U.S. patent application number 16/035319 was filed with the patent office on 2018-11-08 for kexin-derived vaccines to prevent or treat fungal infections.
This patent application is currently assigned to UNIVERSITY OF PITTSBURGH - OF THE COMMONWEALTH SYSTEM OF HIGHER EDUCATION. The applicant listed for this patent is Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College, UNIVERSITY OF PITTSBURGH - OF THE COMMONWEALTH SYSTEM OF HIGHER EDUCATION. Invention is credited to Heather Kling, Jay K. Kolls, Karen Norris, Michael Zheng.
Application Number | 20180318406 16/035319 |
Document ID | / |
Family ID | 44304916 |
Filed Date | 2018-11-08 |
United States Patent
Application |
20180318406 |
Kind Code |
A1 |
Kolls; Jay K. ; et
al. |
November 8, 2018 |
KEXIN-DERIVED VACCINES TO PREVENT OR TREAT FUNGAL INFECTIONS
Abstract
A vaccine is disclosed that promotes CD4+ T cell-independent
host defense mechanisms to defend against infection by fungi such
as Pneumocystis spp. The vaccine may be used to prevent or to treat
fungal infections. The novel vaccine can provide protective
immunity, even for immunocompromised individuals such as HIV
patients having reduced levels of CD4+ T cells.
Inventors: |
Kolls; Jay K.; (Pittsburgh,
PA) ; Zheng; Michael; (Pittsburgh, PA) ;
Norris; Karen; (Pittsburgh, PA) ; Kling; Heather;
(Pittsburgh, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITY OF PITTSBURGH - OF THE COMMONWEALTH SYSTEM OF HIGHER
EDUCATION
Board Of Supervisors Of Louisiana State University And Agricultural
And Mechanical College |
PITTSBURGH
Baton Rouge |
PA
LA |
US
US |
|
|
Assignee: |
UNIVERSITY OF PITTSBURGH - OF THE
COMMONWEALTH SYSTEM OF HIGHER EDUCATION
PITTSBURGH
PA
Board Of Supervisors Of Louisiana State University And
Agricultural And Mechanical College
Baton Rouge
LA
|
Family ID: |
44304916 |
Appl. No.: |
16/035319 |
Filed: |
July 13, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15443976 |
Feb 27, 2017 |
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16035319 |
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13959691 |
Aug 5, 2013 |
9580704 |
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15443976 |
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13521621 |
Nov 12, 2012 |
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PCT/US2011/020170 |
Jan 5, 2011 |
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13959691 |
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61294252 |
Jan 12, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/53 20130101;
A61K 2039/545 20130101; C07K 2319/40 20130101; C07K 14/70575
20130101; A61K 38/191 20130101; A61K 39/00 20130101; C12N 9/58
20130101; A61P 31/10 20180101; C12Y 304/21061 20130101; C12N 9/60
20130101; A61P 37/04 20180101; A61K 2039/5256 20130101; A61K
2039/55516 20130101; A61K 39/0002 20130101; A61K 2039/55
20130101 |
International
Class: |
A61K 39/00 20060101
A61K039/00; A61K 38/19 20060101 A61K038/19; C07K 14/705 20060101
C07K014/705; C12N 9/58 20060101 C12N009/58; C12N 9/60 20060101
C12N009/60 |
Goverment Interests
[0002] This invention was made with government support under grant
P01-HL076100 awarded by the National Institutes of Health. The
United States Government has certain rights in this invention.
Claims
1. The protein mini-Kexin (SEQ ID NO 5).
2. An isolated nucleic acid encoding the protein of claim 1.
3. The nucleic acid of claim 2, wherein said nucleic acid has
sequence SEQ ID NO 1 or SEQ ID NO 2.
4. The nucleic acid of claim 2, wherein the codons of said nucleic
acid are optimized to enhance expression in mammalian cells.
5. The nucleic acid of claim 4, wherein said nucleic acid has
sequence SEQ ID NO 3 or SEQ ID NO 4.
6. A fusion protein that comprises mini-Kexin (SEQ ID NO 5), but
that does not comprise the entire Kexin protein; and that further
comprises a leader that promotes secretion of said fusion protein
from a mammalian cell.
7. An isolated nucleic acid encoding the fusion protein of claim
6.
8. The fusion protein of claim 6, wherein said leader is an IgGC
leader, and wherein the fusion protein has sequence SEQ ID NO
6.
9. An isolated nucleic acid encoding the fusion protein of claim
8.
10. The fusion protein of claim 6, wherein said fusion protein
comprises mini-Kexin and further comprises CD40L.
11. An isolated nucleic acid encoding the fusion protein of claim
10.
12. A vaccine comprising the protein of claim 1, and additionally
comprising an adjuvant.
13. The vaccine of claim 12, wherein the adjuvant comprises
CD40L.
14. A vaccine comprising the nucleic acid of claim 2, and
additionally comprising an isolated nucleic acid that encodes an
adjuvant.
15. The vaccine of claim 14, wherein the encoded adjuvant comprises
CD40L.
16. A vaccine that comprises a live virus containing the nucleic
acid of claim 2.
17. A method of immunizing a mammalian patient against infection by
Pneumocystis, said method comprising administering to the patient
the vaccine of claim 12.
18. A method of immunizing a mammalian patient against infection by
Pneumocystis, said method comprising administering to the patient
the vaccine of claim 14.
19. A method of immunizing a mammalian patient against infection by
Pneumocystis, said method comprising administering to the patient
the fusion protein of claim 10.
20. A method of immunizing a mammalian patient against infection by
Pneumocystis, said method comprising administering to the patient
the nucleic acid of claim 11.
21. The method of claim 20, wherein the patient is a human.
22. The method of claim 21, wherein the patient is
immunocompromised.
23. The method of claim 21, wherein the patient is not suffering
adverse effects of Pneumocystis infection, and wherein said method
affords protection against future infection by Pneumocystis.
24. The method of claim 21, wherein the patient is suffering
adverse effects of Pneumocystis infection, and wherein said method
ameliorates the Pneumocystis infection.
25. The method of claim 21, wherein the patient is immunized a
plurality of times to provide a stronger immune response than would
be provided by a single immunization.
26. The method of claim 21, wherein said method comprises one or
more prime vaccinations and one or more boost vaccinations; and
wherein the prime and boost vaccinations differ from one another in
the location of administration, or the composition of the vaccine,
or both.
27. The method of claim 26, wherein the prime vaccination comprises
intramuscular vaccination with a plasmid vaccine; and wherein the
boost vaccination comprises intranasal vaccination or other mucosal
vaccination with an adenovirus vaccine.
28. A method of immunizing a mammalian patient against fungal
infection, said method comprising administering to the patient the
vaccine of claim 12.
29. A method of immunizing a mammalian patient against fungal
infection, said method comprising administering to the patient the
vaccine of claim 14.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/521,621, filed Nov. 12, 2012, which is a
.sctn. 371 National Stage Application of PCT application
PCT/US11/20170, filed Jan. 5, 2011, which claims the benefit of the
Jan. 12, 2010 filing date of U.S. provisional patent application
Ser. No. 61/294,252 is claimed under 35 U.S.C. .sctn. 119(e) in the
United States, and is claimed under applicable treaties and
conventions in all countries.
[0003] The specification incorporates by reference the Sequence
Listing submitted herewith via EFS on Aug. 5, 2013. Pursuant to 37
C.F.R. .sctn. 1.52(e)(5), the Sequence Listing text file,
identified as 072396_0528_Sequence_Listing.txt, is 9,546 bytes and
was created on Aug. 5, 2013. The Sequence Listing, electronically
filed herewith, does not extend beyond the scope of the
specification and thus does not contain new matter.
TECHNICAL FIELD
[0004] This invention pertains to certain proteins derived from
kexin, nucleic acids encoding those proteins, and the use of the
proteins or nucleic acids as vaccines, for example as vaccines
against Pneumocystis jerovici or other Pneumocystis spp.
BACKGROUND ART
Epidemiology of Pneumocystis Infection
[0005] Despite advances in highly active anti-retroviral therapy
(HAART), opportunistic pulmonary infection with Pneumocystis (PC)
remains the most common opportunistic infection for HIV patients.
Indeed, Pneumocystis pneumonia (PCP) is the index infection for
25-40 percent of AIDS cases. In patients with established AIDS,
prophylactic regimens have decreased the overall incidence of PCP,
but in most patients this means that PCP is delayed rather than
eliminated. For example, in patients with CD4 counts below
200/.mu.l who are on recommended prophylactic regimens, there is
still approximately an 18% risk of active PCP infection over a 36
month period. The widespread use of PCP prophylaxis also means that
more than 80% of PCP cases in the U.S. are now "breakthrough"
cases. Moreover, one study of high-risk children found that the
incidence of PCP had not declined despite efforts to identify
HIV-infected infants and to initiate PCP prophylaxis for them.
[0006] There is a strong correlation between a higher CD4+ T cell
count and a lower risk of PCP. Where HAART is successful, as shown
by an increase in a patient's CD4+ T-cell count above 200/.mu.l,
available data suggest that PCP prophylaxis can be safely
discontinued. Unfortunately, not all AIDS patients respond to
HAART, and drug resistance is emerging. PCP is still a serious
clinical problem in the third decade of the HIV epidemic. There is
an unfilled need for improved methods for PCP prevention and
treatment.
[0007] In AIDS the depletion or dysfunction of CD4+ lymphocytes not
only hinders the patient's immune response to infection, it also
reduces or eliminates the ability to safely and effectively
vaccinate a patient against PCP. Pneumocystis is a genus of fungi
that is found in the respiratory tracts of many mammals and humans
Pneumocystis infection is easily defended by a healthy immune
system. The symptoms of PCP infection include pneumonia, fever, and
respiratory symptoms such as dry cough, chest pain and dyspnea.
Currently, antibiotics are the preferred method of treatment, along
with corticosteroids in some severe cases. The most popular
antibiotic, and the accepted benchmark for efficacy is
Trimethoprim-sulfamethoxazole (TMP-SMX). Alternative antibiotics
are also available due to the severe allergic reactions that some
people have to TMP-SMX. Studies have shown that individuals who are
on highly active antiretroviral therapies (HAART), and who have
CD4+ T-cell counts above a threshold of about 200 cells/mm.sup.3
have a sufficient immune response to defend against PCP infection
without antibiotics. Prophylaxis is recommended for HIV-positive
individuals once their CD4+ T cell count falls below 200
cells/mm.sup.3, and is also recommended for other severely
immunocompromised patients such as transplant patients or leukemia
patients. Drug prophylaxis reduces the incidence of PCP and
lengthens the disease free intervals between episodes. However, the
most effective prophylactic treatment, TMP-SMX, has a high rate of
adverse effects. Second-line drugs may be used, but they typically
have serious side effects and generally are less effective.
[0008] PCP infection will occur in approximately 15%-28% of
individuals with AIDS in a given year. Within the population of
HIV/AIDS patients with PCP, the mortality rate is between 10%-20%.
An estimated 1 million people worldwide suffer from PCP, while
another 5 million people are treated prophylactically to prevent
the disease. Definitive diagnosis of Pneumocystis pneumonia is
relatively complex, requiring microscopy of tissues or fluids. As
PCP prophylaxis and HAART become more widespread, the incidence of
PCP has declined in populations where infection can be properly
diagnosed and treatment can be administered. Studies suggest that
the low prevalence figures reported from developing countries may
simply reflect the lack of adequate infrastructure to properly
diagnose PCP.
[0009] Organ transplant recipients are also at risk for PCP
infection. Transplant recipients take regimens of anti-rejection
drugs that function by suppressing the immune system. The overall
incidence of PCP in solid organ transplant recipients not taking
PCP prophylaxis is about 5%, with the highest incidence following
liver, heart, and lung transplants. The most common prophylaxis
currently used for organ transplant patients is TMP-SMX, or
aerosolized pentamidine if TMP-SMX is not tolerated by the
patient.
[0010] Most currently available antibiotic treatments have mild to
severe side effects, leaving an unfilled need for alternative
treatments. Additionally, antibiotic-resistant Pneumocystis are
emerging, in part because some patients cease treatment due to
allergic reaction or other adverse effects.
[0011] Host Defense and Pneumocystis Infection
[0012] The inability to reliably culture Pneumocystis organisms in
vitro has limited experimental work with the pathogen to clinical
specimens and animal models of infection. Human Pneumocystis
infection is associated more with defects in cell-mediated immunity
than with neutrophil dysfunction. Pneumocystis infections are a
particular clinical problem in AIDS patients, whose progressive
loss of CD4+ helper T lymphocytes results in profound suppression
of cell-mediated immunity. The risk of an HIV-infected adult
acquiring PCP shows an inverse, and almost linear correlation with
the number of circulating CD4+ lymphocytes. A similar relationship
has also been seen for in pediatric PCP infection, although the
relative CD4+ count may be higher in children. The importance of
CD4+ T lymphocytes in host defense against PCP is further supported
by work with animal models. For example, experimental work from our
laboratory shows that normal mice inoculated with P. murina are
able to resolve the infection without treatment, while mice that
have been specifically and selectively depleted of CD4+ T
lymphocytes with an anti-CD4 monoclonal antibody develop
progressive PCP. When administration of the antibody cease and CD4+
lymphocytes are restored, P. murina organisms are cleared from lung
tissue and the PCP infection resolves.
[0013] CD4+ T-Cell Factors in Pneumocystis Infection
[0014] Among the mechanisms used by CD4+ lymphocytes to mediate
host defense against Pneumocystis is the secretion of cytokines
such as interferon (IFN). Lymphocytes exposed to PC organisms or to
the major surface glycoprotein of PC in vitro will secrete IFN.
However, lymphocytes from AIDS patients have a reduced capacity to
produce IFN after antigenic or mitogenic stimulation. Although IFN
is not directly lethal to Pneumocystis, it can activate macrophages
in vitro to kill the organism. However, evidence for an in vivo
role for IFN in host defense is conflicting. In vivo neutralization
of IFN with an antibody has been reported not to alter clearance of
P. murina in reconstituted SCID mice. Also, SCID mice that had been
reconstituted with splenocytes from mice with a homozygous deletion
of the IFN gene were nevertheless able to reduce levels of P.
murina infection.
[0015] It has been postulated that a potential target cell for
exogenous IFN is the alveolar macrophage cell, because aerosolized
IFN will augment expression of these cells. It has been
demonstrated that depletion of alveolar macrophages leads to
delayed clearance of P. carinii from the rat lung.
[0016] Possible mechanisms for IFN bolstering of host defense
include up-regulation of TNF production, increased generation of
superoxide, and increased release of reactive nitrogen
intermediates.
[0017] Overexpression of interferon by gene delivery results in
augmented clearance of P. murina, which depends in part on enhanced
recruitment of CXCR3+ CD8+ T-cells. Although IFN is clearly
therapeutic, endogenous IFN is not required; for example, IFN-gamma
knockout (KO) mice can clear P. murina infection.
[0018] CD40L and T- and B-Cell Immune Responses and Host Defense
Against PC
[0019] CD40L is another factor that is expressed on CD4+ T cells,
and that is critical for host defense against PCP. CD40L (also
known as CD154) is a 33 kDa, type II membrane protein. It is a
member of the tumor necrosis factor (TNF) gene family, and it is a
ligand for CD40 on antigen presenting cells (APC) such as dendritic
cells (DCs) and B cells. It has been recently shown that CD40L
expression in CD4+ T cells is critical for T cell "help," and
permits direct interactions between APCs and CD8+ cytotoxic T
cells. Moreover, as CD40 is also expressed on B cells,
up-regulation of CD40L on CD4+ T cells also is a critical component
of T helper function to induce B cell proliferation.
[0020] CD40L:CD40 interactions appear critical for effective host
defense against PC. Patients with missense or nonsense mutations in
CD40L often have hyper-IgM syndrome. Hyper-IgM syndrome results
from a lack of B-cell differentiation. Patients with hyper-IgM
syndrome are often infected with PC. Antibody blockage of
CD40L:CD40 interactions prevents splenocyte-reconstituted scid mice
from clearing PCP infection. Indeed, 4-6 week old CD40L knockout
mice from a respected laboratory have been inadvertently shipped
infected with PC. Soluble CD40L has been reported to have a
beneficial effect against PCP in a steroid-induced immunosuppressed
rat model.
[0021] DCs genetically engineered to express CD40L have been
reported to present antigens (from Pseudomonas aeruginosa) to
B-cells both in vitro and in vivo in a CD4-independent manner. The
resulting antibodies conferred protection against in vivo challenge
with the bacteria.
[0022] Our laboratory has previously reported the use of kexin,
which is a PC antigen, in a DNA vaccine with or without CD40L. See
M. Zheng et al., "CD4+ T cell-independent DNA vaccination against
opportunistic infections," J. Clin. Invest., vol. 115, pp.
3536-3544 (2005). Despite the promise of Kex1 DNA vaccination,
there remains an unfilled need for improvements to the earlier
vaccine. Vaccination with the Kex1 DNA resulted in only a 2-3 log
improvement in protection as compared to controls; mice challenged
after Kex1 vaccination still have detectable infection
histologically at 28 days post-PC challenge.
Rationale for a Pneumocystis Vaccine
[0023] The pathogenesis of HIV infection involves profound
immunosuppression, which leads to greatly increased susceptibility
to infections. Most opportunistic infections in HIV patients
involve the respiratory tract. Pneumonia caused by the fungal
pathogen Pneumocystis jirovecii remains the most common
AIDS-defining opportunistic infection. Antimicrobial therapies are
available, but emerging antimicrobial resistance is making
treatments less effective. Furthermore, high drug costs can
preclude antimicrobial therapy in many third world countries have
high rates of HIV infection. Even in developed countries, 20-30% of
eligible patients do not receive prophylaxis, either because of
noncompliance or because of the cost of the medications Also,
Pneumocystis colonization is no longer confined to the HIV-infected
population. Pneumocystis spp. are incredibly successful pathogens,
being found in all areas of the world and in numerous animal
species. PCP infection carries a high mortality rate. There remains
a pressing, unfilled need for new vaccines and vaccination
approaches to prevent or treat HIV-associated pulmonary
infections.
[0024] Molecular techniques have recently shown that Pneumocystis
colonization of the respiratory tract is common in many
non-HIV-associated pulmonary diseases, such as emphysema, where PCP
can lead to a systemic inflammatory response and accelerated
progression of obstructive airway disease. Thus, a vaccine against
Pneumocystis can prevent not just the development of pneumonia, but
may also limit co-morbidities of HIV infection, emphysema, and
other diseases.
[0025] Potential candidates to receive a Pneumocystis vaccine would
include individuals who are currently candidates for PCP
prophylaxis, such as HIV-infected persons with a CD4 count below
200; and patients receiving immunosuppressive drugs including
high-dose corticosteroids, and receiving anti-inflammatory agents
such as anti-TNF and anti-B-lymphocyte agents. Such patients would
include transplant recipients, cancer patients (including leukemia
and lymphoma patients), and patients with inflammatory and
autoimmune diseases such as rheumatoid arthritis, lupus, or Crohn's
disease.
