# Patent

## Title
Adenovirus isolated from Titi Monkeys

## Abstract

Provided is a Titi Monkey Adenovirus (TMAdV) that can infect both human and non-human primates. Further provided are nucleic acid sequences, proteins, expression vectors and host cells, anti-TMAdV antibodies, vaccines, compositions, methods of detecting TMAdV, methods for assaying for anti-TMAdV compounds, and methods for treating or preventing a TMAdV infection.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims benefit under 35 USC 119(e) to U.S. Provisional Application No. 61/484,536 filed May 10, 2011, which is incorporated herein by reference in its entirety for all purposes.


STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
This invention was made with government support under grant nos. R56AI089532, K08 AI074913, P51 RR000169 and R01 HD053555 awarded by the National Institutes of Health. The government has certain rights in the invention.


FIELD OF THE INVENTION
The present invention relates to the discovery of a new Titi Monkey Adenovirus (TMAdV), nucleic acids, proteins, vaccines, compositions, kits, methods of detecting and diagnosing TMAdV infection, methods of treating or preventing TMAdV infection, and methods for identifying anti-TMAdV compounds.
BACKGROUND OF THE INVENTION
Adenoviruses, first isolated in the 1950s from explanted adenoid tissue, are double-stranded nonenveloped DNA viruses that naturally infect many vertebrates, including nonhuman primates and humans. The human adenoviruses in the Mastadenovirus genus (comprised of all mammalian adenoviruses) are classified into 7 species groups A-G and 54 different serotypes (Harrach, B., et al. Virus Taxonomy (9th Report of the International Committee on Taxonomy of Viruses) (eds. King, A., Carstens, E., Adams, M. & Lefkowitz, E.) (Elsevier, N.Y., 2011)). Adenoviruses are the cause of an estimated 5-10% of febrile illnesses in children worldwide (Fox, J. P., Hall, C. E. & Cooney, M. K. Am J Epidemiol 105, 362-386 (1977)). Some serotypes, such as human adenovirus type 14 (hAd14), have been associated with severe and potentially fatal outbreaks of pneumonia in residential facilities and military bases (Lewis, P. F., et al., J Infect Dis 199, 1427-1434 (2009)). Adenoviruses have also been associated with other clinical syndromes including conjunctivitis, hepatitis, and diarrhea. In nonhuman primates, most epidemiologic studies of adenoviruses have focused on their identification in fecal samples from asymptomatic animals (Banyai, K., et al. Vet Microbiol 142, 416-419 (2010); Roy, S., et al. PLoS Pathog 5, e1000503 (2009); Wevers, D., et al, Virol J 7, 303 (2010)). Overt respiratory disease associated with simian adenoviruses has also been observed (Tong, S., et al., Am J Trop Med Hyg 82, 967-970 (2010)). Although adenoviruses are significant pathogens, genetically modified strains are being actively explored as potential vectors for vaccines and gene therapy (Ghosh, S. S., Gopinath, P. & Ramesh, A. Appl Biochem Biotechnol 133, 9-29 (2006)).
It is unclear whether or not infection by adenoviruses is species-specific. Human adenoviruses do not usually replicate in monkey cells (or vice versa). Studies of sera from animal handlers and zoo workers exposed to chimpanzees in captivity fail to detect antibodies to chimpanzee adenoviruses (Basnight, M., Jr., Rogers, N. G., Gibbs, C. J., Jr. & Gajdusek, D. C., Am J Epidemiol 94, 166-171 (1971); Xiang, Z., et al., Emerg Infect Dis 12, 1596-1599 (2006)). However, recent serological surveys have found antibodies to New World or Old World monkey adenoviruses in donor human sera from regions where the monkeys are endemic (Xiang, Z., et al., supra; Ersching, J., et al., Virology 407, 1-6 (2010); Roy et al., supra). In addition, phylogenetic analyses of adenoviruses from greater apes reveal that they fall precisely into “human” adenoviral groups B, C, and E. The high degree of sequence relatedness within members of each group suggests that at least some adenoviral strains may be capable of infecting both nonhuman primates and humans.
BRIEF SUMMARY OF THE INVENTION
The claimed subject matter relates to the Applicants' discovery of a Titi Monkey Adenovirus (TMAdV) during a fulminant pneumonia outbreak at a primate research center. A human researcher in close contact with the monkey colony tested positive, demonstrating the potential for cross-species outbreak.
Accordingly, the claimed subject matter provides compositions and methods useful in the detection, treatment and prevention, and modulation of TMAdV infection.
In an embodiment, an isolated nucleic acid is provided. The isolated nucleic acid comprises a nucleotide sequence at least 100 nucleotides in length that has at least 90% sequence identity over its length to SEQ ID NO:1 or its complement. In an embodiment, the nucleic acid comprises at least 95% identity over its length to SEQ ID NO:1. In an embodiment, the nucleic acid comprises at least 90% identity over the full length of SEQ ID NO:1. In an embodiment, the nucleic acid comprises at least 95% identity over the full length SEQ ID NO:1. In yet another embodiment, the nucleotide sequence comprises SEQ ID NO:1.
In an embodiment, an isolated Titi Monkey Adenovirus (TMAdV) is provided. The TMAdV comprises a nucleotide sequence that has at least 90% sequence identity over the full length of SEQ ID NO:1. In an embodiment, the TMAdV comprises at least 95% identity over the full length of SEQ ID NO:1. In an embodiment, the TMAdV comprises SEQ ID NO:1.
In an embodiment, an isolated nucleic acid is provided. The isolated nucleic acid comprises a nucleotide sequence at least 100 nucleotides in length and has at least 90% sequence identity to an open reading frame selected from the group consisting of SEQ ID NOs:2-37. In an embodiment, the isolated nucleotide sequence comprises at least 95% identity to the open reading frame encoded by the nucleotide sequence selected from the group consisting of SEQ ID NOs:2-37. In an embodiment, the nucleotide sequence comprises the open reading frame encoded by the nucleotide sequence selected from the group consisting of SEQ ID NOs:2-37.
In an embodiment, an expression vector is provided. In one aspect, expression vector comprises an isolated nucleic acid comprising a nucleotide sequence at least 100 nucleotides in length that has at least 90% sequence identity over its length to SEQ ID NO:1 or its complement. In another aspect, the expression vector comprises an isolated nucleic acid comprising a nucleotide sequence at least 100 nucleotides in length and has at least 90% sequence identity to an open reading frame selected from the group consisting of SEQ ID NOs:2-37. In another embodiment, a host cell is provided comprising the expression vectors.
In another embodiment, a composition (i.e., pharmaceutical or physiological) is provided. In one aspect, the composition comprises an isolated nucleic acid comprising a nucleotide sequence at least 100 nucleotides in length that has at least 90% sequence identity over its length to SEQ ID NO:1 or its complement. In another aspect, the composition comprises an isolated nucleic acid comprising a nucleotide sequence at least 100 nucleotides in length and has at least 90% sequence identity to an open reading frame selected from the group consisting of SEQ ID NOs:2-37.
In an embodiment, a protein is provided. The protein is encoded by an isolated nucleic acid comprising a nucleotide sequence at least 100 nucleotides in length and has at least 90% sequence identity to an open reading frame selected from the group consisting of SEQ ID NOs:2-37.
In an embodiment, a composition (i.e., pharmaceutical or physiological) is provided. The composition comprises a protein encoded by an isolated nucleic acid comprising a nucleotide sequence at least 100 nucleotides in length and has at least 90% sequence identity to an open reading frame selected from the group consisting of SEQ ID NOs:2-37.
In an embodiment, an isolated antibody is provided. The antibody specifically binds to a protein encoded by an isolated nucleic acid comprising a nucleotide sequence at least 100 nucleotides in length and has at least 90% sequence identity to an open reading frame selected from the group consisting of SEQ ID NOs:2-37. In one aspect, the antibody is a monoclonal antibody. In another aspect, the antibody is a polyclonal antibody. In yet another aspect, purified serum comprising the polyclonal antibodies is provided. In another aspect, a method of producing a TMAdV antibody is provided.
In an embodiment, a method is provided. The method comprises detecting a TMAdV nucleic acid comprising the steps of contacting a sample suspected of comprising a TMAdV nucleic acid with a nucleotide sequence at least 100 nucleotides in length that has 90% identity over its length to the corresponding segment of SEQ ID NO:1, and detecting the presence or absence of specific binding of the nucleotide sequence to a TMAdV nucleic acid.
In another embodiment a method is provided comprising detecting a TMAdV nucleic acid, the method comprising the steps of contacting a sample suspected of comprising the TMAdV nucleic acid with at least one primer that hybridizes to a nucleotide sequence of SEQ ID NO:1, performing a PCR reaction; and detecting the presence or absence of the TMAdV nucleic acid.
In another embodiment a method is provided comprising detecting a TMAdV infection in a sample. The method comprises the steps of contacting a sample suspected of comprising a TMAdV antibody with a TMAdV protein, and detecting the presence or absence of the TMAdV antibody. In another embodiment, a detectable moiety detects the presence or absence of the TMAdV antibody.
In another embodiment a method is provided comprising detecting a TMAdV infection in a sample. The method comprises the steps of contacting a sample suspected of comprising a TMAdV protein with a TMAdV antibody, and detecting the presence or absence of the TMAdV protein. In another embodiment, a detectable moiety detects the presence or absence of the TMAdV protein.
In an embodiment a method is provided comprising assaying for an anti-TMAdV compound, the method comprising the steps of contacting a sample comprising a TMAdV, the TMAdV comprising a genome that has at least 90% identity over its length to the corresponding segment of SEQ ID NO:1; and determining whether the compound has a functional effect on TMAdV.
In an embodiment, a method is provided. The method comprises treating or preventing a TMAdV infection in a subject comprising the step of administering to the subject a therapeutically effective dose of a compound that has a functional effect on TMAdV. In one aspect, the compound comprises an antigen capable of providing an immune response. In another aspect, the antigen is a protein encoded by an isolated nucleic acid comprising a nucleotide sequence at least 100 nucleotides in length and has at least 90% sequence identity to an open reading frame selected from the group consisting of SEQ ID NOs:2-37. In another aspect, the compound comprises an antibody that specifically binds to a protein encoded by an isolated nucleic acid comprising a nucleotide sequence at least 100 nucleotides in length and has at least 90% sequence identity to an open reading frame selected from the group consisting of SEQ ID NOs:2-37. In another aspect, the compound comprises a small organic molecule. In another aspect, the compound comprises an aptamer. In one aspect, the aptamer is an siRNA or antisense molecule comprising a double-stranded region of about 15 to about 60 nucleotides in length and has at least 90% identity over its length to the corresponding segment of SEQ ID NO:1. In another aspect, the compound is administered by means comprising oral, topical, intraarticular, intravenous, intramuscular, intradermal, intraperitoneal or subcutaneous routes.
In an embodiment, a vaccine is provided. In an aspect, the vaccine comprises an isolated protein encoded by an isolated nucleic acid comprising a nucleotide sequence at least 100 nucleotides in length and has at least 90% sequence identity to an open reading frame selected from the group consisting of SEQ ID NOs:2-37. In another aspect, the vaccine comprises an antibody that specifically binds to a protein encoded by an isolated nucleic acid comprising a nucleotide sequence at least 100 nucleotides in length and has at least 90% sequence identity to an open reading frame selected from the group consisting of SEQ ID NOs:2-37.
In an embodiment, a kit is provided. In one aspect, the kit detects a TMAdV nucleic acid, the kit comprising a nucleotide sequence at least 100 nucleotides in length that has at least 90% identity over its length to the corresponding segment of SEQ ID NO:1. In another aspect, the kits detects a TMAdV nucleic acid, the kit comprising at least one primer that hybridizes to a nucleotide sequence comprising SEQ ID NO:1 under highly stringent PCR conditions comprising a denaturation phase of 90° C.-95° C. for 30 sec-2 min., an annealing phase of 50° C. to about 65° C. lasting 30 sec.-2 min., and an extension phase of about 72° C. for 1-2 min., and an extension phase of about 72° C. for 1-2 min for 20-40 cycles. In another aspect, the kit detects TMAdV in a sample and comprises an antibody that specifically binds to a protein encoded by an isolated nucleic acid comprising a nucleotide sequence at least 100 nucleotides in length and has at least 90% sequence identity to an open reading frame selected from the group consisting of SEQ ID NOs:2-37. In one aspect, the antibody is a monoclonal antibody. In another aspect, the antibody is a polyclonal antibody. In another aspect, the kit comprises a protein encoded by the nucleotide sequences selected from the group consisting of SEQ ID NOs:2-37.
In an embodiment, a method of expressing a protein is provided. The method comprises a host cell further comprising an expression vector comprising the nucleotide sequences selected from the group consisting of SEQ ID NOs:2-37. In some embodiments the host cell is a human cell.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates the clinical and epidemiologic features of the titi monkey outbreak. (A) Map of the titi monkey cages situated in one quadrant of a building, showing the locations of asymptomatic, at-risk monkeys (brown or green), affected surviving monkeys (black), and monkeys who died from their illness (skeleton). 3 monkeys were moved into the building (arrows pointing down and to the left) and 4 monkeys out of the building (arrows pointing up and to the right) during the 3rd week of the outbreak. The upper left photograph shows an image of an adult male titi monkey and his infant. The upper right inset shows the location of the titi monkey cages relative to other rhesus monkey cages in the building. Asymptomatic monkeys with positive serum antibody titers to TMAdV 4 months after the outbreak are shown in green. (B) Epidemic curve of the outbreak, with the number of cases in blue and cumulative attack rate in red. (C) Anteroposterior chest radiograph of an affected titi monkey, showing bilateral basilar infiltrates and a prominent right middle lobe consolidation. (D) 1—gross photograph of lungs at necropsy; the lungs failed to fully collapse upon opening the chest, and a single  ̃1.5 cm focus of dark red discoloration (hemorrhage) can be seen in the left caudal lobe. 2—photomicrograph of H&E stained lung tissue showing a severe diffuse necrotizing bronchopneumonia characterized by the presence of hemorrhage and intranuclear inclusions (arrows). 3—photomicrograph of H&E stained liver tissue showing a multifocal necrotizing hepatitis with numerous intranuclear inclusions (arrows). 4—transmission electron micrograph of an affected lung alveolus (scale bar=1 μm) filled with adenovirus-like particles (inset, scale bar=0.1 μm).
FIG. 2 illustrates the discovery and whole-genome characterization of the novel adenovirus TMAdV. (A) The locations of the 4 Virochip probes derived from adenovirus sequences and used to detect TMAdV are mapped onto the  ̃37 kB genome. The 4 Virochip probe sequences are also aligned with the corresponding sequence in the TMAdV genome, with mismatches highlighted in pink. (B) Coverage map of deep sequencing reads corresponding to TMAdV using BLASTN (blue) and TBLASTX (transparent blue) alignments to the simian adenovirus SAdV-18. The actual coverage achieved by deep sequencing as determined by alignments to the fully sequenced genome of TMAdV is much higher (red). (C) Genome organization of TMAdV. Predicted protein coding regions are shown as boxes. Boxes above the central black line represent open reading frames (ORFs) that are encoded on the forward strand, while boxes underneath the black line represent reverse-strand encoded ORFs. Early region ORFs are shaded in gray. The x-axis refers to the nucleotide position along the  ̃37 k genome of TMAdV. Abbreviations: FAdV, fowl adenovirus; SAdV, simian adenovirus; PAdV, porcine adenovirus; HAdV, human adenovirus, TMAdV, titi monkey adenovirus.
FIG. 3 illustrates the whole-genome phylogenetic analysis of TMAdV. The whole-genome nucleotide phylogenetic tree is reconstructed from a multiple sequence alignment of all 95 unique, fully-sequenced adenovirus genomes in GenBank and TMAdV. Both rectangular cladogram and radial tree layouts are displayed. The branch corresponding to TMAdV is highlighted in boldface red. Abbreviations: HAdV, human adenovirus; SAdV, simian adenovirus; MAdV, mouse adenovirus, FrAdV, frog adenovirus; TAdV, turkey adenovirus; SnAdV, snake adenovirus; DAdV, duck adenovirus; OAdV, ovine adenovirus; BAdV, bovine adenovirus; PAdV, porcine adenovirus; TSAdV, tree shrew adenovirus; CAdV, canine adenovirus.
FIG. 4 shows scanning pairwise alignment of representative adenoviruses with TMAdV. The scanning nucleotide pairwise identities of TMAdV relative to representative human (yellow) or simian (brown) adenoviruses in species A-G, porcine adenovirus (red), or fowl adenovirus (green) are shown. The window size is 400 bp with a step size of 40 bp. The x-axis refers to the nucleotide position along the  ̃37 k genome of TMAdV. Abbreviations: HAdV, human adenovirus; SAdV, simian adenovirus; PAdV, porcine adenovirus; FAdV, fowl adenovirus.
FIG. 5 demonstrates growth and propagation of TMAdV in cell culture. The flow chart displays up to 10 passages (P1-P10) of TMAdV in cell culture in human lung adenocarcinoma (A549, orange), primary rhesus macaque kidney (PMK, brown), or established African green monkey kidney (BSC-1, green) cells.
FIG. 6 shows the seroprevalence of TMAdV in humans and monkeys. Sera from titi monkeys (circles), rhesus macaques (squares), and humans (triangles) were tested for antibodies to TMAdV by virus neutralization. Arrows designate pre-outbreak and post-outbreak serum samples from the same individual monkey. Pre-outbreak serum samples were previously banked in 2007. Sera from CNPRC personnel and close contacts (orange triangles) were collected 4 months post-outbreak, except for the two family members of the clinically ill researcher, whose sera were collected 1 year post-outbreak. * denotes a clinically ill researcher; ** denotes a family member of the researcher, who was also sick. Abbreviations: CNPRC, California National Primate Research Center; NEG, negative.
FIG. 7 illustrates phylogenetic analysis of the hexon, polymerase, penton base, and fiber genes of TMAdV. A multiple sequence alignment of selected genes from all 95 unique, fully-sequenced adenovirus genomes in GenBank and TMAdV is performed and the results displayed as a radial phylogenetic tree. The branch corresponding to TMAdV is highlighted in boldface red. Abbreviations: HAdV, human adenovirus, SAdV, simian adenovirus; PAdV, porcine adenovirus; FAdV, fowl adenovirus.
FIG. 8 demonstrates bootscanning recombination analysis of TMAdV. Bootscanning analysis was initially performed with all 95 unique, fully-sequenced adenovirus genomes in GenBank (data not shown), then, after removal of similar viral genomes, with a selected subset representing human/simian adenoviruses in species A-G and all non-primate vertebrate adenoviruses (whole genome, hexon, polymerase, penton base, and fiber). The window size is 400 bp with a step size of 40 bp for the whole genome and 200 bp with a step size of 20 bp for the individual genes. The x-axis refers to the nucleotide position. Please refer to FIG. 3 for definition of abbreviations.
FIG. 9 is the TMAdV sequence. The entire TMAdV sequence is provided with identification of putative open reading frames.



