CN1208463C - Differential cytotoxicity of alternative forms of rotavirus nonstructural protein 4 - Google Patents

Abstract

The nonstructural protein 4 (NSP4) in the SA11 ATCC rotavirus strain has a histidine at amino acid position 47. This substituted form is more cytotoxic than the NSP4 of the Australia rotavirus strain, which has an asparagine at amino acid position 47. The histidine at amino acid position 47 is mutagenized to another amino acid to produce an alternative form of NSP4 which has reduced toxicity, while retaining its antigenicity and immunogenicity. NSP4 having a glutamic acid at amino acid position 48 is more cytotoxic than NSP4 having a lysine at amino acid position 48. The lysine at amino acid position 48 is mutagenized to another amino acid other than glutamic acid to produce an alternative form of NSP4 which has reduced toxicity, while retaining its antigenicity and immunogenicity.

Description

Differential cytotoxicity of variant forms of rotavirus nonstructural protein 4

field of invention

The present invention relates to the identification of sequence differences in alternative forms of rotavirus nonstructural protein 4 that lead to significant changes in cytotoxicity.

Background of the invention

Rotavirus is considered to be the most important cause of severe viral gastroenteritis in humans and animals (Literature Citation 1). Rotaviruses are non-enveloped, three-layered particles with a genome composed of 11 double-stranded RNA segments. Group A rotaviruses are the major cause of rotavirus disease in humans and animals.

The prototype group A rotavirus, designated SA11, was discovered 10 years before the identification of human rotaviruses. Malherbe et al. (2) describe the isolation of a virus, simian medium 11 (SA11), from the rectum of healthy East African monkeys, and its cytopathic effect in kidney cell cultures of East African monkeys. The virus was distributed to Holmes et al., (Dept. Microbiology, University of Melbourne, Australia) (3), Estes et al., (Division of Molecular Virology, Baylor College of Medicine, Houston, TX, USA) (4), and the United States Type Culture Collection. Unfortunately, the exact passage history of each virus sample is not known.

Nonstructural protein 4, called NPS4, is encoded by gene 10 of group A rotaviruses. Among several putative functions of the multifunctional rotavirus nonstructural protein 4 (NSP4) is the causative agent of disease (5-11). This argument is based on several past studies (5-8) of NSP4 encoded by the prototype rotavirus, the SA11 strain.

In 1983, Both et al. reported the nucleotide sequence (12) of gene 10 of rotavirus SA11 strain (group A rotavirus prototype) according to the analysis of the plaque-purified virus stocks provided by Australia Holmes et al. By combining several methods. Portions of gene 10 were subcloned into bacteriophage M13 and sequenced by the Sanger method (13); other regions of the cDNA clone were sequenced by the Maxam and Gilbert method (14).

Previous studies of NSP4 (5-8, 11, 12, 15-23) were based on cDNA clones of NSP4 gene versions provided by Both et al. The original NSP4 sequence of the Australian strain had an asparagine (NSP4(Asp)) at amino acid position 47 in the deduced amino acid sequence (12).

NSP is a transmembrane protein with multiple functions. It functions as an intracellular receptor for subviral particle budding into the endoplasmic reticulum (ER) of infected cells (16, 21). Expression of NSP4 leads to an increase in intracellular calcium levels ([Ca ²⁺ ] _i ) in Sf9 insect cells (5). A similar effect on changes in intracellular calcium levels was observed for NSP4 of the OSU strain (a different group A rotavirus) (9). When NSP4 protein or the synthetic peptide NSP4 114-135 was exogenously added to insect (6) or mammalian (8) cells, the phospholipase C (PLC)-mediated pathway was activated.

A role for calcium in cytopathic effect (CPE) and cell death has been proposed for many virus-induced pathogenic processes (24). Increased [Ca2 ⁺ ] _i levels have been linked to cytotoxicity in group A rotavirus-infected MA104 cells (monkey kidney cells; Biowhittaker Inc., Walkersville, MD) (25). It has been proposed that NSP4-induced cytotoxicity is due to increased intracellular calcium levels in infected cells (5). NSP4 has been found to be cytotoxic to mammalian cells (5, 11). NSP4 has also been shown to act as a diarrhea-causing enterotoxin in young mice (7). Antibodies against NSP4 provided passive protection against rotavirus in the same animal model (7).

There is a need to develop alternative forms of NSP4 that have reduced cytotoxicity, but retain a similar conformation to the prototype rotavirus strain and thus remain antigenic and immunogenic. Such antigenic and immunogenic forms are candidates for inclusion in antigenic compositions to protect the body against rotavirus disease.

Brief description of the invention

Therefore, it is an object of the present invention to identify the region of NPS4 responsible for cytotoxicity and increased intracellular calcium levels.

Yet another object of the present invention is to identify and develop alternative forms of NSP4 that have reduced cytotoxicity but retain antigenicity and immunogenicity due to conformational similarity to the prototype rotavirus strain.

As described herein, NSP4 in the SA11 ATCC rotavirus strain (NSP4(His)) was compared to asparagine in the Australian rotavirus strain (NSP4(Asn)) obtained by Holmes et al. and sequenced by Both et al. There is a histidine at amino acid position 47 of . This substituted form, NSP4(His), retains the main functions of NSP4(Asn), ie, cytotoxicity and intracellular calcium altering functions, but it is more cytotoxic to cells.

To generate an alternative form of NSP4 with reduced toxicity while retaining its antigenicity and immunogenicity, the histidine and asparagine at amino acid position 47 of NSP4 were mutagenized to another amino acid. For example, the histidine codon CAT encoding amino acid 47 or the asparagine codon AAT is mutagenized to GAA encoding aspartic acid. Other attenuating mutations of this amino acid can also be used to reduce toxicity.

