# Patent

## Title
Compositions and methods for the treatment or prevention of human adenovirus-36 infection

## Abstract

Disclosed are fusion proteins, recombinant nucleic acid molecules, and therapeutic compositions, including yeast-based immunotherapy compositions, for use in the diagnosis, prevention and treatment of adenovirus-36 (Ad-36) infection and sequela thereof.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a national stage application under 35 U.S.C. 371 of PCT Application No. PCT/US2011/65868, having an international filing date of Dec. 19, 2011, which designated the United States, which PCT application claims the benefit of priority under 35 U.S.C. §119(e) from U.S. Provisional Application No. 61/424,472, filed Dec. 17, 2010, the entire disclosure of which is hereby incorporated by reference.


REFERENCE TO A SEQUENCE LISTING
This application contains a Sequence Listing submitted electronically as a text file by EFS-Web. The text file, named “3923-33-PCT_ST25”, has a size in bytes of 211 KB, and was recorded on 16 Dec. 2011. The information contained in the text file is incorporated herein by reference in its entirety pursuant to 37 CFR §1.52(e)(5).
FIELD OF THE INVENTION
The present invention generally relates to immunotherapeutic compositions and methods for the prevention and/or treatment of human adenovirus-36 infection, as well as the prevention and/or treatment of obesity and/or obesity-associated disorders or other sequela related to human adenovirus-36 infection.
BACKGROUND OF THE INVENTION
The terms “obesity” and “overweight” or “pre-obese” define ranges of weights that are greater than weights that are generally considered to be healthy for a person of a given height. According to a report in August 2010 by the Centers for Disease Control (CDC), “no state met the Healthy people 2010 obesity target of 15%, and the self-reported prevalence of obesity among U.S. adults had increased 1.1 percentage points from 2007” (Sherry et al., Morbidity and Mortality Weekly Report (MMWR), 59; 1-5; Aug. 3, 2010). In children and teens, excess weight represents a very serious health issue. The 2007-2008 National Health and Nutrition Examination Survey (NHANES) estimated that 17% of individuals age 2-19 are obese (CDC). Indeed, the CDC and the WHO have referred to an “obesity epidemic” in many populations worldwide. Overweight and obese individuals have a higher likelihood of developing a variety of health problems including, but not limited to, cardiovascular diseases and associated conditions (e.g., high blood pressure, high cholesterol), type 2 diabetes, respiratory disorders, cancer, reproductive disorders, hepatic dysfunction, and osteoarthritis.
Several different factors can contribute to obesity or being overweight, and the condition can be a complex health issue for many individuals. Behavioral factors, environmental factors, genetics, illness, and/or infectious agents may play a role in the condition. Lack of sufficient physical activity and excess calorie intake in the diet, i.e., caloric imbalance, are the most apparent and common causes of being overweight or obese. However, there appear to be several genetic factors that may predispose certain individuals to weight gain, including mutations in genes related to control of feeding behavior, and various genetic mutations or correlations of genotype with obesity in individuals and populations. In addition to these factors, various illnesses and drugs can also impact an individual's weight. More recently, infectious agents have been identified as contributing to some cases of obesity.
A few infectious agents have been associated with obesity in non-human animals, and one in particular has been associated with human obesity. Human adenovirus-36 (also denoted Ad-36, Adv-36, or hAdv-36) was first described in a child with diabetes in 1980 (Wigand et al., 1980, Arch. Viol. 64(3):225-233). Beginning in the early 1990's, experiments by Dhurandhar and colleagues first showed that Ad-36 increased adiposity in chickens and in mice ((Dhurandhar et al., 1990, J. Bombay Vet. College 2:131-132; Dhurandhar et al., 1992, Vet. Microbiol., 31:101-107; Dhurandhar et al., 2000, Int J Obes Relat Metab Disord 24:989-996; Dhurandar et al., 2001, Int. J. Obes. Relat. Metab. Disord. 25(7):990-996), as well as in monkeys (Dhurandhar, et al., 2002, J. Nutr. 132(10):3155-3160). In mice and chickens, infection with Ad-36 resulted in viremia, infection of adipose tissue, increased visceral fat, total body fat, and/or body weight, and reduced serum cholesterol and triglycerides. In monkeys, Ad-36 promoted weight gain and lowered serum cholesterol. Pasarica and colleagues have shown that human Ad-36 induces adiposity, increases insulin sensitivity, and alters hypothalamic monoamines in rats (Pasarica et al., 2006, Obesity 14(11):1905-1913).
In humans, Ad-36 has been shown to have a high probability of being associated with obesity, where a unique phenotype of low serum cholesterol and triglyceride levels was present in about 30% of obese humans subjects having anti-Ad-36 antibodies, whereas only 5% of the non-obese humans tested had antibodies to Ad-36 (Dhurandhar et al., 1997, FASEB J, 3:A230; Atkinson et al., 1998, Int J Obes Relat Metab Disord 22(Suppl): S57). An epidemiological study showed that 30% of obese people were infected with Ad-36 compared to only 11% of lean people in the study (Atkinson et al., 2005, Int J Obes (Lond), 29(3):281-286). These investigators showed that Ad-36 is associated with increased body weight and the reduction of serum lipids in humans. Additional researchers have reported an association between human Ad-36 and lipid disorders or obesity rates in children and adolescents worldwide (Na et al., 2010, Int. J. Obes. 34:89-93; Gabbert et al., 2010, Pediatrics 2010; 126:721-726; and Atkinson et al., 2010, Int. J. Ped. Obes. 5:157-160). Further work by Pasarica and Dhurandhar and colleagues showed that Ad-36 induces commitment, differentiation, and lipid accumulation in human adipose-derived stem cells (Pasarica et al., 2008, Stem Cells 26:969-978). Moreover, in vitro adipogenesis was shown to be accelerated by infection of preadipocytes with human Ad-36 (Vangipuram et al., 2004, Obes. Res. 12(5):770-777), and infection was also shown to increase insulin sensitivity and suppress the expression of leptin mRNA (Vangipuram et al., 2007, Int. J. Obes. (Lond.) 31(1):87-96. The activity of the E4 orf1 gene of Ad-36 has been suggested to be responsible for this adipogenesis (Rogers et al., 2008, International Journal of Obesity 32:397-406).
In 2010, Arnold and colleagues reported the complete characterization of the human Ad-36 genome (Arnold et al., 2010, Virus Res. 149:152-161). Diagnostic assays have been described for the identification of Ad-36 infection in human tissues, via identification or use of anti-Ad-36 antibodies (see, e.g., WO 98/44946, WO 2007/120362), and a diagnostic test for Ad-36 is in commercial development (Scandivir AB). However, a treatment for the viral infection, once identified, is lacking; no preventative or therapeutic treatment that directly targets Ad-36 infection is currently commercially available. Accordingly, there remains a need in the art for an effective prophylactic and/or therapeutic treatment for adenovirus-36 infection, in order to reduce or eliminate Ad-36-associated obesity and overweight conditions.
SUMMARY OF THE INVENTION
One embodiment of the invention relates to an immunotherapeutic composition comprising: (a) a yeast vehicle; and (b) an adenovirus-36 (Ad-36) antigen comprising one or more Ad-36 proteins and/or immunogenic domains of such proteins. In one aspect, the Ad-36 proteins include at least one protein selected from, but is not limited to: hexon, fiber, CR1α, and CR1γ, and/or at least one immunogenic domain of at least one of the proteins. In one aspect, the Ad-36 proteins include at least one immunogenic domain of CR1α and at least one immunogenic domain of CR1γ.
In one aspect, the Ad-36 antigen comprises Ad-36 sequences, wherein the Ad-36 sequences consist of: positions 71-136 of Ad-36 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain; positions 145-169 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain; positions 290-313 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain; and positions 334-363 of Ad-36 SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain. For example, such an Ad-36 antigen can include, but is not limited to, an amino acid sequence selected from the group consisting of: SEQ ID NO:42 or a corresponding sequence from another Ad-36 strain, SEQ ID NO:48 or a corresponding sequence from another Ad-36 strain and SEQ ID NO:49 or a corresponding sequence from another Ad-36 strain.
In one aspect, the Ad-36 antigen comprises Ad-36 sequences, wherein the Ad-36 sequences consist of: positions 136-218 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain; positions 235-285 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain; positions 297-308 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain; and positions 410-450 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain. For example, such an Ad-36 antigen can include, but is not limited to, SEQ ID NO:43 or a corresponding sequence from another Ad-36 strain, SEQ ID NO:50 or a corresponding sequence from another Ad-36 strain and SEQ ID NO:51 or a corresponding sequence from another Ad-36 strain.
In another aspect, the Ad-36 antigen comprises Ad-36 sequences, wherein the Ad-36 sequences consist of positions 2-944 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain. For example, such an Ad-36 antigen can include, but is not limited to, SEQ ID NO:44 or a corresponding sequence from another Ad-36 strain, SEQ ID NO:52 or a corresponding sequence from another Ad-36 strain and SEQ ID NO:53 or a corresponding sequence from another Ad-36 strain.
In yet another aspect, the Ad-36 antigen comprises Ad-36 sequences, wherein the Ad-36 sequences consist of: positions 71-136 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain; positions 145-169 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain; positions 290-313 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain; positions 334-363 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain; positions 136-218 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain; positions 235-285 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain; positions 297-308 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain; and positions 410-450 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain. For example, such an Ad-36 antigen can include, but is not limited to, SEQ ID NO: 45 or a corresponding sequence from another Ad-36 strain, and positions 7 to 418 of SEQ ID NO:45 or a corresponding sequence from another Ad-36 strain.
In another aspect, the Ad-36 antigen comprises Ad-36 sequences, wherein the Ad-36 sequences consist of: positions 136-218 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain; positions 235-285 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain; positions 297-308 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain; positions 410-450 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain; positions 71-136 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain; positions 145-169 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain; positions 290-313 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain; and positions 334-363 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain. For example, such an Ad-36 antigen can include, but is not limited to, SEQ ID NO:46 or a corresponding sequence from another Ad-36 strain, and positions 7 to 418 of SEQ ID NO:46 or a corresponding sequence from another Ad-36 strain.
In another aspect, the Ad-36 antigen comprises Ad-36 sequences, wherein the Ad-36 sequences consist of: positions 18-60 of SEQ ID NO:26 or a corresponding sequence from another Ad-36 strain; positions 123-157 SEQ ID NO:26 or a corresponding sequence from another Ad-36 strain; positions 19-60 of SEQ ID NO:29 or a corresponding sequence from another Ad-36 strain; and positions 83-116 of SEQ ID NO:29 or a corresponding sequence from another Ad-36 strain. For example, such an Ad-36 antigen can include, but is not limited to, SEQ ID NO:47 or a corresponding sequence from another Ad-36 strain, SEQ ID NO:54 or a corresponding sequence from another Ad-36 strain, and SEQ ID NO:55 or a corresponding sequence from another Ad-36 strain.
In any of the aspects or embodiments of the invention described above or elsewhere herein, in one aspect, the Ad-36 antigen is expressed by the yeast vehicle. In one aspect, the yeast vehicle is a whole yeast. In one aspect, the yeast is killed. In one aspect, the yeast is heat-inactivated. In one aspect, the yeast vehicle is from a genus selected from: Saccharomyces, Candida, Cryptococcus, Hansenula, Kluyveromyces, Pichia, Rhodotorula, Schizosaccharomyces and Yarrowia. In one aspect, the yeast vehicle is from Saccharomyces. In one aspect, the yeast vehicle is from Saccharomyces cerevisiae. 
In any of the aspects or embodiments of the invention described above or elsewhere herein, in one aspect, a composition of the invention is formulated in a pharmaceutically acceptable excipient suitable for administration to an individual.
Another embodiment of the invention relates to a fusion protein comprising two or more Ad-36 proteins and/or immunogenic domains of one or more Ad-36 proteins, wherein the Ad-36 proteins include at least one protein selected from: hexon, fiber, CR1α, and CR1γ, and/or at least one immunogenic domain of at least one of the proteins. In one aspect, the Ad-36 proteins include E4 or at least one immunogenic domain thereof. In one aspect, the Ad-36 proteins include at least one immunogenic domain of CR1α and at least one immunogenic domain of CR1γ. In one aspect, the fusion protein comprises: (a) Ad-36 sequences consisting of: positions 71-136 of Ad-36 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain; positions 145-169 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain; positions 290-313 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain; and positions 334-363 of Ad-36 SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain; (b) Ad-36 sequences consisting of: positions 136-218 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain; positions 235-285 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain; positions 297-308 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain; and positions 410-450 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain; (c) Ad-36 sequences consisting of: positions 2-944 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain; (d) Ad-36 sequences consisting of: positions 71-136 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain; positions 145-169 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain; positions 290-313 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain; positions 334-363 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain; positions 136-218 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain; positions 235-285 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain; positions 297-308 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain; and positions 410-450 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain; (e) Ad-36 sequences consisting of: positions 136-218 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain; positions 235-285 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain; positions 297-308 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain; positions 410-450 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain; positions 71-136 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain; positions 145-169 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain; positions 290-313 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain; and positions 334-363 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain; or (f) Ad-36 sequences consisting of: positions 18-60 of SEQ ID NO:26 or a corresponding sequence from another Ad-36 strain; positions 123-157 SEQ ID NO:26 or a corresponding sequence from another Ad-36 strain; positions 19-60 of SEQ ID NO:29 or a corresponding sequence from another Ad-36 strain; and positions 83-116 of SEQ ID NO:29 or a corresponding sequence from another Ad-36 strain. In one aspect, the fusion protein is selected from the group of: SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, and SEQ ID NO:55.
Yet another embodiment of the invention relates to a recombinant nucleic acid molecule encoding any of the fusion proteins described above or elsewhere herein.
Another embodiment of the invention relates to an isolated cell transfected with the recombinant nucleic acid molecule above. In one aspect, the cell is a yeast cell.
Further embodiments of the invention relate to a composition comprising any of the fusion proteins, recombinant nucleic acid molecules, or isolated cells, described above or elsewhere herein. In any of these embodiments, in one aspect, the composition further comprises at least one biological response modifier.
Another embodiment of the invention relates to a method to treat adenovirus-36 (Ad-36) infection in a subject. The method includes the step of administering to a subject that has been infected with Ad-36 any of the compositions described above or elsewhere herein, wherein administration of the composition to the subject reduces Ad-36 infection in the subject. In one aspect, administration of the composition to the subject reduces Ad-36 viral load in the subject.
Yet another embodiment of the invention relates to a method to treat adenovirus-36 (Ad-36) infection in a subject. The method includes the step of administering to a subject that has been infected with Ad-36 any of the compositions described above or elsewhere herein, wherein administration of the composition to the subject reduces the rate of weight gain in the subject.
Another embodiment of the invention relates to a method to treat adenovirus-36 (Ad-36)-associated obesity or excess weight in a subject. The method includes the step of administering to a subject that has been infected with Ad-36 and has a body mass index (BMI) of at least 25, any of the compositions described above or elsewhere herein, wherein administration of the composition to the subject reduces the BMI in the subject.
Yet another embodiment of the invention relates to a method to treat adenovirus-36 (Ad-36)-associated obesity or excess weight in a subject. The method includes the step of administering to a subject that has been infected with Ad-36 and has a body mass index (BMI) of less than 25, any of the compositions described above or elsewhere herein, wherein administration of the composition to the subject reduces the BMI in the subject or reduces the rate of weight gain in the subject.
Another embodiment of the invention relates to a method to elicit an antigen-specific, T cell-mediated immune response against an Ad-36 antigen. The method includes the step of administering to a subject any of the compositions described above or elsewhere herein.
Yet another embodiment of the invention relates to a method to prevent Ad-36 infection in a subject or to reduce the rate of weight gain in a subject. The method includes the step of administering to a subject that has not been infected with Ad-36 any of the compositions described above or elsewhere herein. In one aspect, the subject has a BMI of less than 25. In one aspect, the subject has a BMI of 25 or greater. In one aspect, the subject is between age 2 and age 19. In one aspect, the subject is an adult.
Another embodiment of the invention relates to a method to immunize a population of individuals against Ad-36 infection, comprising administering to the population of individuals any of the compositions described above or elsewhere herein. In one aspect, the individuals are adults. In one aspect, the individuals are age 2 to 19. In one aspect, the individuals have a BMI of 25 or greater. In one aspect, the individuals have a BMI of less than 25.
Another embodiment of the invention relates to any of the compositions described above or elsewhere herein for use to treat Ad-36 infection.
Yet another embodiment of the invention relates to any of the compositions described above or elsewhere herein for use to prevent Ad-36 infection.
Another embodiment of the invention relates to any of the compositions described above or elsewhere herein for use to reduce the rate of weight gain in an individual infected with Ad-36.
Another embodiment of the invention relates to any of the compositions described above or elsewhere herein for use to elicit an Ad-36 immune response in an individual.
Yet another embodiment of the invention relates to the use of any of the compositions described above or elsewhere herein in the preparation of a medicament to treat Ad-36 infection.
Another embodiment of the invention relates to the use of any of the compositions described above or elsewhere herein in the preparation of a medicament to prevent Ad-36 infection.
Another embodiment of the invention relates to the use of any of the compositions described above or elsewhere herein in the preparation of a medicament for reducing the rate of weight gain in an individual infected with Ad-36.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a digitized image of a western blot showing expression of: (1) a yeast-based immunotherapy composition expressing an Ad-36 fusion protein comprising fiber (FIB) (SEQ ID NO:42) under the control of a TEF2 promoter; (2) a yeast-based immunotherapy composition expressing an Ad-36 fusion protein comprising hexon (HEX) (SEQ ID NO:43) under the control of a TEF2 promoter; and (3) a yeast-based immunotherapy composition expressing an Ad-36 fusion protein comprising CR1α and CR1γ (CRAG) (SEQ ID NO:47) under the control of a TEF2 promoter.
FIG. 2 is a digitized image of a western blot showing expression of: (1) a yeast-based immunotherapy composition expressing an Ad-36 fusion protein comprising fiber (Ad-aFL-FIB) (SEQ ID NO:48) under the control of a Cup1 promoter; (2) a yeast-based immunotherapy composition expressing an Ad-36 fusion protein comprising hexon (Ad-aFL-HEX) (SEQ ID NO:50) under the control of a Cup1 promoter; (3) a yeast-based immunotherapy composition expressing an Ad-36 fusion protein comprising CR1α and CR1γ (Ad-aFL-CRAG) (SEQ ID NO:54) under the control of a Cup1 promoter; and (4) a yeast-based immunotherapy composition expressing an Ad-36 fusion protein comprising full length hexon (Ad-aFL-Hexon-Full) (SEQ ID NO:52) under the control of a TEF2 promoter.
FIG. 3 is a bar graph showing the expression of genes encoding Ad-36 E1A, Ad-36 E4orf1, and Ad-36 hexon in rat adipose-derived stem cells (ADS) 15 hours after Ad-36 infection in vitro.
FIG. 4 is a bar graph showing the expression of genes encoding Ad-36 E1A, Ad-36 E4orf1 and Ad-36 hexon in A549 cells (natural host cell for human adenoviruses) 15 hours after Ad-36 infection in vitro.
FIG. 5 is a graph showing the mock control for the early virus particles (V.P.) kinetics study.
FIG. 6 is a graph showing early virus particles (V.P.) kinetics after 107 PFU Ad-36 challenge.
FIG. 7 is a graph showing early virus particles (V.P.) kinetics after 108 PFU Ad-36 challenge.
FIG. 8 is a graph showing early virus particles (V.P.) kinetics after 109 PFU Ad-36 challenge.
FIG. 9 is a digitized image of nested PCR detecting Ad-36 DNA in visceral adipose tissue from rats two weeks after infection with various doses of the virus in vivo.
FIG. 10 is a scatter graph showing body weight gain 18 weeks after Ad-36 infection in rats which were injected with PBS (PBS), control yeast (YVEC), a yeast-based immunotherapy composition expressing a fusion protein comprising Ad-36 CR1α and Ad-36 CR1γ (aFL-Crag), and a yeast-based immunotherapy composition expressing a fusion protein comprising Ad-36 hexon (aFL-Hex).
FIG. 11 is a line graph plotting the time course of median body weight gain over baseline in Ad-36 infected rats which were injected with PBS (PBS), control yeast (YVEC), a yeast-based immunotherapy composition expressing a fusion protein comprising Ad-36 CR1α and Ad-36 CR1γ (aFL-Crag), and a yeast-based immunotherapy composition expressing a fusion protein comprising Ad-36 hexon (aFL-Hex).
FIG. 12 is a bar graph comparing the median body weight gain at week 4 and week 12 after Ad-36 infection in rats which were injected with PBS (PBS, white bars), control yeast (YVEC, gray bars), a yeast-based immunotherapy composition expressing a fusion protein comprising Ad-36 CR1α and Ad-36 CR1γ (aFL-CRAG, black bars), and a yeast-based immunotherapy composition expressing a fusion protein comprising Ad-36 hexon (aFL-HEX, checkered bars).
FIG. 13 is a line graph showing the Ad-36 viral kinetics in the blood for rats that were infected with Ad-36 and injected with PBS (PBS), control yeast (YVEC), a yeast-based immunotherapy composition expressing a fusion protein comprising Ad-36 CR1α and Ad-36 CR1γ (aFL-Crag), and a yeast-based immunotherapy composition expressing a fusion protein comprising Ad-36 hexon (aFL-Hex).
FIG. 14 is a bar graph comparing the total diet consumption (by weight) over 12 weeks of non-Ad-36-infected rats injected with a yeast-based immunotherapeutic expressing an Ad-36 fiber protein and rats which were mock-injected (no immunotherapeutic).
FIG. 15 is a line graph comparing the weight gain over 12 weeks of non-Ad-36-infected rats injected with a yeast-based immunotherapeutic expressing an Ad-36 fiber protein and rats which were mock-injected (no immunotherapeutic).
FIG. 16 is a digitized image of PCR showing Ad-36 DNA in organs and tissues of a rat 15 weeks after intraperitoneal inoculation with the Ad-36 virus.