DETAILED DESCRIPTION OF THE INVENTION
Described herein is the identification of a novel adenovirus associated with a fulminant pneumonia outbreak in a titi monkey colony. Although the absence of an animal model precludes a strict fulfillment of Koch's postulates, there are several lines of evidence implicating this novel adenovirus, TMAdV, as the cause of the outbreak. First, conventional testing for other pathogens was negative, and affected monkeys did not respond to empiric therapy with antibiotics or antivirals. Second, the clinical presentation of pneumonia and hepatitis is consistent with the known spectrum of disease associated with adenoviral infections. Third, TMAdV sequence was recovered by PCR in various body fluids and tissues from affected monkeys, including blood, respiratory secretions, and lung/liver tissue (Table 1). Fourth, the finding of intranuclear inclusions in diseased tissues, as well as direct visualization of adenoviral-like particles (TMAdV) in lung alveoli by electron microscopy (FIGS. 1D-2 to 1D-4) support a role for TMAdV in the pathogenesis of tissue injury in affected monkeys. Finally, the most compelling data for TMAdV as the etiology of this outbreak is the marked neutralizing Ab response to the virus in 2 surviving animals, undetectable prior to the outbreak but >1:512 at convalescence (FIG. 6).
Although TMAdV retains the core genomic features common to all adenoviruses (FIG. 2C), phylogenetic analysis clearly places TMAdV within a separate group, with no closely related neighbors (FIG. 3). A phylogenetic distance of >10% combined with the lack of cross-neutralization defines TMAdV as a new species. Emerging human adenovirus strains such as hAd14 and hAdV-D53 are known to arise from recombination events among related ancestral strains (Kajon, A. E., et al., J Infect Dis 202, 93-103 (2010); Walsh, M. P., et al., PLoS ONE 4, e5635 (2009)). However, bootscanning analysis to look for such events in TMAdV was uninformative because closely related and/or ancestral strains to TMAdV have not yet been identified. Although the presence of an RGD motif in the TMAdV penton base suggests that the virus uses αv integrins for internalization (Wickham, T. J. et al., Cell 73, 309-319 (1993)), the high sequence divergence in the fiber protein (Table 2) implies that the fiber knob of TMAdV does not use human CAR (coxsackievirus-adenovirus receptor) as its primary receptor for attachment (Bergelson, J. M., et al., Science 275, 1320-1323 (1997)). This is significant because the attachment of the fiber knob to its receptor determines adenoviral cell tropism (Renaut, L. et al., Virology 321, 189-204 (2004)), and choice of receptor may define both the extent and virulence of disease caused by TMAdV. The TMAdV genome is approximately 35 kB with 35 open reading frames.
The virus grew efficiently in human A549 lung adenocarcinoma cells, and a fully adapted strain of TMAdV (after 10 passages in human A549 cells) exhibits an extended host range with the ability to infect all 3 cell lines, both monkey and human. This observation suggests that TMAdV possesses an inherent capacity to cross the species barrier and infect both humans and nonhuman primates.
The virulence of TMAdV in healthy, apparently immunocompetent titi monkeys (83% case fatality rate) is highly unusual for infections by adenovirus. In humans, deaths due to adenovirus infections or outbreaks are generally low (up to 18% for hAd14-associated pneumonia, see e.g., Lewis, P. F., et al., 14. J Infect Dis 199, 1427-1434 (2009)). Furthermore, severe infections from adenoviruses are closely associated with older age, immunosuppression, and chronic underlying conditions such as kidney failure (Lewis et al., supra; Carrigan, D. R., Am J Med 102, 71-74 (1997)). Young, healthy individuals are much less likely to succumb to adenoviral-related illness. The severity of TMAdV-related illness in affected titi monkeys indicates that this species of monkey may lack pre-existing immunity, and, thus, may not be a native host for the virus. The failure to detect asymptomatic fecal shedding of TMAdV (Table 1) also suggests that the virus does not normally circulate in titi monkeys.
Although the exact origin of TMAdV remains unclear, a cross-species “jump” from captive rhesus macaques to titi monkeys is the most likely precipitant for the outbreak. First, there have been no new introductions of monkeys into the closed colony for the past 6 years. Second, CNPRC personnel who visited macaque areas would occasionally enter titi rooms with no change in personal protective equipment (anecdotal evidence), thus providing a route of transmission for TMAdV. Third, neutralizing Abs to TMAdV were detected in rhesus macaques, but not in asymptomatic titi monkeys, within the same building (FIG. 6). Fourth, the virulence of TMAdV in titi monkeys makes them unlikely to be native hosts for the virus. Finally, the closest known phylogenetic relatives to TMAdV appear to be Old World monkey adenoviruses such as sAd3 and sAd18 (FIG. 4; Table 2). Serological evidence for cross-species adenoviral transmission events between different nonhuman primate species has also been reported in the literature (Mwenda, J. M. et al., East Afr Med J 82, 371-375 (2005)).
The decreased levels of neutralizing Abs to TMAdV in t two human cases relative to those in some affected titi monkeys (>1:512) may be secondary to adenovirus-specific T-cell responses in humans, which were found to be largely absent in nonhuman primates. Because 2.5% of random adult blood donors have neutralizing antibody to TMAdV, it can be implied that cross-species transmission of TMAdV may be ongoing in the human population. The newly discovered capacity of adenoviruses such as TMAdV to cross species barriers highlights the need to monitor adenoviruses closely for outbreak or even pandemic potential.
Described herein is the potential for zoonotic transmission of TMAdV. Provided are compositions and methods useful for the identification, isolation, expression, purification, detection, treatment, preventions, and modulation of TMAdV.
Definitions
Unless otherwise noted, the technical terms used herein are according to conventional usage as understood by persons skilled in the art. Definitions of common terms in molecular biology may be found in standard texts (e.g. Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd, 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8)).
The term “nucleic acid” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, and complements thereof. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide. A particular nucleotide sequence also implicitly encompasses “splice variants,” which as the name suggests, are products of alternative splicing of a gene. After transcription, an initial nucleic acid transcript can be spliced such that different (alternate) nucleic acid splice products encode different polypeptides. Mechanisms for the production of splice variants vary, but include alternate splicing of exons. Alternate polypeptides derived from the same nucleic acid by read-through transcription are also encompassed by this definition. Any products of a splicing reaction, including recombinant forms of the splice products, are included in this definition.
The term “isolated” as used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs, or RNAs, respectively that are present in the natural source of the macromolecule. Isolated is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state. The term isolated as used herein also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors, or other chemicals when chemically synthesized.
The term “identical” or “identity” or “percent identity,” or “sequence identity” in the context of two or more nucleic acids or polypeptide sequences that correspond to each other refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection. Such sequences are then said to be “substantially identical” and are embraced by the term “substantially identical.’ This definition also refers to, or can be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists for a specified entire sequence or a specified portion thereof or over a region of the sequence that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length. A corresponding region is any region within the reference sequence.
For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. A comparison window includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted (e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).
A preferred example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J Mol. Biol. 215:403-410 (1990), respectively. BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=−4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands.
“Titi Monkey Adenovirus” or “TMAdV” refers to both the genetic components of the virus, e.g., the genome and RNA transcripts thereof, proteins encoded by the genome (including structural and nonstructural proteins), and viral particles. The term “comprising a nucleic acid sequence” as it refers to TMAdV refers to nucleic acids and polypeptide polymorphic variants, alleles, mutants, and interspecies homologs that: (1) have a nucleotide sequence that has greater than about 60% nucleotide sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater nucleotide sequence identity, preferably over a region of over a region of at least about 25, 50, 100, 200, 500, 1000, or more nucleic acids, up to the full length sequence, to the nucleotide sequence of SEQ ID NO:1; (2) bind to antibodies, e.g., polyclonal or monoclonal antibodies, raised against an immunogen comprising an amino acid sequence of a protein encoded by an open reading frame (ORF) of SEQ ID NOs:2-37; and conservatively modified variants thereof; (3) specifically hybridize under stringent hybridization conditions to an anti-sense strand corresponding to a nucleic acid sequence of SEQ ID NO:1 and conservatively modified variants thereof; (4) encoding a protein having an amino acid sequence that has greater than about 60% nucleotide sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater nucleotide sequence identity, preferably over a region of over a region of at least about 25, 50, 100, 200, 500, 1000, or more amino acids, to a protein (e.g., a N, X, P, M, G, L protein) encoded by an open reading frame of SEQ ID NOs:2-37, and also those of FIG. 2.
“Protein encoded by TMAdV” or “protein encoded by the nucleotide sequence” comprising identity to a TMAdV open reading frame (ORF) refers to structural and non-structural Titi Monkey adenovirus proteins encoded by nucleic acids that: (1) have a nucleotide sequence that has greater than about 60% nucleotide sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater nucleotide sequence identity, preferably over a region of over a region of at least about 25, 50, 100, 200, 500, 1000, or more nucleic acids, up to the full length sequence, to the nucleotide sequence of SEQ ID NO:1; (2) bind to antibodies, e.g., polyclonal or monoclonal antibodies, raised against an immunogen comprising an amino acid sequence of a protein encoded by an open reading frame of SEQ ID NOs:2-37; and conservatively modified variants thereof; (3) specifically hybridize under stringent hybridization conditions to an anti-sense strand corresponding to a nucleic acid sequence of SEQ ID NOs:2-37; and (4) encoding a protein having an amino acid sequence that has greater than about 60% nucleotide sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater nucleotide sequence identity, preferably over a region of at least about 25, 50, 100, 200, 500, 1000 or more amino acids, to a protein encoded by an open reading frame of SEQ ID NOs:2-37. The amino acid sequence of the structural and non-structural viral proteins encoded by TMAdV can be easily identified by one of skill in the art, using the algorithms disclosed herein, by aligning the TMAdV sequence with other adenovirus sequences, including simian adenovirus 3 (SAdV-3), simian adenovirus 18 (SAdV-18), and the Group D human adenoviruses (e.g HAdV-9).
The term “open reading frame” or “ORF” refers to a length of DNA or RNA sequence capable of being translated into a peptide normally located between a start or initiation signal and a termination signal.
The term “expression vector” indicates a plasmid, a virus or another medium, known in the art, into which a nucleic acid sequence for encoding a desired protein can be inserted or introduced.
The term “host cell” is a cell that is susceptible to transformation, transfection, transduction, conjugation, and the like with a nucleic acid construct or expression vector. Host cells can be derived from plants, bacteria, yeast, fungi, insects, animals, etc.
The terms “polypeptide” or “peptide” or “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer. Macromolecular structures such as polypeptide structures can be described in terms of various levels of organization. For a general discussion of this organization (see, e.g., Alberts et al., Molecular Biology of the Cell (3rd ed., 1994) and Cantor and Schimmel, Biophysical Chemistry Part I: The Conformation of Biological Macromolecules (1980)). “Primary structure” refers to the amino acid sequence of a particular peptide. “Secondary structure” refers to locally ordered, three dimensional structures within a polypeptide. These structures are commonly known as domains, e.g., enzymatic domains, extracellular domains, transmembrane domains, pore domains, and cytoplasmic tail domains. Domains are portions of a polypeptide that form a compact unit of the polypeptide and are typically 15 to 350 amino acids long. Exemplary domains include domains with enzymatic activity. Typical domains are made up of sections of lesser organization such as stretches of 3-sheet and a-helices. “Tertiary structure” refers to the complete three dimensional structure of a polypeptide monomer. “Quaternary structure” refers to the three dimensional structure formed by the noncovalent association of independent tertiary units. Anisotropic terms are also known as energy terms.
The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code. Amino acids can be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, can be referred to by their commonly accepted single-letter codes. Amino acid substitutions, deletions or additions to individual or a small percentage of amino acids in the encoded sequence is a conservatively modified variant, where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention. The following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).
The term “antibody” refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen. The term “antigen” refers to any molecule capable of being bound by an antibody or a T cell receptor if presented by MHC molecules. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. Typically, the antigen-binding region of an antibody will be most critical in specificity and affinity of binding. An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kD) and one “heavy” chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VI) and variable heavy chain (VH) refer to these light and heavy chains respectively. Antibodies exist, e.g., as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)′2, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond. The F(ab)′2 can be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)′2 dimer into an Fab′ monomer. The Fab′ monomer is essentially Fab with part of the hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993)). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments can be synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries (see, e.g., McCafferty et al., Nature 348:552-554 (1990))
The phrase “specifically (or selectively) binds” to an antibody or “specifically (or selectively) immunoreactive with,” when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein, often in a heterogeneous population of proteins and other biologics. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times the background and more typically more than 10 to 100 times background. Specific binding to an antibody under such conditions requires an antibody that is selected for its specificity for a particular protein. For example, polyclonal antibodies that are raised to TMAdV, polymorphic variants, alleles, orthologs, and conservatively modified variants, or splice variants, or portions thereof, can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with TMAdV and not with other proteins. This selection can be achieved by subtracting out antibodies that cross-react with other molecules. A variety of immunoassay formats can be used to select antibodies specifically immunoreactive with a particular protein, as described herein.
The term “detectable moiety” or “conjugate” refers to any atom, molecule or a portion thereof, the presence, absence or level of which is directly or indirectly monitorable. A variety of detectable moieties are well known to those skilled in the art, and can be any material detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Such detectable labels can include, but are not limited to, magnetic beads, fluorescent dyes, radiolabels, enzymes, and colorimetric labels such as colloidal gold or colored glass or plastic beads, each of which is described in greater detail herein.
The term “vaccine” refers to a pharmaceutical composition comprising at least one immunologically active component that induces an immunological response in an animal and possibly but not necessarily one or more additional components that enhance the immunological activity of the active component. A vaccine can additionally comprise further components typical to pharmaceutical compositions. The immunologically active component of a vaccine can comprise complete virus particles in either their original form or as attenuated particles in a so called modified live vaccine (MLV) or particles inactivated by appropriate methods in a so called killed vaccine (KV). A vaccine comprising antigenic substances can be administered for the purpose of inducing a specific and active immunity against a disease provoked by a TMAdV infection. A vaccine can also provide passive immunity in the form of antibodies previously generated against TMAdV antigens.
The term “immune response” refers to a reaction of the immune system to an antigen in the body of a host, which includes generation of an antigen-specific antibody and/or cellular cytotoxic response. The term further refers to an immune system response that leads to a condition of induce sensitivity to an immunogenic product.