As described herein, there is a lysine at amino acid position 48 in the SA11 ATCC and Australian rotavirus strains. Conversion of this lysine to glutamate increases intracellular calcium levels and toxicity caused by the virus. Therefore, amino acid 48 was also identified as a position involved in viral toxicity. To generate an alternative form of NSP4 that is less toxic while retaining its antigenic and immunogenic properties, the lysine at amino acid position 48 can be mutagenized to another amino acid than glutamic acid.

Amino acids flanking amino acid positions 47 and 48 can also be mutagenized in a similar manner. Mutations in other regions of NSP4 can also be combined with mutations in amino acid positions 47 and/or 48.

As also described herein, the present invention also relates to an isolated and purified nucleic acid sequence comprising a nucleic acid sequence encoding: (a) rotavirus NSP4 protein, wherein the histidine or asparagine at amino acid position 47 is mutagenized Another amino acid to produce a NSP4 variable form with reduced toxicity while retaining antigenicity and immunogenicity; (b) rotavirus NSP4 protein, wherein the lysine at amino acid position 48 is mutagenized to another amino acid other than glutamic acid an amino acid to produce an alternative form of NSP4 with reduced toxicity while retaining antigenicity and immunogenicity; or (c) a rotavirus NSP4 protein in which histidine or asparagine at amino acid position 47 is mutagenized to another amino acid, and wherein the lysine at amino acid position 48 is mutagenized into another amino acid other than glutamic acid to produce a variable form of NSP4 with reduced toxicity while retaining antigenicity and immunogenicity.

The present invention also relates to the construction of plasmids expressing the above-mentioned variant forms of NSP4.

To obtain expression of alternative forms of NSP4, the isolated and purified nucleic acid sequence is first inserted into a suitable plasmid vector. The plasmid is then used to transform, transfect, or infect a suitable host cell. In one embodiment of the invention, the host cells are Sf9 cells. The host cell is then cultured under conditions that allow the host cell to express the alternative form of NSP4.

In another embodiment of the present invention, a variable form of the NSP protein is used to prepare an antigenic composition capable of inducing a protective immune response against rotavirus in a mammalian host, or capable of inducing Improved diarrhea symptoms in hosts infected with rotavirus. The antigenic composition may also comprise an adjuvant, diluent or carrier. Examples of these adjuvants include aluminum hydroxide, aluminum phosphate, MPL ^™ , Stimulon ^™ QS21, IL-12 and cholera toxin. The antigenic composition is administered to a mammalian host in an immunogenic amount sufficient to protect the host against disease caused by rotavirus, or to ameliorate the symptoms of diarrhea in a host already infected with rotavirus.

Brief description of the drawings

Figure 1 depicts the source of the rotavirus SA11 virus stock. The passage history of the SA11 ATCC strain is indicated. WLVP stands for Wyeth-Lederle Vaccines and Pediatrics, and VL stands for the batch number of the virus used in WLVP.

Figure 2 shows Sf9 cells infected with 0.2MOI of recombinant baculovirus containing rotavirus gene VP6 (square), NSP4 (His) (circle) of amino acid 47 (circle) and NSP4 (Asn) (triangle) of amino acid 47 respectively cell viability. Cell viability was measured at the indicated times using the trypan blue exclusion assay. The mean of each time point represents three independent infections.

Figure 3 shows intracellular calcium levels in Sf9 cells (mock-infected) (left), Sf9 cells expressing NSP4(Asn) at amino acid position 47 (middle) and Sf9 cells expressing NSP4(His) at amino acid 47 (right). Images are from a representative experiment showing the [Ca ²⁺ ] _i ratio (340/380 nm).

Figure 4 depicts the Western blot analysis of NSP4 expression in Sf9 cells: Swimming lane 1 - NSP4 (His) of amino acid 47; Swimming lane 2 - NSP4 (Asn) of amino acid 47; Swimming lane 3 - MA104 cell lysate infected with SA11 as a positive control ; Lane 4 - molecular weight markers.

Figure 5 depicts NSP4 (His) containing amino acid 47 (squares), NSP4 (Asn) of amino acid 47 (crosses), and NSP4 (Glu) of amino acid 48 without rotavirus (mock infection) (circles) (diamonds) Cell viability of recombinant baculovirus-infected Sf9 cells. Sf9 cells were infected with recombinant baculovirus at 10 MOI. Cell viability was measured at the indicated times using the trypan blue exclusion assay. The mean of each time point represents three independent infections.

Figure 6 depicts Sf9 cells (mock infection) (black bars) and NSP4 (Asn) (dark gray bars) expressing amino acid 47, NSP4 (His) (light gray bars) at amino acid 47, and amino acid 48, respectively. Intracellular calcium levels of Sf9 cells for NSP4(Glu) (white bars). Sf9 cells were infected with recombinant baculovirus at 10 MOI. Intracellular calcium was measured with the fluorescent calcium indicator fura-2 48 hours after infection. Columns show intracellular calcium levels compared to mock-infected Sf9 cells. Each bar represents the mean of three experiments.

Detailed description of the invention

Rotavirus SA11 was first isolated in 1963 by Malherbe et al. (2). The SA11 passage history from Dr. Malherbe's laboratory is no longer available. The passage history of the virus used to publish the sequence is also no longer available. Dr. Estes provided SA11 clone 3 with a history of 5 generations.

In RNA viruses, point mutations occur with regular frequency. It has been estimated that rotaviruses have ^10-4 replication errors at each nucleotide site with varying frequency per replication (26). Therefore, it is possible that isolates of the same virus have some nucleotide sequence differences. For example, two versions of VP7 with glycosylation variants have been reported in different clones of SA11 isolated from the same virus (27). A mutation in gene 9 of SA11 resulted in an aglycosylated form of VP7 (clone 28). Therefore, the sequence of each cDNA clone of the virus or a plasmid derived from RT-PCR amplification of viral RNA may not be a completely accurate representation of the consensus sequence of the virus.

