Patent Grant
7273614
On Jun. 13, 2005, a Second Substitute Sequence Listing on two compact discs labeled “Copy 1” and “Copy 2” was submitted pursuant to 37 C.F.R. §§ 1.52(e) and 1.821(c). The compact discs and their contents are incorporated by reference herein in their entireties and are described as follows:
Copy 1: Machine Format: IBM-PC
Copy 2: Machine Format: IBM-PC
The present invention relates, first, to DP178 (SEQ ID NO:1), a peptide corresponding to amino acids 638 to 673 of the HIV-1LAI transmembrane protein (TM) gp41, and portions or analogs of DP178 (SEQ ID NO:1), which exhibit anti-membrane fusion capability, antiviral activity, such as the ability to inhibit HIV transmission to uninfected CD-4+ cells, or an ability to modulate intracellular processes involving coiled-coil peptide structures. Further, the invention relates to the use of DP178 (SEQ ID NO:1) and DP178 portions and/or analogs as antifusogenic or antiviral compounds or as inhibitors of intracellular events involving coiled-coil peptide structures. The present invention also relates to peptides analogous to DP107 (SEQ ID NO: 89), a peptide corresponding to amino acids 558 to 595 of the HIV-1LAI transmembrane protein (TM) gp41, having amino acid sequences present in other viruses, such as enveloped viruses, and/or other organisms, and further relates to the uses of such peptides. These peptides exhibit anti-membrane fusion capability, antiviral activity, or the ability to modulate intracellular processes involving coiled-coil peptide structures. The present invention additionally relates to methods for identifying compounds that disrupt the interaction between DP178 and DP107, and/or between DP107-like and DP178-like peptides. Further, the invention relates to the use of the peptides of the invention as diagnostic agents. For example, a DP178 peptide may be used as an HIV subtype-specific diagnostic. The invention is demonstrated, first, by way of an Example wherein DP178 (SEQ ID:1), and a peptide whose sequence is homologous to DP178 are each shown to be potent, non-cytotoxic inhibitors of HIV-1 transfer to uninfected CD-4+ cells. The invention is further demonstrated by Examples wherein peptides having structural and/or amino acid motif similarity to DP107 and DP178 are identified in a variety of viral and nonviral organisms, and in examples wherein a number of such identified peptides derived from several different viral systems are demonstrated to exhibit antiviral activity.
Membrane fusion is a ubiquitous cell biological process (for a review, see White, J. M., 1992, Science 258:917-924). Fusion events which mediate cellular housekeeping functions, such as endocytosis, constitutive secretion, and recycling of membrane components, occur continuously in all eukaryotic cells.
Additional fusion events occur in specialized cells. Intracellularly, for example, fusion events are involved in such processes as occur in regulated exocytosis of hormones, enzymes and neurotransmitters. Intercellularly, such fusion events feature prominently in, for example, sperm-egg fusion and myoblast fusion.
Fusion events are also associated with disease states. For example, fusion events are involved in the formation of giant cells during inflammatory reactions, the entry of all enveloped viruses into cells, and, in the case of human immunodeficiency virus (HIV), for example, are responsible for the virally induced cell-cell fusion which leads to cell death.
The human immunodeficiency virus (HIV) has been implicated as the primary cause of the slowly degenerative immune system disease termed acquired immune deficiency syndrome (AIDS) (Barre-Sinoussi, F. et al., 1983, Science 220:868-870; Gallo, R. et al., 1984, Science 224:500-503). There are at least two distinct types of HIV: HIV-1 (Barre-Sinoussi, F. et al., 1983, Science 220:868-870; Gallo R. et al., 1984, Science 224:500-503) and HIV-2 (Clavel, F. et al., 1986, Science 233:343-346; Guyader, M. et al., 1987, Nature 326:662-669). Further, a large amount of genetic heterogeneity exists within populations of each of these types. Infection of human CD-4+ T-lymphocytes with an HIV virus leads to depletion of the cell type and eventually to opportunistic infections, neurological dysfunctions, neoplastic growth, and ultimately death.
HIV is a member of the lentivirus family of retroviruses (Teich, N. et al., 1984, RNA Tumor Viruses, Weiss, R. et al., eds., CSH-Press, pp. 949-956). Retroviruses are small enveloped viruses that contain a diploid, single-stranded RNA genome, and replicate via a DNA intermediate produced by a virally-encoded reverse transcriptase, an RNA-dependent DNA polymerase (Varmus, H., 1988, Science 240:1427-1439). Other retroviruses include, for example, oncogenic viruses such as human T-cell leukemia viruses (HTLV-I,-II,-III), and feline leukemia virus.
The HIV viral particle consists of a viral core, composed of capsid proteins, that contains the viral RNA genome and those enzymes required for early replicative events. Myristylated Gag protein forms an outer viral shell around the viral core, which is, in turn, surrounded by a lipid membrane enveloped derived from the infected cell membrane. The HIV enveloped surface glycoproteins are synthesized as a single 160 Kd precursor protein which is cleaved by a cellular protease during viral budding into two glycoproteins, gp41 and gp120. gp41 is a transmembrane protein and gp120 is an extracellular protein which remains non-covalently associated with gp41, possibly in a trimeric or multimeric form (Hammarskjold, M. and Rekosh, D., 1989, Biochem. Biophys. Acta 989:269-280).
HIV is targeted to CD-4+ cells because the CD-4 cell surface protein acts as the cellular receptor for the HIV-1 virus (Dalgleish, A. et al., 1984, Nature 312:763-767; Klatzmann et al. 1984, Nature 312:767-768; Maddon et al. 1986, Cell 47:333-348). Viral entry into cells is dependent upon gp120 binding the cellular CD-4+ receptor molecules (McDougal, J. S. et al., 1986, Science 231:382-385; Maddon, P. J. et al., 1986, Cell 47:333-348) and thus explains HIV's tropism for CD-4+ cells, while gp41 anchors the enveloped glycoprotein complex in the viral membrane.
HIV infection is pandemic and HIV associated diseases represent a major world health problem. Although considerable effort is being put into the successful design of effective therapeutics, currently no curative anti-retroviral drugs against AIDS exist. In attempts to develop such drugs, several stages of the HIV life cycle have been considered as targets for therapeutic intervention (Mitsuya, H. et al., 1991, FASEB J. 5:2369-2381). For example, virally encoded reverse transcriptase has been one focus of drug development. A number of reverse-transcriptase-targeted drugs, including 2′,3′-dideoxynucleoside analogs such as AZT, ddI, ddC, and d4T have been developed which have been shown to been active against HIV (Mitsuya, H. et al., 1991, Science 249:1533-1544). While beneficial, these nucleoside analogs are not curative, probably due to the rapid appearance of drug resistant HIV mutants (Lander, B. et al., 1989, Science 243:1731-1734). In addition, the drugs often exhibit toxic side effects such as bone marrow suppression, vomiting, and liver function abnormalities.
Attempts are also being made to develop drugs which can inhibit viral entry into the cell, the earliest stage of HIV infection. Here, the focus has thus far been on CD4, the cell surface receptor for HIV. Recombinant soluble CD4, for example, has been shown to inhibit infection of CD-4+T-cells by some HIV-1 strains (Smith, D. H. et al., 1987, Science 238:1704-1707). Certain primary HIV-1 isolates, however, are relatively less sensitive to inhibition by recombinant CD-4 (Daar, E. et al., 1990, Proc. Natl. Acad. Sci. USA 87:6574-6579). In addition, recombinant soluble CD-4 clinical trials have produced inconclusive results (Schooley, R. et al., 1990, Ann. Int. Med. 112:247-253; Kahn, J. O. et al., 1990, Ann. Int. Med. 112:254-261; Yarchoan, R. et al., 1989, Proc. Vth Int. Conf. on AIDS, p. 564, MCP 137).
The late stages of HIV replication, which involve crucial virus-specific secondary processing of certain viral proteins, have also been suggested as possible anti-HIV drug targets. Late stage processing is dependent on the activity of a viral protease, and drugs are being developed which inhibit this protease (Erickson, J., 1990, Science 249:527-533). The clinical outcome of these candidate drugs is still in question.
Attention is also being given to the development of vaccines for the treatment of HIV infection. The HIV-1 enveloped proteins (gp160, gp120, gp41) have been shown to be the major antigens for anti-HIV antibodies present in AIDS patients (Barin, et al., 1985, Science 228:1094-1096). Thus far, therefore, these proteins seem to be the most promising candidates to act as antigens for anti-HIV vaccine development. To this end, several groups have begun to use various portions of gp160, gp120, and/or gp41 as immunogenic targets for the host immune system. See for example, Ivanoff, L. et al., U.S. Pat. No. 5,141,867; Saith, G. et al., WO 92/22,654; Shafferman, A., WO 91/09,872; Formoso, C. et al., WO 90/07,119. Clinical results concerning these candidate vaccines, however, still remain far in the future.
Thus, although a great deal of effort is being directed to the design and testing of anti-retroviral drugs, a truly effective, non-toxic treatment is still needed.
The present invention relates, first, to DP178 (SEQ ID: 1), a 36-amino acid synthetic peptide corresponding to amino acids 638 to 673 of the transmembrane protein (TM) gp41 from the HIV-1 isolate LAI (HIV-1 LAI), which exhibits potent anti-HIV-1 activity. As evidenced by the Example presented below, in Section 6, the DP178 (SEQ ID:1) antiviral activity is so high that, on a weight basis, no other known anti-HIV agent is effective at concentrations as low as those at which DP178 (SEQ ID: 1) exhibits its inhibitory effects.
The invention further relates to those portions and analogs of DP178 which also show such antiviral activity, and/or show anti-membrane fusion capability, or an ability to modulate intracellular processes involving coiled-coil peptide structures. The term “DP178 analog” refers to a peptide which contains an amino acid sequence corresponding to the DP178 peptide sequence present within the gp41 protein of HIV-1LAI, but found in viruses and/or organisms other than HIV-1LAI. Such DP178 analog peptides may, therefore, correspond to DP178-like amino acid sequences present in other viruses, such as, for example, enveloped viruses, such as retroviruses other than HIV-1LAI, as well as non-enveloped viruses. Further, such analogous DP178 peptides may also correspond to DP178-like amino acid sequences present in nonviral organisms.
The invention further relates to peptides DP107 (SEQ ID NO: 89) analogs. DP107 is a peptide corresponding to amino acids 558-595 of the HIV-1LAI transmembrane protein (TM) gp41. The term “DP107 analog” as used herein refers to a peptide which contains an amino acid sequence corresponding to the DP107 peptide sequence present within the gp41 protein of HIV-1 LAI, but found in viruses and organisms other than HIV-1LAI. Such DP107 analog peptides may, therefore, correspond to DP107-like amino acid sequences present in other viruses, such as, for example, enveloped viruses, such as retroviruses other than HIV-1LAI, as well as non-enveloped viruses. Further, such DP107 analog peptides may also correspond to DP107-like amino acid sequences present in nonviral organisms.
Further, the peptides of the invention include DP107 analog and DP178 analog peptides having amino acid sequences recognized or identified by the 107×178×4, ALLMOTI5 and/or PLZIP search motifs described herein.
The peptides of the invention may, for example, exhibit antifusogenic activity, antiviral activity, and/or may have the ability to modulate intracellular processes which involve coiled-coil peptide structures. With respect to the antiviral activity of the peptides of the invention, such an antiviral activity includes, but is not limited to the inhibition of HIV transmission to uninfected CD-4+ cells. Additionally, the antifusogenic capability, antiviral activity or intracellular modulatory activity of the peptides of the invention merely requires the presence of the peptides of the invention, and, specifically, does not require the stimulation of a host immune response directed against such peptides.
The peptides of the invention may be used, for example, as inhibitors of membrane fusion-associated events, such as, for example, the inhibition of human and non-human retroviral, especially HIV, transmission to uninfected cells. It is further contemplated that the peptides of the invention may be used as modulators of intracellular events involving coiled-coil peptide structures.
The peptides of the invention may, alternatively, be used to identify compounds which may themselves exhibit antifusogenic, antiviral, or intracellular modulatory activity. Additional uses include, for example, the use of the peptides of the invention as organism or viral type and/or subtype-specific diagnostic tools.
The terms “antifusogenic” and “anti-membrane fusion”, as used herein, refer to an agent's ability to inhibit or reduce the level of membrane fusion events between two or more moieties relative to the level of membrane fusion which occurs between said moieties in the absence of the peptide. The moieties may be, for example, cell membranes or viral structures, such as viral envelopes or pili. The term “antiviral”, as used herein, refers to the compound's ability to inhibit viral infection of cells, via, for example, cell-cell fusion or free virus infection. Such infection may involve membrane fusion, as occurs in the case of enveloped viruses, or some other fusion event involving a viral structure and a cellular structure (e.g., such as the fusion of a viral pilus and bacterial membrane during bacterial conjugation). It is also contemplated that the peptides of the invention may exhibit the ability to modulate intracellular events involving coiled-coil peptide structures. “Modulate”, as used herein, refers to a stimulatory or inhibitory effect on the intracellular process of interest relative to the level or activity of such a process in the absence of a peptide of the invention.
Embodiments of the invention are demonstrated below wherein an extremely low concentration of DP178 (SEQ ID: 1), and very low concentrations of a DP178 homolog (SEQ ID:3) are shown to be potent inhibitors of HIV-1 mediated CD-4+ cell-cell fusion (i.e., syncytial formation) and infection of CD-4+ cells by cell-free virus. Further, it is shown that DP178 (SEQ ID:1) is not toxic to cells, even at concentrations 3 logs higher than the inhibitory DP-178 (SEQ ID:1) concentration.
The present invention is based, in part, on the surprising discovery that the DP107 and DP178 domains of the HIV gp41 protein non-covalently complex with each other, and that their interaction is required for the normal infectivity of the virus. This discovery is described in the Example presented, below, in Section 8. The invention, therefore, further relates to methods for identifying antifusogenic, including antiviral, compounds that disrupt the interaction between DP107 and DP178, and/or between DP107-like and DP178-like peptides.
Additional embodiments of the invention (specifically, the Examples presents in Sections 9-16 and 19-25, below) are demonstrated, below, wherein peptides, from a variety of viral and nonviral sources, having structural and/or amino acid motif similarity to DP107 and DP178 are identified, and search motifs for their identification are described. Further, Examples (in Sections 17, 18, 25-29) are presented wherein a number of the peptides of the invention are demonstrated exhibit substantial antiviral activity or activity predictive of antiviral activity.
Peptides are defined herein as organic compounds comprising two or more amino acids covalently joined by peptide bonds. Peptides may be referred to with respect to the number of constituent amino acids, i.e., a dipeptide contains two amino acid residues, a tripeptide contains three, etc. Peptides containing ten or fewer amino acids may be referred to as oligopeptides, while those with more than ten amino acid residues are polypeptides. Such peptides may also include any of the modifications and additional amino and carboxy groups as are described herein.
Peptide sequences defined herein are represented by one-letter symbols for amino acid residues as follows:
A (alanine)
R (arginine)
N (asparagine)
D (aspartic acid)
C (cysteine)
Q (glutamine)
E (glutamic acid)
G (glycine)
H (histidine)
I (isoleucine)
L (leucine)
K (lysine)
M (methionine)
F (phenylalanine)
P (proline)
S (serine)
T (threonine)
W (tryptophan)
Y (tyrosine)
V (valine)
): 107×178×4; Hearts (♥) ALLMOTI5; Clubs (
): PLZIP; Diamonds (♦): transmembrane region (the putative transmembrane domains were identified using a PC/Gene program designed to search for such peptide regions). Asterisk (*): Lupas method. The amino acid sequences identified by each motif are bracketed by the respective characters. Representative sequences chosen based on 107×178×4 searches are underlined and in bold. DP107 and DP178 sequences are marked, and additionally double-underlined and italicized.
Antiviral activity (AV) is represented by the following qualitative symbols:
“−”, negative antiviral activity;
“+/−”, antiviral activity at greater than 100 μg/ml;
“+”, antiviral activity at between 50-100 μg/ml;
“++”, antiviral activity at between 20-50 μg/ml;
“+++”, antiviral activity at between 1-20 μg/ml;
“++++”, antiviral activity at <1 μg/ml.
CD data, referring to the level of helicity is represented by the following qualitative symbol:
“−”, no helicity;
“+”, 25-50% helicity;
“++”, 50-75% helicity;
“+++”, 75-100% helicity.
IC50 refers to the concentration of peptide necessary to produce only 50% of the number of syncytial relative to infected control cultures containing no peptide. IC50 values were obtained using purified peptides only.
HPIV3 peptide T-184 CD spectrum at 1° C. in 0.1M NaCl 10 mM KPO4, pH 7.0. The data demonstrates the peptide's helical secondary structure (θ222/208=1.2) over a wide range of concentrations (100-1500 μM). This evidence is consistent with the peptide forming a helical coiled-coil structure.
In instances where peptides contained DP178 point mutations, the mutated amino acid residues are shown with a shaded background. In instances in which the test peptide has had an amino and/or carboxy-terminal group added or removed (apart from the standard amido- and acetyl-blocking groups found on such peptides), such modifications are indicated.
The amino acid residue within this region which corresponds to the first amino acid residue of each peptide is listed to the left of each peptide, while the amino acid residue within this region which corresponds to the last amino acid residue of each peptide is listed to the right of each peptide. The length of each test peptide is listed at the far right of each line, under the heading “Res”.
“ACT” refers to a test peptide's ability to inhibit Zebra binding to its response element. “+” refers to a visible, but incomplete, abrogation of the response element/Zebra homodimer complex; “+++” refers to a complete abrogation of the complex; and “−” represents a lack of complex disruption.
Described herein are peptides which may exhibit antifusogenic activity, antiviral capability, and/or the ability to modulate intracellular processes involving coiled-coil peptide structures. The peptides described include, first, DP178 (SEQ ID NO:1), a gp41-derived 36 amino acid peptide and fragments and analogs of DP178.
In addition, the peptides of the invention described herein include peptides which are DP107 analogs. DP107 (SEQ ID NO: 89) is a 38 amino acid peptide corresponding to residues 558 to 595 of the HIV-1LAI transmembrane (TM) gp41 protein. Such DP107 analogs may exhibit antifusogenic capability, antiviral activity or an ability to modulate intracellular processes involving coiled-coil structures.
Further, peptides of the invention include DP107 and DP178 are described herein having amino acid sequences recognized by the 107×178×4, ALLMOTI5, and PLZIP search motifs. Such motifs are also discussed.
Also described here are antifusogenic, antiviral, intracellular modulatory, and diagnostic uses of the peptides of the invention. Further, procedures are described for the use of the peptides of the invention for the identification of compounds exhibiting antifusogenic, antiviral or intracellular modulatory activity.
While not limited to any theory of operation, the following model is proposed to explain the potent anti-HIV activity of DP178, based, in part, on the experiments described in the Examples, infra. In the HIV protein, gp41, DP178 corresponds to a putative α-helix region located in the C-terminal end of the gp41 ectodomain, and appears to associate with a distal site on gp41 whose interactive structure is influenced by the leucine zipper motif, a coiled-coil structure, referred to as DP107. The association of these two domains may reflect a molecular linkage or “molecular clasp” intimately involved in the fusion process. It is of interest that mutations in the C-terminal α-helix motif of gp41 (i.e., the D178 domain) tend to enhance the fusion ability of gp41, whereas mutations in the leucine zipper region (i.e., the DP107 domain) decrease or abolish the fusion ability of the viral protein. It may be that the leucine zipper motif is involved in membrane fusion while the C-terminal α-helix motif serves as a molecular safety to regulate the availability of the leucine zipper during virus-induced membrane fusion.
On the basis of the foregoing, two models are proposed of gp4′-mediated membrane fusion which are schematically shown in
When synthesized as peptides, both DP107 and DP178 are potent inhibitors of HIV infection and fusion, probably by virtue of their ability to form complexes with viral gp41 and interfere with its fusogenic process; e.g., during the structural transition of the viral protein from the native structure to the fusogenic state, the DP178 and DP107 peptides may gain access to their respective binding sites on the viral gp41, and exert a disruptive influence. DP107 peptides which demonstrate anti-HIV activity are described in Applicants' co-pending application Ser. No. 08/264,531, filed Jun. 23, 1994, which is incorporated by reference herein in its entirety.
As shown in the Examples, infra, a truncated recombinant gp41 protein corresponding to the ectodomain of gp41 containing both DP107 and DP178 domains (excluding the fusion peptide, transmembrane region and cytoplasmic domain of gp41) did not inhibit HIV-1 induced fusion. However, when a single mutation was introduced to disrupt the coiled-coil structure of the DP107 domain—a mutation which results in a total loss of biological activity of DP107 peptides—the inactive recombinant protein was transformed to an active inhibitor of HIV-1 induced fusion. This transformation may result from liberation of the potent DP178 domain from a molecular clasp with the leucine zipper, DP107 domain.
For clarity of discussion, the invention will be described primarily for DP178 peptide inhibitors of HIV. However, the principles may be analogously applied to other viruses, both enveloped and nonenveloped, and to other non-viral organisms.
The DP178 peptide (SEQ ID: 1) of the invention corresponds to amino acid residues 638 to 673 of the transmembrane protein gp41 from the HIV-1LAI isolate, and has the 36 amino acid sequence (reading from amino to carboxy terminus):
NH2-YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF-COOH (SEQ ID:1)
In addition to the full-length DP178 (SEQ ID:1) 36-mer, the peptides of the invention may include truncations of the DP178 (SEQ ID:1) peptide which exhibit antifusogenic activity, antiviral activity and/or the ability to modulate intracellular processes involving coiled-coil peptide structures. Truncations of DP178 (SEQ ID:1) peptides may comprise peptides of between 3 and 36 amino acid residues (i.e., peptides ranging in size from a tripeptide to a 36-mer polypeptide), as shown in Tables I and IA, below. Peptide sequences in these tables are listed from amino (left) to carboxy (right) terminus. “X” may represent an amino group (—NH2) and “Z” may represent a carboxyl (—COOH) group. Alternatively, “X” may represent a hydrophobic group, including but not limited to carbobenzyl, dansyl, or T-butoxycarbonyl; an acetyl group; a 9-fluorenylmethoxy-carbonyl (FMOC) group; or a covalently attached macromolecular group, including but not limited to a lipid-fatty acid conjugate, polyethylene glycol, carbohydrate or peptide group. Further, “Z” may represent an amido group; a T-butoxycarbonyl group; or a covalently attached macromolecular group, including but not limited to a lipid-fatty acid conjugate, polyethylene glycol, carbohydrate or peptide group. A preferred “X” or “Z” macromolecular group is a peptide group.
Additionally,
“X” may represent an amino group, a hydrophobic group, including but not limited to carbobenzoxyl, dansyl, or T-butyloxycarbonyl; an acetyl group; a 9-fluorenylmethoxy-carbonyl (FMOC) group; a macromolecular carrier group including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.
“Z” may represent a carboxyl group; an amido group; a T-butyloxycarbonyl group; a macromolecular carrier group including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.
Additionally,
“X” may represent an amino group, a hydrophobic group, including but not limited to carbobenzoxyl, dansyl, or T-butyloxycarbonyl; an acetyl group; a 9-fluorenylmethoxy-carbonyl group; a macromolecular carrier group including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.
“Z” may represent a carboxyl group; an amido group; a T-butyloxycarbonyl group; a macromolecular carrier group including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.
The peptides of the invention also include DP178-like peptides. “DP178-like”, as used herein, refers, first, to DP178 and DP178 truncations which contain one or more amino acid substitutions, insertions and/or deletions. Second, “DP-178-like” refers to peptide sequences identified or recognized by the ALLMOTI5, 107×178×4 and PLZIP search motifs described herein, having structural and/or amino acid motif similarity to DP178. The DP178-like peptides of the invention may exhibit antifusogenic or antiviral activity, or may exhibit the ability to modulate intracellular processes involving coiled-coil peptides. Further, such DP178-like peptides may possess additional advantageous features, such as, for example, increased bioavailability, and/or stability, or reduced host immune recognition.
HIV-1 and HIV-2 enveloped proteins are structurally distinct, but there exists a striking amino acid conservation within the DP178-corresponding regions of HIV-1 and HIV-2. The amino acid conservation is of a periodic nature, suggesting some conservation of structure and/or function. Therefore, one possible class of amino acid substitutions would include those amino acid changes which are predicted to stabilize the structure of the DP178 peptides of the invention. Utilizing the DP178 and DP178 analog sequences described herein, the skilled artisan can readily compile DP178 consensus sequences and ascertain from these, conserved amino acid residues which would represent preferred amino acid substitutions.
The amino acid substitutions may be of a conserved or non-conserved nature. Conserved amino acid substitutions consist of replacing one or more amino acids of the DP178 (SEQ ID:1) peptide sequence with amino acids of similar charge, size, and/or hydrophobicity characteristics, such as, for example, a glutamic acid (E) to aspartic acid (D) amino acid substitution. Non-conserved substitutions consist of replacing one or more amino acids of the DP178 (SEQ ID:1) peptide sequence with amino acids possessing dissimilar charge, size, and/or hydrophobicity characteristics, such as, for example, a glutamic acid (E) to valine (V) substitution.
Amino acid insertions may consist of single amino acid residues or stretches of residues. The insertions may be made at the carboxy or amino terminal end of the DP178 or DP178 truncated peptides, as well as at a position internal to the peptide. Such insertions will generally range from 2 to 15 amino acids in length. It is contemplated that insertions made at either the carboxy or amino terminus of the peptide of interest may be of a broader size range, with about 2 to about 50 amino acids being preferred. One or more such insertions may be introduced into DP178 (SEQ. ID:1) or DP178 truncations, as long as such insertions result in peptides which may still be recognized by the 107×178×4, ALLMOTI5 or PLZIP search motifs described herein, or may, alternatively, exhibit antifusogenic or antiviral activity, or exhibit the ability to modulate intracellular processes involving coiled-coil peptide structures.
Preferred amino or carboxy terminal insertions are peptides ranging from about 2 to about 50 amino acid residues in length, corresponding to gp41 protein regions either amino to or carboxy to the actual DP178 gp41 amino acid sequence, respectively. Thus, a preferred amino terminal or carboxy terminal amino acid insertion would contain gp41 amino acid sequences found immediately amino to or carboxy to the DP178 region of the gp41 protein.
Deletions of DP178 (SEQ ID:1) or DP178 truncations are also within the scope of the invention. Such deletions consist of the removal of one or more amino acids from the DP178 or DP178-like peptide sequence, with the lower limit length of the resulting peptide sequence being 4 to 6 amino acids. Such deletions may involve a single contiguous or greater than one discrete portion of the peptide sequences. One or more such deletions may be introduced into DP178 (SEQ. ID: 1) or DP178 truncations, as long as such deletions result in peptides which may still be recognized by the 107×178×4, ALLMOTI5 or PLZIP search motifs described herein, or may, alternatively, exhibit antifusogenic or antiviral activity, or exhibit the ability to modulate intracellular processes involving coiled-coil peptide structures.
DP178 analogs are further described, below, in Section 5.3.
Further, the peptides of the invention include peptides having amino acid sequences corresponding to DP107 analogs. DP107 is a 38 amino acid peptide which exhibits potent antiviral activity, and corresponds to residues 558 to 595 of HIV-1LAI transmembrane (TM) gp41 protein, as shown here:
NH2-NNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQ-COOH (SEQ ID NO. 89)
In addition to the full-length DP107 (SEQ (SEQ ID NO. 89) 38-mer, the peptides of the invention may include truncations of the DP107 (SEQ (SEQ ID NO. 89) peptide which exhibit antifusogenic activity, antiviral activity and/or the ability to modulate intracellular processes involving coiled-coil peptide structures. Truncations of DP107 (SEQ ID (SEQ ID NO. 89) peptides may comprise peptides of between 3 and 38 amino acid residues (i.e., peptides ranging in size from a tripeptide to a 38-mer polypeptide), as shown in Tables II and IIA, below. Peptide sequences in these tables are listed from amino (left) to carboxy (right) terminus. “X” may represent an amino group (—NH2) and “Z” may represent a carboxyl (—COOH) group. Alternatively, “X” may represent a hydrophobic group, including but not limited to carbobenzyl, dansyl, or T-butoxycarbonyl; an acetyl group; a 9-fluorenylmethoxy-carbonyl (FMOC) group; or a covalently attached macromolecular group, including but not limited to a lipid-fatty acid conjugate, polyethylene glycol, carbohydrate or peptide group. Further, “Z” may represent an amido group; a T-butoxycarbonyl group; or a covalently attached macromolecular group, including but not limited to a lipid-fatty acid conjugate, polyethylene glycol, carbohydrate or peptide group. A preferred “X” or “Z” macromolecular group is a peptide group.
