Animal Health – Louis Picker, Scott Hansen, Klaus Frueh, Daniel Malouli, Oregon Health Science University

Abstract for “Cytomegalovirus vectors enable control of T cell targeting”

“CMV vectors containing a heterologous antigen, an active UL131 or an ortholog thereof, and an active UL128 (or any other form thereof) protein but without an active UL130 (or any other form thereof) are available. CMV vectors containing a heterologous antigen, an activate UL131 protein or an ortholog thereof, and an active UL130 proteins (or an equivalent thereof) are also available. However, they do not contain an active UL128. Methods of using CMV vectors for generating an immune response that is at least 10% CD8+ T cells directed towards epitopes present in MHC Class II are also provided.

Background for “Cytomegalovirus vectors enable control of T cell targeting”

T cell receptor (TCR),-mediated recognition by CD8+ T cells of intracellular pathogens (class I MHC proteins (MHC-1I)) and an exceptional system of intracellular protein sampling and transport (Neefjies M. L. et al., Nat Rev Immunol 11: 823 (2011); incorporated herein). Pathogens may produce thousands of different peptides, which can be used to recognize CD8+ T-cells. However, there are requirements for proteolytic processing and peptide transport as well as matching TCR repertoire. These mechanisms, along with the poorly understood immunoregulatory mechanisms, allow for the narrowing down of potential targets for CD8+ T-cells. The complexity of this process is not surprising. Pathogen-specific CD8-T cell responses that are mounted by individuals with common MHC-I alleles tends to recognize an overlap of so-called immunodominant peptides (Yewdell J W et Al, 2006 supra; Irvine K et Al, Expert Rev Clin Immunol 2, 135 (2006); Assarsson E et. al, J Immunol 178, 7890 (2007); incorporated herein by referenced by reference herein; The vast majority of pathogens can be recognized by CD8+ T cells that target immunodominant epitopes. These immunedominant epitopes can also mount memory and anti-pathogen effectsor responses. Agents with high immune evasion abilities, such as the HIV and its similian counterpart SIV, are not immune-deficient. These viruses’ massive replication, coupled with their high rate for mutation and functional plasticity allow escape from most CD8+T cell responses (Picker U. et al., Ann Rev Med, 63, 95 (2012); incorporated by reference herein). The majority of infected subjects with these viruses have CD8 T cells that fail to target epitopes containing functionally critical, conserved viral sequences. This makes them difficult to control viral replication. Although these viruses can increase the number of CD8+ T cells responding to infection, they also target immunodominant epitopes, which is why these larger responses are still susceptible to immune escape (Picker 2012 supra; Barouch, D H et. al., J Virol 77 7367 (2003); Mudd, P A et. al., Nature 491, 129 (2012); all are incorporated herein). The AIDS vaccine industry has attempted to create strategies that could elicit HIV/SIV-specific CD8+ cell responses. Epitopes can be spread across different MHC-I haplotypes. This is done by increasing recognition breadth, or focusing responses to conserved sequences. However, this effort has not yielded strategies that significantly modify CD8+ T cells immunodominance hierarchies.

“A HIV/AIDS vaccine strategy that uses a recombinant Cytomegalovirus, (CMV), expressing an HIV protein was created to create and maintain HIV-specific effector T cell responses. This would prevent HIV infection from occurring before the viral amplification is required for effective immune evasion. (Picker 2012 supra). Although this strategy was not intended to prevent infection, the approach proved highly effective in animal models of HIV/AIDS, with approximately 50% of CMV/SIV vector-vaccinated rhesus monkeys (RM) being challenged with highly pathogenic, persistent SIV (Hansen 2011 infra).

“These studies revealed that CMV/SIV vectors didn’t elicit immunodominant CD8+ responses to SIV peptides presented in the well-characterized Mamu A1*001:01(A*01) MHC-1 protein. This suggests that CMV vectors stimulate new T cell epitopes that are targeted by these powerful responses, and contributes to vaccine efficacy.”

“It is revealed that heterologous antigens, such as viral or bacterial expressed via cytomegalovirus Vectors, induce a T-cell immunodominance profile which is fundamentally different to that elicited by all other vectors. As an animal model for human CMV, rhesus macaques infected by rhesus CMV virus (RhCMV), carrying SIV antigens was used to demonstrate that the SIVgag specific CD8+ responses elicited from the RhCMV/gag Vector are three times as wide as the gag-specific CD8+ cell responses elicited either by other vaccines or after infection with SIV. It has been shown that CMV-elicited cells target completely different epitopes than other vaccines. This includes a high percentage of epitopes present by MHC-II (class II MHC). These responses are rare, if any, in CD8+ T cells responses to other infectious agents or vaccines. CMV is also responsible for the immunodominance profile. It was shown that the CMV genes UL128 (and UL130) prevent this response from being induced. T cell responses to the CMV vector containing UL128/UL130 are focused on a small number of epitopes, whereas MHC II restricted CD8+ T cells are induced using vectors that do not contain UL128/UL130. This allows for genetic manipulation of a vaccine vector to create distinct patterns in CD8+ T-cell epitope recognition.

“CMV vectors disclosed herein include a heterologous antigen, an active UL131 or an ortholog thereof, an active UL128 (or an equivalent thereof), and a CMV vector that lacks an active UL130. CMV vectors also disclosed include a heterologous antigen, an activate UL131 protein or an ortholog thereof, and an active UL130 proteins (or an equivalent thereof), but the CMV vector is missing an active UL128.

“The method of producing a CD8+ T-cell response to heterologous antigens in a subject is also disclosed. This involves administering a CMV vector to the subject. CMV vectors are distinguished by having a heterologous antibody, an active UL131 protein, an inactive UL128 protein, an inactive UL130 protein, an inactive UL130 protein or a combination of both. At least 10% of CD8+ T cells are directed against epitopes from MHC Class II.

“Disclosed are vectors of animal or human cytomegalovirus (CMV), capable of infecting multiple organisms. CMV vectors include a nucleic acids sequence that encodes a heterologous antigen protein and a sequence that encodes an active UL131 amino acid protein. The CMV vector, for example, contains a nucleic acids sequence that expresses an activated UL128 protein but not an active UL130. Another example is that the CMV vector encodes an UL130 protein, but not an active UL128 Protein.

“In some cases, the vector doesn’t express an active UL128/UL130 protein because of a deleterious mutation within the nucleic acids sequence encoding UL128/UL130 or their orthologous gene in animal CMVs. Any mutation that causes the expression of UL128 and UL130 proteins to be inactive can be considered a deleterious mutation. These mutations include frameshift mutations and point mutations.

“In additional examples, the vector doesn’t express an active UL128/UL130 protein because it contains a nucleic acids sequence that contains an antisense sequence (siRNA/miRNA) that inhibits expression of the UL128/UL130 proteins.”

“Also described herein are methods for generating CD8+ T cells responses to heterologous antibodies in a subject. These methods involve administering a CMV vector to the subject. CMV vectors are distinguished by having a nucleic acids sequence that encodes a heterologous protein and a sequence that encodes active UL131 proteins. CMV vectors are also distinguished by not encoding active UL128 proteins or active UL130 proteins, or neither active UL128 nor active UL130 proteins. At least 10% of CD8+T cells are directed against epitopes identified by MHC class 2. Further examples include at least 20%, 30%, 40%, at minimum 50%, at most 60%, and at most 60% of CD8+ T cell responses directed against epitopes present in MHC class II.

“In further examples, methods include administering an effective amount CMV vector, which is composed of a nucleic acids sequence that encodes heterologous antigens to the subject. Any CMV vector may be used, including one with an active CMV128 protein and one with an active CMV130 protein. Additional deletions may be included in the second CMV vector to produce different immune responses, such as a US11 or any other deletion. Any heterologous or unidentifiable heterologous antibody can be used as the second heterologous. The administration of the second CMV can occur at any time after administration of the first CMV Vector, including concurrently or following administration of the first CMV Vector. Administration of the second CMV vector can be done at any time, including months, days, hours or minutes before, concurrently with, or after administration of the first vector.

“Human and animal CMV vectors when used as expression vectors in selected subjects, such as humans, are innately not pathogenic in those subjects or have been modified to make them non-pathogenic. For example, replication-defective adenoviruses and alphaviruses are well known and can be used as gene delivery vectors.”

“The heterologous antibody can be any protein, or fragment thereof, that is not derived form CMV, and includes cancer antigens as well as pathogen specific antigens. It also includes model antigens (such lysozyme, ovalbumin) or any other antigen.

“Pathogen-specific antigens can be obtained from any animal or human pathogen. The antigen could be a protein that was derived from the virus. Viruses include, among others, Adenovirus (coxsackievirus), hepatitis A viruses, rhinoviruses, Herpes simplex type 1, Varicella-zostervirus type 2, Epstein-Barrviruses, Epstein-Barrviruses, Kaposi’s sarcoma virus and Kaposi’s sarcoma virus.

The pathogen could be a bacterial infection and the antigen could be a protein that is derived from the bacteria. The pathogenic bacteria include, but are not limited to, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Legionella pneumophila, Leptospira interrogans, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Rickettsia rickettsii, Salmonella typhi, Salmonella typhimurium, Shigella sonnei, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Treponema pallidum, Vibrio cholera and Yersinia pestis.”

The parasite may be the pathogen and the antigen could be a protein that is derived from it. The parasite may be a protozoan organism or a protozoan organism causing a disease such as, but not limited to, Acanthamoeba, Babesiosis, Balantidiasis, Blastocystosis, Coccidia, Dientamoebiasis, Amoebiasis, Giardia, Isosporiasis, Leishmaniasis, Primary amoebic meningoencephalitis (PAM), Malaria, Rhinosporidiosis, Toxoplasmosis-Parasitic pneumonia, Trichomoniasis, Sleeping sickness and Chagas disease. The parasite may be a helminth organism or worm or a disease caused by a helminth organism such as, but not limited to, Ancylostomiasis/Hookworm, Anisakiasis, Roundworm?Parasitic pneumonia, Roundworm?Baylisascariasis, Tapeworm?Tapeworm infection, Clonorchiasis, Dioctophyme renalis infection, Diphyllobothriasis?tapeworm, Guinea worm?Dracunculiasis, Echinococcosis?tapeworm, Pinworm?Enterobiasis, Liver fluke?Fasciolosis, Fasciolopsiasis?intestinal fluke, Gnathostomiasis, Hymenolepiasis, Loa loa filariasis, Calabar swellings, Mansonelliasis, Filariasis, Metagonimiasis?intestinal fluke, River blindness, Chinese Liver Fluke, Paragonimiasis, Lung Fluke, Schistosomiasis?bilharzia, bilharziosis or snail fever (all types), intestinal schistosomiasis, urinary schistosomiasis, Schistosomiasis by Schistosoma japonicum, Asian intestinal schistosomiasis, Sparganosis, Strongyloidiasis?Parasitic pneumonia, Beef tapeworm, Pork tapeworm, Toxocariasis, Trichinosis, Swimmer’s itch, Whipworm and Elephantiasis Lymphatic filariasis. Parasites can be caused by organisms such as Halzoun Syndrome (or bilharziosis or snail fever), Halzoun Syndrome (or worm), Myiasis and Chigoe fleas, Candiru, Candiru, and Candiru. Parasites can be caused by ectoparasites, such as: Bedbug, Head louse??Pediculosis or Body louse??Pediculosis and Demodex?Demodicosis.