DETAILED DESCRIPTION OF THE INVENTION
This invention generally relates to immunotherapeutic compositions and methods for the prevention and/or treatment of adenovirus-36 (Ad-36) infection, as well as the prevention and/or treatment of obesity, obesity-associated disorders related to adenovirus-36 infection, and adipose tissue hypertrophy related to Ad-36 infection. The invention includes a yeast-based immunotherapeutic composition (also referred to as yeast-based immunotherapy) comprising a yeast vehicle and Ad-36 antigen(s) that have been designed to elicit a prophylactic and/or therapeutic immune response against Ad-36 infection in a subject. The invention includes the use of such compositions to prevent and/or treat Ad-36 infection. The invention also includes the recombinant nucleic acid molecules used in the yeast-based compositions of the invention, as well as the proteins encoded thereby, for use in any immunotherapeutic composition and/or therapeutic protocol for Ad-36 infection.
The yeast-based, Ad-36-specific immunotherapeutic compositions of the invention induce innate immune responses, as well as adaptive immune responses that specifically target Ad-36, including CD4-dependent TH17 and TH1 T cell responses and antigen-specific CD8+ T cell responses, which include cytotoxic T lymphocyte (CTL) responses. In addition, yeast-based, Ad-36-specific immunotherapeutic compositions of the invention modulate regulatory T cell (Treg) numbers and/or functionality. The breadth of the immune response elicited by Ad-36-specific yeast-based immunotherapy can be modulated toward the desired type of immune response (e.g., TH1 versus TH17 versus Treg), and is well-suited to target Ad-36. In contrast to vaccines that immunize by generating neutralizing antibody responses, yeast-based immunotherapeutic compositions targeting Ad-36 elicit antigen-specific, broad-based, and potent cellular immune responses, including CD4+ T cell responses that are believed to be particularly effective in providing immunity against adenoviruses, since early adenovirus infection may inhibit MHC class I expression. The ability of yeast-based immunotherapy to enhance TH17 T cell responses is also believed to be useful, since IL-17 blocks differentiation of precursor fat cells into bonafide adipocytes and also promotes lipolysis (Shin et al., 2009).
Yeast-based immunotherapy is also highly adept at activating antigen presenting cells, and has a unique ability to cross-prime the immune response, generating CD8+ CTL responses that are typically effective against viral infections, even in the face of what may otherwise be a suppressive environment. Yeast-based immunotherapy can be designed to target regions of Ad-36 that are specific to this virus, or to target regions that are conserved among many adenovirus serotypes and/or to target a mixture of these regions, making the vaccine highly adaptable to the needs of the infected individual, and to target both protective and therapeutic immunity. Since this type of immunotherapy utilizes the natural ability of the antigen presenting cell to present relevant immunogens, it is not necessary to know the precise identity of CTL epitopes or MHC Class II epitopes to produce an effective immunotherapeutic and indeed, multiple CD4 and CD8 T cell epitopes can be targeted in a single composition. Therefore, yeast-based Ad-36 immunotherapy, by activating both the innate and the adaptive immune response, is expected to effectively target Ad-36-infected cells for non-cytopathic clearance, destruction, or both. In addition to being effective in treating excess weight or controlling the rate of weight gain, as well as in treating conditions related to excess weight or weight gain that are associated with Ad-36 infection, the yeast-based immunotherapeutic compositions of the invention are expected to be effective in cases where adipose tissue displays abnormal growth or hypertrophy that is associated with the presence of Ad-36, such as occurs in patients infected with HIV. Indeed, prior to development of full-blown AIDS, HIV-infected patients and patients experiencing Ad-36-associated abnormal adiposity that may develop in the context of reduced or impaired normal immune function, administration of the yeast-based immunotherapy described herein may be effective to treat such patients by providing a broad-based immune response sufficient to reduce Ad-36 viral load and thereby resolve the abnormal adipose tissue hypertrophy. Yeast-based immunotherapy activates multiple pathways of the immune system, and is expected to be effective where other therapeutic approaches, including other immunotherapeutic approaches, lack efficacy.
The compositions, methods and uses of the invention are directed to the prevention and/or treatment of Ad-36 infection, which may reduce or prevent one or more symptoms or conditions associated with Ad-36 infection, including but not limited to, obesity, being overweight, undesirable or abnormal weight gain, and/or abnormal adipose tissue hypertrophy. By addressing these conditions, downstream sequela of obesity and being clinically overweight, or conditions associated with obesity, excess weight, undesirable or abnormal weight gain, or abnormal adipose tissue hypertrophy, may also be reduced. Such conditions include, but are not limited to, high serum cholesterol, high triglycerides, high blood pressure, respiratory conditions, insulin resistance, and type II diabetes.
Compositions of the Invention
One embodiment of the present invention relates to a yeast-based immunotherapy composition which can be used to prevent and/or treat Ad-36 infection or to alleviate at least one symptom resulting from the Ad-36 infection, including but not limited to, obesity, being overweight, undesired or abnormal weight gain, or the propensity therefore. The composition comprises: (a) a yeast vehicle; and (b) one or more Ad-36 protein(s) and/or immunogenic domain(s) thereof (collectively, “Ad-36 antigens”). In conjunction with the yeast vehicle, the Ad-36 proteins are most typically expressed as recombinant proteins by the yeast vehicle (e.g., by an intact yeast or yeast spheroplast, which can optionally be further processed to a yeast cytoplast, yeast ghost, or yeast membrane extract or fraction thereof), although it is an embodiment of the invention that one or much such Ad-36 proteins are loaded into a yeast vehicle or otherwise complexed with, attached to, mixed with or administered with a yeast vehicle as described herein to form a composition of the present invention. According to the present invention, reference to a “heterologous” protein or “heterologous” antigen, including a heterologous fusion protein, in connection with a yeast vehicle of the invention, means that the protein or antigen is not a protein or antigen that is naturally expressed by the yeast, although a fusion protein may include yeast sequences or proteins or portions thereof that are also naturally expressed by yeast. Ad-36 proteins are heterologous with respect to yeast. Target antigens useful in the present invention are typically Ad-36 proteins and/or immunogenic domains thereof.
Another embodiment of the invention relates to novel Ad-36 fusion proteins described herein. In one aspect, such Ad-36 fusion proteins are useful in an immunotherapeutic composition of the invention, including a yeast-based immunotherapeutic composition of the invention. Such fusion proteins, and/or the recombinant nucleic acid molecules encoding such proteins, can also be used in, in combination with, or to produce, a non-yeast-based immunotherapeutic composition, which may include, without limitation, a DNA vaccine, a protein subunit vaccine, a recombinant viral-based immunotherapeutic composition, and a killed or inactivated pathogen vaccine. In another embodiment, such fusion proteins can be used in a diagnostic assay for Ad-36 and/or to generate antibodies against Ad-36. Described herein are exemplary Ad-36 fusion proteins providing selected portions of Ad-36 which are particularly useful in yeast-based immunotherapeutic compositions of the invention.
Adenovirus-36
Adenovirus-36 (also referred to herein as Ad-36, Adv-36, hAdv-36, or HAdV-D36, or adenovirus serotype 36, all of which may be used interchangeably) is one of 52 currently known serotypes of adenoviruses that infect humans, from the Family Adenoviridae, Genus Mastadenovirus, Species Human Adenovirus D (HAdV-D). The virus was first identified in a child with diabetes and enteritis (Wigand et al., 1980, supra) and was deposited with the ATCC as ATCC® Number VR-1610TH by Wigand. In 2010, Arnold and colleagues sequenced the complete Ad-36 genome (Arnold et al., 2010, supra), which is deposited under GenBank® Accession No. GQ384080.1 (GI:261875889). The nucleotide sequence of this representative adenovirus-36 genomic sequence is represented herein by SEQ ID NO:1.
Ad-36 is a double-stranded DNA virus with a 35,152 bp genome, organized into 39 predicted open reading frames (ORFs). The coding sequences that are most divergent from other adenoviruses are found in the hexon, CR1β, CR1γ, and fiber coding regions. Table 1 indicates the individual protein sequences encoded by the Ad-36 genome (SEQ ID NO:1). It is noted that small variations may occur in the amino acid sequence between different viral isolates of the same protein from Ad-36. However, using the guidance provided herein and the reference to the exemplary Ad-36 sequences, one of skill in the art will readily be able to produce a variety of Ad-36-based proteins, including fusion proteins, from any Ad-36 strain (isolate) or genotype, for use in the compositions and methods of the present invention, and as such, the invention is not limited to the specific sequences disclosed herein. Reference to an Ad-36 protein or antigen anywhere in this disclosure, or to any functional, structural, or immunogenic domain thereof, can accordingly be made by reference to a particular sequence from one or more of the sequences presented in this disclosure, or by reference to the same, similar or corresponding sequence from a different Ad-36 isolate (strain). One of skill in the art will readily be able to identify the position of the corresponding sequence for each protein in Table 1 in a given Ad-36 sequence of any Ad-36 strain/isolate, given the guidance provided below, even though some amino acids may differ from those sequences in Table 1.







TABLE 1







Adenovirus-36 Protein Sequences










Protein Name
Sequence Identifier







E1A 28K
SEQ ID NO: 2



E1A 21K
SEQ ID NO: 3



E1B 19K
SEQ ID NO: 4



E1B 55K
SEQ ID NO: 5



pIX
SEQ ID NO: 6



IVa2
SEQ ID NO: 7



Pol protein
SEQ ID NO: 8



13.6K
SEQ ID NO: 9



pTP
SEQ ID NO: 10



52K
SEQ ID NO: 11



pIIIa
SEQ ID NO: 12



III
SEQ ID NO: 13



pVII
SEQ ID NO: 14



V
SEQ ID NO: 15



pX
SEQ ID NO: 16



pVI
SEQ ID NO: 17



Hexon
SEQ ID NO: 18



Protease
SEQ ID NO: 19



DBP
SEQ ID NO: 20



100K
SEQ ID NO: 21



33K
SEQ ID NO: 22



22K
SEQ ID NO: 23



pVIII
SEQ ID NO: 24



E3 12.5K
SEQ ID NO: 25



E3 CR1α
SEQ ID NO: 26



E3 18.4K
SEQ ID NO: 27



E3 50K (CR1β)
SEQ ID NO: 28



E3B1-2 30.8K (CR1γ)
SEQ ID NO: 29



E3B2-2 10K (RIDα)
SEQ ID NO: 30



E3B2-2 14.6K (RIDβ)
SEQ ID NO: 31



E3B 14.7K
SEQ ID NO: 32



U protein
SEQ ID NO: 33



Fiber
SEQ ID NO: 34



E4 ORF 6/7
SEQ ID NO: 35



E4 34K
SEQ ID NO: 36



E4 17K
SEQ ID NO: 37



E4 ORF4
SEQ ID NO: 38



E4 ORF3
SEQ ID NO: 39



E4 ORF2
SEQ ID NO: 40



E4 ORF1
SEQ ID NO: 41










Adenovirus-36 Target Antigens and Constructs.

