Skin Test Initiative
Background
Over the past 80 years several
attempts have been made to develop a skin test antigen which is sensitive
and specific for all cases of lepromatous and tuberculoid leprosy.
In 1992, in light of the pressing need for a diagnostic test to evaluate
the incidence of leprosy, we started developing two new leprosy skin
test antigens. In our attempts to investigate a new skin test initiative,
we have modified the Rees-type MLSA (M. leprae soluble antigens)
so as to remove immunosuppressive components [such as LAM (lipoarabinomannan),
LM (lipomannan), PIMs (phosphatidylinositolmannosides), and other
lipids]. Secondly, we have also introduced cell wall proteins into
the skin test format because studies have shown that M. leprae
cell wall proteins are powerful immunogens. The proposed skin test
antigens are (1) soluble proteins of M. leprae (i.e., MLSA,
M. leprae soluble antigen with minimal amounts of the immunosuppressive
lipoglycans (mostly LAM) called MLSA-LAM and (2) cell wall-associated
proteins of M. leprae called MLCwA, also devoid of lipoglycans
(mostly LAM).
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Microbiology Building, Colorado
State University.
Location of the Mycobacterial Research laboratories (MRL) and
Leprosy Skin Test Antigen Pilot Plant.
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Antigen
Descriptions
Antigen MLSA-LAM is derived from M.
leprae extract following sonication and centrifugation at 27,000
x g then 100,000 x g, leaving the cytosol (MLSA). This soluble material
is then extracted with detergent (Triton X-114) to remove carbohydrate
and lipid constituents. MLSA-LAM contains soluble protein antigens
of M. leprae; over 100 individual proteins are recognized on
2-dimensional gels. About 30 of the major problems have been sequenced
and immunological responses studied. Foremost among them are the 70
kDa (DnaK), 65 kDa (GroEL), 45 kDa, 38 kDa, 35 kDa major membrane
protein (MMP-I), 22 kDa superoxide dismutase (SOD), 18 kDa small heat
shock protein (SmHSP), 18 kDa bacterioferritin (Bfr), 10 kDa (GroES),
and ribosomal proteins S7/S12.
Antigen MLCwA is the 27,000 x g pellet extracted three times at 56
C with 2% sodium dodecylsulfate (SDS) followed by removal of
SDS by column chromatography. MLCwA is also treated with Triton X-114
to remove the amount of lipoglycans. MLCwA contains many of the same
proteins as MLSA-LAM, particularly the 70 kDa and 65 kDa, the export/secretory
proteins (notably the 30/31 kDa, multigene antigen 85 complex) and
also contains some larger uncharacterized proteins.
Click here for a flow chart detailing the production of MLSA-LAM
and MLCwA.
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Armadillo
Experimentally infected armadillos are the primary source of M.
leprae bacteria.
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| Pre-clinical
Testing
Once the antigens were developed, the protocols
were reviewed to ensure that the reagents used during production
were present in the US Pharmacopeia (USP). Reagents which were not
listed were replaced with others which were on the list. Test batches
of both antigens were then produced for extensive quality control
testing. Tests were run to ensure that detergents used during manufacturing
were no longer present. SDS-PAGE and Immunoblots were also run to
ensure that the major antigens were present and the lipoglycans
were absent. Once the quality control measures were deemed satisfactory,
both antigens were evaluated in M. leprae sensitized guinea pigs
to determine the sensitivity of the antigens. Those were compared
to PPD as a measure of specificity. To determine the stability of
the antigens, several vials were stored at each of four temperatures
(56°C, 37°C, 4°C and -70°C) and tested periodically
over the course of one year. The reactions produced over that period
remained similar, with no statistically significant changes in potency.
Dr. Paul Roche also tested MLSA-LAM and MLCwA
in T-cell assays based on blood from various population groups by
measuring the IFN-g response at 24 hours. The results showed that
there were significantly higher levels of IFN-g produced in exposed
as compared to unexposed Nepali subjects. From these data, we concluded
that there should be distinct differences in the skin test response
of leprosy exposed compared to unexposed individuals.
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Acid Fast bacteria.
Kinyoun acid fast staining of M. leprae purified from armadillo
tissue.
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| Antigen
Manufacturing
Satisfactory completion of the quality control
and the in vivo testing indicated that we were ready to prepare
antigens for use in humans. Needing a dedicated location to prepare
these antigens one of the laboratory suites was converted into a
Pilot Plant, a self-sufficient Laboratory. The Pilot Plant is designed
similar to a GMP laboratory having three rooms, the innermost room
containing a Biological Safety Cabinet in which production takes
place. During construction of the Pilot Plant, standard operating
procedures (SOP's) were developed for every step of the production
phase of the skin test antigens. These SOP's covered everything
from the capturing of the armadillos to environmental testing and
quality control testing. Two Master Production & Control Records
were designed to purify M.Leprae from Armadillo liver and/or spleen
and production of skin test antigens from this M.Leprae.
In May of 1997, batch 23 MLSA-LAM and MLCwA were produced in the
Pilot Plant. The antigens went through extensive testing including
evaluation for sterility (both bacterial and viral), absence of
detergents and endotoxin, protien profile analysis by SDS-PAGE and
Immunoblot, saftey in vivo studies using guinea pigs and mice and
potency testing using guinea pigs. The protocol and accompanying
paperwork were submitted to FDA in the form of an Investigational
New Drug (IND) application to allows us to proceed with human clinical
trials. The IND application (#BB-IND 7938) was granted by FDA in
November of 1998.