Additionally,
“X” may represent an amino group, a hydrophobic group, including but not limited to carbobenzoxyl, dansyl, or T-butyloxycarbonyl; an acetyl group; a 9-fluorenylmethoxy-carbonyl (FMOC) group; a macromolecular carrier group including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.
“Z” may represent a carboxyl group; an amido group; a T-butyloxycarbonyl group; a macromolecular carrier group including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.
Additionally,
“X” may represent an amino group, a hydrophobic group, including but not limited to carbobenzoxyl, dansyl, or T-butyloxycarbonyl; an acetyl group; a 9-fluorenylmethoxy-carbonyl group; a macromolecular carrier group including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.
“Z” may represent a carboxyl group; an amido group; a T-butyloxycarbonyl group; a macromolecular carrier group including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.
The peptides of the invention also include DP107-like peptides. “DP107-like”, as used herein, refers, first, to DP107 and DP107 truncations which contain one or more amino acid substitutions, insertions and/or deletions. Second, “DP-107-like” refers to peptide sequences identified or recognized by the ALLMOTI5, 107×178×4 and PLZIP search motifs described herein, having structural and/or amino acid motif similarity to DP107. The DP107-like peptides of the invention may exhibit antifusogenic or antiviral activity, or may exhibit the ability to modulate intracellular processes involving coiled-coil peptides. Further, such DP107-like peptides may possess additional advantageous features, such as, for example, increased bioavailability, and/or stability, or reduced host immune recognition.
HIV-1 and HIV-2 enveloped proteins are structurally distinct, but there exists a striking amino acid conservation within the DP107-corresponding regions of HIV-1 and HIV-2. The amino acid conservation is of a periodic nature, suggesting some conservation of structure and/or function. Therefore, one possible class of amino acid substitutions would include those amino acid changes which are predicted to stabilize the structure of the DP107 peptides of the invention. Utilizing the DP107 and DP107 analog sequences described herein, the skilled artisan can readily compile DP107 consensus sequences and ascertain from these, conserved amino acid residues which would represent preferred amino acid substitutions.
The amino acid substitutions may be of a conserved or non-conserved nature. Conserved amino acid substitutions consist of replacing one or more amino acids of the DP107 (SEQ (SEQ ID NO. 89) peptide sequence with amino acids of similar charge, size, and/or hydrophobicity characteristics, such as, for example, a glutamic acid (E) to aspartic acid (D) amino acid substitution. Non-conserved substitutions consist of replacing one or more amino acids of the DP107 (SEQ (SEQ ID NO. 89) peptide sequence with amino acids possessing dissimilar charge, size, and/or hydrophobicity characteristics, such as, for example, a glutamic acid (E) to valine (V) substitution.
Amino acid insertions may consist of single amino acid residues or stretches of residues. The insertions may be made at the carboxy or amino terminal end of the DP107 or DP107 truncated peptides, as well as at a position internal to the peptide. Such insertions will generally range from 2 to 15 amino acids in length. It is contemplated that insertions made at either the carboxy or amino terminus of the peptide of interest may be of a broader size range, with about 2 to about 50 amino acids being preferred. One or more such insertions may be introduced into DP107 (SEQ (SEQ ID NO. 89) or DP107 truncations, as long as such insertions result in peptides which may still be recognized by the 107×178×4, ALLMOTI5 or PLZIP search motifs described herein, or may, alternatively, exhibit antifusogenic or antiviral activity, or exhibit the ability to modulate intracellular processes involving coiled-coil peptide structures.
Preferred amino or carboxy terminal insertions are peptides ranging from about 2 to about 50 amino acid residues in length, corresponding to gp41 protein regions either amino to or carboxy to the actual DP107 gp41 amino acid sequence, respectively. Thus, a preferred amino terminal or carboxy terminal amino acid insertion would contain gp41 amino acid sequences found immediately amino to or carboxy to the DP107 region of the gp41 protein.
Deletions of DP107 (SEQ (SEQ ID NO. 89) or DP178 truncations are also within the scope of the invention. Such deletions consist of the removal of one or more amino acids from the DP107 or DP107-like peptide sequence, with the lower limit length of the resulting peptide sequence being 4 to 6 amino acids. Such deletions may involve a single contiguous or greater than one discrete portion of the peptide sequences. One or more such deletions may be introduced into DP107 (SEQ (SEQ ID NO. 89) or DP107 truncations, as long as such deletions result in peptides which may still be recognized by the 107×178×4, ALLMOTI5 or PLZIP search motifs described herein, or may, alternatively, exhibit antifusogenic or antiviral activity, or exhibit the ability to modulate intracellular processes involving coiled-coil peptide structures.
DP107 and DP107 truncations are more fully described in Applicants' co-pending U.S. patent application Ser. No. 08/374,666, filed Jan. 27, 1995, and which is incorporated herein by reference in its entirety. DP107 analogs are further described, below, in Section 5.3.
Peptides corresponding to analogs of the DP178, DP178 truncations, DP107 and DP107 truncation sequences of the invention, described, above, in Sections 5.1 and 5.2 may be found in other viruses, including, for example, non-HIV-1LAI enveloped viruses, non-enveloped viruses and other non-viral organisms.
The term “analog”, as used herein, refers to a peptide which is recognized or identified via the 107×178×4, ALLMOTI5 and/or PLZIP search strategies discussed below. Further, such peptides may exhibit antifusogenic capability, antiviral activity, or the ability to modulate intracellular processes involving coiled-coil structures.
Such DP178 and DP107 analogs may, for example, correspond to peptide sequences present in TM proteins of enveloped viruses and may, additionally correspond to peptide sequences present in non enveloped and non-viral organisms. Such peptides may exhibit antifusogenic activity, antiviral activity, most particularly antiviral activity which is specific to the virus in which their native sequences are found, or may exhibit an ability to modulate intracellular processes involving coiled-coil peptide structures.
DP178 analogs are peptides whose amino acid sequences are comprised of the amino acid sequences of peptide regions of, for example, other (i.e., other than HIV-1 LAI) viruses that correspond to the gp41 peptide region from which DP178 (SEQ ID: 1) was derived. Such viruses may include, but are not limited to, other HIV-1 isolates and HIV-2 isolates. DP178 analogs derived from the corresponding gp41 peptide region of other (i.e., non HIV-1LAI) HIV-1 isolates may include, for example, peptide sequences as shown below.
NH2-YTNTIYTLLEESQNQQEKNEQELLELDKWASLWNWF-COOH (DP-185; SEQ ID: 3);
NH2-YTGIIYNLLEESQNQQEKNEQELLELDKWANLWNWF-COOH (SEQ ID: 4);
NH2-YTSLIYSLLEKSQIQQEKNEQELLELDKWASLWNWF-COOH (SEQ ID: 5).
SEQ ID:3 (DP-185), SEQ ID:4, and SEQ ID:5 are derived from HIV-1SF2, HIV-1RF, and HIV-1MN isolates, respectively. Underlined amino acid residues refer to those residues that differ from the corresponding position in the DP178 (SEQ ID:1) peptide. One such DP178 analog, DP-185 (SEQ ID:3), is described in the Example presented in Section 6, below, where it is demonstrated that DP-185 (SEQ ID:3) exhibits antiviral activity. The DP178 analogs of the invention may also include truncations, as described above. Further, the analogs of the invention modifications such those described for DP178 analogs in Section 5.1., above. It is preferred that the DP178 analogs of the invention represent peptides whose amino acid sequences correspond to the DP178 region of the gp41 protein, it is also contemplated that the peptides of the invention may, additionally, include amino sequences, ranging from about 2 to about 50 amino acid residues in length, corresponding to gp41 protein regions either amino to or carboxy to the actual DP178 amino acid sequence.
Striking similarities, as shown in
NH2-LEANISQSLEQAQIQQEKNMYELQKLNSWDVFTNWL-COOH (SEQ ID:7)
Table III and Table IV show some possible truncations of the HIV-2NIHZ DP178 analog, which may comprise peptides of between 3 and 36 amino acid residues (i.e., peptides ranging in size from a tripeptide to a 36-mer polypeptide). Peptide sequences in these tables are listed from amino (left) to carboxy (right) terminus. “X” may represent an amino group (—NH2) and “Z” may represent a carboxyl (—COOH) group. Alternatively, “X” may represent a hydrophobic group, including but not limited to carbobenzyl, dansyl, or T-butoxycarbonyl; an acetyl group; a 9-fluorenylmethoxy-carbonyl (FMOC) group; or a covalently attached macromolecular group, including but not limited to a lipid-fatty acid conjugate, polyethylene glycol, carbohydrate or peptide group. Further, “Z” may represent an amido group; a T-butoxycarbonyl group; or a covalently attached macromolecular group, including but not limited to a lipid-fatty acid conjugate, polyethylene glycol, carbohydrate or peptide group. A preferred “X” or “Z” macromolecular group is a peptide group.
Additionally,
“X” may represent an amino group, a hydrophobic group, including but not limited to carbobenzoxyl, dansyl, or T-butyloxycarbonyl; an acetyl group; a 9-fluorenylmethoxy-carbonyl (FMOC) group; a macromolecular carrier group including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.
“Z” may represent a carboxyl group; an amido group; a T-butyloxycarbonyl group; a macromolecular carrier group including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.
Additionally,
“X” may represent an amino group, a hydrophobic group, including but not limited to carbobenzoxyl, dansyl, or T-butyloxycarbonyl; an acetyl group; a 9-fluorenylmethoxy-carbonyl (FMOC) group; a macromolecular carrier group including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.
“Z” may represent a carboxyl group; an amido group; a T-butyloxycarbonyl group; a macromolecular carrier group including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.
DP178 and DP107 analogs are recognized or identified, for example, by utilizing one or more of the 107×178×4, ALLMOTI5 or PLZIP computer-assisted search strategies described and demonstrated, below, in the Examples presented in Sections 9 through 16 and 19 through 25. The search strategy identifies additional peptide regions which are predicted to have structural and/or amino acid sequence features similar to those of DP107 and/or DP178.
The search strategies are described fully, below, in the Example presented in Section 9. While this search strategy is based, in part, on a primary amino acid motif deduced from DP107 and DP178, it is not based solely on searching for primary amino acid sequence homologies, as such protein sequence homologies exist within, but not between major groups of viruses. For example, primary amino acid sequence homology is high within the TM protein of different strains of HIV-1 or within the TM protein of different isolates of simian immunodeficiency virus (SIV). Primary amino acid sequence homology between HIV-1 and SIV, however, is low enough so as not to be useful. It is not possible, therefore, to find peptide regions similar to DP107 or DP178 within other viruses, or within non-viral organisms, whether structurally, or otherwise, based on primary sequence homology, alone.
Further, while it would be potentially useful to identify primary sequence arrangements of amino acids based on, for example, the physical chemical characteristics of different classes of amino acids rather than based on the specific amino acids themselves, such search strategies have, until now, proven inadequate. For example, a computer algorithm designed by Lupas et al. to identify coiled-coil propensities of regions within proteins (Lupas, A., et al., 1991 Science 252:1162-1164) is inadequate for identifying protein regions analogous to DP107 or DP178.
Specifically, analysis of HIV-1 gp160 (containing both gp120 and gp41) using the Lupas algorithm does not identify the coiled-coil region within DP107. It does, however, identify a region within DP178 beginning eight amino acids N-terminal to the start of DP178 and ending eight amino acids from the C-terminus. The DP107 peptide has been shown experimentally to form a stable coiled coil. A search based on the Lupas search algorithm, therefore, would not have identified the DP107 coiled-coil region. Conversely, the Lupas algorithm identified the DP178 region as a potential coiled-coil motif. However, the peptide derived from the DP178 region failed to form a coiled coil in solution.
A possible explanation for the inability of the Lupas search algorithm to accurately identify coiled-coil sequences within the HIV-1 TM, is that the Lupas algorithm is based on the structure of coiled coils from proteins that are not structurally or functionally similar to the TM proteins of viruses, antiviral peptides (e.g. DP107 and DP178) of which are an object of this invention.
The computer search strategy of the invention, as demonstrated in the Examples presented below, in Sections 9 through 16 and 19 through 25, successfully identifies regions of proteins similar to DP107 or DP178. This search strategy was designed to be used with a commercially-available sequence database package, preferably PC/Gene.
A series of search motifs, the 107×178×4, ALLMOTI5 and PLZIP motifs, were designed and engineered to range in stringency from strict to broad, as discussed in this Section and in Section 9, with 107×178×4 being preferred. The sequences identified via such search motifs, such as those listed in Tables V-XIV, below, potentially exhibit antifusogenic, such as antiviral, activity, may additionally be useful in the identification of antifusogenic, such as antiviral, compounds, and are intended to be within the scope of the invention.
Coiled-coiled sequences are thought to consist of heptad amino acid repeats. For ease of description, the amino acid positions within the heptad repeats are sometimes referred to as A through G, with the first position being A, the second B, etc. The motifs used to identify DP107-like and DP178-like sequences herein are designed to specifically search for and identify such heptad repeats. In the descriptions of each of the motifs described, below, amino acids enclosed by brackets, i.e., [ ], designate the only amino acid residues that are acceptable at the given position, while amino acids enclosed by braces, i.e., { }, designate the only amino acids which are unacceptable at the given heptad position. When a set of bracketed or braced amino acids is followed by a number in parentheses i.e., ( ), it refers to the number of subsequent amino acid positions for which the designated set of amino acids hold, e.g, a (2) means “for the next two heptad amino acid positions”.
The ALLMOTI5 is written as follows:
{CDGHP}-{CFP}(2)-{CDGHP}-{CFP}(3)-
{CDGHP}-{CFP}(2)-{CDGHP}-{CFP}(3)-
{CDGHP}-{CFP}(2)-{CDGHP}-{CFP}(3)-
{CDGHP}-{CFP}(2)-{CDGHP}-{CFP}(3)-
{CDGHP}-{CFP}(2)-{CDGHP}-{CFP}(3)-
Translating this motif, it would read: “at the first (A) position of the heptad, any amino acid residue except C, D, G, H, or P is acceptable, at the next two (B,C) amino acid positions, any amino acid residue except C, F, or P is acceptable, at the fourth heptad position (D), any amino acid residue except C, D, G, H, or P is acceptable, at the next three (E, F, G) amino acid positions, any amino acid residue except C, F, or P is acceptable. This motif is designed to search for five consecutive heptad repeats (thus the repeat of the first line five times), meaning that it searches for 35-mer sized peptides. It may also be designed to search for 28-mers, by only repeating the initial motif four times. With respect to the ALLMOTI5 motif, a 35-mer search is preferred. Those viral (non-bacteriophage) sequences identified via such an ALLMOTI5 motif are listed in Table V, below, at the end of this Section. The viral sequences listed in Table V potentially exhibit antiviral activity, may be useful in the identification of antiviral compounds, and are intended to be within the scope of the invention. In those instances wherein a single gene exhibits greater than one sequence recognized by the ALLMOTI5 search motif, the amino acid residue numbers of these sequences are listed under “Area 2”, Area 3”, etc. This convention is used for each of the Tables listed, below, at the end of this Section.
The 107×178×4 motif is written as follows:
[EFIKLNQSTVWY]-{CFMP}(2)-[EFIKLNQSTVWY]-{CFMP}(3)-
[EFIKLNQSTVWY]-{CFMP}(2)-[EFIKLNQSTVWY]-{CFMP}(3)-
[EFIKLNQSTVWY]-{CFMP}(2)-[EFIKLNQSTVWY]-{CFMP}(3)-
[EFIKLNQSTVWY]-{CFMP}(2)-[EFIKLNQSTVWY]-{CFMP}(3)-
Translating this motif, it would read: “at the first (A) position of the heptad, only amino acid residue E, F, I, K, L, N, Q, S, T, V, W, or Y is acceptable, at the next two (B,C) amino acid positions, any amino acid residue except C, F, M or P is acceptable, at the fourth position (D), only amino acid residue E, F, I, K, L, N, Q, S, T, V, W, or Y is acceptable, at the next three (E, F, G) amino acid positions, any amino acid residue except C, F, M or P is acceptable. This motif is designed to search for four consecutive heptad repeats (thus the repeat of the first line four times), meaning that it searches for 28-mer sized peptides. It may also be designed to search for 35-mers, by repeating the initial motif five times. With respect to the 107×178×4 motif, a 28-mer search is preferred.
Those viral (non-bacteriophage) sequences identified via such a 107×178×4 motif are listed in Table VI, below, at the end of this Section, with those viral (non-bacteriophage) sequences listed in Table VII, below at the end of this Section, being preferred.
The 107×178×4 search motif was also utilized to identify non-viral procaryotic protein sequences, as listed in Table VIII, below, at the end of this Section. Further, this search motif was used to reveal a number of human proteins. The results of this human protein 107×178×4 search is listed in Table IX, below, at the end of this Section. The sequences listed in Tables VIII and IX, therefore, reveal peptides which may be useful as antifusogenic compounds or in the identification of antifusogenic compounds, and are intended to be within the scope of the invention.
The PLZIP series of motifs are as listed in
Tables X through XIV, below, at the end of this Section, list sequences identified via searches conducted with such PLZIP motifs. Specifically, Table X lists viral sequences identified via PCTLZIP, P1CTLZIP and P2CTLZIP search motifs, Table XI lists viral sequences identified via P3CTLZIP, P4CTLZIP, P5CTLZIP and P6CTLZIP search motifs, Table XII lists viral sequences identified via P7CTLZIP, P8CTLZIP and P9CTLZIP search motifs, Table XIII lists viral sequences identified via P12LZIPC searches and Table XIV lists viral sequences identified via P23TLZIPC search motifs The viral sequences listed in these tables represent peptides which potentially exhibit antiviral activity, may be useful in the identification of antiviral compounds, and are intended to be within the scope of the invention.
The Examples presented in Sections 17, 18, 26 and 27 below, demonstrate that viral sequences identified via the motif searches described herein identify substantial antiviral characteristics. Specifically, the Example presented in Section 17 describes peptides with anti-respiratory syncytial virus activity, the Example presented in Section 18 describes peptides with anti-parainfluenza virus activity, the Example presented in Section 26 describes peptides with anti-measles virus activity and the Example presented in Section 27 describes peptides with anti-simian immunodeficiency virus activity.
The DP107 and DP178 analogs may, further, contain any of the additional groups described for DP178, above, in Section 5.1. For example, these peptides may include any of the additional amino-terminal groups as described above for “X” groups, and may also include any of the carboxy-terminal groups as described, above, for “Z” groups.
Additionally, truncations of the identified DP107 and DP178 peptides are among the peptides of the invention. Further, such DP107 and DP178 analogs and DP107/DP178 analog truncations may exhibit one or more amino acid substitutions, insertion, and/or deletions. The DP178 analog amino acid substitutions, insertions and deletions, are as described, above, for DP178-like peptides in Section 5.1. The DP-107 analog amino acid substitutions, insertions and deletions are also as described, above, for DP107-like peptides in Section 5.2.
Tables XV through XXII, below, present representative examples of such DP107/DP178 truncations. Specifically, Table XV presents Respiratory Syncytial Virus F1 region DP107 analog carboxy truncations, Table XVI presents Respiratory Syncytial Virus F1 region DP107 analog amino truncations, Table XVII presents Respiratory Syncytial Virus F1 region DP178 analog carboxy truncations, Table XVIII presents Respiratory Syncytial Virus F1 region DP178 analog amino truncations, Table XIX presents Human Parainfluenza Virus 3 F1 region DP178 analog carboxy truncations, Table XX presents Human Parainfluenza Virus 3 μl region DP178 analog amino truncations, Table XXI presents Human Parainfluenza Virus 3 μl region DP107 analog carboxy truncations and Table XXII presents Human Parainfluenza Virus 3 μl region DP107 analog amino truncations. Further, Table XXIII, below, presents DP107/DP178 analogs and analog truncations which exhibit substantial antiviral activity. These antiviral peptides are grouped according to the specific virus which they inhibit, including respiratory syncytial virus, human parainfluenza virus 3, simian immunodeficiency virus and measles virus.