One embodiment of the invention relates to novel Ad-36 proteins and fusion proteins which can be used as target antigens in an immunotherapeutic composition of the invention, and recombinant nucleic acid molecules encoding these proteins or antigens. Described herein are several different novel Ad-36 proteins and fusion proteins for use as target antigens in a yeast-based immunotherapeutic composition or other composition (e.g., other immunotherapeutic or diagnostic composition) that provide one, two, or multiple (three, four, five, six, seven, eight, nine, ten, or more) proteins and/or one, two or multiple immunogenic domains from one or more proteins, all contained within the same polypeptide and encoded by the same recombinant nucleic acid construct. The proteins used in the compositions of the invention include at least one Ad-36 antigen for immunizing an animal (prophylactically or therapeutically). The composition can include, one, two, a few, several or a plurality of Ad-36 antigens, including one or more immunogenic domains of one or more Ad-36 proteins, as desired.
According to the present invention, the general use herein of the term “antigen” refers: to any portion of a protein (peptide, partial protein, full-length protein), wherein the protein is naturally occurring or synthetically derived, to a cellular composition (whole cell, cell lysate or disrupted cells), to an organism (whole organism, lysate or disrupted cells) or to a carbohydrate, or other molecule, or a portion thereof. An antigen may elicit an antigen-specific immune response (e.g., a humoral and/or a cell-mediated immune response) against the same or similar antigens that are encountered by an element of the immune system (e.g., T cells, antibodies).
An antigen can be as small as a single epitope, or larger, and can include multiple epitopes. As such, the size of an antigen can be as small as about 5-12 amino acids (i.e., a peptide) and as large as: a full length protein, a multimer, a fusion protein, a chimeric protein, a whole cell, a whole microorganism, or any portions thereof (e.g., lysates of whole cells or extracts of microorganisms. In addition, antigens can include carbohydrates, which can be loaded into a yeast vehicle or into a composition of the invention. It will be appreciated that in some embodiments (i.e., when the antigen is expressed by the yeast vehicle from a recombinant nucleic acid molecule), the antigen is a protein, fusion protein, chimeric protein, or fragment thereof, rather than an entire cell or microorganism.
When the antigen is to be expressed in yeast, an antigen is of a minimum size capable of being expressed recombinantly in yeast, and is typically at least or greater than 25 amino acids in length, or at least or greater than 26, at least or greater than 27, at least or greater than 28, at least or greater than 29, at least or greater than 30, at least or greater than 31, at least or greater than 32, at least or greater than 33, at least or greater than 34, at least or greater than 35, at least or greater than 36, at least or greater than 37, at least or greater than 38, at least or greater than 39, at least or greater than 40, at least or greater than 41, at least or greater than 42, at least or greater than 43, at least or greater than 44, at least or greater than 45, at least or greater than 46, at least or greater than 47, at least or greater than 48, at least or greater than 49, or at least or greater than 50 amino acids in length, or is at least 25-50 amino acids in length, at least 30-50 amino acids in length, or at least 35-50 amino acids in length, or at least 40-50 amino acids in length, or at least 45-50 amino acids in length. Smaller proteins may be expressed, and considerably larger proteins (e.g., hundreds of amino acids in length or even a few thousand amino acids in length) may be expressed. In one aspect, a full-length protein or a structural or functional domain thereof or an immunogenic domain thereof that is lacking one or more amino acids from the N- and/or the C-terminus may be expressed (e.g., lacking between about 1 and about 20 amino acids from the N- and/or the C-terminus). Fusion proteins and chimeric proteins are also antigens that may be expressed in the invention. A “target antigen” is an antigen that is specifically targeted by an immunotherapeutic composition of the invention (i.e., an antigen against which elicitation of an immune response is desired). An “Ad-36 antigen” is an antigen derived, designed, or produced from one or more Ad-36 proteins such that targeting the antigen also targets Adenovirus-36.
When referring to stimulation of an immune response, the term “immunogen” is a subset of the term “antigen”, and therefore, in some instances, can be used interchangeably with the term “antigen”. An immunogen, as used herein, describes an antigen which elicits a humoral and/or cell-mediated immune response (i.e., is immunogenic), such that administration of the immunogen to an individual mounts an antigen-specific immune response against the same or similar antigens that are encountered by the immune system of the individual. In one embodiment, the immunogen elicits a cell-mediated immune response, including a CD4+ T cell response (TH1 and/or TH17) and/or a CD8+ T cell response (e.g., a CTL response).
An “immunogenic domain” of a given antigen can be any portion, fragment or epitope of an antigen (e.g., a peptide fragment or subunit or an antibody epitope or other conformational epitope) that contains at least one epitope that can act as an immunogen when administered to an animal. Therefore, an immunogenic domain is larger than a single amino acid and is at least of a size sufficient to contain at least one epitope. For example, a single protein can contain multiple different immunogenic domains. Immunogenic domains need not be linear sequences within a protein, such as in the case of a humoral immune response, where conformational domains are contemplated.
An epitope is defined herein as a single immunogenic site within a given antigen that is sufficient to elicit an immune response when provided to the immune system in the context of appropriate costimulatory signals and/or activated cells of the immune system. In other words, an epitope is the part of an antigen that is recognized by components of the immune system, and may also be referred to as an antigenic determinant. Those of skill in the art will recognize that T cell epitopes are different in size and composition from B cell or antibody epitopes, and that epitopes presented through the Class I MHC pathway differ in size and structural attributes from epitopes presented through the Class II MHC pathway. For example, T cell epitopes presented by Class I MHC molecules are typically between 8 and 11 amino acids in length, whereas epitopes presented by Class II MHC molecules are less restricted in length and may be up to 25 amino acids or longer. In addition, T cell epitopes have predicted structural characteristics depending on the specific MHC molecules bound by the epitope. Epitopes can be linear sequence epitopes or conformational epitopes (conserved binding regions). Most antibodies recognize conformational epitopes.
A “functional domain” of a given protein is a portion or functional unit of the protein that includes sequence or structure that is directly or indirectly responsible for at least one biological or chemical function associated with, ascribed to, or performed by the protein. For example, a functional domain can include an active site for enzymatic activity, a ligand binding site, a receptor binding site, a binding site for a molecule or moiety such as calcium, a phosphorylation site, or a transactivation domain.
A “structural domain” of a given protein is a portion of the protein or an element in the protein's overall structure that has an identifiable structure (e.g., it may be a primary or tertiary structure belonging to and indicative of several proteins within a class or family of proteins), is self-stabilizing and/or may fold independently of the rest of the protein. A structural domain is frequently associated with or features prominently in the biological function of the protein to which it belongs.
In some embodiments, an Ad-36 antigen useful in the present invention is a fusion protein. In one aspect of the invention, such a fusion protein can include two or more antigens. In one aspect, the fusion protein can include two or more immunogenic domains and/or two or more epitopes of one or more Ad-36 proteins. An immunotherapeutic composition containing such antigens may provide antigen-specific immunization in a broad range of patients. For example, a protein or fusion protein encompassed by the invention can include at least a portion or the full-length of any one or more Ad-36 proteins represented in Table 1 (amino acid sequences represented by SEQ ID NOs:2 through 41) and/or any one or more immunogenic domains of any one or more of these Ad-36 proteins, provided in any combination. In one embodiment, a protein useful in the present invention comprises one or more of the following Ad-36 proteins and/or one or more immunogenic domains of any one of more of the following proteins: hexon, fiber, CR1α, CR1γ, and/or E4. In one embodiment, an antigen useful in an immunotherapeutic composition of the invention is a single Ad-36 protein (full-length, near full-length, or portion thereof comprising at least, one, two, three, four or more immunogenic domains of a full-length protein). In one embodiment of the invention, an immunotherapeutic composition includes one, two, three, four, five or more individual yeast vehicles, each expressing or containing a different Ad-36 antigen.
In one embodiment of the invention, the Ad-36 antigen(s) for use in a composition or method of the invention is an Ad-36 antigen comprising or consisting of hexon, fiber, CR1α, CR1γ, and/or E4 and/or one or more domains (structural, functional or immunogenic) thereof, or any combination thereof. In one aspect, any one or more of these proteins or domains is full-length or near full-length. According to the present invention, reference to a “full-length” protein (or a full-length functional domain or full-length immunological domain) includes the full-length amino acid sequence of the protein or functional domain or immunological domain, as described herein or as otherwise known or described in a publicly available sequence. A protein or domain that is “near full-length”, which is also a type of homologue of a protein, differs from a full-length protein or domain, by the addition or deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from the N- and/or C-terminus of such a full-length protein or full-length domain. General reference to a protein or domain can include both full-length and near full-length proteins, as well as other homologues thereof. In one aspect, one or more of these proteins or domains comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more immunogenic domains. In one aspect, any one or more of these proteins or domains comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the linear sequence of the corresponding full-length sequence or of a specified domain or portion of the full-length sequence. An N-terminal expression sequence and/or a C-terminal tag are optional for use with the Ad-36 antigens, and may be selected from several different sequences described elsewhere herein to improve expression, stability, and/or allow for identification and/or purification of the protein, or one or both of the N- or C-terminal sequences are omitted altogether. In addition, many different promoters suitable for use in yeast are known in the art. Furthermore if two or more Ad-36 proteins or domains thereof are included in an Ad-36 antigen, short intervening linker sequences (e.g., 1, 2, 3, 4, or 5, or larger, amino acid peptides) may optionally be introduced between portions of the protein or between the proteins and other elements (e.g., N-terminal peptides) for a variety of reasons, including the introduction of restriction enzyme sites to facilitate cloning and future manipulation of the constructs. Finally, as discussed elsewhere herein, the sequences described herein are exemplary, and may be modified as described above to substitute, add, or delete sequences in order to accommodate preferences for Ad-36 strain or isolate, or consensus sequences and inclusion of preferred T cell epitopes, including dominant and/or subdominant T cell epitopes.
In one aspect of the invention, the Ad-36 antigens useful in the invention are antigens that are divergent, or less conserved, with respect to other adenoviruses (e.g., have relatively low sequence homology or identity with the same or equivalent proteins from other adenovirus serotypes/genotypes). In one embodiment of the invention, a divergent region of a protein, or reference to a protein or region of a protein that is divergent with respect to other proteins of similar structure and/or function (e.g., a region of an Ad-36 protein as compared to approximately the same or similar region of the same protein or an equivalent protein from another adenovirus serotype/genotype), is defined as a protein region for which there is less than about 60% average amino acid identity between the reference sequence and at least five other sequences from other sources that are equivalent in structure and or function, determined, for example, using a BLAST algorithm (described below). Accordingly, proteins or domains or portions of proteins from Ad-36 that are not highly conserved (are relatively or very non-conserved) with other adenovirus serotypes/genotypes are included in antigens and fusion proteins useful in the invention, in one embodiment of the invention. The inclusion of Ad-36 antigens that are divergent from other adenovirus antigens (e.g., similar or equivalent antigens, with respect to structure and/or function, from other adenovirus serotypes or genotypes) has the advantage of creating an immunotherapeutic composition that is specific for Ad-36 and potentially minimizes off-target effects of the immunotherapeutic or dilution of the specificity of the immunotherapeutic. In another aspect of the invention, antigens from conserved regions of Ad-36 (e.g., regions with higher sequence homology to other similar or equivalent antigens from other adenovirus serotypes/genotypes) may be included in a fusion protein or composition of the invention, which has the advantage, for example, of providing a broad spectrum immunotherapeutic with potential applications beyond the treatment or prevention of obesity and adipose-related conditions.
In one exemplary embodiment of the invention, the Ad-36 antigen(s) for use in a composition or method of the invention is a protein comprising Ad-36 sequences, wherein the Ad-36 sequences comprise or consist of Ad-36 fiber protein and/or one or more immunogenic domains of Ad-36 fiber protein. In one aspect, the Ad-36 fiber antigen is full-length fiber protein or near full-length fiber protein. In one aspect, the Ad-36 fiber antigen comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the linear sequence of a full-length Ad-36 fiber antigen or an immunogenic domain or portion thereof. In one aspect, the Ad-36 fiber antigen is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a full-length Ad-36 fiber antigen or an immunogenic domain or portion thereof. In one embodiment, a protein useful in a composition or method of the invention comprises or consists of divergent domains or portions, i.e., relatively non-conserved domains or portions, with respect to other adenoviruses, of Ad-36 fiber protein. For example, an Ad-36 fiber protein construct according to this embodiment can be comprised of a fusion of one, two, three, four, or more different regions of Ad-36 fiber protein that are poorly conserved across human adenoviral genotypes.
Examples of such fusion proteins are described in Example 1. One Ad-36 antigen comprising fiber protein sequence described in Example 1 is a fusion protein expressed as a single polypeptide with the following sequence elements fused in frame from N- to C-terminus, represented by SEQ ID NO:42: (1) an N-terminal peptide to impart resistance to proteasomal degradation and stabilize expression (positions 1 to 6 of SEQ ID NO:42); (2) positions 71-136 of Ad-36 fiber (positions 71-136 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 7-72 of SEQ ID NO:42; (3) positions 145-169 of Ad-36 fiber (positions 145-169 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 73-97 of SEQ ID NO:42; (4) positions 290-313 of Ad-36 fiber (positions 290-313 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 98-194 of SEQ ID NO:42; (5) positions 334-363 of Ad-36 fiber (positions 334-363 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 195-224 of SEQ ID NO:42; and (6) a hexahistidine tag (positions 225-230 of SEQ ID NO:42). A nucleic acid sequence encoding the fusion protein of SEQ ID NO:42 (codon optimized for yeast expression) is also included in the present invention.
Another Ad-36 antigen comprising fiber protein sequence described in Example 1 is a fusion protein expressed as a single polypeptide with the following sequence elements fused in frame from N- to C-terminus, represented by SEQ ID NO:48: (1) an N-terminal peptide to impart resistance to proteasomal degradation and stabilize or enhance expression (SEQ ID NO:56, or positions 1 to 89 of SEQ ID NO: 48); (2) a two amino acid spacer/linker (Thr-Ser) to facilitate cloning and manipulation of the sequences (positions 90 to 91 of SEQ ID NO:48); (3) positions 71-136 of Ad-36 fiber (positions 71-136 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 92-157 of SEQ ID NO:48; (4) positions 145-169 of Ad-36 fiber (positions 145-169 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 158-182 of SEQ ID NO:48; (5) positions 290-313 of Ad-36 fiber (positions 290-313 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 183-279 of SEQ ID NO:48; (6) positions 334-363 of Ad-36 fiber (positions 334-363 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 280-309 of SEQ ID NO:48; and (7) a hexahistidine tag (positions 310-315 of SEQ ID NO:48). A nucleic acid sequence encoding the fusion protein of SEQ ID NO:48 (codon optimized for yeast expression) is also included in the present invention.
The amino acid segments used in these fusion proteins can be modified by the use of additional amino acids flanking either end of any domain; the examples provided herein are exemplary only. In addition, the N-terminal expression sequence (e.g., positions 1 to 6 of SEQ ID NO:42 or positions 1-89 of SEQ ID NO:48) and the C-terminal tag (e.g., positions 225-230 of SEQ ID NO:42 or positions 310-315 of SEQ ID NO:48) are optional, and may be selected instead from other different sequences described elsewhere herein or known in the art to improve expression, stability, and/or allow for identification and/or purification of the protein, or one or both may be omitted altogether. Furthermore, short intervening linker sequences such as that exemplified in SEQ ID NO:48 (e.g., 1, 2, 3, 4, or 5, or larger, amino acid peptides) may be introduced between portions of the fusion protein for a variety of reasons, including the introduction of restriction enzyme sites to facilitate cloning as cleavage sites for host phagosomal proteases, to accelerate protein or antigen processing, and for future manipulation of the constructs. The amino acid sequence consisting of only the Ad-36 fiber proteins in the fusion proteins described above is represented herein by SEQ ID NO:49. SEQ ID NO:49 is a fusion protein expressed as a single polypeptide: (1) positions 71-136 of Ad-36 fiber (positions 71-136 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 1-66 of SEQ ID NO:49; (2) positions 145-169 of Ad-36 fiber (positions 145-169 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 67-91 of SEQ ID NO:49; (3) positions 290-313 of Ad-36 fiber (positions 290-313 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 92-188 of SEQ ID NO:49; and (4) positions 334-363 of Ad-36 fiber (positions 334-363 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 189-218 of SEQ ID NO:49. A nucleic acid sequence encoding the fusion protein of SEQ ID NO:49 (codon optimized for yeast expression) is also included in the present invention.
In another exemplary embodiment of the invention, the Ad-36 antigen(s) for use in a composition or method of the invention is a protein comprising Ad-36 sequences, wherein the Ad-36 sequences comprise or consist of Ad-36 hexon protein and/or one or more immunogenic domains of Ad-36 hexon protein. In one aspect, the Ad-36 hexon antigen is full-length hexon protein or near full-length hexon protein (full-length and near full-length are defined above). In one aspect, the Ad-36 hexon antigen comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the linear sequence of a full-length Ad-36 hexon protein or an immunogenic domain or portion thereof. In one aspect, the Ad-36 hexon antigen is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a full-length Ad-36 hexon protein or an immunogenic domain or portion thereof. In one embodiment, a protein useful in a composition or method of the invention comprises or consists of divergent domains or portions, i.e., relatively non-conserved domains or portions, with respect to other adenoviruses, of Ad-36 hexon protein. For example, an Ad-36 hexon protein construct according to this embodiment can be comprised of a fusion of one, two, three, four, five, or more different regions of Ad-36 hexon protein that are poorly conserved across human adenoviral genotypes.
Examples of such fusion proteins comprising hexon proteins are described in Example 1. One such Ad-36 antigen comprising hexon protein sequences derived from divergent portions of Ad-36 hexon is a fusion protein expressed as a single polypeptide with the following sequence elements fused in frame from N- to C-terminus, represented by SEQ ID NO:43: (1) an N-terminal peptide to impart resistance to proteasomal degradation and stabilize expression (positions 1 to 6 of SEQ ID NO:43); (2) positions 136-218 of Ad-36 hexon (positions 136-218 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 7-89 of SEQ ID NO:43; (3) positions 235-285 of Ad-36 hexon (positions 235-285 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 90-141 of SEQ ID NO:43; (4) positions 297-308 of Ad-36 hexon (positions 297-308 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 142-153 of SEQ ID NO:43; (5) positions 410-450 of Ad-36 hexon (positions 410-450 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 154-194 of SEQ ID NO:43; and (6) a hexahistidine tag (positions 195-200 of SEQ ID NO:43). A nucleic acid sequence encoding the fusion protein of SEQ ID NO:43 (codon optimized for yeast expression) is also included in the present invention.
Another Ad-36 antigen comprising hexon protein sequence derived from divergent portions of Ad-36 sequence is a fusion protein expressed as a single polypeptide with the following sequence elements fused in frame from N- to C-terminus, represented by SEQ ID NO:50: (1) an N-terminal peptide to impart resistance to proteasomal degradation and stabilize or enhance expression (SEQ ID NO:56, or positions 1 to 89 of SEQ ID NO:50); 2) a two amino acid spacer/linker (Thr-Ser) to facilitate cloning and manipulation of the sequences (positions 90 to 91 of SEQ ID NO:50); (3) positions 136-218 of Ad-36 hexon (positions 136-218 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 92-174 of SEQ ID NO:50; (4) positions 235-285 of Ad-36 hexon (positions 235-285 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 175-226 of SEQ ID NO:50; (5) positions 297-308 of Ad-36 hexon (positions 297-308 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 227-238 of SEQ ID NO:50; (6) positions 410-450 of Ad-36 hexon (positions 410-450 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 239-279 of SEQ ID NO:50; and (7) a hexahistidine tag (positions 280-285 of SEQ ID NO:50). A nucleic acid sequence encoding the fusion protein of SEQ ID NO: 50 (codon optimized for yeast expression) is also included in the present invention.
The amino acid segments used in these hexon-based fusion proteins described above can be modified by the use of additional amino acids flanking either end of any domain; the examples provided herein are exemplary only. In addition, the N-terminal expression sequence (e.g., positions 1 to 6 of SEQ ID NO:43 or positions 1-89 of SEQ ID NO:50) and the C-terminal tag (e.g., positions 195-200 of SEQ ID NO:43 or positions 280-285 of SEQ ID NO:50) are optional, and may be selected instead from other different sequences described elsewhere herein or known in the art to improve expression, stability, and/or allow for identification and/or purification of the protein, or one or both may be omitted altogether. Furthermore, short intervening linker sequences such as that exemplified in SEQ ID NO:48 (e.g., 1, 2, 3, 4, or 5, or larger, amino acid peptides) may be introduced between portions of the fusion protein for a variety of reasons, including the introduction of restriction enzyme sites to facilitate cloning as cleavage sites for host phagosomal proteases, to accelerate protein or antigen processing, and for future manipulation of the constructs. The amino acid sequence consisting of only the Ad-36 hexon proteins in the fusion proteins described above is represented herein by SEQ ID NO:51. SEQ ID NO:51 is a fusion protein expressed as a single polypeptide: (1) positions 136-218 of Ad-36 hexon (positions 136-218 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 1-83 of SEQ ID NO:51; (2) positions 235-285 of Ad-36 hexon (positions 235-285 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 84-135 of SEQ ID NO:51; (3) positions 297-308 of Ad-36 hexon (positions 297-308 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 136-147 of SEQ ID NO:51; and (4) positions 410-450 of Ad-36 hexon (positions 410-450 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 148-188 of SEQ ID NO:51. Any suitable N-terminal and/or C-terminal sequence may be appended to this sequence, as described above for SEQ ID NOs:43 and 50, or one or both may be omitted. A nucleic acid sequence encoding the fusion protein of SEQ ID NO:51 (codon optimized for yeast expression) is also included in the present invention.
An Ad-36 antigen comprising full-length or near full-length hexon protein sequence described in Example 1 is a fusion protein expressed as a single polypeptide with the following sequence elements fused in frame from N- to C-terminus, represented by SEQ ID NO:44: (1) an N-terminal peptide to impart resistance to proteasomal degradation and stabilize expression (positions 1 to 6 of SEQ ID NO:44); (2) positions 2-944 of Ad-36 hexon (positions 2-944 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 7-949 of SEQ ID NO:44; and (3) a hexahistidine tag (positions 950-955 of SEQ ID NO:44). This construct contains demonstrated or putative MHC Class I epitopes (e.g., positions 119-129 of SEQ ID NO:44; positions 319-327 of SEQ ID NO:44; positions 710-718 of SEQ ID NO:44; positions 843-851 of SEQ ID NO:44; or positions 909-915 of SEQ ID NO:44), and demonstrated or putative MHC Class II epitopes (e.g., positions 15-25 of SEQ ID NO:44; positions 31-41 of SEQ ID NO:44; 321-335 of SEQ ID NO:44; positions 373-383 of SEQ ID NO:44; positions 707-718 of SEQ ID NO:44; or positions 862-872 of SEQ ID NO:44). A nucleic acid sequence encoding the fusion protein of SEQ ID NO:44 (codon optimized for yeast expression) is also included in the present invention.
Another Ad-36 antigen comprising full-length or near full-length hexon protein sequence described in Example 1 is a fusion protein expressed as a single polypeptide with the following sequence elements fused in frame from N- to C-terminus, represented by SEQ ID NO:52: (1) an N-terminal peptide to impart resistance to proteasomal degradation and stabilize or enhance expression (SEQ ID NO:56, or positions 1 to 89 of SEQ ID NO:52); 2) a two amino acid spacer/linker (Thr-Ser) to facilitate cloning and manipulation of the sequences (positions 90 to 91 of SEQ ID NO:52); (3) positions 2-944 of Ad-36 hexon (positions 2-944 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 92-1034 of SEQ ID NO:52; and (3) a hexahistidine tag (positions 1035-1040 of SEQ ID NO:52). This construct contains demonstrated or putative MHC Class I epitopes (e.g., positions 204-214 of SEQ ID NO:52; positions 404-412 of SEQ ID NO:52; positions 795-803 of SEQ ID NO:52; positions 928-936 of SEQ ID NO:52; or positions 994-1000 of SEQ ID NO:52), and demonstrated or putative MHC Class II epitopes (e.g., positions 100-110 of SEQ ID NO:52; positions 116-126 of SEQ ID NO:52; 406-420 of SEQ ID NO:52; positions 458-468 of SEQ ID NO:52; positions 792-803 of SEQ ID NO:52; or positions 947-957 of SEQ ID NO:52). A nucleic acid sequence encoding the fusion protein of SEQ ID NO:52 (codon optimized for yeast expression) is also included in the present invention.
The amino acid segments used in these hexon-based fusion proteins described above can be modified by the use of additional amino acids flanking either end of any domain; the examples provided herein are exemplary only. In addition, the N-terminal expression sequence (e.g., positions 1 to 6 of SEQ ID NO:44 or positions 1-89 of SEQ ID NO:52) and the C-terminal tag (e.g., positions 950-955 of SEQ ID NO:44 or positions 1035-1040 of SEQ ID NO:52) are optional, and may be selected instead from other different sequences described elsewhere herein or known in the art to improve expression, stability, and/or allow for identification and/or purification of the protein, or one or both may be omitted altogether. Furthermore, short intervening linker sequences such as that exemplified in SEQ ID NO:48 (e.g., 1, 2, 3, 4, or 5, or larger, amino acid peptides) may be introduced between portions of the fusion protein for a variety of reasons, including the introduction of restriction enzyme sites to facilitate cloning as cleavage sites for host phagosomal proteases, to accelerate protein or antigen processing, and for future manipulation of the constructs. The amino acid sequence consisting of only the Ad-36 hexon protein in the fusion proteins described above is represented herein by SEQ ID NO:53. SEQ ID NO:53 is a fusion protein expressed as a single polypeptide and comprises positions 2-944 of Ad-36 hexon (positions 2-944 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 1-943 of SEQ ID NO:53. This construct contains demonstrated or putative MHC Class I epitopes (e.g., positions 113-123 of SEQ ID NO:53; positions 313-321 of SEQ ID NO:53; positions 704-712 of SEQ ID NO:53; positions 837-845 of SEQ ID NO:53; or positions 903-909 of SEQ ID NO:53), and demonstrated or putative MHC Class II epitopes (e.g., positions 9-19 of SEQ ID NO:53; positions 25-35 of SEQ ID NO:53; 315-329 of SEQ ID NO:53; positions 367-377 of SEQ ID NO:53; positions 701-712 of SEQ ID NO:53; or positions 856-866 of SEQ ID NO:53). Any suitable N-terminal and/or C-terminal sequence may be appended to this sequence, as described above for SEQ ID NOs:44 and 52, or one or both may be omitted. A nucleic acid sequence encoding the fusion protein of SEQ ID NO:53 (codon optimized for yeast expression) is also included in the present invention.
In another exemplary embodiment of the invention, the Ad-36 antigen(s) for use in a composition or method of the invention is a protein comprising Ad-36 sequences, wherein the Ad-36 sequences comprise or consist of Ad-36 hexon protein and fiber protein and/or one or more immunogenic domains of hexon protein and fiber protein. In one aspect, the Ad-36 hexon and/or the Ad-36 fiber antigen are full-length proteins or near full-length proteins (full-length and near full-length are defined above). In one aspect, the Ad-36 hexon and/or fiber antigen comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the linear sequence of a full-length Ad-36 protein or immunogenic domain or portion thereof. In one aspect, the Ad-36 hexon and/or the Ad-36 fiber antigen is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a full-length Ad-36 protein or immunogenic domain or portion thereof. In one embodiment, a protein useful in a composition or method of the invention comprises or consists of divergent domains or portions, i.e., relatively non-conserved domains or portions, with respect to other adenoviruses, of Ad-36 hexon protein and Ad-36 fiber protein. For example, an Ad-36 hexon-fiber or fiber-hexon protein construct according to this embodiment can be comprised of a fusion of one, two, three, four, five, or more different regions of Ad-36 hexon protein that are poorly conserved across human adenoviral genotypes, and one, two, three, four, five or more different regions of Ad-36 fiber protein that are poorly conserved across human adenoviral genotypes.
Examples of such fusion proteins comprising both hexon and fiber proteins are described in Example 1. One such Ad-36 antigen comprising hexon and fiber protein sequences derived from divergent portions of Ad-36 hexon and fiber is a fusion protein expressed as a single polypeptide with the following sequence elements fused in frame from N- to C-terminus, represented by SEQ ID NO:45: (1) an N-terminal peptide to impart resistance to proteasomal degradation and stabilize expression (positions 1 to 6 of SEQ ID NO:45); (2) positions 71-136 of Ad-36 fiber (positions 71-136 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 7-72 of SEQ ID NO:45; (3) positions 145-169 of Ad-36 fiber (positions 145-169 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 73-97 of SEQ ID NO:45; (4) positions 290-313 of Ad-36 fiber (positions 290-313 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 98-194 of SEQ ID NO:45; (5) positions 334-363 of Ad-36 fiber (positions 334-363 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 195-224 of SEQ ID NO:45; (6) positions 136-218 of Ad-36 hexon (positions 136-218 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 225-307 of SEQ ID NO:45; (7) positions 235-285 of Ad-36 hexon (positions 235-285 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 308-359 of SEQ ID NO:45; (8) positions 297-308 of Ad-36 hexon (positions 297-308 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 360-371 of SEQ ID NO:45; (9) positions 410-450 of Ad-36 hexon (positions 410-450 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 372-412 of SEQ ID NO:45; and (10) a hexahistidine tag (positions 413-418 of SEQ ID NO:45).
Another Ad-36 antigen comprising hexon and fiber protein sequences derived from divergent portions of Ad-36 hexon and fiber is a fusion protein expressed as a single polypeptide with the following sequence elements fused in frame from N- to C-terminus, represented by SEQ ID NO:46: (1) an N-terminal peptide to impart resistance to proteasomal degradation and stabilize expression (positions 1 to 6 of SEQ ID NO:46); (2) positions 136-218 of Ad-36 hexon (positions 136-218 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 7-89 of SEQ ID NO:46; (3) positions 235-285 of Ad-36 hexon (positions 235-285 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 90-141 of SEQ ID NO:46; (4) positions 297-308 of Ad-36 hexon (positions 297-308 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 142-153 of SEQ ID NO:46; (5) positions 410-450 of Ad-36 hexon (positions 410-450 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 154-194 of SEQ ID NO:46; (6) positions 71-136 of Ad-36 fiber (positions 71-136 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 195-260 of SEQ ID NO:46; (7) positions 145-169 of Ad-36 fiber (positions 145-169 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 261-285 of SEQ ID NO:46; (8) positions 290-313 of Ad-36 fiber (positions 290-313 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 286-382 of SEQ ID NO:46; (9) positions 334-363 of Ad-36 fiber (positions 334-363 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 383-412 of SEQ ID NO:46; and (10) a hexahistidine tag (positions 413-418 of SEQ ID NO:46). A nucleic acid sequence encoding the fusion protein of SEQ ID NO:45 or SEQ ID NO:46 (codon optimized for yeast expression) is also included in the present invention.
The amino acid segments used in any of the fusion proteins described above can be modified by the use of additional amino acids flanking either end of any domain; the examples provided herein are exemplary only. The N-terminal expression sequence (positions 1 to 6 of SEQ ID NO:45 or 46) and the C-terminal tag (positions 413-418 of SEQ ID NO:45 or 46) are optional, and may be selected instead from other different sequences described elsewhere herein or known in the art to improve expression, stability, and/or allow for identification and/or purification of the protein, or one or both may be omitted altogether. Furthermore, short intervening linker sequences such as that exemplified in SEQ ID NO:48 (e.g., 1, 2, 3, 4, or 5, or larger, amino acid peptides) may be introduced between portions of the fusion protein for a variety of reasons, including the introduction of restriction enzyme sites to facilitate cloning as cleavage sites for host phagosomal proteases, to accelerate protein or antigen processing, and for future manipulation of the constructs. For example, a fusion protein omitting both the N- and C-terminal sequences of SEQ ID NO:45 is represented by positions 7-412 of SEQ ID NO:45 and a fusion protein omitting both the N- and C-terminal sequences of SEQ ID NO:46 is represented by positions 7-412 of SEQ ID NO:46.
In yet another exemplary embodiment of the invention, the Ad-36 antigen(s) for use in a composition or method of the invention is a protein comprising Ad-36 sequences, wherein the Ad-36 sequences comprise or consist of Ad-36 CR1α protein and/or Ad-36 CR1γ and/or one or more immunogenic domains of CR1α and/or CR1γ. In one aspect, the Ad-36 CR1α and/or the Ad-36 CR1γ antigen are full-length proteins or near full-length proteins (full-length and near full-length are defined above). In one aspect, the Ad-36 CR1α and/or the Ad-36 CR1γ antigen comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the linear sequence of a full-length Ad-36 protein or immunogenic domain or portion thereof. In one aspect, the Ad-36 CR1α and/or the Ad-36 CR1γ antigen is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a full-length Ad-36 protein or immunogenic domain or portion thereof. In one embodiment, a protein useful in a composition or method of the invention comprises or consists of divergent domains or portions, i.e., relatively non-conserved domains or portions, with respect to other adenoviruses, of Ad-36 CR1α and/or CR1γ. For example, an Ad-36 CR1α and/or CR1γ protein construct according to this embodiment can be comprised of a fusion of one, two, three, four, five, or more different regions of Ad-36 CR1α and/or CR1γ protein that are poorly conserved across human adenoviral genotypes. In one embodiment, a notably hydrophobic N-terminal region is omitted from CR1α in a protein of the invention (e.g., about positions 1-17 of the mature protein) to minimize the risk of aggregation and/or insolubility when that protein is expressed in yeast. In one embodiment, a C-terminal segment of mature CR1α is omitted from proteins used in the invention because of notable hydrophobicity (positions 158-177) plus high sequence conservation with other adenovirus serotypes/genotypes (positions 158 through C-terminus). In another embodiment, the N-terminal positions 1-18 of CR1γ are omitted from proteins used in the invention as they contain both highly conserved amino acid positions with other adenovirus serotypes/genotypes, and they also contain a very hydrophobic element.
Examples of such fusion proteins comprising both CR1α and CR1γ proteins are described in Example 1. One such Ad-36 antigen comprising CR1α and CR1γ protein sequences derived from divergent and/or selected portions of Ad-36 CR1α and CR1γ is a fusion protein expressed as a single polypeptide with the following sequence elements fused in frame from N- to C-terminus, represented by SEQ ID NO:47: (1) an N-terminal peptide to impart resistance to proteasomal degradation and stabilize expression (positions 1 to 6 of SEQ ID NO:47); (2) positions 18-60 of CR1α (positions 18-60 of SEQ ID NO:26 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 7-49 of SEQ ID NO:47; (3) positions 123-157 of Ad-36 CR1α (positions 123-157 of SEQ ID NO:26 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 50-84 of SEQ ID NO:47; (4) positions 19-60 of Ad-36 CR1γ (positions 19-60 of SEQ ID NO:29 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 85-126 of SEQ ID NO:47; (5) positions 83-116 of Ad-36 CR1γ (positions 83-116 of SEQ ID NO:29 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 127-160 of SEQ ID NO:47; and (6) a hexahistidine tag (positions 161-166 of SEQ ID NO:47). The amino acid segments used in any of the fusion proteins described above can be modified by the use of additional amino acids flanking either end of any domain; the examples provided herein are exemplary only. A nucleic acid sequence encoding the fusion protein of SEQ ID NO:47 (codon optimized for yeast expression) is also included in the present invention.