[0026] Despite the long-standing need for a vaccine against
Pneumocystis or other fungal pathogens, to our knowledge no fungal
vaccine has yet reached Phase III clinical trials.
Disclosure of the Invention
[0027] We have discovered a vaccine that promotes CD4+ T
cell-independent (CD4IND) host defense mechanisms to defend against
infection by Pneumocystis and other fungi. The vaccine may be used
to prevent or to treat fungal infections, including but not limited
to Pneumocystis spp. The novel vaccine can provide protective
immunity, even for immunocompromised individuals with reduced
levels of CD4+ T cells.
[0028] We used an animal model that mimics HIV-induced CD4+ T cell
deficiency: a CD4-depleted mouse treated with GK1.5, which is a
monoclonal antibody that causes 97% or greater depletion of CD4+ T
cells in spleen, blood, thymus, and lung. We have shown that using
CD40L as an adjuvant allows the generation of protective humoral
immune responses, even in CD4-deficient patients. We identified
immunodominant antigens, including Kex1, a subtilisin-like
protease. Mice that were immunized with Kex1 cDNA via a
DNA-adenovirus vaccine showed significant protection against PC
challenge. Surprisingly, when the vaccine was administered with the
molecular adjuvant CD40L, even mice with CD4+ T-cells could develop
a substantial immune response. By contrast, without the CD40L
adjuvant, there was a poor response in CD4+ T-cell deficient
mice.
[0029] We have improved the Kex1 DNA vaccine by defining and
isolating a smaller antigen, which we have named "mini-kexin." This
antigen will confer protective immunity, especially (but not only)
when administered with a CD40L adjuvant. The mini kexin motif
represents a highly conserved segment across Pneumocystis spp., and
homologs are expressed in other fungi. It thus may also provide
some protection against infection by other Pneumocystis spp. or
other fungi, although we have not yet specifically tested efficacy
against other fungal species. Codon optimization is preferred to
enhance the expression of mini kexin DNA in eukaryotic cells;
preliminary studies suggest that vaccine efficacy is improved with
the codon-optimized version.
[0030] We have also constructed recombinant adenoviruses whose DNA
encodes mini-kexin. In preliminary studies these adenovirus-based
vectors have shown greater efficacy and have provoked greater
mucosal IgA and IgG2a responses in the lung, either as compared to
DNA alone, or as compared to systemic boosting with adenovirus. In
SIV-infected macaques we have examined both anti-Kex1 titers and
the rate of PC lung infection, the latter as determined by nested
PCR in BAL fluid. In a cohort of 12 macaques, 75% (9 of 12) became
PCP positive within 3 months of SIV infection. The three animals
that remained PCP-negative (as determined by PCR in BAL fluid) had
mean serum anti-Kex1 Ab levels that were at least 0-fold greater
than those in PCP-positive monkeys.
[0031] CD40 ligand (CD40L) is expressed on activated CD4+ T cells.
CD40L and is critical for host defense against PC as well as
bacterial pneumonia. We have previously shown that bone-marrow
derived dendritic cells (DCs) can be genetically engineered to
express CD40L (using an E1-deleted adenovirus, AdCD40L), resulting
in significant DC activation and maturation. The activated, mature
DCs, when pulsed with PC or bacterial antigens, and then injected
into mice, produce protective, antigen-specific IgG independently
of CD4+ T cells. This strategy was protective against PC both in
primary-vaccinated, CD4+ T cell-deficient mice, and also in CD4+ T
cell-deficient mice receiving adoptive transfer of immune serum or
CD19+ cells from vaccinated mice. These results demonstrated the
critical role of B cells in protecting against PC after DC
vaccination. We have also observed that dendritic cell IL-23 (but
not IL-12) is required for functional recall antibody responses to
PC antigen challenge. DC-based vaccination can suffer from problems
such as scalability, and the cost of producing patient-specific
DC's. To try to avoid these problems we developed a prime boost
vaccination platform that greatly enhanced protection against PC in
CD4-depleted mice, using the immunodominant antigen from PC, Kexin,
and CD40L as a molecular adjuvant. Both components were required
for vaccine efficacy in CD4-deficient hosts; however, the vaccine
only resulted in a 3-log reduction of organism burden and thus did
not afford complete protection.
[0032] We then undertook two approaches to improve the
effectiveness of the kexin DNA vaccine. One was to use antibody
response to map regions of kexin that were particularly
immunogenic. These results showed that over 70% of the antibody
response was directed against a region of PC kexin that is highly
conserved region across mouse, rat, monkey, and human Pneumocystis
spp. We call this 100 amino acid region "mini Kexin." The second
strategy was to perform mucosal boosting rather than systemic
boosting. In preliminary studies, we found that mucosal boosting
with a recombinant adenovirus encoding mini Kexin afforded
significant greater protection against PC challenge as compared to
systemic boosting.
[0033] We discovered that activation of CD40 signaling in vivo, in
conjunction with vaccination with miniKexin, can produce
antigen-specific immune responses, even in the absence of CD4+
T-cells. We have further discovered that mucosal boosting can
provide effective vaccination against PCP, even in the absence of
CD4+ T-cells.
[0034] In one aspect, the vaccine is used for therapeutic purposes
in early HIV infection, when CD4 numbers remain intact, or it is
used in otherwise immunocompetent hosts who are at risk for
infection (e.g., patients with COPD, cystic fibrosis, or
interstitial lung disease). In another aspect, vaccination is
administered to individuals with advanced HIV infection, or to
other immunosuppressed patients having low numbers of circulating
CD4+ T-lymphocytes, to provide protective immunity.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] FIG. 1 depicts serum levels of anti-PC antibodies following
vaccination with different constructs.
[0036] FIG. 2 depicts anti-PC IgG2a titers both with and without
CD40L.
[0037] FIG. 3 depicts PC burden in mice receiving various vaccines,
both with and without CD40L.
[0038] FIG. 4 depicts PC copy number in the lung 28 days after PC
challenge in mice receiving various vaccines, both with and without
CD40L.
[0039] FIG. 5 depicts IgG titers in mice receiving various
vaccines, both with and without CD40L.
[0040] FIG. 6 depicts IgA titers in mice receiving various
vaccines, both with and without CD40L.
[0041] FIG. 7 depicts the induction of IL-12p40, IL-12p70, and
IL-23 in mice transduced with AdCD40L, versus controls.
[0042] FIGS. 8A, 8B, and 8C depict, respectively, anti-PC IgG1
titers, anti-PC IgG2 titers, and percent killing of PC organisms by
anti-PC serum in response to vaccination with various DCs.
[0043] FIG. 9 depicts the results of PC challenge in prime-boost
vaccinated mice that were artificially immunosuppressed.
MODES FOR PRACTICING THE INVENTION
[0044] Preferably, the vaccine comprises a live recombinant
delivery system, such as a bacterium or virus expressing
mycobacteria genes, or an immunogenic delivery system such as a DNA
vaccine, e.g. a plasmid, expressing one or more genes or gene
fragments for mini-Kexin. Alternatively, the vaccine may comprise a
protein vaccine, that is, the mini-Kexin polypeptide itself or a
portion thereof, in a delivery system including a carrier or an
adjuvant.
[0045] In one embodiment, one aspect of the invention is an
isolated nucleic acid, preferably DNA, wherein said isolated
nucleic acid: [0046] (a) comprises a sequence that encodes
mini-Kexin or a portion thereof, or comprises a sequence
complementary thereto; but does not encode the entire Kexin
protein; or [0047] (b) has a length of at least 10 nucleotides, and
preferably at least 20 nucleotides, and hybridizes readily under
stringent hybridization conditions with a nucleotide sequence as
disclosed herein, or with a nucleotide sequence selected from a
sequence described in part (a) above.
[0048] Another embodiment comprises such a nucleic acid fragment
inserted into a vector. The vector-based vaccine causes in vivo
expression of mini-Kexin or a portion thereof by a human or other
mammal to whom the vaccine has been administered, the amount of
expressed antigen being effective to confer substantially increased
resistance to a pathogenic fungus such as Pneumocystis.
[0049] Another embodiment of a vaccine for immunizing a human or
other mammal against a pathogenic fungus such as Pneumocystis
comprises as the effective component a non-pathogenic
microorganism, wherein at least one copy of a DNA fragment
comprising a DNA sequence encoding mini-Kexin or a portion thereof
has been incorporated into the microorganism (e.g., placed on a
plasmid or in the genome) in a manner allowing the microorganism to
express, and optionally to secrete mini-Kexin or a portion
thereof.
[0050] Another embodiment comprises a replicable expression vector
that comprises a nucleic acid fragment according to the invention,
and a transformed cell harboring at least one such vector.
[0051] Another embodiment comprises a method for immunizing a
mammal, including a human being, against a pathogenic fungus such
as Pneumocystis, comprising administering to the mammal an
effective amount of a vaccine a nucleic acid, a polypeptide, a
vector, or a cell as described.
[0052] A further embodiment comprises a pharmaceutical composition
that comprises an immunologically reactive amount of at least one
member selected from the group consisting of: [0053] (a) the
mini-Kexin polypeptide, or an immunogenic portion thereof; [0054]
(b) a polypeptide whose amino acid sequence has an identity of at
least 70%, 75%, 80%, 85%, 90%, or 95% to any one of said
polypeptides in (a); and is immunogenic; [0055] (c) a fusion
polypeptide comprising at least one polypeptide according to (a) or
(b) and at least one fusion partner; [0056] (d) a nucleic acid that
encodes a polypeptide according to (a), (b) or (c); [0057] (e) a
nucleic acid whose sequence is complementary to the sequence of a
nucleic acid according to (d); [0058] (f) a nucleic acid sequence
having a length of at least 10 nucleotides, or at least 20
nucleotides, that hybridizes under stringent conditions with a
nucleic acid according to (d) or (e); and [0059] (g) a
non-pathogenic micro-organism that has incorporated therein (e.g.
placed in a plasmid or chromosome) a nucleic acid sequence
according to (d), (e), or (f) in a manner to permit expression of
the encoded polypeptide.
[0060] A further embodiment comprises a method for stimulating an
immunogenic response in an human or other mammal by administering
to the human or other mammal an effective amount of at least one
member selected from the group consisting of: [0061] (a) the
mini-Kexin polypeptide, or an immunogenic portion thereof; [0062]
(b) a polypeptide whose amino acid sequence has an identity of at
least 70%, 75%, 80%, 85%, 90%, or 95% to any one of said
polypeptides in (a); and is immunogenic; [0063] (c) a fusion
polypeptide comprising at least one polypeptide according to (a) or
(b) and at least one fusion partner; [0064] (d) a nucleic acid that
encodes a polypeptide according to (a), (b) or (c); [0065] (e) a
nucleic acid whose sequence is complementary to the sequence of a
nucleic acid according to (d); [0066] (f) a nucleic acid sequence
having a length of at least 10 nucleotides, or at least 20
nucleotides, that hybridizes under stringent conditions with a
nucleic acid according to (d) or (e); and [0067] (g) a
non-pathogenic micro-organism that has incorporated therein (e.g.
placed in a plasmid or chromosome) a nucleic acid sequence
according to (d), (e), or (f) in a manner to permit expression of
the encoded polypeptide.
Definitions
[0068] Unless context clearly indicates otherwise, the following
definitions should be understood to apply throughout the
specification and claims. Other terms, those for which specific
definitions are not given, should be interpreted as they would be
understood, in context, by a person of skill in the art:
[0069] The word "polypeptide" or "protein" or "peptide" should have
its usual meaning: an amino acid chain of any length, including a
full-length protein, oligopeptide, short peptide, or fragment
thereof, wherein the amino acid residues are linked by covalent
peptide bonds. As used in the present specification and claims,
unless context clearly indicates otherwise, no distinction is
intended between the terms "polypeptide," "peptide," and "protein,"
which should be considered synonymous.
[0070] The polypeptide may be chemically modified by being
glycosylated, phosphorylated, lipidated, by incorporating one or
more prosthetic groups or functional group, or by containing
additional amino acids such as e.g. a his-tag or a signal
sequence.
[0071] Each polypeptide may thus be characterized by specific amino
acids and be encoded by specific nucleic acid sequences. It will be
understood that such sequences include analogues and variants
produced by recombinant or synthetic methods wherein such
polypeptide sequences have been modified by substitution,
insertion, addition or deletion of one or more amino acid residues
in the recombinant polypeptide and are still immunogenic.
Substitutions are preferably conservative.
[0072] A "substantially pure polypeptide fragment" means a
polypeptide preparation that contains at most 5% by weight of other
polypeptide material (lower percentages of other polypeptide
material are preferred, e.g. at most 4%, at most 3%, at most 2%, at
most 1%, and at most 1/2%). It is preferred that the substantially
pure polypeptide is at least 96% pure, i.e. that the specified
polypeptide constitutes at least 96% by weight of total polypeptide
material present in the preparation, and higher percentages are
preferred, such as at least 97%, at least 98%, at least 99%, at
least 99.25%, at least 99.5%, and at least 99.75%. It is especially
preferred that the polypeptide fragment is in "essentially pure
form", i.e. that the polypeptide fragment is essentially free of
any other antigen with which it is natively associated, i.e.
essentially free of any other antigen from the same fungus. This
can be accomplished by preparing the polypeptide fragment by means
of recombinant methods in a non-fungal host cell, or by
synthesizing the polypeptide fragment by the well-known methods of
solid or liquid phase peptide synthesis, e.g. by the method
described by Merrifield or variations thereof.
[0073] The term "nucleic acid fragment" (or "nucleic acid
sequence") means any nucleic acid molecule including DNA, RNA, LNA
(locked nucleic acids), PNA, RNA, dsRNA and RNA-DNA-hybrids. A
preferred nucleic acid for use in this invention is DNA. Also
included are nucleic acid molecules comprising non-naturally
occurring nucleosides. The term includes nucleic acid molecules of
any length, e.g. from 10 to 10,000 nucleotides, depending on the
use and context. The nucleic acid molecule is optionally inserted
into a vector.
[0074] The term "stringent" when used in conjunction with
hybridization conditions has the meaning generally understood in
the art, i.e. the hybridization is performed at a temperature not
more than 15-20.degree. C. under the melting point T.sub.m, cf.
Sambrook et al, 1989, pages 11.45-11.49. Preferably, the conditions
are "highly stringent", i.e. 5-10.degree. C. under the melting
point T.sub.m.
[0075] The term "sequence identity" indicates a quantitative
measure of the degree of homology between two amino acid sequences
of equal length or between two nucleotide sequences of equal
length. The two sequences to be compared are aligned to the best
possible fit, allowing for the insertion of gaps or alternatively,
for truncation at one or both ends. The sequence identity can be
calculated as (N.sub.ref-N.sub.dif)100/N.sub.ref, wherein N.sub.dif
is the total number of non-identical residues in the two sequences
when aligned, and wherein N.sub.ref is the number of residues in
one of the sequences. Hence, the DNA sequence AGTCAGTC has a
sequence identity of 75% with the sequence AATCAATC (N.sub.dif=2
and N.sub.ref=8). A gap is counted as non-identity of the specific
residue(s), i.e. the DNA sequence AGTGTC has a sequence identity of
75% with the DNA sequence AGTCAGTC (N.sub.dif=2 and N.sub.ref=8).
Sequence identity can alternatively be calculated by available
software, such as BLAST, e.g. the BLASTP program (Pearson, 1988, or
available through ncbi.nlm.nih.gov). Alignment may also be
performed with the sequence alignment method ClustalW with default
parameters as described by Thompson J., et al 1994, available at
http://www2.ebi.ac.uk/clustalw/.
[0076] A preferred minimum percentage of sequence identity is at
least 80%, such as at least 85%, at least 90%, at least 91%, at
least 92%, at least 93%, at least 94%, at least 95%, at least 96%,
at least 97%, at least 98%, at least 99%, and at least 99.5%.
[0077] "Variants." A common feature of the polypeptides of the
invention is their capability to induce an immunological response.
It is understood that a variant of mini-Kexin produced by
substitution, insertion, addition or deletion may also be
immunogenic as determined by any of the assays described
herein.
[0078] An "immune individual" is a human or other mammal who has
cleared or controlled an infection with a virulent fungus such as
Pneumocystis, or who has received a vaccination in accordance with
this invention.
[0079] The "immune response" of an individual may be monitored by
any one of several methods known in the art, including for example
one or more of the following:
[0080] A cellular response may be determined in vitro by induction
of the release of a relevant cytokine such as IFN-.gamma. from, or
the induction of proliferation in lymphocytes withdrawn from a
human or other mammal currently or previously infected with
virulent fungus or directly or indirectly immunized with
polypeptide. The induction may be performed by the addition of the
polypeptide or an immunogenic portion of the polypeptide to a
suspension comprising from 2.times.10.sup.5 cells to
4.times.10.sup.5 cells per well. The cells are isolated from blood,
the spleen, the liver, or the lung, and the addition of the
polypeptide or the immunogenic portion results in a concentration
of not more than 20 .mu.g per ml in suspension, with the
stimulation being performed over a period from two to five days. To
monitor cell proliferation the cells are pulsed with
radioactively-labeled thymidine; after 16-22 hours of incubation
liquid scintillation counting is used to assess proliferation. A
positive response is considered to be one that exceeds background
by at least two standard deviations. The release of IFN-.gamma. can
be determined, e.g., by the ELISA method, or other methods known in
the art. Other cytokines besides IFN-T may be used to assess
immunological response to the polypeptide, such as IL-12,
TNF-.alpha., IL-4, IL-5, IL-10, IL-6, or TGF-.beta.. A sensitive
method for detecting an immune response is the ELISpot method for
determining the frequency of IFN-.gamma. producing cells. In an
ELIspot plate (MAHA, Millipore) that is pre-coated with anti-murine
IFN-.gamma. antibodies (PharMingen), graded numbers of cells
isolated from blood, spleen, or lung (typically from 1 to
4.times.10.sup.5 cells/well) are incubated for 24-32 hrs in the
presence of the polypeptide or an immunogenic portion therefor, at
a concentration not more than about 20 .mu.g per ml. The plates are
subsequently incubated with biotinylated anti-IFN-.gamma.
antibodies followed by a streptavidin-alkaline phosphatase
incubation. The cells producing IFN-.gamma. are identified by
adding BCIP/NBT (Sigma), and the relevant substrates develop spots.
These spots can be enumerated with a dissection microscope. It is
also possible to determine the presence of mRNA that encodes the
relevant cytokine by PCR. Usually one or more cytokines will be
measured using, for example, PCR, ELISPOT, or ELISA. It will be
appreciated by a person skilled in the art that the immunological
activity of a particular polypeptide can be evaluated by observing
whether there is a significant increase or decrease in the amounts
of these cytokines.
