Methods of preparing carboxy-terminally truncated recombinant flavivirus envelope glycoproteins employing drosophila melanogaster expression systems

TECHNICAL FIELD
The invention relates to protection against and diagnosis of dengue fever. More specifically, the invention concerns a subunit of the dengue virus envelope protein secreted as a mature recombinantly produced protein from eucaryotic cells which is protective against dengue infection, which raises antibodies useful in passive immunization, and which is useful in diagnosis of infection by the virus.
BACKGROUND ART
The dengue viruses are members of the family Flaviviridae which also includes the Japanese encephalitis (JE) virus, Tick-borne encephalitis (TBE) virus, and the is initially discovered prototype of this class, the yellow fever (YF) virus. The flaviviruses contain a single positive strand genomic RNA and are small enveloped viruses affecting animals, but generally transmitted to vertebrates by chronically infected mosquito or tick vectors. Flaviviruses are enveloped by host cell membrane and contain the three structural proteins capsid (C), membrane (M), and envelope (E). The E and M proteins are found on the surface of the virion where they are anchored in the membrane. Mature E is glycosylated, whereas M is not, although its precursor, preM, is a glycoprotein. Glycoprotein E, the largest structural protein, contains functional domains responsible for cell surface attachment and intraendosomal fusion activities. It is also a major target of the host immune system, inducing neutralizing antibodies, protective immunity, as well as antibodies which inhibit hemagglutination.
Dengue virus is the causative agent of dengue fever and is transmitted to man by Aedes mosquitoes, principally Aedes aegypti and Aedes albopictus. Classic dengue fever is an acute illness marked by fever, headache, aching muscles and joints, and rash. A fraction of cases, typically in children, results in more extreme forms of infection, i.e., dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). Without diagnosis and prompt medical intervention, the sudden onset and rapid progress of DHF/DSS can be fatal.
Dengue is one of the most important virus groups transmitted to man by arthropods in terms of global morbidity; it has been estimated that dengue is responsible for up to 100 million illnesses annually. With the advent of modern jet travel, dengue has spread globally in the tropics and subtropics, and multiple dengue serotypes in a region are common.
Every flavivirus genome is a single positive-stranded RNA of approximately 10,500 nucleotides containing short 5' and 3' untranslated regions, a single long open reading frame (ORF), a 5' cap, and a nonpolyadenylated 3' terminus. The ten gene products encoded by the single, long open reading frame are contained in a polyprotein organized in the order, C (capsid), preM/M (membrane), E (envelope), NS1 (nonstructural), NS2a, NS2b, NS3, NS4a, NS4b, and NS5 (Chambers, T. J. et al. Ann Rev Microbiol (1990) 44:649-688). Processing of the encoded polyprotein is initiated cotranslationally, and full maturation requires both host and viral-specific proteases. The sites of proteolytic cleavage in the YF virus have been determined by comparing the nucleotide sequence and the amino terminal sequences of the viral proteins. Subsequent to initial processing of the polyprotein, preM is converted to M during virus release (Wengler, G. et al. J Virol (1989) 63:2521-2526), and anchored C is processed during virus maturation (Nowak et al. Virology (1987) 156:127-137).
There are four antigenically related dengue viruses which, however, can be recognized as distinct serotypes. The complete genomic sequence for at least one strain of each of the four dengue serotypes has been reported (DEN-1, Fu, J. et al. Virology (1992) 188:953-958; DEN-2, Deubel, V. et al. Virology (1986) 155:365-377; Hahn, Y. S. et al. Virology (1988) 162:167-180; DEN-3, Osatomi, K. et al. Virus Genes (1988) 2:99-108; Osatomi, K. et al. Virology (1990) 176:643-647; DEN-4, Zhao, B. E. et al. Virology (1986) 155:77-88; Mackow, E. et al. Virology (1987) 159:217-228). In addition, the compete genomic sequences of other flaviviruses are known (e.g., YF virus: Rice et al., Science (1985) 229:726-733).
It does not appear that infection by one dengue serotype can confer long-term immunity on the individual with respect to other serotypes. In fact, secondary infections with heterologous serotypes are quite common. In general, antibody responses in infected subjects to primary infection are mostly IgM antibodies and these antibodies are directed against type-specific determinants. On the other hand, secondary infections by heterologous serotypes generate IgG antibodies which are flavivirus crossreactive.
Helpful reviews of the nature of the dengue disease, the history of attempts to develop suitable vaccines, and structural features of flaviviruses in general as well as the molecular structural features of the envelope protein of flaviviruses are found in Halstead, S. B. Science (1988) 239:476-481; Brandt, W. E. J Infect Disease (1990) 162:577-583; Chambers, T. J. et al. Annual Rev Microbiol (1990) 44:649-688; Mandl, C. W. et al. Virology (1989) 63:564-571; and Henchal, E. A. and J. R. Putnak, Clin Microbiol Rev (1990) 3:376-396.
A successful vaccine for protection against dengue infection has never been developed. However, there have been a number of preliminary efforts, many of which focus on the envelope protein, since this protein is exposed at the surface and is believed to be responsible for eliciting immunity.
Monoclonal antibodies (Mabs) directed against purified E of several flaviviruses DEN-2 (Henchal et al. Am J Trop Med Hyg (1985) 34:162-169, TBE (Heinz, F. X. et al. Virology (1983) 126:525-537), St. Louis encephalitis (SLE, Mathews, J. H. et al. J Immunol (1984) 132:1533-1537), Murray Valley encephalitis (MVE, Hawkes, R. A. et al. J Gen Virol (1988) 69:1105-1109), and JE (Takegami, T. et al. Acta Virologica (1982) 26:312-320) are neutralizing in vitro. Some of these Mabs can also passively confer protection in vivo (Heinz, F. X. et al. (1983, supra)); Mathews, J. H. et al. (1984, supra)); Kimuro-Kuroda and Yasui, J Immunol (1988) 141:3603-3610).
Although the primary amino acid sequence of the flavivirus E glycoprotein is variable (45-80% identity), all have twelve conserved cysteine residues, forming six disulfide bridges, and hydrophilicity profiles are nearly superimposable, suggesting that they may all have similar secondary and tertiary structures. Based on the position of the 12 conserved cysteines (determined for West Nile virus, Nowak and Wengler, Virology (1987) 156:127-137), monoclonal antibody competitive binding studies, monoclonal antibody binding to purified proteolytic fragments, and analysis of neutralizing antibody escape mutants of Tick-Borne Encephalitis Virus, glycoprotein E was divided into three antigenic domains (A, B, and C) and two transmembrane segments at its carboxy-terminus. See, for example, Mandl, C. W. et al. J Virol (1989) 63:564-571. FIG. 1, reproduced from this article, shows the locations of these domains.
Domain A was defined as a denaturation sensitive, highly folded, and disulfide stabilized discontinuous domain composed of the amino acids from 50-125 and 200-250 containing five of the six disulfide bridges. Neutralization and hemagglutination inhibition epitopes are found within domain A, and, for dengue viruses, one of the two N-linked glycosylation sites. A conserved hydrophobic amino acid sequence within domain A has been postulated to provide fusogenic properties after low pH treatment. Amino acid sequences conserved among the flavivirus family are located within this region; thus, broadly flavivirus-cross-reactive epitopes lie within this domain.
Domain B was identified as a continuous domain composed of amino acids 301-395 (an approximate region between amino acids 300-400 for all flaviviruses). The domain can be isolated as a single immunoreactive proteolytic fragment. It has been postulated that this domain forms part of the receptor binding site (Heinz, F. X. et al. APMIS (1993) 101:735-745), and attenuating mutations have been mapped to sequences within domain B Heinz et al. (supra). A variety of neutralizing antibodies have been shown to specifically map to Domain B (Heinz et al. (1983, supra)); Trirawatanapong et al., 1992; Megret et al., 1992; Lin et al., 1994). The binding of these neutralizing monoclonal antibodies is dependent on formation of a disulfide bond, and in some cases also is sensitive to detergent denaturation. Species-specific monoclonal antibodies bind this domain.
Domain C represents a hypervariable loop between the two segments of Domain A. Its antigenicity is insensitive to denaturation or reducing agents, and contains one N-linked glycosylation site. Predominantly sub-type specific monoclonal antibodies react with this domain. No specific activity has been assigned to this domain.
Many strategies are currently under investigation to develop an effective and safe dengue vaccine; however, to date, no single strategy has proven completely satisfactory. Attempts to generate live attenuated dengue vaccine strains have not been entirely successful, although research into this area continues. In the absence of effective, live attenuated dengue vaccines, a significant effort has been invested in the development of recombinant, dengue subunit or viral-vectored vaccines.
Recombinant dengue proteins have been expressed in several systems to date (see Putnak, R. A. (1994) Modern Vaccinology, E. Kurstak ed., Plenum Medical, New York, pp. 231-252, for review). Most efforts using Escherichia coli have yielded poor immunogen unable to elicit neutralizing antibodies. This may reflect non-native conformation of dengue proteins expressed in the bacteria and the necessity to process the viral proteins through the secretion pathway in order to form the proper disulfide bonds, glycosylate the proteins, or both.
Several reports have described vaccinia-flavivirus recombinants expressing envelope protein as part of a polyprotein (e.g. C-preM-E-NS1; [Dengue] Zhao, B. G. et al. J Virol (1987) 61:4019-4022; Deubel, V. et al. J Gen Virol (1988) 69:1921-1929; Bray, M. et al. J Virol (1991) 63:2853-2856; [YF] Hahn, Y. S. et al. Arch Virol (1990) 115:251-265), as a single protein (e.g. 100% E; [Dengue] Bray, M. et al., J Virol (1989) 63:2853-2856), or as polypeptides (e.g. 79% E-RKG; Men, R. et al. J Virol (1991) 65:1400-1407). The most successful recombinant vaccinia viruses, those capable of inducing neutralizing antibodies and protecting mice from virus challenge, were the which were secreted E extracellularly or accumulated E on the cell surface.
Men, R. et al. (1991, supra) described the recombinant production of various C-terminal truncations of the DEN-4 envelope protein using a recombinant Vaccinia virus vector and infecting mammalian CV1 cells. The results showed that the recombinants that contain greater than 79% of the coding sequence produced an intracellular protein that could be immunoprecipitated with anti-dengue virus antibodies contained in hyperimmune mouse ascitic fluid (HMAF). Although there was a reduced level of detection for protein which contained 79% of envelope or less, this did not appear to result from reduced production of the protein. It was also found that only truncations which contained 79% of E or less were secreted efficiently; E polypeptides equal to or larger than 81% E were not secreted efficiently.
Men et al. (1991, supra) constructed additional C-terminal truncations between 79% E and 81% E to map the amino acids responsible for the difference in secretion and immunoreactivity with HMAF of these two truncated E polypeptides. The results demonstrated that 79% E containing the additional tripeptide sequence RKG was also secreted. Although both 59% E and 79% E-RKG were secreted, only 79% E-RKG was detected at the cells' surface. The recombinant Vaccinia viruses containing various truncations were also used to immunize mice. Mice immunized with recombinants expressing 79% E-RKG or larger portions of the envelope protein were protected. However, except for 59% E, mice immunized with 79% E or a smaller product were only partially protected. The 59% E elicited high protection rates (>90%) comparable to 79% E-RKG and larger C-terminal truncated E polypeptides. Protection correlated with binding to HMAF.
Combinations of immunogenic structural and nonstructural JE virus, DEN-1, DEN-2, and DEN-4 proteins have been expressed by baculovirus recombinants (Matsuura, Y. et al. Virology (1989) 173:674-682; Putnak, R. A. et al. Am J Trop Med Hyg (1991) 45:159-167; Deubel, V. et al. Virology (1991) 180:442-447). Baculovirus-expressed dengue and JE E glycoprotein elicited neutralizing antibodies, protected mice from a lethal dengue virus challenge, or both. In spite of these successes, the expression levels reported in baculovirus are low and the recombinant protein is less immunogenic than the viral protein (Putnak, R. A. et al. Am J Trop Med Hyg (1991) supra).
Research with purified polypeptides released by proteolysis of flavivirus envelope proteins, with recombinant polypeptides, and with synthetic peptides has indicated where protective epitopes may map. The isolated 9000 dalton domain B trypsin fragment from TBE virus spontaneously refolds and is stabilized by disulfide bridges (Winkler, G. et al. J Gen Virol (1987) 68:2239-2244). This disulfide stabilized fragment elicited neutralizing antibodies in mice (Heinz, F. X. et al. Virology (1984) 130:485-501). In contrast, a 28,000 dalton trypsin fragment from WN virus containing domain B sequences was unable to spontaneously refold and did not elicit neutralizing antibodies (Wengler and Wengler, 1989).
A cyanogen bromide-cleaved 8 kD fragment (amino acids 375-456) overlapping domain B from JE envelope protein was found to induce neutralizing antibodies in mice (Srivastava, A. K. et al. Acta Virol (1990) 34:228-238). Immunization of mice with a larger polypeptide (JE E amino acid 319 to NS1 amino acid 65) spanning the 8 kD peptide expressed in Escherichia coli as a fusion to protein A elicited neutralizing antibodies and protected mice from lethal virus challenge (Srivastava, A. K. et al. Microbiol Immunol (1991) 35:863-870). This polypeptide begins between the two cysteines within domain B, and, therefore, cannot form the stabilizing disulfide bond that earlier reports suggest is necessary for presentation of protective epitopes.
Immunization of mice with synthetic peptides corresponding to amino acids within domain B, aa 356-376 from MVE (Roehrig, J. T. et al. Virology (1989) 171:49-60) or aa 352-368 from DEN-2 virus (Roehrig, J. T. et al. Virology (1990) 177:668-675), elicited low levels of neutralizing antibodies in mice, suggesting the presence in domain B of a weak linear neutralizing epitope (Roehrig, J. T. et al. 1989 and 1990, supra).
Mason, P. W. et al. J Gen Virol (1990) 71:2107-2114 identified two domains of the DEN-1 envelope protein: domain I which includes amino acids 76-93 of the E protein and domain II (equivalent to domain B) which includes amino acids 293-402. These domains were identified from deletion analysis using recombinant fusion proteins expressed in E. coli and reacted with antiviral monoclonal antibodies. Recombinant fusion proteins containing E. coli trpE sequences fused to the envelope protein (amino acids 1 to 412) elicited antibodies in mice which reacted with a portion of the protein containing domain II.
In addition, Mason, P. W. et al. (J Gen Virol (1989) 70:2037-2049) expressed a collection of E. coli trpE fusion proteins to segments of JE virus envelope protein spanning domain B. The trpE fusion proteins containing the smallest JE E fragments that retained immunoreactivity with a panel of neutralizing monoclonal antibodies included amino -acid residues from methionine 303 through tryptophan 396. However, animals immunized with immunoreactive trpE fusion polypeptides did not produce neutralizing antibodies nor were they protected from lethal challenge.
Trirawatanapong, T. et al. Gene (1992) 116:139-150. prepared several truncated forms of dengue 2 envelope proteins in E. coli for epitope mapping, and mapped monoclonal antibody 3H5 to its corresponding epitope. This was first localized between amino acids 255 and 422. Targeted gene deletions in the plasmid constructs encoding the truncated proteins permitted mapping of the binding site to the 12 amino acids between positions 386 and 397. The mapping was apparently confirmed by the ability of a synthetic peptide containing E protein amino acids 386-397 to bind 3H5 specifically.
Megret, F. et 41. Virology (1992) 187:480-491 prepared 16 overlapping fragments of DEN-2 envelope protein as trpE fusion products in E. coli for epitope mapping. The fusion proteins are produced intracellularly and obtained from the lysates. These products were used to map epitopes defined by a panel of 20 monoclonal antibodies. Six antigenic domains were described: non-neutralizing antibodies bound to peptides containing amino acids 22-58, amino acids 304-332, amino acids 60-97, and amino acids 298-397. Neutralizing antibodies bound to peptides containing amino acids 60-135, 60-205, and 298-397.
Significantly, Megret et al. (1992, supra) demonstrated that all MAbs (including 3H5), with two exceptions (below), that recognize "full-length" domain B (amino acids 298-397) are unable to recognize slightly shorter polypeptides. For example, in contrast to the findings of Trirawatanapong et al. Gene (1992, supra), MAb 3H5 was unable to bind to trpE fusion proteins containing DEN-2 E amino acids 304-397, 298-385, or 366-424. The two exceptional MAbs in the findings of Megret et al. are MAbs 5A2 and 9D12. The pattern of binding of MAb 5A2 suggests that it recognizes a linear epitope between amino acids 304 to 332, while MAb 9D12 binds to a polypeptide, amino acids 298-385, which is slightly shorter than the smallest polypeptide (amino acids 298-397) to which MAb 3H5 binds. These results indicate that both the disulfide bond in domain B and the domain B C-terminal amino acids are involved in forming the immunodominant domain B epitopes.
Although it appears established from the art that the B domain of the flavivirus envelope protein contains epitopes which bind neutralizing antibodies, problems have arisen with respect to producing recombinant polypeptides containing the B domain in a form which mimics the native protein and is thus capable of eliciting an immune response. The only recombinantly produced E polypeptides containing the B domain that elicited a protective immune response in mice were expressed from Vaccinia and baculovirus vectors. Generally, recombinantly produced proteins lack the appropriate 100-120 amino acid peptides, wherein the extensions do not interfere with the immunogenic effectiveness or secretion of the B domain. In one embodiment, such extensions are minimal--i.e., not more than six additional amino acids--at either the N-terminus or the C-terminus, or distributed between these termini; preferably no more than four total additional amino acids, and most preferably no more than two.