Another Ad-36 antigen comprising CR1α and CR1γ protein sequences described in Example 1 is a fusion protein expressed as a single polypeptide with the following sequence elements fused in frame from N- to C-terminus, represented by SEQ ID NO:54: (1) an N-terminal peptide to impart resistance to proteasomal degradation and stabilize or enhance expression (SEQ ID NO:56, or positions 1 to 89 of SEQ ID NO:54); 2) a two amino acid spacer/linker (Thr-Ser) to facilitate cloning and manipulation of the sequences (positions 90 to 91 of SEQ ID NO:54); (3) positions 18-60 of CR1α (positions 18-60 of SEQ ID NO:26 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 92-134 of SEQ ID NO:54; (4) positions 123-157 of Ad-36 CR1α (positions 123-157 of SEQ ID NO:26 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 135-169 of SEQ ID NO:54; (5) positions 19-60 of Ad-36 CR1γ (positions 19-60 of SEQ ID NO:29 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 170-211 of SEQ ID NO:54; (6) positions 83-116 of Ad-36 CR1γ (positions 83-116 of SEQ ID NO:29 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 212-245 of SEQ ID NO:54; and (7) a hexahistidine tag (positions 246-251 of SEQ ID NO:54). A nucleic acid sequence encoding the fusion protein of SEQ ID NO:54 (codon optimized for yeast expression) is also included in the present invention.
The amino acid segments used in these CR1α and CR1γ-based fusion proteins described above can be modified by the use of additional amino acids flanking either end of any domain; the examples provided herein are exemplary only. In addition, the N-terminal expression sequence (e.g., positions 1 to 6 of SEQ ID NO:47 or positions 1-89 of SEQ ID NO:54) and the C-terminal tag (e.g., positions 161-166 of SEQ ID NO:47 or positions 246-251 of SEQ ID NO:54) are optional, and may be selected instead from other different sequences described elsewhere herein or known in the art to improve expression, stability, and/or allow for identification and/or purification of the protein, or one or both may be omitted altogether. Furthermore, short intervening linker sequences such as that exemplified in SEQ ID NO:48 (e.g., 1, 2, 3, 4, or 5, or larger, amino acid peptides) may be introduced between portions of the fusion protein for a variety of reasons, including the introduction of restriction enzyme sites to facilitate cloning as cleavage sites for host phagosomal proteases, to accelerate protein or antigen processing, and for future manipulation of the constructs. The amino acid sequence consisting of only the Ad-36 CR1α and CR1γ proteins in the fusion proteins described above is represented herein by SEQ ID NO:55. SEQ ID NO:55 is a fusion protein expressed as a single polypeptide: (1) positions 18-60 of CR1α (positions 18-60 of SEQ ID NO:26 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 1-43 of SEQ ID NO:55; (2) positions 123-157 of Ad-36 CR1α (positions 123-157 of SEQ ID NO:26 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 44-78 of SEQ ID NO:55; (3) positions 19-60 of Ad-36 CR1γ (positions 19-60 of SEQ ID NO:29 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 79-120 of SEQ ID NO:55; and (4) positions 83-116 of Ad-36 CR1γ (positions 83-116 of SEQ ID NO:29 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 121-154 of SEQ ID NO:55. Any suitable N-terminal and/or C-terminal sequence may be appended to this sequence, as described above for SEQ ID NOs:47 and 54, or one or both may be omitted. A nucleic acid sequence encoding the fusion protein of SEQ ID NO:55 (codon optimized for yeast expression) is also included in the present invention.
The invention also includes homologues of any of the above-described fusion proteins, as well as the use of homologues, variants, or mutants of the individual Ad-36 proteins or portions thereof that are part of such fusion proteins. In one aspect, the invention includes the use of fusion proteins having amino acid sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the fusion proteins described herein over the full length of the fusion protein, or with respect to a defined protein or domain thereof (immunological domain or functional domain (domain with at least one biological activity)) that forms part of the fusion protein.
Recombinant nucleic acid molecules useful in a yeast-based composition of the invention do not include the full length genome of Ad-36, but rather include less than the full-length Ad-36 genome. Typically, recombinant nucleic acid molecules useful in a yeast-based composition of the invention include one or more full-length coding sequences and/or one or more coding sequences of domains (immunogenic or functional) for Ad-36 proteins. Proteins included in a single yeast-based composition of the invention do not include all of the proteins encoded by Ad-36. Preferably, a yeast-based composition comprises one, two, three, four, five, six, seven, eight, nine, ten or more proteins encoded by Ad-36, and/or one or more immunogenic domains of any one or more Ad-36 proteins.
Recombinant nucleic acid molecules and the proteins encoded thereby, including fusion proteins, as one embodiment of the invention, may be used in yeast-based immunotherapy compositions, or for any other suitable purpose for an Ad-36 antigen(s), including in an in vitro assay, for the production of antibodies, or in another immunotherapy composition, including another vaccine, that is not based on the yeast-based immunotherapy described herein. Expression of the proteins/antigens by yeast is one preferred embodiment, although other expression systems may be used to produce the proteins/antigens for applications other than a yeast-based immunotherapy composition.
Yeast-Based Immunotherapy Compositions. In various embodiments of the invention, the invention includes the use of at least one “yeast-based immunotherapeutic composition” (which phrase may be used interchangeably with “yeast-based immunotherapy product”, “yeast-based immunotherapy composition”, “yeast-based composition”, “yeast-based immunotherapeutic”, “yeast-based vaccine”, or derivatives of these phrases). An “immunotherapeutic composition” is a composition that elicits an immune response sufficient to achieve at least one therapeutic benefit in a subject. As used herein, yeast-based immunotherapeutic composition refers to a composition that includes a yeast vehicle component and that elicits an immune response sufficient to achieve at least one therapeutic benefit in a subject. More particularly, a yeast-based immunotherapeutic composition is a composition that includes a yeast vehicle component and can elicit or induce an immune response, such as a cellular immune response, including without limitation a T cell-mediated cellular immune response. In one aspect, a yeast-based immunotherapeutic composition useful in the invention is capable of inducing a CD8+ and/or a CD4+ T cell-mediated immune response and in one aspect, a CD8+ and a CD4+ T cell-mediated immune response. A CD4+ immune response can include TH1 immune responses, TH17 immune responses, or both, as yeast-based immunotherapeutics are capable of generating both types of response. A CD8+ immune response can include a cytotoxic T lymphocyte (CTL) response, as yeast-based immunotherapeutics are capable of generating such responses. In one aspect, a yeast-based immunotherapeutic composition modulates the number and/or functionality of regulatory T cells (Tregs) in a subject. Yeast-based immunotherapy can also be modified to promote one type of response over another, e.g., by the addition of cytokines, antibodies, and/or modulating the manufacturing process for the yeast. Optionally, a yeast-based immunotherapeutic composition is capable of eliciting a humoral immune response. A yeast-based immunotherapeutic composition useful in the present invention can, for example, elicit an immune response in an individual such that the individual is protected from Ad-36 infection and/or is treated for Ad-36 infection or for symptoms resulting from Ad-36 infection.
Yeast-based immunotherapy compositions of the invention may be either “prophylactic” or “therapeutic”. When provided prophylactically, the compositions of the present invention are provided in advance of any symptom of Ad-36 infection. Such a composition could be administered at birth, in early childhood, or to adults, and can include obese, overweight, non-obese, and non-overweight subjects. The prophylactic administration of the immunotherapy compositions serves to prevent subsequent Ad-36 infection, to resolve an infection more quickly or more completely if Ad-36 infection subsequently ensues, and/or to prevent or ameliorate the symptoms of Ad-36 infection if infection subsequently ensues. When provided therapeutically, the immunotherapy compositions are provided at or after the onset of Ad-36 infection, with the goal of preventing or ameliorating at least one symptom of the infection (e.g., preventing obesity in non-obese, Ad-36-infected subjects, or reducing weight in obese, Ad-36-infected subjects) and preferably, with a goal of eliminating the infection, providing a long lasting remission of infection, and/or providing long term immunity against subsequent infections or reactivations of the virus.
Typically, a yeast-based immunotherapy composition includes a yeast vehicle and at least one antigen (e.g., an Ad-36 protein) or immunogenic domain thereof expressed by, attached to, or mixed with the yeast vehicle, wherein the antigen is heterologous to the yeast, and wherein the antigen comprises one or more Ad-36 proteins or immunogenic domains thereof. In some embodiments, the antigen or immunogenic domain thereof is provided as a fusion protein. Several Ad-36 fusion proteins suitable for use in the compositions and methods of the invention have been described above. In one aspect of the invention, fusion protein can include two or more antigens. In one aspect, the fusion protein can include two or more immunogenic domains of one or more antigens, or two or more epitopes of one or more antigens.
A TARMOGEN® is one non-limiting example of a yeast-based immunotherapy composition that is useful in the present invention. A TARMOGEN® (TARgeted MOlecular immunoGEN, GlobeImmune, Inc., Louisville, Colo.) generally refers to a yeast vehicle expressing one or more heterologous antigens extracellularly (on its surface), intracellularly (internally or cytosolically) or both extracellularly and intracellularly. TARMOGEN®s have been generally described in the art. See, e.g., U.S. Pat. No. 5,830,463.
Yeast-based immunotherapy compositions, and methods of making and generally using the same, are described in detail, for example, in U.S. Pat. No. 5,830,463, U.S. Pat. No. 7,083,787, U.S. Pat. No. 7,736,642, Stubbs et al., Nat. Med. 7:625-629 (2001), Lu et al., Cancer Research 64:5084-5088 (2004), and in Bernstein et al., Vaccine 2008 Jan. 24; 26(4):509-21, each of which is incorporated herein by reference in its entirety. These yeast-based immunotherapeutic products have been shown to elicit immune responses, including cellular and humoral immune responses. Yeast-based immunotherapeutic products are capable of killing target cells expressing a variety of antigens in vivo, in a variety of animal species, and to do so via antigen-specific, CD4+ and CD8+ T cell-mediated immune responses. Additional studies have shown that yeast are avidly phagocytosed by and directly activate dendritic cells which then present yeast-associated proteins to CD4+ and CD8+ T cells in a highly efficient manner. See, e.g., Stubbs et al. Nature Med. 5:625-629 (2001) and U.S. Pat. No. 7,083,787.
In any of the yeast-based immunotherapy compositions used in the present invention, the following aspects related to the yeast vehicle are included in the invention. According to the present invention, a yeast vehicle is any yeast cell (e.g., a whole or intact cell) or a derivative thereof (see below) that can be used in conjunction with one or more antigens, immunogenic domains thereof or epitopes thereof in a therapeutic composition of the invention, or in one aspect, the yeast vehicle can be used alone or as an adjuvant. The yeast vehicle can therefore include, but is not limited to, a live intact (whole) yeast microorganism (i.e., a yeast cell having all its components including a cell wall), a killed (dead) or inactivated intact yeast microorganism, or derivatives of intact yeast including: a yeast spheroplast (i.e., a yeast cell lacking a cell wall), a yeast cytoplast (i.e., a yeast cell lacking a cell wall and nucleus), a yeast ghost (i.e., a yeast cell lacking a cell wall, nucleus and cytoplasm), a subcellular yeast membrane extract or fraction thereof (also referred to as a yeast membrane particle and previously as a subcellular yeast particle), any other yeast particle, or a yeast cell wall preparation.
Yeast spheroplasts are typically produced by enzymatic digestion of the yeast cell wall. Such a method is described, for example, in Franzusoff et al., 1991, Meth. Enzymol. 194, 662-674., incorporated herein by reference in its entirety.
Yeast cytoplasts are typically produced by enucleation of yeast cells. Such a method is described, for example, in Coon, 1978, Natl. Cancer Inst. Monogr. 48, 45-55 incorporated herein by reference in its entirety.
Yeast ghosts are typically produced by resealing a permeabilized or lysed cell and can, but need not, contain at least some of the organelles of that cell. Such a method is described, for example, in Franzusoff et al., 1983, J. Biol. Chem. 258, 3608-3614 and Bussey et al., 1979, Biochim. Biophys. Acta 553, 185-196, each of which is incorporated herein by reference in its entirety.
A yeast membrane particle (subcellular yeast membrane extract or fraction thereof) refers to a yeast membrane that lacks a natural nucleus or cytoplasm. The particle can be of any size, including sizes ranging from the size of a natural yeast membrane to microparticles produced by sonication or other membrane disruption methods known to those skilled in the art, followed by resealing. A method for producing subcellular yeast membrane extracts is described, for example, in Franzusoff et al., 1991, Meth. Enzymol. 194, 662-674. One may also use fractions of yeast membrane particles that contain yeast membrane portions and, when the antigen or other protein was expressed recombinantly by the yeast prior to preparation of the yeast membrane particles, the antigen or other protein of interest. Antigens or other proteins of interest can be carried inside the membrane, on either surface of the membrane, or combinations thereof (i.e., the protein can be both inside and outside the membrane and/or spanning the membrane of the yeast membrane particle). In one embodiment, a yeast membrane particle is a recombinant yeast membrane particle that can be an intact, disrupted, or disrupted and resealed yeast membrane that includes at least one desired antigen or other protein of interest on the surface of the membrane or at least partially embedded within the membrane.
An example of a yeast cell wall preparation is a preparation of isolated yeast cell walls carrying an antigen on its surface or at least partially embedded within the cell wall such that the yeast cell wall preparation, when administered to an animal, stimulates a desired immune response against a disease target.
Any yeast strain can be used to produce a yeast vehicle of the present invention. Yeast are unicellular microorganisms that belong to one of three classes: Ascomycetes, Basidiomycetes and Fungi Imperfecti. One consideration for the selection of a type of yeast for use as an immune modulator is the pathogenicity of the yeast. In one embodiment, the yeast is a non-pathogenic strain such as Saccharomyces cerevisiae. The selection of a non-pathogenic yeast strain minimizes any adverse effects to the individual to whom the yeast vehicle is administered. However, pathogenic yeast may be used if the pathogenicity of the yeast can be negated by any means known to one of skill in the art (e.g., mutant strains). In accordance with one aspect of the present invention, nonpathogenic yeast strains are used.
Genera of yeast strains that may be used in the invention include but are not limited to Saccharomyces, Candida (which can be pathogenic), Cryptococcus, Hansenula, Kluyveromyces, Pichia, Rhodotorula, Schizosaccharomyces and Yarrowia. In one aspect, yeast genera are selected from Saccharomyces, Candida, Hansenula, Pichia or Schizosaccharomyces, and in one aspect, Saccharomyces is used. Species of yeast strains that may be used in the invention include but are not limited to Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Candida albicans, Candida kefyr, Candida tropicalis, Cryptococcus laurentii, Cryptococcus neoformans, Hansenula anomala, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Kluyveromyces marxianus var. lactis, Pichia pastoris, Rhodotorula rubra, Schizosaccharomyces pombe, and Yarrowia lipolytica. It is to be appreciated that a number of these species include a variety of subspecies, types, subtypes, etc. that are intended to be included within the aforementioned species. In one aspect, yeast species used in the invention include S. cerevisiae, C. albicans, H. polymorpha, P. pastoris and S. pombe. S. cerevisiae is useful as it is relatively easy to manipulate and being “Generally Recognized As Safe” or “GRAS” for use as food additives (GRAS, FDA proposed Rule 62FR18938, Apr. 17, 1997). One embodiment of the present invention is a yeast strain that is capable of replicating plasmids to a particularly high copy number, such as a S. cerevisiae cir° strain. The S. cerevisiae strain is one such strain that is capable of supporting expression vectors that allow one or more target antigen(s) and/or antigen fusion protein(s) and/or other proteins to be expressed at high levels. In addition, any mutant yeast strains can be used in the present invention, including those that exhibit reduced post-translational modifications of expressed target antigens or other proteins, such as mutations in the enzymes that extend N-linked glycosylation.
In one embodiment, a yeast vehicle of the present invention is capable of fusing with the cell type to which the yeast vehicle and antigen/agent is being delivered, such as a dendritic cell or macrophage, thereby effecting particularly efficient delivery of the yeast vehicle, and in many embodiments, the antigen(s) or other agent, to the cell type. As used herein, fusion of a yeast vehicle with a targeted cell type refers to the ability of the yeast cell membrane, or particle thereof, to fuse with the membrane of the targeted cell type (e.g., dendritic cell or macrophage), leading to syncytia formation. As used herein, a syncytium is a multinucleate mass of protoplasm produced by the merging of cells. A number of viral surface proteins (including those of immunodeficiency viruses such as HIV, influenza virus, poliovirus and adenovirus) and other fusogens (such as those involved in fusions between eggs and sperm) have been shown to be able to effect fusion between two membranes (i.e., between viral and mammalian cell membranes or between mammalian cell membranes). It is noted, however, that incorporation of a targeting moiety into the yeast vehicle, while it may be desirable under some circumstances, is not necessary. In the case of yeast vehicles that express antigens extracellularly, this can be a further advantage of the yeast vehicles of the present invention. In general, yeast vehicles useful in the present invention are readily taken up by dendritic cells (as well as other cells, such as macrophages).
In most embodiments of the invention, the yeast-based immunotherapy composition includes at least one antigen, immunogenic domain thereof, or epitope thereof. The antigens contemplated for use in this invention include any Ad-36 antigen or immunogenic domain thereof, including mutants, variants and agonists of Ad-36 proteins or domains thereof, against which it is desired to elicit an immune response for the purpose of prophylactically or therapeutically immunizing a host against Ad-36 infection.
As discussed above, the compositions of the invention include at least one Ad-36 antigen and/or at least one immunogenic domain of at least one Ad-36 antigen for immunizing a subject. In some embodiments, the antigen is a fusion protein, several examples of which have been described above.
Optionally, proteins, including fusion proteins, which are used as a component of the yeast-based immunotherapeutic composition of the invention are produced using constructs that are particularly useful for improving the expression of heterologous antigens in yeast. Typically, the desired antigenic protein(s) or peptide(s) are fused at their amino-terminal end to: (a) a specific synthetic peptide that stabilizes the expression of the fusion protein in the yeast vehicle or prevents posttranslational modification of the expressed fusion protein (such peptides are described in detail, for example, in U.S. Patent Publication No. 2004-0156858 A1, published Aug. 12, 2004, incorporated herein by reference in its entirety); (b) at least a portion of an endogenous yeast protein, wherein either fusion partner provides improved stability of expression of the protein in the yeast and/or a prevents post-translational modification of the proteins by the yeast cells (such proteins are also described in detail, for example, in U.S. Patent Publication No. 2004-0156858 A1, supra); and/or (c) at least a portion of a yeast protein that causes the fusion protein to be expressed on the surface of the yeast (e.g., an Aga protein, described in more detail herein). In addition, the present invention optionally includes the use of peptides that are fused to the C-terminus of the antigen-encoding construct, particularly for use in the selection and identification of the protein. Such peptides include, but are not limited to, any synthetic or natural peptide, such as a peptide tag (e.g., 6×His) or any other short epitope tag. Peptides attached to the C-terminus of an antigen according to the invention can be used with or without the addition of the N-terminal peptides discussed above.
In one embodiment, a synthetic peptide useful in a fusion protein is linked to the N-terminus of the antigen, the peptide consisting of at least two amino acid positions that are heterologous to the antigen, wherein the peptide stabilizes the expression of the fusion protein in the yeast vehicle or prevents posttranslational modification of the expressed fusion protein. The synthetic peptide and N-terminal portion of the antigen together form a fusion protein that has the following requirements: (1) the amino acid residue at position one of the fusion protein is a methionine (i.e., the first amino acid in the synthetic peptide is a methionine); (2) the amino acid residue at position two of the fusion protein is not a glycine or a proline (i.e., the second amino acid in the synthetic peptide is not a glycine or a proline); (3) none of the amino acid positions at positions 2-6 of the fusion protein is a methionine (i.e., the amino acids at positions 2-6, whether part of the synthetic peptide or the protein, if the synthetic peptide is shorter than 6 amino acids, do not include a methionine); and (4) none of the amino acids at positions 2-6 of the fusion protein is a lysine or an arginine (i.e., the amino acids at positions 2-6, whether part of the synthetic peptide or the protein, if the synthetic peptide is shorter than 5 amino acids, do not include a lysine or an arginine) The synthetic peptide can be as short as two amino acids, but in one aspect, is 2-6 amino acids (including 3, 4, 5 amino acids), and can be longer than 6 amino acids, in whole integers, up to about 200 amino acids, 300 amino acids, 400 amino acids, 500 amino acids, or more.
In one embodiment, a fusion protein comprises an amino acid sequence of M-X2-X3-X4-X5-X6, wherein M is methionine; wherein X2 is any amino acid except glycine, proline, lysine or arginine; wherein X3 is any amino acid except methionine, lysine or arginine; wherein X4 is any amino acid except methionine, lysine or arginine; wherein X5 is any amino acid except methionine, lysine or arginine; and wherein X6 is any amino acid except methionine, lysine or arginine. In one embodiment, the X6 residue is a proline. An exemplary synthetic sequence that enhances the stability of expression of an antigen in a yeast cell and/or prevents post-translational modification of the protein in the yeast includes the sequence M-A-D-E-A-P (e.g., SEQ ID NO:58). Another exemplary synthetic sequence with the same properties is M-V. In addition to the enhanced stability of the expression product, this fusion partner does not appear to negatively impact the immune response against the immunizing antigen in the construct. In addition, the synthetic fusion peptides can be designed to provide an epitope that can be recognized by a selection agent, such as an antibody.
In one aspect of the invention, the yeast vehicle is manipulated such that the antigen is expressed or provided by delivery or translocation of an expressed protein product, partially or wholly, on the surface of the yeast vehicle (extracellular expression). One method for accomplishing this aspect of the invention is to use a spacer arm for positioning one or more protein(s) on the surface of the yeast vehicle. For example, one can use a spacer arm to create a fusion protein of the antigen(s) or other protein of interest with a protein that targets the antigen(s) or other protein of interest to the yeast cell wall. For example, one such protein that can be used to target other proteins is a yeast protein (e.g., cell wall protein 2 (cwp2), Aga2, Pir4 or Flo1 protein) that enables the antigen(s) or other protein to be targeted to the yeast cell wall such that the antigen or other protein is located on the surface of the yeast. Proteins other than yeast proteins may be used for the spacer arm; however, for any spacer arm protein, it is most desirable to have the immunogenic response be directed against the target antigen rather than the spacer arm protein. As such, if other proteins are used for the spacer arm, then the spacer arm protein that is used should not generate such a large immune response to the spacer arm protein itself such that the immune response to the target antigen(s) is overwhelmed. One of skill in the art should aim for a small immune response to the spacer arm protein relative to the immune response for the target antigen(s). Spacer arms can be constructed to have cleavage sites (e.g., protease cleavage sites) that allow the antigen to be readily removed or processed away from the yeast, if desired. Any known method of determining the magnitude of immune responses can be used (e.g., antibody production, lytic assays, etc.) and are readily known to one of skill in the art.
Another method for positioning the target antigen(s) or other proteins to be exposed on the yeast surface is to use signal sequences such as glycosylphosphatidyl inositol (GPI) to anchor the target to the yeast cell wall. Alternatively, positioning can be accomplished by appending signal sequences that target the antigen(s) or other proteins of interest into the secretory pathway via translocation into the endoplasmic reticulum (ER) such that the antigen binds to a protein which is bound to the cell wall (e.g., cwp).
In one aspect, the spacer arm protein is a yeast protein. The yeast protein can consist of between about two and about 800 amino acids of a yeast protein. In one embodiment, the yeast protein is about 10 to 700 amino acids. In another embodiment, the yeast protein is about 40 to 600 amino acids. Other embodiments of the invention include the yeast protein being at least 250 amino acids, at least 300 amino acids, at least 350 amino acids, at least 400 amino acids, at least 450 amino acids, at least 500 amino acids, at least 550 amino acids, at least 600 amino acids, or at least 650 amino acids. In one embodiment, the yeast protein is at least 450 amino acids in length. Another consideration for optimizing antigen surface expression, if that is desired, is whether the antigen and spacer arm combination should be expressed as a monomer or as dimer or as a trimer, or even more units connected together. This use of monomers, dimers, trimers, etc. allows for appropriate spacing or folding of the antigen such that some part, if not all, of the antigen is displayed on the surface of the yeast vehicle in a manner that makes it more immunogenic.
Use of yeast proteins can stabilize the expression of fusion proteins in the yeast vehicle, prevents posttranslational modification of the expressed fusion protein, and/or targets the fusion protein to a particular compartment in the yeast (e.g., to be expressed on the yeast cell surface). For delivery into the yeast secretory pathway, exemplary yeast proteins to use include, but are not limited to: Aga (including, but not limited to, Aga1 and/or Aga2); SUC2 (yeast invertase); alpha factor signal leader sequence; CPY; Cwp2p for its localization and retention in the cell wall; BUD genes for localization at the yeast cell bud during the initial phase of daughter cell formation; Flo1p; Pir2p; and Pir4p.
Other sequences can be used to target, retain and/or stabilize the protein to other parts of the yeast vehicle, for example, in the cytosol or the mitochondria or the endoplasmic reticulum or the nucleus. Examples of suitable yeast protein that can be used for any of the embodiments above include, but are not limited to, TK, AF, SECT; phosphoenolpyruvate carboxykinase PCK1, phosphoglycerokinase PGK and triose phosphate isomerase TPI gene products for their repressible expression in glucose and cytosolic localization; the heat shock proteins SSA1, SSA3, SSA4, SSC1, whose expression is induced and whose proteins are more thermostable upon exposure of cells to heat treatment; the mitochondrial protein CYC1 for import into mitochondria; ACT1.
In one embodiment, the Ad-36 antigen is linked at the N-terminus to a yeast protein, such as an alpha factor prepro sequence (also referred to as the alpha factor signal leader sequence, the amino acid sequence of which is exemplified herein by SEQ ID NO:56 or SEQ ID NO:57. Other sequences for yeast alpha factor prepro sequence are known in the art and are encompassed for use in the present invention. Without being bound by theory, the inventors believe that one advantage of utilizing alpha factor prepro sequence in a yeast-based fusion protein is the minimization of proteolysis of the protein, since the protein is sequestered away from cytosolic proteasomes.
Methods of producing yeast vehicles and expressing, combining and/or associating yeast vehicles with antigens and/or other proteins and/or agents of interest to produce yeast-based immunotherapy compositions are contemplated by the invention.
According to the present invention, the term “yeast vehicle-antigen complex” or “yeast-antigen complex” is used generically to describe any association of a yeast vehicle with an antigen, and can be used interchangeably with “yeast-based immunotherapy composition” when such composition is used to elicit an immune response as described above. Such association includes expression of the antigen by the yeast (a recombinant yeast), introduction of an antigen into a yeast, physical attachment of the antigen to the yeast, and mixing of the yeast and antigen together, such as in a buffer or other solution or formulation. These types of complexes are described in detail below.
In one embodiment, a yeast cell used to prepare the yeast vehicle is transfected with a heterologous nucleic acid molecule encoding a protein (e.g., the antigen) such that the protein is expressed by the yeast cell. Such a yeast is also referred to herein as a recombinant yeast or a recombinant yeast vehicle. The yeast cell can then be loaded into the dendritic cell as an intact cell, or the yeast cell can be killed, or it can be derivatized such as by formation of yeast spheroplasts, cytoplasts, ghosts, or subcellular particles, any of which is followed by loading of the derivative into the dendritic cell. Yeast spheroplasts can also be directly transfected with a recombinant nucleic acid molecule (e.g., the spheroplast is produced from a whole yeast, and then transfected) in order to produce a recombinant spheroplast that expresses an antigen or other protein.
In one aspect, a yeast cell or yeast spheroplast used to prepare the yeast vehicle is transfected with a recombinant nucleic acid molecule encoding the antigen(s) or other protein such that the antigen or other protein is recombinantly expressed by the yeast cell or yeast spheroplast. In this aspect, the yeast cell or yeast spheroplast that recombinantly expresses the antigen(s) or other protein is used to produce a yeast vehicle comprising a yeast cytoplast, a yeast ghost, or a yeast membrane particle or yeast cell wall particle, or fraction thereof.
In general, the yeast vehicle and antigen(s) and/or other agents can be associated by any technique described herein. In one aspect, the yeast vehicle was loaded intracellularly with the antigen(s) and/or agent(s). In another aspect, the antigen(s) and/or agent(s) was covalently or non-covalently attached to the yeast vehicle. In yet another aspect, the yeast vehicle and the antigen(s) and/or agent(s) were associated by mixing. In another aspect, and in one embodiment, the antigen(s) and/or agent(s) is expressed recombinantly by the yeast vehicle or by the yeast cell or yeast spheroplast from which the yeast vehicle was derived.
A number of antigens and/or other proteins to be produced by a yeast vehicle of the present invention is any number of antigens and/or other proteins that can be reasonably produced by a yeast vehicle, and typically ranges from at least one to at least about 6 or more, including from about 2 to about 6 heterologous antigens and or other proteins.
Expression of an antigen or other protein in a yeast vehicle of the present invention is accomplished using techniques known to those skilled in the art. Briefly, a nucleic acid molecule encoding at least one desired antigen or other protein is inserted into an expression vector in such a manner that the nucleic acid molecule is operatively linked to a transcription control sequence in order to be capable of effecting either constitutive or regulated expression of the nucleic acid molecule when transformed into a host yeast cell. Nucleic acid molecules encoding one or more antigens and/or other proteins can be on one or more expression vectors operatively linked to one or more expression control sequences. Particularly important expression control sequences are those which control transcription initiation, such as promoter and upstream activation sequences. Any suitable yeast promoter can be used in the present invention and a variety of such promoters are known to those skilled in the art. Promoters for expression in Saccharomyces cerevisiae include, but are not limited to, promoters of genes encoding the following yeast proteins: alcohol dehydrogenase I (ADH1) or II (ADH2), CUP1, phosphoglycerate kinase (PGK), triose phosphate isomerase (TPI), translational elongation factor EF-1 alpha (TEF2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; also referred to as TDH3, for triose phosphate dehydrogenase), galactokinase (GAL1), galactose-1-phosphate uridyl-transferase (GAL7), UDP-galactose epimerase (GAL10), cytochrome c1 (CYC1), Sec7 protein (SECT) and acid phosphatase (PHO5), including hybrid promoters such as ADH2/GAPDH and CYC1/GAL10 promoters, and including the ADH2/GAPDH promoter, which is induced when glucose concentrations in the cell are low (e.g., about 0.1 to about 0.2 percent), as well as the CUP1 promoter and the TEF2 promoter. Likewise, a number of upstream activation sequences (UASs), also referred to as enhancers, are known. Upstream activation sequences for expression in Saccharomyces cerevisiae include, but are not limited to, the UASs of genes encoding the following proteins: PCK1, TPI, TDH3, CYC1, ADH1, ADH2, SUC2, GAL1, GAL7 and GAL10, as well as other UASs activated by the GAL4 gene product, with the ADH2 UAS being used in one aspect. Since the ADH2 UAS is activated by the ADR1 gene product, it may be preferable to overexpress the ADR1 gene when a heterologous gene is operatively linked to the ADH2 UAS. Transcription termination sequences for expression in Saccharomyces cerevisiae include the termination sequences of the α-factor, GAPDH, and CYC1 genes.
Transcription control sequences to express genes in methyltrophic yeast include the transcription control regions of the genes encoding alcohol oxidase and formate dehydrogenase.
Transfection of a nucleic acid molecule into a yeast cell according to the present invention can be accomplished by any method by which a nucleic acid molecule can be introduced into the cell and includes, but is not limited to, diffusion, active transport, bath sonication, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. Transfected nucleic acid molecules can be integrated into a yeast chromosome or maintained on extrachromosomal vectors using techniques known to those skilled in the art. Examples of yeast vehicles carrying such nucleic acid molecules are disclosed in detail herein. As discussed above, yeast cytoplast, yeast ghost, and yeast membrane particles or cell wall preparations can also be produced recombinantly by transfecting intact yeast microorganisms or yeast spheroplasts with desired nucleic acid molecules, producing the antigen therein, and then further manipulating the microorganisms or spheroplasts using techniques known to those skilled in the art to produce cytoplast, ghost or subcellular yeast membrane extract or fractions thereof containing desired antigens or other proteins.
Effective conditions for the production of recombinant yeast vehicles and expression of the antigen and/or other protein by the yeast vehicle include an effective medium in which a yeast strain can be cultured. An effective medium is typically an aqueous medium comprising assimilable carbohydrate, nitrogen and phosphate sources, as well as appropriate salts, minerals, metals and other nutrients, such as vitamins and growth factors. The medium may comprise complex nutrients or may be a defined minimal medium. Yeast strains of the present invention can be cultured in a variety of containers, including, but not limited to, bioreactors, Erlenmeyer flasks, test tubes, microtiter dishes, and Petri plates. Culturing is carried out at a temperature, pH and oxygen content appropriate for the yeast strain. Such culturing conditions are well within the expertise of one of ordinary skill in the art (see, for example, Guthrie et al. (eds.), 1991, Methods in Enzymology, vol. 194, Academic Press, San Diego).
In some aspects of the invention, the yeast are grown under neutral pH conditions, and particularly, in a media maintained at a pH level of at least 5.5, namely the pH of the culture media is not allowed to drop below pH 5.5. In other aspects, the yeast is grown at a pH level maintained at about 5.5. In other aspects, the yeast is grown at a pH level maintained at about 5.6, 5.7, 5.8 or 5.9. In another aspect, the yeast is grown at a pH level maintained at about 6. In another aspect, the yeast is grown at a pH level maintained at about 6.5. In other aspects, the yeast is grown at a pH level maintained at about 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 or 7.0. In other aspects, the yeast is grown at a pH level maintained at about 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. The pH level is important in the culturing of yeast. One of skill in the art will appreciate that the culturing process includes not only the start of the yeast culture but the maintenance of the culture as well. As yeast culturing is known to turn acidic (i.e., lowering the pH) over time, care must be taken to monitor the pH level during the culturing process. Yeast cell cultures whereby the pH level of the medium drops below 6 are still contemplated within the scope of the invention provided that the pH of the media is brought up to at least 5.5 at some point during the culturing process. As such, the longer time the yeast are grown in a medium that is at least pH 5.5 or above, the better the results will be in terms of obtaining yeast with desirable characteristics.
As used herein, the general use of the term “neutral pH” refers to a pH range between about pH 5.5 and about pH 8, and in one aspect, between about pH 6 and about 8. One of skill the art will appreciate that minor fluctuations (e.g., tenths or hundredths) can occur when measuring with a pH meter. As such, the use of neutral pH to grow yeast cells means that the yeast cells are grown in neutral pH for the majority of the time that they are in culture. The use of a neutral pH in culturing yeast promotes several biological effects that are desirable characteristics for using the yeast as vehicles for immunomodulation. In one aspect, culturing the yeast in neutral pH allows for good growth of the yeast without any negative effect on the cell generation time (e.g., slowing down the doubling time). The yeast can continue to grow to high densities without losing their cell wall pliability. In another aspect, the use of a neutral pH allows for the production of yeast with pliable cell walls and/or yeast that are sensitive to cell wall digesting enzymes (e.g., glucanase) at all harvest densities. This trait is desirable because yeast with flexible cell walls can induce unusual immune responses, such as by promoting the secretion of cytokines (e.g., interferon-γ (IFN-γ)) in the cells hosting the yeast. In addition, greater accessibility to the antigens located in the cell wall is afforded by such culture methods. In another aspect, the use of neutral pH for some antigens allows for release of the di-sulfide bonded antigen by treatment with dithiothreitol (DTT) that is not possible when such an antigen-expressing yeast is cultured in media at lower pH (e.g., pH 5). Finally, in another aspect, yeast cultured using the neutral pH methodologies, elicit increased production of at least TH1-type cytokines including, but not limited to, IFN-γ, interleukin-12 (IL-12), and IL-2, and may also elicit increased production of other cytokines, such as proinflammatory cytokines (e.g., IL-6).