[0081] A cellular response may also be determined in vitro with T
cell lines derived from an immune individual, or a
Pneumocystis-infected person, where the T cell lines have been
driven with either live fungus, extracts from the fungus, or
culture filtrate for 10 to 20 days, with the addition of IL-2. The
induction is performed by adding not more than 20 .mu.g polypeptide
per ml suspension to the T cell lines, from 1.times.10.sup.5 cells
to 3.times.10.sup.5 cells per well, with incubation from two to six
days. The induction of IFN-.gamma. or the release of another
relevant cytokine is detected by ELISA. The stimulation of T cells
can also be monitored by detecting cell proliferation using
radioactively labeled thymidine as described above. For both assays
a positive response is considered to be one that is at least two
standard deviations above background.
[0082] A humoral response may be determined in vitro by a specific
antibody response from an immune or infected individual. The
presence of antibodies may be determined through methods known in
the art, e.g., by ELISA or Western blot. The serum is preferably
diluted in PBS from 1:10 to 1:100 and added to the adsorbed
polypeptide, with incubation from 1 to 12 hours. By the use of
labeled secondary antibodies the presence of specific antibodies
can be determined by measuring the OD, e.g. by ELISA, where a
positive response is considered to be one that is at least two
standard deviations above background, or alternatively by a visible
response in a Western blot.
[0083] Protein Vaccine.
[0084] Another aspect of the invention pertains to a vaccine
composition comprising the mini-Kexin polypeptide, or an
immunogenic portion thereof, or a fusion polypeptide thereof. It is
preferred that the vaccine additionally comprise an immunologically
and pharmaceutically acceptable carrier, vehicle or adjuvant.
[0085] Suitable carriers for polypeptides may be selected from the
group consisting of a polymer to which the polypeptides are bound
by a hydrophobic, non-covalent interaction, such as a polystyrene,
or a polymer to which the polypeptides are covalently bound, such
as a polysaccharide, or a polypeptide, e.g. bovine serum albumin,
ovalbumin, or keyhole limpet haemocyanin. Suitable vehicles may be
selected from the group consisting of a diluent and a suspending
agent. The adjuvant is preferably selected from the group
consisting of dimethyldioctadecylammonium bromide (DDA), Quil A,
poly I:C, aluminum hydroxide, Freund's incomplete adjuvant,
IFN-.gamma., IL-2, IL-12, monophosphoryl lipid A (MPL), Trehalose
Dimycolate (TDM), Trehalose Dibehenate, and muramyl dipeptide
(MDP).
[0086] The preparation of vaccines that contain polypeptides as
their active ingredients is generally well understood in the art,
as exemplified by U.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231
and 4,599,230, and published application US2004/0057963, the
complete disclosures of all of which are incorporated herein by
reference.
[0087] Other methods of achieving adjuvant effect for a vaccine
include the use of agents such as aluminum hydroxide or aluminum
phosphate (alum), synthetic polymers of sugars (Carbopol),
aggregation of the polypeptide in the vaccine by heat treatment,
aggregation by reactivating with pepsin-treated (Fab) antibodies to
albumin, mixture with bacterial cells such as C. parvum or
endotoxins or other lipopolysaccharide components of gram negative
bacteria, emulsion in physiologically acceptable oil vehicles such
as mannide mono-oleate (Aracel A), or emulsion with 20 percent
solution of a perfluorocarbon (Fluosol-DA) used as a block
substitute. Other possibilities involve the use of
immune-modulating substances such as cytokines or synthetic
IFN-.gamma. inducers such as poly I:C in combination with an
adjuvant.
[0088] Another possibility for achieving adjuvant effect is to
conjugate the polypeptide or a portion thereof to an antibody (or
antigen binding antibody fragment) against the Fc.gamma. receptors
on monocytes/macrophages.
[0089] The vaccines are administered in a manner that is compatible
with the dosage formulation, and in an effective, immunogenic
amount. The quantity to be administered depends on the subject to
be treated, including, e.g., the capacity of the individual's
immune system to mount an immune response, and the degree of
protection desired. Suitable dosage ranges are of the order of
several hundred micrograms active ingredient per vaccination with a
preferred range from about 0.1 .mu.g to 1000 .mu.g, such as in the
range from about 1 .mu.g to 300 .mu.g, and especially in the range
from about 10 .mu.g to 50 .mu.g, as may readily be determined by
routine experimentation such as is well known in the art. Suitable
regimens for initial administration and booster shots are also
variable but are typified by an initial administration followed by
subsequent inoculations or other administrations.
[0090] As used in the specification and claims, an "effective
amount" or an "effective dosage" of a vaccine is an amount or
dosage, that when administered to a patient (whether as a single
dose or as part of a multi-dose or boosting regimen) provides
protective immunity to a clinically significant degree; or
alternatively, to a statistically significant degree as compared to
control. "Statistical significance" means significance at the
P<0.05 level, or such other measure of statistical significance
as would be used by those of skill in the art of biomedical
statistics in the context of immunization.
[0091] The manner of application may be varied. Any of the
conventional methods for administration of a vaccine are
applicable. These can include oral application on a solid
physiologically acceptable base or in a physiologically acceptable
dispersion, parenterally, by inhalation, by injection or the like.
The dosage of the vaccine will depend on the route of
administration and will vary according to the age of the person to
be vaccinated and, to a lesser degree, the size of the person to be
vaccinated.
[0092] The vaccines are conventionally administered parenterally,
by injection, for example, either subcutaneously or
intramuscularly. Additional formulations which are suitable for
other modes of administration include suppositories and, in some
cases, oral formulations and inhalable aerosols. For suppositories,
traditional binders and carriers may include, for example,
polyalkylene glycols or triglycerides; such suppositories may be
formed from mixtures containing the active ingredient in the range
of 0.5% to 10%, preferably 1-2%. Oral formulations include such
normally employed excipients as, for example, pharmaceutical grades
of mannitol, lactose, starch, magnesium stearate, sodium
saccharine, cellulose, magnesium carbonate, and the like. These
compositions may take the form of solutions, suspensions, tablets,
pills, capsules, sustained release formulations or powders and
advantageously contain 10-95% of active ingredient, preferably
25-70%.
[0093] DNA Vaccine.
[0094] In a preferred embodiment, nucleic acid fragments in
accordance with the invention are used for the in vive expression
of antigens, i.e. in so-called DNA vaccines as reviewed in Ulmer et
al 1993, which is incorporated by reference. Hence, the invention
also relates to a vaccine comprising a nucleic acid fragment
according to the invention, the vaccine causing in vivo expression
of antigen by a human or other mammal, the amount of expressed
antigen being effective to confer substantially increased
resistance to infections caused by virulent fungi, including for
example Pneumocystis jerovici or other Pneumocystis spp.
[0095] Live Recombinant Vaccines; Plasmids.
[0096] Another possibility for effectively activating a cellular
immune response is to express the antigen in a non-pathogenic
microorganism or virus that is then used as a vaccine. Well-known
examples of such microorganisms are Mycobacterium bovis BCG,
Salmonella, and Pseudomonas, and examples of such viruses are
Vaccinia Virus and Adenovirus.
[0097] Accordingly, another aspect of the present invention is to
incorporate one or more copies of a DNA sequence as described into
the genome of the microorganism or virus in a manner allowing the
micro-organism to express and secrete the polypeptide. The
incorporation of more than one copy of a nucleotide sequence of the
invention may enhance the immune response.
[0098] Another possibility is to integrate the DNA encoding the
polypeptide in an attenuated virus such as the vaccinia virus or
Adenovirus (Rolph et al 1997). The genes carried by the recombinant
vaccinia virus are expressed within an infected host cell, and the
expressed polypeptide of interest can induce an immune
response.
[0099] Because the target population for this vaccine will often
have a compromised immune system, even attenuated live vaccines may
be inappropriate vehicles. In such cases, it can be preferred to
administer the DNA sequence in a non-replicating vehicle, such as a
plasmid or a disabled virus that is capable of delivering DNA to a
host cell, but that is incapable of replicating in the host.
Example 1. Co-Administration of CD40L with Mini-Kexin Vaccination
Induces a CD4IND Humoral Response and Protection Against PC In
Vivo
[0100] Four forms of mini-Kexin DNA are used for vaccination:
mini-Kexin-wild type (mKexin-WT); mini-Kexin that has been codon
optimized for mammalian expression (mKexin-CO); miniKexin that has
been engineered to be secreted with an IgGC leader sequence
(smKexin); and smKexin that has been codon optimized (smKexin-CO).
We compared these wild type and codon-optimized forms of the DNA
vaccine. We also compare mucosal boosting with recombinant
adenovirus and recombinant modified vaccinia Ankara strain (MVA)
vectors. Outcome measures include anti-Kexin and anti-PC
isotype-specific antibody responses, as well as anti-Kexin subclass
determinations. Serum is tested in functional assays including
opsonic phagocytosis, and passive transfer protection into scid
mice. We also examine the efficacy of the vaccine against PC
challenge performed at several times after vaccination.
Example 2
[0101] Our hypothesized mechanism predicts that endogenous IL-23 is
required; and results in durable vaccine responses in both CD4+
T-cell deficient mice and CD40L knockout mice. Specifically we
demonstrate the efficacy of CD40L co-transduction in CD40L knockout
mice; and the requirement of IL-12 family members (including
IL-12p35, IL-12p40, and IL-23), and critical activation molecules
that are induced by CD40L-modified DCs to generate effective
primary and memory B-cell responses. Preliminary studies have
suggested that IL-23 production is critical to generate B-cell
memory against PC antigen.
Example 3. CD4IND Pathogen-Specific Immune Responses Against
Pneumocystis Kexin are Generated in an SIV Model of
Immunodeficiency in Macaques
[0102] We expect that the mini-kexin constructs will produce
vaccine-induced immune responses in SIV-infected, CD4 deficient
macaques. Control or SIV infected macaques will undergo DNA
priming, followed by mucosal boosting 4 weeks after mock or live
SIV infection. Outcome measures will include humoral responses to
the vaccine, and the prevention of Pneumocystis colonization as
determined by PCR of BAL fluid. Preliminary studies suggest that
Pneumocystis colonization occurs in up to 80% of SIV infected
macaques, compared to 0% in non-SIV infected monkeys. We will also
challenge SIV-infected monkeys with live Pneumocystis, and
demonstrate vaccine efficacy in the challenge model.
Example 4
[0103] We generated anti-Pneumocystis antibodies in CD4-deficient
mice by vaccination with PC-pulsed, CD40L-transduced, bone
marrow-derived dendritic cells. These antibodies stain the surface
of PC, and enhance opsonic phagocytosis and killing of PC in a
dectin-1-independent but Fc-dependent manner. These antibodies also
confer significant protection against PC when passively transferred
to scid mice prior to PC challenge.
Example 5
[0104] We identified antigen specificities using both 1-dimensional
and 2-dimensional electrophoresis, as well as immunoprecipitation
followed by 2-D gel electrophoresis. Silver-stained spots on 2-D
gels were picked, enzymatically-digested, and analyzed by tandem MS
(Applied Biosystems). We also performed N-terminal sequencing on
proteins. Due to a lack of published data for the entire PC genome,
and in light of the significant homology of many PC genes to those
of Saccharomyces cerevisiae and S. pombe, we performed homology
searches against PC and Saccharomyces spp. One antigen consistently
identified by both MS-MS and N-terminal sequencing was kexin (also
called Kex1). Kex1 is a protease with high homology to furin. Kexin
is presumably involved in processing of pre-pro proteins in yeast.
Monoclonal antibodies raised against Kex1 show protective efficacy
in murine models of PCP.
Example 6
[0105] We cloned the full length Kex1 cDNA, and generated DNA
vaccines, both with and without an additional open reading frame
encoding CD40L as a B-cell adjuvant. CD4-deficient mice that were
immunized by intramuscular DNA encoding Kex1 and CD40L developed
significant anti-Pneumocystis antibody titers, as well as
approximately a three log protection against PC challenge. Moreover
these antibodies stained the surface of PC organisms from mouse and
monkey, and enhanced opsonic phagocytosis and killing of mouse PC
in vitro. However, despite the efficacy of full-length Kex1
vaccination, vaccinated mice still had readily detectable infection
4 and 6 weeks after challenge.
Example 7
[0106] To improve upon our original Kex1 vaccine we tried several
approaches. The first was to examine if mucosal boosting with
recombinant adenovirus would enhance DNA priming. Although
full-length Kex1 could be packaged, the recombinant Ad5-based
vectors grew poorly, with titers of 10.sup.7 or 10 per ml. The Kex1
coding sequence is over 3 kB. We explored whether we could improve
both packaging and expression by truncating the antigen and by
using codon optimization. Our analysis of Kex1 revealed a 100 amino
acid segment of Kex1 with over 75% homology among PC organisms
obtained from mouse, rat, monkey, and human hosts. (See FIG. 1 from
priority application 61/294,252, not reproduced here but
incorporated by reference.)
Example 8
[0107] Expressing peptides from this region of Kex1 in recombinant
E. coli, we demonstrated that antibodies recognizing epitopes in
this region account for a significant amount of the opsonic killing
of PC. PC organisms were opsonized with naive serum (control), or
with serum from mice vaccinated with full length kexin/CD40L, and
then incubated with peritoneal macrophages to assess opsonic
killing in vitro. To assess viability of PC organisms 24 hours
later, we measured the integrity of the PC mitochondrial large
subunit mRNA by real time PCR. Opsonization of PC with serum from
Kexin/CD40L-vaccinated mice markedly increased PC killing in vitro.
Absorption of the serum against Kexin peptides or against miniKexin
markedly decreased opsonic phagocytosis, suggesting that
recognition of epitopes in this 100 as stretch of Kexin is
important to activity. (See FIG. 2 from priority application
61/294,252, not reproduced here but incorporated by reference.)
Example 9
[0108] We next performed passive transfer experiments into scid
mice using control serum, serum from Kexin/CD40L vaccinated mice,
and serum from vaccinated mice that had been pre-adsorbed against
recombinant Kexin. The mice were then challenged with PC
(2.times.10.sup.5 cysts) intratracheally. Mice were sacrificed at
day 28, and PC burden in the lung was assessed by real-time PCR.
Transfer of 300 .mu.L of serum from Kexin/CD40L-vaccinated mice
resulted in significantly reduced PC burden as compared to control
serum. Adsorption of serum against recombinant Kexin significantly
attenuated the protection of the transferred serum. (See FIG. 3
from priority application 61/294,252, not reproduced here but
incorporated by reference.)
Example 10
[0109] We modified the vaccine by constructing vectors encoding the
100 amino acid conserved region of Kex1 that we identified, a
region that we have named "mini-Kexin." We constructed 4 DNA
vaccines: (1) wild-type mini Kexin without a leader sequence, (2)
wild type mini Kexin with an IgGk leader sequence to facilitate
secretion, (3) codon-optimized mini Kexin with no leader sequence,
and (4) codon optimized mini Kexin with a IgGk leader sequence.
These vectors were called, respectively: (1) pmini-Kexin WT, (2)
psec-mini-Kexin-WT, (3) pmini-Kexin CO, and (4) psec-mini-Kexin-CO.
To assess the secretion of mini-Kexin we transfected 293 cells with
these constructs and assayed for Kexin by direct ELISA in cell
lysates or in cell supernatants 48 hours after transfection. (See
FIG. 4 from priority application 61/294,252, not reproduced here
but incorporated by reference.) The addition of the IgG-kappa
leader sequence in the psec-mini Kexin constructs resulted in
higher levels of Kexin in cell supernatants. Moreover, codon
optimization was associated with higher expression. Thus a
preferred embodiment uses both a leader sequence and codon
optimization.
Example 11
[0110] We next examined the efficacy of these constructs in DNA
vaccination of CD4-depleted mice. For these studies, mice were
vaccinated with pmini-Kexin WT (K-wt), psec-mini-Kexin-WT (sK-wt),
pmini-Kexin CO (K-co), or psec-mini-Kexin-CO (sK-co). The mice were
vaccinated with constructs either lacking CD40L or with a sequence
encoding CD40L cloned into the second open reading frame of the
plasmids, to act as a B-cell adjuvant. Mice were vaccinated by two
injections of 100 .mu.g DNA, given intramuscularly three weeks
apart. Anti-PC antibodies were measured by ELISA 7 days after the
second injection of DNA. FIG. 1 shows serum levels for anti-PC
antibodies, measured as end point dilutions (1:64). Mice vaccinated
with psec-mini-Kexin-CO (leader sequence, codon optimized) had the
highest levels of anti-PC IgG1. Interestingly, the presence or
absence of CD40L seemed to have little effect on anti-PC IgG1
titers (FIG. 1, * denotes p<0.05 compared to SK-wt, K-wt, and
K-co, ANOVA, n=6-8 per group). By contrast, the presence of CD40L
was associated with significant increases in anti-PC IgG2a titers
as compared to constructs lacking CD40L (FIG. 2, * denotes
p<0.05 compared to the non-CD40L group, ANOVA, n=6-8 per
group).
Example 12
[0111] To test the protective effect of the antibodies against PC
infection, we performed a PC challenge following the second dose of
DNA. The mice were sacrificed 28 days later to assess PC organism
burden in lung tissue (FIG. 3). Mice vaccinated with
psec-kexin-Co-CD40L had the lowest organism burden in the lung
compared to all other groups (*p<0.01 ANOVA, n=6 per group).
Furthermore, the addition of CD40L was associated with lower
organism burdens in all vaccine groups compared to mice vaccinated
without CD40L. Nevertheless, there were still between 10.sup.6 and
10.sup.7 PC organisms present, even in the psec-kexin-Co-CD40L
group.
Example 13
[0112] We examined whether mucosal boosting can augment protection
against PC. We constructed recombinant Ad5-based vectors encoding
all four of the mini-Kexin constructs described above. Mice were
primed with 2 IM injections of DNA, followed either by no mucosal
boost or by intranasal boosting with 10 PFU of Ad5 encoding the
same construct as was used for the DNA prime vaccination. FIG. 4
depicts PC copy number in the lung 28 days after PC challenge in
these mice. The mucosal boost with AdCD40L resulted in nearly a
three log improvement in both the sK-co and k-Co groups (p<0.01
ANOVA, n=6 per group, compared to SK-wt or K-wt with CD40L). Immune
response was enhanced with CD40L, both when used in the DNA prime
as well as when used in the adenovirus boost. The improved
protection against PC challenge was associated with higher anti-PC
IgG titers (FIG. 5, n=6 each group, * p<0.05 ANOVA) as well as
higher anti-PC IgA titers in BAL (FIG. 6, n=6 each group, *
p<0.05 ANOVA). These preliminary data suggest that psecKexin-co
and pmini-Kexin-co with CD40L are superior vaccines.
Example 14
[0113] Our hypothesis predicts that this strategy: (1) should
require endogenous IL-23, and (2) should result in durable vaccine
responses both in CD4+ T-cell deficient mice and in CD40L knockout
mice. We have demonstrated that AdCd40L is a potent inducer of
IL-12p40, IL-12p70, and IL-23 (FIG. 7). For these experiments, bone
marrow-derived dendritic cells were grown from hematopoietic
progenitors, and transduced with AdLuc or AdCD40L at a dose of 100
viral particles per cell. Supernatants were collected 24 hours
later and assayed for IL-12p40 or IL-12p70 by Luminex, or for IL-23
by ELISA (n=5 per group, * denotes p<0.05 compared to AdLuc
controls).
