U.S. patent application number 15/931213 was filed with the patent office on 2021-05-20 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 | 20210145948 15/931213 |
Document ID | / |
Family ID | 1000005373896 |
Filed Date | 2021-05-20 |
![](/patent/app/20210145948/US20210145948A1-20210520-D00001.png)
![](/patent/app/20210145948/US20210145948A1-20210520-D00002.png)
![](/patent/app/20210145948/US20210145948A1-20210520-D00003.png)
![](/patent/app/20210145948/US20210145948A1-20210520-D00004.png)
![](/patent/app/20210145948/US20210145948A1-20210520-D00005.png)
![](/patent/app/20210145948/US20210145948A1-20210520-D00006.png)
![](/patent/app/20210145948/US20210145948A1-20210520-D00007.png)
![](/patent/app/20210145948/US20210145948A1-20210520-D00008.png)
![](/patent/app/20210145948/US20210145948A1-20210520-D00009.png)
United States Patent
Application |
20210145948 |
Kind Code |
A1 |
Kolls; Jay K. ; et
al. |
May 20, 2021 |
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: |
1000005373896 |
Appl. No.: |
15/931213 |
Filed: |
May 13, 2020 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
16035319 |
Jul 13, 2018 |
|
|
|
15931213 |
|
|
|
|
15443976 |
Feb 27, 2017 |
|
|
|
16035319 |
|
|
|
|
13959691 |
Aug 5, 2013 |
9580704 |
|
|
15443976 |
|
|
|
|
13521621 |
Nov 12, 2012 |
|
|
|
PCT/US2011/020170 |
Jan 5, 2011 |
|
|
|
13959691 |
|
|
|
|
61294252 |
Jan 12, 2010 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/191 20130101;
A61K 2039/5256 20130101; A61K 39/0002 20130101; C12Y 304/21061
20130101; A61K 39/00 20130101; C12N 9/58 20130101; A61K 2039/55516
20130101; C07K 14/70575 20130101; A61K 2039/55 20130101; A61K
2039/53 20130101; C07K 2319/40 20130101; C12N 9/60 20130101; A61K
2039/545 20130101 |
International
Class: |
A61K 39/00 20060101
A61K039/00; C12N 9/58 20060101 C12N009/58; A61K 38/19 20060101
A61K038/19; C07K 14/705 20060101 C07K014/705; 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 (MN). 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 WV-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
STY-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 STY 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 10-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.
[0068] Definitions. 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-.gamma. may be used to assess
immunological response to the polypeptide, such as IL-12,
TNF-.alpha., TL-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 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. 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.
[0084] 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).
[0085] 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.
[0086] 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.
[0087] 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.
[0088] 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.
[0089] 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.
[0090] 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.
[0091] 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 faun of solutions, suspensions, tablets,
pills, capsules, sustained release formulations or powders and
advantageously contain 10-95% of active ingredient, preferably
25-70%.
[0092] DNA Vaccine. In a preferred embodiment, nucleic acid
fragments in accordance with the invention are used for the in vivo
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.
[0093] Live Recombinant Vaccines; Plasmids. 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.
[0094] 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.
[0095] 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.
[0096] 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
[0097] 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
[0098] 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
[0099] 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
[0100] 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 Fe-dependent manner. These antibodies also
confer significant protection against PC when passively transferred
to scid mice prior to PC challenge.
Example 5
[0101] 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
[0102] 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
[0103] 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.sup.8 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
[0104] 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 aa 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
[0105] 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
[0106] 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
[0107] 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
[0108] 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
[0109] 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.sup.7 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
[0110] 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
[0111] To determine the role of IL-12 and IL-23 in
AdCD40L-transduced, DC-based vaccine responses, we generated DCs
from IL-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
[0112] CD41ND, 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/4 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 STY-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
[0113] 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
[0114] 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 ease, 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.
[0115] Manipulations: There will be two doses of plasmid
injections, three weeks apart.
[0116] Measures and Outcomes: 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.
[0117] Expected Results and Interpretations: 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.
[0118] Alternative Approaches: 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
[0119] Hypothesis: We hypothesize that the co-administration of
CD40L with antigen allows for CD4-independent (CD4IND) B-cell
responses in vivo, and that mucosal boosting will enhance mucosal
antigen-specific B-cell responses, as well as overall protective
immunity.
[0120] Rationale: 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.
[0121] 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 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.
[0122] Manipulations: 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.
[0123] Measures and Outcomes: 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.
[0124] Expected Results and Interpretations: 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.