HERPES SIMPLEX VIRUS
HERPES SIMPLEX VIRUS
HERPES SIMPLEX VIRUS
HERPES SIMPLEX VIRUS
HERPES SIMPLEX VIRUS
HERPES SIMPLEX VIRUS
HERPES SIMPLEX VIRUS
HERPES SIMPLEX VIRUS
CHORISTONEURA BIENNIS
PARAMECIUM BURSARIA
HERPES SIMPLEX VIRUS (TYPE
HERPES SIMPLEX VIRUS (TYPE
HERPES SIMPLEX VIRUS (TYPE
HERPES SIMPLEX VIRUS (TYPE
HERPES SIMPLEX VIRUS (TYPE
AUTOGRAPHA CALIFORNICA
SACCHAROMYCES CEREVISIAE
HERPES SIMPLEX VIRUS (TYPE
HERPES SIMPLEX VIRUS (TYPE
HERPES SIMPLEX VIRUS (TYPE
HERPES SIMPLEX VIRUS (TYPE
HERPES SIMPLEX VIRUS (TYPE
HERPES SIMPLEX VIRUS (TYPE
AMSACTA MOOREI
HERPES SIMPLEX VIRUS (TYPE
HERPES SIMPLEX VIRUS (TYPE
HERPES SIMPLEX VIRUS (TYPE
HERPES SIMPLEX VIRUS (TYPE
HERPES SIMPLEX VIRUS (TYPE
HERPES SIMPLEX VIRUS (TYPE
HERPES SIMPLEX VIRUS (TYPE
PARAMECIUM BURSARIA
AMSACTA MOOREI
CHORISTONEURA BIENNIS
HERPES SIMPLEX VIRUS (TYPE
AUTOGRAPHA CALIFORNICA
ORGYIA PSEUDOTSUGATA
AUTOGRAPHA CALIFORNICA
ORGYIA PSEUDOTSUGATA
AUTOGRAPHA CALIFORNICA
ORGYIA PSEUDOTSUGATA
AUTOGRAPHA CALIFORNICA
AGROTIS SEGETU M NUCLEAR
BOMBYX MORI NUCLEAR
LYMANTRIA DISPAR
MAMESTRA BPASSICAE NUCLEAR
ORGYIA PSEUDOTSUGATA
ORGYIA PSEUDOTSUGATA
PANOLIS FLAMMEA
SPODOPTERA EXIGUA
HERPES SIMPLEX VIRUS (TYPE
SACCHAROMYCES CEREVISIAE
AMSACTA MOOREI
PARAMECIUM BURSARIA
AUTOGRAPHA CALIFORNICA
BOMBYX MORI NUCLEAR
ORGYIA PSEUDOTSUGATA
HERPES SIMPLEX VIRUS (TYPE
HERPES SIMPLEX VIRUS (TYPE
HERPES SIMPLEX VIRUS (TYPE
ORGYIA PSEUDOTSUGATA
HERPES SIMPLEX VIRUS (TYPE
HERPES SIMPLEX VIRUS (TYPE
HERPES SIMPLEX VIRUS (TYPE
HERPES SIMPLEX VIRUS (TYPE
HERPES SIMPLEX VIRUS (TYPE
HERPES SIMPLEX VIRUS (TYPE
HERPES SIMPLEX VIRUS (TYPE
HERPES SIMPLEX VIRUS (TYPE
HERPES SIMPLEX VIRUS (TYPE
HERPES SIMPLEX VIRUS (TYPE
HERPES SIMPLEX VIRUS (TYPE
HERPES SIMPLEX VIRUS (TYPE
HERPES SIMPLEX VIRUS (TYPE
HERPES SIMPLEX VIRUS (TYPE
HERPES SIMPLEX VIRUS (TYPE
HERPES SIMPLEX VIRUS (TYPE
HERPES SIMPLEX VIRUS (TYPE
HERPES SIMPLEX VIRUS (TYPE
HERPES SIMPLEX VIRUS (TYPE
HERPES SIMPLEX VIRUS (TYPE
HERPES SIMPLEX VIRUS (TYPE
HERPES SIMPLEX VIRUS (TYPE
HERPES SIMPLEX VIRUS (TYPE
AUTOGRAPHA CALIFORNICA
AUTOGRAPHA CALIFORNICA
HERPES SIMPLEX VIRUS (TYPE
HERPES SIMPLEX VIRUS
AUTOGRAPHA CALIFORNICA
AUTOGRAPHA CALIFORNICA
AUTOGRAPHA CALIFORNICA
ORGYIA PSEUDOTSUGATA
AUTOGRAPHA CALIFORNICA
AMSACTA MOOREI
AMSACTA MOOREI
AMSACTA MOOREI
HERPES SIMPLEX VIRUS (TYPE 1
HERPES SIMPLEX VIRUS (TYPE 1
HERPES SIMPLEX VIRUS (TYPE 1
HERPES SIMPLEX VIRUS (TYPE 1
HERPES SIMPLEX VIRUS (TYPE 2
HERPES SIMPLEX VIRUS (TYPE 2
HERPES SIMPLEX VIRUS (TYPE 2
HERPES SIMPLEX VIRUS (TYPE 6
HERPES SIMPLEX VIRUS (TYPE
HERPES SIMPLEX VIRUS (TYPE
HERPES SIMPLEX VIRUS (TYPE 2)
HERPES SIMPLEX VIRUS (TYPE
HERPES SIMPLEX VIRUS
HERPES SIMPLEX VIRUS (TYPE 2)
HERPES SIMPLEX VIRUS (TYPE
HERPES SIMPLEX VIRUS (TYPE
HERPES SIMPLEX VIRUS (TYPE
ORGYIA PSEUDOTSUGATA
AUTOGRAPHA CALIFORNICA
HERPES SIMPLEX VIRUS (TYPE
AUTOGRAPHA CALIFORNICA
ORGYIA PSEUDOTSUGATA
HERPES SIMPLEX VIRUS (TYPE
ORGYIA PSEUDOTSUGATA
AUTOGRAPHA CALIFORNICA
BOMBYX MORI NUCLEAR
HERPES SIMPLEX VIRUS (TYPE 1
AUTOGRAPHA CALIFORNICA
AUTOGRAPHA CALIFORNICA
ORGYIA PSEUDOTSUGATA
AUTOGRAPHA CALIFORNICA
GALLERIA MELLONELLA NUCLEAR
AUTOGRAPHA CALIFORNICA
CHORISTONEURA FUMIFERANA
AUTOGRAPHA CALIFORNICA
AUTOGRAPHA CALIFORNICA
ORGYIA PSEUDOTSUGATA
AUTOGRAPHA CALIFORNICA
ORGYIA PSEUDOTSUGATA
HERPES SIMPLEX VIRUS (TYPE
ORGYIA PSEUDOTSUGATA
AUTOGRAPHA CALIFORNICA
THERMOPROTEUS TENAX VIRUS
THERMOPROTEUS TENAX VIRUS
THERMOPROTEUS TENAX VIRUS
THERMOPROTEUS TENAX VIRUS
THERMOPROTEUS TENAX VIRUS
THERMOPROTEUS TENAX VIRUS
THERMOPROTEUS TENAX VIRUS
THERMOPROTEUS TENAX VIRUS
AUTOGRAPHA CALIFORNICA
AUTOGRAPHA CALIFORNICA
AMSACTA MOOREI
AMSACTA MOOREI
HERPES SIMPLEX VIRUS
HERPES SIMPLEX VIRUS
HERPES SIMPLEX VIRUS
ORGYIA PSEUDOTSUGATA
HERPES SIMPLEX
HERPES SIMPLEX
THERMOPROTEUS TENAX
PARAMECIUM BURSARIA
CHORISTONEURA BIENNIS
PARAMECIUM BURSARIA
AUTOGRAPHA
CALIFORNICA
LYMANTRIA DISPAR
SACCHAROMYCES
66-
HERPES SIMPLEX
HERPES SIMPLEX
AMSACTA MOOREI
PARAMECIUM BURSARIA
AMSACTA MOOREI
HERPES SIMPLEX
AUTOGRAPHA
CALIFORNICA
ORGYIA PSEUDOTSUGATA
AUTOGRAPHA
CALIFORNICA
AUTOGRAPHA
CALIFORNICA
BOMBYX MORI CYTOPLASMIC
AUTOGRAPHA
CALIFORNICA
AGROTIS SEGETUM
BOMBYX MORI
BUZURA SUPPRESSARIA
HYPHANTRIA CUNEA NUCLEAR
LYMANTRIA DISPAR
MAMESTRA BRASSICAE
ORGYIA PSEUDOTSUGATA
ORGYIA PSEUDOTSUGATA
PANOLIS FLAMMEA
SPODOPTERA EXIGUA NUCLEAR
SPODOPTERA FRUGIPERDA
4l-
SACCHAROMYCES CEREVISIAE
AMSACTA MOOREI
PARAMECIUM BURSARIA
AUTOGRAPHA
CALIFORNICA
BOMBYX MORI NUCLEAR
ORGYIA PSEUDOTSUGATA
HERPES SIMPLEX
HERPES SIMPLEX
HERPES SIMPLEX
HERPES SIMPLEX
HERPES SIMPLEX
HERPES SIMPLEX
HERPES SIMPLEX
HERPES SIMPLEX
HERPES SIMPLEX
HERPES SIMPLEX
AUTOGRAPHA
CALIFORNICA
AUTOGRAPHA
CALIFORNICA
HERPES SIMPLEX
AUTOGRAPHA
CALIFORNICA
AUTOGRAPHA
CALIFORNICA
AUTOGRAPHA
CALIFORNICA
AMSACTA MOOREI
AMSACTA MOOREI
AMSACTA MOOREI
HERPES SIMPLEX
HERPES SIMPLEX
HERPES SIMPLEX
HERPES SIMPLEX
ORGYIA PSEUDOTSUGATA
AUTOGRAPHA
CALIFORNICA
AUTOGRAPHA
CALIFORNICA
ORGYIA PSEUDOTSUGATA
SPODOPTERA EXIGUA
AUTOGRAPHA
CALIFORNICA
BOMBYX MORI
AUTOGRAPHA
CALIFORNICA
ORGYIA PSEUDOTSUGATA
AUTOGRAPHA
CALIFORNICA
ORGYIA PSEUDOTSUGATA
AUTOGRAPHA
CALIFORNICA
AUTOGRAPHA
CALIFORNICA
AUTOGRAPHA
CALIFORNICA
AUTOGRAPHA
CALIFORNICA
ORGYIA PSEUDOTSUGATA
AUTOGRAPHA
CALIFORNICA
ORGYIA PSEUDOTSUGATA
HERPES SIMPLEX
THERMOPROTEUS TENAX
THERMOPROTEUS TENAX
AUTOGRAPHA
CALIFORNICA
AUTOGRAPHA
CALIFORNICA
AUTOGRAPHA
CALIFORNICA
THERMOPROTEUS TENAX
THERMOPROTEUS TENAX
THERMOPROTEUS TENAX
THERMOPROTEUS TENAX
AUTOGRAPHA
CALIFORNICA
ORGYIA PSEUDOTSUGATA
AUTOGRAPHA
CALIFORNICA
AMSACTA MOOREI
HERPES SIMPLEX
HERPES SIMPLEX
HERPES SIMPLEX
HERPES SIMPLEX
14!–
RICKETTSIA
RICKETTSII
RICKETTSIA
TYPHI
RICKETTSIA
RICKETTSII
DESULFUROCOCCUS
MOBILIS
VIBRIO
ANGUILLARUM
ESCHERICHIA
COLI
PROTEUS
MIRABILIS
ZYMOMONAS
MOBILIS
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
STAPHYLOCOCCUS
AUREUS
BACILLUS SP
ESCHERICHIA
COLI
ESCHERICHIA
COLI
LACTOCOCCUS
LACTIS
AGROBACTERIUM
TUMEFACIENS
ESCHERICHIA
COLI
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
ALCALIGENES
EUTROPHUS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
STREPTOMYCES
COELICOLOR
LISTERIA
MONOCYTOGENES
NOCARDIA
LACTAMDURANS
BACILUS
SUBTILIS
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
CLOSTRIDIUM
ACETOBUTYLICUM
CLOSTRIDIUM
ACETOBUTYLICUM
CLOSTRIDIUM
ACETOBUTYLICUM
CLOSTRIDIUM
ACETOBUTYLICUM
ESCHERICHIA
COLI
ESCHERICHIA
COLI
MYCOPLASMA
GENITALIUM
MYCOPLASMA
PNEUMONIAE
RICKETTSIA
PROWAZEKII
AEROMONAS
HYDROPHILA
STREPTOCOCCUS
MUTANS
PSEUDOMONAS
ATLANTICA
STAPHYLOCOCCUS
AUREUS
YERSINIA
ENTEROCOLITICA
ESCHERICHI
COLI
ESCHERICHIA
COLI
BACILLUS
SUBTILIS
CORYNEBACTERIUM
GLUTMICUM
ESCHERICHIA
COLI
PSEUDOMONAS
AERUGINOSA
PSEUDOMONAS
AERUGINOSA
PSEUDOMONAS
AERUGINOSA
PSEUDOMONAS
OLEOVORANS
PSEUDOMONAS
OLEOVORANS
ESCHERICHIA
COLI
BACILLUS
STEAROTHERMORPHILUS
BACILLUS
SUBTILIS
BACILLUS SP
BACILLUS
SUBTILIS
STAPHYLOCOCCUS
AUREUS
STREPTOCOCCUS
PNEUMONIAE
PSEUDOMONAS
CHLORORAPHIS
TREPTOCOCCUS
PNEUMONIAE
ESCHERICHIA
COLI
SERRATIA
MARCESCENS
RICKETTSIA
PROWAZEKII
ESCHERICHIA
COLI
ESCHERICHIA
COLI
THERMUS
AQUATICUS
DICTYOGLOMUS
THERMOPHILUM
DICTYOGLOMUS
THERMOPHILUM
SALMONELLA
TYPHIMURIUM
DICTYOGLOMUS
THERMOPHILUM
BACILLUS
CIRCULANS
BACILLUS
POLYMYXA
CLOSTRIDIUM
THERMOSULFUROGENES
CLOSTRIDIUM SP
BACILLUS
STEAROTHERMOPHILUS
BACILLUS SP
AEROMONAS
HYDROPHILA
ALTEROMONAS
HALOPLANKTIS
BACILLUS
AMYLOLIQUEFACIENS
BACILLUS
CIRCULANS
BACILLUS
MEGATERIUM
BACILLUS
SUBTILIS
BUTYRIVIBRIO
FIBRISOLVENS
CLOSTRIDIUM
ACETOBUTYLICUM
CLOSTRIDIUM
THERMOSULFUROGENES
STREPTOMYCES
LIMOSUS
AZOTOBACTER
VINELANDII
AZOTOBACTER
VINELANDII
AZOTOBACTER
VINELANDII
VIBRIO
ANGUILLARUM
FREMYELLA
DIPLOSIPHON
SYNECHOCOCCUS SP
SYNECHOCYSTIS SP
SALMONELLA
TYPHIMURIUM
ACHROMOBACTER
LYTICUS
ESCHERICHIA
COLI
PSEUDOMONAS
AERUGINOSA
PSEUDOMONAS
AERUGINOSA
ESCHERICHIA
COLI
THERMOANAEROBACTER
ETHANOLICUS
MYCOPLASMA
ARGININI
ESCHERICHIA
COLI
PSEUDOMONAS
AERUGINOSA
ESCHERICHIA
COLI
ESCHERICHIA
COLI
STAPHYLOCOCCUS
AUREUS
ESCHERICHIA
COLI
SALMONELLA
TYPHI
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
STREPTOCOCCUS
PYOGENES
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
STAPHYLOCOCCUS
AUREUS
STAPHYLOCOCCUS
XYLOSUS
STAPHYLOCOCCUS
AUREUS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ENTEROCOCCUS
FAECALIS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
SERRATIA
MARCESCENS
BACILLUS
LICHENIFORMIS
ERWINIA
CHRYSANTHEMI
ACINETOBACTER
GLUTAMINASIFIANS
ESCHERICHIA
COLI
METHANOSARCINA
BARKERI
STAPHYLOCOCCUS
AUREUS
ENTEROCOCCUS
FAECALIS
ENTEROCOCCUS
FAECALIS
SALMONELLA
TYPHIMURIUM
SYNECHOCOCCUS SP
VIBRIO
ALGINOLYTICUS
ANABAENA SP
BACILLUS
MEGATERIUM
ESCHERICHIA
COLI
ENTEROCOCCUS
FAECALIS
MYCOPLASMA
GALLISEPTICUM
PROPIONIGENIUM
MODESTUM
RHODOSPIRILLUM
RUBRUM
SULFOLOBUS
ACIDOCALDARIUS
SYNECHOCOCCUS SP
SYNECHOCOCCUS SP
SYNECHOCYSTIS SP
THERMOPHILIC
BACTERIUMPS-3
VIBRIO
ALGINOLYTICUS
ANABAENA SP
BACILLUS
FIRMUS
MYCOPLASMA
GALLISEPTICUM
RHODOSPIRILLUM
RUBRUM
SULFOLOBUS
ACIDOCALDARIUS
SYNECHOCOCCUS SP
SYNECHOCOCCUS SP
SYNECHOCYSTIS SP
ANABAENA SP
BACILLUS
FIRMUS
BACILLUS
MEGATERIUM
ENTEROCOCCUS
FAECALIS
PROPIONIGENIUM
MODESTUM
RHODOPSEUDOMONAS
BLASTICA
RHODOSPIRILLUM
RUBRUM
SYNECHOCOCCUS SP
SYNECHOCYSTIS SP
VIBRIO
ALGINOLYTICUS
BACILLUS
FIRMUS
MYCOPLASMA
GALLISEPTICUM
PROPIONIGENIUM
MODESTUM
SYNECHOCOCCUS SP
ANABAENA SP
BACILLUS
FIRMUS
BACILLUS
MEGATERIUM
MYCOPLASMA
GALLISEPTICUM
SYNECHOCOCCUS SP
SYNECHOCOCCUS SP
THERMOPHILIC
BACTERIUM PS-3
ANABAENA SP
ESCHERICHIA
COLI
MYCOPLASMA
GALLISEPTICUM
RHODOSPIRILLUM
RUBRUM
SYNECHOCOCCUS SP
SYNECHOCYSTIS SP
MYCOPLASMA
GALLISEPTICUM
ANABAENA SP
BACILLUS
FIRMUS
RHODOSPIRILLUM
RUBRUM
SYNECHOCOCCUS SP
SYNECHOCOCCUS SP
SYNECHOCYSTIS SP
BACILLUS
MEGATERIUM
SYNECHOCOCCUS SP
PSEUDOMONAS
SYRINGAE
EUBACTERIUM SP
EUBACTERIUM SP
HALOBACTERIUM
HALOBIUM
HALOBACTERIUM SP
ESCHERICHIA
COLI
STREPTOCOCCUS
AGALACTIAE
VITREOSCILLA SP
EUBACTERIUM SP
ESCHERICHIA
COLI
ESCHERICHIA
COLI
HALOBACTERIUM
HALOBIUM
ESCHERICHIA
COLI
ESCHERICHIA
COLI
RHODOBACTER
CAPSULATUS
RHODOBACTER
CAPSULATUS
CLOSTRIDIUM
PERFRINGENS
PROSTHECOCHLORIS
AESTUARII
ACETOBACTER
XYLINUM
ACETOBACTER
XYLINUM
ACINETOBACTER
CALCOACETICUS
ESCHERICHIA
COLI
HAEMOPHILUS
INFLUENZAE
HAEMOPHILUS
INFLUENZAE
HAEMOPHILUS
INFLUENZAE
NITROBACTER
WINOGRADSKYI
ESCHERICHIA
COLI
BACILLUS
STEAROTHERMOPHILUS
CLOSTRIDIUM
ACETOBUTYLICUM
CLOSTRIDIUM
THERMOSULFUROGENES
KLEBSIELLA
PNEUMONIAE
LACTOBACILLUS
DELBRUECKII
STREPTOCOCCUS
THERMOPHILUS
SULFOLOBUS
SOLFATARICUS
LEUCONOSTOC
LACTIS
SULFOLOBUS
SOLFATARICUS
CLOSTRIDIUM
THERMOCELLUM
CLOSTRIDIUM
THERMOCELLUM
ESCHERICHIA
COLI
AGROBACTERIUM SP
BUTYRIVIBRIO
FIBRISOLVENS
STAPHYLOCOCCUS
AUREUS
STAPHYLOCOCCUS
AUREUS
STAPHYLOCOCCUS
AUREUS
BACILLUS
SPHAERICUS
BACILLUS
SPHAERICUS
ESCHERICHIA
COLI
BACILLUS
SPHAERICUS
BACILLUS
CEREUS
HAEMOPHILUS
INFLUENZAE
BACILLUS
CEREUS
BACILLUS SP
BACILLUS
CEREUS
PSEUDOMONAS
AERUGINOSA
BACILLUS
CEREUS
BACTEROIDES
FRAGILIS
BACILLUS
CEREUS
BACILLUS
LICHENIFORMIS
PROTEUS
MIRABILIS
PROTEUS
VULGARIS
STREPTOMYCES
ALBUS G
KLEBSIELLA
PNEUMONIAE
STAPHYLOCOCCUS
AUREUS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
BACILLUS
LICHENIFORMIS
STAPHYLOCOCCUS
AUREUS
TREPONEMA
PALLIDUM
BACILLUS
SUBTILIS
PSEUDOMONAS
PUTIDA
PSEUDOMONAS
PUTIDA
PSEUDOMONAS
PUTIDA
DESULFUROLOBUS
AMBIVALENS
PSEUDOMONAS
AERUGINOSA
PSEUDOMONAS
AERUGINOSA
PSEUDOMONAS
AERUGINOSA
ESCHERICHIA
COLI
ESCHERICHIA
COLI
BORDETELLA
PERTUSSIS
BORDETELLA
BRONCHISEPTICA
BORDETELLA
PERTUSSIS
BORDETELLA
PERTUSSIS
BORDETELLA
BRONCHISEPTICA
CLOSTRIDIUM
BOTULINUM
CLOSTRIDIUM
BOTULINUM
CLOSTRIDIUM
BOTULINUM
CLOSTRIDIUM
BOTULINUM
CLOSTRIDIUM
BOTULINUM
CLOSTRIDIUM
BUTYRICUM
CLOSTRIDIUM
BOTULINUM
MICROCYSTIS
AERUGINOSA
BACILLUS
FIRMUS
STAPHYLOCOCCUS
AUREUS
ESCHERICHIA
COLI
YERSINIA
530–
PESTIS
BACILLUS
ANTHRACIS
BACILLUS
ANTHRACIS
ANACYSTIS
NIDULANS
ANABAENA SP
CORYNEBACTERIUM
GLUTMICUM
ESCHERICHIA
COLI
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
STAPHYLOCOCCUS
AUREUS
STAPHYLOCOCCUS
AUREUS
ACINETOBACTER
CALCOACETICUS
BACILLUS
STEAROTHERMOPHILUS
ESCHERICHIA
COLI
MICROCOCCUS
LUTEUS
SALMONELLA
TYPHIMURIUM
ESCHERICHIA
COLI
CAMPYLOBACTER
COLI
CLOSTRIDIUM
BUTYRICUM
ESCHERICHIA
COLI
PROTEUS
MIRABILIS
STAPHYLOCOCCUS
INTERMEDIUS
STREPTOCOCCUS
AGALACTIAE
COXIELLA
BURNETII
THERMOACTINOMYCES
VULGARIS
ESCHERICHIA
COLI
SYNECHOCOCCUS SP
SYNECHOCOCCUS SP
THERMOANAEROBACTER
ETHANOLICUS
BACILLUS
MACERANS
BACILLUS
MACERANS
BACILLUS
CIRCULANS
BACILLUS
LICHENIFORMIS
BACILLUS
OHBENSIS
BACILLUS SP
BACILLUS SP
BACILLUS SP
BACILLUS SP
BACILLUS SP
BACILLUS
STEAROTHERMOPHILUS
KLEBSIELLA
PNEUMONIAE
ESCHERICHIA
COLI
SHIGELLA
SONNEI
ESCHERICHIA
3)4–
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
CITROBACTER
FREUNDII
STREPTOMYCES
CLAVULIGERUS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ACETOBACTER
XYLINUM
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ACYRTHOSIPHON
PISUM
SYMBIOTIC
BACTERIU
BACILLUS
SUBTILIS
CHLAMYDIA
TRACHOMATIS
ESCHERICHIA
COLI
HAEMOPHILUS
DUCREYI
LEGIONELLA
MICDADEI
RICKETTSIA
TSUTSUGAMUSHI
THERMOPHILIC
BACTERIUM PS-3
ACYRTHOSIPHON
PISUM
SYMBIOTIC
BACTERIU
AGROBACTERIUM
TUMEFACIENS
AMOEBA
PROTEUS
SYMBIOTIC
BACTERIUM
BACILLUS
SUBTILIS
BORRELIA
BURGDORFERI
BRUCELLA
ABORTUS
CHLAMYDIA
PNEUMONIAE
CHLAMYDIA
TRACHOMATIS
CHROMATIUM
VINOSUM
CLOSTRIDIUM
ACETOBUTYLICUM
CLOSTRIDIUM
PERFRINGENS
COXIELLA
BURNETII
HAEMOPHILUS
DUCREYI
LEGIONELLA
MICDADEI
LEGIONELLA
PNEUMOPHILA
MYCOBACTERIUM
LEPRAE
MYCOBACTERIUM
TUBERCULOSIS &
BOVIS
PSEUDOMONAS
AERUGINOSA
RHIZOBIUM
LEGUMINOSARUM
RICKETTSIA
TSUTSUGAMUSHI
SYNECHOCOCCUS SP
SYNECHOCYSTIS SP
THERMORHILIC
BACTERIUM PS-3
STREPTOMYCES
ALBUS G
VIBRIO
HARVEYI
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
BACILLUS
CIRCULANS
ALTEROMONAS SP
SERRATIA
MARCESCENS
BACILLUS
CIRCULANS
SACCHAROPOLYSPORA
ERYTHRAEA
STREPTOMYCES
PLICATUS
BACILLUS
SUBTILIS
BREVIBACTERIUM
STEROLICUM
VIBRIO
CHOLERAE
AGROBACTERIUM
TUMEFACIENS
AGROBACTERIUM
TUMEFACIENS
CITROBACTER
FREUNDII
ESCHERICHIA
COLI
BACILLUS
SUBTILIS
ACINETOBACTER
ANITRATUM
BACILLUS
COAGULANS
SALMONELLA
TYPHIMURIUM
KLEBSIELLA
PNEUMONIAE
SALMONELLA
DUBLIN
SALMONELLA
PULLORUM
PSEUDOMONAS
PUTIDA
ESCHERICHIA
COLI
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
CLOSTRIDIUM
HISTOLYTICUM
ESCHERICHIA
COLI
RHODOPSEUDOMONAS
BLASTICA
BACTEROIDES
NODOSUS
ESCHERICHIA
COLI
AZOTOBACTER
VINELANDII
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
STAPHYLOCOCCUS
AUREUS
PSEUDOMONAS
SYRINGAE
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
BACILLUS
SUBTILIS
BRADYRHIZOBIUM
JAPONICUM
PARACOCCUS
DENITRIFICANS
RHODOBACTER
SPHAEROIDES
BACILLUS
FIRMUS
BACILLUS
SUBTILIS
NEISSERIA
GONORRHOEAE
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
ESCHERICHIA
COLI
STREPTOMYCES SP
SACCHAROPOLYSPORA
ERYTHRAEA
BACILLUS
SUBTILIS
ANABAENA SP
BACILLUS
THURINGIENSIS
BACILLUS
THURINGIENSIS
BACILLUS
SPHAERICUS
BACILLUS
SPHAERICUS
BACILLUS
SPHAERICUS
BACILLUS
THURINGIENSIS
BACILLUS
THURINGIENSIS
BACILLUS
THURINGIENSIS
BACILLUS
THURINGIENSIS
BACILLUS
THURINGIENSIS
BACILLUS
THURINGIENSIS
BACILLUS
THURINGIENSIS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
SHIGELLA
FLEXNERI
SALMONELLA
TYPHIMURIUM
KLEBSIELLA
AEROGENES
ERWINIA
HERBICOLA
RHODOBACTER
CAPSULATUS
RHODOBACTER
CAPSULATUS
BACILLUS
THURINGIENSIS
BACILLUS
THURINGIENSIS
BACILLUS
THURINGIENSIS
BACILLUS
THURINGIENSIS
BACILLUS
THURINGIENSIS
BACILLUS
THURINGIENSIS
BACILLUS
THURINGIENSIS
BACILLUS
THURINGIENSIS
BACILLUS
THURINGIENSIS
BACILLUS
THURINGIENSIS
BACILLUS
THURINGIENSIS
BACILLUS
THURINGIENSIS
BACILLUS
THURINGIENSIS
BACILLUS
THURINGIENSIS
BACILLUS
THURINGIENSIS
BACILLUS
THURINGIENSIS
BACILLUS
THURINGIENSIS
BACILLUS
THURINGIENSIS
BACILLUS
THURINGIENSIS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
HALOBACTERIUM
HALOBIUM
HALOBACTERIUM
VOLCANII
METHANOTHERMUS
FERVIDUS
METHANOTHERMUS
SOCIABILIS
ESCHERICHIA
COLI
CLOSTRIDIUM
ACETOBUTYLICUM
CLOSTRIDIUM
ACETOBUTYLICUM
NEISSERIA
MENINGITIDIS
PSEUDOMONAS
AERUGINOSA
ESCHERICHIA
COLI
ESCHERICHIA
COLI
BACILLUS
BREVIS
BACILLUS
BREVIS
BORDETELLA
PERTUSSIS
YERSINIA
INTERMEDIA
BORDETELLA
PERTUSSIS
BORDETELLA
PERTUSSIS
BORDETELLA
PERTUSSIS
RHODOBACTER
CAPSULATUS
ESCHERICHIA
COLI
NOSTOC SP
ACINETOBACTER SP
SYNECHOCOCCUS SP
ESCHERICHIA
COLI
ESCHERICHIA
COLI
SYNECHOCOCCUS SP
ESCHERICHIA
COLI
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
ESCHERICHIA
COLI
ESCHERICHIA
COLI
SYNECHOCOCCUS SP
ALCALIGENES
EUTROPHUS
ALCALIGENES
EUTROPHUS
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
ALTEROMONAS
HALOPLANKTIS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
CLOSTRIDIUM
PASTEURIANUM
ESCHERICHIA
COLI
CORYNEBACTERIUM
GLUTMICUM
PSEUDOMONAS
AERUGINOSA
ESCHERICHIA
COLI
ENTEROBACTER
AEROGENES
KLEBSIELLA
PLANTICOLA
MORGANELA
MORGANII
BACILLUS
SUBTILIS
HAFNIA
ALVEI
KLEBSIELLA
PNEUMONIAE
SALMONELLA
TYPHIMURIUM
SALMONELLA
TYPHIMURIUM
RHIZOBIUM
LEGUMINOSARUM
RHIZOBIUM
MELILOTI
ESCHERICHIA
COLI
KLEBSIELLA
PNEUMONIAE
ESCHERICHIA
COLI
BACILLUS
SUBTILIS
MORAXELLA SP
ESCHERICHIA
COLI
PSEUDOMONAS
OLEOVORANS
BACILLUS
SUBTILIS
CORYNEBACTERIUM
GLUTMICUM
ESCHERICHIA
COLI
VIBRIO
CHOLERAE
BACILLUS
SPHAERICUS
BACILLUS
STEAROTHERMOPHILUS
CLOSTRIDIUM
DIFFICILE
PEPTOSTREPTOCOCCUS
ASACCHAROLYTICUS
SULFOLOBUS
SOLFATARICUS
CORYNEBACTERIUM
GLUTMICUM
ACINETOBACTER
CALCOACETICUS
BACILLUS
MEGATERIUM
ESCHERICHIA
COLI
STREPTOMYCES
VIOLACEORUBER
BACILLUS
STEAROTHERMOPHILUS
AGROBACTERIUM
TUMEFACIENS
METHYLOBACTERIUM
EXTORQUENS
METHYLOBACTERIUM
ORGANOPHILUM
PARACOCCUS
DENITRIFICANS
BACILLUS SP
ESCHERICHIA
COLI
BACILLUS
SUBTILIS
CORYNEBACTERIUM
GLUTMICUM
BACILLUS
SPHAERICUS
ESCHERICHIA
COLI
ANABAENA
CYLINDRICA
SYNECHOCOCCUS SP
METHYLOTROPHUS
METHYLOPHILUS
ESCHERICHIA
COLI
BACILLUS
SUBTILIS
PSEUDOMONAS
PUTIDA
AZOTOBACTER
VINELANDII
BACILLUS
STEAROTHERMOPHILUS
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
PSEUDOMONAS
FLUORESCENS
PSEUDOMONAS
PUTIDA
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
BORRELIA
BURGDORFERI
BUCHNERA
APHIDICOLA
ESCHERICHIA
COLI
MICROCOCCUS
LUTEUS
MYCOPLASMA
CAPRICOLUM
PROTEUS
MIRABILIS
PSEUDOMONAS
PUTIDA
SPIROPLASMA
CITRI
CHLAMYDIA
TRACHOMATIS
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
ESCHERICHIA
COLI
BACILLUS
MEGATERIUM
BORRELIA
BURGDORFERI
BRUCELLA
OVIS
CAULOBACTER
CRESCENTUS
CLOSTRIDIUM
ACETOBUTYLICUM
CLOSTRIDIUM
PERFRINGENS
METHANOSARCINA
MAZEI
MYCOBACTERIUM
TUBERCULOSIS
STREPTOMYCES
COELICOLOR
ESCHERICHIA
COLI
ZYMOMONAS
MOBILIS
STREPTOMYCES
PEUCETIUS
SULFOLOBUS
SOLFATARICUS
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
SACCHAROPOLYSPORA
ERYTHRAEA
SALMONELLA
TYPHIMURIUM
BACILLUS
SUBTILIS
BORRELIA
BURGDORFERI
BUCHNERA
APHIDICOLA
MICROCOCCUS
LUTEUS
MYCOPLASMA
CAPRICOLUM
PSEUDOMONAS
PUTIDA
SPIROPLASMA
CITRI
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
STREPTOCOCCUS
PNEUMONIAE
BACILLUS
CALDOTENAX
STREPTOCOCCUS
PNEUMONIAE
THERMUS
AQUATICUS
THERMUS
AQUATICUS
ESCHERICHIA
COLI
PYROCOCCUS
FURIOSUS
SULFOLOBUS
SOLFATARICUS
THERMOCOCCUS
LITORALIS
LACTOCOCCUS
LACTIS
LACTOCOCCUS
LACTIS
ESCHERICHIA
COLI
STREPTOCOCCUS
EQUISIMILIS
STREPTOMYCES
PEUCETIUS
STAPHYLOCOCCUS
AUREUS
BACILLUS
CIRCULANS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
STAPHYLOCOCCUS
AUREUS
RHODOBACTER
CAPSULATUS
ESCHERICHIA
COLI
ZYMOMONAS
MOBILIS
STAPHYLOCOCCUS
AUREUS
DESULFUROCOCCUS
MOBILIS
HALOBACTERIUM
HALOBIUM
METHANOCOCCUS
VANNIELII
SULFOLOBUS
ACIDOCALDARIUS
THERMOPLASMA
ACIDOPHILUM
ANACYSTIS
NIDULANS
ESCHERICHIA
COLI
MYCOBACTERIUM
LEPRAE
SALMONELLA
TYPHIMURIUM
SPIRULINA
PLATENSIS
SYNECHOCYSTIS SP
STREPTOMYCES
RAMOCISSIMUS
STREPTOMYCES
RAMOCISSIMUS
