Viral obesity methods and compositions

Abstract

A source of viral induced obesity has been discovered. A virus known as AD-36P adenovirus type 36 has been found to be associated with obesity in both animals and humans. Diagnostic DNA sequences are presented so that DNA based tests for the presence of the obesity associated virus can be conducted.

Description

RELATED APPLICATION

This application is a continuation of patent application Ser. No. 09/056,153 filed Apr. 6, 1998, now U.S. Pat. No. 6,127,113, which claims the benefit of the provisional U.S. application Ser. No. 60/042,942 filed Apr. 4, 1997.

TECHNICAL FIELD

This invention concerns obesity in humans caused by viruses and methods and compositions for diagnosing, treating and preventing this disease.

The invention also concerns methods and compositions for reducing levels of triglycerides and cholesterol in humans.

More particularly, the invention concerns methods and compositions for diagnosing whether obesity in a human is caused by a virus or whether a person is susceptible to becoming obese because of having been infected with

EXAMPLE 2

Ad-36 virus was obtained from the ATCC (American Type Culture Collection), Accession No. VR-913. The virus was grown in A549 bronchial human carcinoma cells (Wisconsin State Laboratory of Hygiene, University of Wisconsin, Madison, Wis., USA). Plaques were grown, and a single plaque was removed and used to reinfect a fresh batch of A549 cells. Plaques of this second passage were grown and again a single plaque was picked and used to reinfect another fresh batch of A549 cells. The resulting virus is Ad-36P.

In virus neutralization assays of 5 human sera known to have antibodies to Ad-36 (ATTC VR-913), Ad-36p showed a 2- to 4-fold greater titer for neutralizing antibodies than did Ad-36. This demonstrates the greater sensitivity of Ad-36p, as compared to Ad-36, in assaying serum for antibody against obesity-causing adenoviruses.

EXAMPLE 3

Minimum Essential Media Eagle (MEM)(Sigma Chemical Co. Cat. No. M-0643) with non-essential amino acids, Earle's salts, 1-glutamine, 10% fetal bovine serum and 2.9% sodium bicarbonate (v/v), pH 7.4, is used for growing A549 cells. 5 to 10 microliters of virus stock solution is mixed with 8 ml of the medium and the mixture is pipetted into flasks with growing cells. The flasks are incubated for 1 h at 37 deg. C. while shaking gently every 15 min. After 1 h, the medium is removed and replaced with fresh medium. The flasks are then incubated at 37 deg. C. The cells are grown over 7 to 8 days, until 90% of the cells show CPE. The cell material and medium are then centrifuged at 1000 rpm for 15 min to eliminate cell debris. The supernatant with virus (e.g., Ad-36 or Ad-36P), is again centrifuged at 1000 rpm for 10 min and then aliquoted into 2 ml cryovials for storage at −70 deg. C. until use.

EXAMPLE 4

Fiber protein is isolated from virus in a two step procedure. First, virus is purified from supernatant (see Example 3) using CsCl gradient centrifugation, following the procedure described by Graham and Ludvik in Chapter 11 of Methods in Molecular Biology, Vol. 7: Gene transfer and Expression Protocols, E. J. Murray, ed., The Human Press, Inc., Clifton, N.J. (1991). Then the fiber protein is isolated from the virus electrophoretically following Oostrum and Burnett, J. Virology, 56,439448 (1985) and Maizel et aL, Virology, 36, 115-125 (1968).

The electrophoretic band of fiber protein (approx. mol. wt. 60,000 daltons) is cut from the gel and the fiber protein can be isolated from the band by standard techniques.

EXAMPLE 5

A virus neutralization assay (serum neutralization assay) is used to assay serum for antibody reactive with adenovirus in serum of test subjects.

Serum is thawed and heat-inactivated for 30 min. at 56 deg. C.

The assay is carried out in standard 96-well microtiter plates. Serial two fold dilutions (1:2 to 1:1024) are made with the medium that is the A549 growth medium described in Example 3 but lacks the fetal calf serum and sodium bicarbonate. 50 microliters of each dilution is added in duplicate to the wells of the plate. 50 microliters of virus suspension (100 TCID₅₀ is then added to each well. (TCID₅₀ is calculated by serially diluting viral stock solution and inoculating A549 cells with the dilutions to determine the reciprocal of the highest dilution of virus which causes CPE in 50% of the material inoculated). The plates are then incubated at 37 deg. C. for 1 hr. Then 100 microliters of A549 cell suspension, containing approximately 20,000 cells, is added to each well and the plate is further incubated at 37 deg. C. for 12 days. Crystal violet-ethanol is then added to each of the wells to fix and stain the cells and the plates are examined macroscopically for CPE;. The highest serum dilution with no CPE is the titer. Controls used in the procedure are wells with no virus and wells with virus but no serum. A back titration is carried out to confirm that appropriate virus dilutions were used. Positive control is antisera to chicken adenovirus and human adenovirus. Presence of CPE with the virus and no CPE in the presence of serum is considered an indication of effective neutralization of the virus with antibody in serum, such that the serum is considered to have antibody against the virus. A titer of 1:8 or greater is considered positive.