The form of domain B which spans positions of about 296-395 is referred to herein as "classical" domain B. When the B domain includes at least portions of the region extending to amino acid 413, the additional region may confer additional functions, e.g., enhancing immunogenicity by providing a helper T cell epitope. The form of domain B which includes positions about 296-413 is referred to herein as DomB+T. The domain B of the invention includes these two specific embodiments; "classical" domain B and DomB+T, as well as those forms which span positions approximately 296 to position between position 395 and 413.
Other portions of the E protein illustrated below are self-explanatory. 80% E is the N-terminal 80% of the protein from residue 1 to residue 395. 60% E represents the corresponding shorter sequence. These subunits are produced from vectors containing the DNA encoding the mature protein, or along with the prM fusion which results in secretion of the 80% or 60% per se.
For practical large-scale production of the subunits used as active ingredients in the vaccine of the invention, recombinant techniques provide the most practical approach. However, to be useful as active glycosylation, folding, and disulfide bond formation for producing a proper immune response.
It has now been found that the B domain of the envelope protein can be successfully secreted from yeast in a form which elicits the production of neutralizing antibodies. This permits, for the first time, the production of a successful recombinantly produced subunit dengue vaccine.
DISCLOSURE OF THE INVENTION
The invention provides vaccines containing as an active ingredient, a secreted recombinantly produced the dengue envelope protein or a subunit thereof. The vaccines are capable of eliciting the production of neutralizing antibodies against-dengue virus. In the illustrations below, the B domain of the envelope protein (E) is secreted from yeast by producing it in an expression vector containing the .alpha.-mating factor prepropeptide leader sequence (preproMF.alpha..sub.L). Peptide subunits representing 60% E and 80% E are secreted from Drosophila cells using the human tissue plasminogen activator secretion signal sequence for the propeptide (tPA.sub.L) or from the homologous premembrane (prM) leader. The secreted products can easily be purified and prepared as a vaccine.
Thus, in one aspect, the invention is directed to a vaccine for protection of a subject against infection by dengue virus. The vaccine contains, as active ingredient, the envelope protein of a dengue virus serotype or a subunit thereof. The E or subunit is secreted as a recombinantly produced protein from eucaryotic cells. The vaccine may further contain portions of additional dengue virus serotype E proteins similarly produced.
In other aspects, the invention is directed to methods to protect subjects against infection by administering the invention vaccines, to antibodies generated in a mammalian subject administered an immunogenic amount of the vaccine; immortalized B cell lines which generate monoclonal antibodies of this type; methods to effect passive immunization by administering the antibodies of the invention; methods to detect the presence or absence of antidengue virus immunoglobulins utilizing the secreted recombinantly produced peptides of the invention and the recombinant materials important in the secretion of the B domain and methods for its production.

BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a drawing reproduced from Mandl, et al. (supra) showing a model of the envelope protein of flaviviruses. Model of the TBE virus protein E. Open circles represent hydrophilic amino acid residues (Arg, Lys, Asn, Asp, Gln, Glu, His), dotted circles show intermediate amino acid residues (Pro, Tyr, Ser, Trp, Thr, Gly), and solid s circles showed hydrophobic amino acid residuess (Ile, Val, Leu, Phe, Cys, Met, Ala). Amino acids were classified by the scale of Kyte and Doolittle (22). Position numbers are shown every 50 amino acids. Cysteine residues forming disulfide bridges are connected by solid lines. Arrows depict cleavage sites that liberate 1RF1 (tryp.) and 1RF3 (CNBr), respectively. Small arrows indicate potential cleavage sites within these fragments that are not utilized. Two solid lines stand for the lipid membrane that is spanned by two transmembrane regions of protein E. The polypeptide chain is folded to indicate the antigenic domains A, B, and C, which are designated by large capital letters. Arrows together with the names of neutralizing MAbs depicts the locations of the mutations identified in the respective antigenic variants of TBE virus by sequence analysis. A line of solid triangle indicates the almost perfectly conserved sequence with in domain A. A line of open triangles marks the region of a potential T-cell determinant. A solid diamond represents the carbohydrate side chain of TBE virus. The Murray Valley (MVE), St. Louis (SLE) and Japanese (JE) encephalitis viruses and DEN viruses have potential N-glycosylation sites at the homologous position. Yellow fever (YE) and St. Louis encephalitis viruses have such a site within domain B. DEN viruses within domain A. The homologous positions of TBE virus are shown as open diamonds.
FIG. 2 (SEQ ID NO:1) shows the partial nucleotide sequence for DEN-2 PR159 S1 mutant strain and differences from the wild-type strain reported by Hahn (1988, supra). DEN-2 PR159/S1 cDNA coding strand sequence for Capsid, preMembrane, Envelope, and NS1 is given (Hahn etr al. 1988). The start of genes are indicated. "*" indicates correction to published sequence. Difference in the wild-type DEN-2 PR159 sequence are indicated above the PR159/S1 sequence.
FIG. 3 (SEQ ID NO:2 and SEQ ID NO:3) shows the partial nucleotide sequence and deduced amino acid sequence of the genome of DEN-2 PR159/S1 strain in comparison with wild-type.
FIG. 4 (Residues 1693-1752 of SEQ ID NO:1 and SEQ ID NO:4 through SEQ ID NO:7) shows the oligonucleotide used to mutagenize an 80% E cDNA clone to obtain the domain B coding sequence. Shown are the necleotide sequence and the corresponding translation of DEN-2 PR159/S1 E sequences between genomic nucleotides 1789 and 1848, the sequence of the oligonucleotide used in mutagenesis, and the resulting sequence and corresponding translations following mutagenesis. Dengue nucleotide sequence is uppercase, non-dengue nucleotides are longer case, inserted restriction endonuclease sites are indicated, and conserved cysteines are underlined.
FIG. 5 shows the construction of a cloning vector containing the nucleotide sequence encoding domain B.
FIG. 6 shows the insertion of the domain B coding sequence into the yeast expression vector pLS5.
FIG. 7 (SEQ ID NO:8 and SEQ ID NO:9) shows the nucleic acid and amino acid sequence of mating factor .alpha. leader, preproMF.alpha..sub.L /domain B fusion protein.
FIG. 8 shows the construction of an expression vector for domain B in Pichia.
FIG. 9 (SEQ ID NO:10 through SEQ ID NO:12) shows the nucleotide and deduced amino acid sequence for the tPA.sub.L -DomB fusion protein.
FIG. 10 shows the survival times of mice immunized with recombinant domain B and challenged with Dengue-2.
FIG. 11 shows the construction of an expression vector for production of domain B in Drosophila melanogaster tissue cultured Schneider cells.
FIG. 12 shows the standard curve for a DomB sandwich assay.

MODES OF CARRYING OUT THE INVENTION
The invention provides, for the first time, a subunit vaccine that can be efficiently produced recombinantly and secreted and that is effective in protecting subjects against infection with dengue virus. Although many attempts have been made to obtain such a subunit vaccine, either the subunit itself is resistant to recombinant production techniques which permit it to be secreted in a processed form so as to render it effective as an immunogen, or, if its recombinant production is facile, it fails to elicit neutralizing antibodies. The present inventors have found that certain portions of the envelope protein of dengue virus type 2, such as domain B representing approximately 100 amino acids of the envelope protein extending approximately from the Gly at position 296 to the Gly at position 395, and optionally including additional E sequence through position 413 of the protein, and other portions of E, i.e., 60% E and 80% E are effectively secreted by certain convenient eucaryotic recombinant hosts, in a form that permits processing to mimic the native conformation of the protein. The secretion of the protein into the culture medium facilitates purification. Furthermore, this form is able, when administered, especially in the presence of adjuvant, to raise neutralizing antibodies in animals. Thus, this subunit represents a useful component of a vaccine for protecting subjects against dengue infection.
As used herein, "B domain" refers to a peptide which spans from approximately Gly 296 to Gly 395 of the DEN-2 envelope protein, and optionally including additional E sequence through position 413 of the envelope protein. These positions are approximate; for example, Mandl (1989, supra) describes the generation of a tryptic fragment containing domain B which spans the amino acids of the TBE E protein from position 301 to 396. The sequences described in the present application represent the envelope protein from dengue Type 2 virus (DEN-2); three additional distinct dengue serotypes have been recognized. Therefore, "Domain B" also refers to the corresponding peptide region of the envelope protein of these serotypes, and to any naturally occurring variants. In addition, B domain includes extended forms of the about ingredients, the subunits as produced must assume a conformation and undergo processing under conditions which, render them similar to the native envelope portion as it exists in the envelope protein of the virus. In order to achieve this, the recombinant production must be conducted in eucaryotic cells, preferably yeast or Drosophila cells. In addition to the S. cerevisiae in P. pastoris yeasts illustrated below, other yeasts, such as Kluveromyces sp Yarrowia lipolytica, and Schizosaccharomyces pombe may be used. Other insect cells besides the Drosophila melongaster Schneider cells illustrated below may also be employed. Additional appropriate eucaryotic cells include mammalian expression cells such as Chinese hamster ovary cells. Other insect cells may also be used in conjunction with baculovirus based vectors. The B domain or 60% E or 80% E must be produced as a correctly processed protein and secreted.
It has been found, as demonstrated hereinbelow, that particularly efficient secretion of biologically active mature protein can be achieved in several ways. First, this can be done by expressing the B domain in yeast in operable linkage with the .alpha.-mating factor signal sequence. Constructs which place the nucleotide sequence encoding the B domain disposed so as to encode a fusion protein with an upstream .alpha.-mating factor signal sequence are therefore included within the scope of the invention. An additional preferred embodiment employs Drosophila cells and the human tissue plasminogen activator leader sequence for secretion of 60% E or 80% E as well as domain B. Envelope protein subunits that represent N-terminal portions of truncated protein may also be secreted from the homologous prM fusion. Other secretion signal peptides or secretion leader pre/pro peptides, such as those associated with invertase or acid phosphatase of S. cerevisiae or with glucoamylase of C. albicans or of A. niger or the bovine chymosin prepropeptide secretion leader can also be used. Secretion leaders in general that occur at the amino terminus of secreted proteins and function to direct the protein into the cellular secretion pathway generically can be used. In general, the invention includes expression systems that are operable in eucaryotic cells and which result in the formation of envelope protein or a subunit secreted into the medium. Thus, useful in the invention are cells and cell cultures which contain expression systems resulting in the production and secretion of mature B domain.
The properly processed E protein or subunit is recovered from the cell culture medium, purified, and formulated into vaccines. Purification and vaccine formulation employ standard techniques and are matters of routine optimization. Suitable formulations are found, for example, in Remington's Pharmaceutical Sciences, latest edition, Mack Publishing Company, Easton, Pa. In particular, formulations will include an adjuvant, such as alum or other effective adjuvant. Alternatively, the active ingredient and the adjuvant may be coadministered in separate formulations.
The active vaccines of the invention can be used alone or in combination with other active vaccines such as those containing attenuated forms of the virus or those containing other active subunits to the extent that they become available. The vaccines may contain only one subunit as an active ingredient, or additional isolated active components may be added. Corresponding or different subunits from one or several serotypes may be included in a particular formulation.
To immunize subjects against dengue fever, the vaccines containing the subunit are administered to the subject in conventional immunization protocols involving, usually, multiple administrations of the vaccine. Administration is typically by injection, typically intramuscular or subcutaneous injection; however, other systemic modes of administration may also be employed. Although the technology is not as well developed, transmucosal and transdermal formulations are included within the scope of the invention as are effective means of oral administration. The efficacy of these formulations is a function of the development of formulation technology rather than the contribution of the present invention.
Since the subunit vaccines containing B domain have been shown to elicit the production of neutralizing antibodies, the antibodies thus raised can themselves be used as passive vaccines. For production of passive vaccine, a suitable mammalian subject is immunized with the E protein or subunit of the invention and antibodies are either recovered directly as a composition from the antisera or indirectly as monoclonal antibodies from immortalized B cells of the subject. For production of monoclonal antibodies, the conventional techniques of Kohler & Milstein, for example, or treatment with Epstein Barr virus, are used in immortalizing peripheral blood lymphocytes or spleen cells and screening for antibodies immunoreactive with the immunogen. These antibodies may further be screened for their ability to effect plaque reduction in infected sera or cultures.
The polyclonal antisera are generally subjected to purification techniques such as standard size separation and chromatographic techniques to obtain purified immunoglobulins. The recombinantly produced proteins of the invention are particularly useful affinity ligands for chromatographic purification. A multiplicity of techniques is available for the purification of immunoglobulins for use in passive vaccines. If monoclonal antibodies are to be purified from small volumes of a medium ascites fluid, a protein A affinity column is particularly useful. For larger volumes, additional standard chemical techniques, such as ammonium sulfate precipitation and DEAE chromatography can be used. These immunoglobulins or the monoclonal antibodies generated by the immortalized B cells are then used in vaccine formulations as is understood in the art.
As is the case with active vaccines, the passive vaccines of the invention may be used in combination with additional antibodies or tandem administration of the antibodies of the invention and additional antibodies may be employed.
In the event that the passive vaccine is intended for use in a species other than that of the subject in which the antibodies were prepared, it may be desirable to modify the antibodies to minimize any immunogenic response. For example, it may be possible to use only the variable regions of these antibodies, such as the F.sub.ab, F.sub.ab', or F.sub.(ab').sbsb.2 regions. These fragments can be prepared either from polyclonal antisera or from the supernatants of hybridoma cultures by treating with proteolytic enzymes and recovering the desired fragments. The fragments are readily separated by using the relevant protein of the invention as an affinity reagent.
Alternatively, chimeric antibodies can be produced wherein the constant region corresponding to the species to be protected is substituted for the constant region characteristic of the species of antibody origin. The availability of recombinant techniques makes the production of chimeric antibodies a relatively trivial exercise. Briefly, a hybridoma or cell line producing the antibody of interest is used as a source for the genes encoding the antibody. The genes are recovered from, for example, the hybridoma using standard cloning procedures. The genes are then manipulated in vitro to remove the constant region and replace it with a constant region of a different species origin. The modified genes are then ligated into expression systems and expressed in recombinant host cells, such as CHO cells, monkey cells, yeast cells, and the like.
Further modifications in the variable regions can also reduce immunogenicity. Again, since recovery of the genes encoding the antibody is within the skill of the art, the variable regions, too, can be manipulated to replace the framework regions with framework regions more representative of the desired species, leaving intact the complementarily determining regions responsible for antigen specificity. In still another embodiment, the variable heavy chain and variable light chain regions can be linked through a peptide linker and produced as a single chain F.sub.v molecule.
Thus, if the passive vaccines are intended for humans, the foregoing various techniques of humanizing antibodies can be employed to minimize any immunogenic response even though the original antibodies are raised in nonhuman species.
In addition to use in vaccines or in the generation of passive vaccines, the mature recombinant E protein and subunits of the invention may be used as analytical reagents in assessing the presence or absence of antidengue antibodies in samples. The interest in doing this may be diagnosis of infection with dengue, monitoring response to dengue infection or may simply reside in the use of immunoassays as part of standard laboratory procedures in the study of the progress of antibody formation or in epitope mapping and the like. The antigen is employed in standard immunoassay formats with standard detection systems such as enzyme-based, fluorescence-based, or isotope-based detection systems. Preferably, the antigen is used coupled to solid support or in sandwich assays, but a multiplicity of protocols is possible and standard in the art.
Thus, the secreted protein, such as 60% E, 80% E or B domain may be adsorbed onto solid support and the support then treated with a sample to be tested for the presence of antidengue antibodies. Unbound sample is removed, and any bound antibodies are detected using standard detection systems, for example, by treating the support with an antispecies antibody with respect to the species represented in the sample to be tested, the antispecies antibody coupled to a detection reagent, for example, horseradish peroxidase (HRP). The presence of the HRP conjugated antispecies antibody is then detected by supplying a suitable chromogenic substrate.
Alternatively, the anti-subunit or antidengue antibody may be adsorbed to the solid support and detected by treating the solid support with the recombinant domain B, either directly labeled, or labeled with an additional antibody in a sandwich-type assay.
In addition, both the mature peptides, such as domain B and 60% E or 80% E of the invention and the antibodies immunoreactive with it can be used in standard purification procedures as affinity reagents. Thus, purification of the subunits from recombinantly produced cultures can be effected by affinity columns using antibodies raised against these antigens. Conversely, immunoglobulins useful in passive vaccines can be readily purified from antisera using the peptides of the invention.
The mature domain B or other subunit of the invention may be used to detect the presence or absence of antibodies of various isotypes, including IgG and IgM isotypes. As set forth above, detection of IgM isotypes is significant since this is an index of primary infection.
In the examples below, particular subunits of the dengue Type 2 envelope protein, in particular 60% E, 80% E and domain B are illustrated as representative of effective subunits of the envelope protein. For the 60% E and 80% E constructs in general, secretion can be obtained from constructions designed to express the prME subunit fusion. The mature N-terminus of the envelope protein is then secreted into the culture medium. Whether the N-terminus of the envelope protein subunits were fused to a heterologous leader, such as the human tissue plasminogen activator leader sequence, or to the homologous prM sequence, the mature form of the truncated envelope protein is secreted. The secreted truncated Es are expressed at high levels in Drosophila, efficiently processed, and secreted into the medium. The products are glycosylated and processed to an endo-H resistant form. The secreted form of truncated E produced cotranslationally with prM generally represents about 20-30% of the total protein in the medium. Furthermore, based upon reactivity with conformationally sensitive monoclonal antibodies, using a ELISA and immunofluorescence formats, the secreted E products are shown to have a native conformation. Immunization of mice with crude medium from transformed cells expressing prM-truncated E induces a potent virus-neutralizing response.
The following examples are intended to illustrate but not to limit the invention.