In one embodiment, control of the amount of yeast glycosylation is used to control the expression of antigens by the yeast, particularly on the surface. The amount of yeast glycosylation can affect the immunogenicity and antigenicity of the antigen expressed on the surface, since sugar moieties tend to be bulky. As such, the existence of sugar moieties on the surface of yeast and its impact on the three-dimensional space around the target antigen(s) should be considered in the modulation of yeast according to the invention. Any method can be used to reduce the amount of glycosylation of the yeast (or increase it, if desired). For example, one could use a yeast mutant strain that has been selected to have low glycosylation (e.g. mnn1, och1 and mnn9 mutants), or one could eliminate by mutation the glycosylation acceptor sequences on the target antigen. Alternatively, one could use a yeast with abbreviated glycosylation patterns, e.g., Pichia. One can also treat the yeast using methods that reduce or alter the glycosylation.
In one embodiment of the present invention, as an alternative to expression of an antigen or other protein recombinantly in the yeast vehicle, a yeast vehicle is loaded intracellularly with the protein or peptide, or with carbohydrates or other molecules that serve as an antigen and/or are useful as immunomodulatory agents or biological response modifiers according to the invention. Subsequently, the yeast vehicle, which now contains the antigen and/or other proteins intracellularly, can be administered to an individual or loaded into a carrier such as a dendritic cell. Peptides and proteins can be inserted directly into yeast vehicles of the present invention by techniques known to those skilled in the art, such as by diffusion, active transport, liposome fusion, electroporation, phagocytosis, freeze-thaw cycles and bath sonication. Yeast vehicles that can be directly loaded with peptides, proteins, carbohydrates, or other molecules include intact yeast, as well as spheroplasts, ghosts or cytoplasts, which can be loaded with antigens and other agents after production. Alternatively, intact yeast can be loaded with the antigen and/or agent, and then spheroplasts, ghosts, cytoplasts, or subcellular particles can be prepared therefrom. Any number of antigens and/or other agents can be loaded into a yeast vehicle in this embodiment, from at least 1, 2, 3, 4 or any whole integer up to hundreds or thousands of antigens and/or other agents, such as would be provided by the loading of a microorganism or portions thereof, for example.
In another embodiment of the present invention, an antigen and/or other agent is physically attached to the yeast vehicle. Physical attachment of the antigen and/or other agent to the yeast vehicle can be accomplished by any method suitable in the art, including covalent and non-covalent association methods which include, but are not limited to, chemically crosslinking the antigen and/or other agent to the outer surface of the yeast vehicle or biologically linking the antigen and/or other agent to the outer surface of the yeast vehicle, such as by using an antibody or other binding partner. Chemical cross-linking can be achieved, for example, by methods including glutaraldehyde linkage, photoaffinity labeling, treatment with carbodiimides, treatment with chemicals capable of linking di-sulfide bonds, and treatment with other cross-linking chemicals standard in the art. Alternatively, a chemical can be contacted with the yeast vehicle that alters the charge of the lipid bilayer of yeast membrane or the composition of the cell wall so that the outer surface of the yeast is more likely to fuse or bind to antigens and/or other agent having particular charge characteristics. Targeting agents such as antibodies, binding peptides, soluble receptors, and other ligands may also be incorporated into an antigen as a fusion protein or otherwise associated with an antigen for binding of the antigen to the yeast vehicle.
When the antigen or other protein is expressed on or physically attached to the surface of the yeast, spacer arms may, in one aspect, be carefully selected to optimize antigen or other protein expression or content on the surface. The size of the spacer arm(s) can affect how much of the antigen or other protein is exposed for binding on the surface of the yeast. Thus, depending on which antigen(s) or other protein(s) are being used, one of skill in the art will select a spacer arm that effectuates appropriate spacing for the antigen or other protein on the yeast surface. In one embodiment, the spacer arm is a yeast protein of at least 450 amino acids. Spacer arms have been discussed in detail above.
In yet another embodiment, the yeast vehicle and the antigen or other protein are associated with each other by a more passive, non-specific or non-covalent binding mechanism, such as by gently mixing the yeast vehicle and the antigen or other protein together in a buffer or other suitable formulation (e.g., admixture).
In one embodiment of the invention, the yeast vehicle and the antigen or other protein are both loaded intracellularly into a carrier such as a dendritic cell or macrophage to form the therapeutic composition or vaccine of the present invention. Alternatively, an antigen or other protein can be loaded into a dendritic cell in the absence of the yeast vehicle.
In one embodiment, intact yeast (with or without expression of heterologous antigens or other proteins) can be ground up or processed in a manner to produce yeast cell wall preparations, yeast membrane particles or yeast fragments (i.e., not intact) and the yeast fragments can, in some embodiments, be provided with or administered with other compositions that include antigens (e.g., DNA vaccines, protein subunit vaccines, killed or inactivated pathogens) to enhance immune responses. For example, enzymatic treatment, chemical treatment or physical force (e.g., mechanical shearing or sonication) can be used to break up the yeast into parts that are used as an adjuvant.
In one embodiment of the invention, yeast vehicles useful in the invention include yeast vehicles that have been killed or inactivated. Killing or inactivating of yeast can be accomplished by any of a variety of suitable methods known in the art. For example, heat inactivation of yeast is a standard way of inactivating yeast, and one of skill in the art can monitor the structural changes of the target antigen, if desired, by standard methods known in the art. Alternatively, other methods of inactivating the yeast can be used, such as chemical, electrical, radioactive or UV methods. See, for example, the methodology disclosed in standard yeast culturing textbooks such as Methods of Enzymology, Vol. 194, Cold Spring Harbor Publishing (1990). Any of the inactivation strategies used should take the secondary, tertiary or quaternary structure of the target antigen into consideration and preserve such structure as to optimize its immunogenicity.
Yeast vehicles can be formulated into yeast-based immunotherapy compositions or products of the present invention, including preparations to be administered to a subject directly or first loaded into a carrier such as a dendritic cell, using a number of techniques known to those skilled in the art. For example, yeast vehicles can be dried by lyophilization. Formulations comprising yeast vehicles can also be prepared by packing yeast in a cake or a tablet, such as is done for yeast used in baking or brewing operations. In addition, yeast vehicles can be mixed with a pharmaceutically acceptable excipient, such as an isotonic buffer that is tolerated by a host or host cell. Examples of such excipients include water, saline, Ringer's solution, dextrose solution, Hank's solution, and other aqueous physiologically balanced salt solutions. Nonaqueous vehicles, such as fixed oils, sesame oil, ethyl oleate, or triglycerides may also be used. Other useful formulations include suspensions containing viscosity-enhancing agents, such as sodium carboxymethylcellulose, sorbitol, glycerol or dextran. Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability. Examples of buffers include phosphate buffer, bicarbonate buffer and Tris buffer, while examples of preservatives include thimerosal, m- or o-cresol, formalin and benzyl alcohol. Standard formulations can either be liquid injectables or solids which can be taken up in a suitable liquid as a suspension or solution for injection. Thus, in a non-liquid formulation, the excipient can comprise, for example, dextrose, human serum albumin, and/or preservatives to which sterile water or saline can be added prior to administration.
In one embodiment of the present invention, a composition can include additional agents, which may also be referred to as biological response modifier compounds, or the ability to produce such agents/modifiers. For example, a yeast vehicle can be transfected with or loaded with at least one antigen and at least one agent/biological response modifier compound, or a composition of the invention can be administered in conjunction with at least one agent/biological response modifier. Biological response modifiers include adjuvants and other compounds that can modulate immune responses, which may be referred to as immunomodulatory compounds, as well as compounds that modify the biological activity of another compound or agent, such as a yeast-based immunotherapeutic, such biological activity not being limited to immune system effects. Certain immunomodulatory compounds can stimulate a protective immune response whereas others can suppress a harmful immune response, and whether an immunomodulatory is useful in combination with a given yeast-based immunotherapeutic may depend, at least in part, on the disease state or condition to be treated or prevented, and/or on the individual who is to be treated. Certain biological response modifiers preferentially enhance a cell-mediated immune response whereas others preferentially enhance a humoral immune response (i.e., can stimulate an immune response in which there is an increased level of cell-mediated compared to humoral immunity, or vice versa.). Certain biological response modifiers have one or more properties in common with the biological properties of yeast-based immunotherapeutics or enhance or complement the biological properties of yeast-based immunotherapeutics. There are a number of techniques known to those skilled in the art to measure stimulation or suppression of immune responses, as well as to differentiate cell-mediated immune responses from humoral immune responses, and to differentiate one type of cell-mediated response from another (e.g., a TH17 response versus a TH1 response).
Agents/biological response modifiers useful in the invention may include, but are not limited to, cytokines, chemokines, hormones, lipidic derivatives, peptides, proteins, polysaccharides, small molecule drugs, antibodies and antigen binding fragments thereof (including, but not limited to, anti-cytokine antibodies, anti-cytokine receptor antibodies, anti-chemokine antibodies), vitamins, polynucleotides, nucleic acid binding moieties, aptamers, and growth modulators. Some suitable agents include, but are not limited to, IL-1 or agonists of IL-1 or of IL-1R, anti-IL-1 or other IL-1 antagonists; IL-6 or agonists of IL-6 or of IL-6R, anti-IL-6 or other IL-6 antagonists; IL-12 or agonists of IL-12 or of IL-12R, anti-IL-12 or other IL-12 antagonists; IL-17 or agonists of IL-17 or of IL-17R, anti-IL-17 or other IL-17 antagonists; IL-21 or agonists of IL-21 or of IL-21R, anti-IL-21 or other IL-21 antagonists; IL-22 or agonists of IL-22 or of IL-22R, anti-IL-22 or other IL-22 antagonists; IL-23 or agonists of IL-23 or of IL-23R, anti-IL-23 or other IL-23 antagonists; IL-25 or agonists of IL-25 or of IL-25R, anti-IL-25 or other IL-25 antagonists; IL-27 or agonists of IL-27 or of IL-27R, anti-IL-27 or other IL-27 antagonists; type I interferon (including IFN-α) or agonists or antagonists of type I interferon or a receptor thereof; type II interferon (including IFN-γ) or agonists or antagonists of type II interferon or a receptor thereof; anti-CD40, CD40L, anti-CTLA-4 antibody (e.g., to release anergic T cells); T cell co-stimulators (e.g., anti-CD137, anti-CD28, anti-CD40); alemtuzumab (e.g., CAMPATH®), denileukin diftitox (e.g., ONTAK®); anti-CD4; anti-CD25; anti-PD-1, anti-PD-L1, anti-PD-L2; agents that block FOXP3 (e.g., to abrogate the activity/kill CD4+/CD25+ T regulatory cells); Flt3 ligand, imiquimod (ALDARATM), granulocyte-macrophage colony stimulating factor (GM-CSF); granulocyte-colony stimulating factor (G-CSF), sargramostim (Leukine®); hormones including without limitation prolactin and growth hormone; Toll-like receptor (TLR) agonists, including but not limited to TLR-2 agonists, TLR-4 agonists, TLR-7 agonists, and TLR-9 agonists; TLR antagonists, including but not limited to TLR-2 antagonists, TLR-4 antagonists, TLR-7 antagonists, and TLR-9 antagonists; anti-inflammatory agents and immunomodulators, including but not limited to, COX-2 inhibitors (e.g., Celecoxib, NSAIDS), glucocorticoids, statins, and thalidomide and analogues thereof including IMiDTMs (which are structural and functional analogues of thalidomide (e.g., REVLIMID® (lenalidomide), ACTIMID® (pomalidomide)); proinflammatory agents, such as fungal or bacterial components or any proinflammatory cytokine or chemokine; immunotherapeutic vaccines including, but not limited to, virus-based vaccines, bacteria-based vaccines, or antibody-based vaccines; and any other immunomodulators, immunopotentiators, anti-inflammatory agents, and/or pro-inflammatory agents. Any combination of such agents is contemplated by the invention, and any of such agents combined with or administered in a protocol with (e.g., concurrently, sequentially, or in other formats with) a yeast-based immunotherapeutic is a composition encompassed by the invention. Such agents are well known in the art. These agents may be used alone or in combination with other agents described herein.
Agents can include agonists and antagonists of a given protein or peptide or domain thereof. As used herein, an “agonist” is any compound or agent, including without limitation small molecules, proteins, peptides, antibodies, nucleic acid binding agents, etc., that binds to a receptor or ligand and produces or triggers a response, which may include agents that mimic the action of a naturally occurring substance that binds to the receptor or ligand. An “antagonist” is any compound or agent, including without limitation small molecules, proteins, peptides, antibodies, nucleic acid binding agents, etc., that blocks or inhibits or reduces the action of an agonist.
Compositions of the invention can further include or can be administered with (concurrently, sequentially, or intermittently with) any other compounds or compositions that are useful for preventing or treating Ad-36 infection or any compounds that treat or ameliorate any symptom of Ad-36 infection. In addition, compositions of the invention can be used together with other immunotherapeutic compositions, including prophylactic and/or therapeutic immunotherapy.
The invention also includes a kit comprising any of the compositions described herein, or any of the individual components of the compositions described herein. Kits may include additional reagents and written instructions or directions for using any of the compositions of the invention to prevent or treat Ad-36 infection and/or obesity or being overweight that is or may be associated with such an infection.
Methods for Administration or Use of Compositions of the Invention
Compositions of the invention, which in one embodiment, include yeast-based immunotherapeutic compositions described above, as well as Ad-36 fusion proteins described herein and recombinant nucleic acid molecules encoding such Ad-36 fusion proteins, and other compositions comprising such yeast-based compositions, fusion proteins, or recombinant molecules described herein, can be used in a variety of in vivo and in vitro methods, including, but not limited to, methods and uses to treat and/or prevent Ad-36 infection and/or obesity, excess weight (e.g., being clinically overweight), or abnormal adipose tissue hypertrophy associated with Ad-36 infection, other symptoms and conditions associated with Ad-36 infection and/or excess weight or abnormal adipose tissue hypertrophy, in diagnostic assays for Ad-36, or to produce antibodies against Ad-36.
One embodiment of the invention relates to a method to treat Ad-36 infection, and/or to prevent, ameliorate or treat at least one symptom or sequela of chronic Ad-36 infection, in an individual or population of individuals. In one aspect, the invention relates to a method to reduce or prevent obesity, excess weight, or abnormal adipose tissue hypertrophy that is associated with Ad-36 infection, by reducing, halting, or preventing, Ad-36 infection. The method includes the step of administering to an individual or a population of individuals who are, may be, or may become, infected with Ad-36, an immunotherapeutic composition of the invention. In one aspect, the composition is an immunotherapeutic composition comprising one or more Ad-36 antigens (Ad-36 proteins and/or immunogenic domains thereof), including any of the Ad-36 antigens (including any fusion protein) as described herein. In one aspect, the immunotherapeutic composition is a yeast-based immunotherapeutic composition. In one aspect, the composition includes a fusion protein comprising Ad-36 antigens as described herein, or recombinant nucleic acid molecule encoding such antigens. In one embodiment, the individual or population of individuals has Ad-36 infection (is currently infected with Ad-36 or at least has evidence of being infected). In one embodiment, the individual or population of individuals is overweight or obese, and in another embodiment, the individual or population of individuals is not overweight or is not obese. In one aspect, the individual or population of individuals is additionally treated with at least one other therapeutic compound or therapeutic protocol useful for the treatment of Ad-36 infection, or useful for the treatment of a condition associated with Ad-36 infection, including, but not limited to, obesity, being overweight, abnormal adipose tissue hypertrophy, type II diabetes, or symptoms of these conditions. Suitable additional therapeutic compounds include, but are not limited to, direct-acting antiviral drugs and/or interferons and/or other immunotherapeutic or immunomodulatory agents and/or insulin. Suitable additional therapeutic protocols include, but are not limited to, the administration of such agents, diet programs, and exercise programs.
Another embodiment of the invention relates to a method to immunize an individual or population of individuals against Ad-36 in order to prevent Ad-36 infection, prevent chronic Ad-36 infection, and/or reduce the severity of Ad-36 infection in the individual or population of individuals. The method includes the step of administering to an individual or population of individuals that is not infected with Ad-36 (or believed not to be infected with Ad-36 or not known to be or have been infected with Ad-36), a composition of the invention. In one aspect, the composition is an immunotherapeutic composition comprising one or more Ad-36 antigens as described herein, including a yeast-based immunotherapeutic composition. In one aspect, the composition includes a fusion protein comprising Ad-36 antigens as described herein, or recombinant nucleic acid molecule encoding such fusion protein.
As used herein, the phrase “treat” Ad-36 infection, or any permutation thereof (e.g., “treated for Ad-36 infection”, etc.) generally refers to applying or administering a composition of the invention once the infection (acute or chronic) has occurred, with the goal of reduction or elimination of detectable viral titer, reaching seroconversion as measured by development of antibodies against Ad-36 that are reflective of an elimination of the virus, reduction in at least one symptom resulting from the infection in the individual (e.g., reduction in BMI, reduction in body weight, reduced rate of weight gain, reduced adiposity, etc.), delaying or preventing the onset and/or severity of symptoms and/or downstream sequela caused by the infection, reduction of organ or physiological system damage resulting from the infection, improvement in organ or system function that was negatively impacted by the infection, improvement of immune responses against the virus, improvement of long term memory immune responses against the virus, and/or improved general health of the individual or population of individuals. To “prevent” Ad-36 infection, or any permutation thereof (e.g., “prevention of Ad-36 infection”, etc.), generally refers to applying or administering a composition of the invention before an infection with Ad-36 has occurred, with the goal of preventing infection by Ad-36, preventing chronic infection by Ad-36 (i.e., enabling an individual to clear an acute Ad-36 infection without further intervention), or at least reducing the severity, and/or length of infection and/or the physiological damage caused by the chronic infection, and/or reducing the rate of weight gain, in an individual or population of individuals should the infection later occur.
According to the present invention, body mass index, or BMI, is routinely used to determine a degree of weight excess (e.g., being overweight) and obesity, although it is not a direct measure of body fat. It is a measure of weight in relation to height of an individual and can be calculated in English or metric units. According to the Centers for Disease Control and Prevention (CDC), an adult who has a body mass index (BMI) between 25 and 29.9 is considered to be overweight. An adult who has a BMI of 30 or higher is considered to be obese. For children and teens, BMI ranges above a normal weight have different labels and take into account normal differences in body fat between boys and girls and differences in body fat at various ages. Being “overweight” in children and teens ages 2-19 years is defined as a BMI at or above the 85th percentile and lower than the 95th percentile for children of the same age and sex. Obesity in children and teens ages 2-19 is defined as a BMI at or above the 95th percentile for children of the same age and sex. As used herein, the phrase “excess weight” is generally used to refer to a weight that is greater than that considered to be healthy for an individual of a given age, gender, and/or height, which is typically at least “overweight” as defined by the CDC or other public health institution and as set forth herein. Accordingly, the reference to “excess weight” can be used interchangeably with reference to “overweight” or “being overweight”. BMI calculators for children and teens, as well as adults, are publicly available through the Centers for Disease Control and Prevention, for example, and can be used to determine BMI for a specific age, height, gender and weight (for children and teens, for adults, height and weight are considered), and advise the weight percentile for the individual if child or teen, and further advise whether the individual is considered to be potentially overweight or obese according to current standards for children, teens and adults.
According to the invention, reference to “abnormal adipose tissue”, “hypertrophic adipose tissue” or “abnormal adipose tissue hypertrophy”, refers to an increase in adipose tissue (adiposity) or adipocyte growth that is abnormal and typically presents as a benign lipoma or a deposit of adipose tissue in an unusual anatomical location. Abnormal adipose tissue is therefore distinguished from obesity, as an individual may not be clinically obese, but may have areas of abnormal adipose tissue or adipose tissue hypertrophy. Abnormal adipose tissue is, for example, a condition associated with HIV infection.
Preferably, the use of an immunotherapeutic composition of the invention results in the prevention of obesity or excess weight gain, in a reduction in weight gained or a reduced rate of weigh gain in individuals who are or become infected with Ad-36, and/or in a reduction in the likelihood of becoming obese or overweight, in an individual who is or becomes infected with the virus but is not currently overweight or obese. In an adult individual with a BMI of 30 or higher, or in a child or teen aged 2-19 years with a BMI at or above the 95th percentile for children/teens of the same age and sex, in one aspect of the invention, the use of an immunotherapeutic composition of the invention results in a reduction of BMI in the individual to less than 30 for such adults, or less than the 95th percentile for such children or teens. In an adult individual with a BMI of between 25 and 29.9, or in a child or teen aged 2-19 years with a BMI at or above the 85th percentile for children/teens of the same age and sex, in one aspect of the invention, the use of an immunotherapeutic composition of the invention results in a reduction of BMI to below 25 for such adults, and to less than the 85th percentile for such children or teen.
The efficacy, or effectiveness, of an immunotherapeutic composition of the invention can also be defined as a statistically significant change, or statistical trend, toward patient benefit in any one or more measurable or detectable parameter associated with Ad-36 infection or conditions linked to such infection, in an individual receiving the immunotherapeutic composition, as compared to a control value for the parameter being evaluated. In one aspect of the invention, a clinically relevant change can be measured as a percentage change toward patient benefit as compared to a prior evaluation, and can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or greater. Benefit can also be measured as a change in the slope of a curve over time as compared to a control (e.g., a change in the slope of body weight or the rate of weight gain plotted over time before, during and after treatment).
Parameters to be evaluated for determination of effectiveness of a composition or method of the invention include, but are not limited to, viral load, viral clearance, adipose tissue hypertrophy, body weight, BMI, rate of weight gain, total body fat, serum cholesterol, triglycerides, blood pressure, glucose tolerance, insulin sensitivity, and immune responses, including Ad-36-specific T cell responses and neutralizing antibody responses. The control value can be selected from any suitable control value, including, but not limited to, one or more prior measurements of the parameter in the same individual; a measurement of the parameter as an average or mean in a population of individuals meeting similar criteria for gender, age, weight, and or other clinical status; or a reference value provided in the form of stored information regarding a previously determined baseline level for the given parameter. Such a form of stored information can include, for example, but is not limited to, a reference chart, listing or electronic file of population or individual data regarding “healthy” individuals (negative control), or obese or overweight individuals or individuals infected with Ad-36 that have not been cured or treated (positive control); a medical chart for the individual recording data from previous evaluations; or any other source of data regarding baseline levels that are useful for the evaluation of the efficacy of the treatment.
According to the invention, a “baseline level” is a control level, and in some embodiments (but not all embodiments, depending on the method), a normal level, of a given clinical endpoint or parameter against which a test level of the given clinical endpoint or parameter can be compared. The term “negative control” used in reference to a baseline level of such a clinical endpoint or parameter typically refers to a baseline level established in a sample from the patient or from a population of individuals that is believed to be normal (i.e., not infected with Ad-36, not overweight, not obese, not being abnormal with respect to the endpoint being tested). In one embodiment, a baseline level or control can be established from an individual at the onset of therapeutic or preventative treatment so that the status of the individual can be monitored over time and/or so that the efficacy of a given therapeutic or prophylactic protocol can be evaluated over time (continuously or intermittently). A “positive control” can include any control that confirms the positive detection of the parameter or clinical endpoint that is associated with Ad-36 infection and/or obesity or excess weight, or other associated endpoint.
Methods for detection of Ad-36 virus are known in the art and are described, for example, in WO 2007/120362, WO 2010 011440, WO 2007/064836, and WO 98/44946. The presence of viral DNA can be determined by conventional methods including, but not limited to, DNA sequencing, oligonucleotide hybridization, or PCR amplification. Detection of Ad-36 antibodies or proteins that bind to Ad-36 antibodies have also been described and such methods are encompassed by the invention. Binding can be measured using a variety of methods standard in the art, including, but not limited to: Western blot, immunoblot, enzyme-linked immunosorbant assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, surface plasmon resonance, chemiluminescence, fluorescent polarization, phosphorescence, immunohistochemical analysis, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, microcytometry, microarray, microscopy, fluorescence activated cell sorting (FACS), and flow cytometry.
The present invention includes the delivery (administration, immunization) of an immunotherapeutic composition of the invention, including a yeast-based immunotherapy composition, to a subject. The administration process can be performed ex vivo or in vivo, but is typically performed in vivo. Ex vivo administration refers to performing part of the regulatory step outside of the patient, such as administering a composition of the present invention to a population of cells (dendritic cells) removed from a patient under conditions such that a yeast vehicle, antigen(s) and any other agents or compositions are loaded into the cell, and returning the cells to the patient. The therapeutic composition of the present invention can be returned to a patient, or administered to a patient, by any suitable mode of administration.
Administration of a composition can be systemic, mucosal and/or proximal to the location of the target site (e.g., near a site of infection or target tissue, such as adipose tissue). Suitable routes of administration will be apparent to those of skill in the art, depending on the type of condition to be prevented or treated, the antigen used, and/or the target cell population or tissue. Various acceptable methods of administration include, but are not limited to, intravenous administration, intraperitoneal administration, intramuscular administration, intranodal administration, intracoronary administration, intraarterial administration (e.g., into a carotid artery), subcutaneous administration, transdermal delivery, intratracheal administration, intraarticular administration, intraventricular administration, inhalation (e.g., aerosol), intracranial, intraspinal, intraocular, aural, intranasal, oral, pulmonary administration, impregnation of a catheter, and direct injection into a tissue. In one aspect, routes of administration include: intravenous, intraperitoneal, subcutaneous, intradermal, intranodal, intramuscular, transdermal, inhaled, intranasal, oral, intraocular, intraarticular, intracranial, and intraspinal. Parenteral delivery can include intradermal, intramuscular, intraperitoneal, intrapleural, intrapulmonary, intravenous, subcutaneous, atrial catheter and venal catheter routes. Aural delivery can include ear drops, intranasal delivery can include nose drops or intranasal injection, and intraocular delivery can include eye drops. Aerosol (inhalation) delivery can also be performed using methods standard in the art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA 189:11277-11281, 1992). Other routes of administration that modulate mucosal immunity may be useful in the treatment of viral infections. Such routes include bronchial, intradermal, intramuscular, intranasal, other inhalatory, rectal, subcutaneous, topical, transdermal, vaginal and urethral routes. In one aspect, an immunotherapeutic composition of the invention is administered subcutaneously. In one aspect, the immunotherapeutic composition is administered directly to adipose tissue.
With respect to the yeast-based immunotherapy compositions of the invention, in general, a suitable single dose is a dose that is capable of effectively providing a yeast vehicle and an antigen (if included) to a given cell type, tissue, or region of the patient body in an amount effective to elicit an antigen-specific immune response against one or more Ad-36 antigens or epitopes, when administered one or more times over a suitable time period. For example, in one embodiment, a single dose of a yeast vehicle of the present invention is from about 1×105 to about 5×107 yeast cell equivalents per kilogram body weight of the organism being administered the composition. In one aspect, a single dose of a yeast vehicle of the present invention is from about 0.1 Y.U. (1×106 cells) to about 100 Y.U. (1×109 cells) per dose (i.e., per organism), including any interim dose, in increments of 0.1×106 cells (i.e., 1.1×106, 1.2×106, 1.3×106 . . . ). In one embodiment, doses include doses between 1 Y.U and 40 Y.U. or 80 Y.U. and in one aspect, between 10 Y.U. and 40 Y.U. or 80 Y.U. In one embodiment, the doses are administered at different sites on the individual but during the same dosing period. For example, a 40 Y.U. dose may be administered via by injecting 10 Y.U. doses to four different sites on the individual during one dosing period, or a 20 Y.U. dose may be administered by injecting 5 Y.U. doses to four different sites on the individual, or by injecting 10 Y.U. doses to two different sites on the individual, during the same dosing period. The invention includes administration of an amount of the yeast-based immunotherapy composition (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 Y.U. or more) at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different sites on an individual to form a single dose. One Yeast Unit (Y.U.) is 1×107 yeast cells.
“Boosters” or “boosts” of a therapeutic composition are administered, for example, when the immune response against the antigen has waned or as needed to provide an immune response or induce a memory response against a particular antigen or antigen(s). Boosters can be administered from about 1, 2, 3, 4, 5, 6, 7, or 8 weeks apart, to monthly, to bimonthly, to quarterly, to annually, to several years after the original administration. In one embodiment, an administration schedule is one in which from about 1×105 to about 5×107 yeast cell equivalents of a composition per kg body weight of the organism is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times over a time period of from weeks, to months, to years. In one embodiment, the doses are administered weekly for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses, followed by monthly doses as needed to achieve the desired inhibition or elimination of the Ad-36 virus.
In one aspect of the invention, one or more additional therapeutic agents are administered sequentially with the yeast-based immunotherapy composition (e.g., a direct-acting antiviral, a nutraceutical composition, or the like). In another embodiment, one or more additional therapeutic agents are administered before the yeast-based immunotherapy composition is administered. In another embodiment, one or more additional therapeutic agents are administered after the yeast-based immunotherapy composition is administered. In one embodiment, one or more additional therapeutic agents are administered in alternating doses with the yeast-based immunotherapy composition, or in a protocol in which the yeast-based composition is administered at prescribed intervals in between or with one or more consecutive doses of the additional agents, or vice versa. In one embodiment, the yeast-based immunotherapy composition is administered in one or more doses over a period of time prior to commencing the administration of the additional agents. In other words, the yeast-based immunotherapeutic composition is administered as a monotherapy for a period of time, and then the agent administration is added, either concurrently with new doses of yeast-based immunotherapy, or in an alternating fashion with yeast-based immunotherapy. Alternatively, the agent may be administered for a period of time prior to beginning administration of the yeast-based immunotherapy composition. In one aspect, the yeast is engineered to express or carry the agent, or a different yeast is engineered or produced to express or carry the agent.
In the method of the present invention, compositions and therapeutic compositions can be administered to animal, including any vertebrate, and particularly to any member of the Vertebrate class, Mammalia, including, without limitation, primates, rodents, livestock and domestic pets. Livestock include mammals to be consumed or that produce useful products (e.g., sheep for wool production). Mammals to treat or protect include humans, dogs, cats, mice, rats, goats, sheep, cattle, horses and pigs.
An “individual” is a vertebrate, such as a mammal, including without limitation a human. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, mice and rats. The term “individual” can be used interchangeably with the term “animal”, “subject” or “patient”.
General Techniques Useful in the Invention
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those skilled in the art. Such techniques are explained fully in the literature, such as, Methods of Enzymology, Vol. 194, Guthrie et al., eds., Cold Spring Harbor Laboratory Press (1990); Biology and activities of yeasts, Skinner, et al., eds., Academic Press (1980); Methods in yeast genetics: a laboratory course manual, Rose et al., Cold Spring Harbor Laboratory Press (1990); The Yeast Saccharomyces: Cell Cycle and Cell Biology, Pringle et al., eds., Cold Spring Harbor Laboratory Press (1997); The Yeast Saccharomyces Gene Expression, Jones et al., eds., Cold Spring Harbor Laboratory Press (1993); The Yeast Saccharomyces: Genome Dynamics, Protein Synthesis, and Energetics, Broach et al., eds., Cold Spring Harbor Laboratory Press (1992); Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) and Molecular Cloning: A Laboratory Manual, third edition (Sambrook and Russel, 2001), (jointly referred to herein as “Sambrook”); Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987, including supplements through 2001); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Harlow and Lane (1988), Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York; Harlow and Lane (1999) Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (jointly referred to herein as “Harlow and Lane”), Beaucage et al. eds., Current Protocols in Nucleic Acid Chemistry, John Wiley & Sons, Inc., New York, 2000); Casarett and Doull's Toxicology The Basic Science of Poisons, C. Klaassen, ed., 6th edition (2001), and Vaccines, S. Plotkin, W. Orenstein, and P. Offit, eds., Fifth Edition (2008).
GENERAL DEFINITIONS
As used herein, the term “analog” refers to a chemical compound that is structurally similar to another compound but differs slightly in composition (as in the replacement of one atom by an atom of a different element or in the presence of a particular functional group, or the replacement of one functional group by another functional group). Thus, an analog is a compound that is similar or comparable in function and appearance, but has a different structure or origin with respect to the reference compound.
The terms “substituted”, “substituted derivative” and “derivative”, when used to describe a compound, means that at least one hydrogen bound to the unsubstituted compound is replaced with a different atom or a chemical moiety.
Although a derivative has a similar physical structure to the parent compound, the derivative may have different chemical and/or biological properties than the parent compound. Such properties can include, but are not limited to, increased or decreased activity of the parent compound, new activity as compared to the parent compound, enhanced or decreased bioavailability, enhanced or decreased efficacy, enhanced or decreased stability in vitro and/or in vivo, and/or enhanced or decreased absorption properties.
In general, the term “biologically active” indicates that a compound (including a protein or peptide) has at least one detectable activity that has an effect on the metabolic or other processes of a cell or organism, as measured or observed in vivo (i.e., in a natural physiological environment) or in vitro (i.e., under laboratory conditions).