STREPTOMYCES
RAMOCISSIMUS
ESCHERICHIA
COLI
SPIROPLASMA
CITRI
BACTEROIDES
FRAGILIS
BACILLUS
SUBTILIS
BURKHOLDERIA
CEPACIA
CHLAMYDIA
TRACHOMATIS
DEINONEMA SP
FLEXISTIPES
SINUSARABICI
HALOARCULA
MARISMORTUI
MICROCOCCUS
LUTEUS
MYCOPLASMA
HOMINIS
MYCOBACTERIUM
LEPRAE
MYCOBACTERIUM
TUBERCULOSIS
SHEWANELLA
PUTREFACIENS
STREPTOCOCCUS
ORALIS
PSEUDOMONAS
AERUGINOSA
ESCHERICHIA
COLI
ESCHERICHIA
COLI
CLOSTRIDIUM
PERFRINGENS
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
STAPHYLOCOCCUS
EPIDERMIDIS
STAPHYLOCOCCUS
EPIDERMIDIS
STAPHYLOCOCCUS
EPIDERMIDIS
STAPHYLOCOCCUS
EPIDERMIDIS
STAPHYLOCOCCUS
EPIDERMIDIS
ESCHERICHIA
COLI
SACCHAROPOLYSPORA
ERYTHRAEA
ESCHERICHIA
COLI
SACCHAROPOLYSPORA
ERYTHRAEA
STREPTOMYCES
SCABIES
PSEUDOMONAS
FLUORESCENS
STAPHYLOCOCCUS
AUREUS
STAPHYLOCOCCUS
AUREUS
STAPHYLOCOCCUS
AUREUS
STAPHYLOCOCCUS
AUREUS
CLOSTRIDIUM
PERFRINGENS
STAPHYLOCOCCUS
AUREUS
STAPHYLOCOCCUS
AUREUS
STAPHYLOCOCCUS
AUREUS
SALMONELLA
TYPHIMURIUM
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
RHIZOBIUM
MELILOTI
STREPTOCOCCUS
PNEUMONIAE
RHIZOBIUM
MELILOTI
RHIZOBIUM
MELILOTI
RHIZOBIUM
MELILOTI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
PSEUDOMONAS
FRAGI
WOLINELLA
SUCCINOGENES
ESCHERICHIA
COLI
PSEUDOMONAS SP
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
STAPHYLOCOCCUS
AUREUS
SYNECHOCOCCUS SP
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ANABAENA SP
ANABAENA SP
BORDETELLA
PERTUSSIS
BORDETELLA
PERTUSSIS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
SPIROPLASMA
CITRI
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
BORDETELLA
PERTUSSIS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
ESCHERICHIA
COLI
RICKETTSIA
RICKETTSII
AZORHIZOBIUM
CAULINODANS
AZORHIZOBIUM
CAULINODANS
BRADYRHIZOBIUM
JAPONICUM
BORRELIA
BURGDORFERI
HALOBACTERIUM
HALOBIUM
METHANOCOCCUS
VOLTAE
METHANOCOCCUS
VOLTAE
HALOBACTERIUM
HALOBIUM
METHANOCOCCUS
VOLTAE
HALOBACTERIUM
HALOBIUM
HALOBACTERIUM
HALOBIUM
BACILLUS
SUBTILIS
CAMPYLOBACTER
COLI
CAMPYLOBACTER
JEJUNI
METHANOCOCCUS
VOLTAE
PSEUDOMONAS
AERUGINOSA
RHIZOBIUM
MELILOTI
SPIROCHAETA
AURANTIA
TREPONEMA
HYODYSENTERIAE
TREPONEMA
PALLIDUM
CAMPYLOBACTER
COLI
CAMPYLOBACTER
JEJUNI
RHIZOBIUM
MELILOTI
CAULOBACTER
CRESCENT
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
SALMONELLA
TYPHIMURIUM
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
ESCHERICHIA
COLI
PSEUDOMONAS
AERUGINOSA
COLI
SALMONELLA
CHOLERAE-SUIS
SALMONELLA
MUENCHEN
SALMONELLA
PARATYPHI-A
SALMONELLA
RUBISLAW
SALMONELLA
TYPHIMURIUM
SERRATIA
MARCESCENS
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
CAULOBACTER
CRESCENTUS
SALMONELLA
TYPHIMURIUM
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
SALMONELLA
TYPHIMURIUM
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
CAULOBACTER
CRESCENTUS
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
PSEUDOMONAS
AERUGINOSA
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ACTINOMYCES
VISCOSUS
ESCHERICHIA
COLI
BACTEROIDES
NODOSUS
BACTEROIDES
NODOSUS
BACTEROIDES
NODOSUS
BACTEROIDES
NODOSUS
BACTEROIDES
NODOSUS
BACTEROIDES
NODOSUS
BACTEROIDES
NODOSUS
BACTEROIDES
NODOSUS
BACTEROIDES
NODOSUS
PSEUDOMONAS
AERUGINOSA
BACTEROIDES
NODOSUS
BACTEROIDES
NODOSUS
ESCHERICHIA
COLI
NEISSERIA
MENINGITIDIS
NEISSERIA
GONORRHOEAE
MORAXELLA
NONLIQUEFACIENS
PSEUDOMONAS
AERUGINOSA
PSEUDOMONAS
AERUGINOSA
ESCHERICHIA
COLI
ESCHERICHIA
COLI
HAEMOPHILUS
INFLUENZAE
STAPHYLOCOCCUS
AUREUS
ESCHERICHIA
COLI
LACTOBACILLUS
CASEI
BACILLUS
FIRMUS
COLI
WOLINELLA
SUCCINOGENES
MYXOCOCCUS
XANTHUS
CLOSTRIDIUM
THERMOACETUM
METHANOBACTERIUM
THERMOAUTOTROPHICU
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
BACILLUS
STEAROTHERMOPHILUS
BACILLUS
SUBTILIS
YERSINIA
PESTIS
ESCHERICHIA
COLI
ANABAENA
VARIABILIS
ANABAENA
VARIABILIS
ESCHERICHIA
COLI
BACILLUS
MEGATERIUM
BACILLUS
SUBTILIS
PYROCOCCUS
WOESEI
THERMOTOGA
MARITIMA
BACILLUS
STEAROTHERMOPHILUS
ESCHERICHIA
COLI
ZYMOMONAS
MOBILIS
PSEUDOMONAS
FLUORESCENS
SALMONELLA
TYPHIMURIUM
HAEMOPHILUS
INFLUENZAE
LACTOBACILLUS
HELVETICUS
SALMONELLA
TYPHIMURIUM
HAEMOPHILUS
INFLUENZAE
PSEUDOMONAS
FLUORESCENS
ESCHERICHIA
COLI
PHOTOBACTERIUM
LEIOGNATHI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
STAPHYLOCOCCUS
HAEMOLYTICUS
STAPHYLOCOCCUS
HAEMOLYTICUS
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
PSEUDOMONAS
PUTIDA
BACILLUS
SUBTILIS
PSEUDOMONAS
PUTIDA
SYNECHOCYSTIS SP
BACILLUS
STEAROTHERMOPHILUS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
ESCHERICHIA
COLI
METHANOCOCCUS
THERMOLITHOTROPHICUS
ANABAENA SP
BACILLUS
SUBTILIS
CLOSTRIDIUM
ACETOBUTYLICUM
ESCHERICHIA
COLI
METHANOCOCCUS
VOLTAE
PROTEUS
VULGARIS
PYROCOCCUS
FURIOSUS
SALMONELLA
TYPHIMURIUM
STREPTOMYCES
COELICOLOR
AZOSPIRILLUM
URASILENSE
RHODOBACTER
CAPSULATUS
SYNECHOCOCCUS SP
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
ESCHERICHIA
COLI
ESCHERICHIA
COLI
BACILLUS
STEAROTHERMOPHILUS
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
SHIGELLA
FLEXNERI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
BRADYRHIZOBIUM
JAPONICUM
CAMPYLOBACTER
JEJUNI
HYPHOMICROBIUM
METHYLOVORUM
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
CHLAMYDIA
TRACHOMATIS
CHLAMYDIA
TRACHOMATIS
CHLAMYDIA
TRACHOMATIS
CHLAMYDIA
TRACHOMATIS
CHLAMYDIA
TRACHOMATIS
CHLAMYDIA
TRACHOMATIS
RICKETTSIA
PROWAZEKII
BACILLUS
SUBTILIS
BORRELIA
BURGDORFERI
CLOSTRIDIUM
ACETOBUTYLICUM
BACILLUS
BREVIS
BACILLUS
BREVIS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
PSEUDOMONAS
AERUGINOSA
BACILLUS
SUBTILIS
ERWINIA
CAROTOVORA
ERWINIA
CHRYSANTHEMI
KLEBSIELLA
PNEUMONIAE
ERWINIA
CAROTOVORA
ERWINIA
CHRYSANTHEMI
KLEBSIELLA
PNEUMONIAE
PSEUDOMONAS
AERUGINOSA
XANTHOMONAS
CAMPESTRIS
PSEUDOMONAS
AERUGINOSA
AEROMONAS
HYDROPHILA
ERWINIA
CAROTOVORA
KLEBSIELLA
PNEUMONIAE
ERWINIA
CAROTOVORA
ERWINIA
CHRYSANTHEMI
KLEBSIELLA
PNEUMONIAE
PSEUDOMONAS
AERUGINOSA
ERWINIA
CHRYSANTHEMI
XANTHOMONAS
CAMPESTRIS
ERWINIA
CAROTOVORA
ERWINIA
CHRYSANTHEMI
STREPTOCOCCUS
DOWNEI
STREPTOCOCCUS
DOWNEI
STREPTOCOCCUS
MUTANS
STREPTOCOCCUS
MUTANS
STREPTOCOCCUS
MUTANS
STREPTOCOCCUS
DOWNEI
METHANOBACTERIUM
THERMOFORMICICUM
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
BACILLUS
CIRCULANS
BACILLUS
LICHENIFORMIS
BACILLUS
MACERANS
BACILLUS SP
BACILLUS
SUBTILIS
BUTYRIVIBRIO
FIBRISOLVENS
BACILLUS
SUBTILIS
THERMOMONOSPORA
FUSCA
BACILLUS SP
BACILLUS
SUBTILIS
FIBROBACTER
SUCCINOGENES
THERMOMONOSPORA
FUSCA
THERMOMONOSPORA
FUSCA
BACILLUS
LAUTUS
CLOSTRIDIUM
THERMOCELLUM
PSEUDOMONAS
FLUORESCENS
RUMINOCOCCUS
ALDUS
RUMINOCOCCUS
FLAVEFACIENS
BACILLUS
LAUTUS
CALDOCELLUM
SACCHAROLYTICUM
CELLULOMONAS
FIMI
CLOSTRIDIUM
CELLULOVORANS
CLOSTRIDIUM
THERMOCELLUM
CELLULOMONAS
FIMI
PSEUDOMONAS
FLUORESCENS
CLOSTRIDIUM
CELLULOLYTICUM
CLOSTRIDIUM
CELLULOLYTICUM
CLOSTRIDIUM
THERMOCELLUM
CLOSTRIDIUM
THERMOCELLUM
ERWINIA
CAROTOVORA
CLOSTRIDIUM
THERMOCELLUM
CLOSTRIDIUM
STERCORARIUM
BACILLUS
POLYMYXA
BACILLUS SP
BACILLUS SP
COLI
HALOBACTERIUM
HALOBIUM
HALOBACTERIUM
HALOBIUM
APHANIZOMENON
FLOS-AQUAE
FREMYELLA
DIPLOSIPHON
HALOBACTERIUM
MEDITERRANEI
MICROCYSTIS SP
PSEUDOANABAENA SP
APHANIZOMENON
FLOS-AQUAE
HALOBACTERIUM
HALOBIUM
HALOBACTERIUM
MEDITERRANEI
HALOBACTERIUM
HALOBIUM
HALOBACTERIUM
MEDITERRANEI
HALOBACTERIUM
HALOBIUM
HALOBACTERIUM
MEDITERRANEI
HALOBACTERIUM
SALINARIUM
HALOBACTERIUM
HALOBIUM
HALOBACTERIUM
MEDITERRANEI
HALOBACTERIUM
HALOBIUM
HALOBACTERIUM
MEDITERRANEI
HALOBACTERIUM
HALOBIUM
HALOBACTERIUM
MEDITERRANEI
HALOBACTERIUM
SALINARIUM
HALOBACTERIUM
MEDITERRANEI
HALOBACTERIUM
HALOBIUM
HALOBACTERIUM
MEDITERRANEI
HALOBACTERIUM
MEDITERRANEI
BACILLUS
SUBTILIS
CAMPYLOBACTER
JEJUNI
ESCHERICHIA
COLI
KLEBSIELLA
PNEUMONIAE
MYCOPLASMA
PNEUMONIAE
STAPHYLOCOCCUS
AUREUS
BACILLUS
SUBTILIS
BORRELIA
BURGDORFERI
ESCHERICHIA
COLI
HALOFERAX SP
MYCOPLASMA
PNEUMONIAE
NEISSERIA
GONORRHOEAE
PSEUDOMONAS
PUTIDA
SPIROPLASMA
CITRI
STAPHYLOCOCCUS
AUREUS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
HAEMOPHILUS
INFLUENZAE
CHLOROBIUM
VIBRIOFORMES
ESCHERICHIA
COLI
RHODOBACTER
SPHAEROIDES
SALMONELLA
TYPHIMURIUM
SYNECHOCYSTIS SP
METHANOTHERMUS
SOCIABILIS
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
YERSINIA
ENTEROCOLITICA
ESCHERICHIA
COLI
BACILLUS
SUBTILIS
SUBTILIS
ANABAENA SP
STREPTOCOCCUS
PNEUMONIAE
STREPTOCOCCUS
PNEUMONIAE
CAULOBACTER
CRESCENTUS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
HAEMOPHILUS
INFLUENZAE
LACTOCOCCUS
LACTIS
ESCHERICHIA
COLI
LACTOCOCCUS
LACTIS
METHANOCOCCUS
VANNIELII
SALMONELLA
TYPHIMURIUM
LACTOCOCCUS
LACTIS
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
ESCHERICHIA
COLI
HALOBACTERIUM
VOLCANII
LACTOCOCCUS
LACTIS
SALMONELLA
TYPHIMURIUM
SALMONELLA
TYPHIMURIUM
ESCHERICHIA
COLI
LACTOCOCCUS
LACTIS
MYCOBACTERIUM
SMEGMATIS
SALMONELLA
TYPHIMURIUM
STAPHYLOCOCCUS
AUREUS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ACTINOBACILLUS
PLEUROPNEUMONIAE
ACTINOBACILLUS
SUIS
ESCHERICHIA
COLI
PROTEUS
MIRABILIS
SERRATIA
MARCESCENS
VIBRIO
CHOLERAE
ACTINOBACILLUS
PLEUROPNEUMONIAE
ESCHERICHIA
COLI
PROTEUS
MIRABILIS
PROTEUS
VULGARIS
SERRATIA
MARCESCENS
VIBRIO
CHOLERAE
ACTINOBACILLUS
PLEUROPNEUMONIAE
ACTINOBACILLUS
PLEUROPNEUMONIAE
ESCHERICHIA
COLI
DESULFOVIBRIO
VULGARIS
METHANOPYRUS
KANDLERI
SERRATIA
MARCESCENS
ESCHERICHIA
COLI
BRADYRHIZOBIUM
JAPONICUM
NOCARDIA
OPACA
ALCALIGENES
EUTROPHUS
BRADYRHIZOBIUM
JAPONICUM
DEINOCOCCUS
RADIODURANS
LACTOCOCCUS
LACTIS
STREPTOMYCES
COELICOLOR
BURKHOLDERIA
SOLANACEARUM
PSEUDOMONAS
SYRINGAE
PSEUDOMONAS
SYRINGAE
CLOSTRIDIUM
ACETOBUTYLICUM
HALOARCULA
MARISMORTUI
MYCOBACTERIUM
LEPRAE
MYCOBACTERIUM
PARATUBERCULOSIS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
HALOBACTERIUM
HALOBIUM
HALOBACTERIUM
SALINARIUM
BACILLUS
SUBTILIS
LACTOBACILLUS
HELVETICUS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
ESCHERICHIA
COLI
PSEUDOMONAS SP
PSEUDOMONAS SP
PSEUDOMONAS
SYRINGAE
ESCHERICHIA
COLI
ERWINIA
ANANAS
ERWINIA
HERBICOLA
PSEUDOMONAS
FLUORESCENS
PSEUDOMONAS
SYRINGAE
XANTHOMONAS
CAMPESTRIS
SHIGELLA
FLEXNERI
BACILLUS
STEAROTHERMOPHILUS
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
ENTEROCOCCUS
FAECIUM
BACILLUS
STEAROTHERMOPHILUS
ESCHERICHIA
COLI
KLEBSIELLA
PNEUMONIAE
MYCOPLASMA
FERMENTANS
PROTEUS
VULGARIS
SALMONELLA
TYPHIMURIUM
SERRATIA
MARCESCENS
NEISSERIA
GONORRHOEAE
STREPTOCOCCUS SP
STREPTOCOCCUS SP
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
LACTOCOCCUS
LACTIS
SALMONELLA
TYPHIMURIUM
ACINETOBACTER
CALCOACETICUS
BACILLUS
SUBTILIS
BACILLUS
THURINGIENSIS
LISTERIA
MONOCYTOGENES
LISTERIA
MONOCYTOGENES
YERSINIA
ENTEROCOLITICA
SHIGELLA
FLEXNERI
SHIGELLA
FLEXNERI
SHIGELLA
DYSENTERIAE
SHIGELLA
FLEXNERI
SHIGELLA
DYSENTERIAE
SHIGELLA
FLEXNERI
SHIGELLA
DYSENTERIAE
SHIGELLA
FLEXNERI
SHIGELLA
DYSENTERIAE
SHIGELLA
FLEXNERI
PSEUDOMONAS
SYRINGAE
ESCHERICHIA
COLI
VIBRIO
CHOLERAE
VIBRIO
CHOLERAE
SYNECHOCOCCUS SP
SHIGELLA
DYSENTERIAE
SHIGELLA
DYSENTERIAE
ESCHERICHIA
COLI
SHIGELLA
FLEXNERI
SHIGELLA
SONNEI
BACILLUS
SUBTILIS
BACILLUS
POLYMYXA
ESCHERICHIA
COLI
SHIGELLA
SONNEI
ESCHERICHIA
COLI
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
BACILLUS
SUBTILIS
LACTOCOCCUS
LACTIS
BACILLUS SP
STAPHYLOCOCCUS
AUREUS
ESCHERICHIA
COLI
ERWINIA
CHRYSANTHEMI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
BACILLUS
SUBTILIS
FREMYELLA
DIPLOSIPHON
BACILLUS
CIRCULANS
ESCHERICHIA
COLI
SPIROPLASMA
CITRI
BACILLUS
STEAROTHERMOPHILUS
STAPHYLOCOCCUS
AUREUS
STREPTOCOCCUS
MUTANS
STREPTOCOCCUS
MUTANS
LACTOBACILLUS
CASEI
ESCHERICHIA
COLI
KLEBSIELLA
PNEUMONIAE
STAPHYLOCOCCUS
AUREUS
STREPTOCOCCUS
MUTANS
LACTOBACILLUS
DELBRUCKII
VIBRIO
PARAHAEMOLYTICUS
KLEBSIELLA
PNEUMONIAE
CLOSTRIDIUM
THERMOCELLUM
PSEUDOMONAS
AERUGINOSA
LEUCONOSTOC
GELIDUM
LACTOCOCCUS
LACTIS
YERSINIA
ENTEROCOLITICA
YERSINIA
PESTIS
YERSINIA
PESTIS
YERSINIA
PSEUDOTUBERCULOSIS
BACILLUS
CALDOTENAX
BACILLUS
STEAROTHERMOPHILUS
LACTOBACILLUS
PLANTARUM
BACILLUS
PSYCHROSACCHAROLYTICUS
BACILLUS
PSYCHROSACCHAROLYTICUS
BACILLUS
MEGATERIUM
BACILLUS
STEAROTHERMOPHILUS
BACILLUS
SUBTILIS
BIFIDOBACTERIUM
LONGUM
LACTOBACILLUS
PLANTARUM
LISTERIA
MONOCYTOGENES
MYCOPLASMA
HYOPNEUMONIAE
THERMUS
AQUATICUS
BACILLUS
ANTHRACIS
PSEUDOMONAS
FLUORESCENS
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
LACTOCOCCUS
LACTIS
BACILLUS
COAGULANS
CLOSTRIDIUM
PASTEURIANUM
LACTOCOCCUS
LACTIS
BACILLUS
SUBTILIS
ERWINIA
CAROTOVORA
MORAXELLA SP
MORAXELLA SP
ESCHERICHIA
COLI
BURKHOLDERIA
CEPACIA
PSEUDOMONAS
FLUORESCENS
PSEUDOMONAS SP
STAPHYLOCOCCUS
AUREUS
SALMONELLA
TYPHIMURIUM
SALMONELLA
TYPHIMURIUM
SALMONELLA
TYPHIMURIUM
ESCHERICHIA
COLI
CITROBACTER
FREUNDII
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ACTINOBACILLUS
ACTINOMYCETEMCOMITANS
PASTEURELLA
HAEMOLYTICA
ACTINOBACILLUS
ACTINOMYCETEMCOMITANS
PASTEURELLA
HAEMOLYTICA
ACTINOBACILLUS
ACTINOMYCETEMCOMITANS
PASTEURELLA
HAEMOLYTICA
ACTINOBACILLUS
ACTINOMYCETEMCOMITANS
PASTEURELLA
HAEMOLYTICA
ESCHERICHIA
COLI
RICKETTSIA
RICKETTSII
ESCHERICHIA
COLI
STAPHYLOCOCCUS
AUREUS
STAPHYLOCOCCUS
AUREUS
STAPHYLOCOCCUS
AUREUS
KRYPTOPHANARON
ALFREDI
PHOTOBACTERIUM
PHOSPHOREUM
VIBRIO
HARVEYI
PHOTOBACTERIUM
LEIOGNATHI
PHOTOBACTERIUM
PHOSPHOREUM
VIBRIO
FISCHERI
XENORHABDUS
LUMINESCENS
PHOTOBACTERIUM
LEIOGNATHI
VIBRIO
HARVEYI
PHOTOBACTERIUM
LEIOGNATHI
PHOTOBACTERIUM
PHOSPHOREUM
VIBRIO
FISCHERI
VIBRIO
HARVEYI
VIBRIO
FISCHERI
VIBRIO
FISCHERI
PHOTOBACTERIUM
PHOSPHOREUM
VIBRIO
HARVEYI
PHOTOBACTERIUM
LEIOGNATHI
PHOTOBACTERIUM
LEIOGNATHI
BACILLUS
SUBTILIS
CLOSTRIDIUM
ACETOBUTYLICUM
ESCHERICHIA
COLI
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
STREPTOCOCCUS
PYOGENES
STREPTOCOCCUS
PYOGENES
STREPTOCOCCUS
PYOGENES
STREPTOCOCCUS
PYOGENES
STREPTOCOCCUS
PYOGENES
ESCHERICHIA
COLI
ENTEROBACTER
AEROGENES
ENTEROBACTER
AEROGENES
ESCHERICHIA
COLI
STREPTOCOCCUS
PNEUMONIAE
BACILLUS SP
CALDOCELLUM
SACCHAROLYTICUM
BACILLUS
STEAROTHERMOPHILUS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
WOLINELLA
SUCCINOGENES
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
ESCHERICHIA
COLI
ESCHERICHIA
COLI
CAULOBACTER
CRESCENTUS
SALMONELLA
TYPHIMURIUM
ENTEROBACTER
AEROGENES
ENTEROBACTER
AEROGENES
ESCHERICHIA
COLI
METHANOSARCINA
BARKERI
METHANOCOCCUS
VANNIELII
METHANOCOCCUS
VOLTAE
METHANOTHERMUS
FERVIDUS
METHANOCOCCUS
VOLTAE
ESCHERICHIA
COLI
METHANOCOCCUS
VOLTAE
ESCHERICHIA
COLI
METHANOTHERMUS
FERVIDUS
SALMONELLA
TYPHIMURIUM
ESCHERICHIA
COLI
ESCHERICHIA
COLI
STAPHYLOCOCCUS
EPIDERMIDIS &
AUREUS
STAPHYLOCOCCUS
EPIDERMIDIS &
AUREUS
METHYLOCOCCUS
CAPSULATUS
METHYLOSINUS
TRICHOSPORIUM
ESCHERICHIA
COLI
STREPTOMYCES
LIVIDANS
BACILLUS SP
STAPHYLOCOCCUS
AUREUS
STAPHYLOCOCCUS
AUREUS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
CHLAMYDIA
TRACHOMATIS
LEGIONELLA
MICDADEI
ENTEROCOCCUS
FAECALIS
STAPHYLOCOCCUS
AUREUS
ENTEROCOCCUS
FAECALIS
BACTEROIDES
FRAGILIS
ESCHERICHIA
COLI
STREPTOCOCCUS
PNEUMONIAE
STREPTOCOCCUS
SANGUIS
BACTEROIDES
FRAGILIS
METHYLOCOCCUS
CAPSULATUS
ESCHERICHIA
COLI
THIOBACILLUS
FERROOXIDANS
THIOBACILLUS
FERROOXIDANS
THIOBACILLUS
FERROOXIDANS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
CLOSTRIDIUM
PASTEURIANUM
CLOSTRIDIUM
PASTEURIANUM
PARACOCCUS
DENITRIFICANS
SYNECHOCOCCUS SP
SYNECHOCOCCUS SP
ESCHERICHIA
COLI
BACILLUS
SUBTILIS
BACILLUS
CEREUS
BACILLUS
SUBTILIS
KLEBSIELLA
PNEUMONIAE
KLEBSIELLA
PNEUMONIAE
KLEBSIELLA
PNEUMONIAE
KLEBSIELLA
PNEUMONIAE
STREPTOCOCCUS
PYOGENES
STREPTOCOCCUS
SUIS
ESCHERICHIA
COLI
STAPHYLOCOCCUS
EPIDERMIDIS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ACINETOBACTER
CALCOACETICUS
SYNECHOCOCCUS SP
BREVIBACTERIUM
EPIDERMIDIS
HERPETOSIPHON
AURANTIACUS
BACILLUS
AMYLOLIQUEFACIENS
BACILLUS
ANEURINOLYTICUS
BACILLUS
ANEURINOLYTICUS
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
CITROBACTER
FREUNDII
HERPETOSIPHON
AURANTIACUS
HERPETOSIPHON
AURANTIACUS
ESCHERICHIA
COLI
HERPETOSIPHON
AURANTIACUS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ENTEROBACTER
CLOACAE
FLAVOBACTERIUM
OKEANOKOITES
FUSOBACTERIUM
NUCLEATUM
HAEMOPHILUS
GALLINARUM
HAEMOPHILUS
INFLUENZAE
METHANOBACTERIUM
THERMOFORMICICUM
KLEBSIELLA
PNEUMONIAE
STREPTOCOCCUS
MUTANS
MORAXELLA SP
NEISSERIA
LACTAMICA
PROTEUS
VULGARIS
SULFOLOBUS
ACIDOCALDARIUS
PROVIDENCIA
STUARTII
ESCHERICHIA
COLI
STREPTOCOCCUS
SANGUIS
SHIGELLA
SONNEI
STAPHYLOCOCCUS
AUREUS
LACTOCOCCUS
LACTIS
LACTOCOCCUS
LACTIS
SPIROPLASMA SP
SERRATIA
MARCESCENS
THERMUS
AQUATICUS
VIBRIO
ESCHERICHIA
COLI
ERWINIA
AMYLOVORA
MORGANELA
MORGANII
PROTEUS
MIRABILIS
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ENTEROBACTER
CLOACAE
STREPTOMYCES
CINNAMONENSIS
PROPIONIBACTERIUM
FREUDENREICHII
SALMONELLA
TYPHIMURIUM
STREPTOMYCES
CINNAMONENSIS
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
VIBRIO
CHOLERAE
ESCHERICHIA
COLI
STREPTOMYCES
AMBOFACIENS
PSEUDOMONAS
MEVALONII
STREPTOCOCCUS
PYOGENES
STREPTOMYCES
CACAOI
YERSINIA
ENTEROCOLITICA
SALMONELLA
TYPHIMURIUM
SALMONELLA
TYPHIMURIUM
ESCHERICHIA
COLI
CLOSTRIDIUM
PERFRINGENS
ESCHERICHIA
COLI
CLOSTRIDIUM
SEPTICUM
CLOSTRIDIUM
SORDELLII
SALMONELLA
TYPHIMURIUM
ENTEROCOCCUS
HIRAE
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
RHIZOBIUM
MELILOTI
STREPTOMYCES
CYANOGENUS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ENTEROBACTER
CLOACAE
ESCHERICHIA
COLI
ESCHERICHIA
COLI
PSEUDOMONAS
CHLORORAPHIS
RHODOCOCCUS
RHODOCHROUS
AZOSPIRILLUM
BRASILENSE
BRADYRHIZOBIUM
JAPONICUM
HERBASPIRILLUM
SEROPEDICAE
RHIZOBIUM
LEGUMINOSARUM
RHIZOBIUM
MELILOTI
RHODOBACTER
CAPSULATUS
AZOTOBACTER
VINELANDII
KLEBSIELLA
PNEUMONIAE
ANABAENA SP
AZOSPIRILLUM
BRASILENSE
PLECTONEMA
BORYANUM
THIOBACILLUS
FERROOXIDANS
CLOSTRIDIUM
PASTEURIANUM
FRANKIA SP
PLECTONEMA
BORYANUM
AZOSPIRILLUM
BRASILENSE
BRADYRHIZOBIUM
JAPONICUM
BRADYRHIZOBIUM SP
CLOSTRIDIUM
PASTEURIANUM
THIOBACILLUS
FERROOXIDANS
AZOTOBACTER
CHROOCOCCUM MCD 1
BRADYRHIZOBIUM
JAPONICUM
ANABAENA SP
LACTOBACILLUS
DELBRUECKII
AZOTOBACTER
VINELANDII
ANABAENA SP
ANABAENA SP
KLEBSIELLA
PNEUMONIAE
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
PSEUDOMONAS
STUTZERI
LACTOCOCCUS
LACTIS
LACTOCOCCUS
LACTIS
LACTOCOCCUS
LACTIS
CLOSTRIDIUM
PASTEURIANUM
CLOSTRIDIUM
PASTEURIANUM
ESCHERICHIA
COLI
BRADYRHIZOBIUM SP
RHIZOBIUM
LOTI
RHIZOBIUM
LEGUMINOSARUM
RHIZOBIUM
LEGUMINOSARUM
RHIZOBIUM
MELILOTI
RHIZOBIUM
MELILOTI
RHIZOBIUM
MELILOTI
RHIZOBIUM
LEGUMINOSARUM
AZOSPIRILLUM
BRASILENSE
RHIZOBIUM
LEGUMINOSARUM
RHIZOBIUM
LEGUMINOSARUM
RHIZOBIUM
FREDII
BRADYRHIZOBIUM
JAPONICUM
RHIZOBIUM
LEGUMINOSARUM
RHIZOBIUM
FREDII
RHIZOBIUM
MELILOTI
PSEUDOMONAS
STUTZERI
PSEUDOMONAS
STUTZERI
PSEUDOMONAS
AERUGINOSA
PSEUDOMONAS
STUTZERI
BACILLUS
AMYLOLIQUEFACIENS
BACILLUS
POLYMYXA
BACILLUS
SUBTILIS
PARACOCCUS
DENITRIFICANS
PARACOCCUS
DENITRIFICANS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
RHODOCOCCUS
RHODOCHROUS
LACTOCOCCUS
LACTIS
ANABAENA SP
SYNECHOCOCCUS SP
SYNECHOCYSTIS SP
VIBRIO
ALGINOLYTICUS
PROTEUS
VULGARIS
RHIZOBIUM
MELILOTI
SYNECHOCOCCUS SP
SYNECHOCYSTIS SP
SYNECHOCOCCUS SP
SYNECHOCYSTIS SP
SYNECHOCYSTIS SP
SYNECHOCYSTIS SP
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
THERMOTOGA
MARITIMA
FLAVOBACTERIUM SP
FLAVOBACTERIUM SP
BACILLUS
CEREUS
AGROBACTERIUM
TUMEFACIENS
AZOTOBACTER
VINELANDII
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
BACILLUS
SUBTILIS
PSEUDOMONAS
PUTIDA
ESCHERICHIA
COLI
AZOTOBACTER
VINELANDII
ESCHERICHIA
COLI
BACILLUS
STEAROTHERMOPHILUS
BACILLUS
SUBTILIS
BACILLUS
STEAROTHERMOPHILUS
BACILLUS
SUBTILIS
CHLAMYDIA
TRACHOMATIS
NEISSERIA
GONORRHOEAE
NEISSERIA
MENINGITIDIS
NEISSERIA
MENINGITIDIS
NEISSERIA
GONORRHOEAE
NEISSERIA
MENINGITIDIS
NEISSERIA
GONORRHOEAE
NEISSERIA
MENINGITIDIS
NEISSERIA
MENINGITIDIS
NEISSERIA
MENINGITIDIS
NEISSERIA
LACTAMICA
NEISSERIA
SICCA
ACTINOBACILLUS
PLEUROPNEUMONIAE
HAEMOPHILUS
INFLUENZAE
HAEMOPHILUS
INFLUENZAE
NEISSERIA
GONORRHOEAE
STAPHYLOCOCCUS
