The Infectious Disease Research Institute (IDRI, Seattle,
WA) is a not-for-profit corporation that has developed
the first defined vaccine for leishmaniasis, which
is now in clinical trials, in partnership with Corixa
Corp. (Seattle, WA). IDRI is currently involved
in the development of adjuvants that increase the
effectiveness of this vaccine with a focus on monophosphoral
lipid A (MLA), which has proved safe and
effective when used with other vaccines.
The development process requires frequent analysis
of various forms of MLA. The traditional analysis
method, which uses HPLC with a fluorescence detector, has long been a bottleneck because of
lengthy required sample preparation procedures.
IDRI researchers Darrick Carter, Director of Process
Sciences, and Mary Wallace, Development Associate,
are in the process of validating an HPLC detector—
the Corona® charged aerosol detector (CAD) (ESA Biosciences, a division of Magellan Biosciences,
Chelmsford, MA)—that has demonstrated
the potential to substantially reduce analysis times.
IDRI researchers expect the detector to reduce the
time required to prepare 200 MLA samples from
between one and two weeks to just one day.
Fighting a deadly tropic
disease
Figure 1 - Phlebotamine sand fly, which transmits
leishmaniasis.
Members of the genus Leishmania infect many vertebrates,
including humans, dogs, and rodents. The
life cycles of members of the genus involve a vertebrate
host and a vector, a sand fly, which transmits
the parasite between vertebrate hosts (see Figure 1).
The most common forms are cutaneous leishmaniasis,
which causes skin sores, and visceral leishmaniasis,
which affects some of the internal organs of the
body, such as the spleen, liver, and bone marrow.
The number of new cases of leishmaniasis each year
in the world is thought to be about 1.5 million of the
cutaneous and 500,000 of the visceral form. Hundreds
of thousands of people, mostly children, die
from the disease every year. Leishmaniasis is found
in parts of about 88 countries. Most of the affected
countries are in the tropics and subtropics. The settings
in which leishmaniasis is found range from
rain forests in Central and South America to deserts
in West Asia. More than 90% of the world’s cases
of visceral leishmaniasis are in India, Bangladesh,
Nepal, Sudan, and Brazil.
Drugs are available to treat leishmaniasis, but the
cost, at about $100 per dose, is high enough to put it
out of reach of most residents of the affected areas.
IDRI’s goal is to develop a vaccine that can be
delivered at a much lower cost to developing areas.
Protective immunity against leishmaniasis requires
the specific induction of T-helper type 1 (Th1)
cells that recognize the parasite. IDRI researchers
have identified proteins of Leishmania that stimulate
protective Th1 responses and are effective as
vaccines in animal models. In fact, these proteins
have even been used successfully to treat mucosal
leishmaniasis—one of the most disfiguring and difficult
forms to cure—in humans. Three vaccine
proteins have been genetically engineered for production
as a trifusion protein.
Adjuvants aid antigen
delivery
Appropriate antigen delivery to induce the right type
of immune response against Leishmania is another
critical component of an effective vaccine. The two
adjuvants approved for human use, alum and squalene,
induce potent antibody responses but are poor
inducers of antigen-specific Th1 responses. MLA has
been used as an adjuvant in several trials for vaccines
for malaria, genital herpes, and allergy desensitization
as well as being approved for use in hepatitis B.
Recently, scientists at Corixa developed synthetic
lipid A mimetics that are chemically unique, acylated
monosaccharides called aminoalkyl glucosaminide
4-phosphates (AGPs). In contrast to the
complex family of lipid A congeners found in MLA,
synthetic AGPs represent mimetics of MLA that can
be synthesized as highly pure chemical entities. The
AGPs can be designed to accommodate molecular
changes for improved biological and pharmacological
activities. These unique molecules are well suited
for use as adjuvants for the Leishmania and other
vaccines. IDRI researchers have determined that
combining their trifusion proteins with an MLA
adjuvant produced an even more potent vaccine.
This is the first defined candidate vaccine developed
for leishmaniasis. Clinical development of the vaccine
is being supported by a generous grant from the
Bill & Melinda Gates Foundation.
Importance of qualitative and
quantitative analysis
Understanding the importance of the adjuvant to
the vaccine effectiveness and safety, IDRI researchers
are continuing to develop new MLA molecules
in an effort to deliver further improvements. The
development of novel adjuvants naturally requires
frequent qualitative analysis during the development
process. When the time comes to develop
manufacturing processes, quantitative analysis will
be required as well. The current state-of-the-art for
analysis of MLA and similar lipids involves the use
of HPLC with fluorescence detectors. The first step
is a conjugation process that involves attachment of
hydrazone to the lipid sample. This manual process takes about a half-day for a single sample or about a
week for 200 samples. Then the sample is run on an
HPLC system with fluorescence detection. Interpretation
of the results is complicated by the fact that
some forms of MLA fluoresce more than others, primarily
depending on the number of acyl chains. This
means that correction factors need to be applied to
each peak, which adds about half a day to the time
required to analyze a typical batch of samples.