Gene 10 (also known as gene NS28) encoding NSP4 of SA11 rotavirus (ATCC Accession No. VR-899) obtained from the American Type Culture Collection was sequenced as described in Example 2 below. Based on the nucleotide sequence, the histidine (NSP4(His)) at amino acid position 47 in the NSP4 version of SA11 was identified, which differs only by this one amino acid from the previously published sequence of the Australian strain (12), which is at this position Dot has an asparagine residue (NSP4(Asn)). The sequence of the SA11 gene 10 described herein, ie, the gene encoding NSP4(His), is believed to be the prototype sequence.

Although it is difficult to definitively deduce which is the true prototype sequence of gene 10 due to the uncertainty of the generation history of SA11 rotavirus, it is believed that the sequence encoding NSP4(His) is representative of the consensus sequence because: i) in the SA11 strain It has been observed in different passage numbers, including the very early one (Lot 1N); ii) it has been demonstrated by direct sequencing of two RT-PCR products (representatives of virus populations) and individual clones; iii) it is still present in It was found in different SA11 stocks obtained from other laboratories (Estes); and iv) 43 out of 44 published NSP4 sequences of different rotavirus strains had His at position 47.

Since most NSP4 studies (5-8, 12, 15-23) are based on expression cloned from the cDNA gene10 of Both et al., it is known that in two NSP4 variants, namely, NSP4(Asn) and NSP4(His) It is important whether NSP function is fully conserved.

To test whether the biological function of the NSP4 protein was altered by the amino acid substitution of histidine to aspartic acid, the codon encoding histidine at position 47 was replaced with the codon encoding asparagine. A change from A to C at nucleotide 139 results in a mutation from asparagine to histidine. Specifically, the codon (residues 139-141, where the initiation codon is residues 1-3) was changed from AAT to CAT. Both versions of the NPS4 gene were cloned into a baculovirus transfer vector and then expressed in insect cells.

The expression of NSP4 in Sf9 cells infected with recombinant baculovirus vector was detected by Western blot and SDS-PAGE. Synthesis of three forms of NSP4 in infected Sf9 cells was detected with an NSP4 anti-peptide antibody: two glycosylated forms of 26 and 28 kD and one non-glycosylated form of 20 kD (see Figure 5).

Expression of SA11 NSP4 in mammalian cells appears to be cytotoxic (5). Aberrant morphological changes have been observed in cells transfected with plasmids expressing NSP4. Recently, Newton et al. have shown that expression of NSP4 in mammalian cells infected with a recombinant poxvirus leads to cell killing (11). Here it is demonstrated that NSP4 expression is also cytotoxic to insect (Sf9) cells. Expression of NSP4 in Sf9 cells infected with recombinant baculovirus resulted in significantly reduced cell viability compared to cells expressing other rotavirus proteins, including VP2, VP4, VP6 and VP7.

As described in Example 3 below, expression of NSP4(His) in insect cells induced major cytopathic effects associated with cytotoxicity. However, this amino acid change significantly affected the degree of cytotoxicity of SA11 NSP4. Expression of NSP4(His) in Sf9 cells resulted in a significant increase in cell death compared to NSP4(Asn). Thus, NSP4(His) is more cytotoxic than NSP4(Asn) when expressed in Sf9 cells. This result was confirmed as described in Example 7 below.

Recent data (5) showed that expression of NSP4 in Sf9 cells resulted in a marked increase in intracellular calcium levels. It has been suggested (5, 6) that elevated intracellular calcium may contribute to NSP4-induced pathogenesis. As shown in Example 4 below, both NSP4(His) and NSP4(Asn) lead to a significant increase in intracellular calcium levels. However, expression of NSP4(His) resulted in higher intracellular calcium levels than NSP4(Asn). This result was confirmed as described in Example 8 below. The higher calcium levels observed in cells expressing NSP4(His) were not due to overexpression of this protein, as in Sf9 cells infected with recombinant baculovirus NSP4(Asn), Higher amounts of NSP4 were expressed in (His) Sf9 cells (see Example 5 below). Thus, a single amino acid change at position 47 significantly affects the effect of NSP4 on intracellular calcium mobilization.

The functional NSP4 domains responsible for cytopathicity and toxicity have not been fully defined. However, a functional domain of NSP4 spanning amino acids 14 to 140 is known to be important for intracellular calcium motility, as shown below: (i) Synthetic peptides and intact proteins corresponding to amino acids 114 to 135 of NSP4 are important for intracellular calcium motility (6) Functionally similar to causing diarrhea (7); (ii) Zhang et al. reported that the virulent OSU virus differed from the non-virulent form of the same virus due to changes in amino acids 135, 136, 138 (9). However, other functional domains may also be important for the function of NSP4. It has been shown that N-linked glycosylation of NSP4 (amino acids 8 and 18) may be required for removal of the temporary envelope during viral morphogenesis (28). Recently, Newton et al. reported that the H3 domain (amino acids 55 to 85) is critical for the cytotoxic and membrane destabilizing activities of NSP4 (11). Here we describe that an amino acid change (from histidine to aspartic acid at position 47 adjacent to the H2 functional domain) significantly alters the function of NSP4. These results suggest that functional domains other than amino acids 114-140 are important for NSP4 cytotoxicity and calcium motility.

NSP4 is an ER transmembrane protein (Fig. 5). Hydrophobicity analysis revealed three putative hydrophobic regions (H1-H3). Two modes of NSP4 topologies have been proposed. Chan et al proposed that a third hydrophobic region (H3, amino acids 67 to 85) spans the ER membrane, while a second hydrophobic region (H2, amino acids 28 to 47) is located in the ER lumen (15, 27). Bergmann et al proposed that the H2 region (amino acids 30 to 54) traverses the ER membrane, whereas the H3 region (amino acids 63 to 80) is located on the cytoplasmic side of the ER (20). In Bergmann's model, part of the H2 region (amino acids 30 to 44) spans the ER membrane, while amino acid 47 (and amino acid 48) extend into the cytoplasmic location of the ER. Without being limited below, amino acid 47 (and amino acid 48) may be important for the interaction of NSP4 and ER membranes.