The foregoing assay was carried out on serum samples from 155 obese patients and 45 non-obese volunteers. 15-20% of the obese patients were positive for antibody. All of these had TG, CHOL, and LDL-CHOL within the normal ranges for non-obese people. The remaining obese people (antibody-negative) had, on the average, TG, CHOL and LDL-CHOL levels above the normal ranges for non-obese people. None of the non-obese volunteers was positive for antibody. The non-obese volunteers had, on the average, TG, CHOL and LDL-CHOL levels that were in the normal ranges for non-obese people.

EXAMPLE 6

Ad-36P virus is killed by adding 42 microliters of 37% formalin to 150 microliters of virus stock, then incubating the resulting composition at room temperature for 72 hours, and finally adding 15 microliters of 35% sodium bisulfite. Confirmation that the virus was killed is carried out by inoculating a culture of A549 cells with the final solution and determining that virus does not grow in the culture.

EXAMPLE 7

Nucleic acid from adenovirus Ad-36p was isolated and sequenced by a standard sequencing method.

The cDNA sequence encoding the fiber protein is as follows (SEQ ID NO:1): 5′-ATGTCAAAGAGGCTCCGGGTGGAAGATGACTT CAACCCCGTCT ACCCCTATGGCTACGCGCGGAAT-CAGAATATCCCCTTCCTCACT CCCCCCTTTGTCTC-CTCCGATGGATTCCAAAACTTCCCCCCTGG GGTC-CTGTCACTCAAACTGGCTGATCCATGTCTCACTC AAGGTG GGAGGGGGACTCACTGTAGAACAA-CAGTCTGGAAAACTGAGTG TGGATACTAAGGCAC-CCTTGCAAGTTGCAAATGACAACAAATT GGAGC-TATCTTATGATGATCCATTTAAGGTAGAGAGAATAA CAAA CTTGGAATTAAAGCTGGCCATGGTTTAG-CAGTTGTAACTAAAGA AAACACAAGTCTTC-CTAGTCTAGTTGGAACACTTGTAGTTTTAA CTG-GAAAAGGAATAGGTACTGGATCAAGTGCACATG GAGGAAC TATTGATGTAAGACTTGGTGAAGGAG-GTGGGTATCATTTGATG AAAAAGGAGACTTAG-TAGCTTGGGACAAAAAAAATGATACACG CAC-CCTTTGGACAACACCTGATCCTTCTCCAAATTGC AAGTTG AAACAGCAAGAGACTCAAAGCTAACCT-TAGCACTTACAAATTG TGGTAGTCAAATTTTGGC-CACTGTATCTTTACTTGTTGTTACGGG CAAATATGC-TATTATAAGTGACACAGTCAACCCAAAGCAGTTCT CTATTAAGTTACTGTTTAATGA-CAAGGGTGTTTTGTTAAGTGAC TCAAATCT-TGATGGGACATATTGGAACTATAGAAGCAACAATA ACAACATAGGCACTCCTTATAAAGAG-GCTGTTGGTTTTATGCCA AGCACAACAGCTTATC-CTAAGCCAACCAACAACACCAGCACAG ATCCG-GATAAAAAAGTGAGTCAAGGTAAAAATAAAAT TGTAAG CAATATATCTTGGAGGAGAGGTATAT-CAACCAGGATTTATTG TTGTTAAATTTAATCAG-GAAACTGATGCCAATTGTGCATACTCT ATTA-CATTTGATTTGGATGGGGTAAGGTGTATAAGGA TCCTAT ACCATATGATACCTCTTCTACTTTCT-CATATATCGCTCAAGA ATGA

EXAMPLE 8

The cDNA sequence of the Ad-36p genome was screened against all known cDNA sequences and two 25-base sequences and one 28-base sequence were found, all lying in the fiber-encoding sequence provided above in Example 7, that were unique to Ad-36p. These three sequences are as follows:

• • SEQ ID NO:2: 5′-AGTTGAAACAGCAAGAGACTCA AAG
  • SEQ ID NO:3 5′-GGTACTGGATCAAGTGCACATGG AG
  • SEQ ID NO:4 5′-TTGAAACAGCAAGAGACTCAAA GCTAAC

Sequence 3 above was employed a a probe for Ad-36p in a conventional nuclei acid probe hybridizaton assay of DNA isolated from four chickens, two of which had been infected with the virus and became obese and two of which had not been infected and were not obese. DNA hybridizing to the probe was observed with only the DNA from the two infected chickens. The assay involved direct detection and was by capillary electrophoresis using laser-induced fluoresence for detection. More particularly, a replaceable polyacrylamide matrix was employed in the electrophoretic separation and detection employed a dual system with 5′-labeling of the oligo and thiazole orange intercalator in the buffer system. See Kolestar et al., J. Chromatography B, 697, 189-194 (1997).

The skilled will understand that probes, and primers when amplification is also used, of between about 15 and 30 bases in length are advantageously employed to provide suitable specificity and sensitivity.