Example 15
[0114] To determine the role of IL-12 and IL-23 in
AdCD40L-transduced, DC-based vaccine responses, we generated DCs
from IG-12p40-/-, IL-12p35-/-, or IL-23p19-/- mice; transduced each
DC genotype with AdCD40L; pulsed the DCs with PC antigen; and then
administered the DCs intravenously to CD4-depleted mice. Primary
antibody responses were measured after 4 weeks. To assess recall
responses, mice were re-challenged with PC antigen by IP injection,
and serum antibody responses were measured 10 days later. As shown
in FIG. 8A, primary IgG1 responses were similar regardless of the
DC expression of IL-12p40, IL-12p35, or IL-23p19. Mice vaccinated
with IL-23-deficient DCs (either IL-12p40-/- or IL-23p19-/- DCs)
had reduced primary IgG2a responses to PC (FIG. 8B). Furthermore,
recall response to PC antigen, as a measure of B-cell memory, was
significantly diminished both in IL-12p40-/- and in IL-23p19
deficient DCs, but not in IL-12p35-/- DCs (FIGS. 8A and 8B, *
denotes p<0.05 as compared to the other groups, ANOVA, n=5-6 per
group). These data demonstrated that IL-23 is an important mediator
of CD40L-induced B cell expansion and antigen-specific recall
responses. The defect in functional B-cell memory was also
associated with diminished opsonic killing activity of anti-PC
serum from CD4-depleted mice vaccinated with DCs from either
IL-12p40/- or IL-23p19-/- mice (FIG. 8C, * denotes p<0.05
compared to the other groups, ANOVA, n=5-6 per group).
Example 16
[0115] CD4IND, pathogen-specific immune responses against
Pneumocystis kexin are produced in an SIV model of immunodeficiency
in macaques. We have assessed spontaneous PC infection in macaques
infected with SIV/Delta B670. CD4 counts below 500 cells/.mu.L have
been strongly associated with an increase in PC colonization in the
lung, as assessed by nested PCR in BAL fluid. Five of five
SIV-infected monkeys developed detectable PC colonization, as
assayed by nested PCR. Interestingly, several monkeys had an
initial increase in anti-Kexin antibody titers, followed by a fall
in titers prior to the development of a positive PCR response for
PC. In a second cohort of animals, preliminary studies suggested
that SIV-infected monkeys with high baseline anti-Kex1 titers were
protected against PC infection, as measured by PCR in BAL. We will
determine the rate of PC infection at necropsy in 25 SIV-infected
and 25 non-SIV-infected macaques. For these studies we will assess
PC colonization by nested PCR, and real-time PCR on lung tissue and
BAL. These data will also be compared to standard histological
detection of PC by GMS staining of lung tissue.
Example 17
[0116] Additional studies confirm that CD 40L co-administration
results in CD4IND immune responses after IM DNA vaccination,
through B-cell responses. Our preliminary results suggest that
codon-optimized, secreted antigen is a better driver of the B cell
response as compared to non-secreted or wild-type forms of Kexin.
We have cloned the kexin constructs and CD40L into pBUDCE4.1
(Invitrogen), which contains two expression cassettes (one driven
by CMV and one driven by the human EF-1 alpha promoter), allowing
both genes to be effectively expressed in transduced cells
Example 18
[0117] Experimental Groups. Male 6-8 week old BALB/c mice will be
CD4-depleted by the administration of 0.3 mg GK1.5 IP by weekly
injection, or given rat IgG as a control. 48 hours later, mice will
be randomized to be vaccinated by the IM injection of pmini-Kexin
WT, psec-mini-Kexin-WT, pmini-Kexin CO, or psec-mini-Kexin-CO; and
in each case, either with or without CD40L. One group of mice will
be injected with pBudCD40L with no PC antigen as a control. Sample
size will be 10 mice per group, which should give sufficient
statistical resolution to detect a .about.30% difference in anti-PC
or Kexin IgG between different routes of vaccination.
[0118] Manipulations:
[0119] There will be two doses of plasmid injections, three weeks
apart.
[0120] Measures and Outcomes:
[0121] Sera will be assayed for anti-PC and anti-Kexin antibodies,
both IgM and IgG, by ELISA at 3, 6, and 9 weeks. Isotypes will be
determined by using appropriate anti-mouse IgG isotypes: IgG1,
IgG2a, IgG2b, IgG3 (Pierce, Rockford, Ill.). At 9 weeks, mice will
be sacrificed and lungs will be lavaged for anti-PC and anti-Kexin
IgA levels. Kexin-specific, IgG-expressing B cells in the spleen
and mediastinal lymph nodes will be assayed by Elispot. Serum
antibodies will also be tested for complement-dependent killing,
and for opsonic phagocytosis and killing of PC in vitro. Briefly,
serial dilutions of sera will be incubated with PC cysts and
cultured in RPM1640+ 10% FCS (with or without heat inactivation)
for 24 hours, followed by assessment of PC mtLSU rRNA integrity by
real-time PCR. To assess macrophage-dependent killing the assay is
performed in the presence of 50,000 alveolar macrophages obtained
by lung lavage. If we observe high antibody titers and augmentation
of PC killing in vitro, we will also perform passive transfer
experiments in scid mice with 300 .mu.L of serum, followed by
pulmonary PC challenge. Scid mice will be sacrificed 28 days later
to determine if the passive transfer of serum prevents PC
infection.
[0122] Expected Results and Interpretations:
[0123] We expect to observe significant induction of anti-PC and
anti-Kexin IgG in CD4-depleted mice vaccinated with pmini-Kexin WT,
psec-mini-Kexin-WT, pmini-Kexin CO, or
psec-mini-Kexin-CO--particularly when CD40L is included in the
plasmid. We also expect that CD40L will be required for efficient
IgA production in BAL in these mice. Our preliminary data also
suggest that IgG levels, and perhaps Kexin-specific IgG- and
IgA-producing B-cells are enhanced in the secreted Kexin/codon
optimized, CD40L groups in the spleen. We expect the precursor
frequency to be significantly lower in the mediastinal lymph nodes
as compared to spleen in this stage of the vaccine. We also expect
to observe vaccine-induced increases in opsonic activity and
killing of PC in vitro, as well as protection in passive transfer
experiments.
[0124] Alternative Approaches:
[0125] We expect to observe a mixed TH1 and TH2 antibody response,
since IL-4 has been reported to have synergy with IL-12 p70 and
CD40L in activating B-cells to a TH2 response. Alternatively,
activated DC's have been shown to induce preferential TH1 responses
in B-cells. Thus we will observe the roles of specific endogenous
cytokines such as IL-12, IL-23, in IL-12p40, IL-12p35, and IL-23
knockout mice. If we observe only a modest anti-PC/Kexin IgG
response in the IM regimen, we will investigate electroporation
after DNA administration. Electroporation enhances CD8+ T-cell
responses. Its effect on humoral immune response is unclear, but
and we are already achieving significant B-cell responses, and
therefore see electroporation as being less preferred. The titer or
half life of the antibody in the scid mice will be confirmed by
measuring the titer both immediately after transfer and on day 28.
Prior studies with DC-based vaccines suggest that 300 .mu.L of
serum should provide sufficient Ab to protect during the 28 day
study period.
Example 19. The Effect of Mucosal Boosting with Recombinant
Adenovirus Virus-Based Vectors Following DNA Priming
[0126] Hypothesis:
[0127] We hypothesize that the co-administration of CD40L with
antigen allows for CD4-independent (CD41ND) B-cell responses in
vivo, and that mucosal boosting will enhance mucosal
antigen-specific B-cell responses, as well as overall protective
immunity.
[0128] Rationale:
[0129] Preliminary studies have demonstrated that CD40L
co-administered with Kexin antigen resulted in CD4IND B-cell
responses. To optimize the in vivo response, we postulate that
mucosal boosting with recombinant adenovirus vectors will enhance
mucosal IgA and IgG B-cell responses.
[0130] Experimental Groups.
[0131] Male 6-8 week old BALB/c mice will be CD4-depleted by the
administration of 0.3 mg GK1.5 IP by weekly injection or given rat
IgG as a control for the CD4-replete group. 48 hours later, mice
will be randomized to be vaccinated by the IM injection of
pmini-Kexin WT, psec-mini-Kexin-WT, pmini-Kexin CO, or
psec-mini-Kexin-CO, in each case with or without CD40L. One group
of mice will be injected with pBudCD40L with no PC antigen as a
control. After 6 weeks mice will be further randomized for mucosal
boosting with 10.sup.7 adenovirus encoding the same antigen
construct as the prime vaccination, with or without an equal dose
of AdCD40L. Sample sizes will be 10 mice per group, to give the
statistical resolution to detect a 30% difference in anti-PC or
Kexin IgG between different routes of vaccination.
[0132] Manipulations:
[0133] Plasmid injections will be repeated every three weeks for
two doses as otherwise previously described by Ramsay et al. We
will administer 10.sup.7 of E1-deleted, Ad5-based vectors encoding
antigen, with or without AdCD40L by intranasal administration.
[0134] Measures and Outcomes:
[0135] Sera will be assayed for anti-PC and anti-Kexin IgM, and IgG
isotypes by ELISA at 3, 6, and 9 weeks using appropriate anti-mouse
IgG isotypes: IgG1, IgG2a, IgG2b, IgG3 (Pierce, Rockford, Ill.). At
9 weeks, mice will be sacrificed and lungs will be lavaged for
anti-PC and anti-Kexin IgA levels. B cells expressing
Kexin-specific IgG and IgA in the spleen and mediastinal lymph
nodes will be assayed by Elispot. Serum and BAL antibodies will
also be tested for complement-dependent killing as well as opsonic
phagocytosis and killing of PC in vitro. In brief, serial dilutions
of sera will be incubated with PC cysts and cultured in RPM1640+10%
FCS (with or without heat inactivation) for 24 hours, followed by
assessment of PC mtLSU rRNA integrity by real-time PCR. To assess
macrophage-dependent killing the assay is performed in the presence
of 50,000 alveolar macrophages obtained by lung lavage. If we
observe high titers of Ab and augmentation of PC killing in vitro,
we will also perform passive transfer experiments in scid mice with
300 .mu.L of serum followed by pulmonary PC challenge. Scid mice
will be sacrificed 28 days later to determine if the passive
transfer of serum prevents PC infection.
[0136] Expected Results and Interpretations:
[0137] We expect to observe significant enhancement in both BAL
anti-PC and anti-Kexin IgG and IgA in animals that are mucosally
boosted as compared to those in Example 18. Moreover we expect to
observe increases in Kexin-specific IgG and IgA B-cells in the
mediastinal lymph nodes in Ad-boosted mice. We also expect that
CD40L will be required in both the priming and the boosting regimen
to achieve strong mucosal IgG and IgA anti-PC and anti-Kexin
antibody responses. We also expect to observe vaccine-induced
increases in opsonic activity and killing of PC in vitro, as well
as protection in passive transfer experiments.
[0138] Alternative Approaches:
[0139] The dosage of the boost will be optimized, following initial
proof of concept. The initial dose of 10.sup.7 has been validated
in preliminary studies, but could be increased, e.g., to 10.sup.8
for both antigen-containing Ad as well as for AdCD40L. The titer
and half life of the antibody in the scid mice will be assayed by
measuring titer immediately after transfer and on day 28. Prior
studies with DC-based vaccines suggest that 300 .mu.L of serum
should provide sufficient Ab to protect throughout the 28 day study
period. The relative importance of mucosal Ab and serum Ab for
protective immunity can also be assayed, e.g., by transferring
concentrated BAL to supply 100 .mu.g of protein. Controls will
consist of BAL that is brought up to 100 .mu.g of protein with
naive mouse serum.
Example 20. The Effect of Mucosal Boosting with Recombinant
Adenovirus Virus-Based Vectors after DNA Priming in Conferring
Protection Against a PC Challenge
[0140] Hypothesis:
[0141] We hypothesize that the co-administration of CD40L with
antigen allows for CD4IND B-cell responses in vivo, and that
mucosal boosting will enhance mucosal antigen-specific B-cell
responses and confer protection against PCP.
[0142] Rationale:
[0143] Preliminary studies demonstrated that CD40L co-administered
with Kexin antigen results in CD4IND B-cell responses and
protection against PCP. These studies will confirm these
preliminary results.
[0144] Experimental Groups.
[0145] The groups will be similar to those used in Example 19, but
this study will assess responses to PC challenge. Male 6-8 week old
BALB/c mice will be CD4-depleted by the administration of 0.3 mg
GK1.5 IP by weekly injection, or given rat IgG as a control. 48
hours later, mice will be randomized to be vaccinated by IM
injection of pmini-Kexin WT, psec-mini-Kexin-WT, pmini-Kexin CO, or
psec-mini-Kexin-CO, in each case either with or without CD40L. A
control group of mice will be injected with pBudCD40L with no PC
antigen. After 6 weeks mice will be further randomized, and either
given no boosting or mucosal boosting with .about.10.sup.7
adenovirus, encoding the same amount of antigen as the prime,
again, with or without an equal dose of AdCD40L. At 9 weeks mice
will be challenged with 2.times.10.sup.5 PC cysts and followed for
6 weeks to determine PC lung burden by quantitative real time PCR.
Sample Sizes will consist of 10 mice per group to give the power to
resolve a 30% difference in PC burdens.
[0146] Manipulations:
[0147] Plasmid injections will be repeated every after weeks for
two doses as otherwise previously described by Ramsay et al. We
will administer 10.sup.7 of E1-deleted Ad5 based vectors encoding
antigen, with or without AdCD40L, by intranasal administration.
Mice will be sacrificed 6 weeks after PC challenge to assay for
serum and BAL anti-PC and anti-Kexin antibodies, PC burden by real
time PCR, and GMS staining of lung tissue.
[0148] Measures and Outcomes:
[0149] Sera will be assayed for anti-PC and anti-Kexin IgM, and IgG
isotypes by ELISA at 3, 6, 9 and 15. Isotypes will be determined by
using appropriate anti-mouse IgG isotypes: IgG1, IgG2a, IgG2b, IgG3
(Pierce, Rockford, Ill.). At sacrifice one lung will be inflated
with 10% neutral buffered formalin and sent for morphology
examination using H & E and GMS staining. The other lung will
be placed in TRIzol.TM. reagent prior to assaying for PC burden by
real time PCR. Serum and BAL antibodies will also be tested for
complement-dependent killing, opsonic phagocytosis, and killing of
PC in vitro as otherwise described above.
[0150] Expected Results and Interpretations:
[0151] We expect to observe significant protection in mice
vaccinated and boosted with adenovirus carrying DNA that encodes
kexin antigens. Based on preliminary studies we expect to observe
the greatest protection in the codon-optimized, secreted Kexin
group. We expect to achieve a 6-log level of protection compared to
CD40L vaccinated control mice without antigen. During the challenge
studies we also will incorporate a scid mouse control group to
verify infection with the dose of PC used. Both the scid group and
the control, CD4-depleted mice typically have over 10.sup.9 PC copy
number in their lung by week 6. Thus in the effective vaccine group
we expect to observe levels of 10.sup.3 PC copy number or lower. We
also expect to observe vaccine-induced increases in opsonic
activity and killing of PC in vitro as well as protection in
passive transfer experiments.
[0152] Alternative Approaches:
[0153] The dosage of the boost will be optimized. The initial dose
of 10.sup.7 has been validated in preliminary studies, but could be
increased, e.g., to 10.sup.8 for both antigen-containing Ad, as
well as for AdCD40L. We will also determine the duration of
protection. We will choose the two most effective vaccine boost
combinations (with and without CD40L), and challenge at week 16 or
week 26 to determine whether protection still exists. We will also
assess anti-PC and anti-Kexin recall responses in the lung and
serum as well as B-cell Elipsots to assess functional B cell
memory. Here we expect that CD40L will be required for long term
functional B-cell responses. To confirm that the titer and half
life of the antibody suffice to protect the scid mice, we will
measure the titer immediately after transfer, and on day 28. Prior
studies with DC-based vaccines suggest that 300 .mu.L of serum
should provide sufficient Ab to protect during the 28 day study
period The relative importance of mucosal Ab and serum Ab to
protective immunity can also be assayed, e.g., we could transfer
concentrated BAL containing 100 .mu.g of protein. Controls will
consist of BAL that is brought up to 100 .mu.g of protein with
naive mouse serum.
Example 21. Effect of Mucosal Boosting with Recombinant MVA
Virus-Based Vectors after DNA Priming in Conferring Protection
Against a PC Challenge
[0154] Hypothesis:
[0155] We hypothesize that the co-administration of CD40L with
antigen allows for CD4IND B-cell responses in vivo, and that
mucosal boosting with modified vaccinia Ankara strain (MVA)-based
vectors will enhance mucosal antigen specific B-cell responses and
confer protection against PCP.
[0156] Rationale:
[0157] Our preliminary studies have demonstrated that CD40L
co-administered with Kexin antigen resulted in CD4IND B-cell
responses and protection against PCP. However there could be a
concern that an Ad5-based vector might itself exacerbate HIV
disease in patients with pre-existing Ad5 antibodies. Therefore,
although our pre-clinical data support the efficacy of Ad5-based
vectors in rodents, it is possible that an alternative approach
could be a useful option for at least some patients with HIV
disease. We chose MVA vectors to explore such an alternative, as
MVA elicits strong mucosal immune responses.
[0158] Experimental Groups.
[0159] Male 6-8 week old BALB/c mice will be CD4-depleted by the
administration of 0.3 mg GK1.5 IP by weekly injection, or given rat
IgG as a control. 48 hours later mice will be randomized to be
vaccinated by the IM injection of pmini-Kexin WT,
psec-mini-Kexin-WT, pmini-Kexin CO, or psec-mini-Kexin-CO, in each
case with or without CD40L. A control group of mice will be
injected with pBudCD40L with no PC antigen. After 6 weeks mice will
be further randomized, and given either no boost or mucosal
boosting with 10.sup.7 MVA, encoding the same antigen construct as
the prime, in each case either with or without an equal dose of MVA
CD40L. A subgroup of mice will be sacrificed to determine
pre-challenge antibody and B cell responses as outlined in Example
20, and the other mice will be challenged with 2.times.10.sup.5 PC
cysts and followed for 6 weeks. Sample sizes will consist of 10
mice per group to give the power to resolve a 30% difference in PC
lung burden by quantitative real time PCR.
[0160] Manipulations:
[0161] Plasmid injections will be repeated with a second dose after
three weeks, as otherwise previously described by Ramsay et al. We
will administer 10.sup.7 of MVA vectors encoding antigen, either
with or without MVA CD40L, by intranasal administration. Mice will
be sacrificed 6 weeks after PC challenge to determine serum and BAL
anti-PC and anti-Kexin antibodies, PC burden by real time PCR, and
GMS staining of lung tissue.