A “biological sample” or “sample” includes sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histologic purposes. Such samples include blood and blood fractions or products (e.g., serum, plasma, platelets, red blood cells, and the like), sputum, cloacal swabs, mucosa, tissue, cultured cells, e.g., primary cultures, explants, and transformed cells, biological fluids, stool, urine, etc. A biological sample is typically obtained from a eukaryotic organism. The tissue sampled can be, for instance, skin, brain (e.g., cerebrum, cerebellum, optic lobe), spinal cord, adrenals, pectoral muscle, lung, heart, liver, crop, proventriculus, ventriculus, duodenum, small intestine, large intestine, cloaca, kidney, bursa of fabricus, spleen, pancreas, adrenal gland, bone marrow, lumbosacral spinal cord, or blood. Contacting a sample refers to the plain and ordinary meaning to refer to exposing the sample.
The term “detecting” when in reference to detecting the presence of TMAdV refers to determining the presence, using any method, of the virus or viral particles including viral peptides, inside cells, on cells, and/or in medium with which cells or the virus have come into contact. The methods are exemplified by, but not limited to, the observation of cytopathic effect, detection of viral protein, such as by immunofluorescence, ELISA, or Western blot hybridization, detection of viral nucleic acid sequence, such as by PCR, RT-PCR, Southern blots, and Northern blots, nucleic acid hybridization, nucleic acid arrays, and the like.
The phrase “TMAdV infection” refers to the invasion by, multiplication and/or presence of TMAdV in a cell or a subject with or without symptoms.
The phrase “functional effect” in the context of assays for testing compounds that modulate activity of TMAdV, or for treating or preventing TMAdV infection, includes the determination of a parameter that is indirectly or directly under the influence of TMAdV, e.g., a phenotypic or chemical effect, such as the ability to increase or decrease viral genome replication, viral RNA and protein production, virus packaging, viral particle production (particularly replication competent viral particle production), cell receptor binding, viral transduction, cellular infection, antibody binding, inducing a cellular or humoral immune response, viral protein enzymatic activity, etc. “Functional effects” include in vitro, in vivo, and ex vivo activities. Such functional effects can be measured by any means known to those skilled in the art, e.g., changes in spectroscopic characteristics (e.g., fluorescence, absorbance, refractive index); hydrodynamic (e.g., shape); chromatographic; or solubility properties for a protein; measuring inducible markers or transcriptional activation of a protein; measuring binding activity or binding assays, e.g. binding to antibodies; measuring changes in ligand or substrate binding activity; measuring viral replication; measuring cell surface marker expression; measurement of changes in protein levels; measurement of RNA stability; identification of downstream or reporter gene expression (CAT, luciferase, 0-gal, GFP and the like), e.g., via chemiluminescence, fluorescence, colorimetric reactions, antibody binding, and inducible markers.
The term “test compound” or “compound” or “drug candidate” or “modulator” or grammatical equivalents as used herein describes any molecule, either naturally occurring or synthetic, e.g., protein, oligopeptide (e.g., from about 5 to about 25 amino acids in length, preferably from about 10 to 20 or 12 to 18 amino acids in length, preferably 12, 15, or 18 amino acids in length), small organic molecule, polysaccharide, lipid, fatty acid, polynucleotide, oligonucleotide, etc., to be tested for the capacity to directly or indirectly modulation tumor cell proliferation. The test compound can be in the form of a library of test compounds, such as a combinatorial or randomized library that provides a sufficient range of diversity. Test compounds are optionally linked to a fusion partner, e.g., targeting compounds, rescue compounds, dimerization compounds, stabilizing compounds, addressable compounds, and other functional moieties. Conventionally, new chemical entities with useful properties are generated by identifying a test compound (called a “lead compound”) with some desirable property or activity, e.g., inhibiting activity, creating variants of the lead compound, and evaluating the property and activity of those variant compounds. Often, high throughput screening (HTS) methods are employed for such an analysis. Compounds can be inhibitors, activators, or modulators of TMAdV nucleic acid and polypeptide sequences, and are used to refer to activating, inhibitory, or modulating molecules identified using in vitro and in vivo assays of the TMAdV nucleic acid and polypeptide sequences. Inhibitors are compounds that, e.g., bind to, partially or totally block activity, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity or expression of TMAdV, e.g., antagonists. Activators are compounds that increase, open, activate, facilitate, enhance activation, sensitize, agonize, or up regulate TMAdV activity, e.g., agonists. Inhibitors, activators, or modulators also include genetically modified versions of TMAdV, e.g., versions with altered activity, as well as naturally occurring and synthetic ligands, substrates, antagonists, agonists, antibodies, peptides, cyclic peptides, nucleic acids, antisense molecules, ribozymes, or small chemical molecules for example.
The phrase “small organic molecule” refers to an organic molecule, either naturally occurring or synthetic, that has a molecular weight of more than about 50 daltons and less than about 2500 daltons, preferably less than about 2000 daltons, preferably between about 100 to about 1000 daltons, more preferably between about 200 to about 500 daltons.
The term “aptamer” refers to a non-naturally occurring nucleic acid having a desirable action on a target. A desirable action includes, but is not limited to, binding of the target, catalytically changing the target, reacting with the target in a way which modifies/alters the target or the functional activity of the target, covalently attaching to the target as in a suicide inhibitor, facilitating the reaction between the target and another molecule. Aptamer action can be specific binding affinity for a target molecule, such target molecule being a three dimensional chemical structure other than a polynucleotide that binds to the nucleic acid ligand through a mechanism which predominantly depends on Watson/Crick base pairing or triple helix binding, wherein the nucleic acid ligand is not a nucleic acid having the known physiological function of being bound by the target molecule.
An “siRNA” molecule or an “RNAi” molecule refers to a nucleic acid that forms a double stranded RNA, which double stranded RNA has the ability to reduce or inhibit expression of a gene or target gene when the siRNA expressed in the same cell as the gene or target gene. “siRNA” thus refers to the double stranded RNA formed by the complementary strands. The complementary portions of the siRNA that hybridize to form the double stranded molecule typically have substantial or complete identity. In one embodiment, an siRNA refers to a nucleic acid that has substantial or complete identity to a target gene and forms a double stranded siRNA. The sequence of the siRNA can correspond to the full length target gene, or a subsequence thereof. Typically, the siRNA is at least about 15-50 nucleotides in length (e.g., each complementary sequence of the double stranded siRNA is 15-50 nucleotides in length, and the double stranded siRNA is about 15-50 base pairs in length, preferable about preferably about 20-30 base nucleotides, preferably about 20-25 or about 24-29 nucleotides in length, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. See also PCT/US03/07237, herein incorporated by reference in its entirety.
The term “antisense” refers to an oligomeric compound or molecule that is at least partially complementary to a target nucleic acid molecule to which it hybridizes. Antisense compounds or molecules can include, but are not limited to, oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics, and chimeric combination.
An siRNA or antisense molecule or RNAi molecule is “specific” for a target nucleic acid if it reduces expression of the nucleic acid by at least about 10% when the siRNA or RNAi is expressed in a cell that expresses the target nucleic acid.
The term “treating” or “treatment” includes the application or administration of a composition to a subject, or application or administration of a composition to a cell or tissue from a subject who has been infected with TMAdV, or has symptoms of TMAdV infection, with the purpose of curing, healing, alleviating, relieving, altering, remedying, ameliorating, improving, or affecting the disease or condition, the symptom of the disease or condition, or the risk of the disease or condition.
The term “preventing” or “prevention” includes stopping or hindering a disease, disorder, or symptoms associated with TMAdV infection.
The term “subject” refers to any animal, including, but not limited to, humans, Titi Monkeys, and other non-human primates, that presents one or more symptoms indicative of TMAdV infection.
The term “administering” or “administration” refers to therapeutically or prophylactically administering an effective amount of a composition or medicament during the course of therapy. Prophylactic administration can occur prior to manifestation of symptoms characteristic of a TMAdV infection.
The phrase “therapeutically effective dose” herein is meant a dose that produces effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); and Pickar, Dosage Calculations (1999)).
The phrase “stringent conditions” refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acids, but to no other sequences. The term “hybridize” refers to the process by which single strands of nucleic acid sequences form double-helical segments through hydrogen bonding between complementary nucleotides. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Probes, “Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5-10° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The T. is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 times background hybridization. Exemplary stringent hybridization conditions can be as following: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions. Exemplary “moderately stringent hybridization conditions” include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 1×SSC at 45° C. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency. Additional guidelines for determining hybridization parameters are provided in numerous reference (e.g., and Current Protocols in Molecular Biology, ed. Ausubel, et al.).
Isolation, Expression, Purification, and Detection of TMAdV
The subject matter described herein relies on routine techniques in the field of recombinant genetics. Recombinant when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.
Basic texts disclose general methods of use in this invention (e.g. Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994)).
TMAdV Expression
To obtain high level expression of a cloned gene or genome, one typically subclones the nucleic acid into an expression vector that contains a strong promoter to direct transcription, a transcription/translation terminator, and if for a nucleic acid encoding a protein, a ribosome binding site for translational initiation. Suitable bacterial promoters are well known in the art and described (e.g., in Sambrook et al., and Ausubel et al, supra. Bacterial expression systems for expressing the protein are available in, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al., Gene 22:229-235 (1983); Mosbach et al., Nature 302:543-545 (1983)). Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available. Retroviral expression systems can be used in the present invention.
Selection of the promoter used to direct expression of a heterologous nucleic acid depends on the particular application. The promoter is preferably positioned about the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function. Heterologous refers to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).
In addition to the promoter, the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the nucleic acid in host cells. A typical expression cassette thus contains a promoter operably linked to the nucleic acid sequence encoding the nucleic acid of choice and signals required for efficient polyadenylation of the transcript, ribosome binding sites, and translation termination. Additional elements of the cassette can include enhancers and, if genomic DNA is used as the structural gene, introns with functional splice donor and acceptor sites.
In addition to a promoter sequence, the expression cassette should also contain a transcription termination region downstream of the structural gene to provide for efficient termination. The termination region can be obtained from the same gene as the promoter sequence or can be obtained from different genes.
The particular expression vector used to transport the genetic information into the cell is not particularly critical. Any of the conventional vectors used for expression in eukaryotic or prokaryotic cells can be used. Standard bacterial expression vectors include plasmids such as pBR322 based plasmids, pSKF, pET23D, and fusion expression systems such as MBP, GST, and LacZ. Epitope tags can also be added to recombinant proteins to provide convenient methods of isolation, e.g., c-myc. Sequence tags can be included in an expression cassette for nucleic acid rescue. Markers such as fluorescent proteins, green or red fluorescent protein, 13-gal, CAT, and the like can be included in the vectors as markers for vector transduction.
Expression vectors containing regulatory elements from eukaryotic viruses are typically used in eukaryotic expression vectors, e.g., SV40 vectors, papilloma virus vectors, retroviral vectors, and vectors derived from Epstein-Barr virus. Other exemplary eukaryotic vectors include pMSG, pAV009/A+, pMT010/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the CMV promoter, SV40 early promoter, SV40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
Expression of proteins from eukaryotic vectors can also be regulated using inducible promoters. With inducible promoters, expression levels are tied to the concentration of inducing agents, such as tetracycline, by the incorporation of response elements for these agents into the promoter. Generally, high level expression is obtained from inducible promoters only in the presence of the inducing agent; basal expression levels are minimal.
Vectors can have a regulatable promoter, e.g., tet-regulated systems and the RU-486 system (see, e.g., Gossen & Bujard, PNAS 89:5547 (1992); Oligino et al., Gene Ther. 5:491-496 (1998); Wang et al., Gene Ther. 4:432-441 (1997); Neering et al., Blood 88:1147-1155 (1996); and Rendahl et al., Nat. Biotechnol. 16:757-761 (1998)). These impart small molecule control on the expression of the candidate target nucleic acids. This beneficial feature can be used to determine that a desired phenotype is caused by a transfected cDNA rather than a somatic mutation.
Some expression systems have markers that provide gene amplification such as thymidine kinase and dihydrofolate reductase. Alternatively, high yield expression systems not involving gene amplification are also suitable, such as using a baculovirus vector in insect cells, with a sequence of choice under the direction of the polyhedrin promoter or other strong baculovirus promoters.
The elements that are typically included in expression vectors also include a replicon that functions in E. coli, a gene encoding antibiotic resistance to permit selection of bacteria that harbor recombinant plasmids, and unique restriction sites in nonessential regions of the plasmid to allow insertion of eukaryotic sequences. The particular antibiotic resistance gene chosen is not critical, as any of the many resistance genes known in the art are suitable. The prokaryotic sequences are preferably chosen such that they do not interfere with the replication of the DNA in eukaryotic cells, if necessary.
Standard transfection methods are used to produce bacterial, mammalian, yeast or insect cell lines that express large quantities of protein, which are then purified using standard techniques (see, e.g., Colley et al., J. Biol. Chem. 264:17619-17622 (1989); Guide to Protein Purification, in Methods in Enzymology, vol. 182 (Deutscher, ed., 1990)). Transformation of eukaryotic and prokaryotic cells are performed according to standard techniques (see, e.g., Morrison, J Bact. 132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology 101:347-362 (Wu et al., eds, 1983)).
Any of the well-known procedures for introducing foreign nucleotide sequences into host cells can be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, biolistics, liposomes, microinjection, plasma vectors, viral vectors and any of the other well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Sambrook et al., supra). It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one gene into the host cell capable of expressing TMAdV proteins and nucleic acids.
After the expression vector is introduced into the cells, the transfected cells are cultured under conditions favoring expression of the protein of choice, which is recovered from the culture using standard techniques identified below.
Either naturally occurring or recombinant TMAdV proteins can be purified for use in diagnostic assays, for making antibodies (for diagnosis and therapy) and vaccines, and for assaying for anti-viral compounds. Naturally occurring protein can be purified, e.g., from primate tissue samples. Recombinant protein can be purified from any suitable expression system.
TMAdV Proteins
The protein can be purified to substantial purity by standard techniques, including selective precipitation with such substances as ammonium sulfate; column chromatography, immunopurification methods, and others (see, e.g., Scopes, Protein Purification: Principles and Practice (1982); U.S. Pat. No. 4,673,641; Ausubel et al., supra; and Sambrook et al., supra).
A number of procedures can be employed when recombinant protein is being purified. For example, proteins having established molecular adhesion properties can be reversible fused to the protein. With the appropriate ligand or substrate, a specific protein can be selectively adsorbed to a purification column and then freed from the column in a relatively pure form. The fused protein is then removed by enzymatic activity. Finally, protein could be purified using immunoaffinity columns. Recombinant protein can be purified from any suitable source, include yeast, insect, bacterial, and mammalian cells.