AUREUS
THERMOTOGA
MARITIMA
ESCHERICHIA
COLI
NEISSERIA
GONORRHOEAE
SALMONELLA
TYPHI
ESCHERICHIA
COLI
PHOTOBACTERIUM SP
ESCHERICHIA
COLI
NEISSERIA
GONORRHOEAE
NEISSERIA
GONORRHOEAE
NEISSERIA
GONORRHOEAE
NEISSERIA
GONORRHOEAE
NEISSERIA
GONORRHOEAE
NEISSERIA
GONORRHOEAE
NEISSERIA
GONORRHOEAE
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
PSEUDOMONAS
AERUGINOSA
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
NEISSERIA
MENINGITIDIS
NEISSERIA
MENINGITIDIS
ESCHERICHIA
COLI
BORRELIA
BURGDORFERI
BORRELIA
BURGDORFERI
BACILLUS
SUBTILIS
PSEUDOMONAS
AERUGINOSA
PSEUDOMONAS
PUTIDA
BACILLUS
SUBTILIS
ERWINIA
CAROTOVORA
STREPTOCOCCUS
PARASANGUIS
STREPTOCOCCUS
SANGUIS
LACTOCOCCUS
LACTIS
MYCOPLASMA
HYORHINIS
LACTOCOCCUS
LACTIS
LACTOBACILLUS
PARACASEI
ESCHERICHIA
COLI
RICKETTSIA
RICKETTSII
MYCOPLASMA
HYORHINIS
LACTOCOCCUS
LACTIS
PSEUDOMONAS
CHLORORAPHIS
ENTEROCOCCUS
FAECIUM
LISTERIA
GRAYI
LISTERIA
INNOCUA
LISTERIA
IVANOVII
LISTERIA
MONOCYTOGENES
LISTERIA
SEELIGERI
LISTERIA
WELSHIMERI
MYCOPLASMA
HYORHINIS
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
STREPTOMYCES
GRISEUS
ARTHROBACTER
VISCOSUS
BACILLUS
SPHAERICUS
STREPTOCOCCUS
MUTANS
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
AGROBACTERIUM
TUMEFACIENS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
BACILLUS
ANTHRACIS
ESCHERICHIA
COLI
NEISSERIA
GONORRHOEAE
NEISSERIA
MENINGITIDIS
STREPTOCOCCUS
PNEUMONIAE
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
STREPTOCOCCUS
PNEUMONIAE
STAPHYLOCOCCUS
AUREUS
PSEUDOMONAS
PUTIDA
ERWINIA
CAROTOVORA
ERWINIA
CAROTOVORA
ERWINIA
CAROTOVORA
ERWINIA
CAROTOVORA
ERWINIA
CHRYSANTHEMI
ERWINIA
CAROTOVORA
YERSINIA
PSEUDOTUBERCULOSIS
ERWINIA
CAROTOVORA
ERWINIA
CHRYSANTHEMI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
BORDETELLA
BRONCHISEPTICA
BORDETELLA
PARAPERTUSSIS
BORDETELLA
PERTUSSIS
CORYNEBACTERIUM
GLUTMICUM
ESCHERICHIA
COLI
METHANOBACTERIUM
BRYANTII
THERMUS
AQUATICUS
ERWINIA
CAROTOVORA
SALMONELLA
TYPHIMURIUM
FREMYELLA
DIPLOSIPHON
FREMYELLA
DIPLOSIPHON
PSEUDOMONAS
OLEOVORANS
ANABAENA
CYLINDRICA
ANABAENA
VARIABILIS
FREMYELLA
DIPLOSIPHON
MASTIGOCLADUS
LAMINOSUS
SYNECHOCOCCUS SP
SYNECHOCOCCUS SP
FREMYELLA
DIPLOSIPHON
FREMYELLA
DIPLOSIPHON
ALCALIGENES
EUTROPHUS
SYNECHOCYSTIS SP
SYNECHOCOCCUS SP
SYNECHOCOCCUS SP
SYNECHOCYSTIS SP
ESCHERICHIA
COLI
ERWINIA
HERBICOLA
PSEUDOMONAS SP
MASTIGOCLADUS
LAMINOSUS
PSEUDOMONAS SP
SYNECHOCOCCUS SP
ESCHERICHIA
COLI
CLOSTRIDIUM
PASTEURIANUM
BACILLUS
CEREUS
BACILLUS
CEREUS
BACILLUS
CEREUS
BACILLUS
CEREUS
CLOSTRIDIUM
BIFERMENTANS
CLOSTRIDIUM
PERFRINGENS
LISTERIA
MONOCYTOGENES
PSEUDOMONAS
AERUGINOSA
STAPHYLOCOCCUS
AUREUS
BACILLUS
CEREUS
LEPTOSPIRA
INTERROGANS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
CITROBACTER
FREUNDII
ESCHERICHIA
COLI
KLEBSIELLA
OXYTOCA
KLEBSIELLA
PNEUMONIAE
SALMONELLA
TYPHIMURIUM
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
SYNECHOCOCCUS SP
SYNECHOCOCCUS SP
ESCHERICHIA
COLI
ESCHERICHIA
COLI
NEISSERIA
GONORRHOEAE
PSEUDOMONAS
AERUGINOSA
PSEUDOMONAS
AERUGINOSA
NEISSERIA
GONORRHOEAE
PSEUDOMONAS
AERUGINOSA
PSEUDOMONAS
AERUGINOSA
ESCHERICHIA
COLI
MORAXELLA
BOVIS
MORAXELLA
LACUNATA
BACILLUS
CEREUS
BACILLUS
THURINGIENSIS
LISTERIA
MONOCYTOGENES
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ERWINIA
CAROTOVORA
ESCHERICHIA
COLI
ERWINIA
CHRYSANTHEMI
ESCHERICHIA
COLI
ZYMOMONAS
MOBILIS
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
BACTEROIDES
SYMBIOSIS
PSEUDOMONAS
SYRINGAE
PSEUDOMONAS
AERUGINOSA
PSEUDOMONAS
AERUGINOSA
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
ESCHERICHIA
FERGUSONII
FLAVOBACTERIUM
MENINGOSEPTICUM
FLAVOBACTERIUM
MENINGOSEPTICUM
ESCHERICHIA
COLI
CLOSTRIDIUM
PERFRINGENS
ESCHERICHIA
COLI
ACINETOBACTER
CALCOACETICUS
ANABAENA
VARIABILIS
THERMOPLASMA
ACIDOPHILUM
ESCHERICHIA
COLI
STAPHYLOCOCCUS
AUREUS
STAPHYLOCOCCUS
AUREUS
LACTOBACILLUS
PLANTARUM
BACILLUS
LICHENIFORMIS
BACILLUS SP
STREPTOCOCCUS
AGALACTIAE
LISTERIA
MONOCYTOGENES
ESCHERICHIA
COLI
BACILLUS
SUBTILIS
BUCHNERA
APHIDICOLA
CLOSTRIDIUM
ACETOBUTYLICUM
LACTOCOCCUS
LACTIS
RICKETTSIA
PROWAZEKII
DESULFOVIBRIO
DESULFURICANS
ACHROMOBACTER
LYTICUS
LYSOBACTER
ENZYMOGENES
LISTERIA
MONOCYTOGENES
LISTERIA
MONOCYTOGENES
SERRATIA
MARCESCENS
STAPHYLOCOCCUS
AUREUS
SERRATIA
MARCESCENS
STREPTOCOCCUS
AGALACTIAE
PSEUDOMONAS
AERUGINOSA
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
BACILLUS
SUBTILIS
STREPTOMYCES
GRISEUS
ERWINIA
CHRYSANTHEMI
PORPHYROMONAS
GINGIVALIS
ERWINIA
CHRYSANTHEMI
BACTEROIDES
NODOSUS
ERWINIA
CHRYSANTHEMI
ERWINIA
CHRYSANTHEMI
LACTOCOCCUS
LACTIS
LACTOCOCCUS
LACTIS
LACTOBACILLUS
PARACASEI
SERRATIA
MARCESCENS
SERRATIA
MARCESCENS
ERWINIA
CHRYSANTHEMI
SYNECHOCOCCUS
ELONGATUS
NAEGEL
SYNECHOCOCCUS SP
SYNECHOCOCCUS
VULCANUS
SYNECHOCYSTIS SP
YERSINIA
PESTIS
SYNECHOCOCCUS SP
YERSINIA
PESTIS
ESCHERICHIA
COLI
WOLINELLA
SUCCINOGENES
ESCHERICHIA
COLI
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
STAPHYLOCOCCUS
CARNOSUS
STREPTOCOCCUS
SALIVARIUS
ERWINIA
CHRYSANTHEMI
BACILLUS
SUBTILIS
LACTOBACILLUS
CASEI
LACTOCOCCUS
LACTIS
STAPHYLOCOCCUS
AUREUS
ESCHERICHIA
COLI
STAPHYLOCOCCUS
CARNOSUS
ESCHERICHIA
COLI
STREPTOCOCCUS
MUTANS
SALMONELLA
TYPHIMURIUM
LACTOBACILLUS
CASEI
ESCHERICHIA
COLI&
SALMONELLA
TYPHIMURIUM
KLEBSIELLA
PNEUMONIAE
ESCHERICHIA
COLI
KLEBSIELLA
AEROGENES
KLEBSIELLA
PNEUMONIAE
KLEBSIELLA
PNEUMONIAE
KLEBSIELLA
PNEUMONIAE
PSEUDOMONAS
PUTIDA
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
BACILLUS
SUBTILIS
ANABAENA SP
MASTIGOCLADUS
LAMINOSUS
ANABAENA SP
MASTIGOCLADUS
LAMINOSUS
MASTIGOCLADUS
LAMINOSUS
ANABAENA SP
ANABAENA SP
FREMYELLA
DIPLOSIPHON
FREMYELLA
DIPLOSIPHON
FREMYELLA
DIPLOSIPHON
FREMYELLA
DIPLOSIPHON
BACILLUS
SUBTILIS
SERRATIA
MARCESCENS
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
BACILLUS
SUBTILIS
FREMYELLA
DIPLOSIPHON
ESCHERICHIA
COLI
ESCHERICHIA
COLI
CLOSTRIDIUM
PASTEURIANUM
ESCHERICHIA
COLI
STREPTOCOCCUS
THERMOPHILUS
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
KLEBSIELLA
AEROGENES
ESCHERICHIA
COLI
ERWINIA
AMYLOVORA
ERWINIA
STEWARTII
KLESIELLA
AEROGENES
ESCHERICHIA
COLI
LEGIONELLA
PNEUMOPHILA
ACHOLEPLASMA
LAIDLAWII
AGROBACTERIUM
TUMEFACIENS
ANABAENA
VARIABILIS
AQUIFEX
PYROPHILUS
BACTEROIDES
FRAGILIS
BACILLUS
SUBTILIS
BRUCELLA
ABORTUS
BURKHOLDERIA
CEPACIA
ERWINIA
CAROTOVORA
LACTOBACILLUS
DELBRUECKII
LACTOBACILLUS
HELVETICUS
LACTOCOCCUS
LACTIS
METHYLOMONAS
CLARA
METHYLOBACILLUS
FLAGELLATUM
MYCOPLASMA
PULMONIS
MYCOBACTERIUM
TUBERCULOSIS
NEISSERIA
GONORRHOEAE
PROTEUS
MIRABILIS
PSEUDOMONAS
AERUGINOSA
RHIZOBIUM
LEGUMINOSARUM
RHIZOBIUM
LEGUMINOSARUM
RHIZOBIUM
MELILOTI
RHODOBACTER
SPHAEROIDES
STREPTOCOCCUS
PNEUMONIAE
SYNECHOCOCCUS SP
VIBRIO
CHOLERAE
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
PROTEUS
MIRABILIS
PSEUDOMONAS
PUTIDA
SALMONELLA
TYPHIMURIUM
ESCHERICHIA
COLI
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
BACILLUS
SUBTILIS
STAPHYLOCOCCUS
AUREUS
STAPHYLOCOCCUS
EPIDERMIDIS
ESCHERICHIA
COLI
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
NEISSERIA
GONORRHOEAE
LACTOBACILLUS
PLANTARUM
STAPHYLOCOCCUS
AUREUS
STAPHYLOCOCCUS
AUREUS
STREPTOCOCCUS
AGALACTIAE
STREPTOCOCCUS
PYOGENES
STAPHYLOCOCCUS
AUREUS
ESCHERICHIA
COLI
CLOSTRIDIUM
PERFRINGENS
ESCHERICHIA
COLI
LACTOBACILLUS
PLANTARUM
CLOSTRIDIUM
PERFRINGENS
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
SALMONELLA
TYPHIMURIUM
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
SALMONELLA
TYPHIMURIUM
SALMONELLA
TYPHI
VIBRIO
ANGUILLARUM
ESCHERICHIA
COLI
BACILLUS
THURINGIENSIS
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
ESCHERICHIA
COLI
ESCHERICHIA
COLI
RHIZOBIUM
LEGUMINOSARUM
ESCHERICHIA
COLI
BORRELIA
BURGDORFERI
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
PHOTOBACTERIUM
LEIOGNATHI
BACILLUS
SUBTILIS
PHOTOBACTERIUM
LEIOGNATHI
STREPTOMYCES
ANTIBIOTICUS
SYNECHOCYSTIS SP
BACILLUS
STEAROTHERMOPHILUS
M1CROCOCCUS
LUTEUS
MYCOPLASMA
CAPRICOLUM
BACILLUS
LICHENIFORMIS
BACILLUS
STEAROTHERMOPHILUS
BACILLUS
SUBTILIS
CHLAMYDIA
TRACHOMATIS
ESCHERICHIA
COLI
LACTOCOCCUS
LACTIS
METHANOCOCCUS
VANNIELII
MYCOPLASMA
CAPRICOLUM
BACILLUS
STEAROTHERMOPHILUS
CHLAMYDIA
TRACHOMATIS
HALOARCULA
MARISMORTUI
MYCOPLASMA
CAPRICOLUM
ESCHERICHIA
COLI
HALOARCULA
MARISMORTUI
METHANOCOCCUS
VANNIELII
PROTEUS
VULGARIS
SULFOLOBUS
SOLFATARICUS
ESCHERICHIA
COLI
MYCOPLASMA
FERMENTANS
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
METHANOCOCCUS
VANNIELII
MYCOPLASMA
CAPRICOLUM
HALOARCULA
MARISMORTUI
METHANOCOCCUS
VANNIELII
MICROCOCCUS
LUTEUS
CHLAMYDIA
TRACHOMATIS
ESCHERICHIA
COLI
MYCOPLASMA
CAPRICOLUM
BACILLUS
STEAROTHERMOPHILUS
MYCOPLASMA
CAPRICOLUM
THERMUS
AQUATICUS
BACILLUS
STEAROTHERMOPHILUS
ESCHERICHIA
COLI
METHANOCOCCUS
VANNIELII
BACILLUS
STEAROTHERMOPHILUS
ESCHERICHIA
COLI
HALOBACTERIUM
CUTIRUBRUM
HALOBACTERIUM
HALOBIUM
HALOARCULA
MARISMORTUI
METHANOCOCCUS
VANNIELII
HALOPHILIC
EUBACTERIUM NRCC 41227
HALOBACTERIUM
HALOBIUM
HALOARCULA
MARISMORTUI
METHANOCOCCUS
VANNIELII
MICROCOCCUS
LUTEUS
SALMONELLA
TYPHIMURIUM
STAPHYLOCOCCUS
AUREUS
SALMONELLA
TYPHIMURIUM
STAPHYLOCOCCUS
AUREUS
STAPHYLOCOCCUS
AUREUS
SULFOLOBUS
SOLFATARICUS
BACILLUS
AMYLOLIQUEFACIENS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
BUCHNERA
APHIDICOLA
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
BACILLUS
CIRCULANS
BACILLUS
INTERMEDIUS
BACILLUS
THURINGIENSIS
CITROBACTER
FREUNDII
BACILLUS
THURINGIENSIS
ALCALIGENES
EUTROPHUS
AZORHIZOBIUM
CAULINODANS
BACILLUS
SUBTILIS
BRADYRHIZOBIUM
JAPONICUM
KLEBSIELLA
PNEUMONIAE
RHODOBACTER
CAPSULATUS
BRADYRHIZOBIUM
JAPONICUM
ALCALIGENES
EUTROPHUS
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
BUCHNERA
APHIDICOLA
CHLAMYDIA
TRACHOMATIS
ESCHERICHIA
COLI
PSEUDOMONAS
AERUGINOSA
RICKETTSIA
PROWAZEKII
MYXOCOCCUS
XANTHUS
SYNECHOCOCCUS SP
BACILLUS
SUBTILIS
ESCHERICHIA
COLI&
SALMONELLA
TYPHIMURIUM
HALOBACTERIUM
HALOBIUM
HALOCOCCUS
MORRHUAE
METHANOBACTERIUM
THERMOAUTOTROPHICU
SULFOLOBUS
ACIDOCALDARIUS
THERMOCOCCUS
CELER
ESCHERICHIA
COLI
MYCOBACTERIUM
LEPRAE
SALMONELLA
TYPHIMURIUM
SULFOLOBUS
ACIDOCALDARIUS
THERMOTOGA
MARITIMA
ANABAENA SP
ESCHERICHIA
COLI
HALOBACTERIUM
HALOBIUM
HALOCOCCUS
MORRHUAE
METHANOBACTERIUM
THERMOAUTOTROPHICU
MYCOBACTERIUM
LEPRAE
NOSTOC
COMMUNE
SULFOLOBUS
ACIDOCALDARIUS
THERMOCOCCUS
CELER
NOSTOC
COMMUNE
ESCHERICHIA
COLI
ESCHERICHIA
COLI
HALOBACTERIUM
HALOBIUM
AGROBACTERIUM
TUMEFACIENS
ANABAENA SP
CLOSTRIDIUM
ACETOBUTYLICUM
STREPTOMYCES
AUREOFACIENS
ANABAENA SP
BACILLUS
SUBTILIS
MYXOCOCCUS
XANTHUS
STIGMATELLA
AURANTIACA
ANABAENA SP
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
CLOSTRIDIUM
ACETOBUTYLICUM
BACILLUS
LICHENIFORMIS
BACILLUS
MEGATERIUM
BACILLUS
SUBTILIS
BACILLUS
LICHENIFORMIS
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
STAPHYLOCOCCUS
AUREUS
STREPTOMYCES
COELICOLOR
BACILLUS
THURINGIENSIS
ESCHERICHIA
COLI
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
METHANOCOCCUS
VANNIELII
ESCHERICHIA
COLI
PROVIDENCIA SP
RHIZOBIUM
MELILOTI
BACILLUS
STEAROTHERMOPHILUS
SPIROPLASMA
CITRI
ACHOLEPLASMA
LAIDLAWII
MYCOPLASMA
CAPRICOLUM
ESCHERICHIA
COLI
HALOARCULA
MARISMORTUI
MYCOPLASMA
CAPRICOLUM
THERMUS
AQUATICUS
METHANOCOCCUS
VANNIELII
MYCOBACTERIUM
LEPRAE
MICROCOCCUS
LUTEUS
MYCOPLASMA
CAPRICOLUM
ESCHERICHIA
COLI
ESCHERICHIA
COLI
BACILLUS
AMYLOLIQUEFACIENS
BACILLUS
SUBTILIS
STREPTOCOCCUS
MUTANS
BACILLUS
LICHENIFORMIS
BACILLUS
SUBTILIS
STREPTOCOCCUS
PYOGENES
BACILLUS SP
CLOSTRIDIUM
BIFERMENTANS
BACILLUS
CEREUS
BACILLUS
STEAROTHERMOPHILUS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
BACILLUS
SUBTILIS
STREPTOCOCCUS
PYOGENES
KLEBSIELLA
PNEUMONIAE
LACTOCOCCUS
LACTIS
STREPTOCOCCUS
MUTANS
SALMONELLA
THOMPSON
SALMONELLA
TYPHIMURIUM
KLEBSIELLA
PNEUMONIAE
SALMONELLA
TYPHIMURIUM
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
LACTOCOCCUS
LACTIS
METHANOCOCCUS
VANNIELII
STAPHYLOCOCCUS
CARNOSUS
SALMONELLA
ENTERITIDIS
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
SERRATIA
MARCESCENS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
BACILLUS
LICHENIFORMIS
BACILLUS
SUBTILIS
ACETOGENIUM
KIVUI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
SYNECHOCOCCUS SP
COXIELLA
BURNETII
ESCHERICHIA
COLI
METHANOBACTERIUM
THERMOAUTOTROPHICU
PHOTOBACTERIUM
LEIOGNATHI
PROPIONIBACTERIUM
FREUDENREICHII
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
BACILLUS
SUBTILIS
BACILLUS
MEGATERIUM
BACILLUS
SUBTILIS
BACILLUS
LICHENIFORMIS
BACILLUS
MEGATERIUM
BACILLUS
SUBTILIS
BACILLUS
THURINGIENSIS
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
STREPTOCOCCUS
DOWNEI
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
STREPTOCOCCUS
MUTANS
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
STREPTOCOCCUS
PYOGENES
SPIROPLASMA
CITRI
SPIROPLASMA
MELLIFERUM
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ZYMOMONAS
MOBILIS
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
MYCOPLASMA
MYCOIDES
PASTEURELLA
HAEMOLYTICA
STREPTOCOCCUS
PARASANGUIS
STREPTOCOCCUS
SANGUIS
ESCHERICHIA
COLI
PROTEUS
MIRABILIS
SERRATIA
MARCESCENS
STREPTOCOCCUS
SANGUIS
STREPTOMYCES
AVIDINII
ESCHERICHIA
COLI
STAPHYLOCOCCUS
AUREUS
STAPHYLOCOCCUS
AUREUS
CLOSTRIDIUM
BEIJERINCKII
ESCHERICHIA
COLI
STREPTOMYCES
GRISEUS
STREPTOCOCCUS
EQUISIMILIS
STREPTOCOCCUS
PYOGENES
STREPTOCOCCUS SP
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
SALMONELLA
TYPHIMURIUM
SYNECHOCOCCUS SP
SYNECHOCYSTIS SP
BACILLUS
LICHENIFORMIS
BACILLUS
MESENTERICUS
BACILLUS SP
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
BACILLUS
STEAROTHERMOPHILUS
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
AGROBACTERIUM
VITIS
ENTEROBACTER
AEROGENES
ESCHERICHIA
COLI
ESCHERICHIA
COLI
BACILLUS
STEAROTHERMOPHILUS
BACILLUS
SUBTILIS
THERMUS
AQUATICUS
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
STREPTOCOCCUS
EQUISIMILIS
METHANOBACTERIUM
THERMOAUTOTROPHICU
ESCHERICHIA
COLI
ESCHERICHIA
COLI
BACILLUS
STEAROTHERMOPHILUS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
PSEUDOMONAS
SYRINGAE
ESCHERICHIA
COLI
BACILLUS
SUBTILIS
BACILLUS
STEAROTHERMOPHILUS
ESCHERICHIA
COLI
BACILLUS
STEAROTHERMOPHILUS
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
BACILLUS
CALDOTENAX
BACILLUS
STEAROTHERMOPHILUS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
SALMONELLA
POTSDAM
SALMONELLA
TYPHIMURIUM
ESCHERICHIA
COLI
ACINETOBACTER
CALCOACETICUS
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
CITROBACTER
FREUNDII
HERPETOSIPHON
AURANTIACUS
HERPETOSIPHON
AURANTIACUS
DESULFOVIBRIO
DESULFURICANS
STREPTOCOCCUS
PNEUMONIAE
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
FLAVOBACTERIUM
OKEANOKOITES
HAEMOPHILUS
INFLUENZAE
HAEMOPHILUS
PARAINFLUENZAE
HAEMOPHILUS
HAEMOLYTICUS
HAEMOPHILUS
INFLUENZAE
KLEBSIELLA
PNEUMONIAE
MORAXELLA
BOVIS
MORAXELLA
BOVIS
METHANOBACTERIUM
THERMOFORMICICUM
NEISSERIA
GONORRHOEAE
STREPTOCOCCUS
SANGUIS
SHIGELLA
SONNEI
STAPHYLOCOCCUS
AUREUS
SALMONELLA
INFANTIS
SERRATIA
MARCESCENS
THERMUS
AQUATICUS
ESCHERICHIA
COLI
BACILLUS
CEREUS
TREPONEMA
PALLIDUM
TREPONEMA
DENTICOLA
BACILLUS
ALVEI
CLOSTRIDIUM
PERFRINGENS
LISTERIA
IVANOVII
LISTERIA
MONOCYTOGENES
LISTERIA
SEELIGERI
STREPTOCOCCUS
PNEUMONIAE
STREPTOCOCCUS
PYOGENES
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
NEISSERIA
GONORRHOEAE
PSEUDOMONAS
PICKETTII
SALMONELLA
TYPHIMURIUM
VIBRIO
CHOLERAE
VIBRIO
CHOLERAE
VIBRIO
CHOLERAE
VIBRIO
CHOLERAE
VIBRIO
CHOLERAE
VIBRIO
CHOLERAE
VIBRIO
CHOLERAE
VIBRIO
CHOLERAE
VIBRIO
CHOLERAE
BACILLUS
SUBTILIS
BACILLUS
STEAROTHERMOPHILUS
STAPHYLOCOCCUS
AUREUS
STREPTOCOCCUS
AGALACTIAE
STREPTOCOCCUS
PNEUMONIAE
ESCHERICHIA
COLI
ESCHERICHIA
COLI
STREPTOCOCCUS
PYOGENES
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ALCALIGENES SP
ESCHERICHIA
COLI
ENTEROCOCCUS
FAECALIS
ENTEROCOCCUS
FAECALIS
ESCHERICHIA
COLI
STREPTOMYCES
LIVIDANS
UREAPLASMA
UREALYTICUM
CAMPYLOBACTER
COLI
CAMPYLOBACTER
JEJUNI
STREPTOCOCCUS
MUTANS
BACTEROIDES
FRAGILIS
CLOSTRIDIUM
TETANI
PYROCOCCUS
WOESEI
ALCALIGENES
EUTROPHUS
ESCHERICHIA
COLI
LACTOCOCCUS
LACTIS
ESCHERICHIA
COLI
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
PSEUDOMONAS
PUTIDA
BACILLUS
CEREUS
BACILLUS
STEAROTHERMOPHILUS
BACILLUS
THERMOPROTEOLYTICUS
THERMOACTINOMYCES
VULGARIS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
SULFOLOBUS
ACIDOCALDARIUS
BREVIBACTERIUM
LACTOFERMENTUM
SACCHAROPOLYSPORA
ERYTHRAEA
ESCHERICHIA
COLI
TREPONEMA
PALLIDUM
TREPONEMA
PALLIDUM
TREPONEMA
PHAGEDENIS
ESCHERICHIA
COLI
STAPHYLOCOCCUS
AUREUS
ENTEROCOCCUS
FAECALIS
STAPHYLOCOCCUS
AUREUS
STAPHYLOCOCCUS
AUREUS
BACILLUS
THURINGIENSIS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
PSEUDOMONAS
PUTIDA
PSEUDOMONAS
PUTIDA
PSEUDOMONAS
PUTIDA
PSEUDOMONAS
PUTIDA
ESCHERICHIA
COLI
ESCHERICHIA
COLI
SYNECHOCOCCUS SP
ESCHERICHIA
COLI
BORDETELLA
PERTUSSIS
BORDETELLA
PERTUSSIS
CLOSTRIDIUM
DIFFICILE
PSEUDOMONAS
AERUGINOSA
CLOSTRIDIUM
DIFFICILE
VIBRIO
CHOLERAE
TREPONEMA
PALLIDUM
ESCHERICHIA
COLI
MORAXELLA SP
PORPHYROMONAS
GINGIVALIS
AGROBACTERIUM
TUMEFACIENS
AGROBACTERIUM
TUMEFACIENS
PSEUDOMONAS
SYRINGAE
STAPHYLOCOCCUS
AUREUS
STAPHYLOCOCCUS
AUREUS
ESCHERICHIA
COLI
RHIZOBIUM
MELILOTI
STAPHYLOCOCCUS
AUREUS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
SHIGELLA
SONNEI
ESCHERICHIA
COLI
MYCOBACTERIUM
TUBERCULOSIS
BACILLUS
THURINGIENSIS
BACILLUS
THURINGIENSIS
STAPHYLOCOCCUS
AUREUS
BACILLUS
THURINGIENSIS
BACILLUS
THURINGIENSIS
PSEUDOMONAS
AERUGINOSA
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
BACILLUS
SUBTILIS
CAULOBACTER
CRESCENTUS
PSEUDOMONAS
AERUGINOSA
ACINETOBACTER
CALCOACETICUS
BACILLUS
SUBTILIS
BREVIBACTERIUM
LACTOFERMENTUM
LACTOBACILLUS
CASEI
LACTOCOCCUS
LACTIS
VIBRIO
PARAHAEMOLYTICUS
BREVIBACTERIUM
LACTOFERMENTUM
ESCHERICHIA
COLI
LACTOCOCCUS
LACTIS
VIBRIO
PARAHAEMOLYTICUS
ACINETOBACTER
CALCOACETICUS
PSEUDOMONAS
AERUGINOSA
PSEUDOMONAS
PUTIDA
VIBRIO
PARAHAEMOLYTICUS
BACILLUS
PUMILUS
CLOSTRIDIUM
THERMOCELLUM
LACTOCOCCUS
LACTIS
LEPTOSPIRA
BIFLEXA
RHIZOBIUM
MELILOTI
SALMONELLA
TYPHIMURIUM
SULFOLOBUS
SOLFATARICUS
VIBRIO
PARAHAEMOLYTICUS
ACINETOBACTER
CALCOACETICUS
AZOSPIRILLUM
BRASILENSE
ESCHERICHIA
COLI
LACTOCOCCUS
LACTIS
PSEUDOMONAS
AERUGINOSA
SALMONELLA
TYPHIMURIUM
SERRATIA
MARCESCENS
SHIGELLA
DYSENTERIAE
PSEUDOMONAS
AERUGINOSA
ESCHERICHIA
COLI
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
STREPTOMYCES
GRISEUS
STREPTOMYCES
AZUREUS
STAPHYLOCOCCUS
AUREUS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
BACILLUS
BREVIS
TREPONEMA
PERTENUE
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
LACTOBACILLUS
CASEI
LACTOCOCCUS
LACTIS
STAPHYLOCOCCUS
AUREUS
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
SALMONELLA
TYPHIMURIUM
ESCHERICHIA
COLI
ESCHERICHIA
COLI
HELICOBACTER
PYLORI
PROTEUS
MIRABILIS
PROTEUS
VULGARIS
UREAPLASMA
UREALYTICUM
HELICOBACTER
PYLORI
HELICOBACTER
PYLORI
PROTEUS
MIRABILIS
KLEBSIELLA
AEROGENES
LACTOCOCCUS
LACTIS
ESCHERICHIA
COLI
SALMONELLA
TYPHIMURIUM
ESCHERICHIA
COLI
MICROCOCCUS
LUTEUS
PARACOCCUS
DENITRIFICANS
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ENTEROCOCCUS
FAECIUM
ENTEROCOCCUS
GALLINARUM
AGROBACTERIUM
TUMEFACIENS
AGROBACTERIUM
TUMEFACIENS
AGROBACTERIUM
TUMEFACIENS
AGROBACTERIUM
TUMEFACIENS
AGROBACTERIUM
TUMEFACIENS
AGROBACTERIUM
TUMEFACIENS
AGROBACTERIUM
TUMEFACIENS
AGROBACTERIUM
RHIZOGENES
TUMEFACIENS
AGROBACTERIUM
TUMEFACIENS
AGROBACTERIUM
TUMEFACIENS
AGROBACTERIUM
RHIZOGENES
AGROBACTERIUM
TUMEFACIENS
AGROBACTERIUM
TUMEFACIENS
AGROBACTERIUM
TUMEFACIENS
SHIGELLA
FLEXNERI
YERSINIA
ENTEROCOLITICA
AGROBACTERIUM
RHIZOGENES
AGROBACTERIUM
TUMEFACIENS
ESCHERICHIA
COLI
MYCOPLASMA
HYORHINIS
BORRELIA
HERMSII
BORRELIA
HERMSII
BORRELIA
HERMSII
BORRELIA
HERMSII
BORRELIA
HERMSII
AZOTOBACTER
VINELANDII
AZOTOBACTER
CHROOCOCCUM
AZOTOBACTER
VINELANDII
SALMONELLA