[0125] Alternative Approaches: 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
[0126] Hypothesis: 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.
[0127] Rationale: 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.
[0128] Experimental Groups. 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.
[0129] Manipulations: 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.
[0130] Measures and Outcomes: 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.
[0131] Expected Results and Interpretations: 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.
[0132] Alternative Approaches: 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
[0133] Hypothesis: 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.
[0134] Rationale: 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.
[0135] 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 1M injection of pmini-Kexin
WT, psec-mini-Kexin-WT, pmini-Kexin CO, or psec-mini-Kexin-CO, in
each ease 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.
[0136] Manipulations: 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.
[0137] Measures and Outcomes: 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.
[0138] Expected Results and Interpretations: 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.
[0139] Alternative Approaches: 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
[0140] 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
[0141] 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.
[0142] Hypothesis: 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.
[0143] Rationale: 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.
[0144] Experimental Groups. 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.
[0145] Manipulations: 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.
[0146] Measures and Outcomes: 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.
[0147] Expected Results and Interpretations: 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.
[0148] Alternative Approaches: 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
[0149] Hypothesis: 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.
[0150] Rationale: 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.
[0151] 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. 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.
[0152] Measures and Outcomes: 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.
[0153] Expected Results, Interpretations and Alternative
Approaches: 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
[0154] We will confirm that the mini-kexin constructs produce
vaccine-induced immune responses in SIV-infected, CD4-deficient
macaques. Control or STY-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 STY-infected
(untreated) macaques, compared to 0% in non-SIV infected
monkeys.
Example 26. Evaluate the Effect of CD40L on Monkey DCs
[0155] Hypothesis: 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.
[0156] Rationale: Preliminary studies have demonstrated similar
results in the mouse model. We therefore expect similar results in
the macaques.
[0157] Experimental Groups. 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.
[0158] Manipulations: 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.++, CD80.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).
[0159] Expected Results and Interpretations: 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.
[0160] Alternative Approaches: 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
[0161] Hypothesis: 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.
[0162] Rationale: 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.
[0163] Experimental Groups. 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.
[0164] Manipulations, Measurements, and Outcomes: 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-Kex1 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 hCDCl40L, 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.
[0165] Expected Results and Interpretations: 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.
[0166] Alternative Approaches: 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 STY-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
[0167] 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.
[0168] 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).
[0169] 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
[0170] 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
[0171] Except as otherwise stated, the following materials and
procedures have been used or will be used in the experiments
described above:
[0172] 1. Animals. 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-HCl (10 mg/kg, i.m.) for bronchoscopy and
blood sampling. Bronchoscopy will be performed with topical
anesthesia with 2% xylocaine.
[0173] 2. Monitoring Animal Health. 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.
[0174] 3. Maintenance of P. carinii in scid or CD40L knockout mice.
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 CS7BL/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.
[0175] 4. Inoculation of Mice with P. carinii organisms. 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.
[0176] 5. Examination of Lung Tissue for P. carinii Infection. Lung
tissue will be fixed in formalin and stained with Gomori's
methenamine silver and hematoxylinkosin.
[0177] 6. Controls for Bacterial/Fungal/Viral Infection. 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.
[0178] 7. Depletion of host CD4+ lymphocytes. 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.
[0179] 8. RNA isolation and TaqMan.TM. probes and primers for PC
rRNA.
[0180] Total RNA is isolated from the right lung of infected mice
by a single step method using TRIzol.TM. reagent (Life
Technologies, C A, 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 Tag 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.
[0181] 9. Pneumocystis viability assay. Macrophages (10.sup.6/ml)
suspended in a volume of 100 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.
[0182] 10. PC Kex1 ELISA. To determine anti-PC or Kex1 IgG titers,
ELISA plates (Corning, N.Y.) are coated with 100 ng of PC antigen
or Kex1 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.
[0183] 11. Bronchoalveolar lavage. Lavaged lymphocytes will be
obtained by bronchoalveolar 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.
[0184] 12. Retrieval of hilar and paratracheal lymph nodes. 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 DiffQuik.TM. staining. Cells
will be processed for flow cytometry as outlined above.
[0185] 13. B-cell Elipsots. 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.
[0186] 14. DNA Vaccination. 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.
[0187] 15. Statistical Analysis. 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
[0188] 1. Justification for the Use of Experimental Animals: 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.
[0189] 2. Veterinary Care of Experimental Animals: 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.