EXAMPLE 1
Preparation of Envelope Proteins in Saccharoimyces cerevisiae
A cDNA clone derived from dengue serotype 2 (DEN-2) described by Hahn, Y. S. et al. Virology (1988, supra) was used as the starting material. This cDNA derives from strain PR159/S1. This strain has a small plaque, temperature-sensitive phenotype; the complete sequence of the CDNA derived from the genomic RNA for PR159/S1 is set forth in this publication.
FIG. 2 shows the sequence of the CDNA derived from genomic RNA of DEN-2 PR159/S1 for the Capsid, preMembrane, Envelope, and NS1 genes. Shown in bold at nucleotides 103, 1940, 1991, and 2025 are corrections to the Hahn published sequence. Differences in the S1 sequence from the wild-type sequence are noted above the wild-type sequence. There are no nucleotide differences in the Capsid and preMembrane protein-encoding portions and there are four in the E encoding portion.
FIG. 3 shows the cDNA sequence of DEN-2 PR159/S1 for the Capsid, preMembrane, Envelope, and NS1 genes and the inferred translation of those four genes, which is part of the larger dengue polyprotein. The four differences between wild-type DEN-2 PR159 and the S1 strain are shown above the S1 nucleotide sequence. Also shown is the context of the codons in which the nucleotide differences occur and the encoded amino acid.
In the E gene, three of the four mutations are silent; S1 has G instead of A at position 1314, T rather than G at position 1356, and C rather than T at position 2025. The mutation at position 1858, a G in S1 rather than A, results in a coding change from Ile in wild-type to Val in S1. There is, therefore, a one amino acid difference in the B domain in the S1 strain as compared to wild-type. Although the amino acid substitution conferred by the mutation at 1858 is conservative, mutagenesis studies of other viral structural proteins (Coller, B. G. et al. (1994) Mutagenesis Studies on the Predicted VP2 E-F Loop of Coxsackievirus B3, Abstract, 13th Annual Meeting of the American Society for Virology) have demonstrated that even relatively conservative single amino acid changes in surface loops can have profound effects on viral biology, including infectivity and viral stability. Thus, it is not without foundation that the conservative mutation in domain B of DEN-2 PR159/S1 may be involved in the attenuation of this small-plaque, temperature-sensitive variant.
Subsequent determinations on the S1 strain used herein (S1/HBG) showed that the A at position 103 in wild-type was retained in position 103 in S1/HBG and C was substituted for T at position 2025 (a silent mutation). A deduced amino acid sequence from DEN-2 PR159/S1 and differences occurring in the wild-type are shown in FIG. 3.
Various E gene subclones were obtained which represented the amino-terminal 90% of the envelope, 80% of the envelope, 60% of the envelope and classical domain B. Using the assignment of Mandl, C. W. et al. J Virol (1989) 63:564-571, classical domain B starts at Gly296 and ends at Gly395. Classical domain B contains only one disulfide bond and is encoded by the sequence following domains A and C. This classical domain B does not contain the potential T cell epitope described by Mandl et al. at its carboxy end which can be included in some forms of the domain B of the invention, e.g., DomB+T.
The portion of the genome that encodes 80% of the envelope protein (80% E) was amplified using the Polymerase Chain Reaction, primers D2E937p and D2E2121m, and plasmid pC8 (Hahn et al. (1988, supra) as template.
In this notation of the primers, the virus serotype is first indicated (D2 for DEN-2), then the corresponding dengue gene--i.e., in this case envelope, E, is noted. Then is noted the number in the dengue cloned sequences of FIGS. 2 or 3 for the first dengue nucleotide in the 5'-3' direction of the oligonucleotide, i.e., using the numbering of Hahn et al. (1988, supra), and finally the notation shows whether the oligonucleotide primes the plus (p) or the minus (m) strand synthesis. The sequence in the primers corresponding to dengue cDNA are written in uppercase letters; nondengue sequence is written in lowercase letters. D2E937p2 - BglII 5'-cttctagatctcgagtacccgggacc ATG CGC TGC ATA GGA ATA TC-3'(SEQ ID NO:13) XbaI XhoI SmaI - D2E2121m - SalI 5'-gctctagagtcga cta tta TCC TTT CTT GAA CCA G-3'(SEQ ID NO:14) XbaI End End
The D2E2121m primer placed two stop codons after the 395th codon of E. The 80% E amplified cDNA fragment was digested at the XbaI sites in the cloning adapters and cloned into the NheI site of pBR322 to obtain p29D280E. Double-strand sequence for 80% E was determined, which identified a single silent PCR-introduced mutation at nucleotide 2001 (AAC/Asn to AAT/Asn).
A subclone representing domain B was obtained from the 80% E subclone by oligonucleotide-directed mutagenesis. In the mutagenesis, stop codons and restriction endonuclease sites were inserted between domain C- and domain B-encoding sequences. The stop codons were positioned to terminate domain A+C translation and SalI and PvuII restriction sites were added to facilitate subcloning of domains A+C and domain B fragments, respectively, into yeast expression vectors. As shown in FIG. 4, to avoid a high AT content in the mutagenic oligonucleotide, the stop codons defining the carboxy-terminus of 60% E containing domains A and C were positioned four codons upstream of the beginning of domain B, i.e., following Lys291. The original and altered nucleotide sequences of the mutagenized region and the corresponding amino acid translation are shown in FIG. 4.
To perform the mutagenesis, a 580 bp BamHI fragment spanning domain B from the pBR322-80% E clone p29D280E was subcloned into pGEM3Zf (Promega) to yield p29GEB2. (See FIG. 5.) This BamHI fragment encodes the 3' end of domains A and C and all of domain B. The plasmid obtained following mutagenesis, p29GEB24PS, was confirmed by DNA sequencing. As shown in FIG. 6, the introduced PvuII site permits direct subcloning of domain B CDNA into yeast expression vectors as a fusion with yeast secretion leader.
The cloned cDNA fragments encoding B domain and 80% E were inserted into expression vectors so as to maintain the translational frame of fusions to secretion leaders as described below. The sequence of the fusions were confirmed by DNA sequencing.
EXAMPLE 2
Secretion of Domain B from Saccharomyces cerevisiae
The expression vector constructed to secrete classical domain B from Saccharomyces cerevisiae to include envelope protein amino acids 296-395 was constructed so that processing by the proteases normally involved in preproMF.alpha..sub.L processing would yield a precisely processed domain B. One of these proteases, Kex2p, the gene product of the Kex2 gene, cleaves following dibasic peptides, such as LysArg, LysLys, ArgArg, and ArgLys. A second protease involved, a dipeptidyl amino peptidase, removes GluAla and AspAla dipeptides from the amino terminus after Kex2p processing.
The MF.alpha. prepropeptide (MF.alpha..sub.L)-domain B fusion was operably linked to the constitutive promoter from the gene (TDH3) encoding glyceraldehyde phosphate dehydrogenase (GAPDH) contained in pLS5 and, alternatively, to the copper sulfate-inducible CUPI gene promoter contained in pLS6. These vectors were provided by SmithKline Beecham. Both contain the MF.alpha..sub.L sequence, and StuI, BglII, and SalI cloning sites, and use the TRP1 gene as a selectable marker. They contain sequences derived from pBR322 to provide an E. coli origin of replication, the ampicillin resistance gene, and sequences derived from the 2-micron plasmid of S. cerevisiae to enable replication in S. cerevisiae. The insertion of the classical domain B coding sequence into the appropriate reading frame in pLS5 was accomplished by digesting pLS5 with StuI and SalI and by digesting p29GEB24PS with PvuII and SalI and gel purifying the small PvuII-SalI domain B fragment and ligating it into the thus opened pLS5 as shown in FIG. 6. A domain B expression vector derived from pLS6 was constructed in a similar manner.
The nucleotide and amino acid sequence of the MF.alpha..sub.L -domain B fusion is shown in FIG. 7.
The resulting expression vectors were transfected into spheroplasts of Saccharomyces cerevisiae strain GL43 (MATatrp1.DELTA.1 ura3-52 proA::URA3) and transformants were selected by their ability to grow on minimal medium without tryptophan supplementation. S. cerevisiae strain GL43 was supplied by Smith-Kline Beecham. Strain GL43 pLS5-DomB and pLS6-DomB transformants were grown in small (<5 ml) cultures i Casamino acid-supplemented minimal medium, and proteins secreted into the culture medium and cellular proteins were analyzed by Coomassie-stained SDS-PAGE and Western blot. A novel Coomassie-staining band of approximately 12 kD, which was weakly immunoreactive with DEN-2 HMAF, was observed in the culture media of domain B transformants. No novel Coomassie-staining bands and little or no immunoreactive protein not found in negative controls was observed in cellular protein extracts of domain B transformants. When cultured in Casamino acid supplemented minimal medium, comparable levels of domain B were secreted into the medium by pLS5-DomB and pLS6-DomB transformants. About 20% of the total secreted protein was domain B as estimated by the intensity of Coomassie brilliant blue staining of protein in an SDS-PAGE polyacrylamide gel.
The composition of the medium was optimized; when S. cerevisiae containing pLS5-DomB were grown in minimal medium (SD) or Casamino acids (Difco)-supplemented. minimal medium, the yields of protein were less than when cultured in rich (YEPD) medium. The pLS5-domain B transformant was preferred since this transformant can be cultured in rich medium whereas pLS6-domain B transformants require the use of the less preferred supplemented minimal medium in order to induce with copper sulfate.
The secreted protein was confirmed as domain B having a Glu-Ala dipeptide appendage to the N-terminus, designated herein Glu-Ala-Domain B or Glu-Ala-DomB. For this demonstration, proteins secreted by a pLS5-DomB transformant were separated by SDS-PAGE and electroblotted to Immobilon P membrane (Millipore), and the amino terminal amino acid sequence was determined by microsequencing. That sequence is: H.sub.2 N-Glu Ala Gly Met Ser Tyr Ser Met Xxx Thr Gly Lys Phe Xxx Val Val (SEQ ID NO:15). The persistence of the GluAla dipeptide indicates that the dipeptidyl aminopeptidase incompletely processed the domain B N-terminus following proteolysis by Kex2p.
EXAMPLE 3
Purification of Domain B
A. Initial Purification Method:
Pilot purifications of domain B from S. cerevisiae culture medium used size exclusion chromatography on acrylamide Biogel P100.
For these purifications, an S. cerevisiae GL43 pLS5-DomB transformant was cultured in minimal medium overnight and then transferred to either Casamino acid-supplemented minimal (SD) medium or rich (YEPD) medium for expression. After culturing at 30.degree. C. for 2-3 days and feeding daily with buffered glucose to final concentrations of 0.4% glucose and 5 mM sodium phosphate (pH 6.7), the cells were pelleted by centrifugation and the medium was clarified by filtration through a 0.45 .mu.m pore filter. The filtered medium was concentrated about 30-fold either by tangential flow or by centrifugal ultrafiltration using Minitan (Millipore) or Centricon (Amicon) devices, and the concentrate was exchanged into PBS azide buffer (2.7 mM KCl, 1.2 mM KH.sub.2 PO.sub.4, 138 mM NaCl, 4.3 mM Na.sub.2 HPO.sub.4 .multidot.7H.sub.2 O, 0.02% azide) by diafiltration or dialysis.
In one example, the concentrated secreted proteins from approximately 500 ml of culture of GL43 pLS5-DomB grown in SD plus Casamino acids were loaded onto a 2.5.times.75 cm Biogel P100 column and domain B was eluted from the column at approximately 1 bed volume. Domain B was pooled based on SDS-PAGE analysis of column fractions. The pooled domain B fractions were brownish in color and could not be decolorized by dialysis. The brown colored preparation also was not immunoreactive in ELISAs. DEAE chromatography of this sample resulted in binding of the brown color and a mostly colorless flow-through containing domain B. For DEAE chromatography, the brown-colored pooled fractions were dialyzed against 0.1 M acetic acid, pH 5.1 and loaded onto a 1.4.times.15 cm DEAE (Biorad) column. Domain B was eluted using a 0.01 M acetic acid, pH 5.1, 0.1 M NaCl step gradient.
The purified domain B was of high purity as assessed by silver stain on SDS-PAGE. The resulting Glu-Ala-DomB also gained weak reactivity with antidengue mouse polyclonal HMAF and with antidengue MAbs 3H5 and 9D12 in an ELISA format. The reactivities of earlier colorless preparations from smaller cultures were greater than this initial large-scale preparation. For the ELISA, 96-well microtiter plates were coated with domain B by overnight incubation at 4.degree. C. and then blocked with BSA in 50 mM tris-HCl, pH 7.0, 0.15 M NaCl, 0.05% Tween for 1 hour at room temperature. The plates were then treated with either DEN-2 HMAF or monoclonal antibody. Bound antibody was detected with alkaline phosphatase conjugated goat antimouse IgG and measured by the increase in optical density at 414 nm using the alkaline phosphatase substrate paranitrophenylphosphate.
The domain B frequently eluted from the Biogel P100 in two equal fractions, the first with the bulk of the secreted yeast proteins and the second as 70-90% pure domain B. Treatment with 1M NaCl, 1 and 2 M urea, and 1% DDAPS, a zwitterionic detergent, were ineffective in completely disaggregating domain B during size exclusion chromatography. Since the domain B of both fractions had the same electrophoretic mobility in the presence of detergent and absence of DTT, this polypeptide was neither covalently cross-linked to another protein nor self-polymerized via disulfide bonds.
B. Improved Purification Method:
A standard culture and purification method was developed that has reproducibly yielded immunoreactive domain B expressed and secreted by S. cerevisiae GL43 pLS5-DomB. For that method, a primary culture is prepared by inoculating 60 ml of SD medium in a 250 ml baffled flask with a single colony of yeast strain GL43 pLS5-DomB grown on an SD plate. SD medium is prepared by autoclaving 0.67% Bacto yeast nitrogen base without amino acids, cooling and providing 2.0% w/v dextrose and 12 mM K.sub.2 HPO.sub.4 using autoclave stock solutions. Solid SD medium is prepared by including Bacto agar at 1.8%. The culture is grown at 30.degree. C. for 18-24 hours with shaking at 240 rpm.
Secondary cultures are then grown in 300 ml of SD medium supplemented with 0.2% casamino acids in 2 L baffled flasks. The secondary cultures are inoculated with 30 ml of the primary culture. The secondary cultures are incubated at 30.degree. C. for 18-24 hours with shaking at 300 rpm.
Large-scale tertiary cultures are then prepared by inoculating 1 L of YEPD medium in a 4 L baffled flask with 100 ml of the secondary culture. YEPD is prepared by autoclaving 1% yeast extract plus 2% Bactopeptone, to which sterile dextrose is added to a final concentration of 2% after cooling. The large-scale cultures are incubated at 30.degree. C. for 48 hours with shaking at 300 rpm. After 24 hours, the culture is supplemented with 0.01 vol of sterile 40% w/v glucose and 0.02 vol of sterile 1 M phosphate buffer, pH 6.7.
After growth in the 1 L culture, the cells are removed by centrifugation at 5,000 rpm at 4.degree. C. for 5 min. in a Sorvall GS-3 rotor. EDTA and EGTA are added to final concentrations of 1 mM each to the cleared medium, and the resulting solution is filter sterilized using a 045 .mu.m pore filter membrane (Millipack-20 or Opticap-50, Millipore). Glycerol is added to 10% v/v to the filtrate which is then concentrated 20-30 fold using tangential flow ultrafiltration (Millipore Minitan System) with two membrane cartridges (four regenerated cellulose membranes) of a 10 kD MW cutoff. The retentate is kept on ice during ultrafiltration in the coldroom at 4.degree. C. for about 10-15 hours.
The concentrated medium is directly used or rapidly frozen on dry ice/ethanol in 10 ml aliquots in 15 ml polypropylene or polystyrene tubes with a screw cap and stored at -70.degree. C.
Concentrated medium of the tertiary culture is dialyzed at 4.degree. C. using a membrane with a 6-8 kD cutoff (Spectra/Pore-1 Membrane) against 10 mM acetate, pH 4.5 (4.times.4 liters; 2-3 hours each; 1 overnight).
For purification, the concentrated, buffer-exchanged medium is loaded onto a 2.5.times.22 cm CM Biogel A (Biorad) column at 0.8 ml/min. at 4.degree. C. The column is washed with 150-250 ml of 10 mM acetate, pH 4.5 until the optical density at 280 nm of the eluent drops to baseline. Domain B is then eluted with 10 mM acetate, 300 mM NaCl, pH 4.5 at 0.8 ml/min. Fractions of approximately 8 ml are collected and analyzed by SDS-PAGE (15%) and visualized by silver staining. Domain B migrates on the trailing edge of the main peak of eluted material as monitored by 280 nm.
The domain B-containing fractions are pooled and concentrated about 11-fold by centrifugal ultrafiltration using, for example, a Centriprep-10.RTM. (brand membrane ultrafiltration system) (Amicon) and loaded onto a 5.times.60 cm Sephadex G-75.RTM. (brand polysepharose) (Superfine, Pharmacia) column at 4.degree. C. The column is eluted using phosphate-buffered saline (PBS: 8 g/l NaCl, 0.2 g/l KCl, 1.44 g/l Na.sub.2 HPO.sub.4, 0.24 g/l KH.sub.2 PO.sub.4) at a flow rate of 0.8 ml/min. Nine ml fractions are collected and analyzed by SDS-PAGE as above and by indirect ELISA as above using Mab 9D12.
The Sephadex G-75.RTM. (brand polysepharose) fractions containing pure immunoreactive domain B from 2-4 purification runs are concentrated about 50-fold by centrifugal ultrafiltration using a Centriprep-10.RTM. (brand membrane ultrafiltration system) (Amicon; final volume 2-5 ml). The pooled material is stored at 4.degree. C.