[0162] Measures and Outcomes:
[0163] Sera will be assayed for anti-PC and anti-Kexin IgM, and IgG
isotypes by ELISA at 3, 6, 9 and 15 weeks. Isotypes will be
determined by using appropriate anti-mouse IgG isotypes: IgG1,
IgG2a, IgG2b, IgG3 (Pierce, Rockford, Ill.). At sacrifice one lung
will be inflated with 10% neutral buffered formalin and sent for
morphology examination using H & E and GMS staining. The other
lung will be placed in TRIzol.TM. reagent to assay PC burden by
real time PCR.
[0164] Expected Results and Interpretations:
[0165] We expect to observe significant protection in mice
vaccinated and boosted with MVA vectors encoding kexin antigens.
Based on our preliminary studies we expect the greatest protection
will occur in the codon-optimized, secreted Kexin group. Our goal
is to achieve a 6-log level of protection as compared to the
control mice vaccinated with CD40L without antigen. During the
challenge studies we also will incorporate a scid mouse control
group to verify infection with the dose of PC used.
[0166] Alternative Approaches:
[0167] The dosage of the boost will be optimized. The initial dose
of 10.sup.7 has been validated in preliminary studies, but could be
increased, e.g., to 10.sup.8 for both antigen and CD40L. If we
observe significant protection, we will next determine the duration
of protection. We will choose the two most effective vaccine boost
combinations (with and without CD40L) and challenge at week 16 or
week 26 to determine whether protection still exists. In these
studies we will also assess anti-PC and anti-Kexin recall responses
in lung and serum, as well as B-cell Elipsots to assess functional
B cell memory. Here we expect that CD40L will be required for long
term functional B-cell responses.
Example 22
[0168] Our hypothesis predicts that effective vaccination requires
endogenous IL-23, and results in durable vaccine responses in both
CD4+ T-cell deficient mice and CD40L knockout mice. We will examine
the efficacy of CD40L co-transduction in CD40L knockout mice; and
the requirement of IL-12 family members (including IL-12p35,
IL-12p40, and IL-23), all critical activation molecules that are
induced by CD40L-modified DCs in generating effective primary and
memory B-cell responses. Our preliminary studies have suggested
that IL-23 production is critical to generate B-cell memory against
PC antigen.
Example 23
[0169] We investigate whether co-administration of CD40L with
prime-boost vaccination can induce Ig class switching in CD40L
knockout mice, and result in antigen-specific B-cell responses.
[0170] Hypothesis:
[0171] We hypothesize that CD40L co-transduction with Kexin will
result in class switching of B cells in CD40L KO mice and the
generation of anti-Kexin IgG.
[0172] Rationale:
[0173] Patients with mutations in CD40L resulting in Hyper-IgM
syndrome are often infected by PC. There is an unfilled need for
effective vaccines for these individuals.
[0174] Experimental Groups.
[0175] Male PC-free 6-8 week old CD40L knockout or control mice
will be randomized to be vaccinated by the IM injection of pKexin
based on the two most efficacious constructs identified above,
followed by randomization for no boost, or boosting with AdKexin,
or boosting with AdKexin/CD40L, or injection with AdCD40L alone as
a control. Sample sizes will consist of 10 mice per group to give
the resolution to detect a 30% difference in anti-Kexin or anti-PC
IgG between different routes of vaccination. The optimal dose and
route of the DNA prime and boost vector will be chosen based on the
experimental results above.
[0176] Manipulations:
[0177] Based on our preliminary studies we expect that plasmid
injections will be repeated after three weeks for two doses,
followed by a single boost with the viral vector three weeks
later.
[0178] Measures and Outcomes:
[0179] Sera will be assayed for anti-PC/Kexin IgM, and IgG isotypes
by ELISA at 3, 6, and 9 weeks. Isotypes will be determined by using
appropriate anti-mouse IgG isotypes: IgG1, IgG2a, IgG2b, or IgG3
(Pierce, Rockford, Ill.). At 9 weeks, a subgroup will be boosted
with an IN injection of 10.sup.7 particles of AdKexin (a
recombinant adenovirus expressing Kexin). A control group will be
left un-boosted, or boosted with AdCD40L. Three weeks after the
boost, mice will be sacrificed and we will measure anti-PC/Kexin
antibodies in serum and BAL fluid. Splenocytes and mediastinal
lymph node cells will be harvested to determine the precursor
frequency of antigen-specific B-cells by Elipsot. Serum antibodies
will also be tested for complement-dependent killing, as well as
opsonic phagocytosis and killing of PC in vitro as described
above.
[0180] Expected Results and Interpretations:
[0181] Based on our preliminary studies showing that AdCD40L
delivery to the lungs resulted in a significant increase in
anti-PC/Kexin mucosal IgA and IgG, we expect that CD40L will most
likely be optimal when included in both the prime and the boost. We
also expect to observe an increase in opsonic killing of PC.
[0182] Alternative Approaches:
[0183] If CD40L co-transduction in vivo is less effective than we
expect, then we will perform co-culture experiments of irradiated
AdCD40L (or control) modified DCs, pulsed with OVA to induce B cell
proliferation and class switching in CD19+ B cells from CD40L KO
mice. Particularly we will investigate the ratio of DC's to B cells
to induce differentiation and generation of anti-OVA IgG in vitro.
If higher ratios of DCs are required to induce B cell proliferation
in CD40L KO mice as compared to control C57BL/6 mice, then we will
investigate the effects of higher doses of DNA priming, boosting,
or both in vivo. Secondly, if we observe a significant amount of
class switched antibody against PC in CD40L KO mice, we will repeat
the experiment with a PC challenge as outlined above, to examine
the efficacy and duration of protection in these mice. If we
observe efficacy with the MVA platform, we will also repeat this
experiment substituting MVA vectors for Ad vectors to examine their
efficacy and duration of protection.
Example 24. Examine the Role of IL-12p35, IL-12p40, and IL-23 in
Mucosal Prime Boosting with Recombinant Adenovirus Virus-Based
Vectors Against PC
[0184] Hypothesis:
[0185] We hypothesize that the co-administration of CD40L with
antigen allows for CD4IND B-cell responses in vivo, and that
mucosal boosting will enhance mucosal antigen specific B-cell
responses and enhance protection against PCP.
[0186] Rationale:
[0187] Our preliminary studies demonstrated that CD40L-modified DCs
required IL-23 for a functional B-cell recall response to PC
antigen when the DCs were pulsed with PC organisms. However, these
preliminary results do not in themselves show whether IL-23 is also
required for the prime boost regimen in vivo. If IL-23 is indeed
required in vivo, then IL-23 could be used as a mucosal adjuvant.
Secondly, up to 3-7% of the population is heterozygous for a
non-synonymous SNP in the IL-23R coding region. It is possible that
this polymorphism could reduce the immunogenicity of the novel
vaccine platform in heterozygous individuals, or in individuals
homozygous for the less frequent allele.
[0188] Experimental Groups.
[0189] Male 6-8 week old BALB/c mice will be CD4-depleted by the
administration of 0.3 mg GK1.5 IP by weekly injection. 48 hours
later, mice will be randomized to be vaccinated by the IM injection
of the two most efficacious Kexin constructs as identified above:
pmini-Kexin WT, psec-mini-Kexin-WT, pmini-Kexin CO, or
psec-mini-Kexin-CO with CD40L. Control mice will be injected with
pBudCD40L with no PC antigen. After 6 weeks the mice will be
further randomized to no boost or mucosal boosting with 10.sup.7
adenovirus encoding the same antigen construct as the prime, in
each case with or without an equal dose of AdCD40L or 10.sup.7 of
fowl pox vectors encoding the same antigen (with our without
CD40L). Either an adenovirus vector or an MVA vector will be used,
based on their relative efficacy as determined in the experiments
described above. At 9 weeks 50% of the mice will be sacrificed to
examine serum and mucosal anti-Kexin/PC antibodies as well as
antigen-specific B-cells. The other 50% will be challenged with
2.times.10.sup.5 PC cysts intratracheally and sacrificed 7 days
later to assess the role of IL-23 in regulating antigen specific
B-cell recall responses. Sample sizes will consist of 10 mice per
group to give the statistical resolution to detect a 30% difference
in antigen specific B-cell responses.
[0190] Measures and Outcomes:
[0191] At the time of sacrifice (either week 9 or week 10 in PC
challenged mice) lungs will be lavaged for anti-PC and anti-Kexin
IgA levels. Kexin-specific, IgG-expressing B cells in the spleen
and mediastinal lymph nodes will be assayed by Elispot. One lung
will be placed in TRIzol.TM. reagent for measuring PC mtLSU copy
number by real time PCR. Serum antibodies will be tested for
complement-dependent killing, as well as opsonic phagocytosis and
killing of PC in vitro as described above.
[0192] Expected Results, Interpretations and Alternative
Approaches:
[0193] We expect to observe the induction of anti-PC and anti-Kexin
primary antibody responses in the serum of all mice. However, we
expect to observe a defect in mucosal boost responses as well as PC
recall response in the lungs of IL-23- and IL-12p40-deficient mice.
We also expect to observe an increase in opsonic killing of PC in
IL-23 intact mice (only). Such results would be consistent with the
expected role of IL-23 in expanding the B-cell memory pool. If this
is indeed the case, then we will repeat the experiment and examine
the effect of adding AdIL-23 (or MVA IL-23) in the boost along with
AdCD40L to determine whether exogenous IL-23 can restore B-cell
expansion in IL-12p40-/- or IL-23p19-/- mice. We have previously
prepared the AdIL-23 vector. Preliminary studies suggest that the
AdIL-23 vector will restore B-cell memory responses, at least in
the context of ex vivo, pulsed DC-based vaccination with
IL-23p19-/- DCs.
Example 25. Testing In Vivo. CD4IND, Pathogen-Specific Immune
Responses Against Pneumocystis Kexin are Generated in an SIV Model
of Immunodeficiency in Macaques
[0194] We will confirm that the mini-kexin constructs produce
vaccine-induced immune responses in SIV-infected, CD4-deficient
macaques. Control or SIV-infected macaques will undergo DNA priming
followed by mucosal boosting 4 weeks after mock or live SIV
infection. Outcome measures will include humoral responses to the
vaccine and the prevention of Pneumocystis colonization as
determined by PCR in BAL fluid. Preliminary studies suggest that
Pneumocystis colonization occurs in up to 80% of SIV-infected
(untreated) macaques, compared to 0% in non-SIV infected
monkeys.
Example 26. Evaluate the Effect of CD40L on Monkey DCs
[0195] Hypothesis:
[0196] We hypothesize that AdhCD40L-transduced (or MVA hCD40L
transduced) monkey DC's will demonstrate maturation by an increase
in Class II MHC expression, and that they will demonstrate
activation by enhanced elaboration of IL-12 and IL-23 in
culture.
[0197] Rationale:
[0198] Preliminary studies have demonstrated similar results in the
mouse model. We therefore expect similar results in the
macaques.
[0199] Experimental Groups.
[0200] Monkey DC's from juvenile macaques with normal CD4 counts
will be purified by CD11c+ beads (Mitenyi Biotech) from peripheral
blood. Monkey DC's will be grown in RPMI 1640 and then mock
transfected with PBS; or transfected with AdEGFP or with AdhCD40L
at MOIs of 5, 10, 50, and 100. We will carry out similar
experiments with MVA CD40L. Sample sizes will consist of 4-6
control macaques; and transductions will be carried out in
triplicate.
[0201] Manipulations:
[0202] CD11c+ DCs will be cultured in RPMI 1640 growth medium
supplemented with 1% monkey plasma. 50,000 to 100,000 cells will be
analyzed by four-color FACS for each of the following DC markers:
anti-HLA-DR, CD80, CD86, and CD25 from B-D Pharmingen, and CD83
from Coulter. Immature DC's are typically HLA-DR.sup.++,
CD86.sup.++, CD8.sup.+/low, CD83.sup.-/weak, and CD25-, whereas
mature DC's are HLA-DR.sup.+++, CD86.sup.+++, CD25.sup.++,
CD80.sup.++, and CD83.sup.++. The remaining DC's will be transduced
with MOIs of 0, 5, 10, 50, or 100 of AdEGFP or AdhCD40L (or MVA
vectors) and cultured for 24 hours. The supernatant will be
harvested for determinations of monkey IL-12 (p40 and p70) and
TNF-alpha (Biosource, Camarillo, Calif.). The cells will be stained
for maturation markers as outlined above, as well as for CD40L with
clone TRAP-1-PE from Immunotech (Westbrook ME). IL-23 will be
measured by ELISA (Bender MedSystems).
[0203] Expected Results and Interpretations:
[0204] We expect to observe dose-dependent transduction of Monkey
DC's by both AdEGFP and AdhCD40L, as measured by an increase in the
mean channel fluorescence of GFP in AdEGFP-transduced cells, and by
an increase in CD40L as measured by TRAP-1 PE staining. Bioactivity
of AdhCD40L will be assessed by the ability of the vector to
selectively induce IL-12, IL-23, and TNF-alpha in supernatants from
AdhCD40L-transduced DCs. We also expect to observe the maturation
of DC's transduced with AdhCD40L as measured by an increase in mean
channel fluorescence in HLA-DR, CD86, and CD83 expression. There
may also be some increase in HLA-DR, CD86, and CD83 expression in
AdEGFP-transduced cells.
[0205] Alternative Approaches:
[0206] We expect efficient transduction of DCs with an MOI of 100.
Moreover, human CD40L (h CD40L) has 99% homology with CD40L from
monkey. Preliminary studies show that hCD40L induces IL-12p70 in
monkey DCs. Thus we expect that hCD40L should have bioactivity in
this system. If, however, we observe a defect in DC activation, we
will assess hCD40L at the protein level by FASC and at the
transcript level by RT-PCR to verify its expression in DCs with the
Ad vector or the MVA vector.
Example 27. Generating Kex1 Antigen-Specific IgG in SIV-Infected
Macaques by Heterologous, Prime-Boost Immunizations
[0207] Hypothesis:
[0208] We hypothesize that DNA priming followed by heterologous
adenovirus or MVA virus boosting will elicit potent anti-Kex1
systemic and mucosal antibody responses and protection against PC
colonization.
[0209] Rationale:
[0210] The results of the prior studies will help us choose which
Kexin DNA construct to use in macaques, and whether to use
adenovirus or MVA as the boost.
[0211] Experimental Groups.
[0212] There will be five vaccine groups:
1. SIV-uninfected, pneumocystis vaccine with CD40L (n=8) 2.
SIV-infected, pneumocystis vaccine with CD40L (n=8) 3.
SIV-uninfected, pneumocystis vaccine without CD40L (n=4) 4.
SIV-infected, sham vaccine, without PC challenge (n=3) 5.
SIV-uninfected, sham vaccine, without PC challenge (n=3) Due to
expense and the likelihood that the pneumocystis vaccine alone
(without CD40L) will not elicit strong humoral immunity, the
SIV-uninfected, pneumocystis vaccine without CD40L group (Group 3)
may be omitted from the study. Groups 4 and 5 will be used as
shared controls.
[0213] Manipulations, Measurements, and Outcomes:
[0214] Animals (male juvenile Rhesus macaques) will be pre-screened
by ELISA for anti-PC Kexin titers and anti-adenovirus titers (the
latter, if we choose an adenovirus platform). Only animals with
negligible titers, defined as less than 1:64 (OD450 cutoff of 0.1),
will be enrolled. At -4 weeks monkeys will be infected with
SIVmac251, or mock infected with saline injection. The dose of
virus will be 50 TCID50. Inoculations will be made intravenously,
via the saphenous vein. At week 0, the macaques will undergo a
baseline bronchoscopy, they will be assessed for serum and BAL
anti-Kex 1 antibodies, and they will be assessed for PC
colonization by nested and real time PCR. BAL fluid will be
obtained by bronchoscopy in anesthetized animals. Plasmid DNA will
be administered at a dose of 2 mg IM (2 sites [quadriceps], at 1 mg
each in 0.5 ml saline). The plasmid DNA will encode the Kex1
construct that has shown the greatest efficacy in mice, with or
without CD40L, as appropriate for each of the group assignments
listed above. DNA will be administered at weeks 0 and 3 (i.e., 4
and 7 weeks after mock or live SIV infection). At week 6 monkeys
will undergo another bronchoscopy, and blood samples will be taken
to determine pre-boost anti-Kex1 antibodies in serum and BAL.
Following the bronchoscopy, a boost immunization will be
administered intranasally with adenovirus (10.sup.10 pfu) or MVA
(10.sup.8) encoding Kex1 and hCDC40L, or encoding Kex1 alone for
monkeys in the no-CD40L group. A third bronchoscopy will be
performed at week 8 to assess the effect of the boost on anti-Kex1
IgG in serum, and anti-Kex1 IgA in BAL. Two more bronchoscopies
will be performed at weeks 20 and 32 to assess longevity of the
antibody responses, as well as the level of Pneumocystis
colonization as determined by both nested and real time PCR. At
week 32 we will harvest mediastinal lymph nodes for B-cell Elispot
assays, and lung tissue for histology. Real-time PCR will also be
used to assess PC colonization in the lungs. To monitor the safety
of the approach in the macaque model we will also obtain complete
blood counts and serum chemistries, including liver transaminases,
at weeks 6, 8, 20, and 32.
[0215] Expected Results and Interpretations:
[0216] We expect that the Kex1/CD40L vaccination procedure will be
safe and will not have associated hematological or liver toxicity.
We expect to observe significant increases in antigen-specific IgG
in both SIV+ and SIV-animals receiving hCD40L with Kex1. Moreover,
we expect higher anti-Kex1 IgG and IgA levels in SIV-animals
receiving the Kex1/CD40L combination as compared to Kex1 alone.
Moreover we expect the addition of a mucosal boost will
significantly augment the levels of anti-Kex1 IgG and IgA in BAL
fluid (particularly in the Kex1/CD40L groups). We expect to observe
that the Kex1/CD40L vaccine will be associated with reduced PC
colonization in the BAL at week 20, and in the BAL and lung tissue
at week 32 in SIV-infected monkeys as compared to the group 4 or 5
animals.
[0217] Alternative Approaches:
[0218] If we observe significant increases in anti-Kex1 IgG in
SIV-animals but not in SIV+ animals, such an outcome could be due
to inadequate DC homing or an inadequate dose of the boost. If that
should be the case, then we will repeat a mucosal boost with one
log higher virus dose. Our primary measurement in these experiments
will be the level of PC colonization, as determined by nested and
real time PCR. In humans these two assays have over a 93%
concordance rate. Our expected figure of 80% spontaneous PC
colonization in SIV-infected macaques is based on observations of
animals housed in Pittsburgh, and we are not yet sure whether a
similar level of infection will be seen in macaques housed at the
Tulane Primate Center. To assess, 25 SIV-positive and 25
SIV-negative lung tissue samples and paired BAL samples will be
tested to determine the prevalence of PC colonization at necropsy
in these monkeys. Assuming the figure is at least 60% for the
SIV-positive cohort, then this set of experiments should provide a
meaningful outcome. If the figure should be lower, then we will
identify lung samples with high burdens of CP organisms, and
isolate organisms from those samples to use for inoculation at week
20. Lungs from identified animals will be disrupted and processed
as has been previously described for murine lung; cysts will be
purified by sucrose gradient purification.