Changing the amino acid at position 47 from asparagine (which is uncharged and polar) to histidine (which is basic and positively charged) results in a change in charge in the H2 region at physiological pH. Without being limited below, as a result of this charge change, NSP4(His) can destabilize the ER membrane directly due to the additional charge, or indirectly by forming the structure of the NSP4 C-terminal membrane destabilizing region (amino acids 114-140). Damages the ER membrane, thereby promoting the membrane destabilizing activity of NSP4 (18).

Thus, NSP4(His) possesses the major functions of NSP4(Asn) in pathogenic effects such as cytotoxicity and alteration of intracellular calcium. However, this one amino acid change at position 47 significantly affected the cytotoxicity of SA11 NSP4.

The histidine at amino acid position 47 of NSP4 is mutagenized to another amino acid by conventional recombinant DNA techniques to produce a variant form of NSP4 with reduced toxicity while retaining its antigenicity and immunogenicity. For example, at amino acid 47, the codon CAT encoding histidine or the codon AAT encoding asparagine were mutagenized to GAA encoding aspartic acid.

Other attenuating mutations can also be used to attenuate the toxicity of NSP4. Because of the limited number of possible amino acids at any given position, one skilled in the art can conveniently construct a series of plasmids, each containing a different codon encoding a different amino acid at position 47. The resulting expressed NSP4 protein can then readily be tested in the cell viability and intracellular calcium level systems described herein to assess whether toxicity is attenuated. Alternative forms of NSP4 that resulted in attenuated toxicity were then tested in the suckling (young) mouse animal model system (7) to confirm that they were indeed attenuated. The attenuated alternative forms of NSP4 were then tested as follows to confirm that they retained antigenicity and immunogenicity. Immunization of pregnant mice with NSP4 variants. Suckling mice born to these immunized dams were then challenged with native NSP4 or whole rotavirus (29). Those suckling mice that did not develop diarrhea were passively protected by acquired maternal antibodies produced by dams immunized with the variant form of NSP4.

A residue at amino acid position 48 of NSP4 was also associated with toxicity. Both the Australian and SA11 rotavirus strains have a lysine at amino acid position 48. When lysine was mutagenized to glutamate by conventional recombinant DNA techniques as described below in Examples 6-8, this variant was markedly cytotoxic and resulted in high levels of intracellular calcium in Sf9 cells .

Therefore, in order to attenuate the cytotoxicity of NSP4, those skilled in the art can easily construct a series of plasmids, each containing a codon encoding a different amino acid other than glutamic acid at position 48. The resulting expressed NSP4 protein is then conveniently tested in the cell survival and intracellular calcium level systems described herein to assess whether toxicity is attenuated. Those NSP4 variant forms that resulted in reduced toxicity were then tested in the animal model systems described above to confirm that they were reduced, and retained antigenicity and immunogenicity.

NSP4 variant forms include those having a mutation at amino acid position 47, or a mutation at amino acid position 48, or both amino acid positions 47 and 48, so long as each variant is less toxic while retaining its antigenicity and immunogenicity.

In yet another embodiment of the invention, the amino acids flanking amino acid positions 47 and 48 may be mutagenized in a similar manner. Mutations in other regions of NSP4 can also be combined with mutations in amino acid positions 47 and/or 48.

As mentioned above, Estes et al. have focused on the functional domain between amino acids 114-140. However, as shown herein, other regions are also important for NSP4 cytotoxicity. Regions within or near the H2 region - specifically at amino acid position 47 and/or amino acid 48 - were targeted for the generation of genetically detoxified NSP4 proteins. The introduction of a proline in the H2 region and/or H3 region alters the secondary/tertiary structure and may thus attenuate cytotoxicity. These further mutations may be combined with the amino acid 47 mutation described above.

These variant forms of NSP4 have reduced cytotoxicity, yet remain antigenic and immunogenic. Such detoxified antigenic and immunogenic forms are candidates for inclusion in antigenic compositions to protect the body against rotavirus disease.

The present invention also relates to an isolated and purified nucleic acid sequence, which comprises a nucleic acid sequence encoding: (a) rotavirus NSP4 protein, wherein the histidine or aspartic acid at amino acid position 47 is mutagenized into another amino acid , to produce a variable form of NSP4 with reduced toxicity while retaining antigenicity and immunogenicity; (b) rotavirus NSP4 protein, wherein the lysine at amino acid position 48 is mutagenized into another amino acid other than glutamic acid, to produce an alternative form of NSP4 with reduced toxicity while retaining antigenicity and immunogenicity; or (c) a rotavirus NSP4 protein in which the histidine or asparagine at amino acid position 47 is mutagenized to another amino acid, and The lysine at amino acid position 48 is mutagenized into another amino acid other than glutamic acid to produce a variable form of NSP4 with reduced toxicity while retaining antigenicity and immunogen.

Various host cell-vector systems are suitable for expressing the variant forms of NSP4 described herein. The vector system is compatible with the host cell used. Suitable host cells include: bacteria transformed with plasmid DNA, cosmid DNA, or bacteriophage DNA; viruses such as vaccinia virus and adenovirus; yeast such as Pichia cells; insect cells such as Sf9 or Sf21 cells; or mammalian cells such as Chinese hamster ovary cells animal cell lines; and other conventional organisms. A variety of conventional transcription and translation elements can be used in host cell-vector systems.

Plasmids are introduced into host cells by transformation, transduction, transfection or infection, depending on the host cell-vector system used. The host cells are then cultured under conditions that allow the host cells to express the alternative form of NSP4.

Alternative forms of the NSP4 protein can be used to prepare antigenic compositions to confer protection to a mammalian host against disease caused by rotavirus, or to ameliorate diarrhea in a host infected with rotavirus.