The antigen could be a protein that is derived from cancer. The cancers, include, but are not limited to, Acute lymphoblastic leukemia; Acute myeloid leukemia; Adrenocortical carcinoma; AIDS-related cancers; AIDS-related lymphoma; Anal cancer; Appendix cancer; Astrocytoma, childhood cerebellar or cerebral; Basal cell carcinoma; Bile duct cancer, extrahepatic; Bladder cancer; Bone cancer, Osteosarcoma/Malignant fibrous histiocytoma; Brainstem glioma; Brain tumor; Brain tumor, cerebellar astrocytoma; Brain tumor, cerebral astrocytoma/malignant glioma; Brain tumor, ependymoma; Brain tumor, medulloblastoma; Brain tumor, supratentorial primitive neuroectodermal tumors; Brain tumor, visual pathway and hypothalamic glioma; Breast cancer; Bronchial adenomas/carcinoids; Burkitt lymphoma; Carcinoid tumor, childhood; Carcinoid tumor, gastrointestinal; Carcinoma of unknown primary; Central nervous system lymphoma, primary; Cerebellar astrocytoma, childhood; Cerebral astrocytoma/Malignant glioma, childhood; Cervical cancer; Childhood cancers; Chronic lymphocytic leukemia; Chronic myelogenous leukemia; Chronic myeloproliferative disorders; Colon Cancer; Cutaneous T-cell lymphoma; Desmoplastic small round cell tumor; Endometrial cancer; Ependymoma; Esophageal cancer; Ewing’s sarcoma in the Ewing family of tumors; Extracranial germ cell tumor, Childhood; Extragonadal Germ cell tumor; Extrahepatic bile duct cancer; Eye Cancer, Intraocular melanoma; Eye Cancer, Retinoblastoma; Gallbladder cancer; Gastric (Stomach) cancer; Gastrointestinal Carcinoid Tumor; Gastrointestinal stromal tumor (GIST); Germ cell tumor: extracranial, extragonadal, or ovarian; Gestational trophoblastic tumor; Glioma of the brain stem; Glioma, Childhood Cerebral Astrocytoma; Glioma, Childhood Visual Pathway and Hypothalamic; Gastric carcinoid; Hairy cell leukemia; Head and neck cancer; Heart cancer; Hepatocellular (liver) cancer; Hodgkin lymphoma; Hypopharyngeal cancer; Hypothalamic and visual pathway glioma, childhood; Intraocular Melanoma; Islet Cell Carcinoma (Endocrine Pancreas); Kaposi sarcoma; Kidney cancer (renal cell cancer); Laryngeal Cancer; Leukemias; Leukemia, acute lymphoblastic (also called acute lymphocytic leukemia); Leukemia, acute myeloid (also called acute myelogenous leukemia); Leukemia, chronic lymphocytic (also called chronic lymphocytic leukemia); Leukemia, chronic myelogenous (also called chronic myeloid leukemia); Leukemia, hairy cell; Lip and Oral Cavity Cancer; Liver Cancer (Primary); Lung Cancer, Non-Small Cell; Lung Cancer, Small Cell; Lymphomas; Lymphoma, AIDS-related; Lymphoma, Burkitt; Lymphoma, cutaneous T-Cell; Lymphoma, Hodgkin; Lymphomas, Non-Hodgkin (an old classification of all lymphomas except Hodgkin’s); Lymphoma, Primary Central Nervous System; Marcus Whittle, Deadly Disease; Macroglobulinemia, Waldenstrim; Malignant Fibrous Histiocytoma of Bone/Osteosarcoma; Medulloblastoma, Childhood; Melanoma; Melanoma, Intraocular (Eye); Merkel Cell Carcinoma; Mesothelioma, Adult Malignant; Mesothelioma, Childhood; Metastatic Squamous Neck Cancer with Occult Primary; Mouth Cancer; Multiple Endocrine Neoplasia Syndrome, Childhood; Multiple Myeloma/Plasma Cell Neoplasm; Mycosis Fungoides; Myelodysplastic Syndromes; Myelodysplastic/Myeloproliferative Diseases; Myelogenous Leukemia, Chronic; Myeloid Leukemia, Adult Acute; Myeloid Leukemia, Childhood Acute; Myeloma, Multiple (Cancer of the Bone-Marrow); Myeloproliferative Disorders, Chronic; Nasal cavity and paranasal sinus cancer; Nasopharyngeal carcinoma; Neuroblastoma; Non-Hodgkin lymphoma; Non-small cell lung cancer; Oral Cancer; Oropharyngeal cancer; Osteosarcoma/malignant fibrous histiocytoma of bone; Ovarian cancer; Ovarian epithelial cancer (Surface epithelial-stromal tumor); Ovarian germ cell tumor; Ovarian low malignant potential tumor; Pancreatic cancer; Pancreatic cancer, islet cell; Paranasal sinus and nasal cavity cancer; Parathyroid cancer; Penile cancer; Pharyngeal cancer; Pheochromocytoma; Pineal astrocytoma; Pineal germinoma; Pineoblastoma and supratentorial primitive neuroectodermal tumors, childhood; Pituitary adenoma; Plasma cell neoplasia/Multiple myeloma; Pleuropulmonary blastoma; Primary central nervous system lymphoma; Prostate cancer; Rectal cancer; Renal cell carcinoma (kidney cancer); Renal pelvis and ureter, transitional cell cancer; Retinoblastoma; Rhabdomyosarcoma, childhood; Salivary gland cancer; Sarcoma, Ewing family of tumors; Sarcoma, Kaposi; Sarcoma, soft tissue; Sarcoma, uterine; Sezary syndrome; Skin cancer (nonmelanoma); Skin cancer (melanoma); Skin carcinoma, Merkel cell; Small cell lung cancer; Small intestine cancer; Soft tissue sarcoma; Squamous cell carcinoma?see Skin cancer (nonmelanoma); Squamous neck cancer with occult primary, metastatic; Stomach cancer; Supratentorial primitive neuroectodermal tumor, childhood; T-Cell lymphoma, cutaneous (Mycosis Fungoides and Sezary syndrome); Testicular cancer; Throat cancer; Thymoma, childhood; Thymoma and Thymic carcinoma; Thyroid cancer; Thyroid cancer, childhood; Transitional cell cancer of the renal pelvis and ureter; Trophoblastic tumor, gestational; Unknown primary site, carcinoma of, adult; Unknown primary site, cancer of, childhood; Ureter and renal pelvis, transitional cell cancer; Urethral cancer; Uterine cancer, endometrial; Uterine sarcoma; Vaginal cancer; Visual pathway and hypothalamic glioma, childhood; Vulvar cancer; Waldenstrm macroglobulinemia and Wilms tumor (kidney cancer.)”

“The CMV vectors discussed herein provide a vector to clone or express heterologous DNA. They contain recombinant human and animal CMV. A heterologous DNA could encode an expression product that includes an epitope, a biological response modator, a growth factor or recognition sequence, as well as a therapeutic gene or fusion protein.

“The CMV vectors described herein can be used to create an immunogenic, immunological, or vaccine composition containing a recombinant CMVvirus or vector and a pharmaceutically approved carrier or diluent. A composition that contains the recombinant CMV vector or an expression product thereof can elicit an immunological response. This response may be protective, but it does not have to be. A vaccine composition containing the recombinant virus CMV vector or an expression product thereof can also elicit a local or systemic immune response that may be protective, but not necessarily. A vaccine composition triggers a protective response that is either local or systemic. The terms “immunological composition” and “immunogenic composition” are used interchangeably. ?immunogenic composition? include a ?vaccine composition? (The two terms above can also be used to refer to protective compositions.

“The CMV vectors described herein provide methods for inducing an immune response in a subject. This includes administering to the subject an immunogenic or immunological composition consisting of the recombinant CMVvirus or vector and a pharmaceutically approved carrier or diluent. The term “subject” is used in this specification. All animals, non-human primates, and humans are included in the definition of?subject? All vertebrate species are included, except humans. All vertebrates are included, even animals (as ‘animal?). are used herein). A subset of “animal” is, of course. A subset of?animal? is?mammal?”, which, for the purposes of this specification, includes all mammals except humans.

The CMV vectors described herein can be used to create therapeutic compositions that contain the recombinant CMV viruses or vector and a pharmaceutically approved carrier or diluent. The therapeutic composition can be used in gene therapy and immunotherapy embodiments according to the invention. This includes administering the composition to the host and transferring genetic data.

The CMV vectors described herein can be used to express a protein, gene product, or expression product. This involves infecting or transfecting cells in vitro with the recombinant CMV viruses or vectors of the invention, and then extracting, purifying, or isolating the DNA, protein, gene products, or expression product from the cell. The invention also provides a method of cloning or reproducing heterologous DNA sequences. This involves infecting or transfecting cells in vitro and in vivo using a recombinant CMVvirus or vector of invention, as well as optionally extracting, purifying, or isolating DNA from the cell.

The CMV vectors described herein can be made by inserting DNA containing a sequence that encodes heterologous antigen in a non-essential area of the CMV genome. This method may also include deleting one or several regions of the CMV genome. In vivo recombination is possible. The method may include transfecting cells with CMV DNA in a cell compatible medium with donor DNA that contains the heterologous sequences and DNA sequences homologous to portions of the CMV genome. Optionally, the CMV can then be recovered by in vivo Recombination. This method may also include cleaving CMVDNA to obtain CMV-cleaved DNA. Then, the donor DNA can be ligated to the CMV DNA to create hybrid CMV Heterologous, which is then transfected to a cell with hybrid CMVHeterologous DNA. Finally, the option to recover CMV that has been modified by the heterologous. In vivo recombination can be understood so the method also includes a plasmid containing donor DNA. This donor DNA encodes a polypeptide that is foreign to CMV. The donor DNA is located within a segment CMV DNA that would otherwise not be co-linear. To generate recombinant CMV, the heterologous DNA may be placed into CMV in any orientation that allows for stable integration and expression of that DNA.

The promoter can be found in DNA encoding heterologous antigens in the recombinant vector CMV. A promoter can come from any source, including a herpesvirus, rhesus macaque CMV(RhCMV), murine or another CMV promoter. A promoter who is not viral, such as the EF1 can also be used. promoter. A promoter can be a transcriptionally active truncated promoter that contains a region activated with a virus transactivating protein and the minimal promoter area of the full-length enhancer from which the truncated transcriptionally activ promoter was derived. A promoter is a combination of DNA sequences that correspond to the minimal promoter or upstream regulatory sequences. A minimal promoter is made up of the CAP site and TATA box (minimum sequences to support basic transcription; unregulated transcription); and,?upstream regulator sequences? are made up of the enhancer sequences and/or the upstream element. The term “truncated” also refers to the fact that some of the full-length promoter(s) may not be present. The term?truncated? indicates that the promoter’s full length is not present. This means that some of the promoter’s full-length has been removed. The truncated promotor can also be derived from a herpesvirus like MCMV or HCMV.

The inventive promoter could be, like the one above, a herpesvirus such as MCMV or HPV. There can also be a 40 to 90% reduction in size compared to a full-length promoter based on base pairs. A modified non-viral promoter is also possible.