Example 28
[0219] Pneumocystis is also a common infection following
medically-induced immunosuppression, for example in cancer
chemotherapy, or in suppressing host rejection of transplanted
tissues or organs. Vaccination with mini-kexin can protect against
PCP in such instances.
[0220] As a demonstration in a mouse model, we performed DNA
plasmid mini-Kexin prime-boost vaccination in wild type mice, and
then depleted CD4+ T-cells. Depletion of CD4+ T-cells makes
unvaccinated mice susceptible to PCP. Female 6- to 8-week-old
C57BL/6 and BALB/c wild type (wt) mice were immunized
intramuscularly with a mini Kexin-encoded, pBUD plasmid vector
twice, two-weeks apart. The four mini Kexin vectors encoded
secreted (s) or non-secreted versions of mini kexin, either
codon-optimized (co) or wild type (wt). Two weeks after the second
plasmid DNA prime vaccination, the mice were intranasally boosted
with recombinant adenovirus encoding the same type of mini-Kexin as
had been used in the prime vaccination. Because the vaccinated mice
initially had normal levels of CD4+ T-cells, we did not include
CD40L in either the priming or boosting vector. To artificially
induce an immunosuppressed state, the mice were then depleted of
CD4+ T cells by administration of monoclonal antibody GK1.5,
repeated weekly, starting two weeks after the boost. After one week
of CD4+ depletion, the mice were challenged intratracheally with
2.times.10.sup.5 PC organisms per mouse. Four weeks later the mice
were euthanized, and lung tissues were collected for PC organism
burden (assayed by real-time PCR).
[0221] As shown in FIG. 9, all four miniKexin plasmid vaccines
provided significant protection against PC infection, as compared
to Rag2/gamma chain double-knockout mice, which lack B, T, and NK
cells (n=5-6 per group, * denotes P<0.05, Student's-t-test as
compared to RAG2/gamma C KO).
Example 29
[0222] Following successful completion of animal trials, vaccines
in accordance with the present invention are tested in human
patients in clinical trials conducted in compliance with applicable
laws and regulations.
Example 30. Detailed Methodology
[0223] Except as otherwise stated, the following materials and
procedures have been used or will be used in the experiments
described above:
[0224] 1. Animals.
[0225] Virus-free BALB/c mice, aged 6-8 weeks, will be purchased
from NCI/Charles River. Preliminary experiments have shown that
animals from this supplier are not chronically infected with P.
carinii. IL-12p35, IL-12p40, and IL-23p19 mice on a C57BL/6
background are maintained in our laboratory. Homozygous C.B17
scid/scid (scid) mice will be purchased from NCI/Charles River or
Taconic Laboratories in Germantown, N.Y. All animals will be housed
in separate rooms at the LSU Medical Center Animal Care Facility in
HEPA-filtered ventilated racks. Mice are fed autoclaved chow and
water ad libitum, and are held in the facility for least 2 days
before initiating treatment. Changes of animal cages, bedding,
water bottles, and food will be performed in a laminar flow hood.
Access to the room is limited to specific laboratory personnel and
animal care personnel; gown and gloves are required for all workers
entering the room. It is estimated that we will use a total of 1368
mice in the experiments described. Macaques are housed at the
Tulane National Primate Research Center. Where applicable, monkeys
are pre-anesthetized with acepromazine (0.2 mg/kg, i.m.) and
sedated with ketamine-HCI (10 mg/kg, i.m.) for bronchoscopy and
blood sampling. Bronchoscopy will be performed with topical
anesthesia with 2% xylocaine.
[0226] 2. Monitoring Animal Health.
[0227] Sentinel DBA mice are co-housed in the same room as the
experimental mice, with bedding regularly taken from the cages of
P. carinii-infected and scid mice. The sentinel mice are sacrificed
quarterly and tested for antibody titers to a variety of murine
viruses and pathogens. Regular consultation with veterinary staff
is used to assure and to confirm specific-pathogen-free conditions
for the experimental animals.
[0228] 3. Maintenance of P. carinii in Scid or CD40L Knockout
Mice.
[0229] To assure a consistent supply of P. carinii, the P. carinii
organisms will be passaged through the lungs of C.B17 scid/scid (on
a BALB/c background) or CD40L KO mice (on a C57BL/6 background). We
presently maintain a breeding colony of PC-free and PC-infected
CD40L KO mice in separate rooms. Groups of scid or CD40L KO mice
will be inoculated with P. carinii organisms as described below.
Inoculated mice will be housed in ventilated racks for 4-6 weeks
before being sacrificed to harvest P. carinii from lung tissue.
[0230] 4. Inoculation of Mice with P. carinii Organisms.
[0231] P. carinii organisms used for inoculation will be prepared
from homogenized lungs of chronically infected scid or CD40L KO
mice. We have previously used athymic mice, but have found that
scid mice develop more consistent and intense infections. The C.B17
scid mouse strain is allotype co-isogenic to BALB/c mice. Briefly,
lung tissue will be obtained from scid or CD40L KO mice chronically
infected with P. carinii. The lungs will be frozen for 30 minutes,
and then disrupted mechanically in a Stomacher.TM. 80 Biomaster.
The disrupted lung tissue will be filtered through gauze and
adjusted to a level of 2.times.10.sup.6 cysts/ml, as assessed by
DiffQuik.TM. Romanowski staining. P. carinii will then be injected
into the trachea of lightly anesthetized BALB/c mice by passing a
blunt needle into the trachea per os and then threading a catheter
through the needle into the low trachea. Each mouse will receive
0.1 ml of inoculum (2.times.10.sup.5 cysts), followed by 0.8 ml of
air. To assure viability of the organisms, the inoculum will be
injected into recipient mice on the day of preparation. Lung
homogenates containing P. carinii will be routinely checked for
endotoxin contamination using the Whitaker endotoxin assay. The
inoculum will also be quantified using a TaqMan.TM.-based assay for
rRNA copy number. For antigen preparation, PC organisms will
isolated from lung tissue of C.B-17 scid mice (for experiments in
BALB/c mice) or CD40L KO mice (for experiments in C57BL/6 mice)
that were previously inoculated with PC. PC organisms will be
purified by differential centrifugation, and protein antigen will
be produced by sonication for 5 minutes.
[0232] 5. Examination of Lung Tissue for P. carinii Infection.
[0233] Lung tissue will be fixed in formalin and stained with
Gomori's methenamine silver and hematoxylin/eosin.
[0234] 6. Controls for Bacterial/Fungal/Viral Infection.
[0235] Random samples of lung tissue from control and experimental
mice will be cultured to exclude the possibility of intercurrent
bacterial or fungal infection. In addition, when the experimental
design permits, touch preps will be made of lung tissue prior to
formalin fixation and gram staining to look for bacterial
infection. Also, co-housed sentinel DBA mice are routinely
monitored for serologic titers against common viral pathogens,
including Sendai and Mouse Hepatitis Virus.
[0236] 7. Depletion of Host CD4+ Lymphocytes.
[0237] The hybridoma GK1.5 (rat antiCD4) was originally obtained
from the American Type Culture Collection (Manassas, Va.) and is
maintained in the LSU Monoclonal Antibody Facility. GK1.5 is a rat
IgG2b monoclonal antibody. Antibodies from this hybridoma are
prepared from ascites in athymic mice. The antibodies are partially
purified by ammonium sulfate precipitation, dialyzed against
phosphate buffered saline, and quantified by protein
electrophoresis and optical density. To deplete mice of CD4+ T
lymphocytes, mice will receive an intraperitoneal injection of 0.3
mg anti-CD4 monoclonal antibody in 0.2 ml PBS. Control mice will
receive an equal volume of rat IgG. Depletion of CD4+ lymphocytes
will be checked by flow cytometric analysis of splenocytes or
peripheral blood as described below. Depletion of the appropriate
lymphocyte subset will be maintained by weekly administration of
the antibodies for the course of the experiment.
[0238] 8. RNA Isolation and TaqMan.TM. Probes and Primers for PC
rRNA.
[0239] Total RNA is isolated from the right lung of infected mice
by a single step method using TRIzol.TM. reagent (Life
Technologies, CA, USA). As a standard for the assay, a portion of
PC muris rRNA (GenBank Accession No. AF257179) is cloned into
PCR2.1 (Invitrogen, Carlsbad, Calif.), and PC rRNA is produced by
in vitro transcription using T7 RNA polymerase. The template is
digested with RNase-free DNase, quantitated by spectrophotometry
and aliquoted at -80.degree. C. until used. The TaqMan.TM. PCR
primers for mouse PC rRNA are 5'-ATG AGG TGA AAA GTC GAA AGG G-3'
(SEQ ID NO. 7) and 5'-TGA TTG TCT CAG ATG AAA AAC CTC TT-3' (SEQ ID
NO. 8). The probe is labeled with a fluorescent reporter dye,
6-carboxyfluorescein (FAM), and the sequence is
6FAM-ACAGCCCAGAATAATGAATAAAGTTCCTCAATTGTTAC-TAMRA (SEQ ID NO. 9).
(TAMRA=tetramethyl-6-Carboxyrhodamine.) Real-time PCR is carried
out using one-step TaqMan.TM. RT-PCR reagents (Applied Biosystems,
Foster City, Calif.). The PCR amplification is performed for 40
cycles: 94.degree. C. for 20 s and 60.degree. C. for 1 min, in
triplicate using the ABI Prism 7700 SDS. The threshold cycle CT
values are averaged from the values obtained from each reaction,
and data are converted to rRNA copy number using a standard curve.
This assay has a correlation coefficient greater than 0.98 over
8-logs of PC RNA concentration, and is known to correlate with
viable PC since either heat killing or exposure to
Trimethoprim/Sulfamethoxazole ablates the signal.
[0240] 9. Pneumocystis Viability Assay.
[0241] Macrophages (10.sup.6/ml) suspended in a volume of 100 .mu.l
of RPMI 1640 medium containing FCS are co-cultured in round-bottom
96-well plates with PC (2.times.10.sup.4 cysts/ml, 50 .mu.l),
yielding an effector-to-total-PC organism ratio of 1:1 (estimated
1:10 cyst to trophozoite ratio). Before addition of PC, organisms
will be preopsonized with 50 .mu.l of serially diluted serum or
normalized BAL, or 50 .mu.l of DMEM plus 10% FCS. Included as a
viability control are PC organisms incubated with control medium,
DMEM plus 10% FCS. The plates are spun at 2500 rpm to pellet the PC
organisms. The supernatants and cell pellets are collected, and
total RNA is isolated using TRIzol.TM. LS reagent (Invitrogen Life
Technologies). Viability of the PC is analyzed with real-time PCR
measurement of PC large subunit rRNA copy number (GenBank accession
number AF257179), and quantified against a standard curve. This
method detects viable PC organisms, as evidenced by loss of
detectable PC rRNA in heat-killed organisms or those exposed to
trimethoprim/sulfamethoxazole.
[0242] 10. PC Kex 1 ELISA.
[0243] To determine anti-PC or Kex1 IgG titers, ELISA plates
(Corning, N.Y.) are coated with 100 ng of PC antigen or Kex 1
antigen (provided by Dr. Karen Norris, University of Pittsburgh)
per well in carbonate buffer at pH 9.5, and held overnight. Plates
are washed with PBS+0.05% Tween-20 (wash buffer) and blocked with
bovine serum albumin and 2% milk. After washing, serial dilutions
of serum will be added to each well and incubated for one hour at
room temperature. Then, after washing, 100 .mu.l of 1:1000 alkaline
phosphatase conjugated goat anti-mouse IgG or IgA (Bio-RAD,
Hercules, Calif.) will be added and incubated for one hour at room
temperature. Then, after washing, the plates are developed using
Sigma 104 substrate tablets in diethanolamine buffer, and
absorbance is measured at 490 nm. Anti-PC and anti-Kex1 specific
mouse IgG isotypes will be assayed. For macaque antibodies we use
anti-Rhesus IgA and IgG.
[0244] 11. Bronchioalveolar Lavage.
[0245] Lavaged lymphocytes will be obtained by bronchioalveolar
lavage of mice anesthetized with intraperitoneal pentobarbital.
This technique has been previously used in our laboratory to
recover lung cells from mice, rats, and monkeys. For mouse studies,
the lungs will be lavaged through an intratracheal catheter with
warm (37.degree. C.) calcium- and magnesium-deficient PBS
supplemented with 0.5 mM EDTA. A total of 11 ml will be used for
each mouse in 0.5 ml increments, with a 30 second dwell time. This
technique recovers 0.5-1.times.10.sup.6 cells from normal animals,
of which greater than 95% are alveolar macrophages, with greater
than 95% viability as measured by trypan blue exclusion. In mice
inoculated with P. carinii, total cell count can be as high as
4-6.times.10.sup.6, and the percentage of lymphocytes contained
within the lavaged cells is as high as 50%. For some studies, the
first 1.0 ml of BAL fluid may be frozen for cytokine analysis, or
BAL fluid may be concentrated to recover detectable cytokine or
IgA.
[0246] 12. Retrieval of Hilar and Paratracheal Lymph Nodes.
[0247] Hilar lymph nodes and paratracheal (mediastinal) lymph nodes
will be resected under sterile conditions from mice given a lethal
dose of pentobarbital. This method has been used to study draining
lymph node cells from mice challenged with antigen. The lymph nodes
will be passed through a sterile mesh screen into culture medium,
and adjusted for cell number with a hemacytometer. Using this
technique, approximately 12-15.times.10.sup.6 cells are recovered
from a mouse inoculated with P. carinii. More than 90% of these
cells are lymphocytes as measured by Diff-Quik.TM. staining. Cells
will be processed for flow cytometry as outlined above.
[0248] 13. B-Cell Elipsots.
[0249] To determine precursor frequency of Kex1 specific IgG
B-cells, we will perform ELISPOT assays using FACS-sorted B cell
populations. 96-well PVDF filter plates will be coated with Kex1,
and serial dilutions of sorted B cells will be applied to the
wells. Anti-Kex1 antibodies captured on the filter will be
visualized by staining with an AP-conjugated anti-IgG or IgA
secondary antibody. After developing with chromogenic substrate,
the plates will be counted using an automated plate reader, and the
percentage of antibody producing cells in each subset will be
calculated.
[0250] 14. DNA Vaccination.
[0251] For intramuscular delivery, mice are anesthetized with
isoflurane. Then 100 microgram of the DNA vaccine is delivered in
100 .mu.l of normal saline to the tibialis muscle (i.e., 50 .mu.l
per hind leg) using a fine-needle (30G) tuberculin syringe. If
needed, we will follow immediately by mild electroporation using a
BTX ECM 830 electroporator apparatus with caliper electrodes
(Harvard Biosciences). Immediately following injection of DNA to
each leg, the calipers will be set to 4-5 mm and placed tightly on
either side of the tibialis. The machine will be discharged twice,
resulting in 2.times.20 millisecond pulses of 150V at an interval
of 1 sec.
[0252] 15. Statistical Analysis.
[0253] Data will be analyzed using StatView statistical software
(Brainpower Inc., Calabasas, Calif.). Comparisons between groups
will be made with the Student's t-test, and comparisons among
multiple groups will be made with analyses of variance and
appropriate follow-up testing. The Mann-Whitney test or the
Wilcoxson paired sample test will make ordinal comparisons.
Significance will be taken as p<0.05.
Example 31. Use and Care of Vertebrate Animals
[0254] 1. Justification for the Use of Experimental Animals:
[0255] There are no alternatives to the use of live animals to
study host defense mechanisms, nor to study vaccine responses
against Pneumocystis. Pneumocystis cannot be reliably maintained in
vitro, so research with this pathogen requires the use of animal
models of infection. Rhesus monkeys are being used because of the
similarities between infection of this species with simian
immunodeficiency virus (SIV) and human infection with HIV/AIDS.
[0256] 2. Veterinary Care of Experimental Animals:
[0257] Mice will be housed in a separate room at the LSU Medical
Center Animal Care Facility in ventilated rack caging. This
facility is State-licensed and fully accredited by the American
Association for Accreditation of Laboratory Animal Care (AAALAC).
Animals are housed under specific-pathogen-free conditions.
Personnel access to the animal room is limited. All food, cages,
water, and bedding is autoclaved prior to use. Gowns, gloves, and
mask are required to handle the animals. All cage, food, and
bedding changes take place in a laminar flow biosafety hood in the
same room. Sentinel animals are housed in the same room with sample
bedding from the immunosuppressed animals. Serum from these
sentinel animals is routinely screened for a battery of murine
pathogens. A veterinarian oversees the facility.
[0258] Veterinary care of the macaques at the Tulane National
Primate Research Center will be handled similarly. The animals
undergoing study will be monitored closely for food and water
intake, and routine blood work will be limited to 40 cc/month. The
primate center has strict protocols in place to euthanize
SIV-infected animals when weight loss or other clinical parameters
indicate significant morbidity. Animals to be infected with SIV
will receive 50 TCID50 of strain SIVmac251, a pathogenic strain to
which animals have had a median survival of 210 days in past
studies. Basic monitoring will include: 1) twice daily observations
by a trained animal care technician, 2) physical examinations
including blood sampling prior to SIV inoculation, after SIV
inoculation, and at bi-monthly intervals thereafter. All physical
examinations and invasive procedures will be performed on
anesthetized animals. The anesthetic used will either be a
combination of ketamine-HCI (10 mg/kg, i.m.) and acepromazine (0.2
mg/kg, i.m.), or Telazol (5 mg/kg of Tiletamine and Zolazepam).
[0259] Containment practices at the Tulane Primate Center are in
accordance with the recommended guidelines in the Center for
Disease Control Morbidity and Mortality Weekly Report, vol. 31
(#43) "AIDS: Precautions for Clinical and Laboratory Staff, pp.
577-580 (1982); vol. 32 (#34) "AIDS: Precautions for Health Care
Workers and Applied Professionals" pp. 577-580 (1983); and the
Biosafety in Microbiological and Biomedical Laboratories
Guidelines, First edition (1984).
[0260] 3. Experimental Procedures Involving Live Animals:
[0261] a. Intratracheal Inoculation with P. carinii Organisms:
[0262] Pneumocystis carinii organisms will be obtained from the
lungs of chronically infected scid or CD40L KO mice. Organisms for
this colony of infected mice were originally obtained from the Fox
Chase Cancer Center in Philadelphia, Pa. Mice will be sacrificed by
a lethal (400 mg/kg) dose of IP pentobarbital, followed by
exsanguination once they are deeply asleep. The lungs will then be
removed aseptically, and Pneumocystis carinii organisms will be
recovered for injection into other scid mice (to maintain the
organisms) or into BALB/c mice (to conduct the proposed
experiments). The Pneumocystis carinii organisms will be injected
in a volume of 0.1 ml into the tracheas of mice lightly
anesthetized with inhaled isoflurane. Once the animals are asleep,
the animals are briefly suspended by their teeth, the tongue is
gently pulled forward with tweezers, and the inoculum is injected
into the lungs using a blunt 18 g needle. These inoculations do not
appear to cause undue discomfort or pain, and are (themselves)
associated with minimal mortality. Once the injected animals have
recovered from the anesthesia, they (initially) appear healthy.
