Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Fungus – except allergen – or component thereof or substance...
Reexamination Certificate
2001-11-01
2004-12-21
Minnifield, N. M. (Department: 1645)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Fungus, except allergen, or component thereof or substance...
C424S184100, C424S278100, C424S093500, C514S002600, C514S020800
Reexamination Certificate
active
06833136
ABSTRACT:
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
Not Applicable.
Reference to a “Computer Listing Appendix Submitted on a Compact Disc”
Not Applicable.
BACKGROUND OF THE INVENTION
(1) Summary of the Invention
The present invention relates to antigen vaccines and methods of use thereof for the treatment of
Pythium insidiosum
infections, as well as prophylaxis against these infections, in humans and mammals. Further, the present invention relates to a method for preparing the preferred vaccine for the treatment which contains the intracellular and extracellular antigens of
Pythium insidiosum
. The present invention further relates to a mammal model for evaluating
Pythium insidiosum
vaccines and a method for monitoring the Th1 and Th2 response of a mammal in response to
Pythium insidiosum
vaccines.
(2) Description of Related Art
Infections caused by fungal and parafungal organisms are occurring with increasing frequency in patients with debilitating illnesses such as leukemia and AIDS, as well as those undergoing immunosuppressive therapy. Within this group of organisms are the traditional pathogenic fungi and a long list of newly recognized emerging opportunistic fungal and parafungal organisms. Among these emerging pathogens is the oomycete
Pythium insidiosum
, a fungal-like organism in the Kingdom Kromista,
Phylum Pseudofungi. Pythium insidiosum
is not only physiologically distinct from members of the Kingdom Fungi, but also differs physiologically. This may explain why anti-fungal drugs do not have any effect on pythiosis.
Pythiosis insidiosi particularly occurs in humans and lower animals in the tropical, subtropical, and temperate areas of the world (de Cock, W.A.W., et al., J. Clin. Microbiol. 25: 344-349 (1987)). The disease was first described in the beginning of the 20
th
century in equids of tropical and subtropical countries such as India and Indonesia as well as the USA. Soon, however, it was evident that the disease not only affected equids but other mammalian species such as humans. In lower animals, infections of the cutaneous tissues, lymphatic vessels, intestines, lungs, and bones have been found. In humans, a deadly arteritis infection, subcutaneous invasion, and keratitis occurs.
The drugs currently available to treat fungal infections have had little or no effect on
Pythium insidiosum.
Reports of treatment with either amphotericin B or surgery, which are commonly used to treat this disease in humans and lower animals, have indicated that 60% of the patients died of their infections. In cases of arterial invasion in humans, amphotericin B did not eliminate the infection (Rinaldi, M. G., et al., Mycol. Obser. 9:7 (1989); and Thianprasit, M., Trop. Dermathol. 4: 1-4 (1990)), whereas in surgery the main problem has been to determine how much of the infected tissues has to be removed. Thus, relapses are common in surgically treated patients, who must also endure the pain and distress that such an invasive traumatic procedure inflicts on them.
The curative properties of
Pythium insidiosum
antigens was first noticed when some Costa Rican horses with pythiosis, which had been injected with
Pythium insidiosum
antigens in a skin test, were cured (Mendoza, L., et al., Equine pythiosis in Costa Rica: report of 39 cases. Mycopathologia 94: 123-126 (1986)). Simultaneously, another vaccine with similar curative properties was successfully used in horses with the disease in Australia (Miller, R. I., Aust. Vet. J. 57:3 77-382 (1981)). These two vaccines have been referred to in the literature as the Mendoza vaccine and the Miller vaccine, respectively (Newton, J. C., et al., The Compendium 15: 491-493 (1993)) Early reports indicated that the antigens used in the
Pythium insidiosum
-vaccine possessed unique characteristics, somewhat similar to the features of those reported in
Trichophyton verrucosum
(Gudding R., et al., Can. Vet. J. 36: 302-306 (1994)) and other immunotherapeutic vaccines (Foster, J. S., et al., Vet. Med. Small Ani. Clin. 71: 920 (1976); Pier, A. C., et al., Equine Pract. 15: 23-27 (1993)).