CHOLERAE-SUIS
SALMONELLA
DUBLIN
SALMONELLA
DUBLIN
VIBRIO
VULNIFICUS
STREPTOCOCCUS
MUTANS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ANABAENA SP
ESCHERICHIA
COLI
STAPHYLOCOCCUS
XYLOSUS
KLEBSIELLA
AEROGENES
LACTOBACILLUS
PENTOSUS
STAPHYLOCOCCUS
XYLOSUS
BACILLUS
SUBTILIS
LACTOBACILLUS
PENTOSUS
STAPHYLOCOCCUS
XYLOSUS
PSEUDOMONAS
PUTIDA
CALDOCELLUM
SACCHAROLYTICUM
BACILLUS
CIRCULANS
BACILLUS SP
BACILLUS
SUBTILIS
CALDOCELLUM
SACCHAROLYTICUM
PSEUDOMONAS
FLUORESCENS
BACILLUS
PUMILUS
CALDOCELLUM
SACCHAROLYTICUM
PSEUDOMONAS
FLUORESCENS
PSEUDOMONAS
FLUORESCENS
STREPTOMYCES
LIVIDANS
HALOCOCCUS
MORRHUAE
STREPTOCOCCUS
ORALIS
METHANOBREVIBACTER
SMITHII
ESCHERICHIA
COLI
PSEUDOMONAS
FLUORESCENS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
PSEUDOMONAS
AERUGINOSA
CLOSTRIDIUM
ACETOBUTYLICUM
CLOSTRIDIUM
ACETOBUTYLICUM
CLOSTRIDIUM
ACETOBUTYLICUM
YERSINIA
ENTEROCOLITICA
YERSINIA
PSEUDOTUBERCULOSIS
RICKETTSIA
RICKETTSII
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
FERGUSONII
SALMONELLA
TYPHIMURIUM
SYNECHOCYSTIS SP
MYCOBACTERIUM
LEPRAE
BACILLUS
FIRMUS
MYCOPLASMA
GALLISEPTICUM
MYCOPLASMA
GALLISEPTICUM
XANTHOMONAS
CAMPESTRIS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
BACTEROIDES
UNIFORMIS
BACILLUS
LAUTUS
ESCHERICHIA
COLI
ALCALIGENES
EUTROPHUS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
SYNECHOCOCCUS SP
SYNECHOCYSTIS SP
SYNECHOCYSTIS SP
MASTIGOCLADUS
LAMINOSUS
PSEUDANABAENA SP
BACILLUS
THURINGIENSIS
ESCHERICHIA
COLI
BACILLUS
SUBTILIS
HERPETOSIPHON
AURANTIACUS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
BORRELIA
BURGDORFERI
SULFOLOBUS
SOLFATARICUS
SULFOLOBUS
SOLFATARICUS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
BACILLUS
STEAROTHERMOPHILUS
BRADYRHIZOBIUM
JAPONICUM
BACILLUS
MEGATERIUM
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
BACILLUS
THURINGIENSIS
PSEUDOMONAS
PUTIDA
ESCHERICHIA
COLI
BACILLUS
STEAROTHERMOPHILUS
BACILLUS
STEAROTHERMOPHILUS
BACILLUS
CEREUS
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
CLOSTRIDIUM
ACETOBUTYLICUM
STREPTOCOCCUS
MUTANS
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
PSEUDOMONAS
PUTIDA
THIOBACILLUS
FERROOXIDANS
ESCHERICHIA
COLI
BACILLUS
SUBTILIS
ANABAENA SP
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
LACTOCOCCUS
LACTIS
LACTOCOCCUS
LACTIS
LACTOCOCCUS
LACTIS
LACTOCOCCUS
LACTIS
STAPHYLOCOCCUS
AUREUS
VIBRIO
CHOLERAE
METHANOTHERMUS
FERVIDUS
CLOSTRIDIUM
ACETOBUTYLICUM
CLOSTRIDIUM
ACETOBUTYLICUM
CLOSTRIDIUM
ACETOBUTYLICUM
LACTOBACILLUS
HELVETICUS
PSEUDOMONAS SP
HALOBACTERIUM
HALOBIUM
MYCOBACTERIUM
TUBERCULOSIS
PSEUDOMONAS
AMYLODERAMOSA
METHANOBREVIBACTER
SMITHII
HALOBACTERIUM
HALOBIUM
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ERWINIA
CHRYSANTHEMI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
LISTERIA
MONOCYTOGENES
SHIGELLA
SONNEI
STREPTOMYCES
COELICOLOR
SHIGELLA
SONNEI
BACILLUS SP
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
BACILLUS
FIRMUS
LACTOBACILLUS
ACIDOPHILUS
LACTOBACILLUS
ACIDOPHILUS
LACTOBACILLUS
ACIDOPHILUS
SULFOLOBUS
SOLFATARICUS
PSEUDOMONAS
PUTIDA
YERSINIA
ENTEROCOLITICA
ANABAENA
VARIABILIS
LACTOCOCCUS
LACTIS
BACILLUS
SUBTILIS
MYCOPLASMA
GENITALIUM
CLOSTRIDIUM
PERFRINGENS
CLOSTRIDIUM
PERFRINGENS
METHANOCOCCUS
THERMOLITHOTROPHICUS
PARACOCCUS
DENITRIFICANS
ANABAENA SP
ANABAENA SP
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
ESCHERICHIA
COLI
PHOTOBACTERIUM SP
YERSINIA
ENTEROCOLITICA
YERSINIA
PSEUDOTUBERCULOSIS
YERSINIA
ENTEROCOLITICA
YERSINIA
PSEUDOTUBERCULOSIS
YERSINIA
ENTEROCOLITICA
HAEMOPHILUS
INFLUENZAE
LISTERIA
MONOCYTOGENES
PYROCOCCUS
WOESEI
HAEMOPHILUS
INFLUENZAE
HAEMOPHILUS
INFLUENZAE
HAEMOPHILUS
INFLUENZAE
HAEMOPHILUS
INFLUENZAE
HAEMOPHILUS
INFLUENZAE
HAEMOPHILUS
INFLUENZAE
BACILLUS
SUBTILIS
PYROCOCCUS
WOESEI
LISTERIA
MONOCYTOGENES
STAPHYLOCOCCUS
AUREUS
BACILLUS
SUBTILIS
STAPHYLOCOCCUS
AUREUS
STAPHYLOCOCCUS
AUREUS
STAPHYLOCOCCUS
AUREUS
STAPHYLOCOCCUS
AUREUS
AGROBACTERIUM
TUMEFACIENS
LEGIONELLA
PNEUMOPHILA
ENTEROCOCCUS
FAECALIS
BACILLUS
ANTHRACIS
ESCHERICHIA
COLI
BACILLUS
SUBTILIS
ESCHERICHIA
COLI
SYNECHOCOCCUS SP
CLOSTRIDIUM
PERFRINGENS
CLOSTRIDIUM
PERFRINGENS
CLOSTRIDIUM
PERFRINGENS
PSEUDOMONAS
AERUGINOSA
BACILLUS
SUBTILIS
KLEBSIELLA
PNEUMONIAE
PLECTONEMA
BORYANUM
METHANOBACTERIUM
THERMOFORMICICUM
METHANOBACTERIUM
THERMOFORMICICUM
METHANOBACTERIUM
THERMOFORMICICUM
METHANOBACTERIUM
THERMOFORMICICUM
BACILLUS
SUBTILIS
METHANOBACTERIUM
THERMOFORMICICUM
METHANOBACTERIUM
THERMOFORMICICUM
METHANOBACTERIUM
THERMOFORMICICUM
METHANOBACTERIUM
THERMOFORMICICUM
THERMOFILUM
PENDENS
HALOBACTERIUM
CUTIRUBRUM
SYNECHOCOCCUS SP
SALMONELLA
TYPHIMURIUM
SALMONELLA
TYPHIMURIUM
SALMONELLA
TYPHIMURIUM
SALMONELLA
TYPHIMURIUM
LACTOCOCCUS
LACTIS
METHANOCOCCUS
VANNIELII
METHANOCOCCUS
VANNIELII
SULFOLOBUS
ACIDOCALDARIUS
SULFOLOBUS
ACIDOCALDARIUS
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
YERSINIA
ENTEROCOLITICA
YERSINIA
ENTEROCOLITICA
YERSINIA
ENTEROCOLITICA
YERSINIA
ENTEROCOLITICA
YERSINIA
PSEUDOTUBERCULOSIS
YERSINIA
ENTEROCOLITICA
YERSINIA
PSEUDOTUBERCULOSIS
YERSINIA
ENTEROCOLITICA
YERSINIA
PSEUDOTUBERCULOSIS
YERSINIA
ENTEROCOLITICA
YERSINIA
PSEUDOTUBERCULOSIS
SERRATIA
MARCESCENS
DESULFUROLOBUS
AMBIVALENS
DESULFUROLOBUS
AMBIVALENS
LEPTOSPIRA
INTERROGANS
MYCOPLASMA
MYCOIDES
BACILLUS
SUBTILIS
METHANOTHERMUS
FERVIDUS
STREPTOMYCES
FRADIAE
LEPTOSPIRA
BIFLEXA
LACTOCOCCUS
LACTIS
BACILLUS
SUBTILIS
SPIROPLASMA
CITRI
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
BACILLUS
SUBTILIS
ANABAENA SP
CALDOCELLUM
SACCHAROLYTICUM
CALDOCELLUM
SACCHAROLYTICUM
ESCHERICHIA
COLI
SACCHAROMYCES CEREVISIAE VIRUS
409–
55I–
Nov–
Additionally,
“X” may represent an amino group, a hydrophobic group, including but not limited to carbobenzoxyl, dansyl, or T-butyloxycarbonyl; an acetyl group; a 9-fluorenylmethoxy-carbonyl (FMOC) group; a macromolecular carrier group including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.
“Z” may represent a carboxyl group; an amido group; a T-butyloxycarbonyl group; a macromolecular carrier group including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.
Additionally,
“X” may represent an amino group, a hydrophobic group, including but not limited to carbobenzoxyl, dansyl, or T-butyloxycarbonyl; an acetyl group; a 9-fluorenylmethoxy-carbonyl (FMOC) group; a macromolecular carrier group including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.
“Z” may represent a carboxyl group; an amido group; a T-butyloxycarbonyl group; a macromolecular carrier group including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.
Additionally,
“X” may represent an amino group, a hydrophobic group, including but not limited to carbobenzoxyl, dansyl, or T-butyloxycarbonyl; an acetyl group; a 9-fluorenylmethoxy-carbonyl (FMOC) group; a macromolecular carrier group including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.
“Z” may represent a carboxyl group; an amido group; a T-butyloxycarbonyl group; a macromolecular carrier group including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.
Additionally,
“X” may represent an amino group, a hydrophobic group, including but not limited to carbobenzoxyl, dansyl, or T-butyloxycarbonyl; an acetyl group; a 9-fluorenylmethoxy-carbonyl (FMOC) group; a macromolecular carrier group including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.
“Z” may represent a carboxyl group; an amido group; a T-butyloxycarbonyl group; a macromolecular carrier group including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.
Additionally,
“X” may represent an amino group, a hydrophobic group, including but not limited to carbobenzoxyl, dansyl, or T-butyloxycarbonyl; an acetyl group; a 9-fluorenylmethoxy-carbonyl (FMOC) group; a macromolecular carrier group including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.
“Z” may represent a carboxyl group; an amido group; a T-butyloxycarbonyl group; a macromolecular carrier group including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.
Additionally,
“X” may represent an amino group, a hydrophobic group, including but not limited to carbobenzoxyl, dansyl, or T-butyloxycarbonyl; an acetyl group; a 9-fluorenylmethoxy-carbonyl (FMOC) group; a macromolecular carrier group including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.
“Z” may represent a carboxyl group; an amido group; a T-butyloxycarbonyl group; a macromolecular carrier group including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.
Additionally,
“X” may represent an amino group, a hydrophobic group, including but not limited to carbobenzoxyl, dansyl, or T-butyloxycarbonyl; an acetyl group; a 9-fluorenylmethoxy-carbonyl (FMOC) group; a macromolecular carrier group including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.
“Z” may represent a carboxyl group; an amido group; a T-butyloxycarbonyl group; a macromolecular carrier group including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.
Additionally,
“X” may represent an amino group, a hydrophobic group, including but not limited to carbobenzoxyl, dansyl, or T-butyloxycarbonyl; an acetyl group; a 9-fluorenylmethoxy-carbonyl (FMOC) group; a macromolecular carrier group including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.
“Z” may represent a carboxyl group; an amido group; a T-butyloxycarbonyl group; a macromolecular carrier group including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.
Additionally,
“X” may represent an amino group, a hydrophobic group, including but not limited to carbobenzoxyl, dansyl, or T-butyloxycarbonyl; an acetyl group; a 9-fluorenylmethoxy-carbonyl (FMOC) group; a macromolecular carrier group including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.
“Z” may represent a carboxyl group; an amido group; a T-butyloxycarbonyl group; a macromolecular carrier group including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.
The peptides of the invention may be synthesized or prepared by techniques well known in the art. See, for example, Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman and Co., NY, which is incorporated herein by reference in its entirety. Short peptides, for example, can be synthesized on a solid support or in solution. Longer peptides may be made using recombinant DNA techniques. Here, the nucleotide sequences encoding the peptides of the invention may be synthesized, and/or cloned, and expressed according to techniques well known to those of ordinary skill in the art. See, for example, Sambrook, et al., 1989, Molecular Cloning, A Laboratory Manual, Vols. 1-3, Cold Spring Harbor Press, NY.
The peptides of the invention may alternatively be synthesized such that one or more of the bonds which link the amino acid residues of the peptides are non-peptide bonds. These alternative non-peptide bonds may be formed by utilizing reactions well known to those in the art, and may include, but are not limited to imino, ester, hydrazide, semicarbazide, and azo bonds, to name but a few. In yet another embodiment of the invention, peptides comprising the sequences described above may be synthesized with additional chemical groups present at their amino and/or carboxy termini, such that, for example, the stability, bioavailability, and/or inhibitory activity of the peptides is enhanced. For example, hydrophobic groups such as carbobenzoxyl, dansyl, or t-butyloxycarbonyl groups, may be added to the peptides' amino termini. Likewise, an acetyl group or a 9-fluorenylmethoxy-carbonyl group may be placed at the peptides' amino termini. (See “X” in Tables I to IV, above.) Additionally, the hydrophobic group, t-butyloxycarbonyl, or an amido group may be added to the peptides' carboxy termini. (See “Z” in Tables I to IV, above.)
Further, the peptides of the invention may be synthesized such that their steric configuration is altered. For example, the D-isomer of one or more of the amino acid residues of the peptide may be used, rather than the usual L-isomer.
Still further, at least one of the amino acid residues of the peptides of the invention may be substituted by one of the well known non-naturally occurring amino acid residues. Alterations such as these may serve to increase the stability, bioavailability and/or inhibitory action of the peptides of the invention.
Any of the peptides described above may, additionally, have a macromolecular carrier group covalently attached to their amino and/or carboxy termini. Such macromolecular carrier groups may include, for example, lipid-fatty acid conjugates, polyethylene glycol, carbohydrates or additional peptides. “X”, in Tables I to IV, above, may therefore additionally represent any of the above macromolecular carrier groups covalently attached to the amino terminus of a peptide, with an additional peptide group being preferred. Likewise, “Z”, in Tables I to IV, may additionally represent any of the macromolecular carrier groups described above.
Described herein, are methods for ability of a compound, such as the peptides of the invention, to inhibit membrane fusion events. Specifically, assays for cell fusion events are described in Section 5.5.1, below, and assays for antiviral activity are described in Section 5.5.2, below.
5.5.1. Assays for Cell Fusion Events
Assays for cell fusion events are well known to those of skill in the art, and may be used in conjunction, for example, with the peptides of the invention to test the peptides' antifusogenic capabilities.
Cell fusion assays are generally performed in vitro. Such an assay may comprise culturing cells which, in the absence of any treatment would undergo an observable level of syncytial formation. For example, uninfected cells may be incubated in the presence of cells chronically infected with a virus that induces cell fusion. Such viruses may include, but are not limited to, HIV, SIV, or respiratory syncytial virus.
For the assay, cells are incubated in the presence of a peptide to be assayed. For each peptide, a range of peptide concentrations may be tested. This range should include a control culture wherein no peptide has been added.
Standard conditions for culturing cells, well known to those of ordinary skill in the art, are used. After incubation for an appropriate period (24 hours at 37° C., for example) the culture is examined microscopically for the presence of multinucleated giant cells, which are indicative of cell fusion and syncytial formation. Well known stains, such as crystal violet stain, may be used to facilitate the visualization of syncytial formation.
5.5.2. Assays for Antiviral Activity
The antiviral activity exhibited by the peptides of the invention may be measured, for example, by easily performed in vitro assays, such as those described below, which can test the peptides' ability to inhibit syncytia formation, or their ability to inhibit infection by cell-free virus. Using these assays, such parameters as the relative antiviral activity of the peptides, exhibit against a given strain of virus and/or the strain specific inhibitory activity of the peptide can be determined.
A cell fusion assay may be utilized to test the peptides' ability to inhibit viral-induced, such as HIV-induced, syncytia formation in vitro. Such an assay may comprise culturing uninfected cells in the presence of cells chronically infected with a syncytial-inducing virus and a peptide to be assayed. For each peptide, a range of peptide concentrations may be tested. This range should include a control culture wherein no peptide has been added. Standard conditions for culturing, well known to those of ordinary skill in the art, are used. After incubation for an appropriate period (24 hours at 37° C., for example) the culture is examined microscopically for the presence of multinucleated giant cells, which are indicative of cell fusion and syncytia formation. Well known stains, such as crystal violet stain, may be used to facilitate syncytial visualization. Taking HIV as an example, such an assay would comprise CD-4+ cells (such as Molt or CEM cells, for example) cultured in the presence of chronically HIV-infected cells and a peptide to be assayed.
Other well known characteristics of viral infection may also be assayed to test a peptide's antiviral capabilities. Once again taking HIV as an example, a reverse transcriptase (RT) assay may be utilized to test the peptides' ability to inhibit infection of CD-4+ cells by cell-free HIV. Such an assay may comprise culturing an appropriate concentration (i.e., TCID50) of virus and CD-4+ cells in the presence of the peptide to be tested. Culture conditions well known to those in the art are used. As above, a range of peptide concentrations may be used, in addition to a control culture wherein no peptide has been added. After incubation for an appropriate period (e.g., 7 days) of culturing, a cell-free supernatant is prepared, using standard procedures, and tested for the present of RT activity as a measure of successful infection. The RT activity may be tested using standard techniques such as those described by, for example, Goff et al. (Goff, S. et al., 1981, J. Virol. 38:239-248) and/or Willey et al. (Willey, R. et al., 1988, J. Virol. 62:139-147). These references are incorporated herein by reference in their entirety.
Standard methods which are well-known to those of skill in the art may be utilized for assaying non-retroviral activity. See, for example, Pringle et al. (Pringle, C. R. et al., 1985, J. Medical Virology 17:377-386) for a discussion of respiratory syncytial virus and parainfluenza virus activity assay techniques. Further, see, for example, “Zinsser Microbiology”, 1988, Joklik, W. K. et al., eds., Appleton & Lange, Norwalk, Conn., 19th ed., for a general review of such techniques. These references are incorporated by reference herein in their entirety. In addition, the Examples presented below, in Sections 17, 18, 26 and 27 each provide additional assays for the testing of a compound's antiviral capability.
In vivo assays may also be utilized to test, for example, the antiviral activity of the peptides of the invention. To test for anti-HIV activity, for example, the in vivo model described in Barnett et al. (Barnett, S. W. et al., 1994, Science 266:642-646) may be used.
Additionally, anti-RSV activity can be assayed in vivo via well known mouse models. For example, RSV can be administered intranasally to mice of various inbred strains. Virus replicates in lungs of all strains, but the highest titers are obtained in P/N, C57L/N and DBA/2N mice. Infection of BALB/c mice produces an asymptomatic bronchiolitis characterized by lymphocytic infiltrates and pulmonary virus titers of 104 to 105 pfu/g of lung tissue (Taylor, G. et al., 1984, Infect. Immun. 43:649-655).
Cotton rat models of RSV are also well known. Virus replicates to high titer in the nose and lungs of the cotton rat but produces few if any signs of inflammation.
The peptides of the invention may be utilized as antifusogenic or antiviral compounds, or as compounds which modulate intracellular processes involving coiled coil peptide structures. Further, such peptides may be used to identify agents which exhibit antifusogenic, antiviral or intracellular modulatory activity. Still further, the peptides of the invention may be utilized as organism or viral type/subtype-specific diagnostic tools.