[0263] Mice will not receive analgesics after intratracheal
inoculations for the following reasons: a) Many analgesics are
known to alter the host response to infection and endotoxin. b)
There is no evidence that mice undergoing this procedure experience
pain or discomfort. c) Most analgesics have an extremely short half
life in rodents, which would necessitate multiple injections, that
could become a stress in themselves.
[0264] b. Depletion of CD4+ Lymphocytes:
[0265] Mice will be depleted of lymphocytes by weekly
intraperitoneal injections (0.2 ml) with an anti-CD4 monoclonal
antibody. This procedure effectively depletes treated animals of
targeted T lymphocytes in blood and lymphoid tissue with minimal
morbidity and no mortality (in itself). Treated animals do not lose
weight and they (initially) appear healthy.
[0266] c. DNA Vaccination and Mucosal Boosting:
[0267] Mice will be injected under isoflurane anesthesia with
endotoxin-free plasmid DNA, 100 .mu.g, split in two injections, one
into each tibialis anterior muscle. Mucosal boosting is performed
by the intranasal administration of virus under isoflurane
anesthesia.
Example 32. Uses Against Other Pathogenic Fungi
[0268] The methods and constructs of this invention are also
expected to be effective in conferring immunity against at least
some other pathogenic fungi, for example Candida glabrata and
Candida albicans, both of which are human pathogens. A BLAST
comparison of the kexin amino acid sequences in these two species
versus that of P. carinii showed 53% homology with that of C.
glabrata and 50% with that of C. albicans. Effectiveness against
other fungal species with .about.40% or more amino acid sequence
homology is expected.
REFERENCES
[0269] 1. Zheng, M., Ramsay, A. J., Robichaux, M. B., Norris, K.
A., Kliment, C., Crowe, C., Rapaka, R. R., Steele, C., McAllister,
F., Shellito, J. E. et al 2005. CD4 T cell-independent DNA
vaccination against opportunistic infections. J Clin Invest. [0270]
2. Murray, J. F., Felton, C. P., Garay, S. M., Gottlieb, M. S.,
Hopewell, P C, Stover, D. E., and Teirstein, A. S. 1984. Pulmonary
complications of the acquired immunodeficiency syndrome. Report of
a National Heart, Lung, and Blood Institute workshop. N. Engl. J.
Med. 310:1682-1688. [0271] 3. Ives, N. J., Gazzard, B. G., and
Easterbrook, P. J. 2001. The changing pattern of aids-defining
illnesses with the introduction of highly active antiretroviral
therapy (haart) in a london clinic. J Infect. 42:134-139. [0272] 4.
Hoover, D. R., Saah, A. J., Bacellar, H., Phair, J., Detels, R.,
Anderson, R., and Kaslow, R. A. 1993. Clinical manifestations of
AIDS in the era of pneumocystis prophylaxis. Multicenter AIDS
Cohort Study. N. Engl. J. Med. 329:1922-1926. [0273] 5. Bozzette,
S. A., Finkelstein, D. M., Spector, S. A., Frame, P., Powderly, W.
G., He, W., Phillips, L., Craven, D., van, d.H., and Feinberg, J.
1995. A randomized trial of three antipneumocystis agents in
patients with advanced human immunodeficiency virus infection.
NIAID AIDS Clinical Trials Group. N. Engl. J. Med 332:693-699.
[0274] 6. Wallace, J. M., Hansen, N. I., Lavange, L., Glassroth,
J., Browdy, B L, Rosen, M. J., Kvale, P. A., Mangura, B. T.,
Reichman, L. B. et al 1997. Respiratory disease trends in the
Pulmonary Complications of HIV Infection Study cohort. Pulmonary
Complications of HIV Infection Study Group. American Journal of
Respiratory & Critical Care Medicine 155:72-80. [0275] 7.
Simonds, R. J., Hughes, W. T., Feinberg, J., and Navin, T. R. 1995.
Preventing Pneumocystis carinii pneumonia in persons infected with
human immunodeficiency virus. [Review] [41 refs]. Clinical
Infectious Diseases 21 Suppl 1:S44-S48. [0276] 8. Ledergerber, B.,
Mocroft, A., Reiss, P., Furrer, H., Kirk, O., Bickel, M.,
Uberti-Foppa, C., Pradier, C., d'Arminio, M. A., Schneider, M. M.
et al 2001. Discontinuation of secondary prophylaxis against
Pneumocystis carinii pneumonia in patients with HIV infection who
have a response to antiretroviral therapy. Eight European Study
Groups. N. Engl. J Med. 344:168-174. [0277] 9. Lopez Bernaldo de
Quiros J C, Miro, J. M., Pena, J. M., Podzamczer, D., Alberdi, J.
C., Martinez, E., Cosin, J., Claramonte, X., Gonzalez, J., Domingo,
P. et al 2001. A randomized trial of the discontinuation of primary
and secondary prophylaxis against Pneumocystis carinii pneumonia
after highly active antiretroviral therapy in patients with HIV
infection. Grupo de Estudio del SIDA 04/98. N. Engl. J Med.
344:159-167. [0278] 10. Kenyon, G. 2001. Resistance study to
re-evaluate HAART. Nat. Med 7:515. [0279] 11. Richman, D. D. 2001.
HIV chemotherapy. Nature 410:995-1001. [0280] 12. Cushion, M. T.,
Stringer, J. R., and Walzer, P. D. 1991. Cellular and molecular
biology of Pneumocystis carinii. International Review of Cytology
131:59-107. [0281] 13. Stansell, J. D., Osmond, D. H., Charlebois,
E., Lavange, L., Wallace, J M, Alexander, B. V., Glassroth, J.,
Kvale, P. A., Rosen, M. J. et al 1997. Predictors of Pneumocystis
carinii pneumonia in HIV-infected persons. Pulmonary Complications
of HIV Infection Study Group. American Journal of Respiratory &
Critical Care Medicine 155:60-66. [0282] 14. Beck, J. M., Warnock,
M. L., Curtis, J. L., Sniezek, M. J., Arraj-Peffer, S. M.,
Kaltreider, H. B., and Shellito, J. E. 1991. Inflammatory Responses
to Pneumocystis Carinii in Mice Selectively Depleted of Helper T
Lymphocytes. Am. J Respir. Cell Mol. Biol. 5:186-197. [0283] 15.
Shellito, J., Suzara, V. V., Blumenfeld, W., Beck, J. M., Steger,
H. J., and Ermak, T. H. 1990. A new model of Pneumocystis carinii
infection in mice selectively depleted of helper T lymphocytes. J.
Clin. Invest. 85:1686-1693. [0284] 16. Harmsen, A. G., and
Stankiewicz, M. 1990. Requirement for CD4+ cells in resistance to
Pneumocystis carinii pneumonia in mice. J Exp. Med 172:937-945.
[0285] 17. Roths, J. B., and Sidman, C. L. 1992. Both immunity and
hyperresponsiveness to Pneumocystis carinii result from transfer of
CD4+ but not CD8+ T cells into severe combined immunodeficiency
mice. J. Clin. Invest. 90:673-678. [0286] 18. Theus, S. A., Linke,
M. J., Andrews, R. P., and Walzer, P. D. 1993. Proliferative and
cytokine responses to a major surface glycoprotein of Pneumocystis
carinii. Infect. Immun. 61:4703-4709. [0287] 19. Theus, S. A.,
Smulian, A. G., Sullivan, D. W., and Walzer, P. D. 1997. Cytokine
responses to the native and recombinant forms of the major surface
glycoprotein of Pneumocystis carinii. Clinical & Experimental
Immunology 109:255-260. [0288] 20. Murray, H. W., Rubin, B. Y.,
Masur, H., and Roberts, R. B. 1984. Impaired production of
lymphokines and immune (gamma) interferon in the acquired
immunodeficiency syndrome. N. Engl. J. Med. 310:883-889. [0289] 21.
Rudy, T., Opelz, G., Gerlach, R., Daniel, V., and Schimpf, K. 1988.
Correlation of in vitro immune defects with impaired gamma
interferon response in human-immunodeficiency-virus-infected
individuals. Vox Sanguinis 54:92-95. [0290] 22. Pesanti, E. L.
1991. Interaction of cytokines and alveolar cells with Pneumocystis
carinii in vitro. J. Infect. Dis. 163:611-616. [0291] 23. Chen, W.,
Havell, E. A., and Harmsen, A. 1992. Importance of endogenous tumor
necrosis factor-alpha and gamma interferon in host resistance
against Pneumocystis carinii infection. Infect. Immun.
60:1279-1284. [0292] 24. Garvy, B. A., Ezekowitz, R. A., and
Harmsen, A. G. 1997. Role of gamma interferon in the host immune
and inflammatory responses to Pneumocystis carinii infection.
Infect. Immun. 65:373-379. [0293] 25. Shear, H. L., Valladares, G.,
and Narachi, M. A. 1990. Enhanced treatment of Pneumocystis carinii
pneumonia in rats with interferon-gamma and reduced doses of
trimethoprim/sulfamethoxazole. Journal of Acquired Immune
Deficiency Syndromes 3:943-948. [0294] 26. Beck, J. M., Liggit, H.
D., Brunette, E. N., Fuchs, H. J., Shellito, J. E., and Debs, R. J.
1991. Reduction in intensity of Pneumocystis carinii pneumonia in
mice by aerosol administration of interferon-gamma. Infect. Immun.
59:3859-3862. [0295] 27. Debs, R. J., Fuchs, H. J., Philip, R.,
Montgomery, A. B., Brunette, E. N., Liggitt, D., Patton, J. S., and
Shellito, J. E. 1988. Lung-specific delivery of cytokines induces
sustained pulmonary and systemic immunomodulation in rats. J.
Immunol. 140:3482-3488. [0296] 28. Burchett, S. K., Weaver, W. M.,
Westall, J. A., Larsen, A., Kronheim, and Wilson, C. B. 1988.
Regulation of tumor necrosis factor/cachectin and IL-1 secretion in
human mononuclear phagocytes. J. Immunol. 140:3473-3481. [0297] 29.
Drath, D. B. 1986. Modulation of pulmonary macrophage superoxide
release and tumoricidal activity following activation by biological
response modifiers. Immunopharmacology 12:117-126. [0298] 30.
Sherman, M. P., Loro, M. L., Wong, V. Z., and Tashkin, D. P. 1991.
Cytokine- and Pneumocystis carinii-induced L-arginine oxidation by
murine and human pulmonary alveolar macrophages. Journal of
Protozoology 38:234S-236S. [0299] 31. Limper, A. H., Hoyte, J. S.,
and Standing, J. E. 1997. The role of alveolar macrophages in
Pneumocystis carinii degradation and clearance from the lung. J.
Clin. Invest. 99:2110-2117. [0300] 32. Kolls, J. K., Habetz, S.,
Shean, M. K., Vazquez, C., Brown, J. A., Lei, D., Schwarzenberger,
P., Ye, P., Nelson, S., Summer, W. R. et al 1999. IFN-gamma and
CD8+ T Cells Restore Host Defenses Against Pneumocystis carinii in
Mice Depleted of CD4+ T Cells. J Immunol 162:2890-2894. [0301] 33.
Kolls, J. K., Ye, P., and Shellito, J. E. 2001. Gene therapy to
modify pulmonary host defenses. Semin. Respir Infect. 16:18-26.
[0302] 34. McAllister, F., Steele, C., Zheng, M., Shellito, J. E.,
and Kolls, J. K. 2005. In Vitro Effector Activity of Pneumocystis
murina-Specific T-Cytotoxic-1 CD8+ T Cells: Role of
Granulocyte-Macrophage Colony-Stimulating Factor. Infect Immun
73:7450-7457. [0303] 35. Garvy, B. A., Wiley, J. A., Gigliotti, F.,
and Harmsen, A. G. 1997. Protection against Pneumocystis carinii
pneumonia by antibodies generated from either T helper 1 or T
helper 2 responses. Infection & Immunity 65:5052-5056. [0304]
36. Lund, F. E., Hollifield, M., Schuer, K., Lines, J. L., Randall,
T. D., and Garvy, B. A. 2006. B cells are required for generation
of protective effector and memory CD4 cells in response to
Pneumocystis lung infection. J Immunol. 176:6147-6154. [0305] 37.
Ledbetter, J. A., Shu, G., Gallagher, M., and Clark, E. A. 1987.
Augmentation of normal and malignant B cell proliferation by
monoclonal antibody to the B cell-specific antigen BP50 (CDW40). J
Immunol. 138:788-794. [0306] 38. Levy, J., Espanol-Boren, T.,
Thomas, C., Fischer, A., Tovo, P., Bordigoni, P., Resnick, I.,
Fasth, A., Baer, M., Gomez, L. et al 1997. Clinical spectrum of
X-linked hyper-IgM syndrome. J Pediatr. 131:47-54. [0307] 39.
Schoenberger, S. P., Toes, R. E., van der Voort, E. I., Offringa,
R., and Melief, C. J. 1998. T-cell help for cytotoxic T lymphocytes
is mediated by CD40-CD40L interactions. Nature 393:480-483. [0308]
40. Bennett, S. R., Carbone, F. R., Karamalis, F., Flavell, R. A.,
Miller, J. F., and Heath, W. R. 1998. Help for cytotoxic-T-cell
responses is mediated by CD40 signalling. Nature 393:478-480.
[0309] 41. Ridge, J. P., Di Rosa, F., and Matzinger, P. 1998. A
conditioned dendritic cell can be a temporal bridge between a CD4+
T-helper and a T-killer cell. Nature 393:474-478. [0310] 42. Lane,
P., Brocker, T., Hubele, S., Padovan, E., Lanzavecchia, A., and
McConnell, F. 1993. Soluble CD40 ligand can replace the normal T
cell-derived CD40 ligand signal to B cells in T cell-dependent
activation. J Exp. Med. 177:1209-1213. [0311] 43. Wiley, J. A., and
Harmsen, A. G. 1995. CD40 ligand is required for resolution of
Pneumocystis carinii pneumonia in mice. J Immunol. 155:3525-3529.
[0312] 44. Grewal, I. S., Borrow, P., Pamer, E. G., Oldstone, M.
B., and Flavell, R. A. 1997. The CD40-CD154 system in
anti-infective host defense. Curr. Opin. Immunol. 9:491-497. [0313]
45. Guo, L., Johnson, R. S., and Schuh, J. C. 2000. Biochemical
characterization of endogenously formed eosinophilic crystals in
the lungs of mice. J Biol Chem. 275:8032-8037. [0314] 46. Oz, H.
S., Hughes, W. T., Rehg, J. E., and Thomas, E. K. 2000. Effect of
CD40 ligand and other immunomodulators on Pneumocystis carinii
infection in rat model. Microb. Pathog. 29:187-190. [0315] 47.
Kikuchi, T., Worgall, S., Singh, R., Moore, M. A., and Crystal, R.
G. 2000. Dendritic cells genetically modified to express CD40
ligand and pulsed with antigen can initiate antigen-specific
humoral immunity independent of CD4+ T cells. Nat. Med.
6:1154-1159. [0316] 48. Marcotte, H., Levesque, D., Delanay, K.,
Bourgeault, A., de la, D. R., Brochu, S., and Lavoie, M. C. 1996.
Pneumocystis carinii infection in transgenic B cell-deficient mice.
J Infect. Dis. 173:1034-1037. [0317] 49. Theus, S. A., Smulian, A.
G., Steele, P., Linke, M. J., and Walzer, P. D. 1998. Immunization
with the major surface glycoprotein of Pneumocystis carinii elicits
a protective response. Vaccine 16:1149-1157. [0318] 50. Gigliotti,
F., Wiley, J. A., and Harmsen, A. G. 1998. Immunization with
Pneumocystis carinii gpA is immunogenic but not protective in a
mouse model of P. carinii pneumonia. Infect. Immun. 66:3179-3182.
[0319] 51. Pascale, J. M., Shaw, M. M., Durant, P. J., Amador, A.
A., Bartlett, M. S., Smith, J. W., Gregory, R. L., and McLaughlin,
G. L. 1999. Intranasal immunization confers protection against
murine Pneumocystis carinii lung infection. Infect. Immun.
67:805-809. [0320] 52. Smulian, A. G., Sullivan, D. W., and Theus,
S. A. 2000. Immunization with recombinant Pneumocystis carinii p55
antigen provides partial protection against infection:
characterization of epitope recognition associated with
immunization. Microbes. Infect. 2:127-136. [0321] 53. Zheng, M.,
Shellito, J. E., Marrero, L., Zhong, Q., Julian, S., Ye, P.,
Wallace, V., Schwarzenberger, P., and Kolls, J. K. 2001. CD4(+) T
cell-independent vaccination against Pneumocystis carinii in mice.
J Clin. Invest 108:1469-1474. [0322] 54. Steele, C., Marrero, L.,
Swain, S., Harmsen, A. G., Zheng, M., Brown, G. D., Gordon, S.,
Shellito, J. E., and Kolls, J. K. 2003. Alveolar
Macrophage-mediated Killing of Pneumocystis carinii f. sp. muris
Involves Molecular Recognition by the Dectin-1 {beta}-Glucan
Receptor. J. Exp. Med 198:1677-1688. [0323] 55. Numasaki, M.,
Watanabe, M., Suzuki, T., Takahashi, H., Nakamura, A., McAllister,
F., Hishinuma, T., Goto, J., Lotze, M. T., Kolls, J. K. et al 2005.
IL-17 Enhances the Net Angiogenic Activity and In Vivo Growth of
Human Non-Small Cell Lung Cancer in SCID Mice through Promoting
CXCR-2-Dependent Angiogenesis. J Immunol 175:6177-6189. [0324] 56.
Lee, L. H., Gigliotti, F., Wright, T. W., Simpson-Haidaris, P. J.,
Weinberg, G. A., and Haidaris, C. G. 2000. Molecular
characterization of KEX1, a kexin-like protease in mouse
Pneumocystis carinii. Gene 242:141-150. [0325] 57. Gigliotti, F.,
Garvy, B. A., Haidaris, C. G., and Harmsen, A. G. 1998. Recognition
of Pneumocystis carinii antigens by local antibody-secreting cells
following resolution of P. carinii pneumonia in mice. J Infect.
Dis. 178:235-242. [0326] 58. Kling, H. M., Shipley, T. W., Patil,
S., Morris, A., and Norris, K. A. 2009. Pneumocystis colonization
in immunocompetent and simian immunodeficiency virus-infected
cynomolgus macaques. J. Infect. Dis. 199:89-96. [0327] 59.
Estcourt, M. J., Ramsay, A. J., Brooks, A., Thomson, S. A.,
Medveckzy, C. J., and Ramshaw, I. A. 2002. Prime-boost immunization
generates a high frequency, high-avidity CD8(+) cytotoxic T
lymphocyte population. Int. Immunol. 14:31-37. [0328] 60. Cox, K.