According to the present invention, the term “modulate” can be used interchangeably with “regulate” and refers generally to upregulation or downregulation of a particular activity. As used herein, the term “upregulate” can be used generally to describe any of: elicitation, initiation, increasing, augmenting, boosting, improving, enhancing, amplifying, promoting, or providing, with respect to a particular activity. Similarly, the term “downregulate” can be used generally to describe any of: decreasing, reducing, inhibiting, ameliorating, diminishing, lessening, blocking, or preventing, with respect to a particular activity.
In one embodiment of the present invention, any of the amino acid sequences described herein can be produced with from at least one, and up to about 20, additional heterologous amino acids flanking each of the C- and/or N-terminal ends of the specified amino acid sequence. The resulting protein or polypeptide can be referred to as “consisting essentially of” the specified amino acid sequence. According to the present invention, the heterologous amino acids are a sequence of amino acids that are not naturally found (i.e., not found in nature, in vivo) flanking the specified amino acid sequence, or that are not related to the function of the specified amino acid sequence, or that would not be encoded by the nucleotides that flank the naturally occurring nucleic acid sequence encoding the specified amino acid sequence as it occurs in the gene, if such nucleotides in the naturally occurring sequence were translated using standard codon usage for the organism from which the given amino acid sequence is derived. Similarly, the phrase “consisting essentially of”, when used with reference to a nucleic acid sequence herein, refers to a nucleic acid sequence encoding a specified amino acid sequence that can be flanked by from at least one, and up to as many as about 60, additional heterologous nucleotides at each of the 5′ and/or the 3′ end of the nucleic acid sequence encoding the specified amino acid sequence. The heterologous nucleotides are not naturally found (i.e., not found in nature, in vivo) flanking the nucleic acid sequence encoding the specified amino acid sequence as it occurs in the natural gene or do not encode a protein that imparts any additional function to the protein or changes the function of the protein having the specified amino acid sequence.
According to the present invention, the phrase “selectively binds to” refers to the ability of an antibody, antigen-binding fragment or binding partner of the present invention to preferentially bind to specified proteins. More specifically, the phrase “selectively binds” refers to the specific binding of one protein to another (e.g., an antibody, fragment thereof, or binding partner to an antigen), wherein the level of binding, as measured by any standard assay (e.g., an immunoassay), is statistically significantly higher than the background control for the assay. For example, when performing an immunoassay, controls typically include a reaction well/tube that contain antibody or antigen binding fragment alone (i.e., in the absence of antigen), wherein an amount of reactivity (e.g., non-specific binding to the well) by the antibody or antigen-binding fragment thereof in the absence of the antigen is considered to be background. Binding can be measured using a variety of methods standard in the art including enzyme immunoassays (e.g., ELISA, immunoblot assays, etc.).
Reference to a protein or polypeptide used in the present invention includes full-length proteins, fusion proteins, or any fragment, domain (structural, functional, or immunogenic), conformational epitope, or homologue of such proteins. An isolated protein, according to the present invention, is a protein (including a polypeptide or peptide) that has been removed from its natural milieu (i.e., that has been subject to human manipulation) and can include purified proteins, partially purified proteins, recombinantly produced proteins, and synthetically produced proteins, for example. As such, “isolated” does not reflect the extent to which the protein has been purified. Preferably, an isolated protein of the present invention is produced recombinantly. According to the present invention, the terms “modification” and “mutation” can be used interchangeably, particularly with regard to the modifications/mutations to the amino acid sequence of proteins or portions thereof (or nucleic acid sequences) described herein.
As used herein, the term “homologue” is used to refer to a protein or peptide which differs from a naturally occurring protein or peptide (i.e., the “prototype” or “wild-type” protein) by minor modifications to the naturally occurring protein or peptide, but which maintains the basic protein and side chain structure of the naturally occurring form. Such changes include, but are not limited to: changes in one or a few amino acid side chains; changes one or a few amino acids, including deletions (e.g., a truncated version of the protein or peptide) insertions and/or substitutions; changes in stereochemistry of one or a few atoms; and/or minor derivatizations, including but not limited to: methylation, glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitation, amidation and/or addition of glycosylphosphatidyl inositol. A homologue can have either enhanced, decreased, or substantially similar properties as compared to the naturally occurring protein or peptide. A homologue can include an agonist of a protein or an antagonist of a protein. Homologues can be produced using techniques known in the art for the production of proteins including, but not limited to, direct modifications to the isolated, naturally occurring protein, direct protein synthesis, or modifications to the nucleic acid sequence encoding the protein using, for example, classic or recombinant DNA techniques to effect random or targeted mutagenesis.
A homologue of a given protein may comprise, consist essentially of, or consist of, an amino acid sequence that is at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% identical, or at least about 95% identical, or at least about 96% identical, or at least about 97% identical, or at least about 98% identical, or at least about 99% identical (or any percent identity between 45% and 99%, in whole integer increments), to the amino acid sequence of the reference protein. In one embodiment, the homologue comprises, consists essentially of, or consists of, an amino acid sequence that is less than 100% identical, less than about 99% identical, less than about 98% identical, less than about 97% identical, less than about 96% identical, less than about 95% identical, and so on, in increments of 1%, to less than about 70% identical to the naturally occurring amino acid sequence of the reference protein.
As used herein, unless otherwise specified, reference to a percent (%) identity refers to an evaluation of homology which is performed using: (1) a BLAST 2.0 Basic BLAST homology search using blastp for amino acid searches and blastn for nucleic acid searches with standard default parameters, wherein the query sequence is filtered for low complexity regions by default (described in Altschul, S. F., Madden, T. L., Schääffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997) “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.” Nucleic Acids Res. 25:3389-3402, incorporated herein by reference in its entirety); (2) a BLAST 2 alignment (using the parameters described below); (3) and/or PSI-BLAST with the standard default parameters (Position-Specific Iterated BLAST. It is noted that due to some differences in the standard parameters between BLAST 2.0 Basic BLAST and BLAST 2, two specific sequences might be recognized as having significant homology using the BLAST 2 program, whereas a search performed in BLAST 2.0 Basic BLAST using one of the sequences as the query sequence may not identify the second sequence in the top matches. In addition, PSI-BLAST provides an automated, easy-to-use version of a “profile” search, which is a sensitive way to look for sequence homologues. The program first performs a gapped BLAST database search. The PSI-BLAST program uses the information from any significant alignments returned to construct a position-specific score matrix, which replaces the query sequence for the next round of database searching. Therefore, it is to be understood that percent identity can be determined by using any one of these programs.
Two specific sequences can be aligned to one another using BLAST 2 sequence as described in Tatusova and Madden, (1999), “Blast 2 sequences—a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250, incorporated herein by reference in its entirety. BLAST 2 sequence alignment is performed in blastp or blastn using the BLAST 2.0 algorithm to perform a Gapped BLAST search (BLAST 2.0) between the two sequences allowing for the introduction of gaps (deletions and insertions) in the resulting alignment. For purposes of clarity herein, a BLAST 2 sequence alignment is performed using the standard default parameters as follows.
For blastn, using 0 BLOSUM62 matrix:
Reward for match=1
Penalty for mismatch=−2
Open gap (5) and extension gap (2) penalties
gap x_dropoff (50) expect (10) word size (11) filter (on)
For blastp, using 0 BLOSUM62 matrix:
Open gap (11) and extension gap (1) penalties
gap x_dropoff (50) expect (10) word size (3) filter (on).
An isolated nucleic acid molecule is a nucleic acid molecule that has been removed from its natural milieu (i.e., that has been subject to human manipulation), its natural milieu being the genome or chromosome in which the nucleic acid molecule is found in nature. As such, “isolated” does not necessarily reflect the extent to which the nucleic acid molecule has been purified, but indicates that the molecule does not include an entire genome or an entire chromosome in which the nucleic acid molecule is found in nature. An isolated nucleic acid molecule can include a gene. An isolated nucleic acid molecule that includes a gene is not a fragment of a chromosome that includes such gene, but rather includes the coding region and regulatory regions associated with the gene, but no additional genes that are naturally found on the same chromosome. An isolated nucleic acid molecule may also include portions of a gene. An isolated nucleic acid molecule can also include a specified nucleic acid sequence flanked by (i.e., at the 5′ and/or the 3′ end of the sequence) additional nucleic acids that do not normally flank the specified nucleic acid sequence in nature (i.e., heterologous sequences). Isolated nucleic acid molecule can include DNA, RNA (e.g., mRNA), or derivatives of either DNA or RNA (e.g., cDNA). Although the phrase “nucleic acid molecule” primarily refers to the physical nucleic acid molecule and the phrase “nucleic acid sequence” primarily refers to the sequence of nucleotides on the nucleic acid molecule, the two phrases can be used interchangeably, especially with respect to a nucleic acid molecule, or a nucleic acid sequence, being capable of encoding a protein or domain of a protein.
A recombinant nucleic acid molecule is a molecule that can include at least one of any nucleic acid sequence encoding any one or more proteins described herein operatively linked to at least one of any transcription control sequence capable of effectively regulating expression of the nucleic acid molecule(s) in the cell to be transfected. Although the phrase “nucleic acid molecule” primarily refers to the physical nucleic acid molecule and the phrase “nucleic acid sequence” primarily refers to the sequence of nucleotides on the nucleic acid molecule, the two phrases can be used interchangeably, especially with respect to a nucleic acid molecule, or a nucleic acid sequence, being capable of encoding a protein. In addition, the phrase “recombinant molecule” primarily refers to a nucleic acid molecule operatively linked to a transcription control sequence, but can be used interchangeably with the phrase “nucleic acid molecule” which is administered to an animal.
A recombinant nucleic acid molecule includes a recombinant vector, which is any nucleic acid sequence, typically a heterologous sequence, which is operatively linked to the isolated nucleic acid molecule encoding a fusion protein of the present invention, which is capable of enabling recombinant production of the fusion protein, and which is capable of delivering the nucleic acid molecule into a host cell according to the present invention. Such a vector can contain nucleic acid sequences that are not naturally found adjacent to the isolated nucleic acid molecules to be inserted into the vector. The vector can be either RNA or DNA, either prokaryotic or eukaryotic, and preferably in the present invention, is a virus or a plasmid. Recombinant vectors can be used in the cloning, sequencing, and/or otherwise manipulating of nucleic acid molecules, and can be used in delivery of such molecules (e.g., as in a DNA composition or a viral vector-based composition). Recombinant vectors are preferably used in the expression of nucleic acid molecules, and can also be referred to as expression vectors. Preferred recombinant vectors are capable of being expressed in a transfected host cell.
In a recombinant molecule of the present invention, nucleic acid molecules are operatively linked to expression vectors containing regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the host cell and that control the expression of nucleic acid molecules of the present invention. In particular, recombinant molecules of the present invention include nucleic acid molecules that are operatively linked to one or more expression control sequences. The phrase “operatively linked” refers to linking a nucleic acid molecule to an expression control sequence in a manner such that the molecule is expressed when transfected (i.e., transformed, transduced or transfected) into a host cell.
According to the present invention, the term “transfection” is used to refer to any method by which an exogenous nucleic acid molecule (i.e., a recombinant nucleic acid molecule) can be inserted into a cell. The term “transformation” can be used interchangeably with the term “transfection” when such term is used to refer to the introduction of nucleic acid molecules into microbial cells, such as algae, bacteria and yeast. In microbial systems, the term “transformation” is used to describe an inherited change due to the acquisition of exogenous nucleic acids by the microorganism and is essentially synonymous with the term “transfection.” Therefore, transfection techniques include, but are not limited to, transformation, chemical treatment of cells, particle bombardment, electroporation, microinjection, lipofection, adsorption, infection and protoplast fusion.
The following experimental results are provided for purposes of illustration and are not intended to limit the scope of the invention.
EXAMPLES
Example 1
Yeast-Based Immunotherapeutic Design and Production
The following example describes the design and production of several different yeast-based immunotherapeutic compositions for the treatment or prevention of adenovirus-36 (Ad-36) infection.
In these experiments, yeast (e.g., Saccharomyces cerevisiae) were engineered to express various Ad-36 fusion proteins under the control of the copper-inducible promoter, CUP1, or the TEF2 promoter. Briefly, to produce each of the yeast-based immunotherapeutics constructed in this Example, DNA encoding the Ad-36 antigen as set forth for each fusion protein below was prepared, codon optimized for expression in yeast, and then digested with SpeI and NotI and inserted behind the CUP1 promoter (pGI-100) or the TEF2 promoter (pTK57-1), as indicated for each construct below, in yeast 2 μm expression vectors. The resulting plasmids were introduced into Saccharomyces cerevisiae W303α yeast by Lithium acetate/polyethylene glycol transfection, and primary transfectants were selected on solid minimal plates lacking Uracil (UDM; uridine dropout medium). Other yeast strains, yeast species or yeast genera can be used in yeast-based immunotherapeutics of the invention; Saccharomyces cerevisiae W303α is an exemplary strain. Colonies were re-streaked onto UDM or ULDM (uridine and leucine dropout medium) and allowed to grow for 3 days at 30° C. Liquid cultures lacking uridine (U2) or lacking uridine and leucine (UL2) were inoculated from plates and starter cultures were grown for 20 h at 30° C., 250 rpm. If desired, although not used for these experiments, pH buffered media containing 4.2 g/L of Bis-Tris (BT-U2; BT-UL2) can be inoculated. Primary cultures were used to inoculate final cultures of the same formulation and growth is continued until a density or 1.1 to 4.0 Y.U./mL is reached.
For TEF2 strains (constitutive expression), cells were then harvested, washed and heat killed at 56° C. for 1 h in PBS. For CUP1 strains (inducible expression), expression was induced in the same medium with 0.375 mM copper sulfate for 5 h at 30° C., 250 rpm. Cells were harvested, washed and heat killed at 56° C. for 1 h in PBS.
After heat kill of TEF2 and CUP1 cultures, cells were washed three times in PBS. Total protein expression was measured by a TCA precipitation/nitrocellulose binding assay and Ad-36 fusion protein expression was measured by western blot using an anti-his tag monoclonal antibody (see FIGS. 1 and 2). As described below, FIGS. 1 and 2 showed that the yeast-based immunotherapy composition of the invention expressed the Ad-36 fusion protein well using both promoters, and using two different N-terminal sequences in the fusion proteins (SEQ ID NO:56 or SEQ ID NO:58), and were readily identified by Western blot.
Recipe for U2 liquid medium:
20 g/L of glucose
6.7 g/L of Yeast nitrogen base containing ammonium sulfate
0.04 mg/mL each of histidine, leucine, tryptophan, and adenine
Recipe for UL2 liquid medium:
20 g/L of glucose
6.7 g/L of Yeast nitrogen base containing ammonium sulfate
0.04 mg/mL each of histidine, tryptophan, and adenine
Several different yeast-based immunotherapeutics expressing Ad-36 fusion proteins were produced in this experiment. One yeast-based immunotherapeutic, denoted in FIG. 1 as “FIB”, was designed to express an Ad-36 fusion protein as a single polypeptide comprising selected portions of the Ad-36 fiber protein (the full Ad-36 fiber protein is represented by SEQ ID NO:34), fused at its N-terminus to a synthetic peptide represented by SEQ ID NO:58. Saccharomyces cerevisiae were engineered to express this protein under the control of the TEF2 promoter. The fusion protein has the following sequence elements fused in frame from N- to C-terminus, represented by SEQ ID NO:42: (1) an N-terminal peptide to impart resistance to proteasomal degradation and stabilize expression (positions 1 to 6 of SEQ ID NO:42); (2) positions 71-136 of Ad-36 fiber (positions 71-136 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 7-72 of SEQ ID NO:42; (3) positions 145-169 of Ad-36 fiber (positions 145-169 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 73-97 of SEQ ID NO:42; (4) positions 290-313 of Ad-36 fiber (positions 290-313 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 98-194 of SEQ ID NO:42; (5) positions 334-363 of Ad-36 fiber (positions 334-363 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 195-224 of SEQ ID NO:42; and (6) a hexahistidine tag (positions 225-230 of SEQ ID NO:42). The amino acid segments used in this fusion protein can be modified by the use of additional amino acids flanking either end of any domain. The nucleic acid sequence encoding the fusion protein of SEQ ID NO:42 was codon optimized for expression in yeast, and the yeast-based immunotherapeutic expressing this fusion protein was produced as described above.
The expression of this Ad-36 fiber fusion protein in yeast is shown in FIG. 1 (FIB). The estimated expression level of the fusion protein was 1704 ng/Y.U.
Another yeast-based immunotherapeutic, denoted in FIG. 2 as “aFL-Fib” was designed to express an Ad-36 fusion protein as a single polypeptide comprising portions of the Ad-36 fiber protein (the full Ad-36 fiber protein is represented by SEQ ID NO:34) fused at its N-terminus to a yeast alpha factor signal leader (SEQ ID NO:56). Saccharomyces cerevisiae were engineered to express this protein under the control of the CUP1 promoter. This fusion protein has the following sequence elements fused in frame from N- to C-terminus, represented by SEQ ID NO:48: (1) an N-terminal peptide to impart resistance to proteasomal degradation and stabilize or enhance expression (SEQ ID NO:56, or positions 1 to 89 of SEQ ID NO: 48); (2) a two amino acid spacer/linker (Thr-Ser) to facilitate cloning and manipulation of the sequences (positions 90 to 91 of SEQ ID NO:48); (3) positions 71-136 of Ad-36 fiber (positions 71-136 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 92-157 of SEQ ID NO:48; (4) positions 145-169 of Ad-36 fiber (positions 145-169 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 158-182 of SEQ ID NO:48; (5) positions 290-313 of Ad-36 fiber (positions 290-313 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 183-279 of SEQ ID NO:48; (6) positions 334-363 of Ad-36 fiber (positions 334-363 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 280-309 of SEQ ID NO:48; and (7) a hexahistidine tag (positions 310-315 of SEQ ID NO:48). The amino acid segments used in this fusion protein can be modified by the use of additional amino acids flanking either end of any domain; the example provided herein is exemplary. The nucleic acid sequence encoding the fusion protein of SEQ ID NO:48 was codon optimized for expression in yeast, and the yeast-based immunotherapeutic expressing this fusion protein was produced as described above.
The expression of this Ad-36 fiber fusion protein in yeast is shown in FIG. 2 (aFL-FIB). The estimated expression level of the fusion protein was 14,854 ng/Y.U.
Another yeast-based immunotherapeutic, denoted in FIG. 1 as “HEX”, was designed to express an Ad-36 fusion protein as a single polypeptide comprising portions of the Ad-36 hexon protein (the full Ad-36 hexon protein is represented by SEQ ID NO:18) fused at its N-terminus to a synthetic peptide represented by SEQ ID NO:58. Saccharomyces cerevisiae were engineered to express this protein under the control of the TEF2 promoter. This fusion protein has the following sequence elements fused in frame from N- to C-terminus, represented by SEQ ID NO:43: (1) an N-terminal peptide to impart resistance to proteasomal degradation and stabilize expression (positions 1 to 6 of SEQ ID NO:43); (2) positions 136-218 of Ad-36 hexon (positions 136-218 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 7-89 of SEQ ID NO:43; (3) positions 235-285 of Ad-36 hexon (positions 235-285 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 90-141 of SEQ ID NO:43; (4) positions 297-308 of Ad-36 hexon (positions 297-308 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 142-153 of SEQ ID NO:43; (5) positions 410-450 of Ad-36 hexon (positions 410-450 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 154-194 of SEQ ID NO:43; and (6) a hexahistidine tag (positions 195-200 of SEQ ID NO:43). The amino acid segments used in this fusion protein can be modified by the use of additional amino acids flanking either end of any domain. The nucleic acid sequence encoding the fusion protein of SEQ ID NO:43 was codon optimized for expression in yeast, and the yeast-based immunotherapeutic expressing this fusion protein was produced as described above.
The expression of this Ad-36 hexon fusion protein in yeast is shown in FIG. 1 (HEX). The estimated expression level of this protein was 1981 ng/Y.U.
Another yeast-based immunotherapeutic, denoted in FIG. 2 as “aFL-Hexon” was designed to express an Ad-36 fusion protein as a single polypeptide comprising portions of the Ad-36 hexon protein (the full Ad-36 hexon protein is represented by SEQ ID NO:18) fused with yeast alpha factor leader signal sequence (SEQ ID NO:56). Saccharomyces cerevisiae were engineered to express this protein under the control of the CUP1 promoter. This fusion protein has the following sequence elements fused in frame from N- to C-terminus, represented by SEQ ID NO:50: (1) an N-terminal peptide to impart resistance to proteasomal degradation and stabilize or enhance expression (SEQ ID NO:56, or positions 1 to 89 of SEQ ID NO:50); 2) a two amino acid spacer/linker (Thr-Ser) to facilitate cloning and manipulation of the sequences (positions 90 to 91 of SEQ ID NO:50); (3) positions 136-218 of Ad-36 hexon (positions 136-218 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 92-174 of SEQ ID NO:50; (4) positions 235-285 of Ad-36 hexon (positions 235-285 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 175-226 of SEQ ID NO:50; (5) positions 297-308 of Ad-36 hexon (positions 297-308 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 227-238 of SEQ ID NO:50; (6) positions 410-450 of Ad-36 hexon (positions 410-450 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 239-279 of SEQ ID NO:50; and (7) a hexahistidine tag (positions 280-285 of SEQ ID NO:50). The amino acid segments used in this fusion protein can be modified by the use of additional amino acids flanking either end of any domain. The nucleic acid sequence encoding the fusion protein of SEQ ID NO:50 was codon optimized for expression in yeast, and the yeast-based immunotherapeutic expressing this fusion protein was produced as described above.
The expression of this Ad-36 hexon fusion protein in yeast is shown in FIG. 2 (aFL-Hexon). The estimated expression level of this protein was 19,695 ng/Y.U.
Another yeast-based immunotherapeutic, denoted in FIG. 2 as “aFL-Hexon-F”, was designed to express an Ad-36 fusion protein as a single polypeptide comprising the full-length Ad-36 hexon protein (the full hexon protein is represented by SEQ ID NO:18), fused at its N-terminus to yeast alpha factor leader sequence (SEQ ID NO:56). Saccharomyces cerevisiae were engineered to express this protein under the control of the TEF2 promoter. This fusion protein has the following sequence elements fused in frame from N- to C-terminus, represented by SEQ ID NO:52: (1) an N-terminal peptide to impart resistance to proteasomal degradation and stabilize or enhance expression (SEQ ID NO:56, or positions 1 to 89 of SEQ ID NO:52); 2) a two amino acid spacer/linker (Thr-Ser) to facilitate cloning and manipulation of the sequences (positions 90 to 91 of SEQ ID NO:52); (3) positions 2-944 of Ad-36 hexon (positions 2-944 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 92-1034 of SEQ ID NO:52; and (3) a hexahistidine tag (positions 1035-1040 of SEQ ID NO:52). This construct contains demonstrated or putative MHC Class I epitopes (e.g., positions 204-214 of SEQ ID NO:52; positions 404-412 of SEQ ID NO:52; positions 795-803 of SEQ ID NO:52; positions 928-936 of SEQ ID NO:52; or positions 994-1000 of SEQ ID NO:52), and demonstrated or putative MHC Class II epitopes (e.g., positions 100-110 of SEQ ID NO:52; positions 116-126 of SEQ ID NO:52; 406-420 of SEQ ID NO:52; positions 458-468 of SEQ ID NO:52; positions 792-803 of SEQ ID NO:52; or positions 947-957 of SEQ ID NO:52). The amino acid segments used in this fusion protein can be modified by the use of additional amino acids flanking either end of any domain. The nucleic acid sequence encoding the fusion protein of SEQ ID NO:44 was codon optimized for expression in yeast, and the yeast-based immunotherapeutic expressing this fusion protein was produced as described above.
The expression of this Ad-36 hexon fusion protein in yeast is shown in FIG. 2 (aFL-Hexon-F). The estimated expression level of this protein was 25,315 ng/Y.U.
Another yeast-based immunotherapeutic, denoted in FIG. 1 as “CRAG”, was designed to express an Ad-36 fusion protein as a single polypeptide comprising portions of the Ad-36 CR1α and CR1γ proteins (the full CR1α protein is represented by SEQ ID NO:26 and the full CR1γ protein is represented by SEQ ID NO:29), fused at its N-terminus to a synthetic peptide represented by SEQ ID NO:58. Saccharomyces cerevisiae were engineered to express this protein under the control of the TEF2 promoter. This fusion protein has the following sequence elements fused in frame from N- to C-terminus, represented by SEQ ID NO:47: (1) an N-terminal peptide to impart resistance to proteasomal degradation and stabilize expression (positions 1 to 6 of SEQ ID NO:47); (2) positions 18-60 of CR1α (positions 18-60 of SEQ ID NO:26 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 7-49 of SEQ ID NO:47; (3) positions 123-157 of Ad-36 CR1α (positions 123-157 of SEQ ID NO:26 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 50-84 of SEQ ID NO:47; (4) positions 19-60 of Ad-36 CR1γ (positions 19-60 of SEQ ID NO:29 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 85-126 of SEQ ID NO:47; (5) positions 83-116 of Ad-36 CR1γ (positions 83-116 of SEQ ID NO:29 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 127-160 of SEQ ID NO:47; and (6) a hexahistidine tag (positions 161-166 of SEQ ID NO:47). The amino acid segments used in this fusion protein can be modified by the use of additional amino acids flanking either end of any domain. The nucleic acid sequence encoding the fusion protein of SEQ ID NO:43 was codon optimized for expression in yeast, and the yeast-based immunotherapeutic expressing this fusion protein was produced as described above.
The expression of this Ad-36 CR1α-CR1γ fusion protein is shown in FIG. 1 (CRAG). The estimated expression level of this protein was 3341 ng/Y.U.
Another yeast-based immunotherapeutic, denoted “aFL-CRAG” in FIG. 2, was designed to express an Ad-36 fusion protein as a single polypeptide comprising portions of the Ad-36 CR1α and CR1γ proteins (the full CR1α protein is represented by SEQ ID NO:26 and the full CR1γ protein is represented by SEQ ID NO:29), fused at its N-terminus to yeast alpha factor leader sequence (SEQ ID NO:56). Saccharomyces cerevisiae were engineered to express this protein under the control of the CUP1 promoter. This fusion protein has the following sequence elements fused in frame from N- to C-terminus, represented by SEQ ID NO:54: (1) an N-terminal peptide to impart resistance to proteasomal degradation and stabilize or enhance expression (SEQ ID NO:56, or positions 1 to 89 of SEQ ID NO:54); 2) a two amino acid spacer/linker (Thr-Ser) to facilitate cloning and manipulation of the sequences (positions 90 to 91 of SEQ ID NO:54); (3) positions 18-60 of CR1α (positions 18-60 of SEQ ID NO:26 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 92-134 of SEQ ID NO:54; (4) positions 123-157 of Ad-36 CR1α (positions 123-157 of SEQ ID NO:26 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 135-169 of SEQ ID NO:54; (5) positions 19-60 of Ad-36 CR1γ (positions 19-60 of SEQ ID NO:29 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 170-211 of SEQ ID NO:54; (6) positions 83-116 of Ad-36 CR1γ (positions 83-116 of SEQ ID NO:29 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 212-245 of SEQ ID NO:54; and (7) a hexahistidine tag (positions 246-251 of SEQ ID NO:54). The amino acid segments used in this fusion protein can be modified by the use of additional amino acids flanking either end of any domain. The nucleic acid sequence encoding the fusion protein of SEQ ID NO:54 was codon optimized for expression in yeast, and the yeast-based immunotherapeutic expressing this fusion protein was produced as described above. The expression of this Ad-36 CR1α-CR1γ fusion protein is shown in FIG. 2 (aFL-CRAG). The estimated expression level of this protein was 16,154 ng/Y.U.
Additional yeast-based immunotherapeutic compositions have been designed by the inventors and are produced using the same protocols described above. For example, another yeast-based immunotherapeutic is designed to express an Ad-36 fusion protein as a single polypeptide comprising the full-length Ad-36 hexon protein (the full hexon protein is represented by SEQ ID NO:18), fused at its N-terminus to a synthetic peptide represented by SEQ ID NO:58. Saccharomyces cerevisiae are engineered to express this protein under the control of the TEF2 or CUP1 promoter. This fusion protein has the following sequence elements fused in frame from N- to C-terminus, represented by SEQ ID NO:44: (1) an N-terminal peptide to impart resistance to proteasomal degradation and stabilize expression (positions 1 to 6 of SEQ ID NO:44); (2) positions 2-944 of Ad-36 hexon (positions 2-944 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 7-949 of SEQ ID NO:44; and (3) a hexahistidine tag (positions 950-955 of SEQ ID NO:44). This construct contains demonstrated or putative MHC Class I epitopes (e.g., positions 119-129 of SEQ ID NO:44; positions 319-327 of SEQ ID NO:44; positions 710-718 of SEQ ID NO:44; positions 843-851 of SEQ ID NO:44; or positions 909-915 of SEQ ID NO:44), and demonstrated or putative MHC Class II epitopes (e.g., positions 15-25 of SEQ ID NO:44; positions 31-41 of SEQ ID NO:44; 321-335 of SEQ ID NO:44; positions 373-383 of SEQ ID NO:44; positions 707-718 of SEQ ID NO:44; or positions 862-872 of SEQ ID NO:44). The amino acid segments used in this fusion protein can be modified by the use of additional amino acids flanking either end of any domain; the example provided herein is exemplary. A nucleic acid sequence encoding the fusion protein of SEQ ID NO:44 is codon optimized for expression in yeast, and a yeast-based immunotherapeutic expressing this fusion protein is produced as described above.
Another yeast-based immunotherapeutic is designed to express an Ad-36 fusion protein as a single polypeptide comprising portions of the Ad-36 fiber and hexon proteins (full protein represented by SEQ ID NO:34 (fiber) and SEQ ID NO:18 (hexon)), fused at its N-terminus to a synthetic peptide represented by SEQ ID NO:58. Saccharomyces cerevisiae are engineered to express this protein under the control of the TEF2 or CUP1 promoter. This fusion protein has the following sequence elements fused in frame from N- to C-terminus, represented by SEQ ID NO:45: (1) an N-terminal peptide to impart resistance to proteasomal degradation and stabilize expression (positions 1 to 6 of SEQ ID NO:45); (2) positions 71-136 of Ad-36 fiber (positions 71-136 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 7-72 of SEQ ID NO:45; (3) positions 145-169 of Ad-36 fiber (positions 145-169 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 73-97 of SEQ ID NO:45; (4) positions 290-313 of Ad-36 fiber (positions 290-313 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 98-194 of SEQ ID NO:45; (5) positions 334-363 of Ad-36 fiber (positions 334-363 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 195-224 of SEQ ID NO:45; (6) positions 136-218 of Ad-36 hexon (positions 136-218 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 225-307 of SEQ ID NO:45; (7) positions 235-285 of Ad-36 hexon (positions 235-285 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 308-359 of SEQ ID NO:45; (8) positions 297-308 of Ad-36 hexon (positions 297-308 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 360-371 of SEQ ID NO:45; (9) positions 410-450 of Ad-36 hexon (positions 410-450 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 372-412 of SEQ ID NO:45; and (10) a hexahistidine tag (positions 413-418 of SEQ ID NO:45). The amino acid segments used in this fusion protein can be modified by the use of additional amino acids flanking either end of any domain; the example provided herein is exemplary. A nucleic acid sequence encoding the fusion protein of SEQ ID NO:45 is codon optimized for expression in yeast, and the yeast-based immunotherapeutic expressing this fusion protein is produced as described above.
Yet another yeast-based immunotherapeutic is designed to express an Ad-36 fusion protein as a single polypeptide comprising portions of the Ad-36 hexon and fiber proteins (full protein represented by SEQ ID NO:18 (hexon) and SEQ ID NO:34 (fiber)) fused at its N-terminus to a synthetic peptide represented by SEQ ID NO:58. Saccharomyces cerevisiae are engineered to express this protein under the control of the TEF2 or CUP1 promoter. This fusion protein has the following sequence elements fused in frame from N- to C-terminus, represented by SEQ ID NO:46: (1) an N-terminal peptide to impart resistance to proteasomal degradation and stabilize expression (positions 1 to 6 of SEQ ID NO:46); (2) positions 136-218 of Ad-36 hexon (positions 136-218 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 7-89 of SEQ ID NO:46; (3) positions 235-285 of Ad-36 hexon (positions 235-285 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 90-141 of SEQ ID NO:46; (4) positions 297-308 of Ad-36 hexon (positions 297-308 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 142-153 of SEQ ID NO:46; (5) positions 410-450 of Ad-36 hexon (positions 410-450 of SEQ ID NO:18 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 154-194 of SEQ ID NO:46; (6) positions 71-136 of Ad-36 fiber (positions 71-136 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 195-260 of SEQ ID NO:46; (7) positions 145-169 of Ad-36 fiber (positions 145-169 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 261-285 of SEQ ID NO:46; (8) positions 290-313 of Ad-36 fiber (positions 290-313 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 286-382 of SEQ ID NO:46; (9) positions 334-363 of Ad-36 fiber (positions 334-363 of SEQ ID NO:34 or a corresponding sequence from another Ad-36 strain or isolate), corresponding to positions 383-412 of SEQ ID NO:46; and (10) a hexahistidine tag (positions 413-418 of SEQ ID NO:46). The amino acid segments used in this fusion protein can be modified by the use of additional amino acids flanking either end of any domain; the example provided herein is exemplary. A nucleic acid sequence encoding the fusion protein of SEQ ID NO:46 is codon optimized for expression in yeast, and the yeast-based immunotherapeutic expressing this fusion protein is produced as described above.
Example 2
Infection/Replication of Ad-36 in Rat Stem Cells and A549 Cells
The following example describes the ability of Ad-36 to infect primary preadipocytes and A549 cells. This experiment demonstrates that viral stock that is intended for use in in vivo experiments described in the examples below is biologically active and can infect target cells of relevance in vitro. Ad-36 is a DNA virus and lacks mRNA. Transcription of Ad-36 genes into mRNA does not occur unless the virus has infected a mammalian host cell. The presence of Ad-36 mRNAs in the target cells is therefore direct evidence of viral infection and replication.
Purified Ad-36 viral stock was added to rat adipose derived stem cells (ASC) and to A549 cells (human lung carcinoma line that is a natural host cell for human adenoviruses) in culture at a multiplicity of infection (MOI) of 5. Fifteen hours post-viral addition, total RNA was isolated from the target cells and was subjected to real-time reverse transcription PCR (RT-PCR) with fluorescent SYBR green designed to specifically measure the rate of PCR amplification of E1A, E4 orf1, and Hexon mRNAs. The relative expression of these genes was determined for mock-infected or Ad-36 infected targets. The results, shown in FIG. 3 (ASC) and FIG. 4 (A549) show the E1A and Hexon genes were expressed in both cellular targets and that the E4 Orf1 was expressed specifically in A549 cells. Gene expression required the addition of Ad-36, since mock infected cells showed only background levels of signal in all reactions.
Example 3
Rat Pilot Study-Ad36 Kinetics, and Infection of Visceral Fat Tissues 
The following example describes the ability of the Ad-36 stock to infect rats in vivo and evaluates: i) the optimal dose of Ad-36 giving rise to successful viral inoculation, otherwise known as ‘viral take’; ii) the kinetics of the blood viremic phase of infection; iii) the ability of the virus to infect the visceral adipose tissue.
Prior to the present invention, to the inventors' knowledge, there were no kinetic or dosing experiments or fat localization studies available that were robust enough or sufficient to establish an optimal animal model of Ad-36 infection that would be useful to evaluate prophylactic and therapeutic vaccine efficacy. Accordingly, the following experiments were designed to provide this information and to establish a relevant and useful model for studying Ad-36 infection (acute and chronic). Briefly, rats were injected intraperitoneally with PBS only or purified Ad-36 viral particles at 3 doses (107′ 108 or 109 plaque forming units (PFU)), according to the protocol shown in Table 2. Blood samples were taken from rats at days 0 (pre-challenge) and days 1 post challenge (20 h), and at days 2 and 4 post-challenge. Virus DNA was prepared from 100 μl plasma using the QIAAMP® MINELUTE® Virus Kit (Qiagen), and the level of viral DNA was estimated by real time quantitative PCR (qPCR) featuring an Ad-36 hexon-DNA specific probe. Estimates of viral copy number were obtained by interpolation against a standard curve produced with purified hexon plasmid of known copy number. At two weeks post-challenge, rats were euthanized, the visceral fat was dissected and total DNA was isolated from the fat tissue using a proteinase K/isopropanol precipitation method. The DNA was subjected to nested two-round PCR featuring hexon DNA-specific PCR primers.