Amplification methods using PCR and variations thereof maybe employed, as well known in the art.

Claims

1. A method of determining whether an obese person is suffering from viral obesity caused by adenovirus type 36p 36, the method comprising the steps of:

isolating a sample from the person a sample selected from the group consisting of a body fluid, feces, a sample of tissue and a sample of an organ from the person ,

obtaining an antibody specific to an adenovirus type 36p 36, and;

assaying the sample using the antibody the adenovirus type 36 to test for the presence of the an antibody reactive with adenovirus type 36p 36 thus testing whether the person has been or is infected with adenovirus type 36p 36, which causes obesity and reduces cholesterol level in humans.

2. The method according to claim 1 wherein the substance sample analyzed is blood.

3. A method for the detection of virally caused obesity in an obese subject comprising the steps of

isolating a sample of biological tissue or fluid from the subject;

providing an antibody specific an to adenovirus type 36p 36 stock solution;

testing the sample, using the antibody diagnostic for adenovirus type 36p adenovirus type 36p 36 stock solution; and

detecting the presence of antibodies specific to adenovirus type 36p 36 to determine if the subject has viral induced obesity.

4. The method of claim 1, wherein said step of obtaining adenovirus type 36 comprises obtaining a purified form of adenovirus type 36 (adenovirus type 36p) and said assaying step comprises assaying the sample using adenovirus type 36p.

5. The method of claim 3, wherein said step of providing adenovirus 36 stock solution comprises providing a purified adenovirus type 36 (adenovirus type 36p) stock solution and said testing step comprises testing the sample using the adenovirus type 36p stock solution.

6. The method of claim 1, wherein the antibody is reactive with one or more peptides encoded by the nucleic acid sequences selected from the group consisting of SEQ ID. NO.:1, SEQ ID. NO.:2, SEQ ID. NO.:3, and SEQ ID. NO.:4.

7. The method of claim 1, wherein said assaying step is performed by using a method selected from the group consisting of a serum neutralization assay and ELISA.

8. The method of claim 1, wherein the sample is selected from the group consisting of a biological sample, a body fluid, a tissue sample, an organ sample, feces, blood, salvia, and any combination thereof.

9. The method of claim 1, wherein the person has a body mass index (BMI) less than about 27 kg/m² and has adipose tissue that is present in an amount greater than about 25% of the body weight of the person.

10. The method of claim 1, wherein the person has a body mass index (BMI) less than about 27 kg/m² and has adipose tissue that is present in an amount greater than about 30% of the body weight of the person.

11. The method of claim 3, the antibody is specific to one or more peptides encoded by the nucleic acid sequences selected from the group consisting of SEQ ID. NO.:1, SEQ ID. NO.:2, SEQ ID. NO.:3, and SEQ ID. NO.:4.

12. The method of claim 3, wherein said testing step is performed by using a method selected from the group consisting of a serum neutralization assay and ELISA.

13. The method of claim 3, wherein the sample is selected from the group consisting of a biological sample, body fluid, a tissue sample, an organ sample, feces, blood, salvia, and any combination thereof.

14. The method of claim 3, wherein the subject has a body mass index (BMI) less than about 27 kg/m² and has adipose tissue that is present in an amount greater than about 25% of the body weight of the subject.

15. The method of claim 3, wherein the subject has a body mass index (BMI) less than about 27 kg/m² and has adipose tissue that is present in an amount greater than about 30% of the body weight of the subject.

16. The method of claim 3, wherein the subject is a human.

17. The method of claim 3, wherein the subject is an animal.

18. A method for the detection of virally caused obesity in an obese subject, said method comprising the steps of:

obtaining a sample from the subject; and

determining the presence of antibodies specific to adenovirus 36 in the subject.

19. The method of claim 18, wherein said step of determining the presence of the antibodies comprises the steps of:

screening for the presence of antibodies specific to adenovirus 36 in the sample; and

determining the presence of antibodies specific to adenovirus 36 in the sample.

20. The method of claim 18, wherein the antibodies in said determining step are specific to one or more peptide encoded by the nucleic acid sequences selected from the group consisting of SEQ ID NO.:1, SEQ ID NO.:2, SEQ ID NO.:3, and SEQ ID NO.:4.

21. The method of claim 18, wherein the subject is a human.

22. The method of claim 18, wherein the subject is an animal.

23. The method of claim 19, wherein said screening step is performed by using a method selected from the group consisting of serum neutralization assay and ELISA.

24. The method of claim 18, wherein the sample is selected from the group consisting of a biological sample, body fluid, a tissue sample, an organ sample, feces, blood, salvia, and any combinations thereof.

25. The method of claim 18, wherein the subject has a body mass index (BMI) less than about 27 kg/m² and has adipose tissue that is present in an amount greater than about 25% of the body weight of the subject.

26. The method of claim 18, wherein the subject has a body mass index (BMI) less than about 27 kg/m² and has adipose tissue that is present in an amount greater than about 30% of the body weight of the subject.