Glu-Ala-DomB purified as above typically is immunoreactive with the virus-neutralizing monoclonal antibodies 3H5 and 9D12. The above-described culturing and purification protocol has been performed several times with reproducible results. The number of cultures of the above-indicated volumes can be increased, and we have grown simultaneously up to six 1 L tertiary cultures. The culture medium from the multiple cultures is pooled for clarification and concentration, and domain B from the concentrate is purified in batches and pooled following verification of immunoreactivity. The amount of protein in the purified domain B preparations may be quantitated by total amino acid analysis using HCl hydrolysis with on-line ninhydrin reaction and optical detection. Table X presents the yield of protein in purified domain B from four cultures.
TABLE A______________________________________Total protein yields in purified domain B preparations Lot No. Culture Volume Final Yield* Yield mg/l______________________________________198.76 850 ml 4 mg 4.7 198.71 1500 ml 35 mg 23 244.68 5750 ml 90 mg 16 244.190 5000 ml 25 mg 5______________________________________ *Based on amino acid composition of total protein.
EXAMPLE 4
Production of Domain B in Pichia pastoris
In addition to Saccharomyces cerevisiae as used in the previous examples, the methylotrophic budding yeast P. pastoris was used as a host. The domain B coding sequence was subcloned into the expression vector pPIC9 (Invitrogen, San Diego, Calif.) which contains the alcohol oxidase (AOX1) promoter sequence from P. pastoris, the MF.alpha. prepropeptide secretion leader from S. cerevisiae, the AOX1 transcriptional termination sequence, and the HIS4 gene from S. cerevisiae as a selectable marker. To construct pPIC9-DomB, the domain B-encoding XhoI-SalI fragment from pLS5DomB was inserted into XhoI-digested pPIC9 as shown in FIG. 8. XhoI cuts at identical positions within the MF.alpha..sub.L -encoding DNA of both pLS5 and pPIC9, and SalI cuts immediately following the translational stop codons following domain B-encoding sequences. After verification by DNA sequence analysis, the expression cassette, consisting of the AOX1 promoter-MF.alpha..sub.L -domain B-AOX1 terminator, plus HIS4 was released from pPIC9-DomB by BglII digestion and the linear DNA was transformed into competent P. pastoris cells according to the supplier's instructions (Invitrogen, San Diego, Calif.). Transformants were selected for the ability to grow on medium lacking histidine (His.sup..+-. phenotype), and specific integration of the transforming DNA at the AOX1 locus was indicated in some transformants by slow growth on medium containing methanol (Mut.sup.s phenotype) as the sole carbon source.
The expression and secretion of domain B from eleven P. pastoris pPIC9-DomB transformants in 3 ml cultures was surveyed by Coomassie-stained SDS-PAGE analysis. A protein of molecular weight equal to domain B secreted by S. cerevisiae was evident in the culture media of ten of the eleven P. pastoris transformants. Immunoblots of a comparable polyacrylamide gel demonstrated that the protein secreted by P. pastoris pPIC9-DomB transformants was immunoreactive with polyclonal antibodies made to domain B secreted by S. cerevisiae (see Example 6). The SDS-PAGE analysis indicated that P. pastoris secreted domain B at a level comparable to or higher than that achieved in S. cerevisiae.
For further comparison of the secreted expression levels of domain B by S. cerevisiae and P. pastoris, the S. cerevisiae transformant and the two best P. pastoris transformants, Nos. 5-8 (Mut.sup..+-.) and 6-16 (Mut.sup.s), were cultured in 100 ml shake flask cultures. S. cerevisiae pLS5-DomB was cultured 48 hrs in YEPD medium, and 100 ml precultures of the P. pastoris pPIC9-DomB transformants were grown in BMGY medium (1.34% yeast nitrogen base without amino acids, 2% Bacto peptone, 0.4 .mu.g/ml biotin, 1% glycerol, and 100 mM potassium phosphate, pH 6.8) for 24 hrs and then harvested and resuspended ni 25 ml of BMMY heterologous protein-inducing medium (the same as BMGY, except with 0.5% methanol replacing the glycerol) and cultured 48 hrs. Culture media with cells removed by sequential centrifugation and filtration through a 0.45 .mu.m pore size membrane were buffer exchanged by diafiltration into TEEN (10 mM Tris, pH 8.0, 1 mM EDTA, 1 mM EGTA, and 150 mM NaCl). The relative levels of domain B secreted by the three cultures were estimated by size fractionating the proteins in 3, 6, 9, and 12 .mu.l of each sample on a 15% SDS-PAGE gel and staining with Coomassie blue. As estimated by the intensity of Coomassie staining, P. pastoris secreted at least four-fold more domain B per volume of induction culture medium than S. cerevisiae in YEPD. Correcting for the four-fold concentration of culture volume of the P. pastoris when transferred into the induction medium, we conclude that P. pastoris secretes slightly more than S. cerevisiae in shake flask cultures on a per volume basis. However, on a per gram cell dry weight basis, P. pastoris (0.80 gm total cell dry weight) secreted approximately 1.6-fold more domain B than S. cerevisiae (1.33 gm total cell dry weight).
EXAMPLE 5
Effect of Extending Domain B
In a manner similar to that set forth in the preceding examples, two plasmid constructs for expression in S. cerevisiae were made that include additional domain B-carboxy terminal sequence downstream of amino acid 395 with the domain B coding sequence. One construct, designated DomB+stem, represents Gly296-Gly445 and was expressed as a fusion with the MF.alpha..sub.L. This construct includes those sequences that lie between domain B and the transmembrane anchor of E. This region contains a potential T cell epitope (Mandl et al. J Virol (1989) 63:564-571) and additional hydrophobic sequences, a peptide from this region elicits virus-recognizing antibodies (Roehrig et al. 1922 in Vaccines 92, pp. 2777-2281), and this region may contribute to the proper folding and presentation of the domain B B-cell epitope to the immune system.
The domain B+stem cDNA fragment was constructed in E. coli cloning vectors by combining the domain B cDNA fragment and the 3' end of a 90% E clone. As introduced in Example 1, an E gene subclone representing the amino terminal 90% of E was constructed from DEN-2 PR159/S1 cDNA plasmid pC8 of Hahn et al. (1988, supra) using the PCR. The 90% E polypeptide contains all of E except for the C-terminal membrane anchor comprising two transmembrane domains. The 90% E cDNA clone was made as follows. The 90% E fragment was amplified by the PCR using pC8 as template and primers D2E937p and D2E2271m. The sequence of D2E937p is given in Example 1. The sequence of D2E2271m is:
D2E2271m:
(SEQ ID NO:16) SalI 5'-gctctagagtcga cta tta CCC GTA GAT TGC TCC G-3' XbaI End End
Primer D2E2271m placed two stop codons after Gly445 of #, and the two primers positioned useful restriction enzyme sites at both ends of the fragment. The PCR-amplified 90% E cDNA fragment was made blunt at both ends and cloned into the SmaI site of a modified pUC13 cloning vector, yielding pVZ90E.
Combining domain B and the 90% E 3' end made use of a unique Af1III restriction enzyme site found in most pUC-like cloning vectors and a unique Af1III site in domain B sequences. This combining was accomplished by first subcloning the 90% E fragment from pVZ90E into pBluescript to reverse the orientation of 90% E relative to the vector sequences, yielding pBS90E. Then, p29GEB24PS, containing domain B sequences in PGEM (Example 1), and pBS90E were digested with Af1III, and the vector-domain-B5' fragment and the domain-B3'-stem-vector fragment from the two digestions, respectively, were purified, ligated, and recovered in E. coli yielding pBS-Bstem.
For expression in S. cerevisiae, the PvuII-SalI domain B+stem fragment from pBS-Bstem was subcloned into the pLS5 yeast expression vector at its StuI and SalI sites, putting the MF.alpha..sub.L and B+stem in translational frame. The sequence of the ligated junctions were verified by DNA sequence determination. The domain B+stem expression vector, pLS5-BStem, was transformed into S. cerevisiae strain GL43. The resulting transformants grew more slowly than did transformants with pLS5-DomB containing classical DomB and the plasmid appeared to be rapidly lost during growth under nonselective conditions. The DomB+stem product may be toxic to the cells, and the hydrophobicity of the stem may inhibit secretion.
However, a similar construct representing Gly296-Ala413 analogously inserted into pLS5 resulted in substantial amounts of secreted domain B protein. This form of domain B, designated herein DomB+T, contains the downstream T cell epitope. As stated above, the DomB proteins of the present invention include classical DomB and all or part of the amino acid sequence downstream from Gly395 to the Ala residue at position 413.
The DomB+T cDNA fragment was amplified by the PCR from pC8 plasmid DNA using oligonucleotide primers D2E1822p and D2E2175m. The primer sequences are:
D2E1822p KpnI XhoI
5'-cta gcg gta ccc tcg aga AAA GGG AGG CCG GGA TGT CAT ACT CCA TGT GC-3' (SEQ ID NO:17)
D2E2175m SalI NotI
5'-cgt gtg tcg acg cgg ccg cta tta GGC CAT TCT TTT CGC TCC-3' (SEQ ID NO:18)
The PCR product was cloned into pUC18 following KpnI and SalI digestion of the product. The DomB+T cDNA fragment was then released from pUC18-DomB+T by XhoI-SalI digestion and cloned into the XhoI site in pLS5 within the MF.alpha..sub.L and Gly395 of domain B+T. S. cerevisiae strain GL43 secrets approximately ten-fold more domain B than domain B+T.
EXAMPLE 6
Preparation of Antibodies to the Recombinant Domain B Protein
The Glu-Ala-DomB peptide which had been column-purified, denatured by heating in SDS, and reduced by treatment with DTT was used to immunize Swiss Webster mice following removal of the excess SDS. The mice yielded high-titre antibodies that were highly immunoreactive when used to probe Western blots displaying the same antigen or DEN-2 viral envelope protein.
EXAMPLE 7
Recombinantly Produced Glu-Ala-Domain B in ELISA Assays
The serological diagnosis for dengue infection is based on the detection of anti-dengue IgM and IgG in primary and secondary viral infection using standard Enzyme Linked Immunosorbent Assay (ELISA) techniques. Current assays are based on the ability of anti-dengue immunoglobulins to recognize semi-purified virus. Primary and secondary infections can be distinguished by the IgM:IgG ratios. (Innis et al., 1989; Kuno et al., 1991).
The recombinantly produced Glu-Ala-domain B purified as in paragraph B of Example 3 was tested as an antigen preparation in both IgM and IgG tests. In these ELISAs, the antigen was coated on plates, followed by sera positive for dengue antibodies and then detection by goat antihuman antibody.
Microtiter plates were coated overnight at 4.degree. C. with 100 .mu.l of a 5 .mu.g/ml solution of domain B. After blocking with 3% normal goat serum, primary infected dengue 2 positive serum at a 1:100 dilution was added per well. The bound IgM was detected by the addition of goat anti-human IgM (Fc) conjugated to horseradish peroxidase. Under these conditions, the recombinant Glu-Ala-domain B did not provide positive results.
For IgG, high titer secondary dengue 2 infected sera were supplied at a 1:270 dilution and detected with goat anti-human IgG (Fc) conjugated to horseradish peroxidase. Two of the five sera gave positive reactions in this assay.
The recombinant Glu-Ala-domain B purified by the improved procedure of Example 3 is not recognized by murine polyclonal hyperimmune ascitic fluid (HMAF) to other dengue serotypes and to other flaviviruses, when assayed using a similar ELISA format. Flavivirus infected murine sera tested include, Japanese Encephalitis virus, Tick-Borne Encephalitis virus, Yellow Fever virus, Saint Louis Encephalitis virus, West Nile virus, three viral isolates of dengue serotype 1, two viral isolates of dengue serotype 3, and two viral isolates of dengue serotype 4.
Sandwich assay for the detection of any domain B-containing envelope antigen.
In an alternative enzyme immunoassay format, anti-domain B or anti-Dengue capture antibody, polyclonal or monoclonal, may be absorbed to the solid support, and sample containing an unknown quantity or serotype of dengue antigen may be added and then detected by reacting with a second anti-domain B or anti-Dengue antibody, either conjugated to a signal-generating enzyme or to be detected using a appropriate signal generating system, of which there are a multitude. This immunoassay is useful for the quantitation of recombinantly produced envelope protein or whole virus by comparing the immunoreactivity of a known concentration of domain B with that of the unknown antigen preparation. In addition, the conformational sensitivity or epitope binding site of the anti-domain B capture antibody can be varied to garner additional information regarding the conformation of dengue protein in the preparation.
To perform the sandwich enzyme immunoassay, 100 .mu.l of anti-Dengue monoclonal antibody 9D12 or 3H5 (Henchal, E. A. et al., Am J Trop Med Hyg (1985) 34:162-169) was used to coat microtiter wells. The monoclonal antibodies were purified by Protein-A affinity chromatography and used at 10 .mu.g/ml concentration (diluted in PBS: 50 mM sodium Phosphate, pH 7.0, 0.15 M NaCl). After a one hour incubation, the wells were washed three times with TBS-T (50 mM Tris-HCl PH 7.0, 0.15 M NaCl, 0.05% Tween-20), and blocked with 200 .mu.l/well of 1% BSA in PBS for 1 hour at room temperature. Following three washes with TBS-T, 100 .mu.l per well of a standard purified solution of domain B or an unknown sample was added to each well and captured by the bound monoclonal antibody. The domain B solution was allowed to bind the antibody for one hour at room temperature, and after three washes with TBS-T, 100 .mu.l of Protein A affinity purified polyclonal rabbit anti-domain B serum (25 .mu.g/Ml) diluted in PBS-T (PBS, 0.25% BSA, 0.05% Tween-20) was added to each well and allowed to bind to the bound antigen. Following three washes with TBS-T, 100 .mu.l of alkaline phosphatase-labeled goat anti-rabbit IgG+IgM (H and L) conjugate (Caltag) diluted 1:2500 in PBS-T (previously established by titration) was added to each well. After incubation for one hour at room temperature, the plates were washed four times with TBS-T and 200 .mu.l/well of 1 mg/ml p-nitrophenylphosphate (pNPP) substrate in alkaline phosphatase substrate buffer (25 mM Trizma base, pH 9.5, 0.15 M NaCl, 5 mM MgCl.sub.2 ; 0.02% NaN.sub.2) was added. The plates were incubated for one hour at room temperature and the absorbance of each well was measured on a Dynatech MR5000 using a sample wavelength of 410 nm and a reference wavelength of 630 nm. A typical domain B standard curve is presented in FIG. 12.
EXAMPLE 8
Immunization of Mice to Raise Neutralizing Antibodies
A. Crude DomB Immunogenicity in Mice:
Media from yeast cultures secreting DomB and a negative control yeast culture were buffer-exchanged with PBS, and total secreted proteins were concentrated for injection into mice. Six groups of five mice each (Outbred Swiss strain, Simonsen) were inoculated with the crude DomB preparation, negative control secreted protein, or saline, as listed below.
______________________________________Group Immunogen Adjuvant______________________________________I Saline Freund's II Saline none III control medium Freund's IV control medium none V DomB medium Freund's VI DomB medium none______________________________________
Primary immunizations consisted of 50 .mu.g of the immunogen diluted in phosphate buffered saline, with or without complete Freund's adjuvant as indicated above. Three boosts of 25 .mu.g each were delivered at two-week intervals with no adjuvant (Groups II, IV, and VI) or Freund's incomplete adjuvant (Groups I, III, and V). Test bleeds were obtained one week after the first and second boosts, and the mice were bled out one week after the third boost.
Western blot and ELISA analysis of the first test bleeds confirmed a strong immune response to DomB among the Group VI (DomB, no adjuvant) mice. After the second bleed, this response was found to be titratable to greater than 1:6,400 by ELISA. Group VI was the only group that developed high titer DomB antibodies. The mice immunized with DomB in Freund's adjuvant (Group V) mounted a weak immune response; based on reactivity to the immunogen in Western blot format and DomB-ELISA antibody titers in the 100-400 range, Group V mice appeared to be immunosuppressed. The control mice (Groups I-IV) were all negative for antibody to DomB in both the Western and ELISA screens. In spite of high DomB antibody titers in Group VI mice, plaque reduction neutralization tests (PRNT) revealed that none of the mice immunized with the crude DomB produced neutralizing antibody (Table 1).
TABLE 1______________________________________Plaque Reduction Neutralization Tests of Sera from Mice Immunized with DomB in Concentrated, Total Secreted Yeast Proteins Dilution Group Trial Plaque Counts______________________________________PBS + 1:100 1:200 1:400 1:4 1:8 1:16 Freund's 1 109, 105 .about.100 .about.100 Adjuvants 2 34, 33 38, 30 44, 44 PBS 1:10 1:20 1:40 1:4 1:8 1:16 1 100, 106 .about.100 .about.100 2 29, 33 33, 40 39, 37 Negative 1:10 1:20 1:40 1:4 1:8 1:16 secreted 1 106, 95 .about.100 .about.100 proteins + 2 40, 35 37, 39 39, 47 Freund's Negative 1:10 1:20 1:40 1:4 1:8 1:16 secreted 1 109, 94 .about.100 .about.100 proteins 2 38, 42 44, 40 45, 42 Crude 1:10 1:20 1:40 1:4 1:8 1:16 DomB + 1 100, 92 .about.100 .about.100 Freund's 2 37 .+-. 5* 35 .+-. 6* 39 .+-. 3* Crude 1:10 1:20 1:40 1:4 1:8 1:16 DomB 1 105, 91 .about.100 .about.100 2 39 .+-. 3* 41 .+-. 3* 39 .+-. 3* DEN-2 1:100 1:200 1:400 1:800 1:1600 1:3200 HMAF 1 0, 0 0, 0 0, 0 11, 8 31, 19 81, 75 1:250 1:1000 1:4000 2 3, 6 12, 15 21, 21______________________________________ PRNT assays performed on VERO cells (Trial 1) or BHK21 C15 cells (Trial 2 with DEN2 NGC strain. All plaque counts indicate the number of plaques obtained with the sera from five animals were pooled and assayed in duplicate, except those indicated (*) where each serum sample was individually assayed in duplicate and the number of plaques averaged (mea .+-. SD).
