Abundant extracellular products and methods for their production and use
View Patent ↗Vaccines based on one or more combinations of majorly abundant extracellular products of pathogens and methods for their use and production are presented. The most prevalent or majorly abundant extracellular products of a target pathogen are selected irrespective of their absolute molecular immunogenicity and used as vaccines to stimulate a protective immune response in mammalian hosts against subsequent infection by the target pathogen. The majorly abundant extracellular products may be characterized and distinguished by their respective N-terminal amino acid, amino acid, or DNA sequences. As the vaccines may comprise different combinations of the extracellular products, subunits thereof, or encoding nucleic acids, a broad range of effective immunotherapeutic compositions are provided by the present invention. In addition to other infectious agents, the vaccines so produced can be used to stimulate an effective immune response against intracellular pathogens and in particular Mycobacterium tuberculosis.
1. A vaccinating agent for use in promoting an effective immune response, in a mammalian host, against an infectious pathogen from the genus Mycobacterium , said vaccinating agent comprising:
at least a portion of at least one majorly abundant extracellular product selected from the group consisting of M. tuberculosis 110 KD protein, 80 KD protein, 71 KD protein, 58 KD protein, 45 KD protein, 32A KD protein, 32B KD protein, 30 KD protein, 24 KD protein, 23.5 KD protein, 23 KD protein, 16 KD protein, 14 KD protein, 12 KD protein and respective analogs, homologs, and subunits thereof; and
an adjuvant selected from the group consisting of IL-12 and MF 59.
2. The vaccinating agent of claim 1 wherein said at least one majorly abundant extracellular product is M. tuberculosis 32A KD protein.
3. The vaccinating agent of claim 1 wherein said at least one majorly abundant extracellular product is a mixture of M. tuberculosis 32A KD protein, 30 KD protein, and 16 KD protein.
4. The vaccinating agent of claim 1 wherein said adjuvant is IL-12.
5. The vaccinating agent of claim 1 wherein said adjuvant is a mixture of IL-12 and MF 59.
6. A method for immunizing a mammalian host against an infectious pathogen of the genus Mycobacterium , said method comprising the steps of:
providing a vaccinating agent comprising at least a portion of at least one majorly abundant extracellular product selected from the group consisting of M. tuberculosis 110 KD protein, 80 KD protein, 71 KD protein, 58 KD protein, 45 KD protein, 32A KD protein, 328 KD protein, 30 KD protein, 24 KD protein, 23.5 KD protein, 23 KD protein, 16 KD protein, 14 KD protein, 12 KD protein and respective analogs, homologs, and subunits thereof, and an adjuvant selected from the group consisting of IL-12 and MF 59; and
introducing said vaccinating agent into said mammalian host to induce an effective immune response to subsequent infection by said infectious pathogen.
7. The method of claim 6 wherein said at least one majorly abundant extracellular product is M. tuberculosis 32A KD protein.
8. The method of claim 6 wherein said at least one majorly abundant extracellular product is a mixture of M. tuberculosis 32A KD protein, 30 KD protein, and 16 KD protein.
9. The method of claim 6 wherein said adjuvant is IL-12.
10. The method of claim 6 wherein said adjuvant is a mixture of IL-12 and MF 59.
11. A vaccinating agent for use in promoting an effective immune response, in a mammalian host, against an infectious pathogen from the genus Mycobacterium , said vaccinating agent comprising:
at least one immunodominant epitope of at least one majorly abundant extracellular product selected from the group consisting of M. tuberculosis 110 KD protein, 80 KD protein, 71 KD protein, 58 KD protein, 45 KD protein, 32A KD protein, 32B KD protein, 30 KD protein, 24 KD protein, 23.5 KD protein, 23 KD protein, 16 KD protein, 14 KD protein, 12 KD protein, and respective analogs, homologs, and subunits thereof.
12. The vaccinating agent of claim 11 wherein said at least one majorly abundant extracellular product is M. tuberculosis 32A KD protein.