Injectable solutions or suspensions can be prepared by mixing antigenic compositions containing alternative forms of NSP4 protein and immunologically acceptable diluents or carriers in a conventional manner. Antibody levels induced by the antigenic composition can be determined using certain adjuvants such as Stimulon ^™ Qs-21 (Aquila Biopharmaceuticals, Inc., Framingham, MA), MPL ^™ (3-O-deacetyl monophospholipid A; RIBI ImmunoChem Research , Inc., Hamilton, MT), aluminum phosphate, aluminum hydroxide, IL-12 (Genetics Institute, Cambridge, MA) and cholera toxin (wild type or mutant, such as wherein the glutamic acid at amino acid position 29 is replaced by another Amino acid, preferably histidine substitutions, are increased according to US Provisional Patent Application No. 60/102,430).

The antigenic compositions of the present invention are administered by conventional means, such as subcutaneous, intraperitoneal or intramuscular injection into mammals, and by oral, mucosal, intranasal or vaginal administration, to induce protection against disease caused by rotavirus immune response, or to improve diarrhea in hosts already infected with rotavirus. The dosage to be administered can be determined by methods known to those skilled in the art. A single dose of the antigenic composition may be used to confer protection or ameliorate symptoms, or several booster doses may need to be administered.

These variants can also be used as ligands to identify a so far unknown receptor for the rotavirus enterotoxin NSP4. They can block NSP4 receptors by forming non-functional heterodimers and thus also have therapeutic use in the treatment of gastroenteritis diseases.

In order that the present invention may be better understood, the following examples are set forth. These examples are for illustration only and are not intended to limit the scope of the invention.

Example

Example 1

Cells, Virus Strains, and Viral Infections

cells and SA11 rotavirus

The history of the SA11 strain used in this study is shown in Figure 1 . SA11 rotavirus was obtained from the American Type Culture Collection. Lot 1N of the SA11 rotavirus stock, which was the original unpassaged bacterial stock submitted in 1980 (2), was a generous gift of Dr. Charles Buck (American Type Culture Collection, Manassas, VA). The passage history of lot 1N is unclear (passage number not known in primate East African simian kidney cells and rhesus fetal kidney cells). Lot 1N is in ATCC as Lot 2, 6, and 7 strains (Dr. Buck, personal contact). VL589 (Lot 5) was purchased from ATCC. Lot5 has an unknown number of passages in the primate East African ape kidney, and 2 passages in CV-1 cells. VL589 was passed in MA104 cells for 1 passage after receipt from ATCC. VL919 was also obtained from ATCC (Lot 7, unknown number of passages in primate East African monkey kidney and rhesus fetal kidney cells. 5 passages in MA104 cells). SA11 clone 3 was kindly provided by Dr.Mary K.Estes (Baylor College of Medicine), and passed through MA104 cells for 5 generations.

Rotavirus infection and viral RNA isolation

MA104 cells were grown and maintained in modified Dulbecco's medium with 10% fetal bovine serum. SA11 rotavirus was activated with L-1-toluenesulfonamide-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin (Sigma) at a final concentration of 10 μg/ml for 30 minutes and expressed as a multiplicity of infection (MOI) 10 was added to MA104 cells. Ten hours after infection, messenger RNA was isolated from infected cells using the Ultraspec RNA Isolation System (Biotecx Laboratories, Inc., Houston TX).

insect cell infection

Cells of the Spodoptera frugiperda insect cell line (designated Sf9, ATCC Accession No. CAL1711) were grown in serum-free SF 900 II (Gibco BRL, Gaithersburg, MD) or Insect-Xpress (Biowhittaker Inc.) medium , at a density of 2.5×10 ⁵ /ml, inoculated in a 250 mL Erlenmeyer flask on an orbital shaker at 120 rpm at 28°C. Cells were infected in shaking flasks with recombinant baculovirus at a low MOI (0.2) at a density of 1.5×10 ⁶ /mL. 1 ml of cell suspension was collected every 24 hours for cell viability studies (by trypan blue exclusion; see Example 3 below) and for Western blot analysis. Cells were grown in two-well culture chambers (Nunc Inc., Naperville, IL) for imaging studies.

Example 2

RT/PCR and Sequencing

NSP4 cDNA was synthesized from purified viral messenger RNA (obtained in Example 1) using Ready to Go First-Strand Beads (Pharmacia Biotech Inc., Piscataway, NJ) followed by PCR amplification. The SA11 NSP4-specific primers used in the PCR reaction were based on the published NSP4 sequence (12). The 5' end of the SA11NSP4-specific primer was ATGGAAAAGCTTACCGACCTC (SEQ ID NO: 1), while the 3' end of the SA11-specific primer was CTCTTACATTGCTGCAGTC (SEQ ID NO: 2). PCR products were directly sequenced using the ABI PRISM ^™ DNA Sequencing System (Perkin-Elmer Corporation, Forest City, CA). In addition, individual clones obtained from TA ^™ cloning reactions (Invitrogen, San Diego, CA) were also sequenced. To sequence the NSP4 gene in recombinant baculoviruses, viral DNA was extracted by benzene followed by ethanol precipitation. The NSP4 gene sequence was amplified by PCR with NSP4-specific primers. PCR products were purified using the Qiaquick ^™ Spin kit (Qiagen, Santa Clarita, CA), and the reactions were sequenced using the ABI PRISM ^™ DyeTerminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer Corporation).

The nucleotide sequence of gene 10 was obtained by direct sequencing of RT-PCR products from SA11-infected cells, or clones of RT-PCR products. A change from A to C at nucleotide 139 results in a codon change from AAT to CAT resulting in a change of amino acid position 47 from asparagine to histidine. In particular, the Australian strain obtained by Holmes et al. and sequenced by Both et al. was found to have an asparagine at amino acid position 47 of NSP4. In contrast, in the SA11 ATCC strain, NSP4 has a histidine at amino acid position 47.

To determine which version of NSP4 represented the true prototype, the nucleotide sequences of gene 10 of other different SA11 virus stocks were determined. The ATCC SA11 master virus (from which all other SA11 stocks are derived), and the SA11 virus of a completely different origin (Dr. Mary K. Estes, Baylor College of Medicine, Houston, TX) have a C at nucleotide position 139 . Thus, most versions of NSP4 of the different SA11 virus stocks have a histidine at amino acid position 47.

