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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 introduced cell wall proteins into the skin test format because studies have shown that they 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).



Microbiology building

Microbiology Building, Colorado State University.
Location of the Mycobacterial Research laboratories (MRL) and Leprosy Skin Test Antigen Pilot Plant.

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 proteins have been sequenced and immunological responses studied.   Foremost among them are the 70 kDa (DnaK), 65 kDa (GroEL), 45 kDa, 38 kDa,, 228 kDa superoxide dismutase (SOD), 18 kDa small heat shock protein (SmHSP),, 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 then treated with Triton X-114 to remove 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 some larger uncharacterized proteins. Click here for a flow chart detailing the production of MLSA-LAM and MLCwA.



Armadillo

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.

 


Acid fast  bacteria

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. To prepare these antigens, one of the laboratory suites in the Microbiology building at Colorado State University was converted into a Pilot Plant, a self-sufficient Laboratory designed to meet regulatory standards for a Good Manufacturing Practice (GMP) facility.  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 documents covered everything from the capturing of the armadillos to environmental testing the Pilot Plant and quality control testing of the final product. Two Master Production & Control Records were developed to purify M.Leprae from Armadillo liver and/or spleen and to produce the skin test antigens from purified 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, safety 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 allow us to proceed with a Phase I human clinical trial. The IND application (#BB-IND 7938) was granted by FDA in November of 1998.

 

 

Leprosy Skin Test Antigen Pilot Plant

Diagram of Leprosy Skin Test Antigen Pilot Plant.
The Pilot Plant is a self-sufficient laboratory designed as 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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Antigens

Packaged vials.
Final containers of skin test antigens and controls.
Skin test antigens and controls were coded and labeled for use in clinical trials.

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 μl 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 employee in the MRL, which could have been exposed to tuberculosis or leprosy antigens while working in the research laboratory. Although this individual did not test positive to PPD, reactions to M. leprae 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 10 mg/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.

 



Hartshorn Health Services

Hartshorn Health Services Center at Colorado State University. Location of the Phase I clinical trial.

 

 


















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View of the Himalayas

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

Phase II Human Study and Future Plans

Since completion of the Phase I Safety study, we have obtained FDA approval to proceed with a Phase II study at Anandaban Hospital in Kathmandu, Nepal. As of March 27, 2002, approval from all regulatory agencies, including the FDA, National Institute of Allergy and Infectious Diseases, CSU Institutional Review Board (IRB) and Nepal Health Research Council (NHRC) has been obtained. The Phase II research plan comprises three separate stages (A, B and C), to be completed successively. The first two stages (A and B) have been successfully completed and stage C has begun.  Stages A and B were designed to establish the concentration of the two antigens inducing an optimal (less than 10mm) induration in healthy Nepali individuals and to re-confirm the U.S. data on the safety of these products. Study participants included 10 subjects (Study A) and 90 subjects (Study B).  Having found an optimal safe dose for each antigen, both reagents are to be tested on 425 subjects, 140 healthy contacts, and 90 healthy non-contacts, 82 tuberculoid leprosy patients, 65 lepromatous leprosy patients, and 48 tuberculosis patients (Study C).  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 data, 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, a Phase III study will be planned.
Parrots
Development of New Skin Test Antigens

At the same time, researchers in our laboratory are in the process of developing new skin test antigen candidates. These new antigens include MLMA (M. leprae membrane antigens) and sub-fractions of MLMA, MLSA-LAM and MLCwA. These sub-fractions will be analyzed by in vitro IFN-gamma 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.
Water lilies


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This page was last updated October 10, 2007