The antifusogenic capability of the peptides of the invention may additionally be utilized to inhibit or treat/ameliorate symptoms caused by processes involving membrane fusion events. Such events may include, for example, virus transmission via cell-cell fusion, abnormal neurotransmitter exchange via cell-fusion, and sperm-egg fusion. Further, the peptides of the invention may be used to inhibit free viral, such as retroviral, particularly HIV, transmission to uninfected cells wherein such viral infection involves membrane fusion events or involves fusion of a viral structure with a cell membrane. Among the intracellular disorders involving coiled coil peptides structures which may be ameliorated by the peptides of the invention are disorders involving, for example, bacterial toxins.
With respect to antiviral activity, the viruses whose transmission may be inhibited by the peptides of the invention include, but are not limited to all strains of the viruses listed above, in Tables V through VII, and 1× through XIV.
These viruses include, for example, human retroviruses, particularly HIV-1 and HIV-2 and the human T-lymphocyte viruses (HTLV-I and II). The non-human retroviruses whose transmission may be inhibited by the peptides of the invention include, but are not limited to bovine leukosis virus, feline sarcoma and leukemia viruses, simian immunodeficiency, sarcoma and leukemia viruses, and sheep progress pneumonia viruses.
Non retroviral viruses whose transmission may be inhibited by the peptides of the invention include, but are not limited to human respiratory syncytial virus, canine distemper virus, newcastle disease virus, human parainfluenza virus, influenza viruses, measles viruses, Epstein-Barr viruses, hepatitis B viruses, and simian Mason-Pfizer viruses.
Non enveloped viruses whose transmission may be inhibited by the peptides of the invention include, but are not limited to picornaviruses such as polio viruses, hepatitis A virus, enterovirus, echoviruses and coxsackie viruses, papovaviruses such as papilloma virus, parvoviruses, adenoviruses and reoviruses.
As discussed more fully, below, in Section 5.5.1 and in the Example presented, below, in Section 8, DP107, DP178, DP107 analog and DP178 analog peptides form non-covalent protein-protein interactions which are required for normal activity of the virus. Thus, the peptides of the invention may also be utilized as components in assays for the identification of compounds that interfere with such protein-protein interactions and may, therefore, act as antiviral agents. These assays are discussed, below, in Section 5.5.1.
As demonstrated in the Example presented below in Section 6, the antiviral activity of the peptides of the invention may show a pronounced type and subtype specificity, i.e., specific peptides may be effective in inhibiting the activity of only specific viruses. This feature of the invention presents many advantages. One such advantage, for example, lies in the field of diagnostics, wherein one can use the antiviral specificity of the peptide of the invention to ascertain the identity of a viral isolate. With respect to HIV, one may easily determine whether a viral isolate consists of an HIV-1 or HIV-2 strain. For example, uninfected CD-4+ cells may be co-infected with an isolate which has been identified as containing HIV the DP178 (SEQ ID: 1) peptide, after which the retroviral activity of cell supernatants may be assayed, using, for example, the techniques described above in Section 5.2. Those isolates whose retroviral activity is completely or nearly completely inhibited contain HIV-1. Those isolates whose viral activity is unchanged or only reduced by a small amount, may be considered to not contain HIV-1. Such an isolate may then be treated with one or more of the other DP178 peptides of the invention, and subsequently be tested for its viral activity in order to determine the identify of the viral isolate. The DP107 and DP178 analogs of the invention may also be utilized in a diagnostic capacity specific to the type and subtype of virus or organism in which the specific peptide sequence is found. A diagnostic procedure as described, above, for DP178, may be used in conjunction with the DP107/DP178 analog of interest.
5.6.1. Screening Assays
As demonstrated in the Example presented in Section 8, below, DP107 and DP178 portions of the TM protein gp41 form non-covalent protein-protein interactions. As is also demonstrated, the maintenance of such interactions is necessary for normal viral infectivity. Thus, compounds which bind DP107, bind DP178, and/or act to disrupt normal DP107/DP178 protein-protein interactions may act as antifusogenic, antiviral or cellular modulatory agents. Described below are assays for the identification of such compounds. Note that, while, for ease and clarity of discussion, DP107 and DP178 peptides will be used as components of the assays described, but it is to be understood that any of the DP107 analog or DP178 analog peptides described, above, in Sections 5.1 through 5.3 may also be utilized as part of these screens for compounds.
Compounds which may be tested for an ability to bind DP107, DP178, and/or disrupt DP107/DP178 interactions, and which therefore, potentially represent antifusogenic, antiviral or intracellular modulatory compounds, include, but are not limited to, peptides made of D- and/or L-configuration amino acids (in, for example, the form of random peptide libraries; see Lam, K. S. et al. 1991, Nature 354:82-84), phosphopeptides (in, for example, the form of random or partially degenerate, directed phosphopeptide libraries; see, for example, Songyang, Z. et al., 1993, Cell 72:767-778), antibodies, and small organic or inorganic molecules. Synthetic compounds, natural products, and other sources of potentially effective materials may be screened in a variety of ways, as described in this Section.
The compounds, antibodies, or other molecules identified may be tested, for example, for an ability to inhibit cell fusion or viral activity, utilizing, for example, assays such as those described, above, in Section 5.5.
Among the peptides which may be tested are soluble peptides comprising DP107 and/or DP178 domains, and peptides comprising DP107 and/or DP178 domains having one or more mutations within one or both of the domains, such as the M41-P peptide described, below, in the Example presented in Section 8, which contains a isoleucine to proline mutation within the DP178 sequence.
In one embodiment of such screening methods is a method for identifying a compound to be tested for antiviral ability comprising:
In a second embodiment of such screening methods is a method for identifying a compound to be tested for antiviral ability comprising:
One method utilizing these types of approaches that may be pursued in the isolation of such DP107-binding or DP178-binding compounds is an assay which would include the attachment of either the DP107 or the DP178 peptide to a solid matrix, such as, for example, agarose or plastic beads, microtiter plate wells, petri dishes, or membranes composed of, for example, nylon or nitrocellulose. In such an assay system, either the DP107 or DP178 protein may be anchored onto a solid surface, and the compound, or test substance, which is not anchored, is labeled, either directly or indirectly. In practice, microtiter plates are conveniently utilized. The anchored component may be immobilized by non-covalent or covalent attachments. Non-covalent attachment may be accomplished simply by coating the solid surface with a solution of the protein and drying. Alternatively, an immobilized antibody, preferably a monoclonal antibody, specific for the protein may be used to anchor the protein to the solid surface. The surfaces may be prepared in advance and stored.
In order to conduct the assay, the labeled compound is added to the coated surface containing the anchored DP107 or DP178 peptide. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the compound is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the labeled component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the compound (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody).
Alternatively, such an assay can be conducted in a liquid phase, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for DP107 or DP178, whichever is appropriate for the given assay, or ab antibody specific for the compound, i.e., the test substance, in order to anchor any complexes formed in solution, and a labeled antibody specific for the other member of the complex to detect anchored complexes.
By utilizing procedures such as this, large numbers of types of molecules may be simultaneously screened for DP107 or DP178-binding capability, and thus potential antiviral activity.
Further, compounds may be screened for an ability to inhibit the formation of or, alternatively, disrupt DP107/DP178 complexes. Such compounds may then be tested for antifusogenic, antiviral or intercellular modulatory capability. For ease of description, DP107 and DP178 will be referred to as “binding partners.” Compounds that disrupt such interactions may exhibit antiviral activity. Such compounds may include, but are not limited to molecules such as antibodies, peptides, and the like described above.
The basic principle of the assay systems used to identify compounds that interfere with the interaction between the DP107 and DP178 peptides involves preparing a reaction mixture containing peptides under conditions and for a time sufficient to allow the two peptides to interact and bind, thus forming a complex. In order to test a compound for disruptive activity, the reaction is conducted in the presence and absence of the test compound, i.e., the test compound may be initially included in the reaction mixture, or added at a time subsequent to the addition of one of the binding partners; controls are incubated without the test compound or with a placebo. The formation of any complexes between the binding partners is then detected. The formation of a complex in the control reaction, but not in the reaction mixture containing the test compound indicates that the compound interferes with the interaction of the DP107 and DP178 peptides.
The assay for compounds that interfere with the interaction of the binding partners can be conducted in a heterogeneous or homogeneous format. Heterogeneous assays involve anchoring one of the binding partners onto a solid phase and detecting complexes anchored on the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction between the binding partners, e.g., by competition, can be identified by conducting the reaction in the presence of the test substance; i.e., by adding the test substance to the reaction mixture prior to or simultaneously with the binding partners. On the other hand, test compounds that disrupt preformed complexes, e.g. compounds with higher binding constants that displace one of the binding partners from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed. The various formats are described briefly below.
In a heterogeneous assay system, one binding partner, e.g., either the DP107 or DP178 peptide, is anchored onto a solid surface, and its binding partner, which is not anchored, is labeled, either directly or indirectly. In practice, microtiter plates are conveniently utilized. The anchored species may be immobilized by non-covalent or covalent attachments. Non-covalent attachment may be accomplished simply by coating the solid surface with a solution of the protein and drying. Alternatively, an immobilized antibody specific for the protein may be used to anchor the protein to the solid surface. The surfaces may be prepared in advance and stored.
In order to conduct the assay, the binding partner of the immobilized species is added to the coated surface with or without the test compound. After the reaction is complete, unreacted components are removed (e.g., by washing) and any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the binding partner was pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the binding partner is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the binding partner (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody). Depending upon the order of addition of reaction components, test compounds which inhibit complex formation or which disrupt preformed complexes can be detected.
Alternatively, the reaction can be conducted in a liquid phase in the presence or absence of the test compound, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for one binding partner to anchor any complexes formed in solution, and a labeled antibody specific for the other binding partner to detect anchored complexes. Again, depending upon the order of addition of reactants to the liquid phase, test compounds which inhibit complex or which disrupt preformed complexes can be identified.
In an alternate embodiment of the invention, a homogeneous assay can be used. In this approach, a preformed complex of the DP107 and DP178 peptides is prepared in which one of the binding partners is labeled, but the signal generated by the label is quenched due to complex formation (see, e.g., U.S. Pat. No. 4,109,496 by Rubenstein which utilizes this approach for immunoassays). The addition of a test substance that competes with and displaces one of the binding partners from the preformed complex will result in the generation of a signal above background. In this way, test substances which disrupt DP-107/DP-178 protein-protein interaction can be identified.
In an alternative screening assay, test compounds may be assayed for the their ability to disrupt a DP178/DP107 interaction, as measured immunometrically using an antibody specifically reactive to a DP107/DP178 complex (i.e., an antibody that recognizes neither DP107 nor DP178 individually). Such an assay acts as a competition assay, and is based on techniques well known to those of skill in the art.
The above competition assay may be described, by way of example, and not by way of limitation, by using the DP178 and M41A178 peptides and by assaying test compounds for the disruption of the complexes formed by these two peptides by immunometrically visualizing DP178/M41Δ178 complexes via the human recombinant Fab, Fab-d, as described, below, in the Example presented in Section 8. M41Δ178 is a maltose binding fusion protein containing a gp41 region having its DP178 domain deleted, and is described, below, in the Example presented in Section 8.
Utilizing such an assay, M41Δ178 may be immobilized onto solid supports such as microtiter wells. A series of dilutions of a test compound may then be added to each M41Δ178-containing well in the presence of a constant concentration of DP-178 peptide. After incubation, at, for example, room temperature for one hour, unbound DP-178 and test compound are removed from the wells and wells are then incubated with the DP178/M41Δ178-specific Fab-d antibody. After incubation and washing, unbound Fab-d is removed from the plates and bound Fab-d is quantitated. A no-inhibitor control should also be conducted. Test compounds showing an ability to disrupt DP178/M41A178 complex formation are identified by their concentration-dependent decrease in the level of Fab-d binding.
A variation of such an assay may be utilized to perform a rapid, high-throughput binding assay which is capable of directly measuring DP178 binding to M41Δ178 for the determination of binding constants of the ligand of inhibitory constants for competitors of DP178 binding.
Such an assay takes advantage of accepted radioligand and receptor binding principles. (See, for example, Yamamura, H. I. et al., 1985, “Neurotransmitter Receptor Binding”, 2nd ed., Raven Press, NY.) As above, M41A178 is immobilized onto a solid support such as a microtiter well. DP178 binding to M41A178 is then quantitated by measuring the fraction of DP178 that is bound as 125I-DP178 and calculating the total amount bound using a value for specific activity (dpm/μg peptide) determined for each labeled DP178 preparation. Specific binding to M41A178 is defined as the difference of the binding of the labeled DP178 preparation in the microtiter wells (totals) and the binding in identical wells containing, in addition, excess unlabeled DP178 (nonspecifics).
The peptides of the invention may be administered using techniques well known to those in the art. Preferably, agents are formulated and administered systemically. Techniques for formulation and administration may be found in “Remington's Pharmaceutical Sciences”, 18th ed., 1990, Mack Publishing Co., Easton, Pa. Suitable routes may include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections, just to name a few. For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. For such transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
In instances wherein intracellular administration of the peptides of the invention or other inhibitory agents is preferred, techniques well known to those of ordinary skill in the art may be utilized. For example, such agents may be encapsulated into liposomes, then administered as described above. Liposomes are spherical lipid bilayers with aqueous interiors. All molecules present in an aqueous solution at the time of liposome formation are incorporated into the aqueous interior. The liposomal contents are both protected from the external microenvironment and, because liposomes fuse with cell membranes, are effectively delivered into the cell cytoplasm. Additionally, due to their hydrophobicity, when small molecules are to be administered, direct intracellular administration may be achieved.
Nucleotide sequences encoding the peptides of the invention which are to be intracellularly administered may be expressed in cells of interest, using techniques well known to those of skill in the art. For example, expression vectors derived from viruses such as retroviruses, vaccinia viruses, adeno-associated viruses, herpes viruses, or bovine papilloma viruses, may be used for delivery and expression of such nucleotide sequences into the targeted cell population. Methods for the construction of such vectors and expression constructs are well known. See, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor N.Y., and Ausubel et al., 1989, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, NY.
With respect to HIV, peptides of the invention, particularly DP107 and DP178, may be used as therapeutics in the treatment of AIDS. In addition, the peptides may be used as prophylacetic measures in previously uninfected individuals after acute exposure to an HIV virus. Examples of such prophylacetic use of the peptides may include, but are not limited to, prevention of virus transmission from mother to infant and other settings where the likelihood of HIV transmission exists, such as, for example, accidents in health care settings wherein workers are exposed to HIV-containing blood products. The successful use of such treatments do not rely upon the generation of a host immune response directed against such peptides.
Effective dosages of the peptides of the invention to be administered may be determined through procedures well known to those in the art which address such parameters as biological half-life, bioavailability, and toxicity. Given the data presented below in Section 6, DP178, for example, may prove efficacious in vivo at doses required to achieve circulating levels of about 1 to about 10 ng per ml of peptide.
A therapeutically effective dose refers to that amount of the compound sufficient to result in amelioration of symptoms or a prolongation of survival in a patient. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit large therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (e.g., the concentration of the test compound which achieves a half-maximal inhibition of the fusogenic event, such as a half-maximal inhibition of viral infection relative to the amount of the event in the absence of the test compound) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography (HPLC).
The peptides of the invention may, further, serve the role of a prophylacetic vaccine, wherein the host raises antibodies against the peptides of the invention, which then serve to neutralize HIV viruses by, for example, inhibiting further HIV infection. Administration of the peptides of the invention as a prophylacetic vaccine, therefore, would comprise administering to a host a concentration of peptides effective in raising an immune response which is sufficient to neutralize HIV, by, for example, inhibiting HIV ability to infect cells. The exact concentration will depend upon the specific peptide to be administered, but may be determined by using standard techniques for assaying the development of an immune response which are well known to those of ordinary skill in the art. The peptides to be used as vaccines are usually administered intramuscularly.
The peptides may be formulated with a suitable adjuvant in order to enhance the immunological response. Such adjuvants may include, but are not limited to mineral gels such as aluminum hydroxide; surface active substances such as lysolecithin, pluronic polyols, polyanions; other peptides; oil emulsions; and potentially useful human adjuvants such as BCG and Corynebacterium parvum. Many methods may be used to introduce the vaccine formulations described here. These methods include but are not limited to oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, and intranasal routes.
Alternatively, an effective concentration of polyclonal or monoclonal antibodies raised against the peptides of the invention may be administered to a host so that no uninfected cells become infected by HIV. The exact concentration of such antibodies will vary according to each specific antibody preparation, but may be determined using standard techniques well known to those of ordinary skill in the art. Administration of the antibodies may be accomplished using a variety of techniques, including, but not limited to those described in this section.
For all such treatments described above, the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g. Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch. 1 p1).
It should be noted that the attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity, or to organ dysfunctions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity). The magnitude of an administrated dose in the management of the oncogenic disorder of interest will vary with the severity of the condition to be treated and the route of administration. The dose and perhaps dose frequency, will also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above may be used in veterinary medicine.
Use of pharmaceutically acceptable carriers to formulate the compounds herein disclosed for the practice of the invention into dosages suitable for systemic administration is within the scope of the invention. With proper choice of carrier and suitable manufacturing practice, the compositions of the present invention, in particular, those formulated as solutions, may be administered parenterally, such as by intravenous injection. The compounds can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration. Such carriers enable the compounds of the invention to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. The preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions.
The pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
Pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added.
In this example, DP178 (SEQ ID: 1) is shown to be a potent inhibitor of HIV-1 mediated CD-4+ cell-cell fusion and infection by cell free virus. In the fusion assay, this peptide completely blocks virus induced syncytia formation at concentrations of from 1-10 ng/ml. In the infectivity assay the inhibitory concentration is somewhat higher, blocking infection at 90 ng/ml. It is further shown that DP178 (SEQ ID:1) shows that the antiviral activity of DP178 (SEQ ID:1) is highly specific for HIV-1. Additionally, a synthetic peptide, DP-185 (SEQ ID:3), representing a HIV-1-derived DP178 homolog is also found to block HIV-1-mediated syncytia formation.
6.1.1. Peptide Synthesis
Peptides were synthesized using Fast Moc chemistry on an Applied Biosystems Model 431A peptide synthesizer. Generally, unless otherwise noted, the peptides contained amidated carboxy termini and acetylated amino termini. Amidated peptides were prepared using Rink resin (Advanced Chemtech) while peptides containing free carboxy termini were synthesized on Wang (p-alkoxy-benzyl-alcohol) resin (Bachem). First residues were double coupled to the appropriate resin and subsequent residues were single coupled. Each coupling step was followed by acetic anhydride capping. Peptides were cleaved from the resin by treatment with trifluoracetic acid (TFA) (10 ml), H2O (0.5 ml), thioanisole (0.5 ml), ethanedithiol (0.25 ml), and crystalline phenol (0.75 g). Purification was carried out by reverse phase HPLC. Approximately 50 mg samples of crude peptide were chromatographed on a Waters Delta Pak C18 column (19 mm×30 cm, 15μ spherical) with a linear gradient; H2O/acetonitrile 0.1% TFA. Lyophilized peptides were stored desiccated and peptide solutions were made in water at about 1 mg/ml. Electrospray mass spectrometry yielded the following results: DP178 (SEQ ID:1):4491.87 (calculated 4491.94); DP-180 (SEQ ID:2):4491.45 (calculated 4491.94); DP-185 (SEQ ID:3):not done (calculated 4546.97).
6.1.2. Virus
The HIV-1LAI virus was obtained from R. Gallo (Popovic, M. et al., 1984, Science 224:497-508) and propagated in CEM cells cultured in RPMI 1640 containing 10% fetal calf serum. Supernatant from the infected CEM cells was passed through a 0.2 μm filter and the infectious titer estimated in a microinfectivity assay using the AA5 cell line to support virus replication. For this purpose, 2511 of serial diluted virus was added to 75 μl AA5 cells at a concentration of 2×105/ml in a 96-well microtitre plate. Each virus dilution was tested in triplicate. Cells were cultured for eight days by addition of fresh medium every other day. On day 8 post infection, supernatant samples were tested for virus replication as evidenced by reverse transcriptase activity released to the supernatant. The TCID50 was calculated according to the Reed and Muench formula (Reed, L. J. et al., 1938, Am. J. Hyg. 27:493-497). The titer of the HIV-1LAI and HIV-1MN stocks used for these studies, as measured on the AA5 cell line, was approximately 1.4×106 and 3.8×104 TCID50/ml, respectively.
6.1.3. Cell Fusion Assay
Approximately 7×104 Molt cells were incubated with 1×104 CEM cells chronically infected with the HIV-1LAI virus in 96-well plates (one-half area cluster plates; Costar, Cambridge, Mass.) in a final volume of 100 μl culture medium as previously described (Matthews, T. J. et al., 1987, Proc. Natl. Acad. Sci. USA 84: 5424-5428). Peptide inhibitors were added in a volume of 10 μl and the cell mixtures were incubated for 24 hr. at 37° C. At that time, multinucleated giant cells were estimated by microscopic examination at a 40× magnification which allowed visualization of the entire well in a single field.
6.1.4. Cell Free Virus Infection Assay
Synthetic peptides were incubated at 37° C. with either 247 TCID50 (for experiment depicted in
6.1.5. Reverse Transcriptase Assay
The micro-reverse transcriptase (RT) assay was adapted from Goff et al. (Goff, S. et al., 1981, J. Virol. 38:239-248) and Willey et al. (Willey, R. et al., 1988, J. Virol. 62:139-147). Supernatants from virus/cell cultures are adjusted to 1% Triton-X100. A 10 μl sample of supernatant was added to 50 μl of RT cocktail in a 96-well U-bottom microtitre plate and the samples incubated at 37° C. for 90 min. The RT cocktail contained 75 mM KCl, 2 mM dithiothreitol, 5 mM MgCl2, 5 μg/ml poly A (Pharmacia, cat. No. 27-4110-01), 0.25 units/ml oligo dT (Pharmacia, cat. No. 27-7858-01), 0.05% NP40, 50 mM Tris-HCl, pH 7.8, 0.5 μM non-radioactive dTTP, and 10 μCi/ml 32P-dTTP (Amersham, cat. No. PB.10167).
After the incubation period, 40 μl of reaction mixture was applied to a Schleicher and Schuell (S+S) NA45 membrane (or DE81 paper) saturated in 2×SSC buffer (0.3M NaCl and 0.003M sodium citrate) held in a S+S Minifold over one sheet of GB003 (S+S) filter paper, with partial vacuum applied. Each well of the minifold washed four times with 200 μl 2×SSC, under full vacuum. The membrane was removed from the minifold and washed 2 more times in a pyrex dish with an excess of 2×SSC. Finally, the membrane was drained on absorbent paper, placed on Whatman #3 paper, covered with Saran wrap, and exposed to film overnight at −70° C.
6.2.1. Peptide Inhibition of Infected Cell-Induced Syncytia Formation
The initial screen for antiviral activity assayed peptides' ability to block syncytium formation induced by overnight co-cultivation of uninfected Molt4 cells with chronically HIV-1 infected CEM cells. The results of several such experiments are presented herein. In the first of these experiments, serial DP178 (SEQ ID: 1) peptide concentrations between 10 μg/ml and 12.5 ng/ml were tested for blockade of the cell fusion process. For these experiments, CEM cells chronically infected with either HIV-1LAI, HIV-1MN, HIV-1RF, or HIV-1SF2 virus were cocultivated overnight with uninfected Molt 4 cells. The results (
The next series of experiments involved the preparation and testing of a DP178 (SEQ ID:1) homolog for its ability to inhibit HIV-1-induced syncytia formation. As shown in
The next series of experiments involved a comparison of DPI 78 (SEQ ID: 1) HIV-1 and HIV-2 inhibitory activity. As shown in
6.2.2. Peptide Inhibition of Infection by Cell-Free Virus
DP178 (SEQ ID:1) was next tested for its ability to block CD-4+ CEM cell infection by cell free HIV-1 virus. The results, shown in
In a separate experiment, the HIV-1 and HIV-2 inhibitory action of DP178 (SEQ ID:1) was tested with CEM cells and either HIV-1LAI or HIV-2NIHZ. 62 TCID50 HIV-1LAI or 25 GCID50 HIV-2NIHZ were used in these experiments, and were incubated for 7 days. As may be seen in
In this Example, the 36 amino acid synthetic peptide inhibitor DP178 (SEQ ID: 1) is shown to be non-cytotoxic to cells in culture, even at the highest peptide concentrations (40 μg/ml) tested.
Cell proliferation and toxicity assay: Approximately 3.8×105 CEM cells for each peptide concentration were incubated for 3 days at 37° C. in T25 flasks. Peptides tested were DP178 (SEQ ID:1) and DP-116 (SEQ ID:9), as described in
Whether the potent HIV-1 inhibitor DP178 (SEQ ID:1) exhibited any cytotoxic effects was assessed by assaying the peptide's effects on the proliferation and viability of cells in culture. CEM cells were incubated in the presence of varying concentrations of DP178 (SEQ ID: 1), and DP-116 (SEQ ID:9), a peptide previously shown to be ineffective as a HIV inhibitor (Wild, C. et al., 1992, Proc. Natl. Acad. Sci. USA 89:10, 537-10,541). Additionally, cells were incubated in the absence of either peptide.
The results of the cytotoxicity study demonstrate that DP178 (SEQ ID: 1) exhibits no cytotoxic effects on cells in culture. As can be seen, below, in Table XXIV, even the proliferation and viability characteristics of cells cultured for 3 days in the presence of the highest concentration of DP178 (SEQ ID: 1) tested (40 μg/ml) do not significantly differ from the DP-116 (SEQ ID:9) or the no-peptide controls. The cell proliferation data is also represented in graphic form in
Soluble recombinant forms of gp41 used in the example described below provide evidence that the DP178 peptide associates with a distal site on gp41 whose interactive structure is influenced by the DP107 leucine zipper motif. A single mutation disrupting the coiled-coil structure of the leucine zipper domain transformed the soluble recombinant gp41 protein from an inactive to an active inhibitor of HIV-1 fusion. This transformation may result from liberation of the potent DP178 domain from a molecular clasp with the leucine zipper, DP107, determinant. The results also indicate that the anti-HIV activity of various gp41 derivatives (peptides and recombinant proteins) may be due to their ability to form complexes with viral gp41 and interfere with its fusogenic process.
8.1.1. Construction of Fusion Proteins and gp41 Mutants
Construction of fusion proteins and mutants shown in
8.1.2. Purification and Characterization of Fusion Proteins
The fusion proteins were purified according to the protocol described in the manufacturer's brochure of protein fusion and purification systems from New England Biolabs (NEB). Fusion proteins (10 ng) were analyzed by electrophoresis on 8% SDS polyacrylamide gels. Western blotting analysis was performed as described by Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2d Ed, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., Ch. 18, pp. 64-75. An HIV-1 positive serum diluted 1000-fold, or a human Fab derived from repertoire cloning was used to react with the fusion proteins. The second antibody was HRP-conjugated goat antihuman Fab. An ECL Western blotting detection system (Amersham) was used to detect the bound antibody. A detailed protocol for this detection system was provided by the manufacturer. Rainbow molecular weight markers (Amersham) were used to estimate the size of fusion proteins.
8.1.3. Cell Fusion Assays for Anti-HIV Activity
Cell fusion assays were performed as previously described (Matthews et al., 1987, Proc. Natl. Acad. Sci. USA 84: 5424-5481). CEM cells (7×104) were incubated with HIV-1IIIB chronically infected CEM cells (104) in 96-well flat-bottomed half-area plates (Costar) in 100 μl culture medium. Peptide and fusion proteins at various concentrations in 10 μl culture medium were incubated with the cell mixtures at 37° C. for 24 hours. Multinucleated syncytia were estimated with microscopic examination. Both M41 and M41-P did not show cytotoxicity at the concentrations tested and shown in
Inhibition of HIV-1 induced cell-cell fusion activity was carried out in the presence of 10 nM DP178 and various concentrations of M41A178 or M41-PΔ178 as indicated in
8.1.4. Elisa Analysis of DP178 Binding to the Leucine Zipper Motif of GP41
The amino acid sequence of DP178 used is: YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF. For enzyme linked immunoassay (ELISA), M41Δ178 or M41-PΔ178 (5 μg/ml) in 0.1M NaHCO3, pH 8.6, were coated on 96 wells Linbro ELISA plates (Flow Lab, Inc.) overnight. Each well was washed three times with distilled water then blocked with 3% bovine serum albumin (BSA) for 2 hours. After blocking, peptides with 0.5% BSA in TBST (40 mM Tris-HCl pH7.5, 150 mM NaCl, 0.05% Tween 20) were added to the ELISA plates and incubated at room temperature for 1 hour. After washing three times with TBST, Fab-d was added at a concentration of 10 ng/ml with 0.5% BSA in TBST. The plates were washed three times with TBST after incubation at room temperature for 1 hour. Horse radish peroxidase (HRP) conjugated goat antihuman Fab antiserum at a 2000 fold dilution in TBST with 0.5% BSA was added to each well and incubated at room temperature for 45 minutes. The plates were then washed four times with TBST. The peroxidase substrate o-phenylene diamine (2.5 mg/ml) and 0.15% H2O2 were added to develop the color. The reaction was stopped with an equal volume of 4.5 N H2SO4 after incubation at room temperature for 10 minutes. The optical density of the stopped reaction mixture was measured with a micro plate reader (Molecular Design) at 490 nm. Results are shown in
8.2.1. The Expression and Characterization of the Ectodomain of gp41
As a step toward understanding the roles of the two helical regions in gp41 structure and function, the ectodomain of gp41 was expressed as a maltose binding fusion protein (M41) (
8.2.2. Anti-HIV Activity of the Recombinant Ectodomain of gp41
The wild type M41 fusion protein was tested for anti-HIV-1 activity. As explained, supra, synthetic peptides corresponding to the leucine zipper (DP107) and the C-terminal putative helix (DP178) show potent anti-HIV activity. Despite inclusion of both these regions, the recombinant M41 protein did not affect HIV-1 induced membrane fusion at concentrations as high as 50 μM (Table XXV, below).