Recognizing that conventional analytical methods
represent perhaps the most time-consuming aspect of
the adjuvant development process, IDRI researchers
considered several alternate detector technologies.
MLAs have boiling points that are too high to separate
and analyze accurately with GC. The commonly
used UV detector is not quantitative for lipids because
analytes must possess at least one carbon–carbon
double bond to be detected, while many lipids are
saturated and invisible to UV or have varying levels
of saturation. Refractive index detectors work well in
a number of HPLC quantitative analysis applications;
however, they are limited to isocratic analysis with a
single solvent, while gradient analysis with multiple
solvents works better with most lipids because it provides
superior separation. Evaporative light scattering detectors (ELSDs) have become the standard method
of quantitative analysis of lipids on HPLCs, and IDRI
researchers seriously considered their use.
Figure 2 - Corona CAD.
The Corona CAD promises to deliver in a single
package the performance benefits of RI, UV, and
ELSD (Figure 2). In the detector, the HPLC column
eluent is first nebulized with nitrogen, and the
droplets are dried to remove the mobile phase, generating
an aerosol stream composed of particles of
all nonvolatile constituents. A secondary stream of
nitrogen becomes positively charged as it passes a
high-voltage, platinum corona wire. The secondary
stream merges with the primary particle stream to
turbulently mix the gas ions with the aerosol particles.
The secondary stream’s charge transfers to the
opposing stream of analyte particles. The charge is
transferred to a collector, where it is measured by a highly sensitive electrometer, generating a signal in
direct proportion to the quantity of analyte present.
Figure 3 - Twenty microliters of monophosphoryl lipid A in water/triethanolamine, pH = 7.2,
from vendor 1 were injected and run over a reversed-phase C-18 column with a gradient from 90%
Buffer A (95% acetonitrile/0.1% triethanolamine/0.25% acetic acid/water) to 85% Buffer B (95%
isopropanol/0.1% triethanolamine/0.25% acetic acid/water) over 90 min followed by a ramp down
to 10% Buffer B over 30 min (total time = 120 min). The stream was fed into a Corona detector
under nitrogen at 35 psi, and data were collected using Agilent Chemstation software (Agilent
Technologies, Santa Clara, CA). A heterogeneous mix of hexa-, penta-, and tetraacyl congeners
was detected using the Corona detector.
Figure 4 - Conditions the same as in Figure 3, except that 20 μL of monophosphoryl lipid A in
water/triethanolamine, pH = 7.2, from vendor 2 were injected and run over a reversed-phase C-18
column with a gradient from 90% Buffer A (95% acetonitrile/0.1% triethanolamine/0.25% acetic
acid/water) to 85% Buffer B (95% isopropanol/0.1% triethanolamine/0.25% acetic acid/water)
over 90 min followed by a ramp down to 10% Buffer B over 30 min (total time = 120 min). The
stream was fed into a Corona detector under nitrogen at 35 psi, and data were collected using Agilent
Chemstation software. A single peak was detected corresponding to the hexaacyl form of MPL.
IDRI researchers are currently in the process of
developing methods for MLA analysis using the
Corona CAD and HPLC. The detector is simple to
set up and operate. One of the few parameters that
need to be adjusted on the detector is gas flow. The
researchers are currently working with two different
forms of MLA. They are seeing a dramatic reduction
in sample preparation time, primarily due to the
elimination of conjugation. For an aqueous formulation,
there is no sample preparation at all. The lipid
sample is merely mixed with solvent and injected
into the HPLC. Solid-phase extraction may still be
required for oil-based samples, and that will take
another half-day or so depending on the number of
samples. The calculation of correction factors has
also been eliminated (see Figures 3 and 4).
The result is that IDRI researchers have demonstrated
the ability to analyze a single sample in about
2 hr and can prepare 200 samples in one day. This
reduction in preparation time has the potential to
substantially decrease both the development and
manufacturing costs of the leishmaniasis vaccine.
IDRI is currently investigating different excipients
that could be added to the MLA adjuvant, and is also
considering reengineering the organism or switching
to a synthetic source to reduce costs. The researchers
are still in the process of validating the CAD
and have taken note of results from Dr. Robert A.
Moreau of the Eastern Regional Research Center
of the Agricultural Research Service, U.S. Department of Agriculture (Washington, DC). Dr. Moreau
evaluated the CAD with several normal-phase and
reversed-phase HPLC systems commonly used for
the quantitative analysis of lipid classes and lipid
molecular species. The CAD detected common lipids
such as triacylglycerols, cholesterol esters, and
free sterols to a minimum limit of detection of about
1 ng per injection.
Mr. Fireman is President, Structured Information, 363
Massachusetts Ave., Ste. 206, Lexington, MA 02420,
U.S.A.; tel.: 781-674-2300; fax: 408-228-8750; e-mail: [email protected]. Dr. Carter is Vice President for R&D,
Dharma Therapeutics Inc., Seattle, WA, U.S.A. Ms.
Wallace is Research Assistant, IDRI, Seattle, WA.