The sequences of NSP4(His) and NSP4(Asn) in the recombinant baculovirus were reconfirmed by sequencing the PCR product of the recombinant baculovirus extract. The NSP4(Asn) gene was constructed using the QuickChange ^™ site-directed mutagenesis kit (Stratagene, LaJolla, CA). Individual NSP4 (His) and NSP4 (Asn) genes were inserted into copies of the pFastBac transfer vector (Life Technologies, Rockville, MD). Recombinant baculoviruses were plaque purified 3 times and amplified at low MOI. The correct sequence of each virus was confirmed by the method described above. Expression of NSP4 was confirmed by immunofluorescence staining of Sf9 cells infected with recombinant baculovirus and Western blot analysis with an NSP4 anti-peptide antibody (18; see Example 4 below).

Example 3

Cell Viability Measurement

Inoculate Sf9 cells at 2.5×10 ⁵ /mL in 250 mL Erlenmeyer flasks of serum-free Sf-900 II SFM ^TM medium or Insect-Xpress medium. When the cell density reaches 1.5×10 ⁶ /mL, use recombinant rod Viruses were infected at MOI 0.2. Cells were grown at 28°C in an orbital shaker at a speed of 120 rpm. Cell viability was determined by trypan blue exclusion assay (11). Briefly, an aliquot of cell suspension (50 microliters) was incubated with an equal volume of 2% trypan blue for 5 minutes; stained and unstained cells were then counted.

The survival rate of Sf9 cells expressing various rotavirus proteins was measured daily for 6 days (D1 to D6), and the results are shown in Table 1. Data are presented as mean ± standard deviation of at least three independent experiments. Recombinant baculoviruses expressing VP2, VP4, VP6 and VP7 were kindly provided by Dr. Mary K. Estes (Baylor College of Medicine, Houston, TX).

Table 1: Cell Viability (%) VP2 VP4 VP6 VP7 NSP4(His) NSP4(Asn) D1 99.5+/-0.87 96.9+/-0.56 98.1+/-0.9 98+/-1.2 95.4+/-1.99 97.6+/-1 D2 96.8+/-1.23 97.5+/-1.8 97.5+/-0.7 96.3+/-0.8 65.9+/-2 83+/-1.6 D3 94.3+/-1.87 90.2+/-1.5 88.1+/-7 80.7+/-3.2 47.2+/-0.5 61.6+/-0.3 D4 81+/-2.6 73+/-1.7 61+/-3.5 43+/-7 21+/-1.4 36.4+/-2.9 D5 57+/-4 31+/-4 22.5+/-6.8 12+/-5.2 3.9+/-1 10.8+/-4.7 D6 38+/-9 11+/-6 7.5+/-2.4 3+/-1.4 0 0

NSP4 expression is cytotoxic to Sf9 cells. As seen in Table 1, NSP4(His) killed cells more rapidly than NSP4(Asn), whereas expression of other rotavirus proteins, including VP2, VP4, VP6 and VP7, did not result in cell death until very late in infection. However, expression of NSP4 led to rapid cell death. 48 hours after infection (D2), for the cells expressing VP2, VP4, VP6 and VP7, the survival rates were 96.8%, 97.5%, 97.5% and 96.3%, respectively, while for the cells expressing NSP4(His) and NSP4(Asn), The survival rates were 65.9% and 83%, respectively. Therefore, NSP4(His) has a more profound effect on cytotoxicity than NSP4(Asn). Sf9 cells expressing NSP4(His) were killed faster than Sf9 cells expressing NSP4(Asn). At 72 hours post-infection (D3), the viability was 94.3%, 90.2%, 88.1% and 80.7% for cells expressing VP2, VP4, VP6 and VP7, respectively. For the cells expressing NSP4(His) and NSP4(Asn), respectively, the survival rates were 47.2% and 61.6% (Table 1 and Figure 2). There were no statistically significant differences in cell viability among cells expressing VP2, VP4, VP6, and VP7 at any time point. However, in addition to the significant difference (p < 0.05) in the survival rate at 48 and 72 hours after infection between cells expressing one of the NSP2 versions and cells expressing other rotavirus proteins, there was no significant difference between cells expressing different versions of NSP4. There were also significant differences in survival rates. NSP(His) is more cytotoxic to Sf9 cells than NSP(Asn).

Example 4

Measurement of intracellular calcium levels

[Ca ²⁺ ] _i was determined using the fluorescent Ca ²⁺ indicator Fura-2/AM, measured with a Nikon Diaphot ^™ fluorescence microscope equipped with a Quantex ^™ QX-7 CCD camera and a digital imaging system. Briefly, Sf9 cells were reacted with Fura-2/AM (Molecular Probes, Eugene, OR) at a final concentration of 2 μM for 30 minutes at room temperature. Intracellular calcium levels in individual cells were calculated as described by Grynkiewicz et al. (32) and Nankova et al. (31) and expressed as the ratio of absorbance measured at 340 nm and 380 nm. Calcium binding switches the absorbance spectrum of fura-2 to shorter wavelengths (from 380 to 340 nm). The absorbance ratio of 340/380 represents the level of intracellular free calcium. For each experiment, 10 pictures were taken. The intracellular calcium concentration was calculated for the 12 cells in each photograph. At least three independent experiments were performed.