S., Clair, J. H., Prokop, M. T., Sykes, K. J., Dubey, S. A.,
Shiver, J. W., Robertson, M. N., and Casimiro, D. R. 2008. DNA
gag/adenovirus type 5 (Ad5) gag and Ad5 gag/Ad5 gag vaccines induce
distinct T-cell response profiles. J Virol. 82:8161-8171. [0329]
61. Hanke, T., Goonetilleke, N., McMichael, A. J., and Dorrell, L.
2007. Clinical experience with plasmid DNA- and modified vaccinia
virus Ankara-vectored human immunodeficiency virus type 1 clade A
vaccine focusing on T-cell induction. J. Gen. Virol. 88:1-12.
[0330] 62. Karkhanis, L. U., and Ross, T. M. 2007. Mucosal vaccine
vectors: replication-competent versus replication-deficient
poxviruses. Curr. Pharm. Des 13:2015-2023. [0331] 63. Duerr, R. H.,
Taylor, K. D., Brant, S. R., Rioux, J. D., Silverberg, M. S., Daly,
M. J., Steinhart, A. H., Abraham, C., Regueiro, M., Griffiths, A.
et al 2006. A Genome-Wide Association Study Identifies IL23R as an
Inflammatory Bowel Disease Gene. Sci 314:1461-1463. [0332] 64.
Happel, K. I., Lockhart, E. A., Mason, C. M., Porretta, E.,
Keoshkerian, E., Odden, A. R., Nelson, S., and Ramsay, A. J. 2005.
Pulmonary interleukin-23 gene delivery increases local T-cell
immunity and controls growth of
Mycobacterium tuberculosis in the lungs. Infect Immun 73:5782-5788.
[0333] 65. Reay, J., Kim, S. H., Lockhart, E., Kolls, J., and
Robbins, P. D. 2009. Adenoviral-mediated, intratumor gene transfer
of interleukin 23 induces a therapeutic antitumor response. Cancer
Gene Ther. [0334] 66. Morelli, A. E., Larregina, A. T., Ganster, R.
W., Zahorchak, A. F., Plowey, J. M., Takayama, T., Logar, A. J.,
Robbins, P. D., Falo, L. D., and Thomson, A. W. 2000. Recombinant
adenovirus induces maturation of dendritic cells via an
NF-kappaB-dependent pathway. J Virol. 74:9617-9628. [0335] 67.
Kikuchi, T., Moore, M. A., and Crystal, R. G. 2000. Dendritic cells
modified to express CD40 ligand elicit therapeutic immunity against
preexisting murine tumors. Blood 96:91-99. [0336] 68. Zhong, L.,
Granelli-Pipemo, A., Pope, M., Ignatius, R., Lewis, M. G., Frankel,
S. S., and Steinman, R. M. 2000. Presentation of SIVgag to monkey T
cells using dendritic cells transfected with a recombinant
adenovirus. Eur. J Immunol. 30:3281-3290. [0337] 69. Neeson, P.,
Boyer, J., Kumar, S., Lewis, M. G., Mattias, L., Veazey, R.,
Weiner, D., and Paterson, Y. 2006. A DNA prime-oral Listeria boost
vaccine in rhesus macaques induces a SIV-specific CD8 T cell
mucosal response characterized by high levels of alpha4beta7
integrin and an effector memory phenotype. Virology 354:299-315.
[0338] 70. Shean, M. K., Baskin, G., Sullivan, D., Schurr, J.,
Cavender, D. E., Shellito, J. E., Schwarzenberger, P. O., and
Kolls, J. K. 2000. Immunomodulation and adenoviral gene transfer to
the lungs of nonhuman primates. Hum. Gene Ther. 11:1047-1055.
[0339] 71. Sullivan, D. E., Dash, S., Du, H., Hiramatsu, N., Aydin,
F., Kolls, J., Blanchard, J., Baskin, G., and Gerber, M. A. 1997.
Liver-Directed Gene Transfer in Non-human Primates. Hum. Gene Ther.
8:1195-1206.
PUBLICATIONS BY THE INVENTORS AND THEIR COLLEAGUES
[0339] [0340] 1. Steele C, Marrero L, Shellito J E, Kolls J K.
Alveolar macrophage-mediated killing of Pneumocystis carinii f. sp.
muris involves pattern recognition by the Dectin-1 beta-glucan
receptor. J. Exp Med. 2003; 198:1677-1688 [0341] 2. Happel K I,
Zheng M, Quinton L J, Lockhart E, Ramsay A J, Shellito J E, Schurr
J R, Bagby G J, Nelson S, Kolls J K. Cutting Edge: Roles of
Toll-Like Receptor 4 and IL-23 in IL-17 Expression in Response to
Klebsiella pneumoniae Infection. J. Immunol 2003; 170:4432-4436.
[0342] 3. Kolls J K, Kanaly S T, Ramsay A J. Interleukin 17: an
emerging role in lung Inflammation. Am J Respir Cell Mol Biol 2003
January; 28(1):9-11 [0343] 4. McAllister F, Steele C, Zheng M,
Young Erana, Shellito J E, Marrero L, Kolls J K. Tcl CD8+ T-cells
are effector cells against Pneumocystis in mice. J Immunol 2004;
172:1132-1138. [0344] 5. Kolls J K and Linden A. IL-17 family
members and Inflammation. Immunity. 2004 October; 21(4):467-76.
[0345] 6. Steele C, Shellito J E, Kolls J K. Immunity against the
opportunistic fungal pathogen Pneumocystis. Medical Mycology 2004;
43:1-19. [0346] 7. Schurr J R, Young E, Byrne P, Steele C, Happel
K, Shellito J E, Kolls J K. Central role of TLR4 signaling and host
defense in experimental gram negative pneumonia. Infection and
Immunity 2005; 73:532-545. [0347] 8. Mc Allister F, Henry A,
Kreindler J L, Dubin P J, Ulrich L, Steele C, Finder J D, Pilewski
J M, Carreno B, Goldman S J, Pirhonen J, and Kolls J K. Role of
IL-17A, IL-17F and the IL-17 receptor in regulating Gro-alpha and
G-CSF in Bronchial Epithelium: implications for airway inflammation
in cystic fibrosis. J Immunol 175(1):404-12, 2005. [0348] 9. Happel
K, Dubin P J, Zheng M, Ghilardi N, Lockhart C, Quinton L J, Odden A
R, Shellito J E, Bagby G J, Nelson S, Kolls J K Divergent roles of
IL-23 and IL-12 in host defense against Klebsiella pneumoniae J Exp
Med 2005; 202:761-769. [0349] 10. Ruan S, Young E, Luce M J, Reiser
J, Kolls J K, Shellito J E. Conditional expression of
interferon-gamma to enhance host responses to pulmonary bacterial
infection. Pulmonary Pharmacology and Therapeutics. 2005;
19:251-257. [0350] 11. Zheng M, Ramsay A J, Robichaux M B, Norris K
A, Kliment C, Crowe C, Rapaka R R, Steele C, McAllister F, Shellito
J E, Marrero L, Schwarzenberger P, Zhong Q, and Kolls J K. CD4+ T
cell-independent DNA vaccination against opportunistic infections
J. Clin. Invest., 2005; 115: 3536-3544 [0351] 12. McAllister F,
Steele C, Zheng M, Shellito J E, Kolls J K. In vitro effector
activity of Pneumocystis-specific T cytotoxic-1 CD8+ T-cells: role
of G M-CSF. Infec Immun 2005; 73:7450-7457. [0352] 13. McAllister
F, Ruan S, Kolls J K, Shellito J E. CXCR3 and IP-10 Pneumocystis
pneumonia. J. Immunology 2006; 177:1846-1854. [0353] 14. McKinley
L, Logar A J, McAllister F, Zheng M, Steele C, and Kolls J K.
Regulatory T Cells Dampen Pulmonary Inflammation and Lung Injury in
an Animal Model of Pneumocystis Pneumonia. J. Immunol.
177(9):6215-6226, 2006. [0354] 15. Rapaka R R, Goetzman E S, Zheng
M, Vockley J, McKinley L, Kolls J K, Steele C. Enhanced defense
against Pneumocystis carinii mediated by a novel dectin-1 receptor
Fc fusion protein. J Immunol. 178(6):3702-12, 2007. [0355] 16. Hsu
H C, Yang P, Wang J, Wu Q, Myers R, Chen J, Yi J, Guentert T,
Tousson A, Stanus A L, Le T V, Lorenz R G, Xu H, Kolls J K, Carter
R H, Chaplin D D, Williams R W, Mountz J D. Interleukin
17-producing T helper cells and interleukin 17 orchestrate
autoreactive germinal center development in autoimmune BXD2 mice.
Nat Immunol. 2008 February; 9(2):166-75 [0356] 17. Aujla S, Chan Y
C, Zheng M, Fei M, Askew D J, Pociask D A, Reinhart T A, McAllister
F, Edeal J, Gaus K, Husain S, Kreindler J L, Dubin P J, Pilewski J
M, Myerburg M M, Mason C A, Iwakura Y, and Kolls J K. IL-22
mediates mucosal host defense against gram negative bacterial
pneumonia. Nat Med. 2008 March; 14(3):275-81. [0357] 18. Raffatellu
M, Santos R L, Verhoeven D, Wilson R P, Winter S E, Godinez I,
Sankaran S, Paixao T, George M D, Gordon M A, Kolls J K, Dandekar
S, and Blumler A J. IL-17 orchestrates a mucosal response against
Salmonella dissemination from the gut. Nat Med. 2008 April;
14(4):421-8. [0358] 19. Ruan S, McKinley L, Zheng M, Rudner X,
Kolls J K, Shellito J E. Interleukin-12 and host defense against
murine Pneumocystis pneumonia. Infection and Immunity 2008; 76:
2130-2137. [0359] 20. Ouyang W, Kolls J K, Zheng Y. The biological
functions of T helper 17 cell effector cytokines in inflammation.
Immunity. 2008 April; 28(4):454-67. [0360] 21. Kolls J K, McCray P
B Jr, Chan Y R. Cytokine-mediated regulation of antimicrobial
proteins. Nat Rev Immunol. 2008 November; 8(11):829-35. [0361] 22.
Chan Y R, Liu J, Pociask D, Zheng M, Mietzner T A, Berger T, Mak T,
Clifton M, Strong R K, Ray P, Kolls J K. Lipocalin 2 is required
for pulmonary host defense against Klebsiella infection. J.
Immunol. 2009, 182:4493-4494.
Miscellaneous
[0362] The complete disclosures of all references and publications
cited in this disclosure are hereby incorporated by reference in
their entirety, as is the entire disclosure of priority application
61/294,252. In the event of an otherwise irreconcilable conflict,
the present specification shall control.
Sequence CWU 1
1
111306DNAArtificial sequencemini-Kexin, wild type 1atgtcttgta
gctggggacc tcgtgatgat ggaaaaacaa ttgaaggagt tccttatagt 60gcatataatt
caattattaa tgggataaat cttggaagga aaggtcttgg ttctatatat
120gtttttggaa gtggaaatgg aggctattat gataattgca attacgatgg
atatgtagtt 180agtccatata ctattactat cggttctata gatgtgagag
gaataagaca ttatttttca 240gagcaatgtt cttccgttct tgcttctaca
tattcgggtt ctattgtaac caatgcacgc 300atttga 3062399DNAArtificial
sequencemini-Kexin, wild type, with leader sequence 2atggacacag
acacactcgt gctatgggta ctgctctggg ttccaggttc cactggtgac 60gcggcccagc
cggccaggcg cgccgtacga agcttgtctt gtagctgggg acctcgtgat
120gatggaaaaa caattgaagg agttccttat agtgcatata attcaattat
taatgggata 180aatcttggaa ggaaaggtct tggttctata tatgtttttg
gaagtggaaa tggaggctat 240tatgataatt gcaattacga tggatatgta
gttagtccat atactattac tatcggttct 300atagatgtga gaggaataag
acattatttt tcagagcaat gttcttccgt tcttgcttct 360acatattcgg
gttctattgt aaccaatgca cgcatttga 3993306DNAArtificial
sequencemini-Kexin, codon optimized 3atgagctgca gctggggacc
tagggacgac ggcaagacca tcgagggcgt gccctacagc 60gcctacaaca gcatcatcaa
cggcatcaac ctgggccgga agggcctggg cagcatctac 120gtgttcggca
gcggcaacgg cggctactac gacaactgca actacgacgg ctacgtggtg
180tccccctaca ccatcaccat cggctccatc gacgtgcggg gcatccggca
ctacttcagc 240gagcagtgca gcagcgtgct ggcttccacc tacagcggca
gcatcgtgac caacgcccgg 300atctga 3064399DNAArtificial
sequencemini-Kexin, with leader sequence, codon optimized
4atggacaccg acaccctggt gctgtgggtg ctgctgtggg tgcccggcag cacaggggat
60gccgcccagc ccgccagacg ggccgtgcgg agcctgagct gcagctgggg acctagggac
120gacggcaaga ccatcgaggg cgtgccctac agcgcctaca acagcatcat
caacggcatc 180aacctgggcc ggaagggcct gggcagcatc tacgtgttcg
gcagcggcaa cggcggctac 240tacgacaact gcaactacga cggctacgtg
gtgtccccct acaccatcac catcggctcc 300atcgacgtgc ggggcatccg
gcactacttc agcgagcagt gcagcagcgt gctggcttcc 360acctacagcg
gcagcatcgt gaccaacgcc cggatctga 3995101PRTArtificial
sequencemini-Kexin, wild type 5Met Ser Cys Ser Trp Gly Pro Arg Asp
Asp Gly Lys Thr Ile Glu Gly 1 5 10 15 Val Pro Tyr Ser Ala Tyr Asn
Ser Ile Ile Asn Gly Ile Asn Leu Gly 20 25 30 Arg Lys Gly Leu Gly
Ser Ile Tyr Val Phe Gly Ser Gly Asn Gly Gly 35 40 45 Tyr Tyr Asp
Asn Cys Asn Tyr Asp Gly Tyr Val Val Ser Pro Tyr Thr 50 55 60 Ile
Thr Ile Gly Ser Ile Asp Val Arg Gly Ile Arg His Tyr Phe Ser 65 70
75 80 Glu Gln Cys Ser Ser Val Leu Ala Ser Thr Tyr Ser Gly Ser Ile
Val 85 90 95 Thr Asn Ala Arg Ile 100 6132PRTArtificial
sequencemini-Kexin, wild type, with leader sequence 6Met Asp Thr
Asp Thr Leu Val Leu Trp Val Leu Leu Trp Val Pro Gly 1 5 10 15 Ser
Thr Gly Asp Ala Ala Gln Pro Ala Arg Arg Ala Val Arg Ser Leu 20 25
30 Ser Cys Ser Trp Gly Pro Arg Asp Asp Gly Lys Thr Ile Glu Gly Val
35 40 45 Pro Tyr Ser Ala Tyr Asn Ser Ile Ile Asn Gly Ile Asn Leu
Gly Arg 50 55 60 Lys Gly Leu Gly Ser Ile Tyr Val Phe Gly Ser Gly
Asn Gly Gly Tyr 65 70 75 80 Tyr Asp Asn Cys Asn Tyr Asp Gly Tyr Val
Val Ser Pro Tyr Thr Ile 85 90 95 Thr Ile Gly Ser Ile Asp Val Arg
Gly Ile Arg His Tyr Phe Ser Glu 100 105 110 Gln Cys Ser Ser Val Leu
Ala Ser Thr Tyr Ser Gly Ser Ile Val Thr 115 120 125 Asn Ala Arg Ile
130 722DNAArtificial sequenceSynthetic primer for mouse PCrRNA
7atgaggtgaa aagtcgaaag gg 22826DNAArtificial sequenceSynthetic
primer for mouse PCrRNA 8tgattgtctc agatgaaaaa cctctt
26938DNAArtificial sequenceSynthetic primer 9acagcccaga ataatgaata
aagttcctca attgttac 3810261PRTHomo sapiensmisc_featureCD40L 10Met
Ile Glu Thr Tyr Asn Gln Thr Ser Pro Arg Ser Ala Ala Thr Gly 1 5 10
15 Leu Pro Ile Ser Met Lys Ile Phe Met Tyr Leu Leu Thr Val Phe Leu
20 25 30 Ile Thr Gln Met Ile Gly Ser Ala Leu Phe Ala Val Tyr Leu
His Arg 35 40 45 Arg Leu Asp Lys Ile Glu Asp Glu Arg Asn Leu His
Glu Asp Phe Val 50 55 60 Phe Met Lys Thr Ile Gln Arg Cys Asn Thr
Gly Glu Arg Ser Leu Ser 65 70 75 80 Leu Leu Asn Cys Glu Glu Ile Lys
Ser Gln Phe Glu Gly Phe Val Lys 85 90 95 Asp Ile Met Leu Asn Lys
Glu Glu Thr Lys Lys Glu Asn Ser Phe Glu 100 105 110 Met Gln Lys Gly
Asp Gln Asn Pro Gln Ile Ala Ala His Val Ile Ser 115 120 125 Glu Ala
Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly 130 135 140
Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln 145
150 155 160 Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln
Val Thr 165 170 175 Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro
Phe Ile Ala Ser 180 185 190 Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu
Arg Ile Leu Leu Arg Ala 195 200 205 Ala Asn Thr His Ser Ser Ala Lys
Pro Cys Gly Gln Gln Ser Ile His 210 215 220 Leu Gly Gly Val Phe Glu
Leu Gln Pro Gly Ala Ser Val Phe Val Asn 225 230 235 240 Val Thr Asp
Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe 245 250 255 Gly
Leu Leu Lys Leu 260 11786DNAHomo sapiensmisc_featureCD40L
11atgatcgaaa catacaacca aacttctccc cgatctgcgg ccactggact gcccatcagc
60atgaaaattt ttatgtattt acttactgtt tttcttatca cccagatgat tgggtcagca
120ctttttgctg tgtatcttca tagaaggttg gacaagatag aagatgaaag
gaatcttcat 180gaagattttg tattcatgaa aacgatacag agatgcaaca
caggagaaag atccttatcc 240ttactgaact gtgaggagat taaaagccag
tttgaaggct ttgtgaagga tataatgtta 300aacaaagagg agacgaagaa
agaaaacagc tttgaaatgc aaaaaggtga tcagaatcct 360caaattgcgg
cacatgtcat aagtgaggcc agcagtaaaa caacatctgt gttacagtgg
420gctgaaaaag gatactacac catgagcaac aacttggtaa ccctggaaaa
tgggaaacag 480ctgaccgtta aaagacaagg actctattat atctatgccc
aagtcacctt ctgttccaat 540cgggaagctt cgagtcaagc tccatttata
gccagcctct gcctaaagtc ccccggtaga 600ttcgagagaa tcttactcag
agctgcaaat acccacagtt ccgccaaacc ttgcgggcaa 660caatccattc
acttgggagg agtatttgaa ttgcaaccag gtgcttcggt gtttgtcaat
720gtgactgatc caagccaagt gagccatggc actggcttca cgtcctttgg
cttactcaaa 780ctctga 786
* * * * *
References