The Third NIAID Workshop in Medical Mycology:
Immunology
in Medical Mycology: Antigenic Peptides,
Glycobiology and Vaccines
Feature Article
Reprinted with permission from
ASM
News, Vol. 62,
No.2, 1995, pp. 81-84.
Researchers Use Molecular
Immunology and Technology to Combat Fungal Pathogens
The current focus
on peptides and cell wall polysaccharides as
candidate vaccines is part of a much broader
NIAID vaccine
development program
Dennis M. Dixon, Rebecca Cox,
Jim Cutler, and George Deepe
Dennis
M. Dixon (corresponding author) is a Mycology
Program officer at
NIAID in Bethesda, Md.; Rebecca
Cox is director, Department of Research
Immunology, Texas Center for
Infectious Diseases, San Antonio; Jim Cutler
is a professor of microbiology at
Montana State University, Bozeman; and George Deepe is the chairman of
the Division of Infectious Diseases at University of Cincinnati College
of Medicine, Cincinnati, Ohio.
Leading
researchers studying a variety of fungal
pathogens say that there is a major shift in
thinking regarding vaccines. Thus, the prevailing
question of whether vaccines should be considered
as a practical way of preventing fungal diseases
is being challenged by the questions of which
ones and when. Key advances in research on
several important fungal diseases, including
histoplasmosis, coccidioido-mycosis,
cryptococcosis, and even candidiasis, could soon
bring vaccines for one or several of these
diseases within reach. These basic advances are
also leading to stepped-up efforts to establish
the vital interdisciplinary framework needed
within the research community if such valuable
public health products are to be developed.
As
part of this accelerated activity, the National
Institute of Allergy and Infectious Diseases
(NIAID) is sponsoring several workshops on
medical mycology, the most recent of which was
held 7-9 September 1995 in Big Sky, Mont. The
Montana workshop brought investigators working on
different fungal pathogens together with
researchers from other fields, enabling the
assembled group to better identify more of the
many technological challenges that need to be
overcome. Participants considered these
challenges in terms of two distinct specialty
areas of research: (i) antigenic peptides and
proteins and (ii) glycobiology.
There
is still much to be done before appropriate
antigens can be isolated from fungal pathogens,
characterized adequately, and administered in
such a way that they elicit a safe and protective
immune response in the host. For example, because
standardization of strains and reagents is needed
for these vaccine development efforts to succeed,
a group of researchers is establishing a
cryptococcal working group as one step toward
achieving that interim requirement (see box, p.
82). In addition, a second workshop on fungal
immunology is being planned for 1997.
The
Importance for Vaccines of Fungal Peptides
Researchers
are identifying a series of antigenic peptides
from fungal pathogens that can generate immune
responses, particularly cellular immune responses
that are considered critical for protective
immunity. As part of this response, T cells
recognize fungal (or other foreign) peptides that
bind to receptor molecules which are part of the
major histocompatibility complex.
Fungal
peptide vaccine candidates afford both advantages
and disadvantages. On the plus side, peptides
induce particular immune responses, including the
induction of immunoprotective cytokines, the
deployment of cytolytic T cells, and the
production of antibodies. These peptides can be
designed to delete suppressive epitopes that
might prevent a useful immune system response.
CWG
Forms
Because nonuniform strains
of Cryptococcus neoformans are widely
used to study pathogenesis and the host
immune response, investigators are
concerned that the results of their
individual research efforts may not be
generalizable. Since the number of
investigators in the field is relatively
small and the resources of individual
laboratories are limited, a group of them
decided during the NIAID Montana workshop
to form the Cryptococcal Working Group
(CWG), which is open to all interested
individuals throughout the world.
The purpose of CWG is to
identify areas in which scientists can
establish collaborative efforts for
resolving important questions of
cryptococcus biology and pathogenesis.
Since the Montana meeting, the Internet
is proving a valuable means for CWG
members to hold frequent informal
discussions over the selection of
representative cryptococcal strains for
future studies. To expedite these
efforts, Christopher Mody of the
University of Calgary set up an
electronic mail server for CWG. He can be
reached by E-mail at
cmody@acs.ucalgary.ca.
Arturo
Casadevall
Arturo Casadevall is an
assistant professor at Albert
Einstein College of
Medicine in Bronx, N.Y.
|
One
of the difficulties in developing peptide-based
vaccines is the poor immunogenicity of peptides
when they are injected into animals. This problem
can be partly overcome by inserting peptides into
lipid complexes composed of palmitic acid.
Furthermore, the immogenicity of certain peptides
can be enhanced by linking them to helper
epitopes.