1The affinity constants of Fab-d binding to the fusion proteins were determined using a protocol described by B. Friguet et al., 1985, J. Immunol, Method, 77:305-319.
Surprisingly, a single amino acid substitution, proline in place of isoleucine in the middle of the leucine zipper motif, yielded a fusion protein (M41-P) which did exhibit antiviral activity (Table XXV and
A specific association between these two domains is also indicated by other human monoclonal Fab-d studies. For example, Fab-d failed to bind either the DP178 peptide or the fusion protein M41Δ178, but its epitope was reconstituted by simply mixing these two reagents together (
A number of known coiled-coil sequences have been well described in the literature and contain heptad repeat positioning for each amino acid. Coiled-coil nomenclature labels each of seven amino acids of a heptad repeat A through G, with amino acids A and D tending to be hydrophobic positions. Amino acids E and G tend to be charged. These four positions (A, D, E, and G) form the amphipathic backbone structure of a monomeric alpha-helix. The backbones of two or more amphipathic helices interact with each other to form di-, tri-, tetrameric, etc., coiled-coil structures. In order to begin to design computer search motifs, a series of well characterized coiled coils were chosen including yeast transcription factor GCN4, Influenza Virus hemagglutinin loop 36, and human proto-oncogenes c-Myc, c-Fos, and c-Jun. For each peptide sequence, a strict homology for the A and D positions, and a list of the amino acids which could be excluded for the B, C, E, F, and G positions (because they are not observed in these positions) was determined. Motifs were tailored to the DP107 and DP178 sequences by deducing the most likely possibilities for heptad positioning of the amino acids of HIV-1 Bru DP-107, which is known to have coiled-coil structure, and HIV-1 Bru DP178, which is still structurally undefined. The analysis of each of the sequences is contained in
[LMNV]-{CFGIMPTW}(2)-[LMNV]-{CFGIMPTW}(3)-
[LMNV]-{CFGIMPTW}(2)-[LMNV]-{CFGIMPTW}(3)-
[LMNV]-{CFGIMPTW}(2)-[LMNV]-{CFGIMPTW}(3)-
[LMNV]-{CFGIMPTW}(2)-[LMNV]-{CFGIMPTW}(3)
Translating or reading the motif: “at the first A position either L, M, N, or V must occur; at positions B and C (the next two positions) accept everything except C, F, G, I, M, P, T, or W; at the D position either L, M, N, or V must occur; at positions E, F, and G (the next 3 positions) accept everything except C, F, G, I, M, P, T, or W.” This statement is contained four times in a 28-mer motif and five times in a 35-mer motif. The basic motif key then would be: [LMNV]-{CFGIMPTW}. The motif keys for the remaining well described coiled-coil sequences are summarized in
The motif design for DP107 and DP178 was slightly different than the 28-mer model sequences described above due to the fact that heptad repeat positions are not defined and the peptides are both longer than 28 residues.
In addition to making highly specific motifs for each type peptide sequence to be searched, it is also possible to make “hybrid” motifs. These motifs are made by “crossing” two or more very stringent motifs to make a new search algorithm which will find not only both “parent” motif sequences but also any peptide sequences which have similarities to one, the other, or both “parents”. For example, in
Hybridizations can be performed on any combination of two or more motifs.
A special set of motifs was designed based on the fact that DP-178 is located only approximately ten amino acids upstream of the transmembrane spanning region of gp41 and just C-terminal to a proline which separates DP107 and DP178. It has been postulated that DP178 may be an amphipathic helix when membrane associated, and that the proline might aid in the initiation of the helix formation. The same arrangement was observed in Respiratory Syncytial Virus; however, the DP178-like region in this virus also had a leucine zipper just C-terminal to the proline. Therefore, N-terminal proline-leucine zipper motifs were designed to analyze whether any other viruses might contain this same pattern. The motifs are summarized in
The PC/Gene protein database contains 5879 viral amino acid sequences (library file PVIRUSES; CD-ROM release 11.0). Of these, 1092 are viral enveloped or glycoprotein sequences (library file PVIRUSE1). Tables V through XIV contain lists of protein sequence names and motif hit locations for all the motifs searched.
Motif hits for Simian immunodeficiency Virus gp41 (AGM3 isolate; PC/Gene protein sequence name PENV_SIVAG) are shown in
The identification of DP178/DP107 analogs for a second SIV isolate (MM251) is demonstrated in the Example presented, below, in Section 19.
Canine Distemper Virus (strain Onderstepoort) fusion glycoprotein F1 (PC/Gene Protein sequence name PVGLF_CDVO) has regions similar to Human RSV which are predicted to be DP107-like and DP178-like (
Both motifs 107×178×4 and ALLMOTI5 exhibit DP107-like hits in the same region, 115-182 and 117-182 respectively, of Human Parainfluenza Virus (strain NIH 47885; PC/Gene protein sequence name PVGLF_p13H4; (
In the Example presented herein, respiratory syncytial virus (RSV) peptides identified by utilizing the computer-assisted search motifs described in the Examples presented in Sections 9 and 11, above, were tested for anti-RSV activity. Additionally, circular dichroism (CD) structural analyses were conducted on the peptides, as discussed below. It is demonstrated that several of the identified peptides exhibit potent antiviral capability. Additionally, it is shown that several of these peptides exhibit a substantial helical character.
Structural analyses: The CD spectra were measured in a 10 mM sodium phosphate, 150 mM sodium chloride, pH 7.0, buffer at approximately 10 mM concentrations, using a 1 cm pathlength cell on a Jobin/Yvon Autodichrograph Mark V CD spectrophotometer. Peptides were synthesized according to the methods described, above, in Section 6.1. Peptide concentrations were determined from A280 using Edlehoch's method (1967, Biochemistry 6:1948).
Anti-RSV antiviral activity assays: The assay utilized herein tested the ability of the peptides to disrupt the ability of HEp2 cells acutely infected with RSV (i.e., cells which are infected with a multiplicity of infection of greater than 2) to fuse and cause syncytial formation on a monolayer of uninfected an uninfected line of Hep-2 cells. The lower the observed level of fusion, the greater the antiviral activity of the peptide was determined to be.
Uninfected confluent monolayers of Hep-2 cells were grown in microtiter wells in 3% EMEM (Eagle Minimum Essential Medium w/o L-glutamine [Bio Whittaker Cat. No. 12-125F], with fetal bovine serum [FBS; which had been heat inactivated for 30 minutes at 56° C.; Bio Whittaker Cat. No. 14-501F) supplemented at 3%, antibiotics (penicillin/streptomycin; Bio Whittaker Cat. No. 17-602E) added at 1%, and glutamine added at 1%.
To prepare Hep2 cells for addition to uninfected cells, cultures of acutely infected Hep2 cells were washed with DPBS (Dulbecco's Phosphate Buffered Saline w/o calcium or magnesium; Bio Whittaker Cat. No. 17-512F) and cell monolayers were removed with Versene (1:5000; Gibco Life Technologies Cat. No. 15040-017). The cells were spun 10 minutes and resuspended in 3% FBS. Cell counts were performed using a hemacytometer. Persistent cells were added to the uninfected Hep-2 cells.
The antiviral assay was conducted by, first, removing all media from the wells containing uninfected Hep-2 cells, then adding peptides (at the dilutions described below) in 3% EMEM, and 100 acutely RSV-infected Hep2 cells per well. Wells were then incubated at 37° C. for 48 hours.
After incubation, cells in control wells were checked for fusion centers, media was removed from the wells, followed by addition, to each well, of either Crystal Violet stain or XTT. With respect to Crystal Violet, approximately 50 μl 0.25% Crystal Violet stain in methanol were added to each well. The wells were rinsed immediately, to remove excess stain, and were allowed to dry. The number of syncytia per well were then counted, using a dissecting microscope.
With respect to XTT (2,3-bis[2-Methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide inner salt), 50 μl XTT (1 mg/ml in RPMI buffered with 100 mM HEPES, pH 7.2-7.4, plus 5% DMSO) were added to each well. The OD450/690 was measured (after blanking against growth medium without cells or reagents, and against reagents) according to standard procedures.
Peptides: The peptides characterized in the study presented herein were:
1) peptides T-142 to T-155 and T-575, as shown in
2) peptides T-120 to T-141 and T-576, as shown in
3) peptides T-67 and T-104 to T-119 and T-384, as shown in
The peptides of group 1 represent portions of the RSV F2 protein DP178/107-like region. The peptides of group 2 represent portions of the RSV F1 protein DP107-like region. The peptides of groups 3 represent portions of the RSV F1 protein DP178-like region.
Each peptide was tested at 2-fold serial dilutions ranging from 100 μg/ml to approximately 100 ng/ml. For each of the assays, a well containing no peptide was also used. The IC50 data for each peptide represents the average of several experiments conducted utilizing that peptide.
The data summarized in
As shown in
The results summarized in
Thus, the computer assisted searches described, hereinabove, successfully identified viral peptide domains that represent highly promising anti-RSV antiviral compounds.
In the Example presented herein, human parainfluenza virus type 3 (HPIV3) peptides identified by utilizing the computer-assisted search motifs described in the Examples presented in Sections 9 and 15, above, were tested for anti-HPIV3 activity. Additionally, circular dichroism (CD) structural analyses were conducted on the peptides, as discussed below. It is demonstrated that several of the identified peptides exhibit potent antiviral capability. Additionally, it is shown that several of these peptides exhibit a substantial helical character.
Structural analyses: Structural analyses consisted of circular dichroism (CD) studies. The CD spectra were measured in a 10 mM sodium phosphate, 150 mM sodium chloride, pH 7.0, buffer at approximately 10 mM concentrations, using a 1 cm pathlength cell on a Jobin/Yvon Autodichrograph Mark V CD spectrophotometer. Peptide concentrations were determined from A280 using Edlehoch's method (1967, Biochemistry 6:1948).
Anti-HPIV3 antiviral activity assays: The assay utilized herein tested the ability of the peptides to disrupt the ability of Hep2 cells chronically infected with HPIV3 to fuse and cause syncytial formation on a monolayer of an uninfected line of CV-1W cells. The more potent the lower the observed level of fusion, the greater the antiviral activity of the peptide.
Uninfected confluent monolayers of CV-1W cells were grown in microtiter wells in 3% EMEM (Eagle Minimum Essential Medium w/o L-glutamine [Bio Whittaker Cat. No. 12-125F], with fetal bovine serum [FBS; which had been heat inactivated for 30 minutes at 56° C.; Bio Whittaker Cat. No. 14-501F) supplemented at 3%, antibiotics/antimycotics (Gibco BRL Life Technologies Cat. No. 15040-017) added at 1%, and glutamine added at 1%.
To prepare Hep2 cells for addition to uninfected cells, cultures of chronically infected Hep2 cells were washed with DPBS (Dulbecco's Phosphate Buffered Saline w/o calcium or magnesium; Bio Whittaker Cat. No. 17-512F) and cell monolayers were removed with Versene (1:5000; Gibco Life Technologies Cat. No. 15040-017). The cells were spun 10 minutes and resuspended in 3% FBS. Cell counts were performed using a hemacytometer. Persistent cells were added to the uninfected CV-1W cells.
The antiviral assay was conducted by, first, removing all media from the wells containing uninfected CV-1W cells, then adding peptides (at the dilutions described below) in 3% EMEM, and 500 chronically HPIV3-infected Hep2 cells per well. Wells were then incubated at 37° C. for 24 hours.
On day 2, after cells in control wells were checked for fusion centers, media was removed from the wells, followed by addition, to each well, of approximately 50 μl 0.25% Crystal Violet stain in methanol. Wells were rinsed immediately, to remove excess stain and were then allowed to dry. The number of syncytia per well were then counted, using a dissecting microscope.
Alternatively, instead of Crystal Violet analysis, cells were assayed with XTT, as described, above, in Section 17.1.
Peptides: The peptides characterized in the study presented herein were:
Each peptide was tested at 2-fold serial dilutions ranging from 500 μg/ml to approximately 500 ng/ml. For each of the assays, a well containing no peptide was also used.
The data summarized in
As shown in
CD analysis reveals that a number of the peptides exhibit detectable to substantial level of helical structure.
The results summarized in
Thus, the computer assisted searches described, hereinabove, have successfully identified viral peptide domains that represent highly promising anti-HPIV3 antiviral compounds.
The peptide regions found by 107×178×4 were located at amino acid residues 156-215 and 277-289. The peptide regions found by ALLMOTI5 were located at amino acid residues 156-219 and 245-286. Both motifs, therefore, identify similar regions.
Interestingly, the first SIV peptide region (i.e., from amino acid residue 156 to approximately amino acid residue 219) correlates with a DP107 region, while the second region identified (i.e., from approximately amino acid residue 245 to approximately amino acid residue 289) correlates with the DP178 region of HIV. In fact, an alignment of SIV isolate MM251 and HIV isolate BRU, followed by a selection of the best peptide matches for HIV DP107 and DP178, reveals that the best matches are found within the peptide regions identified by the 107×178×4 and ALLMOTI5 search motifs.
It should be noted that a potential coiled-coil region at amino acid residues 242-282 is predicted by the Lupas program. This is similar to the observation in HIV in which the coiled-coil is predicted by the Lupas program to be in the DP178 rather than in the DP107 region. It is possible, therefore, that SIV may be similar to HIV in that it may contain a coiled-coil structure in the DP107 region, despite such a structure being missed by the Lupas algorithm. Likewise, it may be that the region corresponding to a DP178 analog in SIV may exhibit an undefined structure, despite the Lupas program's prediction of a coiled-coil structure.
The results presented herein describe the identification of DP178/DP107 analogs within two different Epstein-Barr Virus proteins. Epstein-Barr is a human herpes virus which is the causative agent of, for example, infectious mononucleosis (IM), and is also associated with nasopharyngeal carcinomas (NPC), Burkitt's lymphoma and other diseases. The virus predominantly exists in the latent form and is activated by a variety of stimuli.
Search results demonstrate that the Zebra protein exhibits a single region which is predicted to be either of DP107 or DP178 similarity, and is found between the known DNA binding and dimerization regions of the protein. Specifically, this region is located at amino acid residues 193-220, as shown in
The 107×178×4 motif identifies a single region at amino acid residues 228-262. The ALLMOTI5 search motif identifies three regions, including amino acid residues 116-184, 228-269 and 452-500. Three regions containing proline residues followed by a leucine zipper-like sequence were found beginning at proline residues 214, 286 and 451.
The Lupas program identified two regions it predicted had potential for coiled-coil structure, which include amino acid residues 141-172 and 444-483.
In order to test the potential anti-HBV antiviral activity of these D178/DP107 analog regions, peptides derived from area around the analog regions are synthesized, as shown in
The anti-HBV activity of the peptides is tested by utilizing standard assays to determine the test peptide concentration required to cause an acceptable (e.g., 90%) decrease in the amount of viral progeny formed by cells exposed to an HBV viral inoculum. Candidate antivial peptides are further characterized in model systems such as wood chuck tissue culture and animal systems, prior to testing on humans.
The results depicted herein illustrate the results of search motifs conducted on the Simian Mason-Pfizer monkey virus. The motifs reveal DP178/DP107 analogs within the enveloped (TM) protein GP20, as shown in
The 107×178×4 motifs identifies a region at amino acid residues 422-470. The ALLMOTI5 finds a region at amino acid residues 408-474. The Lupas program predicted a coiled-coil structure a amino acids 424-459.
The results presented herein demonstrate the identification of DP178/DP107 analogs corresponding to sequences present in proteins of a variety of bacterial species.
The ALLMOTI5 motif identified a region at amino acid residues 22-27. The 107×178×4 motif identified two regions, with the first at amino acid residues 26-69 and the second at 88-115. A P12LZIPC motif search identified two regions, at amino acid residues 163-181 and 230-250.
The Lupas program predicted a region with a high propensity for coiling at amino acid residues 25-54. This sequence is completely contained within the first region identified by both ALLMOTI5 and 107×178×4 motifs.
The results presented herein demonstrate the identification of DP178/DP107 analogs corresponding to peptide sequences present within several different human proteins.
In the Example presented herein, measles (MeV) virus DP178-like peptides identified by utilizing the computer-assisted search motifs described in the Examples presented in Sections 9 and 21, above, are tested for anti-MeV activity. Additionally, circular dichroism (CD) structural analyses are conducted on the peptides, as discussed below. It is demonstrated that several of the identified peptides exhibit potent antiviral capability. Additionally, it is shown that none of the these peptides exhibit a substantial helical character.
Structural analyses: The CD spectra were measured in a 10 mM sodium phosphate, 150 mM sodium chloride, pH 7.0, buffer at approximately 10 mM concentrations, using a 1 cm pathlength cell on a Jobin/Yvon Autodichrograph Mark V CD spectrophotometer. Peptide concentrations were determined from A280 using Edlehoch's method (1967, Biochemistry 6:1948).
Anti-MeV antiviral activity syncytial reduction assay: The assay utilized herein tested the ability of the peptides to disrupt the ability of Vero cells acutely infected with MeV (i.e., cells which are infected with a multiplicity of infection of 2-3) to fuse and cause syncytial formation on a monolayer of an uninfected line of Vero cells. The more potent the peptide, the lower the observed level of fusion, the greater the antiviral activity of the peptide.
Uninfected confluent monolayers of Vero cells were grown in microtiter wells in 10% FBS EMEM (Eagle Minimum Essential Medium w/o L-glutamine [Bio Whittaker Cat. No. 12-125F], with fetal bovine serum [FBS; which had been heat inactivated for 30 minutes at 56° C.; Bio Whittaker Cat. No. 14-501F) supplemented at 10%, antibiotics/antimycotics (Bio Whittaker Cat. No. 17-602E) added at 1%, and glutamine added at 1%.
To prepare acutely infected Vero cells for addition to the uninfected cells, cultures of acutely infected Vero cells were washed twice with HBSS (Bio Whittaker Cat. No. 10-543F) and cell monolayers were removed with trypsin (Bio Whittaker Cat. No. 17-161E). Once cells detached, media was added, any remaining clumps of cells were dispersed, and hemacytometer cell counts were performed.
The antiviral assay was conducted by, first, removing all media from the wells containing uninfected Vero cells, then adding peptides (at the dilutions described below) in 10% FBS EMEM, and 50-100 acutely MeV-infected Vero cells per well. Wells were then incubated at 37° C. for a maximum of 18 hours.
On day 2, after cells in control wells were checked for fusion centers, media was removed from the wells, followed by addition, to each well, of approximately 50 μl 0.25% Crystal Violet stain in methanol. Wells were rinsed twice with water immediately, to remove excess stain and were then allowed to dry. The number of syncytia per well were then counted, using a dissecting microscope.
Anti-MeV antiviral activity plaque reduction assay: The assay utilized herein tested the ability of the peptides to disrupt the ability of MeV to infect permissive, uninfected Vero cells, leading to the infected cells' fusing with uninfected cells to produce syncytia. The lower the observed level of syncytial formation, the greater the antiviral activity of the peptide.
Monolayers of uninfected Vero cells are grown as described above.
The antiviral assay was conducted by, first, removing all media from the wells containing uninfected Vero cells, then adding peptides (at the dilutions described below) in 10% FBS EMEM, and MeV stock virus at a final concentration of 30 plaque forming units (PFU) per well. Wells were then incubated at 37° C. for a minimum of 36 hours and a maximum of 48 hours.
On day 2, after cells in control wells were checked for fusion centers, media was removed from the wells, followed by addition, to each well, of approximately 50 μl 0.25% Crystal Violet stain in methanol. Wells were rinsed twice with water immediately, to remove excess stain and were then allowed to dry. The number of syncytia per well were then counted, using a dissecting microscope.
Peptides: The peptides characterized in the study presented herein were peptides T-252A0 to T-256A0, T-257B1/C1, and T-258B1 to T-265B0, and T-266A0 to T-268A0, as shown in
Each peptide was tested at 2-fold serial dilutions ranging from 100 μg/ml to approximately 100 ng/ml. For each of the assays, a well containing no peptide was also used.
The data summarized in
As shown in
Thus, the computer assisted searches described, hereinabove, as in for example, the Example presented in Section 9, for example, successfully identified viral peptide domains that represent highly promising anti-MeV antiviral compounds.
In the Example presented herein, simian immunodeficiency virus (SIV) DP178-like peptides identified by utilizing the computer-assisted search motifs described in the Examples presented in Sections 9, 12 and 19, above, were tested for anti-SIV activity. It is demonstrated that several of the identified peptides exhibit potent antiviral capability.
Anti-SIV antiviral assays: The assay utilized herein were as reported in Langolis et al. (Langolis, A. J. et al., 1991, AIDS Research and Human Retroviruses 7:713-720).
Peptides: The peptides characterized in the study presented herein were peptides T-391 to T-400, as shown in
Each peptide was tested at 2-fold serial dilutions ranging from 100 μg/ml to approximately 100 ng/ml. For each of the assays, a well containing no peptide was also used.
The data summarized in
As shown in
Thus, the computer assisted searches described, hereinabove, as in for example, the Example presented in Section 9, for example, successfully identified viral peptide domains that represent highly promising anti-SIV antiviral compounds.
The Example presented in this Section represents a study of the antiviral activity of DP107 and DP178 truncations and mutations. It is demonstrated that several of these DP107 and DP178 modified peptides exhibit substantial antiviral activity.
Anti-HIV assays: The antiviral assays performed were as those described, above, in Section 6.1. Assays utilized HIV-1/IIIb and/or HIV-2 NIHZ isolates. Purified peptides were used, unless otherwise noted in
Peptides: The peptides characterized in the study presented herein were:
Blocked peptides contained an acyl N-terminus and an amido C-terminus.
Anti-HIV antiviral data was obtained with the group 1 DP178-derived peptides listed in
In
Among the peptides listed in
In
Truncations of the DP107 peptide (referred to in IG. 50 as T21) were also produced and tested, as shown in
Thus, the results presented in this Section demonstrate that not only do the full length DP107 and DP178 peptides exhibit potent antiviral activity, but truncations and/or mutant versions of these peptides can also possess substantial antiviral character.
In the Example presented herein, peptides derived from the Epstein-Barr (EBV) DP-178/DP107 analog region of the Zebra protein identified, above, in the Example presented in Section 20 are described and tested for anti-EBV activity. It is demonstrated that among these peptides are ones which exhibit potential anti-viral activity.
Electrophoretic Mobility Shift Assays (EMSA): Briefly, an EBV Zebra protein was synthesized utilizing SP6 RNA polymerase in vitro transcription and wheat germ in vitro translation systems (Promega Corporation recommendations; Butler, E. T. and Chamberlain, M. J., 1984, J. Biol. Chem. 257:5772; Pelham, H. R. B. and Jackson, R. J., 1976, Eur. J. Biochem. 67:247). The in vitro translated Zebra protein was then preincubated with increasing amounts of peptide up to 250 ng/ml prior to the addition of 10,000 to 20,000 c.p.m. of a 32P-labeled Zebra response element DNA fragment. After a 20 minute incubation in the presence of the response element, the reaction was analyzed on a 4% non-denaturing polyacrylamide gel, followed by autoradiography, utilizing standard gel-shift procedures. The ability of a test peptide to prevent Zebra homodimer DNA binding was assayed by the peptide's ability to abolish the response element gel migration retardation characteristic of a protein-bound nucleic acid molecule.
Peptides: The peptides characterized in this study represent peptide walks through the region containing, and flanked on both sides by, the DP178/DP107 analog region identified in the Example presented in Section 20, above, and shown as shown in
Each of the tested peptides were analyzed at a range of concentrations, with 150 ng/ml being the lowest concentration at which any of the peptides exerted an inhibitory effect.
The EBV Zebra protein transcription factor contains a DP178/DP107 analog region, as demonstrated in the Example presented, above, in Section 20. This protein appears to be the primary factor responsible for the reactivation capability of the virus. A method by which the DNA-binding function of the Zebra virus may be abolished may, therefore, represent an effective antiviral technique. In order to identify potential anti-EBV DP178/DP107 peptides, therefore, peptides derived from the region identified in Section 20, above, were tested for their ability to inhibit Zebra protein DNA binding.
The test peptides' ability to inhibit Zebra protein DNA binding was assayed via the EMSA assays described, above, in Section 28.1. The data summarized in
Those peptides which exhibit an ability to inhibit Zebra protein DNA binding represent potential anti-EBV antiviral compounds whose ability to inhibit EBV infection can be further characterized.
The present invention is not to be limited in scope by the specific embodiments described which are intended as single illustrations of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. Indeed, various modifications of the invention, in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.
This is a continuation of U.S. application Ser. No. 08/484,223 filed Jun. 7, 1995, which is a division of U.S. application Ser. No. 08/470,896 filed Jun. 6, 1995, now U.S. Pat. No. 6,479,055 issued Nov. 12, 2002, which is a continuation-in-part of U.S. application Ser. No. 08/360,107 filed Dec. 20, 1994, now U.S. Pat. No. 6,017,536 issued Jan. 25, 2000, which is a continuation-in-part of U.S. application Ser. No. 08/255,208 filed Jun. 7, 1994, now U.S. Pat. No. 6,440,656 issued Aug. 27, 2002, which is a continuation-in-part of U.S. application Ser. No. 08/073,028 filed Jun. 7, 1993, now U.S. Pat. No. 5,464,933 issued Nov. 7, 1995, each of which is incorporated herein by reference in its entirety.
This invention was made with Government support under Grant No. AI-30411-02 awarded by the National Institutes of Health. The Government has certain rights in the invention.
| Number | Name | Date | Kind |
|---|---|---|---|
| 4659669 | Kleid et al. | Apr 1987 | A |
| 4707358 | Kieff et al. | Nov 1987 | A |
| 4761470 | Emini et al. | Aug 1988 | A |
| 5057211 | Baummer | Oct 1991 | A |
| 5116725 | Vaughan et al. | May 1992 | A |
| 5141867 | Ivanoff et al. | Aug 1992 | A |
| 5444044 | Jiang et al. | Aug 1995 | A |
| 5464933 | Bolognesi et al. | Nov 1995 | A |
| 5840843 | Jiang et al. | Nov 1998 | A |
| 6017536 | Barney et al. | Jan 2000 | A |
| 6248574 | Shaffermann | Jun 2001 | B1 |
| 6440656 | Bolognesi et al. | Aug 2002 | B1 |
| 6479055 | Barney et al. | Nov 2002 | B1 |
| Number | Date | Country |
|---|---|---|
| 0 323 157 | Dec 1988 | EP |
| 0 362 927 | Apr 1990 | EP |
| 0181 150 | Mar 2003 | EP |
| 2677346 | Dec 1992 | FR |
| WO8808429 | Nov 1988 | WO |
| WO8902935 | Apr 1989 | WO |
| WO9007119 | Jun 1990 | WO |
| WO9109872 | Jul 1991 | WO |
| WO9200997 | Jan 1992 | WO |
| WO9222654 | Dec 1992 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20040033235 A1 | Feb 2004 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 08470896 | Jun 1995 | US |
| Child | 08484223 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 08484223 | Jun 1995 | US |
| Child | 10267682 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 08360107 | Dec 1994 | US |
| Child | 08470896 | US | |
| Parent | 08255208 | Jun 1994 | US |
| Child | 08360107 | US | |
| Parent | 08073028 | Jun 1993 | US |
| Child | 08255208 | US |