Recombinant proteins can be expressed and purified by transformed bacteria in large amounts, typically after promoter induction; but expression can be constitutive. Promoter induction with IPTG is one example of an inducible promoter system. Bacteria are grown according to standard procedures in the art. Fresh or frozen bacteria cells are used for isolation of protein.
Proteins expressed in bacteria can form insoluble aggregates (“inclusion bodies”). Several protocols are suitable for purification of protein inclusion bodies. For example, purification of inclusion bodies typically involves the extraction, separation and/or purification of inclusion bodies by disruption of bacterial cells, e.g., by incubation in a buffer of 50 mM TRIS/HCL pH 7.5, 50 mM NaCl, 5 mM MgCl2, 1 mM DTT, 0.1 mM ATP, and 1 mM PMSF. The cell suspension can be lysed using 2-3 passages through a French Press, homogenized using a Polytron (Brinkman Instruments) or sonicated on ice. Alternate methods of lysing bacteria are apparent to those of skill in the art (see, e.g., Sambrook et al., supra; Ausubel et al., supra).
If necessary, the inclusion bodies are solubilized, and the lysed cell suspension is typically centrifuged to remove unwanted insoluble matter. Proteins that formed the inclusion bodies can be renatured by dilution or dialysis with a compatible buffer. Suitable solvents include, but are not limited to urea (from about 4 M to about 8 M), formamide (at least about 80%, volume/volume basis), and guanidine hydrochloride (from about 4 M to about 8 M). Some solvents which are capable of solubilizing aggregate-forming proteins, for example SDS (sodium dodecyl sulfate), 70% formic acid, are inappropriate for use in this procedure due to the possibility of irreversible denaturation of the proteins, accompanied by a lack of immunogenicity and/or activity. Although guanidine hydrochloride and similar agents are denaturants, this denaturation is not irreversible and renaturation can occur upon removal (by dialysis, for example) or dilution of the denaturant, allowing re-formation of immunologically and/or biologically active protein. Other suitable buffers are known to those skilled in the art. Human proteins are separated from other bacterial proteins by standard separation techniques, e.g., with Ni-NTA agarose resin.
Alternatively, it is possible to purify recombinant protein from bacteria periplasm. After lysis of the bacteria, the periplasmic fraction of the bacteria can be isolated by cold osmotic shock in addition to other methods known to skill in the art. To isolate recombinant proteins from the periplasm, the bacterial cells are centrifuged to form a pellet. The pellet is resuspended in a buffer containing 20% sucrose. To lyse the cells, the bacteria are centrifuged and the pellet is resuspended in ice-cold 5 mM MgSO4 and kept in an ice bath for approximately 10 minutes. The cell suspension is centrifuged and the supernatant decanted and saved. The recombinant proteins present in the supernatant can be separated from the host proteins by standard separation techniques well known to those of skill in the art.
Solubility fractionation can be used as a standard protein separation technique for purifying proteins. As an initial step, particularly if the protein mixture is complex, an initial salt fractionation can separate many of the unwanted host cell proteins (or proteins derived from the cell culture media) from the recombinant protein of interest. The preferred salt is ammonium sulfate Ammonium sulfate precipitates proteins by effectively reducing the amount of water in the protein mixture. Proteins then precipitate on the basis of their solubility. The more hydrophobic a protein is, the more likely it is to precipitate at lower ammonium sulfate concentrations. A typical protocol includes adding saturated ammonium sulfate to a protein solution so that the resultant ammonium sulfate concentration is between 20-30%. This concentration will precipitate the most hydrophobic of proteins. The precipitate is then discarded (unless the protein of interest is hydrophobic) and ammonium sulfate is added to the supernatant to a concentration known to precipitate the protein of interest. The precipitate is then solubilized in buffer and the excess salt removed if necessary, either through dialysis or diafiltration. Other methods that rely on solubility of proteins, such as cold ethanol precipitation, are well known to those of skill in the art and can be used to fractionate complex protein mixtures.
The molecular weight of the protein can be used to isolate it from proteins of greater and lesser size using ultrafiltration through membranes of different pore size (for example, Amicon or Millipore membranes). As a first step, the protein mixture is ultrafiltered through a membrane with a pore size that has a lower molecular weight cut-off than the molecular weight of the protein of interest. The retentate of the ultrafiltration is then ultrafiltered against a membrane with a molecular cut off greater than the molecular weight of the protein of interest. The recombinant protein will pass through the membrane into the filtrate. The filtrate can then be chromatographed as described below.
The protein can also be separated from other proteins on the basis of its size, net surface charge, hydrophobicity, and affinity for ligands or substrates using column chromatography. In addition, antibodies raised against proteins can be conjugated to column matrices and the proteins immunopurified. All of these methods are well known in the art. It will be apparent to one of skill that chromatographic techniques can be performed at any scale and using equipment from many different manufacturers (e.g., Pharmacia Biotech).
Detecting the Presence or Absence of TMAdV
Described herein are diagnostic assays to detect TMAdV, TMAdV nucleic acids (genome and genes), TMAdV antibodies in an infected subject, and TMAdV proteins.
Detecting TMAdV Nucleic Acids
TMAdV infection can be detected based on the level of a TMAdV RNA or DNA in a biological sample. Primers from TMAdV can be used for detection of TMAdV, diagnosis, and determination of TMAdV viral load. Any suitable primer can be used to detect the genome, nucleic acid sub sequence, ORF, or protein of choice, using, e.g., methods described in US 20030104009. For example, the subject nucleic acid compositions can be used as single- or double-stranded probes or primers for the detection of TMAdV mRNA or cDNA generated from such mRNA, as obtained can be present in a biological sample (e.g., extracts of human cells). The TMAdV polynucleotides of the invention can also be used to generate additional copies of the polynucleotides, to generate antisense oligonucleotides, and as triple-strand forming oligonucleotides. For example, two oligonucleotide primers can be employed in a polymerase chain reaction (PCR) based assay to amplify a portion of TMAdV cDNA derived from a biological sample, wherein at least one of the oligonucleotide primers is specific for (i.e., hybridizes to) the TMAdV polynucleotide. The primers are preferably at least or about 12, 15, 16, 18, 20, 22, 24, 25, 30, 35, 40, 45, or 50 nt or are, for instance, from about 12 to 50 nt in length, 15 to 30 nt in length, 15 to 25 nt in length, or 20 to 30 nt in length) fragments of a contiguous sequence of SEQ ID NO: 1 or other polynucleotide sequence encoding an TMAdV nucleic acid or polypeptide. The amplified cDNA is then separated and detected using techniques well known in the art, such as gel electrophoresis. Similarly, oligonucleotide probes that specifically hybridize to a TMAdV polynucleotide can be used in a hybridization assay to detect the presence of the TMAdV polynucleotide in a biological sample.
For PCR, a temperature of about 36° C. is typical for low stringency amplification, although annealing temperatures may vary between about 32° C. and 48° C. depending on primer length. For high stringency PCR amplification, a temperature of about 62° C. is typical, although high stringency annealing temperatures can range from about 50° C. to about 65° C., depending on the primer length and specificity. Typical cycle conditions for both high and low stringency amplifications include a denaturation phase of 90° C.-95° C. for 30 sec-2 min., an annealing phase lasting 30 sec.-2 min., and an extension phase of about 72° C. for 1-2 min. Protocols and guidelines for low and high stringency amplification reactions are provided (e.g., in Innis et al. (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y.)).
Nucleic acid probes or primers specific to TMAdV can be generated using the polynucleotide sequences disclosed herein. The probes are preferably at least about 12, 15, 16, 18, 20, 22, 24, or 25 nt fragments of a contiguous sequence of SEQ ID NO: 1 or other polynucleotide sequence encoding a TMAdV nucleic acid or polypeptide. Nucleic acid probes can be less than about 200 bp, 150 bp, 100 bp, 75 bp, 50 bp, 60 bp, 40 bp, 30 bp, 25 by 2 kb, 1.5 kb, 1 kb, 0.5 kb, 0.25 kb, 0.1 kb, or 0.05 kb in length. The probes can be produced by, for example, chemical synthesis, PCR amplification, generation from longer polynucleotides using restriction enzymes, or other methods well known in the art. Preferred primers and probes are identical to a TMAdV nucleic acid sequence and different from a non-TMAdV sequence.
The polynucleotides described herein, particularly where used as a probe in a diagnostic assay, can be detectably labeled. Exemplary detectable labels include, but are not limited to, radiolabels, fluorochromes, (e.g. fluorescein isothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin, 6-carboxyfluorescein (6-FAM), 2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein, 6-carboxy-X-rhodamine (ROX), 6-carboxy-2′,4′,T,4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein (5-FAM) or N,N,N′,N′-tetramethyl-6-carboxyrho-damine (TAMRA)), radioactive labels, (e.g. sup.32p, .sup.35S, and sup.3H), and the like. The detectable label can involve two stage systems (e.g., biotin-avidin, hapten-anti-hapten antibody, and the like).
Non-PCR-based, sequence specific DNA amplification techniques can also be used with the invention to detect TMAdV sequences. An example of such techniques include, but is not necessarily limited to, the Invader assay (see, e.g., Kwiatkowski et al. Mol Diagn. December 1999, 4:353-64. See also U.S. Pat. No. 5,846,717).
The claimed subject matter can also include solid substrates, such as arrays, comprising any of the polynucleotides described herein. The polynucleotides are immobilized on the arrays using methods known in the art. An array can have one or more different polynucleotides.
Any suitable qualitative or quantitative methods known in the art for detecting specific TMAdV nucleic acid (e.g., RNA or DNA) can be used. TMAdV nucleic acid can be detected by, for example, in situ hybridization in tissue sections, using methods that detect single base pair differences between hybridizing nucleic acid (e.g., using the Invader® technology described in, for example, U.S. Pat. No. 5,846,717), by reverse transcriptase-PCR, or in Northern blots containing poly A+mRNA, and other methods well known in the art. For detection of TMAdV polynucleotides in blood or blood-derived samples, the use of methods that allow for detection of single base pair mismatches is preferred.
Using the TMAdV nucleic acid as a basis, nucleic acid probes (e.g., including oligomers of at least about 8 nucleotides or more) can be prepared, either by excision from recombinant polynucleotides or synthetically, which probes hybridize with the TMAdV nucleic acid, and thus are useful in detection of TMAdV virus in a sample, and identification of infected individuals, as well as further characterization of the viral genome(s). The probes for TMAdV polynucleotides (natural or derived) are of a length or have a sequence which allows the detection of unique viral sequences by hybridization. While about 6-8 nucleotides may be useful, longer sequences may be preferred, e.g., sequences of about 10-12 nucleotides, or about 20 nucleotides or more. Preferably, these sequences will derive from regions which lack heterogeneity among TMAdV viral isolates.
Nucleic acid probes can be prepared using routine methods, including automated oligonucleotide synthetic methods. A complement to any unique portion of the TMAdV genome will be satisfactory, e.g., a portion of the TMAdV genome that allows for distinguishing TMAdV from other viruses that may be present in the sample, e.g., other TMAdV such as B19. For use as probes, complete complementarity is desirable, though it can be unnecessary as the length of the fragment is increased.
For use of such probes as diagnostics, the biological sample to be analyzed, such as blood or serum, can be treated, if desired, to extract the nucleic acids contained therein. The resulting nucleic acid from the sample can be subjected to gel electrophoresis or other size separation techniques; alternatively, the nucleic acid sample can be dot blotted without size separation. The probes are usually labeled with a detectable label. Suitable labels, and methods for labeling probes are known in the art, can include, for example, radioactive labels incorporated by nick translation or kinasing, biotin, fluorescent probes, and chemiluminescent probes. The nucleic acids extracted from the sample are then treated with the labeled probe under hybridization conditions of suitable stringencies.
The probes can be made completely complementary to the TMAdV genome or portion thereof (e.g., to all or a portion of a sequence encoding a TMAdV GAG polypeptide). Therefore, usually high stringency conditions are desirable in order to prevent or at least minimize false positives. However, conditions of high stringency should only be used if the probes are complementary to regions of the viral genome which lack heterogeneity among TMAdV viral isolates. The stringency of hybridization is determined by a number of factors during hybridization and during the washing procedure, including temperature, ionic strength, length of time, and concentration of formamide (Sambrook et al. (1989), “Molecular Cloning; A Laboratory Manual,” Second Edition (Cold Spring Harbor Press, Cold Spring Harbor, N.Y.)).
Generally, it is expected that the TMAdV sequences will be present in a biological sample (e.g., blood, cells, and the liked) obtained from an infected individual at relatively low levels, e.g., at approximately 102-104 TMAdV sequences per 106 cells. This level can require that amplification techniques be used in hybridization assays. Such techniques are known in the art.
For example, the Enzo Biochemical Corporation “Bio-Bridge” system uses terminal deoxynucleotide transferase to add unmodified 3′-poly-dT-tails to a DNA probe. The poly dT-tailed probe is hybridized to the target nucleotide sequence, and then to a biotin-modified poly-A. PCT Publication No. WO84/03520 and European application no. EPA124221 describe a DNA hybridization assay in which: (1) analyte is annealed to a single-stranded DNA probe that is complementary to an enzyme-labeled oligonucleotide; and (2) the resulting tailed duplex is hybridized to an enzyme-labeled oligonucleotide. EPA 204510 describes a DNA hybridization assay in which analyte DNA is contacted with a probe that has a tail, such as a poly-dT tail, an amplifier strand that has a sequence that hybridizes to the tail of the probe, such as a poly-A sequence, and which is capable of binding a plurality of labeled strands.
A particularly desirable technique can first involve amplification of the target TMAdV sequences in sera approximately 10,000 fold, e.g., to approximately 10 sequences/mL. This can be accomplished, for example, by the polymerase chain reactions (PCR) technique (Saiki et al. (1986), by Mullis, U.S. Pat. No. 4,683,195, and by Mullis et al. U.S. Pat. No. 4,683,202). Other amplification methods are well known in the art.
The probes, or alternatively nucleic acid from the samples, can be provided in solution for such assays, or can be affixed to a support (e.g., solid or semi-solid support). Examples of supports that can be used are nitrocellulose (e.g., in membrane or microtiter well form), polyvinyl chloride (e.g., in sheets or microtiter wells), polystyrene latex (e.g., in beads or microtiter plates, polyvinylidine fluoride, diazotized paper, nylon membranes, activated beads, and Protein A beads.
Probes (or sample nucleic acid) can be provided on an array for detection. Arrays can be created by, for example, spotting polynucleotide probes onto a substrate (e.g., glass, nitrocellulose, and the like) in a two-dimensional matrix or array. The probes can be bound to the substrate by either covalent bonds or by non-specific interactions, such as hydrophobic interactions. Samples of polynucleotides can be detectably labeled (e.g., using radioactive or fluorescent labels) and then hybridized to the probes. Double stranded polynucleotides, comprising the labeled sample polynucleotides bound to probe polynucleotides, can be detected once the unbound portion of the sample is washed away. Techniques for constructing arrays and methods of using these arrays are described in EP 799 897; WO 97/29212; WO 97/27317; EP 785 280; WO 97/02357; U.S. Pat. No. 5,593,839; U.S. Pat. No. 5,578,832; EP 728 520; U.S. Pat. No. 5,599,695; EP 721 016; U.S. Pat. No. 5,556,752; WO 95/22058; and U.S. Pat. No. 5,631,734. Arrays are particularly useful where, for example a single sample is to be analyzed for the presence of two or more nucleic acid target regions, as the probes for each of the target regions, as well as controls (both positive and negative) can be provided on a single array. Arrays thus facilitate rapid and convenience analysis.
TMAdV Antibodies
Antibodies raised against TMAdV can serve a wide variety of purposes, as described herein, which include, but are not limited to, diagnostic assays for the detection of TMAdV. A number of immunogens comprising portions of a TMAdV protein, virus or nucleic acid can be used to produce antibodies specifically reactive with the TMAdV. For example, a recombinant TMAdV protein or an antigenic fragment thereof, can be isolated as described herein. Recombinant protein can be expressed in eukaryotic or prokaryotic cells as described above, and purified as generally described above. Recombinant protein is the preferred immunogen for the production of monoclonal or polyclonal antibodies. Alternatively, a synthetic peptide derived from the sequences disclosed herein and conjugated to a carrier protein can be used an immunogen. Naturally occurring protein can also be used either in pure or impure form. The product is then injected into an animal capable of producing antibodies. Either monoclonal or polyclonal antibodies can be generated, for subsequent use in immunoassays to measure the protein.