Still
other methods for producing peptide-based
vaccines are being evaluated. For instance,
researchers studying malaria have developed an
experimental multiple-antigen peptide-based
vaccine, and its efficacy is striking in
experiments conducted with mice. This
experimental malaria vaccine, which consists of
linked multiple epitopes, thus may provide a
model for the development of fungal vaccines.
Researchers
are currently studying a number of peptides as
potential vaccines against several fungal
pathogens. Several examples were highlighted at
the Montana workshop.
- Epitopes of a protective
immunogen from Histoplasma capsulatum,
namely, heat shock protein 60, are being
mapped. In addition, considerable
interest is focusing on the product of a
yeast phase-specific gene from H.
capsulatum, yps3, that is not
detected in the mycelial phase. Although
located in either the cell wall or cell
membrane of the yeast, little is known
about the function of this protein.
However, because it stimulates human
lymphocytes to proliferate, this protein
is a target of interest to investigators
interested in stimulating a protective
immune response to this fungal pathogen.
- The gene coding for enolase
from Candida albicans was recently
cloned, sequenced, and expressed in a
eukaryotic system. Although the
recombinant protein elicits a cellular
immune response in animals colonized with
this fungus, the protective efficacy of
this immunogenic protein has not been
evaluated.
- The gene of WI-1, a surface
antigen from Blastomyces dermatitidis,
contains multiple tandem repeats, an
epidermal growth factor-like domain, and
a region that has homology to invasin.
WI-1 appears to be an adhesin molecule
that mediates binding of this fungus to
mononuclear phagocytes. In addition, it
is a target of both cellular and humoral
immune responses. Some of the T-cell
reactive epitopes as well as the major
histocompatibility complex restriction
patterns of WI-1 have been identified.
- Two cloned and sequenced
immunoreactive antigens from Coccidioides
immitis are involved in the cellular
immune response to this fungus. One
antigen, which stimulates a T-cell line,
is homologous to 4-hydroxy-phenylpyruvate
dioxygenase and the mammalian F antigen;
the other is heat shock protein 60. These
antigens are being tested for their
ability to protect mice against this
pathogen.
A
Potential Antigenic Role for Fungal Surface
Polysaccharides
Virtually
all medically important fungi carry an impressive
array of polysaccharides on their cell surfaces,
and these macromolecules play significant roles
in fungus-host interactions. Yet despite more
than 40 years of study of this class of fungal
antigens, we still have little appreciation for
their physical properties or their potential as
vaccines.
"Antibody
responses against carbohydrate epitopes typically
involve only a limited number of (B-cell) clones,
and the immunoglobulin subclass is often
restricted," points out Tom Kozel of
University of Nevada School of Medicine, Reno,
who spoke during the workshop. This general
phenomenon applies to at least some specific
instances in which the immunogenicity of fungal
polysaccharides has been evaluated. For example,
the glucurono-xylomannan polysaccharide capsule
of Cryptococcus neoformans stimulates
antibodies directed against several epitopes,
according to investigators in the laboratories of
Kozel and Arturo Casadevall, who is at Albert
Einstein College of Medicine in Bronx, N.Y. These
antibodies vary widely in their protective
efficacy. Efficacy also depends on the
immunoglobulin isotype that is produced by the
host defense against cryptococcosis, adds
Casadevall.
For
the fungi, the role of natural antibody immunity
in protection is uncertain despite decades of
investigation. However, the identification of
protective monoclonal antibodies has raised hopes
that vaccines that elicit antibody responses of
the correct specificity and isotype for
protective antibody immunity can be designed.
Specialized
antibodies apparently can protect rodents against
particular fungal pathogens, according to
Casadevall and Jim Cutler of Montana State
University, Bozeman, who is studying candidiasis.
In addition to epitopes identified Casadevall
within the capsular glucuronoxylomannan that seem
to be protective against cryptococcosis, Cutler
and his colleagues find that antibodies to an
epitope within the acid-labile mannan portion of
the phosphomannoprotein complex of C. albicans
protect mice against disseminated candidiasis.
These observations underscore the importance of
defining precisely which fungal epitopes can
induce protective responses by the host and then
applying this information to the development of
candidate vaccines.
Reinventing
Fungal Vaccines
Current vaccine development
efforts represent part of a long-term
program aimed at preventing fungal
infections. Now several new technologies
and approaches are being brought to this
challenge.
In the recent past,
extensive work went into developing a
vaccine for coccidioidomycosis. Those
studies led to a phase III clinical trial
involving nearly 3,000 volunteers (D.
Pappagianis and the Valley Fever Vaccine
Study Group, Am. Rev. Respir. Dis.
148:656-660, 1993). That clinical trial,
conducted between 1980 and 1985,
demonstrated a slight but statistically
insignificant reduction in disease in the
group vaccinated with formaldehyde-killed
spherules of Coccidioides immitis compared
to the unvaccinated control group.