[0190] 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
STY-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-HCl (10 mg/kg, i.m.) and acepromazine (0.2
mg/kg, i.m.), or Telazol (5 mg/kg of Tiletamine and Zolazepam).
[0191] 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).
[0192] 3. Experimental Procedures Involving Live Animals:
[0193] a. Intratracheal Inoculation with P. carinii Organisms:
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.
[0194] 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.
[0195] b. Depletion of CD4+ lymphocytes: 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.
[0196] c. DNA vaccination and mucosal boosting: 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
[0197] 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
[0198] 1. Zheng, M., Ramsay, A. J, Robichaux, M. B., Norri s, 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. [0199]
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. [0200] 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. [0201] 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. [0202] 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.
[0203] 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. [0204] 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 1:544-S48. [0205] 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. [0206] 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 April 1998. N. Engl. J Med
344:159-167. [0207] 10. Kenyon, G. 2001. Resistance study to
re-evaluate HAART. Nat. Med. 7:515. [0208] 11. Richman, D. D. 2001.
HIV chemotherapy. Nature 410:995-1001. [0209] 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. [0210] 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. [0211] 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. [0212] 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. [0213] 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.
[0214] 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. [0215] 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. [0216] 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. [0217] 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. [0218] 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. [0219] 22. Pesanti, E. L.
1991. Interaction of cytokines and alveolar cells with Peumocystis
carinii in vitro. J. Infect. Dis. 163:611-616. [0220] 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. [0221] 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. [0222] 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. [0223] 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. [0224] 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. [0225] 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. [0226] 29.
Drath, D. B. 1986. Modulation of pulmonary macrophage superoxide
release and tumoricidal activity following activation by biological
response modifiers. Immunopharmacology 12:117-126. [0227] 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. [0228] 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. [0229] 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. [0230] 33.
Kolls, J. K., Ye, P., and Shellito, J. E. 2001. Gene therapy to
modify pulmonary host defenses. Semin. Respir Infect. 16:18-26.
[0231] 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. [0232] 35. Garvy, B. A., Gigliotti, F., and Hamsen,
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. [0233] 36. Lund,
F. E., Hollifield, M., Schuer, K., 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. [0234] 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. [0235]
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. [0236] 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. [0237] 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
Nature 393:478-480. [0238] 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. [0239] 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. [0240]
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. [0241] 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. [0242] 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. [0243] 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. [0244] 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. [0245] 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. [0246] 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. [0247] 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, [0248] 51. Pascale, J. M.,
Shaw, M. M., Durant, P., 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. [0249] 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. [0250] 53. Zheng, M., Marrero, L., Zhong, Q., 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. [0251] 54. Steele, C., Marrero, L.,
Swain, S., Ha isen, 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. [0252] 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. [0253] 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. [0254] 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. [0255] 58. Kling, H. M., Shipley, T. W., Patil,
S., Morris, A., and Norris, K. A. 2009.
[0256] Pneumocystis colonization in immunocompetent and simian
immunodeficiency virus-infected cynomolgus macaques. J. Infect.
Dis. 199:89-96, [0257] 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.
[0258] 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. [0259] 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. [0260] 62. Karkhanis, L. U., and Ross, T. M. 2007.
Mucosal vaccine vectors: replication-competent versus
replication-deficient poxviruses. Curr. Pharm. Des 13:2015-2023.
[0261] 63. Duerr, R. H., Taylor, K. D., Brant, S. R., 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. [0262] 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.
[0263] 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. [0264] 66. Morelli, A. E., Larregina, A. T., Ganster, R.
W., Zahorchak, A. F., 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. [0265] 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. [0266] 68. Zhong, L., Granelli-Piperno, A.,
Pope, M., Ignatius, R., Lewis, M. G., Frankel, S. S., and Steinman,
R. M. 2000. Presentation of SlVgag to monkey T cells using
dendritic cells transfected with a recombinant adenovirus. Eur. J
Immunol 30:3281-3290. [0267] 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. [0268] 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. [0269] 71. Sullivan, D. E., Dash, S., Du,
H., Hiramatsu, N., Aydin, F., 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: [0270] 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
[0271] 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. [0272] 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 [0273] 4. McAllister F,
Steele C, Zheng M, Young Erana, Shellito J E, Marrero L, Kolls J K.
Tel CD8+ T-cells are effector cells against Pneumocystis in mice. J
Immunol 2004; 172:1132-1138. [0274] 5. Kolls J K and Linden A.