Expression of NSP4 in Sf9 cells has been shown to lead to a significant increase in intracellular calcium levels (5). It has been proposed that an increase in intracellular calcium levels leads to SA11 -mediated cytotoxicity in mammalian cells (25). The same concept has also been proposed for NSP4 expressing Sf9 cells (15). Intracellular calcium levels were measured in Sf9 cells expressing NSP4(His) and NSP4(Asn). The results depicted in Figure 3 demonstrate that significantly higher intracellular calcium levels were observed in NSP4(His) expressing Sf9 cells (right) than in NSP4(Asn) expressing Sf9 cells (middle). At 48 hours after infection, intracellular calcium levels in mock-infected Sf9 cells, NSP4(Asn)-expressing Sf9 cells and NSP4(His)-expressing Sf9 cells were 0.12, 0.32 and 0.48 (after adding Fura-2/AM Read the ratio of 340/380nm). This corresponds to the following concentrations of intracellular calcium levels: 80 nM for mock-infected Sf9 cells, 213 nM for NSP4(Asn) expressing cells and 320 nM for NSP4(His) expressing cells. Thus, expression of NSP4(Asn) resulted in a significant increase in intracellular calcium levels as previously reported (5). However, there were significantly higher intracellular calcium levels in cells expressing NSP(His) compared to cells expressing NSP(Asn) (P<0.05).

Example 5

Western blot analysis

Although the two versions of the gene encoding NSP4 were cloned separately into the same copy of the baculovirus transfer vector, it is possible that the level of protein expression differs for recombinant baculoviruses expressing the two versions of NSP4. To rule out the possibility that the increased toxicity and intracellular calcium accumulation associated with NSP4(His) expression were due to differences in protein expression levels between these two forms, the absolute amount of NSP4 in Sf9 cells was measured. Sf9 cells were infected with recombinant baculovirus at MOI 0.2, and cells were harvested 48 hours after infection. Equal samples (lysates of 2×10 ⁴ cells) were loaded on SDS-PAGE and detected by Western blot analysis using NSP4 anti-peptide antibody ( FIG. 4 ).

Covance Reaseach Products (Denver, PA) produced this NSP4 anti-peptide antibody in two Elite-New rabbits. Briefly, a synthetic peptide having the sequence Cys Asp Lys Leu Thr ArgGlu Ile Glu Gln Val Glu Leu Leu Lys Arg Ile Tyr Lys Asp Leu Thr (SEQ ID NO: 3) was synthesized and cross-linked with KLH. A cysteine added to the N-terminus is used to facilitate specific cross-linking. The remainder of this sequence represents residues 114-135 of NSP4. 500 μg of cross-linked peptide was mixed with 500 μl of FCA and injected intradermally. Rabbits were boosted 4 times every 3 weeks with 250 µg of cross-linked peptide mixed with 250 µl of IFA.

Sample aliquots of baculovirus recombinant-infected Sf9 cells collected 48 hours after infection were analyzed by SDS-PAGE. The separated proteins were electroblotted onto nitrocellulose membranes, then blocked with PBS containing 5% BLOTTO (Bio-Rad, Inc., Hercules, CA) for 30 minutes at room temperature, and NSP-4 diluted 1:100 with PBS Specific anti-peptide antibodies were incubated for 1 hour at room temperature. After washing 3 times in PBS, horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin (1:200 dilution) was added to the membrane and incubated for 1 hour, followed by HRP ^TM substrate reagent Kit (Bio-Rad, Inc.) for color development.

As shown in Figure 4, as measured by the brightness of bands representing glycosylated and non-glycosylated NSP4, the expression of NSP4 (Asn) in recombinant baculovirus-infected Sf9 cells (lane 3) expressed NSP4 ( His) cells (lane 2) expressed more NSP4. When equal amounts of recombinant baculovirus-infected total cellular proteins were compared with Western blot analysis, essentially the same results were obtained. NSP4 expression was detected as early as 24 hours and peaked between 48 and 72 hours. Degradation of NSP4 was observed 72 hours after infection. The expression of NSP4(Asn) was higher than that of NSP4(His) at each time point except 24 hours after infection. Therefore, the cytotoxicity of NSP4(His) is not due to the higher expression level of NSP4(His) than NSP4(Asn).

The results of at least 3 independent experiments for cell viability and calcium concentration determinations were analyzed using Bonferroni (multiplicative comparisons) t-test. Briefly, the method performed a parametric analysis of variables followed by a t-test for pairwise comparisons. P-values are multiplied by comparison numbers used to resolve combined risks for many statistical comparisons.

Example 6

NSP4 protein expression from Sf9 cells infected with recombinant baculovirus

The gene encoding the NSP4 protein (His) was inserted into the pFastBac baculovirus-based transfer plasmid (Gibco BRL, Gaithersburg, MD). This plasmid was mutated by substituting G for A at nucleotide 142. This mutation changes the codon from AAA (lysine) to GAA (glutamic acid). A third recombinant was generated by inserting the gene encoding the NSP4 protein (Asn) into the pFastBac transfer plasmid. Each plasmid was transfected into Escherichia coli DH10Bac competent cells (Gibco BRL). White colonies obtained by screening recombinant baculovirus DNA. The recombinant baculovirus DNA is then purified. Sf9 cells were transfected with the three recombinant baculovirus DNAs, respectively, and the recombinant baculoviruses were plaque-purified as described (34). The sequences of three NSP4 genes were confirmed. These constructs were used in Examples 7 and 8 below.

Example 7

Further measurements of cell viability

In addition to using MOI10, three recombinant forms of NSP4 protein were evaluated according to the protocol of Example 3: amino acid 47 NSP4 (Asn), amino acid 47 NSP4 (His) and amino acid 47 NSP4 (His), amino acid 48 NSP4 (Glu). Means at each time point represent three independent infections.

NSP4 expression is cytotoxic to Sf9 cells. As seen in Figure 5, amino acid 47NSP(His), amino acid 48(Glu) killed cells slightly faster than amino acid 47 NSP4(His), and according to the Dunnett multiplication comparison test, both forms killed cells faster than amino acid 47 NSP4(Asn) Cells were killed at a statistically significantly higher rate. These results indicated that amino acid 47 NSP4 (His), amino acid 48 NSP4 (Glu) were the most toxic, while NSP4 (Asn) was the least cytotoxic of the three forms.