For preparation of antibodies, e.g., recombinant, monoclonal, or polyclonal antibodies, many technique known in the art can be used (see, e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985); Coligan, Current Protocols in Immunology (1991); Harlow & Lane, Antibodies, A Laboratory Manual (1988); and Goding, Monoclonal Antibodies: Principles and Practice (2d ed. 1986)).
Methods of production of polyclonal antibodies are known to those of skill in the art. An inbred strain of mice (e.g., BALB/C mice) or rabbits is immunized with the protein using a standard adjuvant, such as Freund's adjuvant, and a standard immunization protocol. The animal's immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to the beta subunits. When appropriately high titers of antibody to the immunogen are obtained, blood is collected from the animal and antisera are prepared. Further fractionation of the antisera to enrich for antibodies reactive to the protein can be done if desired (see, Harlow & Lane, supra).
Monoclonal antibodies can be obtained by various techniques familiar to those skilled in the art. Briefly, spleen cells from an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma cell (see, Kohler & Milstein, Eur. J. Immunol. 6:511-519 (1976)). Alternative methods of immortalization include transformation with Epstein Barr Virus, oncogenes, or retroviruses, or other methods well known in the art. Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells can be enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host. Alternatively, one can isolate DNA sequences which encode a monoclonal antibody or a binding fragment thereof by screening a DNA library from human B cells according to the general protocol outlined by Huse, et al., Science 246:1275-1281 (1989).
Monoclonal antibodies and polyclonal sera are collected and titered against the immunogen protein in an immunoassay, for example, a solid phase immunoassay with the immunogen immobilized on a solid support. Typically, polyclonal antisera with a titer of 104 or greater are selected and tested for their cross reactivity against non-TMAdV proteins and nucleic acids, using a competitive binding immunoassay. Specific polyclonal antisera and monoclonal antibodies will usually bind with a Kd of at least about 0.1 mM, more usually at least about 1 uM, preferably at least about 0.1 uM or better, and most preferably, 0.01 uM or better. Antibodies specific only for a particular TMAdV protein can also be made by subtracting out other cross-reacting proteins. In this manner, antibodies that bind only to the protein of choice can be obtained.
Phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens (see, e.g., McCafferty et al., Nature 348:552-554 (1990); Marks et al., Biotechnology 10:779-783 (1992)). Antibodies can also be made bispecific, i.e., able to recognize two different antigens (see, e.g., WO 93/08829, Traunecker et al., EMBO J. 10:3655-3659 (1991); and Suresh et al., Methods in Enzymology 121:210 (1986)). Antibodies can also be heteroconjugates, e.g., two covalently joined antibodies, or immunotoxins (see, e.g., U.S. Pat. No. 4,676,980, WO 91/00360; WO 92/200373; and EP 03089).
Chimeric antibodies can be used, which is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.
Humanized or primatized antibodies can be used. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Methods for humanizing or primatizing non-human antibodies are well known in the art. Humanization can be essentially performed following the method of Winter and co-workers (see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988) and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
Once the specific antibodies against a TMAdV protein, virus or nucleic acid in are available, the antigen can be detected and/or quantified using any of a number of well recognized immunological binding assays (see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168). TMAdV viral particles can be detected based on an epitope defined by the viral proteins as presented in a viral particle and/or an epitope defined by a viral protein that is separate from a viral particle. As used in this context, then, “antigen” is meant to refer to a TMAdV polypeptide as well as TMAdV viral particles. For a review of the general immunoassays, see also Methods in Cell Biology: Antibodies in Cell Biology, volume 37 (Asai, ed. 1993); Basic and Clinical Immunology (Stites & Terr, eds., 7th ed. 1991). Immunological binding assays (or immunoassays) typically use an antibody that specifically binds to a protein or antigen of choice. The antibody can be produced by any of a number of means well known to those of skill in the art and as described above. Immunoassays for detecting TMAdV protein, virus and nucleic acid in samples can be either competitive or noncompetitive, and can be either quantitative or non-quantitative.
Immunoassays
Noncompetitive immunoassays are assays in which antigen is directly detected and, in some instances the amount of antigen directly measured. Enzyme mediated immunoassays such as immunofluorescence assays (IFA), enzyme linked immunosorbent assays (ELISA), immunoblotting (western), and capture assays can be readily adapted to accomplish the noncompetitive detection of the TMAdV proteins.
An ELISA method effective for the detection of the virus can, for example, be as follows: (1) bind an antibody or antigen to a substrate; (2) contact the bound receptor with a fluid or tissue sample containing the virus, a viral antigen, or antibodies to the virus; (3) contact the above with an antibody bound to a detectable moiety (e.g., horseradish peroxidase enzyme or alkaline phosphatase enzyme); (4) contact the above with the substrate for the enzyme; (5) contact the above with a color reagent; (6) observe color change. The above method can be readily modified to detect presence of an anti-TMAdV antibody in the sample or a specific TMAdV protein as well as the virus.
Western blot (immunoblot) analysis can be used to detect and quantify the presence of TMAdV antigen in the sample. The technique generally comprises separating sample proteins by gel electrophoresis on the basis of molecular weight, transferring the separated proteins to a suitable solid support, (such as a nitrocellulose filter, a nylon filter, or derivatized nylon filter), and incubating the sample with the antibodies that specifically bind the TMAdV antigen. The anti-TMAdV antigen antibodies specifically bind to the TMAdV antigen on the solid support. These antibodies can be directly labeled or alternatively can be subsequently detected using labeled antibodies (e.g., labeled sheep anti-mouse antibodies) that specifically bind to the anti-TMAdV antigen antibodies.
Other assay formats include liposome immunoassays (LIA), which use liposomes designed to bind specific molecules (e.g., antibodies) and release encapsulated reagents or markers. The released chemicals are then detected according to standard techniques (see Monroe et al., Amer. ClM. Prod. Rev. 5:34-41 (1986)).
A TMAdV antigen and/or a patient's antibodies to the virus can be detected utilizing a capture assay. Briefly, to detect antibodies to TMAdV in a patient sample, antibodies to the patient's immunoglobulin, e.g., anti-IgG (or IgM) are bound to a solid phase substrate and used to capture the patient's immunoglobulin from serum. TMAdV, or reactive fragments of TMAdV, are then contacted with the solid phase followed by addition of a labeled antibody. The amount of patient TMAdV specific antibody can then be quantitated by the amount of labeled antibody binding.
In competitive assays, TMAdV antigen present in a sample can be detected indirectly by detecting a decrease in a detectable signal associated with a known, added (exogenous) TMAdV antigen displaced (competed away) from an anti-TMAdV antigen antibody by the unknown TMAdV antigen present in a sample.
Competitive assays can also be adapted to provide for an indirect measurement of the amount of TMAdV antigen present in the sample. Briefly, serum or other body fluids from the subject is reacted with an antibody bound to a substrate (e.g. an ELISA 96-well plate). Excess serum is thoroughly washed away. A labeled (enzyme-linked, fluorescent, radioactive, etc.) monoclonal antibody is then reacted with the previously reacted TMAdV virus-antibody complex. The amount of inhibition of monoclonal antibody binding is measured relative to a control. MABs can also be used for detection directly in samples by IFA for MABs specifically reactive for the antibody-virus complex.
A hapten inhibition assay is another competitive assay. In this assay the known TMAdV antigen can be immobilized on a solid substrate. A known amount of anti-TMAdV antibody is added to the sample, and the sample is then contacted with the immobilized TMAdV antigen. The amount of anti-TMAdV antibody bound to the known immobilized TMAdV antigen is inversely proportional to the amount of TMAdV antigen present in the sample. The amount of immobilized antibody can be detected by detecting either the immobilized fraction of antibody or the fraction of the antibody that remains in solution. Detection can be direct where the antibody is labeled or indirect by the subsequent addition of a labeled moiety that specifically binds to the antibody as described above.
Immunoassays in the competitive binding format can also be used for crossreactivity determinations. For example, a TMAdV antigen can be immobilized to a solid support. Proteins can be added to the assay that compete for binding of the antisera to the immobilized antigen. The ability of the added proteins to compete for binding of the antisera to the immobilized protein is compared to the ability of the TMAdV antigen to compete with itself. The percent crossreactivity for the above proteins is calculated, using standard calculations. Those antisera with less than 10% crossreactivity with each of the added proteins listed above are selected and pooled. The cross-reacting antibodies are optionally removed from the pooled antisera by immunoabsorption with the added considered proteins, e.g., distantly related homologs.
The immunoabsorbed and pooled antisera can then be used in a competitive binding immunoassay as described above to compare a second protein, thought to be perhaps an allele or polymorphic variant of a TMAdV antigen, to the immunogen protein. In order to make this comparison, the two proteins are each assayed at a wide range of concentrations and the amount of each protein required to inhibit 50% of the binding of the antisera to the immobilized protein is determined. If the amount of the second protein required to inhibit 50% of binding is less than 10 times the amount of the TMAdV antigen that is required to inhibit 50% of binding, then the second protein is said to specifically bind to the polyclonal antibodies generated to TMAdV antigen.
Immunoassays (both competitive and non-competitive) also often use a labeling agent to specifically bind to and label the complex formed by the antibody and antigen. The labeling agent can itself be one of the moieties comprising the antibody/antigen complex. Thus, the labeling agent can be a labeled TMAdV protein nucleic acid or a labeled anti-TMAdV antibody. Alternatively, the labeling agent can be a third moiety, such a secondary antibody that specifically binds to the antibody/antigen complex (a secondary antibody is typically specific to antibodies of the species from which the first antibody is derived). Other proteins capable of specifically binding immunoglobulin constant regions, such as protein A or protein G can also be used as a label agent. These proteins exhibit a strong non-immunogenic reactivity with immunoglobulin constant regions from a variety of species (see, e.g., Kronval et al., J. Immunol. 111:1401-1406 (1973); Akerstrom et al., J. Immunol. 135:2589-2542 (1985)). The labeling agent can be modified with a detectable moiety, such as biotin, to which another molecule can specifically bind, such as streptavidin. A variety of detectable moieties are well known to those skilled in the art, and can be any material detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Such detectable labels have been well-developed in the field of immunoassays and can include, but are not limited to, magnetic beads (e.g., DYNABEADSTM), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g., 3H, 125j 35s, 14, e, or - - - 12P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic beads (e.g., polystyrene, polypropylene, latex, etc.).
The label can be coupled directly or indirectly to the desired component of the assay according to methods well known in the art. As indicated above, a wide variety of labels can be used, with the choice of label depending on sensitivity required, ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions.
Non-radioactive labels are often attached by indirect means. Generally, a ligand molecule (e.g., biotin) is covalently bound to the molecule. The ligand then binds to another molecules (e.g., streptavidin) molecule, which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound. The ligands and their targets can be used in any suitable combination with antibodies that recognize TMAdV antigen, or secondary antibodies that recognize anti-TMAdV antigen.
The molecules can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorophore. Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidotases, particularly peroxidases. Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc. Chemiluminescent compounds include luciferin, and 2,3-dihydrophthalazinediones, e.g., luminol. For a review of various labeling or signal producing systems that can be used, see U.S. Pat. No. 4,391,904.
Means of detecting labels are well known to those of skill in the art. Thus, for example, where the label is a radioactive label, means for detection include a scintillation counter or photographic film as in autoradiography. Where the label is a fluorescent label, it can be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence can be detected visually, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like. Similarly, enzymatic labels can be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product. Colorimetric or chemiluminescent labels can be detected simply by observing the color associated with the label. Thus, in various dipstick assays, conjugated gold often appears pink, while various conjugated beads appear the color of the bead.
Some assay formats do not require the use of labeled components. For instance, a micro-agglutination test can also be used to detect the presence of TMAdV in test samples. Briefly, latex beads are coated with an antibody and mixed with a test sample, such that TMAdV in the tissue or body fluids that is specifically reactive with the antibody crosslink with the receptor, causing agglutination. The agglutinated antibody-virus complexes within a precipitate, visible with the naked eye or by spectrophotometer. Other assays include serologic assays, in which the relative concentrations of IgG and IgM are measured.
One of skill in the art will appreciate that it is often desirable to minimize non-specific binding in immunoassays. Particularly, where the assay involves an antigen or antibody immobilized on a solid substrate it is desirable to minimize the amount of non-specific binding to the substrate. Means of reducing such non-specific binding are well known to those of skill in the art. Typically, this technique involves coating the substrate with a proteinaceous composition. In particular, protein compositions such as bovine serum albumin (BSA), nonfat powdered milk, and gelatin are widely used with powdered milk being most preferred.
In the diagnostic methods described above, the sample can be taken directly from a subject or in a partially purified form. The antibody specific for a particular TMAdV (the primary reaction) reacts by binding to the virus. Thereafter, a secondary reaction with an antibody bound to, or labeled with, a detectable moiety can be added to enhance the detection of the primary reaction. Generally, in the secondary reaction, an antibody or other ligand which is reactive, either specifically or nonspecifically with a different binding site (epitope) of the virus will be selected for its ability to react with multiple sites on the complex of antibody and virus. Thus, for example, several molecules of the antibody in the secondary reaction can react with each complex formed by the primary reaction, making the primary reaction more detectable.
Assays for Modulators of TMAdV
Modulation of a TMAdV can be assessed using a variety of in vitro and in vivo assays, including cell-based models. Such assays can be used to test for inhibitors and activators of TMAdV. Modulators of TMAdV are tested using either recombinant or naturally occurring protein of choice. Modulation can include, but is not limited to, modulation of infection, replication, receptor binding, cell entry, particle formation, and the like.
Measurement of modulation of a TMAdV or a cell expressing TMAdV, either recombinant or naturally occurring, can be performed using a variety of assays, in vitro, in vivo, and ex vivo, as described herein. A suitable physical, chemical or phenotypic change that affects activity, e.g., enzymatic activity, cell surface marker expression, viral replication and proliferation can be used to assess the influence of a test compound on the polypeptide of this invention. When the functional effects are determined using intact cells or animals, one can also measure a variety of effects.
Assays to identify compounds with TMAdV modulating activity can be performed in vitro. Such assays can use full length TMAdV or a variant thereof, or a mutant thereof, or a fragment thereof. Purified recombinant or naturally occurring protein can be used in the in vitro methods of the invention. In addition to purified TMAdV, the recombinant or naturally occurring protein can be part of a cellular lysate or a cell membrane. As described below, the binding assay can be either solid state or soluble. Preferably, the protein or membrane is bound to a solid support, either covalently or non-covalently. Often, the in vitro assays of the invention are substrate or ligand binding or affinity assays, either non-competitive or competitive. Other in vitro assays include measuring changes in spectroscopic (e.g., fluorescence, absorbance, refractive index), hydrodynamic (e.g., shape), chromatographic, or solubility properties for the protein.