IL-17 family members and Inflammation. Immunity. 2004 October;
21(4):467-76. [0275] 6. Steele C, Shellito J E, Kolls J K. Immunity
against the opportunistic fungal pathogen Pneumocystis. Medical
Mycology 2004; 43:1-19. [0276] 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. [0277] 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. [0278] 9. Happel K I, 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. [0279]
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. [0280] 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
[0281] 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 GM-CSF. Infec Immun 2005;
73:7450-7457. [0282] 13. McAllister F, Ruan 5, Kolls J K, Shellito
J E. CXCR3 and IP-10 Pneumocystis pneumonia. J. Immunology 2006;
177:1846-1854. [0283] 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. [0284]
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. [0285] 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
[0286] 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.
[0287] 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 Baumler A J. IL-17 orchestrates a
mucosal response against Salmonella dissemination from the gut. Nat
Med. 2008 April; 14(4):421-8. [0288] 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. [0289] 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. [0290] 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. [0291] 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
[0292] 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 Gly1 5 10 15Val Pro Tyr Ser Ala Tyr Asn Ser
Ile Ile Asn Gly Ile Asn Leu Gly 20 25 30Arg Lys Gly Leu Gly Ser Ile
Tyr Val Phe Gly Ser Gly Asn Gly Gly 35 40 45Tyr Tyr Asp Asn Cys Asn
Tyr Asp Gly Tyr Val Val Ser Pro Tyr Thr 50 55 60Ile Thr Ile Gly Ser
Ile Asp Val Arg Gly Ile Arg His Tyr Phe Ser65 70 75 80Glu Gln Cys
Ser Ser Val Leu Ala Ser Thr Tyr Ser Gly Ser Ile Val 85 90 95Thr Asn
Ala Arg Ile 1006132PRTArtificial sequencemini-Kexin, wild type,
with leader sequence 6Met Asp Thr Asp Thr Leu Val Leu Trp Val Leu
Leu Trp Val Pro Gly1 5 10 15Ser Thr Gly Asp Ala Ala Gln Pro Ala Arg
Arg Ala Val Arg Ser Leu 20 25 30Ser Cys Ser Trp Gly Pro Arg Asp Asp
Gly Lys Thr Ile Glu Gly Val 35 40 45Pro Tyr Ser Ala Tyr Asn Ser Ile
Ile Asn Gly Ile Asn Leu Gly Arg 50 55 60Lys Gly Leu Gly Ser Ile Tyr
Val Phe Gly Ser Gly Asn Gly Gly Tyr65 70 75 80Tyr Asp Asn Cys Asn
Tyr Asp Gly Tyr Val Val Ser Pro Tyr Thr Ile 85 90 95Thr Ile Gly Ser
Ile Asp Val Arg Gly Ile Arg His Tyr Phe Ser Glu 100 105 110Gln Cys
Ser Ser Val Leu Ala Ser Thr Tyr Ser Gly Ser Ile Val Thr 115 120
125Asn Ala Arg Ile 130722DNAArtificial 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 Gly1 5 10 15Leu Pro Ile Ser Met Lys Ile Phe Met
Tyr Leu Leu Thr Val Phe Leu 20 25 30Ile Thr Gln Met Ile Gly Ser Ala
Leu Phe Ala Val Tyr Leu His Arg 35 40 45Arg Leu Asp Lys Ile Glu Asp
Glu Arg Asn Leu His Glu Asp Phe Val 50 55 60Phe Met Lys Thr Ile Gln
Arg Cys Asn Thr Gly Glu Arg Ser Leu Ser65 70 75 80Leu Leu Asn Cys
Glu Glu Ile Lys Ser Gln Phe Glu Gly Phe Val Lys 85 90 95Asp Ile Met
Leu Asn Lys Glu Glu Thr Lys Lys Glu Asn Ser Phe Glu 100 105 110Met
Gln Lys Gly Asp Gln Asn Pro Gln Ile Ala Ala His Val Ile Ser 115 120
125Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly
130 135 140Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly
Lys Gln145 150 155 160Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile
Tyr Ala Gln Val Thr 165 170 175Phe Cys Ser Asn Arg Glu Ala Ser Ser
Gln Ala Pro Phe Ile Ala Ser 180 185 190Leu Cys Leu Lys Ser Pro Gly
Arg Phe Glu Arg Ile Leu Leu Arg Ala 195 200 205Ala Asn Thr His Ser
Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His 210 215 220Leu Gly Gly
Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn225 230 235
240Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe
245 250 255Gly Leu Leu Lys Leu 26011786DNAHomo
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