“An expression cassette can also be disclosed that can be placed into a recombinant viral or plasmid containing the truncated transcriptionally activate promoter. An expression cassette may also include a functional truncated polyadenylation signals, such as an SV40 signal that is truncated but functional. Given that nature provides a stronger signal, it is not surprising that a functional truncated poladenylation signals is functional. A truncated poladenylation signal addresses insert size limitations of recombinant viruses like CMV. An expression cassette may also contain heterologous DNA. This is true regardless of the virus or system it is being inserted into.

“The present invention includes CMV, recombinants that contain viral or non-viral promotors, and a truncated promotor. The invention also covers antibodies that are elicited from the inventive compositions or recombinants, and their uses. The antibodies or the product (epitopes or recombinants) that elicited them or monoclonal antibody from the antibodies can be used in binding assays or tests to determine whether an antigen or antibodies are present or absent.

“Flanking DNA can be used in the invention from the site or a portion adjacent thereto (where?adjacent is). “Flanking DNA can be from the site of insertion or a portion of the genome adjacent thereto (wherein?adjacent?

“As regards antigens for vaccine or immunological compositions (for a listing of antigens used as expression products of the inventive virus or an expression product thereof),”

“As for heterologous Antigens, one skilled enough in the art can choose a heterologous and coding DNA from the knowledge and sequences of the amino acids of the peptide/polypeptide as well as the nature of specific amino acids (e.g. size, charge, etc.). Without undue experimentation, the codon dictionary and the amino acid sequences.

“Regarding the sequence, it is preferable that the DNA sequence encodes at most regions of the antigen that produce an antibody response or a response from a T cells, especially a CD8+ T-cell response. Epitope mapping is one method of determining T and B cell epitopes. Oligo-peptide synthesizers can create overlapping peptides from the heterologous antibody. Each peptide is then tested to determine if it can bind to the antibody generated by the native protein, or induce T cell and B cell activation. This method has proven to be particularly helpful in mapping epitopes of T-cells, as the T cell recognizes short linear and MHC molecules.

“An immune reaction to heterologous antigens is generally as follows: T cells only recognize proteins when they have been cleaved into smaller, more easily digestible peptides. This complex is called the?major hertocompatibility complex (MHC). located on the surface of another cell. There are two types of MHC complexes: class I and class II. Each class contains many different alleles. Individual subjects and species have different types MHC complex alleles. They are known to have a different HLA type.

“It should be noted that the DNA encoding heterologous antibodies can contain a promoter to drive expression in the CMV vector, or the DNA can only include the coding DNA for the heterologous antibody. This construct can be placed in a way that is operably linked with an endogenous CMV enhancer, and it will then be expressed. Multiple copies of the DNA encoding heterologous antigen can be used, as well as a strong, early, or late promoter or combination thereof, to increase or amplify expression. The DNA that encodes the heterologous can be placed in a suitable position relative to a CMV endogenous promoter or can be translocated so that they can be inserted at a different location. CMV vectors can contain nucleic acids that encode more than one heterologous antibody.

“CMV vectors are also disclosed in pharmaceuticals and other compositions. These pharmaceutical and other compositions may be made so that they can be used in any of the administration procedures known to the art. These pharmaceutical compositions may be administered via intradermal, intramuscular or subcutaneous routes, as well as intravenous. You can also administer the medication via a mucosal route (e.g. oral, nasal or genital).

The disclosed pharmaceutical compositions are prepared according to standard pharmaceutical techniques. These compositions can be administered using dosages and techniques that are well-known to medical professionals. This includes factors such as the patient’s age, gender, sex, weight and condition. These compositions can be administered by themselves, or they can be combined with other CMV vectors, other immunological, antigenic, vaccine, or therapeutic compositions. These compositions may contain purified native antigens, epitopes, or antigens and epitopes derived from a recombinant CMV vector system. They are administered considering the above factors.

“Examples include liquid preparations for oral, nasal and genital orifices such as syrups, syrups, or elixirs. Also, preparations for intradermal, intramuscular, parenteral, or subcutaneous administration (e.g. injectable administration), such as sterile suspensions/emulsions. These compositions may contain recombinant in an admixture with a suitable carrier or diluent such as glucose, physiological saline or glucose.

Antigenic, immunological, or vaccine compositions can typically contain an adjuvant as well as a dose of the CMV vector/expression product to elicit desired responses. Alum, also known as aluminum phosphate or aluminium hydroxide, is used in human applications. The toxicities of Saponin and Quil A, Freund?s complete adjuvant, and other adjuvants used for research and veterinary purposes have limited their use in vaccines. Chemically defined preparations like muramyl dipeptide and monophosphoryllipid A, as well as phospholipid conjugates, such as those described in Goodman-Snitkoff, et al. J. Immunol. 147:410-415 (1991), Encapsulation of the protein in a proteoliposome according to Miller et.al, J. Exp. Med. Med.

The composition can be packed in one dosage form for immunization via parenteral (i.e. intradermal, intramuscular or subcutaneous) administration. The effective dosage and route for administration are determined by the composition of the product, the expression level if recombinant CMV has been directly used, and known factors such as breed, species, age, weight, condition, and host nature. LD50 and any other screening procedures that are not subject to undue experimentation are also important. The dosage of the expressed product can vary from a few micrograms to several hundred micrograms (e.g. 5 to 500 mg). CMV vectors can be administered in any amount necessary to obtain expression at these levels. Non-limiting examples: CMV Vectors can be administered in a minimum of 102 pfu. CMV vectors may also be administered in a range between about 102 and about 10. pfu. You can also use water or buffered soap as a carrier or diluent, with or without a preserver. CMV vectors can be either lyophilized to allow for resuspension after administration, or in solution.

“A polymeric delayed-release system may also be used as a carrier. A controlled release composition can be made using synthetic polymers. Kreuter, J. Microcapsules and Nanoparticles In Medicine and Pharmacology by M. Donbrow (Ed.) was an early example. CRC Press. p. 125-148.”

Microencapsulation is a method for injecting microencapsulated pharmaceuticals in controlled quantities. There are many factors that influence the choice of a specific polymer for microencapsulation. Consider the reproducibility of microencapsulation synthesis, cost of microencapsulation materials, toxicological profile, requirements for variable release kinetics, physicochemical compatibility and antigen compatibility. Polycarbonates, polyurethanes and polyorthoesters, as well as polyamides (especially those that can be biodegradable), are all examples of useful polymers.

“A frequent choice of a carrier for pharmaceuticals and more recently for antigens is poly (d,1-lactide-co-glycolide) (PLGA). This biodegradable polyester has been used in medical applications for erodible sutures and bone plates. It has never been toxic. PLGA microcapsules can be formulated with a wide range of pharmaceuticals, including peptides or antigens. Eldridge, J. H., et. al. reviewed a large amount of data on the adaptation of PLGA to controlled release of antigen. Current Topics In Microbiology And Immunology. 1989, 146:59-66. Orally, the adjuvant effect of PLGA microspheres containing antigens that are 1-10 microns in diameter was demonstrated to be remarkable. The PLGA microencapsulation method uses a phase separation process of a water in-oil emulsion. The compound of interest can be prepared in an aqueous solution. The PLGA is then dissolved in a suitable organic solvent such as methylenechloride or ethyl acetate. High-speed stirring is used to combine these two immiscible mixtures. The non-solvent is added to the polymer, which causes precipitation of the polymer around the droplets. This results in embryonic microcapsules. The microcapsules can then be collected and stabilized with any of a variety of agents (polyvinyl Alcohol (PVA), gelatins, alginates (PVP), and methyl cellulose). The solvent is then removed either by solvent extraction or drying in vacuo.

“Regarding HCMV promoters is made to U.S. Patent. Nos. Nos. 5,168,062 & 5,385,839. Feigner and colleagues refer to the transfection of cells with plasmidDNA for expression. (1994), J. Biol. Chem. 269, 2550-2561. Direct injection of plasmidDNA is a simple and effective way to prevent a wide range of infectious diseases. Science, 259, 1745-49, 1993. This invention allows the direct injection or use of vector DNA.

“The terms?”protein?,?peptide?”,??polypeptide?” and?amino acids sequence? are interchangeable. These terms are interchangeable to refer to polymers of any length of amino acid residues. It can be linear or branched and may contain modified amino acids or analogues. It may also be interrupted by chemical moieties that are not amino acids. These terms can also refer to an amino acid polymer that’s been modified by intervention or natural means, for example glycosylation or lipidation, disulfide bond creation, glycosylation or acetylation or any other manipulation or modification such as conjugation with a labeling component or bioactive ingredient.

“As used herein the terms ‘antigen? or ?immunogen? They are often used interchangeably to mean a substance, usually a protein, that is capable of inducing an immunological response in a subject. This term can also be used to describe proteins that are immunologically activated in the sense that they can elicit an immune response from the humoral or cellular type against a protein.

It should be noted that proteins and nucleic acid encoding them might differ from those shown and described in this document. The invention allows for deletions, additions and truncations of sequences as long as they function according to the invention’s methods. Substitutions within the same family of amino acids will be considered conservative. The four main families of amino acids are: (1) acidic?aspartate, glutamate, (2) basic?lysine and arginine; (2) non-polar?alanine and valine; (3) nonpolar?alanine and valine; (4) uncharged?polar?glycines, asparagine and glutamine; and (5) serine threonine and tyrosine. Sometimes, aromatic amino acids are phenylalanine and tryptophan. It is predictable that an individual replacement of leucine with valine or isoleucine, or vice-versa; an aspartate or glutamate with an aspartate or vice versa, a threonine or serine with an amino acid; or a similar conservative substitution of an amino acid with an amino acid structurally related to it, will not have a significant effect on biological activity. The invention covers proteins with substantially the same amino acids sequences as those illustrated and described, but which have minor amino acid substitutions that don’t substantially alter the immunogenicity of a protein.

“As used herein, the terms ‘nucleotide sequencings?” ?nucleic acids sequences? Deoxyribonucleic Acid (DNA) and ribonucleic Acid (RNA) sequences are also known as messenger RNA (mRNA), DNA/RNA combinations, and synthetic nucleic acids. You can have a single-stranded nucleic acid or a partially or fully double-stranded nucleic acids (duplex). You can choose to have heteroduplex or homoduplex duplex nucleic acid.

“Transgene” is the term used herein. can also be used to mean?recombinant? nucleotide sequences which may be derived from any one of the nucleotide sequencing encoding the proteins described in the present invention. “Recombinant” is a term that refers to a nucleotide sequence that has been modified. A nucleotide sequence that has been altered by man. It is a nucleotide sequence that does not exist in nature or is linked with another nucleotide sequencing or found in a different arrangement in the natural world. It is known that manipulating a gene by man can be done. It is understood that manipulation means manipulating by artificial means such as codon optimization, restriction enzymes and machines. A CMV vector that encodes heterologous antigens is, by definition, a recombinant CMV Vector.

Codon optimization can be done to nucleotide sequences, such as codons that are optimized for human use. Any viral or bacterial sequence can be altered. Many viruses, such as HIV and other lentiviruses use a lot of rare codons. By altering these codons so that they correspond to the codons used in the subject, it is possible to increase heterologous antigen expression, as described by Andre et.al., J. Virol. 72:1497-1503, 1998.”

