numbers or other
technical limitations.
[0150] Summary and Conclusions:
[0151] The results of study indicate that immunization of mice with
low doses of HAVRIX.TM. delivered via the intrarectal (IR) route
appears to be highly effective in inducing HAV-specific IgA AFC (in
mucosal lymphocytes) and IgM AFC (in splenocytes), as well as
HAV-specific antibodies of these classes in the serum and
intestinal mucosal secretions. This is desirable for immune
protection as both pathogen blocking and neutralize antibodies are
likely to be of these classes. Similarly, oral administration of
the polio vaccine is known to elicit antibodies of both the IgG and
IgA classes (see, for example; Herremans T M, Reimerink J H,
Buisman A M, Kimman T G, Koopmans M P. Induction of mucosal
immunity by inactivated poliovirus vaccine is dependent on previous
mucosal contact with live virus. J Immunol. 1999 15;162:5011-8).
For viruses that enter through the gut epithelium, mucosal IgA
antibodies that are produced in the intestinal mucosa and secreted
to the gut lumen, play an essential role in neutralizing the virus
at the mucosal interface. Intrarectal immunization with HAV vaccine
also induced strong cellular (as evidenced by lymphocyte
proliferation) immune responses in both systemic (SPLC) and mucosal
(PPC, LPL) lymphocyte populations that were superior to those
responses observed in parenterally (i.p.) immunized animals. This
suggests that rectal immunization has excellent potential for
induction of T-cell help (either for production of specific
antibodies or for induction of a cytotoxic T-lymphocyte response).
Both antibody and cellular responses to HAVRIX.TM. administered by
the rectal route were optimal at lower doses of immunizing antigen
providing some encouragement that low doses of antigen (at least,
by the IR route) should be effective rather than tolerizing. Also,
these superior responses were manifest in both local (gut mucosa)
and systemic (spleen) compartments of the immune system, and hence,
represent a desirable means of inducing protection vs. HAV, a
pathogen which enters the body via the gut mucosa and spreads to
the liver.
[0152] Parenteral administration of vaccines via intradermal (ID)
or intramuscular (IM) routes usually gives rise to only a systemic
immune response consisting of IgM followed by IgG class antibodies
as evidenced from results of IM administration of killed (Salk)
polio vaccines in contrast to the oral (Sabin) polio vaccine that
also induces IgA-class antibodies. Currently used injectable HAV
vaccines that are also routinely delivered by IM injection are
thought to produce only IgG class antibodies. Successful
immunization against pathogens such as poliovirus and HAV that
enter via the gut epithelium clearly requires local production of
neutralizing antibodies of the IgA class at the portal of entry.
Mucosal immunization via the oral or intrarectal, or intranasal
routes induces both local (mucosal) and systemic immune responses
consisting of both IgG and IgA antibodies. Thus, in the case of
pathogens entering via mucosal (respiratory, gut, genitourinary)
routes the latter type of immune response is clearly more
beneficial to the host.
[0153] The method of the present invention, in which the HAV
vaccine is administered to the body through the rectal mucosa,
could have a major advantage over the current vaccination program,
by not only by inducing antibody and cellular responses which block
pathogen entry through the gastrointestinal mucosal but also by
inducing systemic immune responses that would limit viral survival
in the blood stream and/or its replication in liver tissue. In
addition, the method of the present invention is able to induce the
generation of a successful immune response against HAV without
requiring needles or other invasive methods. The present invention
thus enables the rectal administration of an HAV vaccine such as
HAVRIX.TM. for the generation of a protective immune response
against HAV infection.
[0154] Without wishing to limit the present invention, a suitable
dosage of the HAV vaccine is preferably in the range of from about
0.75 to about 7500 EU of the antigen, for each administration more
preferably approximately from about 50 EU to about 125 EU, and most
preferably approximately 75 EU, applied to the rectum in a
suppository cream, liquid, tablet, or any other solid, semi-solid
or liquid dosage form which is suitable for the administration of
HAV antigen to the rectal mucosa. Such a dosage form is well known
in the art, and could easily be selected by one of ordinary skill
in the art. Optionally the dosage form would contain an adjuvant,
although alternatively, no adjuvant would be used.
[0155] The method of the present invention is also optionally and
preferably suitable for the administration of at least one viral
encapsidated gene as described above through the gastrointestinal
mucosa of the subject, and particularly through the rectal mucosa.