B. Pure DomB Immunogenicity in Mice:
In contrast to mice immunized with crude DomB, outbred ICR mice (Charles River) immunized with purified DomB demonstrated high titer DEN-2 virus neutralizing antibodies. Purified DomB, at a concentration of 3.5 mg/ml, was used to immunize mice (three per group) with 175 .mu.g of purified DomB mixed 1:1 with Freund's adjuvant, with alum, or without adjuvant (PBS). Test bleeds taken after three inoculations were assayed by PRNT (Table 2).
TABLE 2__________________________________________________________________________Plaque Reduction Neutralization Tests of Sera from Mice Immunized with Purified DomB 80% Dilution PRNT Group Trial Plaque Counts Titer.sup.-1__________________________________________________________________________Negative 1:10 1:20 1:40 Ascites 1 36, 40 35, 34 40, 38 Fluid 2 39, 38 47, 45 41, 44 DEN-2 1:100 1:200 1:400 1:800 1:1600 1:3200 HMAF 1 0, 0 0, 0 1, 0 2, 1 8, 9 13, 14 1500 2 0, 1 1, 0 1, 2 1, 1 7, 6 16, 17 >1600 DomB 1:10 1:20 1:40 1:80 1:160 1:320 no 1 36, 29 28, 36 29, 27 36, 30 40, 25 28, 31 >10 adjuvant 2 9, 17 20, 15 22, 26 33, 26 41, 36 42, 47 .about.10 DomB 1:10 1:20 1:40 1:80 1:160 1:30 alum 1 8, 6 10, 9 10, 12 21, 20 32, 26 30, 28 10 2 1, 0 5, 4 8, 7 12, 15 20, 25 27, 35 40 DomB 1:40 1:80 1:160 1:320 1:640 1:1200 Freund's 1 0, 0 1, 0 1, 4 9, 2 ND ND >320 2 0, 0 3, 4 5, 5 5, 20 13, 9 23, 20 .about.640__________________________________________________________________________ PRNT assays performed on VERO cells with DEN2 NGC strain. ND = Not Determined.
Mice immunized with purified DomB in the absence of adjuvant lacked neutralizing antibodies. DomB administered in combination with alum elicited low titer (80T PRNT .about.1:10) neutralizing antibodies, and mice receiving purified DomB in combination with Freund's adjuvants had a high PRNT titer (BOT PRNT >1:320).
Three groups of 3 mice each, 5-6 week-old outbred ICR strain (Charles River) were used. Inoculation was intramuscularly in the rump at one site using 10 .mu.g of antigen in 0.1 ml administered solution. Three inoculations were given to each group on days 1, 20, and 43. In one group, inoculation on day 1 incorporated complete Freunds adjuvants, on day 20 incomplete Freunds adjuvant, and that at day 43, no adjuvant. In a separate group, no adjuvant was supplied and in a third group, alum was supplied with all three inoculations. The sera were withdrawn on day 57 and the sera from each group were pooled, heat-inactivated at 56.degree. C. for 30 minutes and tested for their ability to reduce plaques formed from VERO cells.
C. DomB Protection from Virus Challenge:
Suckling mice were immunized with purified DomB in Freund's, Alum, Hunter's TiterMax (Vaxcel), or no adjuvant for protection against an intracerebral injection of DEN-2 New Guinea C (NGC) strain. DomB administered in all adjuvants conferred comparable moderate survival against dengue virus challenges although survival was statistically significant (P<0.5 G test) only for mice immunized with DomB and Hunter's TiterMax. The results are shown in FIG. 10.
D. KLH-DomB Immunogenicity in Mice:
Two series of mouse immunizations were initiated to determine the 50% effective immunizing dose (EID.sub.50) of unconjugated and KLH-conjugated DomB. Effects of alum and Freund's adjuvants to a no-adjuvant control are compared.
DomB was conjugated to KLH via EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl) using the carbodiimide coupling method at a 1.5:1 DomB-to-KLH mass ratio. The amount of unconjugated and conjugated DomB used for these immunizations was normalized, relative to the amount of unconjugated DomB. This normalization was based upon the specific immunoreactivity of each preparation as assayed by indirect ELISA.
Mice were immunized with the priming does of 174, 52, or 5.2 .mu.g (total protein) of the KLH-conjugated DomB in Freund's, alum, or no adjuvant. Additional mice were immunized with 87, 26, or 2.6 .mu.g (total protein) of unconjugated DomB in Freund's adjuvant to allow direct comparison of conjugated and unconjugated material. Boosts consisting of one-half the priming dose are being given at two-week intervals. Test bleeds are assayed for the induction of anti-DomB antibodies by ELISA and Western blot. Final bleeds are tested for induction of a neutralizing immune response by PRNT assay as well as for the production of binding antibodies by ELISA and Western blot. The results are summarized in Table 3.
The response to unconjugated DomB with Freunds adjuvants was low compared to the results in Table 2, which had shown that unconjugated DomB induced a strong virus neutralizing response in outbred ICR mice when administered with Freunds' adjuvants. This apparent difference may be grounded in testing pooled sera for the data in Table 2. Pooling sera may mask individual variability. The variability in Table 3 may be attributed to the limited epitopes in DomB and to differences in the MHC genes for the outbred Swiss mice used in Table 3.
TABLE 3__________________________________________________________________________Mouse Immune Response to Unconjugated and KLH-Conjugated Purified Domain Final Titer Final Titer Final Titer mouse antigen adjuvant Western.sup.c ELISA.sup.c PRNT.sub.10 .sup.c__________________________________________________________________________ 1 saline none N/T.sup.a <1:100 N/T 2 N/T <1:100 <1:10 3 N/T <1:100 <1:10 4 87 .mu.g B none N/T <1:100 <1:10 5 N/T <1:100 N/T 6 N/T <1:100 <1:10 7 87 .mu.g B Freund's >1:100,000 >1:102,400 1:40 Dblt.sup.b 8 N/T <1:100 <1:10 9 1:1,000,000 Dblt >1:409,600 <1:10 10 N/T <1:100 <1:10 11 1:100,000 1:25,600 <1:10 Dblt 12 26 .mu.g B Freund's N/T <100 <1:10 13 1:100,000 Dblt 1:102,400 <1:10 14 1:10,000 Dblt 1:102,400 N/T 15 1:10,000 Dblt >1:25,600 <1:20.sup.c 16 1:10,000 Dblt 1:409,600 <1:10 17 2.6 .mu.g B Freund's 1:1,000 Dblt 1:25,600 <1:10 18 N/T <1:100 <1:46 19 N/T <1:100 <1:48 20 N/T <1:100 <1:24 21 N/T <1:100 <1:10 22 "87 .mu.g" none N/T 1:100 <1:10 23 KLH-B <1:100 1:400 <1:24 24 N/T <1:100 <1:24 25 N/T <1:100 <1:24 26 N/T <1:100 <1:20 27 "26 .mu.g" none N/T <1:100 <1:34 28 KLH-B N/T <1:100 <1:70 29 N/T <1:100 <1:34 30 N/T <1:100 N/T 31 N/T <1:100 <1:10 32 "2.6 .mu.g" none N/T <1.100 N/T 33 KLH-B N/T <1:100 <1:10 34 N/T <1:100 <1:10 35 N/T <1:100 <1:10 36 N/T <1:100 <1:10 37 "87 .mu.g" Freund's N/T >1:1,600 1:20 38 KLH-B N/T <1:100 <1:10 39 N/T <1:100 <1:10 40 1:1,000 >1:1,600 1:20 41 N/T <1:100 1:10 42 "26 .mu.g" Freund's 1:10,000 Dblt >1:25,600 1:10 43 KLH-B 1:10,000 Dblt >1:25,600 1:2560 44 1:10,000 Dblt >1:25,600 1:5120 45 N/T <1:100 <1:10 46 N/T 1:100 <1:10 47 "2.6 .mu.g" Freund's N/T <1:100 <1:10 48 KLH-B N/T <1:100 <1:10 49 <1:50 1:6,400 <1:10 50 N/T <1:100 <1:10 51 <1:50 1:25.600 <1:10 52 "87 .mu.g" Alum <1:100 >1:400 1:60 53 KLH-B N/T <1:100 <1:10 54 <1:100 >1:1,600 1:320 55 <1:100 >1:1,600 1:80 56 1:1000 <1:6,400 1:640 62 "26 .mu.g" Alum <1:100 1:1,600 1:120 63 KLH-B 1:1000 >1:1,600 1:120 64 N/T <1:100 <1:10 65 1:100 >1:1,600 1:80 66 N/T <1:100 <1:10 57 "2.6 .mu.g" Alum N/T <1:100 <1:10 58 KLH-B N/T >1:100 <1:10 59 N/T <1:100 <1:10 60 N/T <1:100 <1:10 61 N/T <1:100 <1:10__________________________________________________________________________ .sup.a N/T not tested .sup.b Dblt a doublet at approximately 12 kD .sup.c If serum was insufficient for testing at 1:10 dilution then higher initial dilutions were used and are indicated.
E. DomB Immunizations for Hybridoma Generation:
Six BALB/c mice were immunized with 87 .mu.g of unconjugated DomB or 174 .mu.g KLH-conjugated DomB in Freund's adjuvant. Upon demonstration of a strong anti-DomB response, the mice are sacrificed and their spleen cells fused to hybridoma cells for monoclonal antibody production.
EXAMPLE 9
Production of Domain B in Drosophila
The cloning vector p29GEB2.4PS, containing the domain B-encoding nucleotide sequence was digested with PvuII and the short fragment ligated into pMttSma, which is derived from pMttbns by digesting with BglII and inserting an adapter oligonucleotide containing an Sma site. The duplex linker adaptor inserted has the sequence PGATCCCGG. pMttsma then contains a unique SmaI site at the 3' end of sequences encoding the tPA leader. The parent vector pMttbns SmithKline contains the Drosophila metalothionein gene (mtn), the human tissue plasminogen activator secretion signal (tPA.sub.L) and the SV40 early polyadenylation signal. The resulting expression vector, pMttDomB is thus obtained as shown in FIG. 11. The sequence of the fusion is shown in FIG. 9.
The resulting pMttDomB vector was transfected into Schneider S2 cells which were maintained in Schneider medium (Life Technologies, Grand Island, N.Y.) supplemented with 10% heat-inactivated FBS at 25.degree. C. The cells were plated the day prior to transfection at 5.times.10.sup.5 cells in 60 mm tissue culture dishes in a total volume of 4 ml Schneider medium plus 10% FBS. The Schneider cells were then cotransfected with pMttDomB and pCOHygro (a hygromycin B phosphotransferase-based selection vector obtained from SmithKline Beecham) using standard calcium phosphate transfection. 48 hours after transfection, the medium was supplemented with hygromycin at 300 .mu.g/ml; hygromycin-resistant cells grew in 2-3 weeks. Varying levels of the cotransformed plasmids were used in the transfection procedure ranging from expression plasmid: PCOHygro, 1:1 to 100:1.
Cells cotransfected at ratios of 1:1, 5:1 and 20:1 were induced with 200 .mu.M copper sulfate and the media and cells were harvested at days 1, 4 and 7. Western blots showed secretion of domain B into the medium.
EXAMPLE 10
Production of 60% E and 80% E in Drosophila
Using the adapted expression vector of Example 9, containing the Drosophila metalothionein gene, the human tissue plasminogen activator signal and the SV-40 early polyadenylation signal, the nucleotide sequences encoding 80% E, prM 80% E, 60% E and prM 60% E are inserted and the resulting vectors used to transform Schneider cells as described in Example 9. The mature truncated forms of the envelope protein are secreted into the medium, or properly processed, and are conformationally correct with respect to the corresponding native portions of the envelope protein.
Furthermore, crude media from these transformants employed in the protocol set forth in paragraphs A and B of Example 8 produce antibodies which are neutralizing against dengue virus.