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Diagram of Leprosy Skin Test Antigen Pilot
Plant.
The Pilot Plant is a self-sufficient laboratory designed like a
GMP laboratory, having three rooms, the innermost room containing
a Biological Safety Cabinet in which the actual production of skin
test antigens takes place.
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Packaged vials.
Final containers of skin test antigens and controls.
Skin test antigens and controls were coded and labeled for use
in clinical trials.
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Phase I Human Study
In January of 1999 the antigens MLSA-LAM
and MLCwA were used in a Phase I human clinical study to test for
antigen safety. The study was performed on ten healthy volunteers
from Fort Collins, Colorado. Five volunteers received three titrated,
100 ml
doses of MLSA-LAM (1 mg
protein/ml, 10 mg/ml,
and 25 mg/ml),
1 dose of a positive control (Rees Antigen at 10 mg/ml),
and one dose of mock antigen (saline). The other five volunteers
had the same injections except the MLSA-LAM doses were replaced
by MLCwA. The injection sites were read at 15 min, 48 h, 72 h, and
28 days by Cheri Lazar, RN, and Jane Higgins, MD, of Hartshorn Health
Services, Colorado State University, Fort Collins, Colorado.
Upon
injection, all ten volunteers commented that all five sites stung
compared to a PPD injection. This could be attributed to tuberculin/PPD
containing phenol which could act as a local anesthetic; MLSA-LAM
and MLCwA do not contain phenol. None of the volunteers mentioned
any residual stinging after Day 0 (day of injection). Of the ten
individuals tested, nine had no induration at any of the five sites.
One volunteer showed induration at the 25 mg/ml
MLCwA and Rees Antigen injection sites at 48 h and 72 h post injection.
It should be noted that this volunteer was an empl;oyee in the MRI
and at the research lab could have been exposed to tuberculosis
or leprosy antigens at work. Although this individual did not test
positive to PPD, reactions to Mlep antigens must be considered when
analyzing the results. All volunteers had varying sizes of erythema
and itching associated with the injection sites, especially at the
higher concentrations (25 mg/ml
MLSA-LAM and 25 mg/ml
MLCwA) and the control antigen (Rees Antigen at 10Fg/ml) sites.
Dr. Higgins noted that in darker pigmented individuals, the erythema
would probably go unrecognized and, therefore, would not play a
part in reading positive reactions. In most volunteers the itching
disappeared after 72 h and can probably be attributed to the lack
of phenol in the skin test antigens. Throughout the study some patients
complained of various symptoms ranging from headaches to cold-like
symptoms. After examining the volunteers and evaluating the symptoms,
Dr. Higgins felt that they were not side effects of the skin test
antigens. Rather, they were due to extraneous causes which coincided
with the study.
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Hartshorn Health Services Center at Colorado
State University. Location of the Phase I clinical trial.
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View of the Himalayas (Annapurna) from Pokhara, Nepal. Near the
location of the Phase II clinical trials.
(Click for a larger view)
Photo courtesy of Dr. Philip Draper
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Phase
II Human Study and Future Plans
Since completion of the Phase I Safety
study, we have been in the process of obtaining FDA approval to proceed
with a Phase II study at Anandaban Leprosy Hospital, Kathmandu, Nepal.
As of March 27, 2002, approval from all regulatory agencies involvedFDA,
National Institute of Allergy and Infectious Diseases, CSU and Nepal
has been obtained. The Phase II research plan comprises three separate
studies (Studies A, B and C) to be completed successively. The first
two studies (A and B) will be performed to establish the concentration
of the two antigens inducing an optimal (less than 10mm) induration
in healthy Nepali individuals; this study will also serve to re-confirm
the U.S. data on the safety of these products. This will be done in
two steps - the first in 10 subjects (Study A) and the second in 90
subjects (Study B). Having found an optimal safe dose for each antigen,
both reagents will then be tested on 425 leprosy patients, 140 healthy
contacts, and 90 healthy non-contacts, 48 tuberculosisi patients (Study
C). To assess the safety of injections in leprosy patients, 10 leprosy
health care workers before enrolling any additional subjects. Biopsies
will be taken from 12 individuals who have given informed consent
with the purpose of confirming that the reactions are due to a delayed-type
hypersensitivity reaction (a cell mediated reaction) as opposed to
an Arthus reaction (a humoral response).
If, upon analysis of this database, it is determined that MLSA-LAM
and/or MLCwA are effective, i.e., specificity and sensitivity is 95%
or greater, in detecting individuals who have been exposed to M.
leprae, further studies will be planned. These future studies
would each include approximately the same number of subjects as the
Nepalese study (270 subjects) and would be performed in other leprosy-endemic
countries such as Brazil. The purpose of these studies would be to
confirm that the results obtained in Nepal could be repeated in other
leprosy-endemic areas.
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Development of
New Skin Test Antigens
At the same time, researchers in our
laboratory are in the process of developing new skin test antigens.
These new antigens include MLMA (M. leprae membrane antigens) and
sub-fractions of MLMA/MLSA-LAM and MLCwA based on molecular weight.
These sub-fractions which have not been tested previously, will be
analyzed by invitro IFN8 production using human whole blood or PBMC's
and guinea pig DTH skin test studies for their immunogenic effects.
For information on Skin Test Studies in Guinea Pigs click
here. |
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