Irritation at the injection
site made it necessary to dilute the
vaccine to 1/1,000 of the dose that
prevented several species of animals from
developing serious infections. According
to the published report of that clinical
study, "A different physical form
other than the whole spherules must be
sought to increase the tolerability of
the immunogenic component."
Because the time seems right
to adapt new methods to this purpose, the
Rotary Americas Valley Fever Foundation
recently launched a drive to raise money
to help in developing and testing a
vaccine for coccidioidomycosis. For more
information about these fundraising
efforts, contact Thomas Larwood, a
physician in Bakersfield, Calif., at
(805) 871-6090.
|
Technically,
such approaches are at least feasible because it
now is possible to determine very precisely the
physical structure of fungal cell wall
carbohydrate components. Several workshop
participants, including Patrick Brennan of
Colorado State University in Ft. Collins, Robert
Cherniak of Georgia State University in Atlanta,
and Roger O’Neill of Perkin-Elmer Applied
Biosystem Division in Foster City, Calif.,
outlined recent developments that should enable
researchers to map fungal component epitopes,
including those contained within complex
polysaccharides, that may induce protective
antibodies.
Indeed,
Brennan and his colleagues have developed a
detailed model of the mycobacterial cell wall,
combining molecular genetics techniques and
sophisticated chemical instrumental analysis.
This effort to define the cell wall structure of
mycobacteria may well serve as an inspiration for
investigators seeking a comparable understanding
of the fungal cell wall. Cherniak, for instance,
is following a similar approach in studying the
cryptococcal capsular polysaccharide.
Insights
from Other Efforts To Develop Vaccines
According
to Milan Blake from the University of Iowa, there
are three major phases in early vaccine
development: (i) the discovery phase, in which
important and perhaps critical immunogens are
identified, purified, and subsequently tested in
appropriate animal models; (ii) the transfer
phase, in which the experimental vaccine is
readied for clinical testing, standard production
procedures are developed, and commercial
development and licensing are considered; and
(iii) the scale-up phase during which issues such
as good manufacturing practices, the design and
conduct of clinical trials for safety and
efficacy, and questions of large-scale vaccine
production and stability need to be
systematically addressed.
Even
with such general procedures in place for the
development of vaccines, the details for any
particular vaccine are important. Critically,
small and seemingly trivial details can threaten
a promising candidate vaccine, according to
Blake. For instance, the development of a
gonococcal vaccine based on the bacterium’s
porin molecule as the key antigen nearly failed
in the early phase because of a trace
contaminant, he says. Thus, the experimental
vaccine contained miniscule amounts of Rmp
(protein III), which induces anti-Rmp blocking
antibodies that nullify the protective effects of
antiporin antibodies. The problem was resolved by
genetically engineering a mutant source of porin
that cannot produce Rmp because its gene is
deleted.
The
appropriate delivery of particular antigens, such
as those derived from fungal pathogens, is
another challenge for researchers developing
vaccines, points out Roy Curtiss from Washington
University in St. Louis, MO. He and his
colleagues are developing recombinant avirulent
bacterial systems as potential vectors for
delivering a wide variety of vaccine components.
For example, engineered versions of Salmonella
spp. That constitutively express gene inserts
induce both humoral and cellular immune responses
when administered orally to rodents, he says.
Moreover, recombinant Salmonella spp. with
two or more attenuating mutations are avirulent
but retain their tissue tropism.
Fungal
Vaccine Studies Are Part of Larger Vaccine
Program
Current
efforts to develop vaccines against fungal
pathogens are part of a far broader vaccine
development program, according to John La
Montagne, director of the Division of
Microbiology and Infectious Diseases at NIAID.
NIAID established its Program for the Accelerated
Development of Vaccines in 1981, and its emphasis
is on the major infectious diseases. Major
achievements funded through that program include
the development of vaccines for hepatitis B,
invasive pneumococcal disease, pertussis, and Haemophilus
influenzae type b meningitis. Considerable
resources are now going toward development of
genetically engineered vaccines.
At
the Montana workshop, a number of key questions
on the need and support for vaccine development
in the fungal diseases were addressed and the
current status of vaccine research in fungal
diseases was summarized. Statistics on extramural
funding in mycology by the National Institutes of
Health (NIH) emphasized that mycology research
applications have become highly competitive and
the area continues to be enthusiastically
supported by NIH.
Over
the course of the workshop, it became clear that
numerous members of the medical mycological,
health science, and biotechnology communities
could identify with the diversity of skills
necessary to formulate and test the hypotheses
related to the basic and applied aspects of
protective immune responses to the medically
important fungi. This understanding encourages a
team approach for answering questions that could
have broad applicability to public health.
Speakers and Facilitators