13. The vaccinating agent of claim 12 wherein said at least one immunodominant epitope is selected from the group consisting of M. tuberculosis 32A KD protein subunits having the amino acid sequences
Seq.
Peptide Sequence
ID No.
G L R A Q D D F S G W D I N T
104
W D I N T P A F E W Y D Q S G
106
P A F E W Y D Q S G L S V V M
107
P V G G Q S S F Y S D W Y Q P
110
G C Q T Y K W E T F L T S E L
114
K W E T F L T S E L P G W L Q
115
A N R H V K P T G S A V V G L
118
A V V G L S M A A S S A L T L
120
S A L T L A I Y H P Q Q F V Y
122
A I Y H P Q Q F V Y A G A M S
123
Q Q F V Y A G A M S G L L D P
124
G L L D P S Q A M G P T L I G
126
S Q A M G P T L I G L A M G D
127
N D P L L N V G K L I A N N T
134
N V G K L I A N N T R V W V Y
135
I A N N T R V W V Y C G N G K
136
C G N G K P S D L G G N N L P
138
and respective analogs, homologs, and subunits thereof including single or multiple amino acid substitutions, deletions, insertions, and inversions.
14. An immunodiagnostic agent for use in promoting a detectable immune response in a mammalian host identifying an infectious pathogen from the genus Mycobacterium , said immunodiagnostic agent comprising:
at least one immunodominant epitope of at least one majorly abundant extracellular product selected from the group consisting of M. tuberculosis 110 KD protein, 80 KD protein, 71 KD protein, 58 KD protein, 45 KD protein, 32A KD protein, 32B KD protein, 30 KD protein, 24 KD protein, 23.5 KD protein, 23 KD protein, 16 KD protein, 14 KD protein, 12 1KD protein and respective analogs, homologs, and subunits thereof.
15. The immunodiagnostic agent of claim 14 wherein said at least one majorly abundant extracellular product is M. tuberculosis 32A KD protein.
16. The immunodiagnostic agent of claim 15 wherein said at least one immunodominant epitope is selected from the group consisting of M. tuberculosis 32A KD protein subunits having the amino acid sequences
Seq.
Peptide Sequence
ID No.
G L R A Q D D F S G W D I N T
104
W D I N T P A F E W Y D Q S G
106
P A F E W Y D Q S G L S V V M
107
P V G G Q S S F Y S D W Y Q P
110
G C Q T Y K W E T F L T S E L
114
K W E T F L T S E L P G W L Q
115
A N R H V K P T G S A V V G L
118
A V V G L S M A A S S A L T L
120
S A L T L A I Y H P Q Q F V Y
122
A I Y H P Q Q F V Y A G A M S
123
Q Q F V Y A G A M S G L L D P
124
G L L D P S Q A M G P T L I G
126
S Q A M G P T L I G L A M G D
127
N D P L L N V G K L I A N N T
134
N V G K L I A N N T R V W V Y
135
I A N N T R V W V Y C G N G K
136
C G N G K P S D L G G N N L P
138
and respective analogs, homologs, and subunits thereof including single or multiple amino acid substitutions, deletions, insertions, and inversions.
17. A method of immunizing a mammalian host against an infectious pathogen of the genus Mycobacterium , said method comprising the steps of:
providing at least one immunodominant epitope of at least one majorly abundant extracellular product selected from the group consisting of M. tuberculosis 110 KD protein, 80 KD protein, 71 KD protein, 58 KD protein, 45 KD protein, 32A KD protein, 32B KD protein, 30 KD protein, 24 KD protein, 23.5 KD protein, 23 KD protein, 16 KD protein, 14 KD protein, 12 KD protein and respective analogs, homologs, and subunits thereof; and
introducing said at least one immunodominant epitope to said mammalian host to induce an effective immune response to subsequent infection by said infectious pathogen.
18. The method of claim 17 wherein said at least one majorly abundant extracellular product is M. tuberculosis 32A KD protein.
19. The method of claim 18 wherein said at least one immunodominant epitope is selected from the group consisting of M. tuberculosis 32A KD protein subunits having the amino acid sequences
Seq.
Peptide Sequence
ID No.