A high throughput binding assay can be performed in which the protein or a fragment thereof is contacted with a potential modulator and incubated for a suitable amount of time. In one embodiment, the potential modulator is bound to a solid support, and the protein is added. In another embodiment, the protein is bound to a solid support. A wide variety of modulators can be used, as described below, including small organic molecules, peptides, antibodies, etc. A wide variety of assays can be used to identify TMAdV-modulator binding, including labeled protein-protein binding assays, electrophoretic mobility shifts, immunoassays, enzymatic assays, and the like. In some cases, the binding of the candidate modulator is determined through the use of competitive binding assays, where interference with binding of a known ligand or substrate is measured in the presence of a potential modulator. Either the modulator, the known ligand, or substrate is bound first; then the competitor is added. After the protein is washed, interference with binding, either of the potential modulator or of the known ligand or substrate, is determined Often, either the potential modulator or the known ligand or substrate is labeled.
A cell-based assay can be used in which the TMAdV is expressed in a cell, and functional, physical, chemical and phenotypic changes are assayed to identify viral modulators. Any suitable functional effect can be measured as described herein, in addition to viral inhibition assays as are well known in the art. The TMAdV can be naturally occurring or recombinant. Also, fragments of the TMAdV or chimeric proteins can be used in cell based assays. In addition, point mutants in essential residues required by the catalytic site can be used in these assays.
In one preferred embodiment, high throughput screening methods involve providing a combinatorial small organic molecule or peptide library containing a large number of potential therapeutic compounds (potential modulator or ligand compounds). Such “combinatorial chemical libraries” or “ligand libraries” are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional “lead compounds” or can themselves be used as potential or actual therapeutics.
A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical “building blocks” such as reagents. For example, a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.
Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493 (1991) and Houghton et al., Nature 354:84-88 (1991)). Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (e.g., PCT Publication No. WO 91/19735), encoded peptides (e.g., PCT Publication No. WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer. Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of small compound libraries (Chen et al., I Amer. Chem. Soc. 116:2661 (1994)), oligocarbamates (Cho et al., Science 261:1303 (1993)), and/or peptidyl phosphonates (Campbell et al., I Org. Chem. 59:658 (1994)), nucleic acid libraries (see Ausubel, Berger and Sambrook, all supra), peptide nucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibody libraries (see, e.g., Vaughn et al., Nature Biotechnology, 14(3):309-314 (1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al., Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), small organic molecule libraries (see, e.g., benzodiazepines, Baum C&EN, January 18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S. Pat. No. 5,288,514, and the like).
Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md., etc.).
A solid state or soluble high throughput assaying using a TMAdV, or a cell or tissue expressing a TMAdV, either naturally occurring or recombinant can be used. A solid phase based in vitro assay can be used in a high throughput format can be used where TMAdV is attached to a solid phase. Any one of the assays described herein can be adapted for high throughput screening.
In high throughput assays, either soluble or solid state, it is possible to screen up to several thousand different modulators or ligands in a single day. This methodology can be used for TMAdV in vitro, or for cell-based or membrane-based assays comprising a TMAdV. In particular, each well of a microtiter plate can be used to run a separate assay against a selected potential modulator, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator. Thus, a single standard microtiter plate can assay about 100 (e.g., 96) modulators. If 1536 well plates are used, then a single plate can easily assay from about 100-about 1500 different compounds. It is possible to assay many plates per day; assay screens for up to about 6,000, 20,000, 50,000, or more than 100,000 different compounds are possible using the integrated systems of the invention.
For a solid state reaction, the protein of interest or a fragment thereof, e.g., an extracellular domain, or a cell or membrane comprising the protein of interest or a fragment thereof as part of a fusion protein can be bound to the solid state component, directly or indirectly, via covalent or non covalent linkage. A tag for covalent or non-covalent binding can be any of a variety of components. In general, a molecule which binds the tag (a tag binder) is fixed to a solid support, and the tagged molecule of interest is attached to the solid support by interaction of the tag and the tag binder.
A number of tags and tag binders can be used, based upon known molecular interactions well described in the literature. For example, where a tag has a natural binder, for example, biotin, protein A, or protein G, it can be used in conjunction with appropriate tag binders (avidin, streptavidin, neutravidin, the Fc region of an immunoglobulin, etc.) Antibodies to molecules with natural binders such as biotin are also widely available and appropriate tag binders (see, SIGMA Immunochemicals 1998 catalogue SIGMA, St. Louis Mo.).
Similarly, any haptenic or antigenic compound can be used in combination with an appropriate antibody to form a tag/tag binder pair. Thousands of specific antibodies are commercially available and many additional antibodies are described in the literature. For example, in one common configuration, the tag is a first antibody and the tag binder is a second antibody which recognizes the first antibody. In addition to antibody-antigen interactions, receptor-ligand interactions are also appropriate as tag and tag-binder pairs. For example, agonists and antagonists of cell membrane receptors (e.g., cell receptor-ligand interactions such as transferrin, c-kit, viral receptor ligands, cytokine receptors, chemokine receptors, interleukin receptors, immunoglobulin receptors and antibodies, the cadherein family, the integrin family, the selectin family, and the like (see, e.g., Pigott & Power, The Adhesion Molecule Facts Book I (1993)). Similarly, toxins and venoms, viral epitopes, hormones (e.g., opiates, steroids, etc.), intracellular receptors (e.g. which mediate the effects of various small ligands, including steroids, thyroid hormone, retinoids and vitamin D; peptides), drugs, lectins, sugars, nucleic acids (both linear and cyclic polymer configurations), oligosaccharides, proteins, phospholipids and antibodies can all interact with various cell receptors.
Synthetic polymers, such as polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, and polyacetates can also form an appropriate tag or tag binder. Many other tag/tag binder pairs are also useful in assay systems described herein, as would be apparent to one of skill upon review of this disclosure.
Common linkers such as peptides, polyethers, and the like can also serve as tags, and include polypeptide sequences, such as poly gly sequences of between about 5 and 200 amino acids. Such flexible linkers are known to persons of skill in the art. For example, polyethylene glycol linkers are available from Shearwater Polymers, Inc. Huntsville, Ala. These linkers optionally have amide linkages, sulfhydryl linkages, or heterofunctional linkages.
Tag binders are fixed to solid substrates using any of a variety of methods currently available. Solid substrates are commonly derivatized or functionalized by exposing all or a portion of the substrate to a chemical reagent which fixes a chemical group to the surface which is reactive with a portion of the tag binder. For example, groups which are suitable for attachment to a longer chain portion would include amines, hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanes and hydroxyalkylsilanes can be used to functionalize a variety of surfaces, such as glass surfaces. The construction of such solid phase biopolymer arrays is well described in the literature (e.g., Merrifield, J. Am. Chem. Soc. 85:2149-2154 (1963) (describing solid phase synthesis of, e.g., peptides); Geysen et al., J. Immun. Meth. 102:259-274 (1987) (describing synthesis of solid phase components on pins); Frank & Doring, Tetrahedron 44:60316040 (1988) (describing synthesis of various peptide sequences on cellulose disks); Fodor et al., Science, 251:767-777 (1991); Sheldon et al., Clinical Chemistry 39(4):718-719 (1993); and Kozal et al., Nature Medicine 2(7):753759 (1996) (all describing arrays of biopolymers fixed to solid substrates)). Non-chemical approaches for fixing tag binders to substrates include other common methods, such as heat, cross-linking by UV radiation, and the like.
The compounds tested as modulators of TMAdV can be any small organic molecule, or a biological entity, such as a protein, e.g., an antibody or peptide, a sugar, a nucleic acid, e.g., an antisense oligonucleotide or a ribozyme or siRNA, or a lipid. Alternatively, modulators can be genetically altered versions of a TMAdV. Typically, test compounds will be small organic molecules, peptides, circular peptides, siRNA, antisense molecules, ribozymes, and lipids.
Essentially any chemical compound can be used as a potential modulator or ligand in the assays of the invention, although most often compounds can be dissolved in aqueous or organic (especially DMSO-based) solutions are used. The assays are designed to screen large chemical libraries by automating the assay steps and providing compounds from any convenient source to assays, which are typically run in parallel (e.g., in microtiter formats on microtiter plates in robotic assays). It will be appreciated that there are many suppliers of chemical compounds, including Sigma (St. Louis, Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Biochemica Analytika (Buchs Switzerland) and the like.
Treating/Preventing TMAdV
Embodiments described herein further relate to the therapeutic, prophylactic and research uses of various techniques to block or modulate the expression of TMAdV viral proteins or propagation of the virus. Modulators of TMAdV useful for treating or preventing TMAdV can include, but is not limited to, genetically modified versions of TMAdV, e.g., versions with altered activity, as well as naturally occurring and synthetic ligands, substrates, antagonists, agonists, antibodies, peptides, cyclic peptides, aptamers, nucleic acids, antisense molecules, ribozymes, siRNA molecules, miRNA molecules, and small chemical molecules, as is well known in the art.
Further described herein are TMAdV vaccines for therapeutic or prophylactic purposes. Within certain aspects, TMAdV virus, proteins or peptides and immunogenic fragments thereof, and/or polynucleotides, as well as anti-TMAdV antibodies and/or T cells, can be incorporated into pharmaceutical compositions or immunogenic compositions. Whole virus vaccines (live and attenuated, or replication incompetent, or killed) or subunit vaccines, such as structural or non-structural TMAdV proteins or immunogenic fragments thereof, can be used to treat or prevent TMAdV infections by eliciting an immune response in a subject. Alternatively, a pharmaceutical composition can comprise an antigen-presenting cell (e.g., a dendritic cell) transfected with a TMAdV polynucleotide such that the antigen-presenting cell expresses a TMAdV peptide.
Nucleic acid vaccines encoding a genome, structural protein or non-structural protein or a fragment thereof of TMAdV can also be used to elicit an immune response to treat or prevent TMAdV infection. Numerous gene delivery techniques are well known in the art, such as those described by Rolland (1998) Crit. Rev. Therap. Drug Carrier Systems 15: 143-198, and references cited therein. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (such as a suitable promoter and terminating signal). In a preferred embodiment, the DNA can be introduced using a viral expression system (e.g., vaccinia, pox virus, retrovirus, or adenovirus), which can involve the use of a non-pathogenic (defective), replication competent virus. Suitable systems are disclosed, for example, in Fisher-Hoch et al. (1989) Proc. Natl. Acad. Sci. USA 86:317-321; Flexner et al., (1989) Ann. N.Y. Acad. Sci. 569:86-103; Flexner et al., (1990) Vaccine 8:17-21; U.S. Pat. Nos. 4,603,112, 4,769,330, 4,777,127 and 5,017,487; WO 89/01973; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner (1988) Biotechniques 6:616-627; Rosenfeld et al. (1991) Science 252:431-434; Kolls et al. (1994) Proc. Natl. Acad. Sci. USA 91:215-219; Kass-Eisler et al. (1993) Proc. Natl. Acad. Sci. USA 90:11498-11502; Guzman et al. (1993) Circulation 88:2838-2848; and Guzman et al. (1993) Cir. Res. 73:1202-1207. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA can also be “naked,” as described, for example, in Ulmer et al. (1993) Science 259:1745-1749 and reviewed by Cohen (1993) Science 259:1691-1692. The uptake of naked DNA can be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells. It will be apparent that a vaccine can comprise both a polynucleotide and a polypeptide component. Such vaccines can provide for an enhanced immune response.
Vaccine preparation is generally described in, for example, Powell and Newman, eds., Vaccine Design (the subunit and adjuvant approach), Plenum Press (NY, 1995). Vaccines can be designed to generate antibody immunity and/or cellular immunity such as that arising from CTL or CD4+ T cells.
A non-specific immune response enhancer can be any substance that enhances an immune response to an exogenous antigen. Examples of non-specific immune response enhancers include adjuvants, biodegradable microspheres (e.g., polylactic galactide) and liposomes (into which the compound is incorporated; see, e.g., U.S. Pat. No. 4,235,877). Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A, Bortadella pertussis or Mycobacterium tuberculosis derived proteins. Suitable adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2 (SmithKline Beecham); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A. Cytokines, such as GM-CSF or interleukin-2, -7, or -12, can also be used as adjuvants.
Pharmaceutical Compositions
Pharmaceutical compositions and vaccines within the scope of the present invention can also contain other compounds, which can be biologically active or inactive. For example, one or more immunogenic portions of other antigens can be present, either incorporated into a fusion polypeptide or as a separate compound, within the composition or vaccine. Polypeptides can, but need not be, conjugated to other macromolecules as described, for example, within U.S. Pat. Nos. 4,372,945 and 4,474,757. Pharmaceutical compositions and vaccines can generally be used for prophylactic and therapeutic purposes.
Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the packaged nucleic acid suspended in diluents, such as water, saline or PEG 400; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable emulsions. Tablet forms can include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers. Lozenge forms can comprise the active ingredient in a flavor, e.g., sucrose, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art.
The compound of choice, alone or in combination with other suitable components, can be made into aerosol formulations (i.e., they can be “nebulized”) to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.
Formulations suitable for parenteral administration, for example, by intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. In the practice of this invention, compositions can be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically or intrathecally. Parenteral administration and intravenous administration are the preferred methods of administration. The formulations of commends can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials.
Such compositions can also comprise buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, bacteriostats, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), solutes that render the formulation isotonic, hypotonic or weakly hypertonic with the blood of a recipient, suspending agents, thickening agents and/or preservatives. Alternatively, compositions of the present invention can be formulated as a lyophilizate. Compounds can also be encapsulated within liposomes using well known technology.
Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. Cells transduced by nucleic acids for ex vivo therapy can also be administered intravenously or parenterally as described above.
The dose administered to a patient, in the context of the present invention should be sufficient to affect a beneficial therapeutic response in the patient over time. The dose will be determined by the efficacy of the particular vector employed and the condition of the patient, as well as the body weight or surface area of the patient to be treated. The size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular vector, or transduced cell type in a particular patient.
Pharmaceutically acceptable carriers are determined in part by the particular composition being administered (e.g., nucleic acid, protein, modulatory compounds or transduced cell), as well as by the particular method used to administer the composition. Accordingly, there are a wide variety of suitable formulations of pharmaceutical compositions of the present invention (see, e.g., Remington's Pharmaceutical Sciences, 17th ed., 1989). Administration can be in any convenient manner, e.g., by injection, oral administration, inhalation, transdermal application, or rectal administration.
For administration, compounds and transduced cells of the present invention can be administered at a rate determined by the LD-50 of the inhibitor, vector, or transduced cell type, and the side-effects of the inhibitor, vector or cell type at various concentrations, as applied to the mass and overall health of the patient. Administration can be accomplished via single or divided doses.
Pharmaceutical and vaccine compositions can be presented in unit-dose or multi-dose containers, such as sealed ampoules or vials. Such containers are preferably hermetically sealed to preserve sterility of the formulation until use. In general, formulations can be stored as suspensions, solutions or emulsions in oily or aqueous vehicles. Alternatively, a vaccine or pharmaceutical composition can be stored in a freeze-dried condition requiring only the addition of a sterile liquid carrier immediately prior to use.
Kits