“Nucleotide sequences that encode functionally and/or antagonically equivalent variants, derivatives, and corresponding CMV vectors are possible. Functionally equivalent variants, derivatives and fragments have the potential to retain antigenic activities. Changes in DNA sequences that don’t alter the encoded amino acids sequence or those that result in conservative substitutes of amino acids residues can be made. However, substitution of amino amino acid residues with amino acid analogs is possible if they do not have a significant impact on the properties of the encoded polypeptide. Conservative amino acid substitutions are glycine/alanine; valine/isoleucine/leucine; asparagine/glutamine; aspartic acid/glutamic acid; serine/threonine/methionine; lysine/arginine; and phenylalanine/tyrosine/tryptophan. One embodiment has variants that contain at minimum 50%, 55%, 60%, and at most 75%.

“Sequence homology or sequence identity is determined by comparing sequences aligned in such a way as to maximize overlap and identify while minimising sequence gaps. A variety of mathematical algorithms can be used to determine sequence identity. The algorithm of Karlin & Altschul (Proc.) is a non-limiting example of a mathematical algorithm that can be used to compare two sequences. Natl. Acad. Sci. USA 1990; 87, 2264-2268. Modified as in Karlin & Altschul. Proc. Natl. Acad. Sci. USA 1993; 90: 5873-5877.

“Another mathematical algorithm that is used to compare sequences is the one of Myers & Miller. CABIOS 1988, 4: 11-17. This algorithm is included in the ALIGN program (version 2.0), which is part the GCG sequence alignment package. A PAM120 weight residue table can be used to compare amino acid sequences. There is a 12 and 4 gap penalties, respectively, when using the ALIGN program. Another useful algorithm to identify regions of alignment and sequence similarity is the FASTA algorithm, as described by Pearson & Lipman in Proc. Natl. Acad. Sci. USA 1988; 85, 2444-2448

The WU-BLAST version 2.0 software is a preferred option for use according the invention. Download WU-BLAST 2.0 executable programs on several UNIX platforms. This program is based upon WU-BLAST 1.4. It can also be downloaded from the public domain NCBI BLAST version 1.4. Natl. Acad. Sci. Sci.

“The invention’s various recombinant sequences, antibodies and/or an antigens are created using standard recombinant and cloning methods. These techniques are well-known to those skilled in the art. For example, see?Molecular Cloning? A Laboratory Manual? second edition (Sambrook and al. 1989).”

“The nucleotide sequences described in the present invention can be used to insert into?vectors. The term “vector” is used interchangeably with the term “vector”. The term “vector” is widely used by skilled artists. As such, the term “vector” herein will be used. It is used in accordance with its meaning for those skilled in the art. The term “vector” is an example of this. “Vector” is a term that is used commonly by those skilled in art to describe a vehicle that permits or facilitates the transfer nucleic acids molecules from one environment or allows or facilitates manipulation of a nucleic Acid molecule.

The present invention allows the use of any vector that permits expression of the viruses. The viruses of the invention can be used in vitro, such as by using cell-free expression systems and/or cultured cells that have been grown in vitro to generate the HIV-antigens or antibodies. These may then be used for various purposes, including the production of proteinaceous vaccinations. Any vector that permits expression of the virus in vitro or in cultured cells can be used for such applications.

“The heterologous antibodies of the invention must be expressed if the heterologous sequence’s protein coding sequence should be?operable linked? to regulate or nucleic acids control sequences that direct transcription/translation of the protein. A coding sequence and a promoter or nucleic acid control sequence are referred to as ‘operably linked’. When they are covalently linked in a manner that places the expression, transcription, and/or translation from the coding sequence under control of the nucleic acids control sequence. What is the ‘nucleic acids control sequence? Any nucleic acids element that controls the expression of a sequence or code sequence can be considered. The term “promoter” is used herein. The term?promoter? will be used herein to describe a group transcriptional control modules clustered around RNA polymerase II’s initiation site. These modules, when operationally linked with the protein coding sequences described in the invention, lead to the expression the encoded protein. A constitutive or inducible promoter can control the expression of the transgenes according to the invention. This induces transcription when the promoter is exposed to a particular stimulus such as antibiotics like tetracycline or hormones like ecdysone or heavy metals. A promoter may also be specific for a particular type of cell, tissue, or organ. There are many suitable enhancers and promoters that can be used to express the transgenes. You can find suitable enhancers and promoters in the Eukaryotic Promoter Database, EPDB.

“In one instance, the epitope refers to an SIV epitope. One of ordinary skill in the art will know that any reference to HIV in the specification applies to SIV. The SIV epitope in an advantageous embodiment is a protein fragment according to the present invention. However, the invention may also include additional SIV antigens and epitopes. The SIV epitope, which is an SIV-antigen, includes but not limited, to the U.S. Pat. SIV antigens. Nos. Nos.

“The vectors according to the present invention should be chosen so that they contain a suitable gene regulation region, such as a enhancer or promoter, so that the antigens can be expressed.”

When the goal is to express the antigens of an invention in vivo in a subject (e.g. to generate an immune response to an HIV-1 antigen or protective immunity against HIV-1), expression vectors that can be used in vivo and are suitable for that subject should be chosen. In some embodiments, it might be desirable to express the antigens and antibodies of the invention in a laboratory animal for preclinical testing of the HIV-1 vaccines and immunogenic compositions. It may be possible to express the antigens in human subjects in other embodiments. This is for clinical trials or actual clinical use. It is possible to use any vector that is suitable for such purposes. The skilled artisan can easily choose a suitable vector. It may be preferable that vectors used in in vivo applications be attenuated so as to prevent amplifying in the subject. If plasmid vectors were used, it would be preferable that they do not have an origin of replication that functions within the subject. This will increase safety for in-vivo use. If viral vectors are used, preferably they are attenuated or replication-defective in the subject, again, so as to enhance safety for in vivo use in the subject.”

“Viral vectors are preferred in the preferred embodiments. Advantageously, the vector can be a CMV Vector, which is lacking at most the glycoprotein UUL128 or CMV Vector lacking at minimum the glycoprotein UUL130. Every CMV vector also contains the glycoprotein Ul131.

The CMV vectors disclosed can be administered in vivo. This is possible, for example, when the goal is to induce an immunogenic response. In some cases, it might be desirable to use the CMV vectors in laboratory animals, such as rhesus monkeys, for pre-clinical testing and development of vaccines using RhCMV. Other embodiments will allow the use of the CMV vectors in humans for clinical trials or clinical use of immunogenic compositions with HCMV.

“The CMV vectors disclosed are used in such in vivo applications as a component of an immunogenic mixture further comprising a pharmaceutically accepted carrier. The immunogenic compositions described in the invention can be used to induce an immune response against heterologous antigens, including pathogen-specific antigens. They may also be used as components of a prophylactic, therapeutic, or diagnostic vaccine against HIV-1. The invention’s vectors and nucleic acid are especially useful in providing genetic vaccines. The invention provides vaccines that deliver the nucleic acid encoding antigens to a subject such as a person to incite an immune response.

“The immunogenic compositions can include additional substances such as buffering agents or wetting agents to increase the effectiveness of vaccines (Remington?s Pharmaceutical Sciences, 18th Edition, Mack Publishing Company). 1980).”

“Immunogenic compositions can be used to deliver the CMV vectors at a desired location of action and then release them at a controlled rate. The art has many methods for preparing controlled-release formulations. Controlled release formulations can be made by using polymers that absorb or complex the immunogen. A controlled-release formulation can be prepared using appropriate macromolecules (for example, polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine sulfate) known to provide the desired controlled release characteristics or release profile. A controlled-release formulation can also be controlled by the incorporation of active ingredients in particles of polymeric materials such as polyesters, polyamino acid, hydrogels or polylactic acid, copolymers, ethylene vinylacetate copolymers, and copolymers. Alternatively, instead of incorporating these active ingredients into polymeric particles, it is possible to entrap these materials into microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacrylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. These techniques are described in New Trends and Developments in Vaccines by Voller et al. (eds. (eds.

“Those skilled in the art can easily determine the appropriate dosage of CMV vectors in immunogenic compositions. The route of administration and size of the subject can affect the dosage of CMV vectors. The art of measuring the immune response of subjects, such as laboratory animals, can help determine the appropriate doses. This can be done using standard immunological techniques and then adjusting the dosages accordingly. These techniques include, but are not limited, chromium release, tetramer binding, and IFN-? ELISPOT and IL-2 ELISPOT tests, intracellular cytokine (IFN-) assays and other immunological detection methods assays are all available. Ed Harlow and David Lane.

The immunogenic compositions may be administered by any method, including intramuscular, intravenous and mucosal. These techniques are well-known to those who have mastered the art. You can also use intramuscular injections, subcutaneous injections, or intradermal injections to deliver the drugs. Delivery does not have to be restricted to injection methods.

“Immunization regimens or schedules are well-known for animals, including humans. They can be easily determined for each subject and their immunogenic composition. The immunogens may be administered to the subject one or more times. It is preferable that there is a time limit between administrations of the immunogenic mixture. Although the interval will vary for each subject, it is usually between 10 days and several weeks. It can be 2, 4, 6, or 8 weeks. The interval for humans is usually between 2 and 6 weeks. The interval can be extended in a particularly beneficial embodiment of the invention. It is typically between 2 and 6 weeks.

“The immunization regimens usually have between 1 and 6 administrations of immunogenic composition. However, it is possible to have only one, two, or even four. Inducing an immune response may also involve the administration of an adjuvant. Sometimes, booster vaccination can be administered at intervals of 5-10 years, or biannually, to supplement the initial protocol.

The present methods include a variety prime-boost regimens such as DNA prime-Adenovirus booster regimens. These methods include one or more priming vaccines, followed by one or several boosting vaccinations. You can vary the composition of the actual immunogenic composition, such as the type and amount of immunogenic composition (e.g. containing protein or expression vector), and also the route and formulation of the immunogens. An expression vector used in the priming or boosting steps can be either the same type or a different one (e.g. DNA, bacterial, or viral). A prime-boost program provides two immunizations four weeks apart followed by two booster immunizations 4 and 8 weeks later. One skilled in the art will also know that the invention includes many combinations and permutations of viral, bacterial, and DNA expression vectors that provide priming or boosting regimens. If the viral vectors contain US2-11, or any of the genes encoded within the US2-11 region, they can be used repeatedly to express different antigens from different pathogens.

“A specific embodiment provides methods for inducing an immune reaction against a pathogen within a subject by administering immunogenic compositions one or more times. The epitopes are sufficiently expressed to induce a specific immune response. These immunizations may be repeated at intervals of at most 2, 4, or 6 weeks (or longer) according to a desired immunization regimen.

The immunogenic compositions can be administered by themselves, or co-administered or sequentially administered with other antigens (e.g., with?other?). Multivalent or ‘cocktail? immunological, antigenic, vaccine or therapeutic compositions are available. The invention may also include combinations of compositions and methods for using them. The ingredients and methods (sequential and co-administration), as well as dosages, can be determined by taking into account such factors as age, gender, weight, species, and condition of the subject and the route of administration.

“When administered in combination, antigens may be administered simultaneously or at different times. This is part of an overall immunization regimen, such as a prime-boost regimen, or any other immunization protocol.

“Although this invention has been described in great detail, it is important to understand that many modifications, substitutions, and alterations are possible without departing from its spirit and scope as set forth in the appended claims.”