__________________________________________________________________________# SEQUENCE LISTING - - - - (1) GENERAL INFORMATION: - - (iii) NUMBER OF SEQUENCES: 18 - - - - (2) INFORMATION FOR SEQ ID NO:1: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3381 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: cDNA - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Dengue vi - #rus (B) STRAIN: Serotype 2 - #(Den-2) - - (vii) IMMEDIATE SOURCE: (B) CLONE: Den-2 PR159/ - #S1 - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- - #feature (B) LOCATION: group(103, - #1940, 1991, 2025) (D) OTHER INFORMATION: - #/note= "Positions in the S1 strain representing - # corrections to the wild type DEN-2 PR159 strain re - #ported by Hahn(Citation #1)" /citation=- # ([1]) - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- - #feature (B) LOCATION: 1218 (D) OTHER INFORMATION: - #/note= "G is replaced by A for Wild-Type - #sequence" - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- - #feature (B) LOCATION: 1260 (D) OTHER INFORMATION: - #/note= "T is replaced by G for Wild-Type - #sequence" - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- - #feature (B) LOCATION: 1762 (D) OTHER INFORMATION: - #/note= "G is replaced by A for Wild-Type - #sequence" - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- - #feature (B) LOCATION: 1929 (D) OTHER INFORMATION: - #/note= "C is replaced by T for Wild-Type - #sequence" - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- - #feature (B) LOCATION: 2310 (D) OTHER INFORMATION: - #/note= "A is replaced by N for Wild-Type - #sequence" - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- - #feature (B) LOCATION: 1 (D) OTHER INFORMATION: - #/note= "Start of coding strand sequence - #for Capsid." - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- - #feature (B) LOCATION: 343 (D) OTHER INFORMATION: - #/note= "Start of coding strand sequence - #for preMembrane" - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- - #feature (B) LOCATION: 616 (D) OTHER INFORMATION: - #/note= "Start of coding strand sequence - #for Membrane" - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- - #feature (B) LOCATION: 841 (D) OTHER INFORMATION: - #/note= "Start of coding strand sequence - #for Envelope" - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- - #feature (B) LOCATION: 2326 (D) OTHER INFORMATION: - #/note= "Start of coding strand sequence - #for NS1" - - (x) PUBLICATION INFORMATION: (A) AUTHORS: Hahn, Y.S. (C) JOURNAL: Virology (D) VOLUME: 162 (F) PAGES: 167-180 (G) DATE: 1988 - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: - - ATGAATAACC AACGGAAAAA GGCGAGAAAC ACGCCTTTCA ATATGCTGAA AC -#GCGAGAGA 60 - - AACCGCGTGT CAACTGTACA ACAGTTGACA AAGAGATTCT CACTTGGAAT GC -#TGCAGGGA 120 - - CGAGGACCAC TAAAATTGTT CATGGCCCTG GTGGCATTCC TTCGTTTCCT AA -#CAATCCCA 180 - - CCAACAGCAG GGATATTAAA AAGATGGGGA ACAATTAAAA AATCAAAGGC TA -#TTAATGTT 240 - - CTGAGAGGCT TCAGGAAAGA GATTGGAAGG ATGCTGAATA TCTTAAACAG GA -#GACGTAGA 300 - - ACTGCAGGCA TGATCATCAT GCTGATTCCA ACAGTGATGG CGTTTCATCT GA -#CCACACGC 360 - - AACGGAGAAC CACACATGAT CGTCAGTAGA CAAGAAAAAG GGAAAAGCCT TC -#TGTTTAAG 420 - - ACAAAGGACG GCACGAACAT GTGTACCCTC ATGGCCATGG ACCTTGGTGA GT -#TGTGTGAA 480 - - GACACAATCA CGTATAAATG TCCCTTTCTC AAGCAGAACG AACCAGAAGA CA -#TAGATTGT 540 - - TGGTGCAACT CCACGTCCAC ATGGGTAACT TATGGGACAT GTACCACCAC AG -#GAGAGCAC 600 - - AGAAGAGAAA AAAGATCAGT GGCGCTTGTT CCACACGTGG GAATGGGATT GG -#AGACACGA 660 - - ACTGAAACAT GGATGTCATC AGAAGGGGCC TGGAAACATG CCCAGAGAAT TG -#AAACTTGG 720 - - ATTCTGAGAC ATCCAGGCTT TACCATAATG GCCGCAATCC TGGCATACAC CA -#TAGGAACG 780 - - ACGCATTTCC AAAGAGTCCT GATATTCATC CTACTGACAG CCATCGCTCC TT -#CAATGACA 840 - - ATGCGCTGCA TAGGAATATC AAATAGGGAC TTTGTGGAAG GAGTGTCAGG AG -#GGAGTTGG 900 - - GTTGACATAG TTTTAGAACA TGGAAGTTGT GTGACGACGA TGGCAAAAAA TA -#AACCAACA 960 - - CTGGACTTTG AACTGATAAA AACAGAAGCC AAACAACCCG CCACCTTAAG GA -#AGTACTGT 1020 - - ATAGAGGCTA AACTGACCAA CACGACAACA GACTCGCGCT GCCCAACACA AG -#GGGAACCC 1080 - - ACCCTGAATG AAGAGCAGGA CAAAAGGTTT GTCTGCAAAC ATTCCATGGT AG -#ACAGAGGA 1140 - - TGGGGAAATG GATGTGGATT ATTTGGAAAA GGAGGCATCG TGACCTGTGC CA -#TGTTCACA 1200 - - TGCAAAAAGA ACATGGAGGG AAAAATTGTG CAGCCAGAAA ACCTGGAATA CA -#CTGTCGTT 1260 - - ATAACACCTC ATTCAGGGGA AGAACATGCA GTCGGAAATG ACACAGGAAA AC -#ATGGTAAA 1320 - - GAAGTCAAGA TAACACCACA GAGCTCCATC ACAGAGGCGG AACTGACAGG CT -#ATGGCACT 1380 - - GTTACGATGG AGTGCTCTCC AAGAACGGGC CTCGACTTCA ATGAGATGGT GT -#TGCTGCAA 1440 - - ATGAAAGACA AAGCTTGGCT GGTGCACAGA CAATGGTTCC TAGACCTACC GT -#TGCCATGG 1500 - - CTGCCCGGAG CAGACACACA AGGATCAAAT TGGATACAGA AAGAGACACT GG -#TCACCTTC 1560 - - AAAAATCCCC ATGCGAAAAA ACAGGATGTT GTTGTCTTAG GATCCCAAGA GG -#GGGCCATG 1620 - - CATACAGCAC TCACAGGGGC TACGGAAATC CAGATGTCAT CAGGAAACCT GC -#TGTTCACA 1680 - - GGACATCTTA AGTGCAGGCT GAGAATGGAC AAATTACAAC TTAAAGGGAT GT -#CATACTCC 1740 - - ATGTGCACAG GAAAGTTTAA AGTTGTGAAG GAAATAGCAG AAACACAACA TG -#GAACAATA 1800 - - GTCATTAGAG TACAATATGA AGGAGACGGC TCTCCATGCA AGATCCCTTT TG -#AGATAATG 1860 - - GATCTGGAAA AAAGACATGT TTTGGGCCGC CTGATCACAG TCAACCCAAT TG -#TAACAGAA 1920 - - AAGGACAGCC CAGTCAACAT AGAAGCAGAA CCTCCATTCG GAGACAGCTA CA -#TCATCATA 1980 - - GGAGTGGAAC CAGGACAATT GAAGCTGGAC TGGTTCAAGA AAGGAAGTTC CA -#TCGGCCAA 2040 - - ATGTTTGAGA CAACAATGAG GGGAGCGAAA AGAATGGCCA TTTTGGGCGA CA -#CAGCCTGG 2100 - - GATTTTGGAT CTCTGGGAGG AGTGTTCACA TCAATAGGAA AGGCTCTCCA CC -#AGGTTTTT 2160 - - GGAGCAATCT ACGGGGCTGC TTTCAGTGGG GTCTCATGGA CTATGAAGAT CC -#TCATAGGA 2220 - - GTTATCATCA CATGGATAGG AATGAACTCA CGTAGCACAT CACTGTCTGT GT -#CACTGGTA 2280 - - TTAGTGGGAA TCGTGACACT GTACTTGGGA GTTATGGTGC AGGCCGATAG TG -#GTTGCGTT 2340 - - GTGAGCTGGA AGAACAAAGA ACTAAAATGT GGCAGTGGAA TATTCGTCAC AG -#ATAACGTG 2400 - - CATACATGGA CAGAACAATA CAAGTTCCAA CCAGAATCCC CTTCAAAACT GG -#CTTCAGCC 2460 - - ATCCAGAAAG CTCATGAAGA GGGCATCTGT GGAATCCGCT CAGTAACAAG AC -#TGGAAAAT 2520 - - CTTATGTGGA AACAAATAAC ATCAGAATTG AATCATATTC TATCAGAAAA TG -#AAGTGAAA 2580 - - CTGACCATCA TGACAGGAGA CATCAAAGGA ATCATGCAGG TAGGAAAACG AT -#CTCTGCGG 2640 - - CCTCAACCCA CTGAGTTGAG GTATTCATGG AAAACATGGG GTAAAGCGAA AA -#TGCTCTCC 2700 - - ACAGAACTCC ATAATCAGAC CTTCCTCATT GATGGTCCCG AAACAGCAGA AT -#GCCCCAAC 2760 - - ACAAACAGAG CTTGGAATTC ACTAGAAGTT GAGGACTACG GCTTTGGAGT AT -#TCACTACC 2820 - - AATATATGGC TAAGATTGAG AGAAAAGCAG GATGCATTTT GTGACTCAAA AC -#TCATGTCA 2880 - - GCGGCCATAA AGGACAACAG AGCCGTCCAT GCTGATATGG GTTATTGGAT AG -#AAAGCGCA 2940 - - CTCAATGATA CATGGAAGAT AGAGAAAGCT TCTTTCATTG AAGTCAAAAG TT -#GCCACTGG 3000 - - CCAAAGTCAC ACACTCTATG GAGTAATGGA GTGCTAGAAA GCGAGATGGT AA -#TTCCAAAG 3060 - - AATTTCGCTG GACCAGTGTC ACAACATAAT AACAGACCAG GCTATCACAC AC -#AAACAGCA 3120 - - GGACCTTGGC ATCTAGGCAA GCTTGAGATG GACTTTGATT TCTGCGAAGG GA -#CTACAGTG 3180 - - GTGGTAACCG AGGACTGTGG AAACAGAGGG CCCTCTTTAA GAACAACCAC TG -#CCTCAGGA 3240 - - AAACTCATAA CGGAATGGTG TTGTCGATCT TGCACACTAC CACCACTAAG AT -#ACAGAGGT 3300 - - GAGGATGGAT GCTGGTACGG GATGGAAATC AGACCATTGA AAGAGAAAGA AG -#AAAATCTG 3360 - - GTCAGTTCTC TGGTCACAGC C - # - # 3381 - - - - (2) INFORMATION FOR SEQ ID NO:2: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3381 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: cDNA - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Dengue vi - #rus (B) STRAIN: Serotype 2( - #DEN-2) - - (vii) IMMEDIATE SOURCE: (B) CLONE: Den-2 PR159/ - #S1 - - (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..3381 - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- - #feature (B) LOCATION: 1216..1218 (D) OTHER INFORMATION: - #/note= "GAG(coding for Glu) is replaced - #by GAA(coding for Glu) for the wild-type DEN-2 PR1 - #59 strain(Citat - #ion #1)" /citation=- # ([1]) - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- - #feature (B) LOCATION: 1258..1260 (D) OTHER INFORMATION: - #/note= "GTG(coding for Val) is replaced - #for GTT(coding for Val) for the wild-type DEN-2 PR1 - #59 Strain(Citation #1)" /citation=- # ([1]) - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- - #feature (B) LOCATION: 1762..1764 (D) OTHER INFORMATION: - #/note= "ATT(coding for Ile) is replaced - #by GTT(coding for Val) for the wild-type DEN-2 PR1 - #59 strain(citation #1)" /citation=- # ([1]) - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- - #feature (B) LOCATION: 1927..1929 (D) OTHER INFORMATION: - #/note= "AGT(Coding for Ser) is replaced - #by AGC(coding for Ser) for the wild-type DEN-2 PR1 - #59 strain(citation #1)" /citation=- # ([1]) - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- - #feature (B) LOCATION: 1 (D) OTHER INFORMATION: - #/note= "Start of coding strand sequence - #for Capsid" - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- - #feature (B) LOCATION: 343 (D) OTHER INFORMATION: - #/note= "Start of coding strand sequence - #for preMembrane" - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- - #feature (B) LOCATION: 616 (D) OTHER INFORMATION: - #/note= "Start of coding strand sequence - #of Membrane" - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- - #feature (B) LOCATION: 841 (D) OTHER INFORMATION: - #/note= "Start of coding strand sequence - #of Envelope" - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- - #feature (B) LOCATION: 2326 (D) OTHER INFORMATION: - #/note= "Start of coding strand sequence - #for NS1" - - (x) PUBLICATION INFORMATION: (A) AUTHORS: Hahn, Y.S. (C) JOURNAL: Virology (D) VOLUME: 162 (F) PAGES: 167-180 (G) DATE: 1988 - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: - - ATG AAT AAC CAA CGG AAA AAG GCG AGA AAC AC - #G CCT TTC AAT ATG CTG 48 Met Asn Asn Gln Arg Lys Lys Ala Arg Asn Th - #r Pro Phe Asn Met Leu 1 5 - # 10 - # 15 - - AAA CGC GAG AGA AAC CGC GTG TCA ACT GTA CA - #A CAG TTG ACA AAG AGA 96 Lys Arg Glu Arg Asn Arg Val Ser Thr Val Gl - #n Gln Leu Thr Lys Arg 20 - # 25 - # 30 - - TTC TCA CTT GGA ATG CTG CAG GGA CGA GGA CC - #A CTA AAA TTG TTC ATG 144 Phe Ser Leu Gly Met Leu Gln Gly Arg Gly Pr - #o Leu Lys Leu Phe Met 35 - # 40 - # 45 - - GCC CTG GTG GCA TTC CTT CGT TTC CTA ACA AT - #C CCA CCA ACA GCA GGG 192 Ala Leu Val Ala Phe Leu Arg Phe Leu Thr Il - #e Pro Pro Thr Ala Gly 50 - # 55 - # 60 - - ATA TTA AAA AGA TGG GGA ACA ATT AAA AAA TC - #A AAG GCT ATT AAT GTT 240 Ile Leu Lys Arg Trp Gly Thr Ile Lys Lys Se - #r Lys Ala Ile Asn Val 65 - # 70 - # 75 - # 80 - - CTG AGA GGC TTC AGG AAA GAG ATT GGA AGG AT - #G CTG AAT ATC TTA AAC 288 Leu Arg Gly Phe Arg Lys Glu Ile Gly Arg Me - #t Leu Asn Ile Leu Asn 85 - # 90 - # 95 - - AGG AGA CGT AGA ACT GCA GGC ATG ATC ATC AT - #G CTG ATT CCA ACA GTG 336 Arg Arg Arg Arg Thr Ala Gly Met Ile Ile Me - #t Leu Ile Pro Thr Val 100 - # 105 - # 110 - - ATG GCG TTT CAT CTG ACC ACA CGC AAC GGA GA - #A CCA CAC ATG ATC GTC 384 Met Ala Phe His Leu Thr Thr Arg Asn Gly Gl - #u Pro His Met Ile Val 115 - # 120 - # 125 - - AGT AGA CAA GAA AAA GGG AAA AGC CTT CTG TT - #T AAG ACA AAG GAC GGC 432 Ser Arg Gln Glu Lys Gly Lys Ser Leu Leu Ph - #e Lys Thr Lys Asp Gly 130 - # 135 - # 140 - - ACG AAC ATG TGT ACC CTC ATG GCC ATG GAC CT - #T GGT GAG TTG TGT GAA 480 Thr Asn Met Cys Thr Leu Met Ala Met Asp Le - #u Gly Glu Leu Cys Glu 145 1 - #50 1 - #55 1 -#60 - - GAC ACA ATC ACG TAT AAA TGT CCC TTT CTC AA - #G CAG AAC GAA CCAGAA 528 Asp Thr Ile Thr Tyr Lys Cys Pro Phe Leu Ly - #s Gln Asn Glu Pro Glu 165 - # 170 - # 175 - - GAC ATA GAT TGT TGG TGC AAC TCC ACG TCC AC - #A TGG GTA ACT TAT GGG 576 Asp Ile Asp Cys Trp Cys Asn Ser Thr Ser Th - #r Trp Val Thr Tyr Gly 180 - # 185 - # 190 - - ACA TGT ACC ACC ACA GGA GAG CAC AGA AGA GA - #A AAA AGA TCA GTG GCG 624 Thr Cys Thr Thr Thr Gly Glu His Arg Arg Gl - #u Lys Arg Ser Val Ala 195 - # 200 - # 205 - - CTT GTT CCA CAC GTG GGA ATG GGA TTG GAG AC - #A CGA ACT GAA ACA TGG 672 Leu Val Pro His Val Gly Met Gly Leu Glu Th - #r Arg Thr Glu Thr Trp 210 - # 215 - # 220 - - ATG TCA TCA GAA GGG GCC TGG AAA CAT GCC CA - #G AGA ATT GAA ACT TGG 720 Met Ser Ser Glu Gly Ala Trp Lys His Ala Gl - #n Arg Ile Glu Thr Trp 225 2 - #30 2 - #35 2 -#40 - - ATT CTG AGA CAT CCA GGC TTT ACC ATA ATG GC - #C GCA ATC CTG GCATAC 768 Ile Leu Arg His Pro Gly Phe Thr Ile Met Al - #a Ala Ile Leu Ala Tyr 245 - # 250 - # 255 - - ACC ATA GGA ACG ACG CAT TTC CAA AGA GTC CT - #G ATA TTC ATC CTA CTG 816 Thr Ile Gly Thr Thr His Phe Gln Arg Val Le - #u Ile Phe Ile Leu Leu 260 - # 265 - # 270 - - ACA GCC ATC GCT CCT TCA ATG ACA ATG CGC TG - #C ATA GGA ATA TCA AAT 864 Thr Ala Ile Ala Pro Ser Met Thr Met Arg Cy - #s Ile Gly Ile Ser Asn 275 - # 280 - # 285 - - AGG GAC TTT GTG GAA GGA GTG TCA GGA GGG AG - #T TGG GTT GAC ATA GTT 912 Arg Asp Phe Val Glu Gly Val Ser Gly Gly Se - #r Trp Val Asp Ile Val 290 - # 295 - # 300 - - TTA GAA CAT GGA AGT TGT GTG ACG ACG ATG GC - #A AAA AAT AAA CCA ACA 960 Leu Glu His Gly Ser Cys Val Thr Thr Met Al - #a Lys Asn Lys Pro Thr 305 3 - #10 3 - #15 3 -#20 - - CTG GAC TTT GAA CTG ATA AAA ACA GAA GCC AA - #A CAA CCC GCC ACCTTA 1008 Leu Asp Phe Glu Leu Ile Lys Thr Glu Ala Ly - #s Gln Pro Ala Thr Leu 325 - # 330 - # 335 - - AGG AAG TAC TGT ATA GAG GCT AAA CTG ACC AA - #C ACG ACA ACA GAC TCG 1056 Arg Lys Tyr Cys Ile Glu Ala Lys Leu Thr As - #n Thr Thr Thr Asp Ser 340 - # 345 - # 350 - - CGC TGC CCA ACA CAA GGG GAA CCC ACC CTG AA - #T GAA GAG CAG GAC AAA 1104 Arg Cys Pro Thr Gln Gly Glu Pro Thr Leu As - #n Glu Glu Gln Asp Lys 355 - # 360 - # 365 - - AGG TTT GTC TGC AAA CAT TCC ATG GTA GAC AG - #A GGA TGG GGA AAT GGA 1152 Arg Phe Val Cys Lys His Ser Met Val Asp Ar - #g Gly Trp Gly Asn Gly 370 - # 375 - # 380 - - TGT GGA TTA TTT GGA AAA GGA GGC ATC GTG AC - #C TGT GCC ATG TTC ACA 1200 Cys Gly Leu Phe Gly Lys Gly Gly Ile Val Th - #r Cys Ala Met Phe Thr 385 3 - #90 3 - #95 4 -#00 - - TGC AAA AAG AAC ATG GAG GGA AAA ATT GTG CA - #G CCA GAA AAC CTGGAA 1248 Cys Lys Lys Asn Met Glu Gly Lys Ile Val Gl - #n Pro Glu Asn Leu Glu 405 - # 410 - # 415 - - TAC ACT GTC GTT ATA ACA CCT CAT TCA GGG GA - #A GAA CAT GCA GTC GGA 1296 Tyr Thr Val Val Ile Thr Pro His Ser Gly Gl - #u Glu His Ala Val Gly 420 - # 425 - # 430 - - AAT GAC ACA GGA AAA CAT GGT AAA GAA GTC AA - #G ATA ACA CCA CAG AGC 1344 Asn Asp Thr Gly Lys His Gly Lys Glu Val Ly - #s Ile Thr Pro Gln Ser 435 - # 440 - # 445 - - TCC ATC ACA GAG GCG GAA CTG