The invention further provides diagnostic reagents and kits comprising one or more such reagents for use in a variety of diagnostic assays, including for example, immunoassays such as ELISA and “sandwich” type immunoassays, as well as nucleic acid assay, e.g., PCR assays. In a related embodiment, the assay is performed in a flow-through or strip test format, wherein the binding agent is immobilized on a membrane, such as nitrocellulose. Such kits can preferably include at least a first peptide, or a first antibody or antigen binding fragment of the invention, a functional fragment thereof, or a cocktail thereof, or a first oligo pair, and means for signal generation. The kit's components can be pre-attached to a solid support, or can be applied to the surface of a solid support when the kit is used. The signal generating means can come pre-associated with an antibody or nucleic acid of the invention or can require combination with one or more components, e.g., buffers, nucleic acids, antibody-enzyme conjugates, enzyme substrates, or the like, prior to use.
Kits can also include additional reagents, e.g., blocking reagents for reducing nonspecific binding to the solid phase surface, washing reagents, enzyme substrates, enzymes, and the like. The solid phase surface can be in the form of microtiter plates, microspheres, or other materials suitable for immobilizing nucleic acids, proteins, peptides, or polypeptides. An enzyme that catalyzes the formation of a chemiluminescent or chromogenic product or the reduction of a chemiluminescent or chromogenic substrate is one such component of the signal generating means. Such enzymes are well known in the art. Where a radiolabel, chromogenic, fluorigenic, or other type of detectable label or detecting means is included within the kit, the labeling agent can be provided either in the same container as the diagnostic or therapeutic composition itself, or can alternatively be placed in a second distinct container means into which this second composition can be placed and suitably aliquoted. Alternatively, the detection reagent and the label can be prepared in a single container means, and in most cases, the kit will also typically include a means for containing the vial(s) in close confinement for commercial sale and/or convenient packaging and delivery.
EXAMPLES
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.
Example 1
Identification of a New Titi Monkey Adenovirus (TMAdV)
Materials and Methods
The California National Primate Research Center (CNPRC)
The California National Primate Research Center (CNPRC) is a part of the National Primate Research Centers At the beginning of 2009, the CNPRC maintained a colony of 74 New World titi monkeys (Callicebus cupreus) for studies of social behavior. Eighty-eight percent of the CNPRC titi monkey population (n=65) occupied 1 quadrant in 1 animal building. The other 3 quadrants housed 133 rhesus macaques (Macaca mulatta). Nearby outdoor enclosures containing rhesus macaques and cynomolgus macaques (Macaca fasicularis) surrounded this building.
An outbreak of fluminant pneumonia lasted from May 14, 2009 until Aug. 5, 2009. All titi monkeys demonstrating clinical signs of the outbreak were housed in the animal building. Three additional titi monkeys were moved into the building on May 28, 2009, reflecting a total at-risk population of 68 animals. Over the past 6 years, there were no new introductions of titi monkeys into the CNPRC; over the past 2 years, there were no new introduction of rhesus macaques.
Microbial Testing
Bacterial, mycoplasma, and fungal cultures were performed at the CNPRC. Clinical samples were also sent to an outside laboratory (Focus Diagnostics, Cypress, Calif.) for respiratory viral testing by centrifugation-enhanced shell vial culture followed by direct fluorescent antibody staining for 8 viruses (respiratory syncytial virus, adenovirus, influenza virus A and B, parainfluenza virus types 1, 2, and 3, and human metapneumovirus).
Gross, Histophathological, and Ultrastructural Analyses
Gross and histopathological analyses of post-mortem tissues were performed by a board-certified veterinary pathologist at the Morphology Core of the CNPRC. At necropsy, tissue samples from the trachea, lung, and liver were collected and fixed in 10% formalin. Tissues were routinely processed and embedded in paraffin. 3-μm sections were stained with hematoxylin and eosin (HE) and examined by light microscopy. For transmission electron microscopy, tissue fragments (2×2 mm) were excised from paraffin blocks of lung, deparaffinized, and processed as previously described (Woods, et al., Vet Pathol 33:125 (1996)).
Nucleic Acid Extraction and cDNA Library Preparation
Total nucleic acid was extracted from body fluid or swab samples using commercially available kits (Qiagen, Valencia, Calif.). 200 μL of sample were passed through a 0.22 μm filter (Millipore, Temecula, Calif.) to remove bacteria and cellular debris and then treated with Turbo DNase (Ambion, Culver City, Calif.) to degrade host genomic DNA prior to extraction. For tissue samples, lung or liver tissue was homogenized in an 15 mL Eppendorf tube using a disposable microtube pestle (Eppendorf, San Diego, Calif.) and scapel; RNA extraction was then performed using TRIzol LS (Invitrogen, Carlsbad, Calif.) followed by isopropanol precipitation and two washes in 70% ethanol. Extracted nucleic acid was amplified using a random PCR method to generate cDNA libraries for Virochip and deep sequencing analyses as previously described (Wang, et al., PNAS, 99:150807 (2002); Greninger, et al., PloS ONE 5:e13381 (2010)).
Virochip Analysis
Virochip analysis was performed as previously described (Wang, et al., PNAS, 99:150807 (2002); Greninger, et al., PloS ONE 5:e13381 (2010)). Briefly, samples were labeled with Cy3 or Cy5 fluorescent dye, normalized to 10 pmol of incorporated dye, and hybridized overnight using the Agilent Gene Expression Hybridization kit (Agilent Technologies, Santa Clara, Calif.). The current 8×60 k Virochip arrays used in this study contain  ̃36,000 probes representing all viral species in GenBank, and combine probes from all previous Virochip designs (Greninger, et al., PloS ONE:e13381 (2010). Slides were scanned at 2 μm resolution using an Agilent DNA Microarray Scanner. Virochip microarrays were analyzed with Z-score analysis (Chiu, et al., Clin Infect Dis, 43:e71 (2006)), hierarchical cluster analysis (Eisen, et al., PNAS 95:14863 (1998)), and E-Predict, an automated computational algorithm for viral species prediction from microarrays (Urisman, et al., Genome Biol 6:R78 (2005)). Only Z-score analysis, a method for assessing the statistical significance of individual Virochip probes, yielded a credible viral signature on the microarray.
PCR Screening
Consensus primers derived initially from a highly conserved region of the hexon gene to confirm the Virochip finding of a novel adenovirus by PCR. From the sequence of a resulting 301-bp amplicon, a set of specific PCR primers was designed for TMAdV. The TMAdV-specific PCR was performed using a reaction mixture consisting of 17 μL of water, 2.5 μL of 10×Taq buffer, 1 μL of MgCl2 (50 mM), 0.5 μL of deoxynucleoside triophosphates (dNTPs; 12.5 mM), 0.5 μL of each primer (10 μM), and 0.5 μL of Taq polymerase (Invitrogen, Carlsbad, Calif.), in a final volume of 25 μL. Conditions for the PCR reaction were 40 cycles of 94° C. for 30 s, 50° C. for 45 s, and 72° C. for 1 min. Amplicons were purified on a 2% agarose gel, cloned into plasmid vectors using TOPO TA (Invitrogen, Carlsbad, Calif.), and sent to an outside company (Elim Biopharmaceuticals, Hayward, Calif.) for Sanger sequencing in both directions using vector primers M13F and M13R.
PCR assays commonly used to detect human adenoviruses in clinical or public health settings were analyzed to determine the ability to detect TMAdV. Adenovirus PCR was performed on 12 TMAdV-positive clinical samples using an additional 6 pairs of primers, according to previously published protocols (Hierholzer, et al., J Clin Microbiol 31:1886 (1993); Lee, et al., J Clin Microbiol 45:2626 (2007); Xu, et al., J Clin Microbiol 38:4114 (2000)). 5 of the 6 primer pairs, including all of the pairs designed to detect human respiratory adenoviruses, completely failed to amplify TMAdV from positive clinical samples, while the remaining pair was only 75% sensitive relative to TMAdV-specific PCR
Whole Genome Sequencing
To facilitate whole-genome sequencing of TMAdV, cDNA libraries were prepared and amplified for deep sequencing from lung tissue and a lung swab sample from two different monkeys using previously published protocols (Sorber, et al., PloS One 3:e495 (2008)). Briefly, libraries were cleaved with a Type IIs restriction endonuclease (GsuI) and truncated adapters were ligated on the resulting strand ends. Full-length adapters were added via an additional 15 cycles of PCR. Amplified libraries were size-selected on a 2% agarose gel at approximately 350 bp average length and then sent an outside company (Elim Biopharmaceuticals, Hayward, Calif.) for deep sequencing on an Illumina Genome Analyzer IIx (Illumina, San Diego, Calif.). Paired-end reads were sequenced for 73 cycles in each direction. Paired-end reads were subsequently filtered to eliminate low-complexity sequences with a Lempel-Ziv-Welch (LZW) compression ratio below 0.4, Welch, T. A. Computer 17, 8-19 (1984), split into individual reads, and stripped of any remaining primer sequences using BLASTN alignments (word size=11, E-value=1×10−5). After low-complexity filtering and primer trimming, 11,950,557 sequence reads remained, with each read consisting of 67 nucleotides, for a total of  ̃800 megabases of sequence. Remaining reads were then aligned to the genome sequence of sAd18, the closest relative to TMAdV (FIG. 2B) and to a database consisting of all adenovirus genomes and partial sequences deposited in GenBank by BLASTN (word size=11, E-value=1×10−5) and TBLASTX (word size=11, E-value=1×10−5). Reads that aligned to adenoviruses were then used to assemble portions of the TMAdV genome with Geneious software (version 3.6.5) Drummond, A., et al. Geneious v5.3.4., employing the sAd18 genome as a reference sequence and requiring a 20-bp minimum overlap and 95% overlap identity. Aligning reads were also used to design PCR primers to fill in remaining gaps in the TMAdV genome. Amplicons derived from specific TMAdV PCR primers were gel-purified, cloned, and sequenced as described above. The 5′ end corresponding to the inverted terminal repeat (ITR) of TMAdV was obtained by PCR with a forward degenerate consensus primer and a reverse TMAdV-specific primer.
Structural Features and Phylogenetic Analysis
To identify coding regions in the TMAdV genome, the fully annotated genomic sequence of simian adenovirus 21 (sAd21) was used as a reference. First, the two genomes and identified all ORFs that were present were aligned with Geneious. An overlapping ORF that best matched the corresponding ORF in the annotated reference genome was considered to be correct. For adenovirus genes that are spliced (e.g. E1A), the identification of a GT-AG intron start-stop signal was used to pinpoint the correct ORF. To verify the accuracy of the coding sequence, the sequence of each identified ORF was aligned to a database containing all adenoviral proteins in GenBank by BLASTX. Alignments were then manually examined to ensure that all ORF assignments were correct.
To generate the phylogeny trees, representative whole-genome sequences from simian and human members of Groups A-G and non-mammalian adenoviruses were first compiled from GenBank. Multiple sequence alignments of the hexon and polymerase genes were then constructed with Geneious. Sliding window analysis (window size, 200 bp; step size 20 bp) was performed with Simplot (Lole, K. S., et al., J Virol 73, 152-160 (1999)). Bootscanning was performed using the Jukes and Cantor method, also with Simplot (window size: 200 bp; step size, 20; 1000 replicates). Pairwise amino acid alignments between TMAdV proteins and corresponding proteins in other adenoviruses (Table 3) were performed using Geneious.
Culturing
A549 (human lung adenocarcinoma), PMK (primary rhesus macaque kidney), and BSC-1 (African green monkey kidney epithelial) cell lines were obtained from the Viral and Rickettsial Disease Laboratory (VRDL) branch of the California Department of Public Health. Cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 1× nonessential amino acids (Invitrogen, Carlsbad, Calif.), 10% fetal bovine serum, 100 U of penicillin/mL, and 100 μg of streptomycin/mL. Anti-SV40 antibodies were added to the cell culture media to prevent growth of SV40 polyomavirus. Clinical samples were clarified by centrifugation for 10 min×4,000×g and passage through a 0.2-μm filter. After achieving 80-90% confluency, cell cultures were inoculated with XXX μL of clinical sample or passaged viral supernatant. Viral replication was monitored over 14 days by visual inspection under light microscopy for cytopathic effect (CPE). To confirm the generation of infectious virus, viral supernatants were quantitated by an end-point dilution assay.
Microarray and Nucleotide Sequence Accession Numbers
All Virochip microarrays used in this study have been submitted to the NCBI GEO database (study accession number GSE26898; microarray accession numbers GSM662370-GSM662391; microarray design accession number GPL 11662). The annotated, whole-genome sequence of TMAdV has been submitted to GenBank (accession number HQ913600). Deep sequencing reads have been submitted to the NCBI Sequence Read Archive (accession number SRA031285).
Results
Outbreak of Fulminant Pneumonia in a Titi Monkey Colony
A healthy adult titi monkey presented on May 14, 2009 with cough, lethargy, and decreased appetite (FIG. 1A, T30). Despite treatment with intravenous fluids and antibiotics, the animal died in 5 days. A second case presented 4 weeks later. In the interim period, 3 healthy titi monkeys were relocated from a separate building, including 2 into the cage formerly occupied by the index case (FIG. 1A, T31 and T32), reflecting an at-risk population of 68. Over the next 2 months, 21 additional monkeys presented with clinical signs similar to those shown by the index case, including one of the relocated monkeys (attack rate=23/68, or 34%) (FIGS. 1A and 1B). Clinical signs in affected animals included cough, lethargy, poor appetite, tachypnea, and abdominal breathing. These symptoms progressed to overt respiratory distress and death within an average of 8 days. Chest radiographs typically revealed diffuse interstitial pulmonary changes and bronchoalveolar consolidation indicative of pneumonia. Right middle lobe involvement was predominant (FIG. 1C). Animals displaying clinical signs were quarantined and aggressively treated by veterinarians with supplemental oxygen, anti-inflammatory medications, broad-spectrum antibiotics, and antivirals (oseltamivir and/or ribavirin). In total, 19 animals died from the illness during the outbreak (case fatality rate=83%). Only 4 monkeys survived, even though the at-risk population consisted of apparently healthy adults and juveniles. Interestingly, none of the 133 rhesus macaques (Macaca mulatta) housed in the same building became sick during the outbreak, and neither did any of the Old World monkeys from surrounding outdoor colonies of rhesus and cynomolgus macaques (Macaca fascicularis).
Gross necropsy findings were similar in all titi monkeys and were characterized primarily by diffuse, consolidated pneumonias, with prominent right middle lobe involvement. Occasional evidence of fibrinous pleuritis, pericardial/pleural edema, and hemorrhage was found (FIG. 1D-1). Some livers, spleens, and lymph nodes were found to be abnormally enlarged. Hepatic necrosis and hemorrhage, along with ascites, were sometimes appreciated. On histologic examination, respiratory epithelium was found to be completely obliterated. Lung architecture was destroyed, and prominent intranuclear inclusion bodies were observed (FIGS. 1D-2 and 1D-3).
A routine microbiological workup for infectious causes of the outbreak, including bacterial, mycoplasma, and fungal cultures, was negative. Respiratory viral testing failed to detect evidence of respiratory syncytial virus, adenovirus, influenza virus A and B, human metapneumovirus, and parainfluenza virus types 1, 2, and 3.
Virochip Identification, PCR Screening and Electron Microscopic (EM) Confirmation of TMAdV
Given the clinical presentation of an acute viral respiratory illness and the appearance of intranuclear inclusion bodies on histological examination, it was suspected that a virus that had eluded detection by conventional assays was the cause of the titi monkey outbreak. Nasal, lung, and liver swab samples collected during necropsy were analyzed using the Virochip. Microarrays were analyzed using ranked Z-scores (Chiu, C. Y., et al. Clin Infect Dis, 43, e71-76 (2006)), with 5 of the top 50 probes on the Virochip corresponding to adenoviruses and mapping to 4 different regions in the adenovirus genome (FIG. 2A). The 5 probes were derived from 3 different Adenoviridae genera, suggesting the presence of a divergent adenovirus that was not a member of any previously known species.
Using consensus primers (Echavarria, et al., J Clin Microbiol 36:3323 (1998)), we amplified a 301 bp fragment of the adenovirus from the hexon gene. The fragment shared  ̃86% nucleotide identity with its closest phylogenetic relatives, the human group D adenoviruses, and the newly identified adenovirus was designated TMAdV, or titi monkey adenovirus. Specific PCR for TMAdV was used to screen body fluids and tissues from affected monkeys. PCR results were positive from post-necropsy liver and lung tissues as well as from sera, oral swabs, and nasal swabs collected at time of quarantine in 8 different affected monkeys, but were negative from nasal swabs in 3 asymptomatic control animals (Table 1). To confirm the presence of virus in diseased tissues, lung tissue from affected monkeys was examined by transmission electron microscopy, revealing abundant icosahedral particles characteristic of adenovirus filling the alveoli (FIG. 1D-4). Next, to assess persistent subclinical infection from TMAdV, convalescent sera from surviving affected monkeys 1 month post-outbreak was analyzed (n=4). All serum samples were negative for TMAdV by PCR. To assess potential TMAdV shedding, stool samples collected from all cages housing titi monkeys (n=22) were analyzed by PCR, and were found to be negative. In addition, TMAdV was analyzed in rectal swab samples from rhesus macaques housed in the same building as the titi monkeys (n=26) and in wild rodent droppings (n=2) from rodents living near the at-risk titi monkey room. All macaque and rodent fecal samples were negative for TMAdV by PCR.