“EXAMPLES”

“The following are examples of disclosed methods. Those skilled in the art will be able to see that there are variations of these and other methods disclosed. This disclosure is intended to show that it is possible to use the method without any undue experimentation.

“Example 1: Immunization with RhCMV vectors and a deletion of UL128 or UL130 results in an immune response characterized by a wide variety of CD8+ T cell epitopes against an SIV antigen.”

“Epitope targeting profiles for SIVgag specific CD8+ T cells responses elicited from RhCMV/gag Vectors derived RhCMV 68-1 strain. These vectors, which are devoid of active UL128 or UL130 (?UL128-135) but contain an active UL131 (Hansen S G et. al., J Virol 77. 6620 (2003); incorporated herein by reference) were compared with those elicites such as well as well as well as well as well as well as well as well as well as well as well as well as well as well as well as ed vectors. Flow cytometric intracellular staining was used for individual CD8+ T cells responses to each of the 125 consecutive 15mer-peptides (with an 11-amino acid overlap). This covered the entire SIVgag proteins. Twenty-nine rhesus monkeys (RM) were used. Fourteen were vaccinated using the?UL128-13 RhCMV/gag virus. Fourteen were vaccinated using electroporated DNA/gag+interleukin (IL-12). Three animals were vaccinated using adenovirus (Ad5/gag) and three others with vaccinia virus(MVA/gag). Five additional animals had been previously infected with SIV (SIVmac239) and were treated with spontaneous viral control.

“Peripheral blood CD8+T cells from?UL128-13 RhCMV/gag vector vaccined RM showed an average response to 46 of the 125 15mer sIVgag peptides. This corresponded with an average of 35 distinct epitopes. (FIG. 1A). SIV-infected controllers and RM vaccinated using electroporated DNA/gag+IL-12, Ad5/gag, and MVA/gag each responded to an average 10-19 peptides. This corresponds to an average 9-15 distinct epitopes. The range of responses from the?UL128-13 RhCMV/gag vector-vaccinated RM was such that many of these SIVgag 15mer peptides were reacted to by CD8+ cells in all or most of the 14 outbred animals (FIG. 1A).”

“To determine if this finding is indicative of promiscuous recognition a single common epitope? T cell recognition of?hotspots? The response to a series truncated propeptides was analysed. These truncated peptides corresponded with 7 of 15mers that were recognized in 3 RM per reaction. These peptides were used to identify core epitopes within each RM (FIG. 1B).”

Two distinct responses were observed with the truncated proteomes. Type 1 is the first. This pattern is characterized by a drop in response frequencies and loss of an essential amino residue. These truncations often resulted in a 9mer epitope (e.g. Gag259-267 or Gag276-284), and Gag482-490. The second type of response, Type 2, is a pattern where response frequencies slowly decline as the optimal sequence is truncated. These truncations often resulted in a 12mer-core epitope (Gag41-52, Gag211-222, Gag290-301 and Gag495-506). These truncation responses patterns and core peptides were identical in each RM. In all cases, core peptides showed superior stimulation (higher frequency response) than the parent 15mer (FIG. 1C).”

These data strongly suggest that many SIVgag epitopes that CD8+ T cell receptors in?UL128/130 RhCMV/gag vector vaccined RM target are specific determinants that can be recognized by MHC haplotypes of different types. A detectable CD8+ response to the core (optimal peptide) for five of these truncated15mers was observed in 100% of 42 RhCMV/gag-vaccinated RM. Responses to six other peptides, two optimal peptides, and four 15mers, were also found in >60% (FIG. 1D). These epitopes were not recognized by CD8+ cells in conventionally SIV infected RM. These CD8+ T cells that are stimulated by?UL128-13 RhCMV/gag vector are three times as wide as those that are infected with SIVgag-specific CD8+ cell responses. They are also unique because they are often targeted at’supertopes’.

“Example 2” Type 1 CD8+ responses are MHC-1 Restricted, Type 2-CD8+ responses are MHC-2 Restricted

MHC-I-restricted epitopes typically have 8-10 amino acid lengths and contain position-specific amino acids which engage binding pockets (anchor residues), so that they can fit in a closed end. The MHC-1 binding groove (Rammensee G et al., Ann Rev Immunol 11 213 (1993); incorporated herein) has characteristics that are consistent with the Type 1 pattern. The Type 2 truncation is more typical for MHC II-restricted epitopes. They are usually longer, have a >12mer core and are more tolerant to length heterogeneity (Southwood S et al., J Immunol 160 (1998) and Chelvanayagam GL, Hum Immunol 58 (1997), both of which are incorporated herein). It was suggested that CD8+ T cells that recognize Type 2 SIVgag epitopes within the RhCMV/gag-vaccinated RM may be MHC-II-restricted. This suggests that class II-restricted CD8 T cells responses might be unusual. However, such responses were previously reported in mice (Mizuochi T et Al, J Exp Med 168.437 (1988); Suzuki E et Al, J Immunol 153.4496 (1994); Matechak E et A, Immunity 4, 337 (1996), Shimizu T and Takeda T, Eur J Immunol 27,500 (1997); Rist M et, Blood 102, 11244 (2002); and J ets (1997), Eur J eta eta, 21.

“To determine if?UL128-135, RhCMVgag might be eliciting an MHCII-restricted CD8+ cell response to gag, we tested the ability of?blocking? Monoclonal antibodies (mAbs), specific for MHC-128 and MHC-1II, as well as the invariant-chain-derived, MHC-2II-specific binding peptide CLIP were tested to block Type 1 and Type 2 epitopes-specific CD8+ T cells responses in RM immunized by?UL130 RhCMVgag. 2A). These reagents inhibited the 5 universal supertope-specific CD8+ cell responses. This corresponded exactly to the Type 1 and 2 truncation patterns, with T cells recognizing the three Type 2 epitopes Gag211-222-301, Gag290-306 and Gag495-506 being blocked by anti?MHCII and CLIP but not anti?MHCII. The reverse was true for the 2 Type 1 epitopes Gag276-284 and Gag482-490

“The epitope-specific responses shown in FIG. 2A in relation to MHC-1 vs MHC-2 blockade (FIG. 2B, FIG. 2C). 2C).

“To confirm that the MHC-II-blocked CD8+ T cell responses were MHC-II-restricted?defined as the epitope in question being recognized in the context of MHC-II?and to investigate the basis of the promiscuity of these responses across MHC-disparate RM, cell lines expressing single rhesus MHC-II allomorphs were constructed. Four RhCMV/gag-vaccinated RM were used to express the MHC-II alleles. They had characterized SIVgag epitope recognition profiles. Flow cytometric ICS tests showed that pulsing the MHCII allomorph transfectants but not the parent MHCII negative line with individual peptides resulted a robust CD8+ T-cell stimulation of only those responses classed as MHCII-associated (FIG. These responses can be blocked by anti-MHCII mAbs or CLIP peptide but not anti-MHCII mAbs. Importantly, MHC-II Allomorphs often presented multiple peptides. Individual peptides were also frequently presented by multiple MHC?II allomorphs (FIG. 3A). Individual allomorphs can present multiple gag peptides, which helps to explain the range of MHC-II-restricted reactions. Multiple MHCII allomorphs can present individual peptides. This suggests that all RM expressing at most one MHCII allomorph are likely to explain the widespread recognition of these peptides across RhCMV/gag vector elicited CD8+ cells.

“As previously reported for MHCII-restricted CD4+ cell responses (Corradin A and Lanzaveccia, Int Rev Immunl 7, 139 (1991); incorporated herein), MHCII-restricted SIVgag specific CD8+ T cells elicited with RhCMV/gag Vectors can respond to the peptide epitope within the context of peptide binding MHCII allomorphs not expressed by the donor (FIGS). 3A and 3B, respectively, indicate that these T cells recognize the bound protein either as a single molecule or in combination with other non-polymorphic structures of the MHC-II molecules.

“Example 3?” Phenotype and Function of the?UL128-133/SIV Vector-Elicited T Cell Responses to CD8+”

“The epitope-specificity of SIV-specific CD8-specific T cells generated and maintained with RhCMV/SIV vector vaccine raises questions about their functional potential, particularly the unusual MHC-II-restricted population that dominates these responses. This is because supertope-specific CD8T cell responses are not due to high peptide levels used in standard ICS assays. Responses to optimal peptides (both Type 1 and 2) can be demonstrated at peptide dilutions greater than 1:105 (FIG. 4A). Second, supertope-specific Type 1 and 2 responses appear immediately following vaccination (FIG. 4B and coordinately distribute throughout the body in the same pattern as previously reported for RhCMV/SIV vector vaccined RM (Hansen, S G et. al., Nature 473/523 (2011); incorporated herein by reference (FIGS. 4C and 4D. Third, as previously reported in RhCMV-specific CD8+ and RhCMV/SIV vector elicited SIV?specific T cells (Hansen S. G. et al., Nat Med 15, 293 (2008); incorporated herein), both Type 1 supertope-specific and Type 2 T cells exhibit an identical phenotype that is indicative of effector-memory T cell differentiation (CCR7, CD28?). This effector-memory profile is consistent with the identical polyfunctional profile (high TNF, IFN, and MIP-1). Production, high CD107 externalization (degranulation), and low IL-2 production. 4E and 4F. These data indicate that CD8+ T cells in vaccinated RM receive the same in vivo exposures to Type 1 and 2 epitopes, since effector memory differentiation is believed to be Ag-driven.

“Example 4?” UL128 and/or UL130 Control Targeting CMV-Elicited CD8+ Cell Responses

“To identify candidate CMV gene associations with this unusual CD8+ immune reaction, it was first questioned whether CD8+ T cells responses to an endogenous CMV instant early (IE protein) also target unorthodox epitopes (in particular supertopes that are restricted by MHC?II). This was done by comparing RM infected naturally with wildtype RhCMV (colony circulating viruses) and RM vaccinated using the exemplary?UL128-13 deficient strain 68.1 RhCMV/SIV virus vector. It was not surprising that RM vaccinated using the?UL128-13 vector showed IE-specific CD8+ cell responses with identical targeting characteristics to the SIVgag specific CD8+ cell responses in the same RM. There were >30 distinct IE epitopes/RM. The majority of these responses were blocked by anti-MHCII and a few blocked with anti?MHCII.

“In striking contrast, however, the IE-specific CD8+ responses in naturally RhCMV infected RM were more targeted (?8 epitopes/RM) and did not show evidence of MHC-2 restriction or epitope promiscuity. These results are consistent with the conventional immunodominance hierarchies. These results likely explain why CMV-specific CD8+ cell responses that are not targeted by CMV have not been reported in naturally exposed CMV+ RM or humans (despite extensive analysis). They also implicate genetic differences in the mechanisms responsible for generating these unconventionally targeted CD8+ cell responses between wildtype RhCMV and the?UL128-130-deficient strain 68.1-based RhCMV vectors.

“To evaluate the role of these genes during the targeting of CD8+ cells during priming, a RhCMV/gag was created in which expressions of the UL128, UL130 orthologs were re-established (Lilja A E et al., Proc Natl Aca Sci USA 105, 19950 (2008). The question was then posed whether this “repair”? The epitope targeting profiles for vector-elicited gag specific CD8+ T cells responses were affected by the UL128 or UL130 ortholog expression. The UL128 and UL130-repaired RhCMV/gag vector-elicited SIVgag specific CD8+ T cells responses, which did not include recognition or any of the MHC?II supertopes, were more targeted than the response elicited from the unrepaired 68.1 strain vector. They were also entirely MHC?I-associated (FIGS). 5D, 5E and 5F.