The Miller vaccine is prepared from sonicated hyphal antigens (Miller, R. E., Aust. Vet. J. 57: 377-382 (1981)), while the Mendoza vaccine is prepared from culture filtrate antigens (Mendoza, L., et al., Mycopathologia 94: 123-129 (1986)). Both the Miller vaccine and the Mendoza vaccine have been used to cure early cases of equine pythiosis, i.e., horses with pythiosis-caused lesions of 0.5 months or less in duration; however, neither vaccine can cure horses that are chronically infected with
Pythium insidiosum
, i.e., horses with pythiosis-caused lesions two or more months old (Mendoza, L., et al., Mycopathologia 119: 89-95 (1992)). While both vaccines have cure rates of about 53% for early cases of pythiosis, the Mendoza vaccine has a longer shelf-life and milder side effects than the Miller vaccine (Miller, R. I., et al., J. Am. Vet. Med. Assoc. 182: 1227-1229 (1983)). The Mendoza vaccine, in addition to its immunotherapeutic properties, also showed some degree of protection against disease caused by
Pythium insidiosum
. However, this protection was later found to be of short duration (Mendoza, L., et al., Mycopathologia 119: 89-95 (1992)).
In over 15 years of use, the Mendoza vaccine has been shown to be safe and consistently efficacious, curing more than 300 horses with pythiosis-caused lesions of short duration. However, the Mendoza vaccine can only cure early equine pythiosis, not chronic cases of this disease (Mendoza, L., et al., Mycopathologia 119: 89-95 (1992)). Aside from the fact that the Mendoza vaccine can only cure early equine pythiosis cases, nothing was known about the immunogens involved in its curative properties nor the immune mechanisms that triggered the killing of
Pythium insidiosum's
hyphae infected tissues.
In a recent study using SDS-PAGE and Western blot analysis, the presence of three immunodominant hyphal proteins were identified (Mendoza, L., et al., J. Clin. Microbiol. 30: 2980-2983 (1992)). While the Western blots revealed that the IgG in sera from horses with active pythiosis recognized most of the proteins of
Pythium insidiosum
, the Western blots also showed that three protein bands of the 32 kDa, 30 kDa, and 28 kDa were particularly prominent. More significant was the finding that antibodies against these three proteins persisted for long periods of time in the sera from successfully cured horses.
Even though there are two vaccines which can be used to treat pythiosis, there remains a need for a vaccine which can cure pythiosis and prevent infection by
Pythium insidiosum
. In particular, there is a need for a vaccine that can be used to treat and cure patients who are in a chronic stage of the disease.
SUMMARY OF THE INVENTION
The present invention provides antigen vaccines and methods of use thereof for the treatment of
Pythium insidiosum
infections, as well as prophylaxis against these infections, in humans and mammals. Further, the present invention provides a method for preparing the preferred vaccine for the treatment which contains the intracellular and extracellular antigens of
Pythium insidiosum
. The present invention further provides a mammal model for evaluating
Pythium insidiosum
vaccines and a method for monitoring the Th1 and Th2 response of a mammal in response to
Pythium insidiosum
vaccines.
Therefore, the present invention provides a method for treatment of pythiosis in a human patient having pythiosis or prophylaxis against pythiosis which comprises (a) providing a vaccine consisting of intracellular cytoplasmic antigens separated from disrupted cells of
Pythium insidiosum
and extracellular antigens secreted into a medium for growing the cells of the
Pythium insidiosum
in a sterile aqueous solution; and (b) vaccinating the patient with the vaccine.
In further embodiment of the method, the vaccination is subcutaneous. Further still, the patient after vaccination is monitored for a change in a Th1 response and a Th2 response, wherein an increase in the Th1 respon
Board of Trustees of Michigan State University
McLeod Ian C.
Minnifield N. M.
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