ACA GGC TAT GG - #C ACT GTT ACG ATG GAG 1392 Ser Ile Thr Glu Ala Glu Leu Thr Gly Tyr Gl - #y Thr Val Thr Met Glu 450 - # 455 - # 460 - - TGC TCT CCA AGA ACG GGC CTC GAC TTC AAT GA - #G ATG GTG TTG CTG CAA 1440 Cys Ser Pro Arg Thr Gly Leu Asp Phe Asn Gl - #u Met Val Leu Leu Gln 465 4 - #70 4 - #75 4 -#80 - - ATG AAA GAC AAA GCT TGG CTG GTG CAC AGA CA - #A TGG TTC CTA GACCTA 1488 Met Lys Asp Lys Ala Trp Leu Val His Arg Gl - #n Trp Phe Leu Asp Leu 485 - # 490 - # 495 - - CCG TTG CCA TGG CTG CCC GGA GCA GAC ACA CA - #A GGA TCA AAT TGG ATA 1536 Pro Leu Pro Trp Leu Pro Gly Ala Asp Thr Gl - #n Gly Ser Asn Trp Ile 500 - # 505 - # 510 - - CAG AAA GAG ACA CTG GTC ACC TTC AAA AAT CC - #C CAT GCG AAA AAA CAG 1584 Gln Lys Glu Thr Leu Val Thr Phe Lys Asn Pr - #o His Ala Lys Lys Gln 515 - # 520 - # 525 - - GAT GTT GTT GTC TTA GGA TCC CAA GAG GGG GC - #C ATG CAT ACA GCA CTC 1632 Asp Val Val Val Leu Gly Ser Gln Glu Gly Al - #a Met His Thr Ala Leu 530 - # 535 - # 540 - - ACA GGG GCT ACG GAA ATC CAG ATG TCA TCA GG - #A AAC CTG CTG TTC ACA 1680 Thr Gly Ala Thr Glu Ile Gln Met Ser Ser Gl - #y Asn Leu Leu Phe Thr 545 5 - #50 5 - #55 5 -#60 - - GGA CAT CTT AAG TGC AGG CTG AGA ATG GAC AA - #A TTA CAA CTT AAAGGG 1728 Gly His Leu Lys Cys Arg Leu Arg Met Asp Ly - #s Leu Gln Leu Lys Gly 565 - # 570 - # 575 - - ATG TCA TAC TCC ATG TGC ACA GGA AAG TTT AA - #A GTT GTG AAG GAA ATA 1776 Met Ser Tyr Ser Met Cys Thr Gly Lys Phe Ly - #s Val Val Lys Glu Ile 580 - # 585 - # 590 - - GCA GAA ACA CAA CAT GGA ACA ATA GTC ATT AG - #A GTA CAA TAT GAA GGA 1824 Ala Glu Thr Gln His Gly Thr Ile Val Ile Ar - #g Val Gln Tyr Glu Gly 595 - # 600 - # 605 - - GAC GGC TCT CCA TGC AAG ATC CCT TTT GAG AT - #A ATG GAT CTG GAA AAA 1872 Asp Gly Ser Pro Cys Lys Ile Pro Phe Glu Il - #e Met Asp Leu Glu Lys 610 - # 615 - # 620 - - AGA CAT GTT TTG GGC CGC CTG ATC ACA GTC AA - #C CCA ATT GTA ACA GAA 1920 Arg His Val Leu Gly Arg Leu Ile Thr Val As - #n Pro Ile Val Thr Glu 625 6 - #30 6 - #35 6 -#40 - - AAG GAC AGC CCA GTC AAC ATA GAA GCA GAA CC - #T CCA TTC GGA GACAGC 1968 Lys Asp Ser Pro Val Asn Ile Glu Ala Glu Pr - #o Pro Phe Gly Asp Ser 645 - # 650 - # 655 - - TAC ATC ATC ATA GGA GTG GAA CCA GGA CAA TT - #G AAG CTG GAC TGG TTC 2016 Tyr Ile Ile Ile Gly Val Glu Pro Gly Gln Le - #u Lys Leu Asp Trp Phe 660 - # 665 - # 670 - - AAG AAA GGA AGT TCC ATC GGC CAA ATG TTT GA - #G ACA ACA ATG AGG GGA 2064 Lys Lys Gly Ser Ser Ile Gly Gln Met Phe Gl - #u Thr Thr Met Arg Gly 675 - # 680 - # 685 - - GCG AAA AGA ATG GCC ATT TTG GGC GAC ACA GC - #C TGG GAT TTT GGA TCT 2112 Ala Lys Arg Met Ala Ile Leu Gly Asp Thr Al - #a Trp Asp Phe Gly Ser 690 - # 695 - # 700 - - CTG GGA GGA GTG TTC ACA TCA ATA GGA AAG GC - #T CTC CAC CAG GTT TTT 2160 Leu Gly Gly Val Phe Thr Ser Ile Gly Lys Al - #a Leu His Gln Val Phe 705 7 - #10 7 - #15 7 -#20 - - GGA GCA ATC TAC GGG GCT GCT TTC AGT GGG GT - #C TCA TGG ACT ATGAAG 2208 Gly Ala Ile Tyr Gly Ala Ala Phe Ser Gly Va - #l Ser Trp Thr Met Lys 725 - # 730 - # 735 - - ATC CTC ATA GGA GTT ATC ATC ACA TGG ATA GG - #A ATG AAC TCA CGT AGC 2256 Ile Leu Ile Gly Val Ile Ile Thr Trp Ile Gl - #y Met Asn Ser Arg Ser 740 - # 745 - # 750 - - ACA TCA CTG TCT GTG TCA CTG GTA TTA GTG GG - #A ATC GTG ACA CTG TAC 2304 Thr Ser Leu Ser Val Ser Leu Val Leu Val Gl - #y Ile Val Thr Leu Tyr 755 - # 760 - # 765 - - TTG GGA GTT ATG GTG CAG GCC GAT AGT GGT TG - #C GTT GTG AGC TGG AAG 2352 Leu Gly Val Met Val Gln Ala Asp Ser Gly Cy - #s Val Val Ser Trp Lys 770 - # 775 - # 780 - - AAC AAA GAA CTA AAA TGT GGC AGT GGA ATA TT - #C GTC ACA GAT AAC GTG 2400 Asn Lys Glu Leu Lys Cys Gly Ser Gly Ile Ph - #e Val Thr Asp Asn Val 785 7 - #90 7 - #95 8 -#00 - - CAT ACA TGG ACA GAA CAA TAC AAG TTC CAA CC - #A GAA TCC CCT TCAAAA 2448 His Thr Trp Thr Glu Gln Tyr Lys Phe Gln Pr - #o Glu Ser Pro Ser Lys 805 - # 810 - # 815 - - CTG GCT TCA GCC ATC CAG AAA GCT CAT GAA GA - #G GGC ATC TGT GGA ATC 2496 Leu Ala Ser Ala Ile Gln Lys Ala His Glu Gl - #u Gly Ile Cys Gly Ile 820 - # 825 - # 830 - - CGC TCA GTA ACA AGA CTG GAA AAT CTT ATG TG - #G AAA CAA ATA ACA TCA 2544 Arg Ser Val Thr Arg Leu Glu Asn Leu Met Tr - #p Lys Gln Ile Thr Ser 835 - # 840 - # 845 - - GAA TTG AAT CAT ATT CTA TCA GAA AAT GAA GT - #G AAA CTG ACC ATC ATG 2592 Glu Leu Asn His Ile Leu Ser Glu Asn Glu Va - #l Lys Leu Thr Ile Met 850 - # 855 - # 860 - - ACA GGA GAC ATC AAA GGA ATC ATG CAG GTA GG - #A AAA CGA TCT CTG CGG 2640 Thr Gly Asp Ile Lys Gly Ile Met Gln Val Gl - #y Lys Arg Ser Leu Arg 865 8 - #70 8 - #75 8 -#80 - - CCT CAA CCC ACT GAG TTG AGG TAT TCA TGG AA - #A ACA TGG GGT AAAGCG 2688 Pro Gln Pro Thr Glu Leu Arg Tyr Ser Trp Ly - #s Thr Trp Gly Lys Ala 885 - # 890 - # 895 - - AAA ATG CTC TCC ACA GAA CTC CAT AAT CAG AC - #C TTC CTC ATT GAT GGT 2736 Lys Met Leu Ser Thr Glu Leu His Asn Gln Th - #r Phe Leu Ile Asp Gly 900 - # 905 - # 910 - - CCC GAA ACA GCA GAA TGC CCC AAC ACA AAC AG - #A GCT TGG AAT TCA CTA 2784 Pro Glu Thr Ala Glu Cys Pro Asn Thr Asn Ar - #g Ala Trp Asn Ser Leu 915 - # 920 - # 925 - - GAA GTT GAG GAC TAC GGC TTT GGA GTA TTC AC - #T ACC AAT ATA TGG CTA 2832 Glu Val Glu Asp Tyr Gly Phe Gly Val Phe Th - #r Thr Asn Ile Trp Leu 930 - # 935 - # 940 - - AGA TTG AGA GAA AAG CAG GAT GCA TTT TGT GA - #C TCA AAA CTC ATG TCA 2880 Arg Leu Arg Glu Lys Gln Asp Ala Phe Cys As - #p Ser Lys Leu Met Ser 945 9 - #50 9 - #55 9 -#60 - - GCG GCC ATA AAG GAC AAC AGA GCC GTC CAT GC - #T GAT ATG GGT TATTGG 2928 Ala Ala Ile Lys Asp Asn Arg Ala Val His Al - #a Asp Met Gly Tyr Trp 965 - # 970 - # 975 - - ATA GAA AGC GCA CTC AAT GAT ACA TGG AAG AT - #A GAG AAA GCT TCT TTC 2976 Ile Glu Ser Ala Leu Asn Asp Thr Trp Lys Il - #e Glu Lys Ala Ser Phe 980 - # 985 - # 990 - - ATT GAA GTC AAA AGT TGC CAC TGG CCA AAG TC - #A CAC ACT CTA TGG AGT 3024 Ile Glu Val Lys Ser Cys His Trp Pro Lys Se - #r His Thr Leu Trp Ser 995 - # 1000 - # 1005 - - AAT GGA GTG CTA GAA AGC GAG ATG GTA ATT CC - #A AAG AAT TTC GCT GGA 3072 Asn Gly Val Leu Glu Ser Glu Met Val Ile Pr - #o Lys Asn Phe Ala Gly 1010 - # 1015 - # 1020 - - CCA GTG TCA CAA CAT AAT AAC AGA CCA GGC TA - #T CAC ACA CAA ACA GCA 3120 Pro Val Ser Gln His Asn Asn Arg Pro Gly Ty - #r His Thr Gln Thr Ala 1025 1030 - # 1035 - # 1040 - - GGA CCT TGG CAT CTA GGC AAG CTT GAG ATG GA - #C TTT GAT TTC TGC GAA 3168 Gly Pro Trp His Leu Gly Lys Leu Glu Met As - #p Phe Asp Phe Cys Glu 1045 - # 1050 - # 1055 - - GGG ACT ACA GTG GTG GTA ACC GAG GAC TGT GG - #A AAC AGA GGG CCC TCT 3216 Gly Thr Thr Val Val Val Thr Glu Asp Cys Gl - #y Asn Arg Gly Pro Ser 1060 - # 1065 - # 1070 - - TTA AGA ACA ACC ACT GCC TCA GGA AAA CTC AT - #A ACG GAA TGG TGT TGT 3264 Leu Arg Thr Thr Thr Ala Ser Gly Lys Leu Il - #e Thr Glu Trp Cys Cys 1075 - # 1080 - # 1085 - - CGA TCT TGC ACA CTA CCA CCA CTA AGA TAC AG - #A GGT GAG GAT GGA TGC 3312 Arg Ser Cys Thr Leu Pro Pro Leu Arg Tyr Ar - #g Gly Glu Asp Gly Cys 1090 - # 1095 - # 1100 - - TGG TAC GGG ATG GAA ATC AGA CCA TTG AAA GA - #G AAA GAA GAA AAT CTG 3360 Trp Tyr Gly Met Glu Ile Arg Pro Leu Lys Gl - #u Lys Glu Glu Asn Leu 1105 1110 - # 1115 - # 1120 - - GTC AGT TCT CTG GTC ACA GCC - # - # 3381 Val Ser Ser Leu Val Thr Ala 1125 - - - - (2) INFORMATION FOR SEQ ID NO:3: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1127 amino - #acids (B) TYPE: amino acid (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: - - Met Asn Asn Gln Arg Lys Lys Ala Arg Asn Th - #r Pro Phe Asn Met Leu 1 5 - # 10 - # 15 - - Lys Arg Glu Arg Asn Arg Val Ser Thr Val Gl - #n Gln Leu Thr Lys Arg 20 - # 25 - # 30 - - Phe Ser Leu Gly Met Leu Gln Gly Arg Gly Pr - #o Leu Lys Leu Phe Met 35 - # 40 - # 45 - - Ala Leu Val Ala Phe Leu Arg Phe Leu Thr Il - #e Pro Pro Thr Ala Gly 50 - # 55 - # 60 - - Ile Leu Lys Arg Trp Gly Thr Ile Lys Lys Se - #r Lys Ala Ile Asn Val 65 - # 70 - # 75 - # 80 - - Leu Arg Gly Phe Arg Lys Glu Ile Gly Arg Me - #t Leu Asn Ile Leu Asn 85 - # 90 - # 95 - - Arg Arg Arg Arg Thr Ala Gly Met Ile Ile Me - #t Leu Ile Pro Thr Val 100 - # 105 - # 110 - - Met Ala Phe His Leu Thr Thr Arg Asn Gly Gl - #u Pro His Met Ile Val 115 - # 120 - # 125 - - Ser Arg Gln Glu Lys Gly Lys Ser Leu Leu Ph - #e Lys Thr Lys Asp Gly 130 - # 135 - # 140 - - Thr Asn Met Cys Thr Leu Met Ala Met Asp Le - #u Gly Glu Leu Cys Glu 145 1 - #50 1 - #55 1 -#60 - - Asp Thr Ile Thr Tyr Lys Cys Pro Phe Leu Ly - #s Gln Asn Glu ProGlu 165 - # 170 - # 175 - - Asp Ile Asp Cys Trp Cys Asn Ser Thr Ser Th - #r Trp Val Thr Tyr Gly 180 - # 185 - # 190 - - Thr Cys Thr Thr Thr Gly Glu His Arg Arg Gl - #u Lys Arg Ser Val Ala 195 - # 200 - # 205 - - Leu Val Pro His Val Gly Met Gly Leu Glu Th - #r Arg Thr Glu Thr Trp 210 - # 215 - # 220 - - Met Ser Ser Glu Gly Ala Trp Lys His Ala Gl - #n Arg Ile Glu Thr Trp 225 2 - #30 2 - #35 2 -#40 - - Ile Leu Arg His Pro Gly Phe Thr Ile Met Al - #a Ala Ile Leu AlaTyr 245 - # 250 - # 255 - - Thr Ile Gly Thr Thr His Phe Gln Arg Val Le - #u Ile Phe Ile Leu Leu 260 - # 265 - # 270 - - Thr Ala Ile Ala Pro Ser Met Thr Met Arg Cy - #s Ile Gly Ile Ser Asn 275 - # 280 - # 285 - - Arg Asp Phe Val Glu Gly Val Ser Gly Gly Se - #r Trp Val Asp Ile Val 290 - # 295 - # 300 - - Leu Glu His Gly Ser Cys Val Thr Thr Met Al - #a Lys Asn Lys Pro Thr 305 3 - #10 3 - #15 3 -#20 - - Leu Asp Phe Glu Leu Ile Lys Thr Glu Ala Ly - #s Gln Pro Ala ThrLeu 325 - # 330 - # 335 - - Arg Lys Tyr Cys Ile Glu Ala Lys Leu Thr As - #n Thr Thr Thr Asp Ser 340 - # 345 - # 350 - - Arg Cys Pro Thr Gln Gly Glu Pro Thr Leu As - #n Glu Glu Gln Asp Lys 355 - # 360 - # 365 - - Arg Phe Val Cys Lys His Ser Met Val Asp Ar - #g Gly Trp Gly Asn Gly 370 - # 375 - # 380 - - Cys Gly Leu Phe Gly Lys Gly Gly Ile Val Th - #r Cys Ala Met Phe Thr 385 3 - #90 3 - #95 4 -#00 - - Cys Lys Lys Asn Met Glu Gly Lys Ile Val Gl - #n Pro Glu Asn LeuGlu 405 - # 410 - # 415 - - Tyr Thr Val Val Ile Thr Pro His Ser Gly Gl - #u Glu His Ala Val Gly 420 - # 425 - # 430 - - Asn Asp Thr Gly Lys His Gly Lys Glu Val Ly - #s Ile Thr Pro Gln Ser 435 - # 440 - # 445 - - Ser Ile Thr Glu Ala Glu Leu Thr Gly Tyr Gl - #y Thr Val Thr Met Glu 450 - # 455 - # 460 - - Cys Ser Pro Arg Thr Gly Leu Asp Phe Asn Gl - #u Met Val Leu Leu Gln 465 4 - #70 4 - #75 4 -#80 - - Met Lys Asp Lys Ala Trp Leu Val His Arg Gl - #n Trp Phe Leu AspLeu 485 - # 490 - # 495 - - Pro Leu Pro Trp Leu Pro Gly Ala Asp Thr Gl - #n Gly Ser Asn Trp Ile 500 - # 505 - # 510 - - Gln Lys Glu Thr Leu Val Thr Phe Lys Asn Pr - #o His Ala Lys Lys Gln 515 - # 520 - # 525 - - Asp Val Val Val Leu Gly Ser Gln Glu Gly Al - #a Met His Thr Ala Leu 530 - # 535 - # 540 - - Thr Gly Ala Thr Glu Ile Gln Met Ser Ser Gl - #y Asn Leu Leu Phe Thr 545 5 - #50 5 - #55 5 -#60 - - Gly His Leu Lys Cys Arg Leu Arg Met Asp Ly - #s Leu Gln Leu LysGly 565 - # 570 - # 575 - - Met Ser Tyr Ser Met Cys Thr Gly Lys Phe Ly - #s Val Val Lys Glu Ile 580 - # 585 - # 590 - - Ala Glu Thr Gln His Gly Thr Ile Val Ile Ar - #g Val Gln Tyr Glu Gly 595 - # 600 - # 605 - - Asp Gly Ser Pro Cys Lys Ile Pro Phe Glu Il - #e Met Asp Leu Glu Lys 610 - # 615 - # 620 - - Arg His Val Leu Gly Arg Leu Ile Thr Val As - #n Pro Ile Val Thr Glu 625 6 - #30 6 - #35 6 -#40 - - Lys Asp Ser Pro Val Asn Ile Glu Ala Glu Pr - #o Pro Phe Gly AspSer 645 - # 650 - # 655 - - Tyr Ile Ile Ile Gly Val Glu Pro Gly Gln Le - #u Lys Leu Asp Trp Phe 660 - # 665 - # 670 - - Lys Lys Gly Ser Ser Ile Gly Gln Met Phe Gl - #u Thr Thr Met Arg Gly 675 - # 680 - # 685 - - Ala Lys Arg Met Ala Ile Leu Gly Asp Thr Al - #a Trp Asp Phe Gly Ser 690 - # 695 - # 700 - - Leu Gly Gly Val Phe Thr Ser Ile Gly Lys Al - #a Leu His Gln Val Phe 705 7 - #10 7 - #15 7 -#20 - - Gly Ala Ile Tyr Gly Ala Ala Phe Ser Gly Va - #l Ser Trp Thr MetLys 725 - # 730 - # 735 - - Ile Leu Ile Gly Val Ile Ile Thr Trp Ile Gl - #y Met Asn Ser Arg Ser 740 - # 745 - # 750 - - Thr Ser Leu Ser Val Ser Leu Val Leu Val Gl - #y Ile Val Thr Leu Tyr 755 - # 760 - # 765 - - Leu Gly Val Met Val Gln Ala Asp Ser Gly Cy - #s Val Val Ser Trp Lys 770 - # 775 - # 780 - - Asn Lys Glu Leu Lys Cys Gly Ser Gly Ile Ph - #e Val Thr Asp Asn Val 785 7 - #90 7 - #95 8 -#00 - - His Thr Trp Thr Glu Gln Tyr Lys Phe Gln Pr - #o Glu Ser Pro SerLys 805 - # 810 - # 815 - - Leu Ala Ser Ala Ile Gln Lys Ala His Glu Gl - #u Gly Ile Cys Gly Ile 820 - # 825 - # 830 - - Arg Ser Val Thr Arg Leu Glu Asn Leu Met Tr - #p Lys Gln Ile Thr Ser 835 - # 840 - # 845 - - Glu Leu Asn His Ile Leu Ser Glu Asn Glu Va - #l Lys Leu Thr Ile Met 850 - # 855 - # 860 - - Thr Gly Asp Ile