“Example 5:CMV Vectors with Single Delitions of UL128 and UL130 Display CD8 Responds Characterized By Class II Restriction. CMV Vectors with one deletion of Ul131 are Incapable of Suprainfection.”

Summary for “Cytomegalovirus vectors enable control of T cell targeting”

T cell receptor (TCR),-mediated recognition by CD8+ T cells of intracellular pathogens (class I MHC proteins (MHC-1I)) and an exceptional system of intracellular protein sampling and transport (Neefjies M. L. et al., Nat Rev Immunol 11: 823 (2011); incorporated herein). Pathogens may produce thousands of different peptides, which can be used to recognize CD8+ T-cells. However, there are requirements for proteolytic processing and peptide transport as well as matching TCR repertoire. These mechanisms, along with the poorly understood immunoregulatory mechanisms, allow for the narrowing down of potential targets for CD8+ T-cells. The complexity of this process is not surprising. Pathogen-specific CD8-T cell responses that are mounted by individuals with common MHC-I alleles tends to recognize an overlap of so-called immunodominant peptides (Yewdell J W et Al, 2006 supra; Irvine K et Al, Expert Rev Clin Immunol 2, 135 (2006); Assarsson E et. al, J Immunol 178, 7890 (2007); incorporated herein by referenced by reference herein; The vast majority of pathogens can be recognized by CD8+ T cells that target immunodominant epitopes. These immunedominant epitopes can also mount memory and anti-pathogen effectsor responses. Agents with high immune evasion abilities, such as the HIV and its similian counterpart SIV, are not immune-deficient. These viruses’ massive replication, coupled with their high rate for mutation and functional plasticity allow escape from most CD8+T cell responses (Picker U. et al., Ann Rev Med, 63, 95 (2012); incorporated by reference herein). The majority of infected subjects with these viruses have CD8 T cells that fail to target epitopes containing functionally critical, conserved viral sequences. This makes them difficult to control viral replication. Although these viruses can increase the number of CD8+ T cells responding to infection, they also target immunodominant epitopes, which is why these larger responses are still susceptible to immune escape (Picker 2012 supra; Barouch, D H et. al., J Virol 77 7367 (2003); Mudd, P A et. al., Nature 491, 129 (2012); all are incorporated herein). The AIDS vaccine industry has attempted to create strategies that could elicit HIV/SIV-specific CD8+ cell responses. Epitopes can be spread across different MHC-I haplotypes. This is done by increasing recognition breadth, or focusing responses to conserved sequences. However, this effort has not yielded strategies that significantly modify CD8+ T cells immunodominance hierarchies.

“A HIV/AIDS vaccine strategy that uses a recombinant Cytomegalovirus, (CMV), expressing an HIV protein was created to create and maintain HIV-specific effector T cell responses. This would prevent HIV infection from occurring before the viral amplification is required for effective immune evasion. (Picker 2012 supra). Although this strategy was not intended to prevent infection, the approach proved highly effective in animal models of HIV/AIDS, with approximately 50% of CMV/SIV vector-vaccinated rhesus monkeys (RM) being challenged with highly pathogenic, persistent SIV (Hansen 2011 infra).

“These studies revealed that CMV/SIV vectors didn’t elicit immunodominant CD8+ responses to SIV peptides presented in the well-characterized Mamu A1*001:01(A*01) MHC-1 protein. This suggests that CMV vectors stimulate new T cell epitopes that are targeted by these powerful responses, and contributes to vaccine efficacy.”

“It is revealed that heterologous antigens, such as viral or bacterial expressed via cytomegalovirus Vectors, induce a T-cell immunodominance profile which is fundamentally different to that elicited by all other vectors. As an animal model for human CMV, rhesus macaques infected by rhesus CMV virus (RhCMV), carrying SIV antigens was used to demonstrate that the SIVgag specific CD8+ responses elicited from the RhCMV/gag Vector are three times as wide as the gag-specific CD8+ cell responses elicited either by other vaccines or after infection with SIV. It has been shown that CMV-elicited cells target completely different epitopes than other vaccines. This includes a high percentage of epitopes present by MHC-II (class II MHC). These responses are rare, if any, in CD8+ T cells responses to other infectious agents or vaccines. CMV is also responsible for the immunodominance profile. It was shown that the CMV genes UL128 (and UL130) prevent this response from being induced. T cell responses to the CMV vector containing UL128/UL130 are focused on a small number of epitopes, whereas MHC II restricted CD8+ T cells are induced using vectors that do not contain UL128/UL130. This allows for genetic manipulation of a vaccine vector to create distinct patterns in CD8+ T-cell epitope recognition.

“CMV vectors disclosed herein include a heterologous antigen, an active UL131 or an ortholog thereof, an active UL128 (or an equivalent thereof), and a CMV vector that lacks an active UL130. CMV vectors also disclosed include a heterologous antigen, an activate UL131 protein or an ortholog thereof, and an active UL130 proteins (or an equivalent thereof), but the CMV vector is missing an active UL128.

“The method of producing a CD8+ T-cell response to heterologous antigens in a subject is also disclosed. This involves administering a CMV vector to the subject. CMV vectors are distinguished by having a heterologous antibody, an active UL131 protein, an inactive UL128 protein, an inactive UL130 protein, an inactive UL130 protein or a combination of both. At least 10% of CD8+ T cells are directed against epitopes from MHC Class II.

“Disclosed are vectors of animal or human cytomegalovirus (CMV), capable of infecting multiple organisms. CMV vectors include a nucleic acids sequence that encodes a heterologous antigen protein and a sequence that encodes an active UL131 amino acid protein. The CMV vector, for example, contains a nucleic acids sequence that expresses an activated UL128 protein but not an active UL130. Another example is that the CMV vector encodes an UL130 protein, but not an active UL128 Protein.

“In some cases, the vector doesn’t express an active UL128/UL130 protein because of a deleterious mutation within the nucleic acids sequence encoding UL128/UL130 or their orthologous gene in animal CMVs. Any mutation that causes the expression of UL128 and UL130 proteins to be inactive can be considered a deleterious mutation. These mutations include frameshift mutations and point mutations.

“In additional examples, the vector doesn’t express an active UL128/UL130 protein because it contains a nucleic acids sequence that contains an antisense sequence (siRNA/miRNA) that inhibits expression of the UL128/UL130 proteins.”

“Also described herein are methods for generating CD8+ T cells responses to heterologous antibodies in a subject. These methods involve administering a CMV vector to the subject. CMV vectors are distinguished by having a nucleic acids sequence that encodes a heterologous protein and a sequence that encodes active UL131 proteins. CMV vectors are also distinguished by not encoding active UL128 proteins or active UL130 proteins, or neither active UL128 nor active UL130 proteins. At least 10% of CD8+T cells are directed against epitopes identified by MHC class 2. Further examples include at least 20%, 30%, 40%, at minimum 50%, at most 60%, and at most 60% of CD8+ T cell responses directed against epitopes present in MHC class II.

“In further examples, methods include administering an effective amount CMV vector, which is composed of a nucleic acids sequence that encodes heterologous antigens to the subject. Any CMV vector may be used, including one with an active CMV128 protein and one with an active CMV130 protein. Additional deletions may be included in the second CMV vector to produce different immune responses, such as a US11 or any other deletion. Any heterologous or unidentifiable heterologous antibody can be used as the second heterologous. The administration of the second CMV can occur at any time after administration of the first CMV Vector, including concurrently or following administration of the first CMV Vector. Administration of the second CMV vector can be done at any time, including months, days, hours or minutes before, concurrently with, or after administration of the first vector.

“Human and animal CMV vectors when used as expression vectors in selected subjects, such as humans, are innately not pathogenic in those subjects or have been modified to make them non-pathogenic. For example, replication-defective adenoviruses and alphaviruses are well known and can be used as gene delivery vectors.”

“The heterologous antibody can be any protein, or fragment thereof, that is not derived form CMV, and includes cancer antigens as well as pathogen specific antigens. It also includes model antigens (such lysozyme, ovalbumin) or any other antigen.

“Pathogen-specific antigens can be obtained from any animal or human pathogen. The antigen could be a protein that was derived from the virus. Viruses include, among others, Adenovirus (coxsackievirus), hepatitis A viruses, rhinoviruses, Herpes simplex type 1, Varicella-zostervirus type 2, Epstein-Barrviruses, Epstein-Barrviruses, Kaposi’s sarcoma virus and Kaposi’s sarcoma virus.

The pathogen could be a bacterial infection and the antigen could be a protein that is derived from the bacteria. The pathogenic bacteria include, but are not limited to, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Legionella pneumophila, Leptospira interrogans, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Rickettsia rickettsii, Salmonella typhi, Salmonella typhimurium, Shigella sonnei, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Treponema pallidum, Vibrio cholera and Yersinia pestis.”

The parasite may be the pathogen and the antigen could be a protein that is derived from it. The parasite may be a protozoan organism or a protozoan organism causing a disease such as, but not limited to, Acanthamoeba, Babesiosis, Balantidiasis, Blastocystosis, Coccidia, Dientamoebiasis, Amoebiasis, Giardia, Isosporiasis, Leishmaniasis, Primary amoebic meningoencephalitis (PAM), Malaria, Rhinosporidiosis, Toxoplasmosis-Parasitic pneumonia, Trichomoniasis, Sleeping sickness and Chagas disease. The parasite may be a helminth organism or worm or a disease caused by a helminth organism such as, but not limited to, Ancylostomiasis/Hookworm, Anisakiasis, Roundworm?Parasitic pneumonia, Roundworm?Baylisascariasis, Tapeworm?Tapeworm infection, Clonorchiasis, Dioctophyme renalis infection, Diphyllobothriasis?tapeworm, Guinea worm?Dracunculiasis, Echinococcosis?tapeworm, Pinworm?Enterobiasis, Liver fluke?Fasciolosis, Fasciolopsiasis?intestinal fluke, Gnathostomiasis, Hymenolepiasis, Loa loa filariasis, Calabar swellings, Mansonelliasis, Filariasis, Metagonimiasis?intestinal fluke, River blindness, Chinese Liver Fluke, Paragonimiasis, Lung Fluke, Schistosomiasis?bilharzia, bilharziosis or snail fever (all types), intestinal schistosomiasis, urinary schistosomiasis, Schistosomiasis by Schistosoma japonicum, Asian intestinal schistosomiasis, Sparganosis, Strongyloidiasis?Parasitic pneumonia, Beef tapeworm, Pork tapeworm, Toxocariasis, Trichinosis, Swimmer’s itch, Whipworm and Elephantiasis Lymphatic filariasis. Parasites can be caused by organisms such as Halzoun Syndrome (or bilharziosis or snail fever), Halzoun Syndrome (or worm), Myiasis and Chigoe fleas, Candiru, Candiru, and Candiru. Parasites can be caused by ectoparasites, such as: Bedbug, Head louse??Pediculosis or Body louse??Pediculosis and Demodex?Demodicosis.