TABLE 2











Fat




Ad36


Blood Draws
Tissue



#
Dose
Total V.P.

(days
Dis-


Group
Rats
(PFU)
injected
Route
post-challenge)
section
























A
2
0
0
i.p.
d0
d1
d2
d4
d14


B
2
107
2.3 × 109 
i.p.
d0
d1
d2
d4
d14


C
2
108
2.3 × 1010
i.p.
d0
d1
d2
d4
d14


D
2
109
2.3 × 1011
i.p.
d0
d1
d2
d4
d14









Results showing the virus particle (V.P.) copies per ml blood at each level of viral infection are provided in FIG. 5 (Group A; mock control); FIG. 6 (Group B; 107 PFU Ad-36); FIG. 7 (Group C; 108 PFU Ad-36); and FIG. 8 (Group D; 109 PFU Ad-36). FIG. 9 shows the results of PCR to detect Ad-36 hexon in visceral fat. Taken together, the results of these experiments demonstrate that: 1) the level of Ad-36 virus in the blood as determined by hexon qPCR is maximal at the 109 PFU dose, and at 20 h post challenge; and 2) the Ad-36 virus is present in the visceral adipose tissue of all rats by 2 weeks post challenge at the 108 and 109 PFU doses, whereas at the 107 PFU dose, virus was robustly detectable in the adipose tissue of only one of the two rats. These data show that the purified Ad-36 stock that was shown to infect primary preadipocytes in culture in Example 2 are also infective in vivo, and confirm published reports (e.g., Pasarica et al, 2008) that Ad-36 infects visceral adipose tissues. Since the maximal levels of viremia occurred with injection of 109 PFU (see FIG. 8), this dose was selected for challenge of rats in the yeast-based immunotherapy vaccination experiments described in the following examples. Accordingly, these data were used to establish an optimized rat model system of Ad-36 infection for the testing of prophylactic and therapeutic vaccines (immunotherapy).
Example 4
Effect of Prophylactic Administration of Ad-36 Tarmogens in the Rat Model of Ad-36 Infection
The following example describes the use of yeast-based adenovirus-36 (Ad-36) immunotherapeutics in rat prophylactic model of adenovirus-related obesity.
A rat model has been studied in the literature (Dhurandhar et al, Obesity 11:1905, 2006) in which Ad36-infected rats attained significantly greater body weight and fat pad weight by 30 weeks post-inoculation than mock infected control rats. Epididymal-inguinal, retroperitoneal, and visceral fat pad weights of the infected group were greater than PBS control rats by 60%, 46%, and 86%, respectively (p<0.00001). The present inventors have improved this rat model for the purposes of evaluating prophylactic and therapeutic vaccines, as described above in Example 3.
The following experiment describes a study to determine if prophylactic administration of the yeast-based immunotherapeutic compositions described in Example 1 prevent or reduce the extent of or rate of Ad-36-induced weight gain.
Cohorts of rats (n=18/group) were immunized subcutaneously (s.c.) with yeast-based Ad-36 immunotherapeutic compositions (vaccines), administered at four different sites with 20 million yeast cells (2 Y.U.) in 0.1 ml per site. In these experiments, two different yeast-based immunotherapeutic compositions were used. “Ad-aFL-CRAG” is the yeast-based immunotherapeutic described in Example 1 above that expresses an Ad-36 fusion protein comprising Ad-36 CR1α and CR1γ antigens, these antigens having an amino acid sequence of SEQ ID NO:55, which are linked at the N-terminus to an alpha factor leader sequence, to form a complete fusion protein having the amino acid sequence of SEQ ID NO:54. “Ad-aFL-HEX-Full” is the yeast-based immunotherapeutic described in Example 1 above that expresses an Ad-36 fusion protein comprising a near full-length hexon antigen, the antigen having an amino acid sequence of SEQ ID NO:53, which is linked at its N-terminus to an alpha factor leader sequence, to form a complete fusion protein having the amino acid sequence of SEQ ID NO:52. Dosing was once per week for 3 weeks and then, after a two week rest, rats were challenged intraperitoneally with Ad-36 (109 PFU), which was established in Example 3 to be the optimal viral dose for evaluating Ad-36 infection. Immunization was then conducted once per month for up to 30 weeks post-challenge. The experimental cohorts are shown in Table 3. Additional control groups include a group of rats receiving PBS only (naïve or “PBS”), and a group of rats immunized with control yeast compositions (“empty vector” yeast or “YVEC”, which are yeast transfected with a vector that does not contain an antigen insert; i.e., these yeast do not express an Ad-36 antigen(s)).