G L R A Q D D F S G W D I N T
104
W D I N T P A F E W Y D Q S G
106
P A F E W Y D Q S G L S V V M
107
P V G G Q S S F Y S D W Y Q P
110
G C Q T Y K W E T F L T S E L
114
K W E T F L T S E L P G W L Q
115
A N R H V K P T G S A V V G L
118
A V V G L S M A A S S A L T L
120
S A L T L A I Y H P Q Q F V Y
122
A I Y H P Q Q F V Y A G A M S
123
Q Q F V Y A G A M S G L L D P
124
G L L D P S Q A M G P T L I G
126
S Q A M G P T L I G L A M G D
127
N D P L L N V G K L I A N N T
134
N V G K L I A N N T R V W V Y
135
I A N N T R V W V Y C G N G K
136
C G N G K P S D L G G N N L P
138
and respective analogs, homologs, and subunits thereof including single or multiple amino acid substitutions, deletions, insertions, and inversions.
20. A method for detecting the presence of an immune response in a mammal against a pathogen of the genus Mycobacterium , said method comprising the steps of:
providing at least one immunodominant epitope of at least one majorly abundant extracellular product selected from the group consisting of M. tuberculosis 110 KD protein, 80 KD protein, 71 KD protein, 58 KD protein, 45 KD protein, 32A KD protein, 32B KD protein, 30 KD protein, 24 KD protein, 23.5 KD protein, 23 KD protein, 16 KD protein, 14 KD protein, 12 KD protein and respective analogs, homologs, and subunits thereof;
administering said at least one immunodominant epitope to said mammal; and
measuring the resultant immune response.
21. The method of claim 20 wherein said at least one majorly abundant extracellular product is M. tuberculosis 32A KD protein.
22. The method of claim 21 wherein said at least one immunodominant epitope is selected from the group consisting of M. tuberculosis 32A KD protein subunits having the amino acid sequences
Peptide Sequence
Seq. ID No.
G L R A Q D D F S G W D I N T
104
W D I N T P A F E W Y D Q S G
106
P A F E W Y D Q S G L S V V M
107
P V G G Q S S F Y S D W Y Q P
110
G C Q T Y K W E T F L T S E L
114
K W E T F L T S E L P G W L Q
115
A N R H V K P T G S A V V G L
118
A V V G L S M A A S S A L T L
120
S A L T L A I Y H P Q Q F V Y
122
A I Y H P Q Q F V Y A G A M S
123
Q Q F V Y A G A M S G L L D P
124
G L L D P S Q A M G P T L I G
126
S Q A M G P T L I G L A M G D
127
N D P L L N V G K L I A N N T
134
N V G K L I A N N T R V W V Y
135
I A N N T R V W V Y C G N G K
136
C G N C K P S D L G G N N L P
138
and respective analogs, homologs, and subunits thereof including single or multiple maino acid substitutions, deletions, insertions, and inversions.
23. A process for producing a majorly abundant extracellular product selected from the group consisting of M. tuberculosis 110 KD protein, 80 KD protein, 71 KD protein, 58 KD protein, 45 KD protein, 32A KD protein, 32B KD protein, 30 KD protein, 24 KD protein, 23.5 KD protein, 23 KD protein, 16 KD protein, 14 KD protein, 12 KD protein and respective analogs, homologs, and subunits thereof, said process comprising the steps of:
transforming a host cell with a vector to form a transformed cell, said vector comprising a nucleic acid molecule encoding one of said majorly abundant extracellular products; and
culturing said transformed cell to thereby produce said majorly abundant extracellular product.
24. The process of claim 23 wherein said nucleic acid molecule encodes for the 32A KD M. tuberculosis protein.
25. The process of claim 24 which includes the additional step of recovering said majorly abundant extracellular product that is produced by culturing of said transformed cell.
26. The process of claim 24 wherein said vector comprises pSMT3.
27. The process of claim 24 wherein said host cell is M. smegmatis or M. vaccae.
28. The process of claim 24 wherein said transformed cell is cultured at a temperature of 28° C.