The antigen could be a protein that is derived from cancer. The cancers, include, but are not limited to, Acute lymphoblastic leukemia; Acute myeloid leukemia; Adrenocortical carcinoma; AIDS-related cancers; AIDS-related lymphoma; Anal cancer; Appendix cancer; Astrocytoma, childhood cerebellar or cerebral; Basal cell carcinoma; Bile duct cancer, extrahepatic; Bladder cancer; Bone cancer, Osteosarcoma/Malignant fibrous histiocytoma; Brainstem glioma; Brain tumor; Brain tumor, cerebellar astrocytoma; Brain tumor, cerebral astrocytoma/malignant glioma; Brain tumor, ependymoma; Brain tumor, medulloblastoma; Brain tumor, supratentorial primitive neuroectodermal tumors; Brain tumor, visual pathway and hypothalamic glioma; Breast cancer; Bronchial adenomas/carcinoids; Burkitt lymphoma; Carcinoid tumor, childhood; Carcinoid tumor, gastrointestinal; Carcinoma of unknown primary; Central nervous system lymphoma, primary; Cerebellar astrocytoma, childhood; Cerebral astrocytoma/Malignant glioma, childhood; Cervical cancer; Childhood cancers; Chronic lymphocytic leukemia; Chronic myelogenous leukemia; Chronic myeloproliferative disorders; Colon Cancer; Cutaneous T-cell lymphoma; Desmoplastic small round cell tumor; Endometrial cancer; Ependymoma; Esophageal cancer; Ewing’s sarcoma in the Ewing family of tumors; Extracranial germ cell tumor, Childhood; Extragonadal Germ cell tumor; Extrahepatic bile duct cancer; Eye Cancer, Intraocular melanoma; Eye Cancer, Retinoblastoma; Gallbladder cancer; Gastric (Stomach) cancer; Gastrointestinal Carcinoid Tumor; Gastrointestinal stromal tumor (GIST); Germ cell tumor: extracranial, extragonadal, or ovarian; Gestational trophoblastic tumor; Glioma of the brain stem; Glioma, Childhood Cerebral Astrocytoma; Glioma, Childhood Visual Pathway and Hypothalamic; Gastric carcinoid; Hairy cell leukemia; Head and neck cancer; Heart cancer; Hepatocellular (liver) cancer; Hodgkin lymphoma; Hypopharyngeal cancer; Hypothalamic and visual pathway glioma, childhood; Intraocular Melanoma; Islet Cell Carcinoma (Endocrine Pancreas); Kaposi sarcoma; Kidney cancer (renal cell cancer); Laryngeal Cancer; Leukemias; Leukemia, acute lymphoblastic (also called acute lymphocytic leukemia); Leukemia, acute myeloid (also called acute myelogenous leukemia); Leukemia, chronic lymphocytic (also called chronic lymphocytic leukemia); Leukemia, chronic myelogenous (also called chronic myeloid leukemia); Leukemia, hairy cell; Lip and Oral Cavity Cancer; Liver Cancer (Primary); Lung Cancer, Non-Small Cell; Lung Cancer, Small Cell; Lymphomas; Lymphoma, AIDS-related; Lymphoma, Burkitt; Lymphoma, cutaneous T-Cell; Lymphoma, Hodgkin; Lymphomas, Non-Hodgkin (an old classification of all lymphomas except Hodgkin’s); Lymphoma, Primary Central Nervous System; Marcus Whittle, Deadly Disease; Macroglobulinemia, Waldenstrim; Malignant Fibrous Histiocytoma of Bone/Osteosarcoma; Medulloblastoma, Childhood; Melanoma; Melanoma, Intraocular (Eye); Merkel Cell Carcinoma; Mesothelioma, Adult Malignant; Mesothelioma, Childhood; Metastatic Squamous Neck Cancer with Occult Primary; Mouth Cancer; Multiple Endocrine Neoplasia Syndrome, Childhood; Multiple Myeloma/Plasma Cell Neoplasm; Mycosis Fungoides; Myelodysplastic Syndromes; Myelodysplastic/Myeloproliferative Diseases; Myelogenous Leukemia, Chronic; Myeloid Leukemia, Adult Acute; Myeloid Leukemia, Childhood Acute; Myeloma, Multiple (Cancer of the Bone-Marrow); Myeloproliferative Disorders, Chronic; Nasal cavity and paranasal sinus cancer; Nasopharyngeal carcinoma; Neuroblastoma; Non-Hodgkin lymphoma; Non-small cell lung cancer; Oral Cancer; Oropharyngeal cancer; Osteosarcoma/malignant fibrous histiocytoma of bone; Ovarian cancer; Ovarian epithelial cancer (Surface epithelial-stromal tumor); Ovarian germ cell tumor; Ovarian low malignant potential tumor; Pancreatic cancer; Pancreatic cancer, islet cell; Paranasal sinus and nasal cavity cancer; Parathyroid cancer; Penile cancer; Pharyngeal cancer; Pheochromocytoma; Pineal astrocytoma; Pineal germinoma; Pineoblastoma and supratentorial primitive neuroectodermal tumors, childhood; Pituitary adenoma; Plasma cell neoplasia/Multiple myeloma; Pleuropulmonary blastoma; Primary central nervous system lymphoma; Prostate cancer; Rectal cancer; Renal cell carcinoma (kidney cancer); Renal pelvis and ureter, transitional cell cancer; Retinoblastoma; Rhabdomyosarcoma, childhood; Salivary gland cancer; Sarcoma, Ewing family of tumors; Sarcoma, Kaposi; Sarcoma, soft tissue; Sarcoma, uterine; Sezary syndrome; Skin cancer (nonmelanoma); Skin cancer (melanoma); Skin carcinoma, Merkel cell; Small cell lung cancer; Small intestine cancer; Soft tissue sarcoma; Squamous cell carcinoma?see Skin cancer (nonmelanoma); Squamous neck cancer with occult primary, metastatic; Stomach cancer; Supratentorial primitive neuroectodermal tumor, childhood; T-Cell lymphoma, cutaneous (Mycosis Fungoides and Sezary syndrome); Testicular cancer; Throat cancer; Thymoma, childhood; Thymoma and Thymic carcinoma; Thyroid cancer; Thyroid cancer, childhood; Transitional cell cancer of the renal pelvis and ureter; Trophoblastic tumor, gestational; Unknown primary site, carcinoma of, adult; Unknown primary site, cancer of, childhood; Ureter and renal pelvis, transitional cell cancer; Urethral cancer; Uterine cancer, endometrial; Uterine sarcoma; Vaginal cancer; Visual pathway and hypothalamic glioma, childhood; Vulvar cancer; Waldenstrm macroglobulinemia and Wilms tumor (kidney cancer.)”

“The CMV vectors discussed herein provide a vector to clone or express heterologous DNA. They contain recombinant human and animal CMV. A heterologous DNA could encode an expression product that includes an epitope, a biological response modator, a growth factor or recognition sequence, as well as a therapeutic gene or fusion protein.

“The CMV vectors described herein can be used to create an immunogenic, immunological, or vaccine composition containing a recombinant CMVvirus or vector and a pharmaceutically approved carrier or diluent. A composition that contains the recombinant CMV vector or an expression product thereof can elicit an immunological response. This response may be protective, but it does not have to be. A vaccine composition containing the recombinant virus CMV vector or an expression product thereof can also elicit a local or systemic immune response that may be protective, but not necessarily. A vaccine composition triggers a protective response that is either local or systemic. The terms “immunological composition” and “immunogenic composition” are used interchangeably. ?immunogenic composition? include a ?vaccine composition? (The two terms above can also be used to refer to protective compositions.

“The CMV vectors described herein provide methods for inducing an immune response in a subject. This includes administering to the subject an immunogenic or immunological composition consisting of the recombinant CMVvirus or vector and a pharmaceutically approved carrier or diluent. The term “subject” is used in this specification. All animals, non-human primates, and humans are included in the definition of?subject? All vertebrate species are included, except humans. All vertebrates are included, even animals (as ‘animal?). are used herein). A subset of “animal” is, of course. A subset of?animal? is?mammal?”, which, for the purposes of this specification, includes all mammals except humans.

The CMV vectors described herein can be used to create therapeutic compositions that contain the recombinant CMV viruses or vector and a pharmaceutically approved carrier or diluent. The therapeutic composition can be used in gene therapy and immunotherapy embodiments according to the invention. This includes administering the composition to the host and transferring genetic data.

The CMV vectors described herein can be used to express a protein, gene product, or expression product. This involves infecting or transfecting cells in vitro with the recombinant CMV viruses or vectors of the invention, and then extracting, purifying, or isolating the DNA, protein, gene products, or expression product from the cell. The invention also provides a method of cloning or reproducing heterologous DNA sequences. This involves infecting or transfecting cells in vitro and in vivo using a recombinant CMVvirus or vector of invention, as well as optionally extracting, purifying, or isolating DNA from the cell.

The CMV vectors described herein can be made by inserting DNA containing a sequence that encodes heterologous antigen in a non-essential area of the CMV genome. This method may also include deleting one or several regions of the CMV genome. In vivo recombination is possible. The method may include transfecting cells with CMV DNA in a cell compatible medium with donor DNA that contains the heterologous sequences and DNA sequences homologous to portions of the CMV genome. Optionally, the CMV can then be recovered by in vivo Recombination. This method may also include cleaving CMVDNA to obtain CMV-cleaved DNA. Then, the donor DNA can be ligated to the CMV DNA to create hybrid CMV Heterologous, which is then transfected to a cell with hybrid CMVHeterologous DNA. Finally, the option to recover CMV that has been modified by the heterologous. In vivo recombination can be understood so the method also includes a plasmid containing donor DNA. This donor DNA encodes a polypeptide that is foreign to CMV. The donor DNA is located within a segment CMV DNA that would otherwise not be co-linear. To generate recombinant CMV, the heterologous DNA may be placed into CMV in any orientation that allows for stable integration and expression of that DNA.

The promoter can be found in DNA encoding heterologous antigens in the recombinant vector CMV. A promoter can come from any source, including a herpesvirus, rhesus macaque CMV(RhCMV), murine or another CMV promoter. A promoter who is not viral, such as the EF1 can also be used. promoter. A promoter can be a transcriptionally active truncated promoter that contains a region activated with a virus transactivating protein and the minimal promoter area of the full-length enhancer from which the truncated transcriptionally activ promoter was derived. A promoter is a combination of DNA sequences that correspond to the minimal promoter or upstream regulatory sequences. A minimal promoter is made up of the CAP site and TATA box (minimum sequences to support basic transcription; unregulated transcription); and,?upstream regulator sequences? are made up of the enhancer sequences and/or the upstream element. The term “truncated” also refers to the fact that some of the full-length promoter(s) may not be present. The term?truncated? indicates that the promoter’s full length is not present. This means that some of the promoter’s full-length has been removed. The truncated promotor can also be derived from a herpesvirus like MCMV or HCMV.

The inventive promoter could be, like the one above, a herpesvirus such as MCMV or HPV. There can also be a 40 to 90% reduction in size compared to a full-length promoter based on base pairs. A modified non-viral promoter is also possible.

“An expression cassette can also be disclosed that can be placed into a recombinant viral or plasmid containing the truncated transcriptionally activate promoter. An expression cassette may also include a functional truncated polyadenylation signals, such as an SV40 signal that is truncated but functional. Given that nature provides a stronger signal, it is not surprising that a functional truncated poladenylation signals is functional. A truncated poladenylation signal addresses insert size limitations of recombinant viruses like CMV. An expression cassette may also contain heterologous DNA. This is true regardless of the virus or system it is being inserted into.