TABLE 3








Pre-challenge

Post-challenge



Group
Immunization
Challenge
Immunization









A
PBS
PBS
PBS



B
PBS
Ad-36
PBS



C
YVEC
Ad-36
YVEC



D
Yeast-Ad-aFL-
Ad-36
Ad-aFL-CRAG




CRAG



E
Yeast-Ad-aFL-
Ad-36
Ad-aFL-HEX-Full




HEX-Full










Animals were weighed pre-immunization, pre-viral challenge and then biweekly for approximately 30 weeks following inoculation with virus. Food and water consumption were monitored throughout the study. Blood was collected at baseline, before viral challenge, and monthly following viral challenge to monitor for Ad-36 DNA, cholesterol, triglyceride levels, corticosterone, neutralizing antibodies to Ad-36, and other parameters (see Example 5). Glucose tolerance testing is performed at selected intervals and urine glucose levels are also measured. Blood (500 μl per timepoint) was obtained under isofluroane anaesthesia from the tail vein. At the end of the study, animals are euthanized and adipose tissue is harvested to measure viral levels by polymerase chain reaction (PCR). PCR may also be performed on biopsies obtained during the course of the study.
This experiment was performed in outbred Wistar rats. If, as expected, weight gain is prevented or reduced (or the rate of weight gain is reduced) in rats immunized with yeast-based Ad-36 immunotherapy as compared to control rats, inbred Wistar Furth rats will be evaluated according to the same or similar protocol, as this rat is expected to be more amenable to evaluation of T cell immunity. Additional experiments can also be conducted to determine the effect of diet or other factors in conjunction with immunotherapy (e.g., by administering a high fat diet versus a normal diet).
Immunization with a yeast-based Ad-36 immunotherapy composition is deemed active in this study if it causes, as compared with empty vector yeast or PBS controls, notable trends towards normalization of or beneficial outcome (more healthy, less characteristic of obesity or being or becoming overweight) in any one or more of the following parameters for Ad-36 infected rats: i) body weight or rate of body weight gain; ii) percent body fat or body mass index); iii) frequency or titer of neutralizing antibodies; iv) cholesterol levels; v) serum triglycerides vi) serum corticosterone; vii) blood and/or urine glucose levels; viii) glucose tolerance; ix) blood Ad-36 viral titer. Certain of these parameters have already been observed as positive indicators of the effectiveness of Ad-36-targeted yeast-based immunotherapy in immunized rats (see following discussion) at 18 weeks post-challenge, and are believed to show that yeast-based immunotherapy targeting Ad-36 is effective for reducing the rate of weight gain in an antigen-specific manner. It is expected that by the end of the study at 30 weeks when the Ad-36 induced phenotype fully emerges, the results will demonstrate that immunization with a yeast-based Ad-36 immunotherapy composition is effective for reducing and/or preventing weight gain, reducing rate of weight gain, and/or reducing or preventing adiposity in rats infected with Ad-36 in an antigen-specific or Ad-36-specific manner, and this may be accompanied by changes in the biochemical parameters mentioned, given their known association with the obesity phenotype.
As discussed above, the present study is currently at week 18 post-viral challenge. Virus-induced weight gain in control rats is not anticipated to be measurable at this early time-point based on work by Dhurandhar (Dhurandhar et al 2006). Consistent with this expectation, the weight gain data through week 18 show that Ad-36 challenge has not yet caused weight gain above PBS injected control rats. However, the aFL-CRAG Tarmogen immunization group already has a lower overall weight gain than rats in the other groups, as shown in FIGS. 10, 11 and 12. Specifically, FIG. 10 is a scatter plot showing individual rats in each of the immunization groups, and revealing a clear trend in the yeast-based immunotherapy groups, and particularly in the rats immunized with a yeast-based immunotherapeutic expressing Ad-36 CR1α and CR1γ, toward a lower rate of weight gain as compared to rats immunized with PBS only (PBS) or with the “empty vector” yeast control (YVEC). FIG. 11 shows the median weight gain for each group of animals over time. Again, the reduced rate of weight gain as compared to controls in the rats immunized with yeast-based immunotherapeutic expressing Ad-36 CR1α and CR1γ is clear. FIG. 12 illustrates two individual time points (4 weeks post-viral challenge and 12 weeks post-viral challenge) and again, the reduced rate of weight gain in rats immunized with yeast-Ad-36 immunotherapy as compared to the PBS control is evident (p values are relative to the PBS control). Error bars in FIG. 12 are generated based on comparison to the PBS-immunized, virus-challenged control group and statistical significance is measured also as compared to this group.
Taken together, these data demonstrate an Ad-36-specific, and particularly, an Ad-36 CR1α-CR1γ-antigen specific effect, of the yeast-based immunotherapeutic on body weight gain, and one that has emerged before an Ad-36 emergent obesity phenotype is even apparent. A plot of the body weight at weeks 4 and 12 shows that this the weight gain of aFL-CRAG immunized rats is statistically significantly lower than the weight gain of YVEC (control yeast) or Naive rats (PBS) at these time-points (FIG. 12). The rats immunized with the yeast expressing a hexon-based fusion protein show a trend toward a similar phenotype, although at this time point, the difference from controls is not as substantial as for the yeast expressing the CR1α-CR1γ-antigen. Therefore, yeast-based immunotherapy targeting Ad-36 reduces the rate of weight gain in an animal model of chronic Ad-36 infection, and is expected to show reduced weight gain and additional benefits, as compared to the controls, with respect to the other parameters discussed above by 30 weeks post-challenge.
Example 5
Viral Kinetics in the Prophylactic Ad-36 Yeast-Based Immunotherapy Study (Rat)
The following experiment demonstrates the use of the method described in Example 4 to test Ad-36 viral kinetics in the bloodstream after Ad-36 viral challenge.
Briefly, blood genomic DNA was extracted from 100 μl of rat blood using Qiagen's QIAamp Kit. Ad-36 DNA was detected by quantitative polymerase chain reaction (qPCR), featuring a unique Hexon-gene specific probe designed by the inventors. The results, illustrated in FIG. 13, show that Ad-36 DNA is present at 106 to 109 copies per mL for up to 9 weeks post-challenge, and was cleared from the blood completely by 13 weeks post-challenge. Interestingly, the inter-rat variability of viral DNA load decreases over time, reaching a minimum just before clearance. Without being bound by theory, the inventors believe that these data could reflect the natural immune response to the virus, the yeast-based immunotherapy-induced immune response to the virus, or some combination of these effects.
Example 6
Rat Therapeutic Experiment
The following example describes the use of yeast-based Ad-36 immunotherapeutics in a rat therapeutic model of adenovirus-related obesity.
In the following experiment, yeast-based Ad-36 immunotherapeutic compositions (vaccines) were evaluated to determine whether immunization against this virus using yeast-based immunotherapy can reverse obesity or at least reduce weight gain or the rate of weight gain and adiposity in rats when immunization with yeast-based Ad-36 compositions is initiated after Ad-36 infection and subsequent weight gain.
Rats were infected with Ad-36 (approximately 1×109 PFU in 1 ml) by intraperitoneal administration, as described in the prophylactic study in Example 4. After an Ad-36 emergent obesity phenotype has been established, groups of rats are immunized subcutaneously (s.c.) with one of the two yeast-based Ad-36 immunotherapeutic compositions (vaccines) described in Example 4 above and in Table 4 below, administered at four different sites, with 20 million cells (2.0 Y.U.) s.c. in 0.1 ml per site. Vaccinations are performed once per week for 2 weeks after challenge, and then monthly for as long as 30 weeks. Additional control groups include a group of rats immunized with control yeast compositions (“empty vector” yeast, or YVEC, that do not express the Ad-36 antigen(s)), and a group of rats receiving PBS only (naïve or PBS). In the present example the control group (B) is PBS.











TABLE 4









Post-challenge



Group
challenge
Immunization









B
Ad-36
PBS



F
Ad-36
Ad-aFL-CRAG



H
Ad-36
Ad-aFL-HEX-Full










Animals are weighed pre-viral infection and then up to biweekly for the up to 30 weeks duration of the study. In addition, food and water consumption are monitored. Blood is collected pre-viral infection and biweekly to monitor for serum viral load, cholesterol, triglyceride levels, corticosterone, neutralizing antibodies, and the other biochemical parameters as described in Example 5. Glucose tolerance testing is performed and glucose levels are measured in the urine. Blood (500 μl per timepoint) is obtained under isofluroane anaesthesia from the tail vein.
At the end of the study, animals are euthanized and adipose tissue is harvested to measure viral levels by polymerase chain reaction (PCR). PCR may also be performed on biopsies obtained during the course of the study.
This experiment was performed in outbred Wistar rats. If, as expected, additional weight gain is prevented or reduced in rats immunized with yeast-based Ad-36 immunotherapy as compared to control rats, inbred Wistar Furth rats will be evaluated according to the same or similar protocol, as these inbred rats are expected to be more amenable to evaluation of T cell immunity. Additional experiments may also determine the effect of diet or other factors in conjunction with immunotherapy (e.g., by administering a high fat diet versus a normal diet).
Immunization with a yeast-based Ad-36 immunotherapy composition is deemed active if it causes, as compared with empty vector yeast or PBS controls, notable trends towards normalization of any of the following parameters for Ad-36 infected rats: i) body weight or a reduced rate of body weight gain; ii) percent body fat or body mass index; iii) frequency or titer of neutralizing antibodies; iv) cholesterol levels; v) serum corticosterone; vi) serum triglycerides; vii) blood and/or urine glucose levels; viii) glucose tolerance; ix) blood Ad-36 viral titer. In summary, it is expected that immunization with a yeast-based Ad-36 immunotherapy composition will be effective for reducing or preventing weight gain and adiposity in rats and this may be accompanied by changes in the biochemical parameters mentioned, given their known association with the obesity phenotype.
Example 7
Effect of Yeast Vector on Rat Appetite and Body Weight Gain
The following example describes an experiment designed to determine if immunization of rats with yeast-based immunotherapeutic compositions of the invention affects the rate of weight gain of naive uninfected (not infected with Ad-36) rats. This experiment was designed to identify whether there is a yeast vector-based effect of Tarmogen vaccination on appetite or body weight gain that is independent of Ad-36 exposure. Such effects on appetite or body weight, if observed, would not be considered to be antigen-specific, since there is no viral antigen in the host, and would be important to determine prior to interpreting the effect of Ad-36 Tarmogen immunization on Ad-36-induced weight gain.
Rats were immunized with one of the yeast-based immunotherapy compositions described in Example 1 (Ad-Fib, the fusion protein of which is represented by SEQ ID NO:42) once per week, on weeks 1, 2, 7, 9, and 11. Vaccination was at 4 s.c sites with 2 Y.U. per site. The animals were weighed pre-immunization and biweekly following vaccination. The diet consumption and body weight of the rats was monitored during this period. The results, shown in FIG. 14, show that there was no difference in food consumption between the yeast-immunized group and control group. Also, Ad-Fiber yeast vaccination did not change the rate of body weight gain as compared to naive control rats, as shown in FIG. 15. These data demonstrate that yeast based immunotherapy vaccinations per se (in the absence of the target antigen) do not alter the appetite or body weight gain of rats. These results are consistent with the observation that the effects of Ad-36 yeast-based immunotherapy on body weight, when observed in the Ad-36 challenge experiments described above, are not believed to be attributable to a generalized effect of the yeast or yeast vector on rat appetite or metabolism.
Example 8
Organ Distribution of Ad-36 after Intraperitoneal Inoculation
The following experiment demonstrates the Ad36 distribution in major organs and tissues after the virus infection.
This experiment is of relevance to the specificity/tropism of the virus and to the best of the inventors' knowledge, such analyses have not been conducted in any study this late after viral challenge. Therefore, the following experiments were designed to confirm that Ad-36 resides in fat compartments after the virus is no longer detectable in the blood, and to further indicate tissues or organs where yeast-based immunotherapy may be active. In one published study (Pasaricia et al, 2008), conducted at 4 days post challenge, Ad-36 was found in nearly all tissues tested including the central nervous system (CNS), heart, lung, liver, spleen, kidney, visceral fat, and other organs. In the present study, the organ/body-wide distribution of Ad-36 was evaluated at 15 weeks virus post-challenge in a non-immunized rat. Briefly, major organs and tissues (include blood and peripheral blood mononuclear cells (PBMC)) were removed and isolated. Organ and tissue genomic DNA was extracted from all samples using the QIAamp Kit, and Ad-36 DNA was detected with a very sensitive nested polymerase chain reaction (PCR) assay. The results, shown in FIG. 16, indicated that 15 weeks after virus inoculation, Ad-36 DNA is detectable in the epididymal, retroperitoneal, omental visceral adipose tissues, and also in the spleen and kidney. However, Ad36 DNA was absent from heart, liver, lung, brain, and subcutaneous fat, as well as the other organs/tissues tested. These results, taken together with the prior results of Pasarica et al., show that Ad-36, although widely distributed in most major organs early after challenge, becomes more localized to fat compartments, as well as kidney and spleen, by 15 weeks post viral challenge.
Example 9
Mouse Model-Prophylactic
The following example describes the use of yeast-based adenovirus-36 (Ad-36) immunotherapeutics in an animal model of adenovirus-related obesity.
A mouse model has been described in the literature whereby infection of animals with human Ad-36 has caused weight gain and increase in adiposity (Dhurandhar et al. Int. J. Obesity 24:989, 2000). In those studies, a statistically significant increase in body fat weight (p<0.02) was elicited in Ad36-infected mice compared to the control group. Additionally, 60% of Ad-36 injected mice vs. 22% of controls were considered obese when obesity was defined as >85th percentile of the control group.
In the following experiment, yeast-based Ad-36 immunotherapeutic compositions (vaccines) are evaluated to determine whether immunization against this virus using yeast-based immunotherapy can prevent obesity or at least reduce weight gain and adiposity associated with Ad-36 infection.
Groups of mice are immunized subcutaneously (s.c.) with a yeast-based Ad-36 immunotherapeutic composition (vaccine) administered at two to four different sites (1 to 20 million cells (or 0.1-2.0 Y.U.) s.c. in 0.1 ml per site), between three and six times at weekly intervals. After the final administration, mice are challenged with Ad-36 (approximately 2×107 PFU in 0.1-0.2 ml) by intraperitoneal administration. Experimental groups of mice (10-20 mice per group) are immunized with a yeast-based Ad-36 immunotherapeutic composition, e.g., one of the yeast-based immunotherapy compositions described in Example 1. Additional control groups include a group of mice immunized with control yeast compositions (“empty vector” yeast that do not express the Ad-36 antigen(s)), and a group of mice receiving PBS only (naïve).
Animals are weighed pre-treatment, pre-viral challenge and then up to twice weekly for approximately 22 weeks following inoculation with virus. In addition, food and water consumption are monitored. Blood is collected at baseline, pre-viral challenge and biweekly following challenge to monitor for cholesterol, triglyceride levels and for neutralizing antibodies to Ad36 in the serum. Glucose tolerance testing is performed and glucose levels are measured in the urine. Blood (200 μl per timepoint) is obtained under isofluroane anaesthesia from the retro-orbital plexus. At the end of the study, animals are euthanized and adipose tissue is harvested to measure viral levels by polymerase chain reaction (PCR). PCR may also be performed on biopsies obtained during the course of the study.
The experiment is initially performed in outbred mice (e.g., ICR or CD-1® mice). If, as expected, weight gain is prevented or reduced in mice immunized with yeast-based Ad-36 immunotherapy as compared to control mice, inbred strain(s) are further evaluated according to the same or similar protocol (e.g., C57BL/6, BALB/c or C3H), as these mice are expected to be more amenable to evaluation of T cell immunity. Additional experiments may also determine the effect of diet or other factors in conjunction with immunotherapy (e.g., by administering a high fat diet versus a normal diet).
Immunization with a yeast-based Ad-36 immunotherapy composition is effective if immunization results in a statistically significant difference in body weight or body weight gain between yeast-Ad-36 immunized mice and control mice (empty vector yeast or PBS-immunized), and/or at least a two-fold difference in neutralizing antibody levels, and/or a greater than 5% reduction in percent body fat, cholesterol, triglycerides, reduction in glucose in the urine or reduced glucose levels by glucose tolerance test and/or reduction in Ad-36 viral titers, between the experimental and either control group (empty vector yeast or PBS-immunized). It is expected that immunization with a yeast-based Ad-36 immunotherapy composition will be effective for reducing or preventing weight gain and adiposity in mice.
Example 10
Mouse Model-Therapeutic
The following example describes the use of yeast-based Ad-36 immunotherapeutics in an animal model of adenovirus-related obesity.
In the following experiment, yeast-based Ad-36 immunotherapeutic compositions (vaccines) are evaluated to determine whether immunization against this virus using yeast-based immunotherapy can reverse obesity or at least reduce weight gain and adiposity in mice when immunization with yeast based Ad-36 compositions is initiated after Ad-36 infection and subsequent weight gain.
Mice are infected with Ad-36 (approximately 2×107 PFU in 0.1-0.2 ml) by intraperitoneal administration. Once weight gain has been established, groups of mice will be immunized subcutaneously (s.c.) with a yeast-based Ad-36 immunotherapeutic composition (vaccine) administered at two to four different sites (1 to 20 million cells (0.1 to 2.0 Y.U.) s.c. in 0.1 ml per site), between three and six times at weekly intervals. Additional control groups include a group of mice immunized with control yeast compositions (“empty vector” yeast that do not express the Ad-36 antigen(s)), and a group of mice receiving PBS only (naïve).
Animals are weighed pre-viral infection and then up to twice weekly for the duration of the study. In addition, food and water consumption are monitored. Blood is collected pre-viral infection and biweekly to monitor for cholesterol, triglyceride levels and for neutralizing antibodies to Ad36 in the serum. Glucose tolerance testing is performed and glucose levels are measured in the urine. Blood (200 μl per timepoint) is obtained under isofluroane anaesthesia from the retro-orbital plexus.
At the end of the study, animals are euthanized and adipose tissue is harvested to measure viral levels by polymerase chain reaction (PCR). PCR may also be performed on biopsies obtained during the course of the study.
The experiment is initially performed in outbred mice (e.g., ICR or CD-1® mice). If, as expected, additional weight gain is prevented or reduced in mice immunized with yeast-based Ad-36 immunotherapy as compared to control mice, inbred strain(s) are further evaluated according to the same or similar protocol (e.g., C57BL/6, BALB/c or C3H), as these mice are expected to be more amenable to evaluation of T cell immunity. Additional experiments may also determine the effect of diet or other factors in conjunction with immunotherapy (e.g., by administering a high fat diet versus a normal diet).
Immunization with a yeast-based Ad-36 immunotherapy composition is effective if immunization results in a statistically significant difference in body weight or body weight gain between yeast-Ad-36 immunized mice and control mice (empty vector yeast or PBS-immunized), and/or at least a two-fold difference in neutralizing antibody levels, and/or a greater than 5% reduction in percent body fat, cholesterol, triglycerides in glucose in the urine or reduced glucose levels by glucose tolerance test and/or reduction in Ad-36 viral titers between the experimental and either control group (empty vector yeast or PBS-immunized). It is expected that immunization with a yeast-based Ad-36 immunotherapy composition will be effective for reducing or preventing additional weight gain and adiposity in mice.
Example 11
Treatment of Ad-36 Infection in Humans
The following example describes a clinical trial for the treatment of Ad-36 infection in human adult subjects.
A randomized phase 1 clinical trial in adult patients and/or in obese pediatric patients testing positive for adenovirus-36 infection and having a BMI of at least 25 (or pediatric patients with analogous/equivalent BMI) will be conducted. Additional groups or trials include non-obese and/or non-overweight adults and/or pediatric patients testing positive for adenovirus infection. Subjects will be randomized into two arms. Arm 1 patients will receive at least 12 weeks of yeast-based Ad-36 immunotherapy (any composition as described in Example 1) and will follow a prescribed diet and exercise regimen. Arm 2 patients will receive a placebo (PBS control injection or empty yeast) and will follow the same prescribed diet and exercise program. One primary endpoint is reduction in Ad-36 viral titer. Another endpoint is immune seroconversion determined by measurement of the presence of Ad-36 antibodies. Another endpoint is Ad-36-specific cellular immune responses (which may include T cell proliferation, induction of CD4+ Th1 and/or Th17 cells, induction of CD8+ T cells as measure by CTL assay or cytokine assay, and/or modulation in regulatory T cell (Treg) numbers or function). Additional secondary endpoints include a reduction in BMI, as well as relative weight loss and absolute weight loss during treatment and during longitudinal follow-up after completion of therapy.
While various embodiments of the present invention have been described in detail, it is apparent that modifications and adaptations of those embodiments will occur to those skilled in the art. It is to be expressly understood, however, that such modifications and adaptations are within the scope of the present invention, as set forth in the following exemplary claims.

## Claims

1. An immunotherapeutic composition comprising: a) a yeast vehicle; and b) a fusion protein comprising an adenovirus-36 (Ad-36) antigen, wherein the Ad-36 antigen comprises the amino acid sequence SEQ ID NO:55.

2. The immunotherapeutic composition of claim 1, wherein the yeast vehicle is a whole yeast.

3. The immunotherapeutic composition of claim 1, wherein the yeast vehicle is from Saccharomyces cerevisiae.

4. A fusion protein, wherein the fusion protein comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:47, SEQ ID NO:54, or SEQ ID NO:55.

5. An immunotherapeutic composition comprising:
a) a yeast vehicle; and
b) a fusion protein comprising an adenovirus-36 (Ad-36) antigen, wherein the fusion protein comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:54 or SEQ ID NO:47.

6. An immunotherapeutic composition comprising:
a) a yeast vehicle; and
b) a fusion protein comprising an adenovirus-36 (Ad-36) antigen, wherein the Ad-36 antigen comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:55.

7. The immunotherapeutic composition of claim 6, wherein the Ad-36 antigen comprises the amino acid sequence SEQ ID NO:55.

8. The immunotherapeutic composition of claim 2, wherein the Ad-36 antigen is expressed by the whole yeast.

9. The immunotherapeutic composition of claim 2, wherein the whole yeast is heat-inactivated.

10. The immunotherapeutic composition of claim 1, wherein the composition is formulated in a pharmaceutically acceptable excipient suitable for administration to an individual.

11. The immunotherapeutic composition of claim 1, further comprising at least one biological response modifier.

12. The immunotherapeutic composition of claim 6, wherein the Ad-36 antigen comprises an amino acid sequence that is at least 97% identical to SEQ ID NO:55.

13. The immunotherapeutic composition of claim 6, wherein the Ad-36 antigen comprises an amino acid sequence that is at least 99% identical to SEQ ID NO:55.

14. The immunotherapeutic composition of claim 5, wherein the fusion protein comprises the amino acid sequence SEQ ID NO:47 or SEQ ID NO:54.

15. The fusion protein of claim 4, wherein the fusion protein comprises an amino acid sequence that is 95% identical to SEQ ID NO:55.

16. The fusion protein of claim 4, wherein the fusion protein comprises an amino acid sequence that is 99% identical to SEQ ID NO:55.

17. The fusion protein of claim 4, wherein the fusion protein comprises the amino acid sequence SEQ ID NO:55.

18. The fusion protein of claim 4, wherein the fusion protein comprises the amino acid sequence SEQ ID NO:47 or SEQ ID NO:54.

19. The immunotherapeutic composition of claim 6, wherein the yeast vehicle is a whole yeast.

20. The immunotherapeutic composition of claim 6, wherein the yeast vehicle is from Saccharomyces cerevisiae.