TABLE 1







PCR screening for TMAdV.














Date Presenting





PCR
with Clinical
Date of


Sample
Sample Type
Result
Signs
Necropsy










Affected, at-risk titi monkeys (died)











T1
serum§
−
May 14, 2009
May 19, 2009


T26
serum¶
+
Jul. 23, 2009
Jul. 30, 2009


T28
conjunctival swab¶
+
Jul. 16, 2009
Jul. 25, 2009



nasal swab¶
+



liver swab¶
+



lung swab¶
+


T29
serum¶
+
Jul. 26, 2009
Jul. 31, 2009


T30
serum¶
−
Jul. 25, 2009
Jul. 30, 3009


T33
lung swab¶
+
Jun. 23, 2009
Jun. 29, 2009



nasal swab¶
+


T36
lung swab¶
+
Jul. 7, 2009
Jul. 14, 2009



lung swab¶
+


T60
serum¶
−
Jul. 15, 2009
Jul. 22, 2009


T63
serum¶
−
Jun. 20, 2009
Aug. 1, 2009


T67
nasal swab#
−
Jul. 7, 2009
Aug. 13, 2009



nasal swab¶
+







Affected, at-risk titi monkeys (survived)











T31
serum*
−
Jul. 10, 2009
N/A


T32
serum*
−
Jul. 12, 2009
N/A


T34
serum*
−
Jun. 23, 2009
N/A


T62
serum*
−
Jul. 8, 2009
N/A







Asymptomatic at-risk and minimal-risk titi monkeys











T27
throat swab (n = 1)¶
−
N/A
N/A


at-risk titi
stool from cages (n = 14)*
−
N/A
N/A


at-risk titi
serum (n = 29)*
−
N/A
N/A


minimal-risk titi
oral swab (n = 3)¶
−
N/A
N/A


minimal-risk titi
stool from cages (n = 5)*
−
N/A
N/A


minimal-risk titi
serum (n = 8)*
−
N/A
N/A


minimal-risk titi
stool from cages (n = 8)*
−
N/A
N/A







Other











rhesus
rectal swabs (n = 26)*
−
N/A
N/A


human
serum (n = 15)∞
−
N/A
N/A


rodent
droppings (n = 2)*
−
N/A
N/A





§= initial case;


#= collected prior to outbreak;


¶= collected during outbreak;


*= collected 2 months after outbreak;


∞= collected 4 months after outbreak.


For titi monkey cage designations (TXX), please refer to FIG. 1.






Whole Genome Sequencing, Features, and Phylogenetic Analysis of TMAdV

To facilitate whole-genome sequencing of TMAdV, deep sequencing of a lung swab from one affected titi monkey and lung tissue from another affected monkey was performed. Out of  ̃11.9 million high-quality reads, 2,782 reads and 3,767 reads aligned to the SAdV-18 genome by BLASTN (FIG. 2B, blue) and TBLASTX (FIG. 2B, transparent blue), respectively, with reads mapping to sites across the genome. De novo assembly of the complete TMAdV genome from reads that aligned to SAdV-18 was not possible due to insufficient sequence coverage (<46%). The poor apparent coverage was the result of high sequence divergence of the TMAdV genome from the genomes of known adenoviruses, which hindered the identification of most of the 16,524 actual deep sequencing reads derived from TMAdV (FIG. 2B, red). Thus, after partial assembly of TMAdV using overlapping reads aligning to the SAdV-18 g TMAdV was found to be 36,842 base pairs in length, with a base composition of 20.8% A, 29.8% C, 29.8% G, and 19.6% T, and a GC content of 59.6%, comparable to that of adenoviral species Groups C, D, and E in the Mastadenovirus genus. The deduced genomic structure of TMAdV was also similar to that of other mastadenoviruses and consists of 34 open reading frames (FIG. 2C).
Whole-genome phylogenetic analysis placed TMAdV in an independent species group separate from the known human adenoviral species A-G (FIG. 3). Among all 95 fully-sequenced adenovirus genomes in GenBank, the closest simian adenoviral relatives to TMAdV were SAdV-3, SAdV-18, and SAdV-21, with pairwise nucleotide identities ranging from 54.0% to 56.3% (FIG. 4). The closest human adenoviral relatives were the species D adenoviruses, which share 54.3% to 55.1% identity to TMAdV, with human adenoviruses of other species slightly less similar (51.1%-54.6%). The placement of TMAdV into a separate group by phylogenetic analysis was also observed when looking individually at the hexon, polymerase, penton base, and fiber genes (FIG. 7). Scanning nucleotide pairwise identity plots revealed that, among the major adenovirus genes, the DNA polymerase and hexon are more conserved, whereas the E1A and fiber are more divergent (FIG. 4). The significant overall sequence divergence of TMAdV from known human and simian adenoviruses is highlighted by the finding that PAdV-A (porcine adenovirus A), a non-primate mammalian adenovirus, shared only a slightly less similar whole-genome pairwise identity to TMAdV of 47.0% (FIG. 4). In fact, in the DNA polymerase gene, TMAdV shared a pairwise identity with PAdV-A of 67.2%, comparable to its pairwise identities with the other human adenoviruses, 59%-71.7% (FIGS. 4 and 7). Although TMAdV was found to be highly divergent from other adenoviruses, different isolates of TMAdV from 3 affected titi monkeys were remarkably conserved, sharing 100% identity across the full-length hexon gene (data not shown).
The high level of sequence divergence in TMAdV held true at the amino acid level as well, with amino acid identities relative to other mastadenoviruses ranging from 20.8% to 27.5% for the fiber, the most divergent protein, to 68.7%-78.2% for the hexon (Table 2). Although bearing low sequence similarity to other adenoviruses, the penton base of TMAdV contained an RGD motif that presumably binds αv integrins. By both nucleotide and amino acid comparisons, the closest phylogenetic relative to TMAdV in GenBank overall was SAdV-3 (FIG. 4; Table 2). Bootscanning analysis revealed no evidence for recombination of TMAdV with other adenoviruses at either the whole-genome or individual gene level (FIG. 8).







TABLE 2







Amino acid divergence of TMAdV from other adenoviruses. For each protein,


the entry corresponding to the adenovirus with the highest percentage


identity relative to TMAdV is highlighted in boldface underline.















fiber



penton

hexon



(IV)
E1A
DBP
polymerase
base (III)
pTP
(II)


















hAd12 (Group A)
26.4%
31.0%
38.0%
58.3%
64.3%
67.5%
76.4%


hAd3 (Group B)
22.3%
31.4%
36.5%
59.9%
65.8%
68.6%
73.9%


sAd21 (Group B)
22.2%
30.6%
36.0%
60.5%
66.1%
68.6%
72.4%


hAd2 (Group C)
25.0%
29.4%
39.5%
60.5%
67.0%
67.8%
71.6%


sAd42 (Group C)
26.4%
30.5%
38.8%
60.0%
66.5%
68.0%
72.6%


hAd9 (Group D)
21.9%
28.4%
38.3%
61.2%
66.1%
68.3%
74.2%


hAd4 (Group E)
26.3%
32.6%
37.1%
61.6%
67.9%
69.7%
72.9%


sAd22 (Group E)
27.5%
31.2%
36.6%
61.4%
67.6%
70.3%
74.4%


hAd40 (Group F)
26.9%
31.0%
40.1%
59.8%
64.1%
65.8%
77.3%


sAd7 (Group F)
25.5%
32.6%
35.7%
61.3%
67.0%
67.9%
76.7%


hAd52 (Group G)
24.1%
30.3%
35.6%
61.5%
67.8%
67.7%
77.2%


sAd3 (rhesus)
26.6%
29.9%
36.8%
59.4%
68.2%
70.5%
78.2%


sAd18 (vervet)
26.0%
30.7%
39.0%
69.9%
66.5%
67.7%
76.9%


hAd49 (Group D)
22.1%
29.2%
38.3%
62.9%
65.6%
67.6%
74.8%


porcine AdA
26.4%
23.6%
37.4%
54.4%
61.7%
57.6%
68.7%


fowl AdA
 1.6%
N/A*
25.6%
36.5%
41.6%
31.7%
48.1%









Culturing of TMAdV in Human and Monkey Cell Lines

After identification and characterization of TMAdV, an attempt to culture the virus was undertaken in 2 monkey kidney cell lines (rhesus macaque BSC-1 and African green monkey PMK cells) and 1 human cell line (A549 lung adenocarcinoma cells) (FIG. 5). Direct inoculation of cell cultures with a lung swab from an affected titi monkey produced a weak initial cytopathic effect in macaque BSC-1 and human A549 cells at day 7. However, despite multiple serial passages, propagation of the infected cell culture supernatant in either BSC-1 or PMK cells was unsuccessful. In contrast, propagation in human A549 cells resulted in viral adaptation by passage 6 and generation of a fully adapted strain of TMAdV by passage 10 that was able to productively infect all three cell lines. Thus, culturing and propagation of TMAdV were successful in a human A549 cell line, but not in established or primary monkey kidney cell lines.
Example 2
Seroprevalence of TMAdV in Monkeys and Humans
Materials and Methods
Virus Neutralization Assay
A virus stock of TMAdV (passage 10) was produced on human A549 cells, aliquoted, and quantitated by end-point dilution. To perform the virus neutralization assay, A549 cells were plated in a 96-well format and allowed to grow to 80-90% confluency. 55 μL of viral supernatant at a concentration of 100 TCID50 and 55 μL of serum (starting at a 1:8 dilution) were mixed and incubated for one hour at 37° C. As a control for each serum sample, 55 μL of culture media and 55 μL of serum were mixed and treated in an identical fashion. After incubation, 100 μL of mixture were inoculated into appropriate wells and the entire plate was placed in a 37° C. 5% CO2 incubator. Cells in the plate wells were observed for evidence of CPE every other day for 1 week. For wells that showed inhibition of viral CPE, the corresponding serum samples were diluted in 6 two-fold steps and then retested. The highest dilution that completely inhibited viral CPE was taken as the neutralizing antibody titer.
Results
Nineteen serum samples from 16 at-risk, affected titi monkeys were tested. Among 4 affected titi monkeys who survived the outbreak, 2 monkeys mounted a vigorous neutralizing Ab response to TMAdV, with negative pre-outbreak Ab titers (<1:8) but convalescent antibody titers of >1:512, while one monkey exhibited a positive but much weaker response. Affected titi monkeys who died during the outbreak exhibited a wide range of neutralizing Ab titers, from <1:8 to >1:512 (those without Ab likely died before mounting a response). To investigate the possibility of colonization or subclinical infection by TMAdV, serum samples were examined from at-risk asymptomatic titi monkeys (n=3) and nearby rhesus macaques (n=6). All samples from asymptomatic titi monkeys were negative for neutralizing Abs to TMAdV, whereas 1 of the 6 rhesus macaque samples exhibited a titer of 1:16.
One individual at the CNPRC reported becoming ill during the titi monkey outbreak, the researcher in closest, daily contact with the animals. Symptoms began near the onset of the outbreak, although whether they began prior to or after identification of the index case is unclear. The researcher, with a past medical history of multiple sclerosis, initially developed symptoms of a viral upper respiratory infection (URI), including fever, chills, headache, and sore throat, followed by a dry cough, intermittent shortness of breath, and a “burning sensation in the lungs” (suggestive of pleurisy) that lasted 4-6 weeks. Medical care was not sought, and no antibiotics were taken during the illness.
Contact tracing to identify family members and other individuals in close contact with the scientist was conducted. The brother and sister-in-law of the scientist also developed flu-like symptoms about 1-2 weeks after the researcher initially became sick. Their symptoms—fever, cough and muscle aches without shortness of breath or pleurisy—appeared milder and completely resolved within 2 weeks. Neither individual sought medical care for these symptoms. Notably, neither the brother nor the sister-in-law had ever visited the CNPRC.
To explore a potential link between the outbreak and associated illness in humans, CNPRC personnel and close contacts were tested for evidence of recent or prior infection by TMAdV by virus neutralization (FIG. 6).
Approximately 6 months after the outbreak, serum samples were collected from CNPRC personnel in direct contact with the titi monkeys, as well as from 2 family members of the clinically ill CNPRC researcher. Two samples were found positive for neutralizing Abs to TMAdV: (1) Ab titers for the clinically ill researcher were 1:32, and (2) Ab titers for the brother of the clinically ill researcher were 1:8.
Among 80 random blood donors from Northern California with a history of recent travel outside of the United States, 2 individuals (2/80, 2.5%) had positive Ab titers of 1:16 and 1:8. Sera containing antibodies to adenovirus serotypes 1 through 52 were unable to neutralize TMAdV (data not shown), indicating that the results of our serological survey were not due to nonspecific cross-reactivity from prior exposure to known human adenoviruses.

## Claims

1. An isolated antibody that specifically binds to an isolated protein encoded by a nucleotide sequence of SEQ ID NO:9, wherein the isolated antibody is bound to a detectable moiety.

2. The antibody of claim 1, wherein the antibody is a polyclonal antibody.

3. A kit for detecting a titi monkey adenovirus (TMAdV) in a sample, the kit comprising the antibody of claim 1.