Example 8

Further measurements of intracellular calcium levels

The protocol of Example 4 was followed except that three recombinant forms of the NSP4 protein were evaluated: amino acid 47 NSP4 (Asn), amino acid 47 NSP4 (His) and amino acid 47 NSP4 (His), amino acid 48 NSP4 (Glu). Figure 6 shows the results. Bars represent the intracellular calcium levels of Sf9 cells infected with three NSP4 recombinants and mock-infected Sf9 cells. Each bar represents the mean of three experiments.

The highest intracellular calcium levels were observed in cells expressing amino acid 47 NSP (His), amino acid 48 (Glu), followed by NSP4 (His) and NSP4 (Asn). These results indicate that amino acid 47 NSP (His), amino acid 48 (Glu) have the most profound effects on intracellular calcium levels, while NSP4 (Asn) has the least effect of the three forms. Differences were considered statistically significant according to Dunnett's multiplicative comparison test.

references

1. Kapikian, AZ and Chanock, RM, vol. 21657-1709, in Fields Virology , edited by BNFields, et al. (3rd ed., Raven Press, 1996).

2. Malherbe, HH, et al., South African Journal of Medicine , 37 , 401-411 (1963).

3. Dyall-Smith, ML, and Holmes, IH, Journal of Virology , 38 , 1099-1103 (1980).

4. Estes, MK, et al., Journal of Virology , 61 , 1488-1494 (1987).

5. Tian, P., et al., Journal of Virology , 68 , 251-257 (1994).

6. Tian, P., et al., Journal of Virology , 69 , 5763-5772 (1995).

7. Ball J., et al., Science , 272 , 101-104 (1996).

8. Dong, Y., et al., Proc. Natl. Acad. Sci. USA , 94 , 3960-3965 (1997).

9. Zhang, M., et al., Journal of Virology , 172 , 3666-3672 (1998).

10. Hoshino, Y., et al., Virology , 209 , 274-280 (1995).

11. Newton, K., et al., Journal of Virology , 71 , 9458-9465 (1997).

12. Both, GW, et al., Journal of Virology , 48 , 335-339 (1983).

13. Sanger, F., et al., Proc. Natl. Acad. Sci. USA , 5463-5467 (1977).

14. Maxam, AM, and Gilbert, W., Proc. Natl. Acad. Scj. USA , 74 , 560-564 (1977).

15. Chan, WK, et al., Virology , 164 , 435-444 (1988).

16. Au, K.-S., et al., Journal of Virology , 63 , 4553-4562 (1989).

17. Au, K.-S., et al., Virology , 194 , 665-673 (1993).

18. Tian, P., et al., Journal of Virology , 70 , 6973-6978 (1996).

19. Boyle DB, et al., Genes , 35 , 169-177 (1985).

20. Bergmann, CC, et al., EMBO. J , 8 , 1695-1703 (1989).

21. Meyer, JC, et al., Virology , 171 , 98-107 (1989).

22. Taylor, JA, Virology , 194 , 807-814 (1993).

23. Taylor, JA, et al., EMBO. J , 15 , 4469-4476 (1996).

24. Nicotera, P., et al., Toxicology , 32 , 449-470 (1992).

25. Michelangeli, F., et al., Virology , 181 , 520-527 (1991).

26. Blackhall, J., et al., Virology , 225 , 181-190 (1996).

27. Chan, WK, et al., Virology , 151 , 243-252 (1986).

28. Petrie, BL, et al., J. Virology , 46 , 270-274 (1983).

29. Sheridan, JF, et al., J. Infectious Diseases , 149 , 434-438 (1984).

30. Published International Application No. WO 97/00099.

31. Nankova, B, et al., Mol. Brain Res . 35 , 164-172 (1996).

32. Grynkiewicz, G., et al., J. Biol. Chem. , 260 , 3440-3450 (1985).

33. Wason, CJ, et al., Statistics for Management and Economics , pp. 499-502, (5th ed., Allynand Bacon, Boston, MA, 1993).

34. Luckow, VA, et al., J. Virology , 67 , 4566-4579 (1993).

Sequence Listing

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<120> Different cytotoxicity of variant forms of rotavirus nonstructural protein 4

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Claims (7)

1. the rotavirus Nonstructural Protein 4 of a sudden change, its perform toxic attenuation but keep antigenicity and immunogenicity is characterized in that Histidine or l-asparagine on this protein 47 position are become aspartic acid by mutagenesis.

2. an antigen composition is characterized in that, this antigen composition comprises a kind of rotavirus Nonstructural Protein 4 of sudden change, this albumen perform toxic attenuation but keep antigenicity and immunogenicity, and Histidine or l-asparagine on its 47 are become aspartic acid by mutagenesis.

3. antigen composition as claimed in claim 2 is characterized in that this antigen composition also comprises adjuvant, diluent or carrier.

4. the nucleotide sequence of separation and purifying, it is characterized in that, this sequence comprises the nucleotide sequence of the rotavirus Nonstructural Protein 4 of encoding mutant, described albumen perform toxic attenuation but keep antigenicity and immunogenicity, and its Histidine or l-asparagine on 47 is become aspartic acid by mutagenesis.

5. one kind contains and separates and the plasmid of purified nucleic acid sequence, it is characterized in that, this plasmid comprises the nucleotide sequence of the viral Nonstructural Protein 4 of coding colyliform sudden change, described albumen perform toxic attenuation but keep antigenicity and immunogenicity, and its Histidine or l-asparagine on 47 is become aspartic acid by mutagenesis.

6. use the host cell of plasmid conversion, transfection or the infection of claim 5.

7. method that produces the rotavirus Nonstructural Protein 4 of sudden change, it is characterized in that this method comprises with plasmid conversion, transfection or host cells infected, and under the condition that allows the described rotavirus Nonstructural Protein 4 of host cell expression, cultivate described host cell, wherein

Described plasmid contains the nucleotide sequence of separation and purification, the rotavirus Nonstructural Protein 4 of this sequence encoding sudden change, and this albumen perform toxic attenuation but keep antigenicity and immunogenicity, its Histidine or l-asparagine on 47 is become aspartic acid by mutagenesis.

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