Lys Gly Ile Met Gln Val Gl - #y Lys Arg Ser Leu Arg 865 8 - #70 8 - #75 8 -#80 - - Pro Gln Pro Thr Glu Leu Arg Tyr Ser Trp Ly - #s Thr Trp Gly LysAla 885 - # 890 - # 895 - - Lys Met Leu Ser Thr Glu Leu His Asn Gln Th - #r Phe Leu Ile Asp Gly 900 - # 905 - # 910 - - Pro Glu Thr Ala Glu Cys Pro Asn Thr Asn Ar - #g Ala Trp Asn Ser Leu 915 - # 920 - # 925 - - Glu Val Glu Asp Tyr Gly Phe Gly Val Phe Th - #r Thr Asn Ile Trp Leu 930 - # 935 - # 940 - - Arg Leu Arg Glu Lys Gln Asp Ala Phe Cys As - #p Ser Lys Leu Met Ser 945 9 - #50 9 - #55 9 -#60 - - Ala Ala Ile Lys Asp Asn Arg Ala Val His Al - #a Asp Met Gly TyrTrp 965 - # 970 - # 975 - - Ile Glu Ser Ala Leu Asn Asp Thr Trp Lys Il - #e Glu Lys Ala Ser Phe 980 - # 985 - # 990 - - Ile Glu Val Lys Ser Cys His Trp Pro Lys Se - #r His Thr Leu Trp Ser 995 - # 1000 - # 1005 - - Asn Gly Val Leu Glu Ser Glu Met Val Ile Pr - #o Lys Asn Phe Ala Gly 1010 - # 1015 - # 1020 - - Pro Val Ser Gln His Asn Asn Arg Pro Gly Ty - #r His Thr Gln Thr Ala 1025 1030 - # 1035 - # 1040 - - Gly Pro Trp His Leu Gly Lys Leu Glu Met As - #p Phe Asp Phe Cys Glu 1045 - # 1050 - # 1055 - - Gly Thr Thr Val Val Val Thr Glu Asp Cys Gl - #y Asn Arg Gly Pro Ser 1060 - # 1065 - # 1070 - - Leu Arg Thr Thr Thr Ala Ser Gly Lys Leu Il - #e Thr Glu Trp Cys Cys 1075 - # 1080 - # 1085 - - Arg Ser Cys Thr Leu Pro Pro Leu Arg Tyr Ar - #g Gly Glu Asp Gly Cys 1090 - # 1095 - # 1100 - - Trp Tyr Gly Met Glu Ile Arg Pro Leu Lys Gl - #u Lys Glu Glu Asn Leu 1105 1110 - # 1115 - # 1120 - - Val Ser Ser Leu Val Thr Ala 1125 - - - - (2) INFORMATION FOR SEQ ID NO:4: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 47 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: - - GGAGTATGAC ATCCCAGCTG TCGACTATCA TTTGTCCATT CTCAGCC - # 47 - - - - (2) INFORMATION FOR SEQ ID NO:5: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 63 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..27 - - (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 37..63 - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- - #feature (B) LOCATION: 1..22 (D) OTHER INFORMATION: - #/note= "Residues 1693 to 1714 of SEQ ID - #NO:1" - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- - #feature (B) LOCATION: 37..63 (D) OTHER INFORMATION: - #/note= "Residues 1726 to 1848 of SEQ ID - #NO:1" - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: - - TGC AGG CTG AGA ATG GAC AAA TGA TAG TCGACAGCT - # GGG ATG TCA TAC 48 Cys Arg Leu Arg Met Asp Lys * * - # Gly Met Ser Tyr 1130 - # 1135 - # 1 - - TCC ATG TGC ACA GGA - # - # - # 63 Ser Met Cys Thr Gly 5 - - - - (2) INFORMATION FOR SEQ ID NO:6: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino - #acids (B) TYPE: amino acid (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: - - Cys Arg Leu Arg Met Asp Lys 1 5 - - - - (2) INFORMATION FOR SEQ ID NO:7: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino - #acids (B) TYPE: amino acid (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: - - Gly Met Ser Tyr Ser Met Cys Thr Gly 1 5 - - - - (2) INFORMATION FOR SEQ ID NO:8: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 564 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Saccharomyce - #s cerevisiae - - (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..564 - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: - - ATGAGATTTC CTTCAATTTT TACTGCAGTT TTATTCGCAG CATCCTCCGC AT -#TAGCTGCT 60 - - CCAGTCAACA CTACAACAGA AGATGAAACG GCACAAATTC CGGCTGAAGC TG -#TCATCGGT 120 - - TACTCAGATT TAGAAGGGGA TTTCGATGTT GCTGTTTTGC CATTTTCCAA CA -#GCACAAAT 180 - - AACGGGTTAT TGTTTATAAA TACTACTATT GCCAGCATTG CTGCTAAAGA AG -#AAGGGGTA 240 - - TCTCTCGAGA AAAGGGAGGC TGGGATGTCA TACTCCATGT GCACAGGAAA GT -#TTAAAGTT 300 - - GTGAAGGAAA TAGCAGAAAC ACAACATGGA ACAATAGTCA TTAGAGTACA AT -#ATGAAGGA 360 - - GACGGCTCTC CATGCAAGAT CCCTTTTGAG ATAATGGATC TGGAAAAAAG AC -#ATGTTTTG 420 - - GGCCGCCTGA TCACAGTCAA TCCAATTGTA ACAGAAAAGG ACAGCCCAGT CA -#ACATAGAA 480 - - GCAGAACCTC CATTCGGAGA CAGCTACATC ATCATAGGAG TGGAACCAGG AC -#AATTGAAG 540 - - CTGGACTGGT TCAAGAAAGG ATAA - # - # 564 - - - - (2) INFORMATION FOR SEQ ID NO:9: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 187 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: peptide - - (ix) FEATURE: (A) NAME/KEY: Cleavage-sit - #e#20) (B) LOCATION: (19 (D) OTHER INFORMATION: - #/note= "Signalase cleavage" - - (ix) FEATURE: (A) NAME/KEY: Cleavage-sit - #e#86) (B) LOCATION: (85 (D) OTHER INFORMATION: - #/note= "Kex2p cleavage" - - (ix) FEATURE: (A) NAME/KEY: Peptide (B) LOCATION: 1..19 (D) OTHER INFORMATION: - #/note= "MF-alpha secretion signal peptide" - - (ix) FEATURE: (A) NAME/KEY: Peptide (B) LOCATION: 20..85 (D) OTHER INFORMATION: - #/note= "MF-alpha propeptide" - - (ix) FEATURE: (A) NAME/KEY: Domain (B) LOCATION: 86..187 (D) OTHER INFORMATION: - #/note= "Domain B" - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: - - Met Arg Phe Pro Ser Ile Phe Thr Ala Val Le - #u Phe Ala Ala Ser Ser 1 5 - # 10 - # 15 - - Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Gl - #u Asp Glu Thr Ala Gln 20 - # 25 - # 30 - - Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser As - #p Leu Glu Gly Asp Phe 35 - # 40 - # 45 - - Asp Val Ala Val Leu Pro Phe Ser Asn Ser Th - #r Asn Asn Gly Leu Leu 50 - # 55 - # 60 - - Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Al - #a Lys Glu Glu Gly Val 65 - #70 - #75 - #80 - - Ser Leu Glu Lys Arg Glu Ala Gly Met Ser Ty - #r Ser Met Cys Thr Gly 85 - # 90 - # 95 - - Lys Phe Lys Val Val Lys Glu Ile Ala Glu Th - #r Gln His Gly Thr Ile 100 - # 105 - # 110 - - Val Ile Arg Val Gln Tyr Glu Gly Asp Gly Se - #r Pro Cys Lys Ile Pro 115 - # 120 - # 125 - - Phe Glu Ile Met Asp Leu Glu Lys Arg His Va - #l Leu Gly Arg Leu Ile 130 - # 135 - # 140 - - Thr Val Asn Pro Ile Val Thr Glu Lys Asp Se - #r Pro Val Asn Ile Glu 145 1 - #50 1 - #55 1 -#60 - - Ala Glu Pro Pro Phe Gly Asp Ser Tyr Ile Il - #e Ile Gly Val GluPro 165 - # 170 - # 175 - - Gly Gln Leu Lys Leu Asp Trp Phe Lys Lys Gl - #y 180 - # 185 - - - - (2) INFORMATION FOR SEQ ID NO:10: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 417 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Drosophila - - (vii) IMMEDIATE SOURCE: (B) CLONE: p29GEB2.4PS - - (ix) FEATURE: (A) NAME/KEY: sig.sub.-- - #peptide (B) LOCATION: 1..60 (D) OTHER INFORMATION: - #/note= "1 tPA Secretion Signal" - - (ix) FEATURE: (A) NAME/KEY: mat.sub.-- - #peptide (B) LOCATION: 94..417 (D) OTHER INFORMATION: - #/note= "1 RGARSP-domB" - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- - #feature (B) LOCATION: 112 (D) OTHER INFORMATION: - #/note= "Beginning of Domain B region" - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- - #feature (B) LOCATION: 61..93 (D) OTHER INFORMATION: - #/note= "1 tPA Propeptide" - - (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..417 - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: - - ATG GAT GCA ATG AAG AGA GGG CTC TGC TGT GT - #G CTG CTG CTG TGT GGA 48 Met Asp Ala Met Lys Arg Gly Leu Cys Cys Va - #l Leu Leu Leu Cys Gly31 -30 - # -25 - # -20 - - GCA GTC TTC GTT TCG CCC AGC CAG GAA ATC CA - #T GCC CGA TTC AGA AGA 96 Ala Val Phe Val Ser Pro Ser Gln Glu Ile Hi - #s Ala Arg Phe Arg Arg15 - - #10 - #-5 - # 1 - - GGA GCC AGA TCC CCT GGG ATG TCA TAC TCC AT - #G TGC ACA GGA AAG TTT 144 Gly Ala Arg Ser Pro Gly Met Ser Tyr Ser Me - #t Cys Thr Gly Lys Phe 5 - # 10 - # 15 - - AAA ATT GTG AAG GAA ATA GCA GAA ACA CAA CA - #T GGA ACA ATA GTC ATT 192 Lys Ile Val Lys Glu Ile Ala Glu Thr Gln Hi - #s Gly Thr Ile Val Ile 20 - # 25 - # 30 - - AGA GTA CAA TAT GAA GGA GAC GGC TCT CCA TG - #C AAG ATC CCT TTT GAG 240 Arg Val Gln Tyr Glu Gly Asp Gly Ser Pro Cy - #s Lys Ile Pro Phe Glu 35 - # 40 - # 45 - - ATA ATG GAT CTG GAA AAA AGA CAT GTT TTG GG - #C CGC CTG ATC ACA GTC 288 Ile Met Asp Leu Glu Lys Arg His Val Leu Gl - #y Arg Leu Ile Thr Val 50 - # 55 - # 60 - # 65 - - AAC CCA ATT GTA ACA GAA AAG GAC AGT CCA GT - #C AAC ATA GAA GCA GAA 336 Asn Pro Ile Val Thr Glu Lys Asp Ser Pro Va - #l Asn Ile Glu Ala Glu 70 - # 75 - # 80 - - CCT CCA TTC GGA GAC AGC TAC ATC ATC ATA GG - #A GTG GAA CCA GGA CAA 384 Pro Pro Phe Gly Asp Ser Tyr Ile Ile Ile Gl - #y Val Glu Pro Gly Gln 85 - # 90 - # 95 - - TTG AAG CTG GAC TGG TTC AAG AAA GGA TAA TA - #G -# 417 Leu Lys Leu Asp Trp Phe Lys Lys Gly * - # * 100 - # 105 - - - - (2) INFORMATION FOR SEQ ID NO:11: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 137 amino - #acids (B) TYPE: amino acid (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: - - Met Asp Ala Met Lys Arg Gly Leu Cys Cys Va - #l Leu Leu Leu CysGly 31 -30 - # -25 - # -20 - - Ala Val Phe Val Ser Pro Ser Gln Glu Ile Hi - #s Ala Arg Phe ArgArg 15 - - #10 - #-5 - # 1 - - Gly Ala Arg Ser Pro Gly Met Ser Tyr Ser Me - #t Cys Thr Gly Lys Phe 5 - # 10 - # 15 - - Lys Ile Val Lys Glu Ile Ala Glu Thr Gln Hi - #s Gly Thr Ile Val Ile 20 - # 25 - # 30 - - Arg Val Gln Tyr Glu Gly Asp Gly Ser Pro Cy - #s Lys Ile Pro Phe Glu 35 - # 40 - # 45 - - Ile Met Asp Leu Glu Lys Arg His Val Leu Gl - #y Arg Leu Ile Thr Val 50 - # 55 - # 60 - # 65 - - Asn Pro Ile Val Thr Glu Lys Asp Ser Pro Va - #l Asn Ile Glu Ala Glu 70 - # 75 - # 80 - - Pro Pro Phe Gly Asp Ser Tyr Ile Ile Ile Gl - #y Val Glu Pro Gly Gln 85 - # 90 - # 95 - - Leu Lys Leu Asp Trp Phe Lys Lys Gly 100 - # 105 - - - - (2) INFORMATION FOR SEQ ID NO:12: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 417 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Drosophila - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: - - CTATTATCCT TTCTTGAACC AGTCCAGCTT CAATTGTCCT GGTTCCACTC CT -#ATGATGAT 60 - - GTAGCTGTCT CCGAATGGAG GTTCTGCTTC TATGTTGACT GGACTGTCCT TT -#TCTGTTAC 120 - - AATTGGGTTG ACTGTGATCA GGCGGCCCAA AACATGTCTT TTTTCCAGAT CC -#ATTATCTC 180 - - AAAAGGGATC TTGCATGGAG AGCCGTCTCC TTCATATTGT ACTCTAATGA CT -#ATTGTTCC 240 - - ATGTTGTGTT TCTGCTATTT CCTTCACAAT TTTAAACTTT CCTGTGCACA TG -#GAGTATGA 300 - - CATCCCAGGG GATCTGGCTC CTCTTCTGAA TCGGGCATGG ATTTCCTGGC TG -#GGCGAAAC 360 - - GAAGACTGCT CCACACAGCA GCAGCACACA GCAGAGCCCT CTCTTCATTG CA - #TCCAT 417 - - - - (2) INFORMATION FOR SEQ ID NO:13: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 46 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Dengue vi - #rus - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- - #feature (B) LOCATION: 27..46 (D) OTHER INFORMATION: - #/note= "Residues 841 to 860 of SEQ ID NO:1" - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: - - CTTCTAGATC TCGAGTACCC GGGACCATGC GCTGCATAGG AATATC - # 46 - - - - (2) INFORMATION FOR SEQ ID NO:14: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- - #feature (B) LOCATION: 20..35 (D) OTHER INFORMATION: - #/note= "Complementary DNA to nucleotides - #2010-2025 of SEQ ID NO:1." - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: - - GCTCTAGAGT CGACTATTAT CCTTTCTTGA ACCAG - # -# 35 - - - - (2) INFORMATION FOR SEQ ID NO:15: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: - - Glu Ala Gly Met Ser Tyr Ser Met Xaa Thr Gl - #y Lys Phe Xaa Val Val 1 5 - # 10 - # 15 - - - - (2) INFORMATION FOR SEQ ID NO:16: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- - #feature (B) LOCATION: 20..35 (D) OTHER INFORMATION: - #/note= "Complementary DNA to nucleotides - #2160-2175 of SEQ ID NO:1." - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: - - GCTCTAGAGT CGACTATTAC CCGTAGATTG CTCCG - # -# 35 - - - - (2) INFORMATION FOR SEQ ID NO:17: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 50 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: - - CTAGCGGTAC CCTCGAGAAA AGGGAGGCCG GGATGTCATA CTCCATGTGC - # 50 - - - - (2) INFORMATION FOR SEQ ID NO:18: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- - #feature (B) LOCATION: 25..42 (D) OTHER INFORMATION: - #/note= "Corresponding DNA to nucleotides - #2062-2079 of SEQ ID NO:1." - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: - - CGTGTGTCGA CGCGGCCGCT ATTAGGCCAT TCTTTTCGCT CC - # - # 42__________________________________________________________________________

Number	Name	Date	Kind
5494671	Lai et al.	Feb 1996
5514375	Paoletti et al.	May 1996

Number	Date	Country
9202548	Feb 1992	WOX
9203545	Mar 1992	WOX

Methods of preparing carboxy-terminally truncated recombinant flavivirus envelope glycoproteins employing drosophila melanogaster expression systems

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