In this optional but preferred method, the viral encapsidated gene
or genes is administered to the gastrointestinal mucosa of the
subject, in a substantially similar manner as for the previously
described HAV vaccine. Thus, the present invention also provides a
method for administering one or more viral encapsidated gene or
genes to the subject through the gastrointestinal, and particularly
the rectal, mucosa.
[0156] Section 4:Future Implementations
[0157] The previously described transgenic plant vaccine contains
non-infectious viral particles. These viral particles may also
optionally be used to package other viral or host genes that have
been engineered into HAV (or other viral) genomes that could
subsequently, be engineered into tomato and or other plants using
the Agrobacterium transformation system as demonstrated in this
model. In this case, eating such transgenic tomatoes or other plant
material, or otherwise applying the transgenes orally and/or
rectally, would permit the transgenes to be specifically targeted
to the liver by using HAV virus. Similarly, other viruses with
specific trophism for bone marrow or nervous system could be
employed in plant engineering systems to deliver specific genes to
these tissues.
[0158] Without wishing to be limited to a single hypothesis, it is
hypothesized that following ingestion of the HAV-containing
transgenic tomatoes, the immune system should recognize the vital
capsid proteins following the uptake of plant-derived HAV virus
like particles (or similar particles applied to the rectal mucosa)
by mucosal M cells which subsequently transport the virus to
mucosal antigen presenting cells which process and present viral
capsid peptides to mucosal helper T-cells which ultimately activate
HAV-specific B and T lymphocytes in the intestinal mucosa. Using
this same concept, new second-generation recombinant chimeric oral
vaccines for polioviruses (also of the picomavirus family) or other
viral pathogens could be developed. These would utilize HAV
capsid's natural tropism for gastrointestinal epithelial cells to
facilitate exposure to the mucosal immune system. These vaccines
could be engineered to eliminate dangerous viral replication
elements, which have, in the past, created safety issues due to
spontaneous pathogenic revertants or recombination with wild type
viruses present in the gut or other tissues. Such vaccines would be
both safer and easier to administer than existing vaccines, as well
as being relatively simple and inexpensive to produce The success
of HAV particle production in transgenic plants would be the proof
of principle for the production of other orally consumed viral and
bacterial vaccines.
[0159] The efficient viral uptake by the gastrointestinal system
could also be applied for targeting of transgenes to specific
organs. The natural tropism of HAV is to the liver, probably
through the expression of a viral receptor on hepatocyte cell
membranes. Encapsulating the engineered HAV genome containing the
desired gene in HAV nucleocapsid particles would enable targeting
of genes to the liver. Similar targeting of genes to the bone
marrow or the nervous system is potentially feasible using a
similar strategy employing viruses displaying natural tropism for
specific tissues or organs.
[0160] Another aspect of the invention pertains to the method of
rectal immunization with whole virus or other immunogens to induce
both local gastrointestinal) and systemic immunity against the
diseases caused by viruses or other pathogens entering the body
through the gastrointestinal mucosa.
[0161] Through the examples herein, mucosal vaccination has been
shown to induce immunity or tolerance to HAV antigens depending
upon the route of delivery (and possibly, antigen dose). Both
potential outcomes of mucosal immunization could be exploited for
two different treatment modalities:
[0162] 1. Generation of viral or bacterial or other structural
elements in Tg plants or through other technologies for mucosal
immunization to protect against infectious diseases. These
genetically engineered immunogens may be used to prevent disease or
to attenuate disease development. The advantage of employing
pathogen structural elements as immunogens could be further
developed to include multiple targets; e.g. chimeric HAV-HCV virus
like particles encoding structural elements of both hepatitis A and
hepatitis C viruses could be engineered in plant expression systems
to serve as dual immunogens to protect against both diseases.
[0163] 2. The development of tolerance could also support various
therapeutic approaches, including but not limited to:
[0164] a. Induction of tolerance to hepatitis C virus could reduce
the liver inflammatory process, and reduce the risk for development
of cirrhosis and hepatocellular carcinoma.
[0165] b. Induction of immune tolerance a particular virus could be
exploited such that the virus could be engineered to serve as a
vehicle for tissue-specific delivery of other genes in gene therapy
(e.g., the use of nonpathogenic poliovirus virus like particles to
target the nervous system). Immune tolerance to the viral vector
would enhance its persistence in the target tissue and therefore,
expression of the delivered gene.
[0166] While the invention has been described with respect to a
limited number of embodiments, it will be appreciated that many
variations, modifications and other applications of the invention
may be made.
* * * * *
References