“The present invention includes CMV, recombinants that contain viral or non-viral promotors, and a truncated promotor. The invention also covers antibodies that are elicited from the inventive compositions or recombinants, and their uses. The antibodies or the product (epitopes or recombinants) that elicited them or monoclonal antibody from the antibodies can be used in binding assays or tests to determine whether an antigen or antibodies are present or absent.

“Flanking DNA can be used in the invention from the site or a portion adjacent thereto (where?adjacent is). “Flanking DNA can be from the site of insertion or a portion of the genome adjacent thereto (wherein?adjacent?

“As regards antigens for vaccine or immunological compositions (for a listing of antigens used as expression products of the inventive virus or an expression product thereof),”

“As for heterologous Antigens, one skilled enough in the art can choose a heterologous and coding DNA from the knowledge and sequences of the amino acids of the peptide/polypeptide as well as the nature of specific amino acids (e.g. size, charge, etc.). Without undue experimentation, the codon dictionary and the amino acid sequences.

“Regarding the sequence, it is preferable that the DNA sequence encodes at most regions of the antigen that produce an antibody response or a response from a T cells, especially a CD8+ T-cell response. Epitope mapping is one method of determining T and B cell epitopes. Oligo-peptide synthesizers can create overlapping peptides from the heterologous antibody. Each peptide is then tested to determine if it can bind to the antibody generated by the native protein, or induce T cell and B cell activation. This method has proven to be particularly helpful in mapping epitopes of T-cells, as the T cell recognizes short linear and MHC molecules.

“An immune reaction to heterologous antigens is generally as follows: T cells only recognize proteins when they have been cleaved into smaller, more easily digestible peptides. This complex is called the?major hertocompatibility complex (MHC). located on the surface of another cell. There are two types of MHC complexes: class I and class II. Each class contains many different alleles. Individual subjects and species have different types MHC complex alleles. They are known to have a different HLA type.

“It should be noted that the DNA encoding heterologous antibodies can contain a promoter to drive expression in the CMV vector, or the DNA can only include the coding DNA for the heterologous antibody. This construct can be placed in a way that is operably linked with an endogenous CMV enhancer, and it will then be expressed. Multiple copies of the DNA encoding heterologous antigen can be used, as well as a strong, early, or late promoter or combination thereof, to increase or amplify expression. The DNA that encodes the heterologous can be placed in a suitable position relative to a CMV endogenous promoter or can be translocated so that they can be inserted at a different location. CMV vectors can contain nucleic acids that encode more than one heterologous antibody.

“CMV vectors are also disclosed in pharmaceuticals and other compositions. These pharmaceutical and other compositions may be made so that they can be used in any of the administration procedures known to the art. These pharmaceutical compositions may be administered via intradermal, intramuscular or subcutaneous routes, as well as intravenous. You can also administer the medication via a mucosal route (e.g. oral, nasal or genital).

The disclosed pharmaceutical compositions are prepared according to standard pharmaceutical techniques. These compositions can be administered using dosages and techniques that are well-known to medical professionals. This includes factors such as the patient’s age, gender, sex, weight and condition. These compositions can be administered by themselves, or they can be combined with other CMV vectors, other immunological, antigenic, vaccine, or therapeutic compositions. These compositions may contain purified native antigens, epitopes, or antigens and epitopes derived from a recombinant CMV vector system. They are administered considering the above factors.

“Examples include liquid preparations for oral, nasal and genital orifices such as syrups, syrups, or elixirs. Also, preparations for intradermal, intramuscular, parenteral, or subcutaneous administration (e.g. injectable administration), such as sterile suspensions/emulsions. These compositions may contain recombinant in an admixture with a suitable carrier or diluent such as glucose, physiological saline or glucose.

Antigenic, immunological, or vaccine compositions can typically contain an adjuvant as well as a dose of the CMV vector/expression product to elicit desired responses. Alum, also known as aluminum phosphate or aluminium hydroxide, is used in human applications. The toxicities of Saponin and Quil A, Freund?s complete adjuvant, and other adjuvants used for research and veterinary purposes have limited their use in vaccines. Chemically defined preparations like muramyl dipeptide and monophosphoryllipid A, as well as phospholipid conjugates, such as those described in Goodman-Snitkoff, et al. J. Immunol. 147:410-415 (1991), Encapsulation of the protein in a proteoliposome according to Miller et.al, J. Exp. Med. Med.

The composition can be packed in one dosage form for immunization via parenteral (i.e. intradermal, intramuscular or subcutaneous) administration. The effective dosage and route for administration are determined by the composition of the product, the expression level if recombinant CMV has been directly used, and known factors such as breed, species, age, weight, condition, and host nature. LD50 and any other screening procedures that are not subject to undue experimentation are also important. The dosage of the expressed product can vary from a few micrograms to several hundred micrograms (e.g. 5 to 500 mg). CMV vectors can be administered in any amount necessary to obtain expression at these levels. Non-limiting examples: CMV Vectors can be administered in a minimum of 102 pfu. CMV vectors may also be administered in a range between about 102 and about 10. pfu. You can also use water or buffered soap as a carrier or diluent, with or without a preserver. CMV vectors can be either lyophilized to allow for resuspension after administration, or in solution.

“A polymeric delayed-release system may also be used as a carrier. A controlled release composition can be made using synthetic polymers. Kreuter, J. Microcapsules and Nanoparticles In Medicine and Pharmacology by M. Donbrow (Ed.) was an early example. CRC Press. p. 125-148.”

Microencapsulation is a method for injecting microencapsulated pharmaceuticals in controlled quantities. There are many factors that influence the choice of a specific polymer for microencapsulation. Consider the reproducibility of microencapsulation synthesis, cost of microencapsulation materials, toxicological profile, requirements for variable release kinetics, physicochemical compatibility and antigen compatibility. Polycarbonates, polyurethanes and polyorthoesters, as well as polyamides (especially those that can be biodegradable), are all examples of useful polymers.

“A frequent choice of a carrier for pharmaceuticals and more recently for antigens is poly (d,1-lactide-co-glycolide) (PLGA). This biodegradable polyester has been used in medical applications for erodible sutures and bone plates. It has never been toxic. PLGA microcapsules can be formulated with a wide range of pharmaceuticals, including peptides or antigens. Eldridge, J. H., et. al. reviewed a large amount of data on the adaptation of PLGA to controlled release of antigen. Current Topics In Microbiology And Immunology. 1989, 146:59-66. Orally, the adjuvant effect of PLGA microspheres containing antigens that are 1-10 microns in diameter was demonstrated to be remarkable. The PLGA microencapsulation method uses a phase separation process of a water in-oil emulsion. The compound of interest can be prepared in an aqueous solution. The PLGA is then dissolved in a suitable organic solvent such as methylenechloride or ethyl acetate. High-speed stirring is used to combine these two immiscible mixtures. The non-solvent is added to the polymer, which causes precipitation of the polymer around the droplets. This results in embryonic microcapsules. The microcapsules can then be collected and stabilized with any of a variety of agents (polyvinyl Alcohol (PVA), gelatins, alginates (PVP), and methyl cellulose). The solvent is then removed either by solvent extraction or drying in vacuo.

“Regarding HCMV promoters is made to U.S. Patent. Nos. Nos. 5,168,062 & 5,385,839. Feigner and colleagues refer to the transfection of cells with plasmidDNA for expression. (1994), J. Biol. Chem. 269, 2550-2561. Direct injection of plasmidDNA is a simple and effective way to prevent a wide range of infectious diseases. Science, 259, 1745-49, 1993. This invention allows the direct injection or use of vector DNA.

“The terms?”protein?,?peptide?”,??polypeptide?” and?amino acids sequence? are interchangeable. These terms are interchangeable to refer to polymers of any length of amino acid residues. It can be linear or branched and may contain modified amino acids or analogues. It may also be interrupted by chemical moieties that are not amino acids. These terms can also refer to an amino acid polymer that’s been modified by intervention or natural means, for example glycosylation or lipidation, disulfide bond creation, glycosylation or acetylation or any other manipulation or modification such as conjugation with a labeling component or bioactive ingredient.

“As used herein the terms ‘antigen? or ?immunogen? They are often used interchangeably to mean a substance, usually a protein, that is capable of inducing an immunological response in a subject. This term can also be used to describe proteins that are immunologically activated in the sense that they can elicit an immune response from the humoral or cellular type against a protein.

It should be noted that proteins and nucleic acid encoding them might differ from those shown and described in this document. The invention allows for deletions, additions and truncations of sequences as long as they function according to the invention’s methods. Substitutions within the same family of amino acids will be considered conservative. The four main families of amino acids are: (1) acidic?aspartate, glutamate, (2) basic?lysine and arginine; (2) non-polar?alanine and valine; (3) nonpolar?alanine and valine; (4) uncharged?polar?glycines, asparagine and glutamine; and (5) serine threonine and tyrosine. Sometimes, aromatic amino acids are phenylalanine and tryptophan. It is predictable that an individual replacement of leucine with valine or isoleucine, or vice-versa; an aspartate or glutamate with an aspartate or vice versa, a threonine or serine with an amino acid; or a similar conservative substitution of an amino acid with an amino acid structurally related to it, will not have a significant effect on biological activity. The invention covers proteins with substantially the same amino acids sequences as those illustrated and described, but which have minor amino acid substitutions that don’t substantially alter the immunogenicity of a protein.

“As used herein, the terms ‘nucleotide sequencings?” ?nucleic acids sequences? Deoxyribonucleic Acid (DNA) and ribonucleic Acid (RNA) sequences are also known as messenger RNA (mRNA), DNA/RNA combinations, and synthetic nucleic acids. You can have a single-stranded nucleic acid or a partially or fully double-stranded nucleic acids (duplex). You can choose to have heteroduplex or homoduplex duplex nucleic acid.

“Transgene” is the term used herein. can also be used to mean?recombinant? nucleotide sequences which may be derived from any one of the nucleotide sequencing encoding the proteins described in the present invention. “Recombinant” is a term that refers to a nucleotide sequence that has been modified. A nucleotide sequence that has been altered by man. It is a nucleotide sequence that does not exist in nature or is linked with another nucleotide sequencing or found in a different arrangement in the natural world. It is known that manipulating a gene by man can be done. It is understood that manipulation means manipulating by artificial means such as codon optimization, restriction enzymes and machines. A CMV vector that encodes heterologous antigens is, by definition, a recombinant CMV Vector.

Codon optimization can be done to nucleotide sequences, such as codons that are optimized for human use. Any viral or bacterial sequence can be altered. Many viruses, such as HIV and other lentiviruses use a lot of rare codons. By altering these codons so that they correspond to the codons used in the subject, it is possible to increase heterologous antigen expression, as described by Andre et.al., J. Virol. 72:1497-1503, 1998.”

“Nucleotide sequences that encode functionally and/or antagonically equivalent variants, derivatives, and corresponding CMV vectors are possible. Functionally equivalent variants, derivatives and fragments have the potential to retain antigenic activities. Changes in DNA sequences that don’t alter the encoded amino acids sequence or those that result in conservative substitutes of amino acids residues can be made. However, substitution of amino amino acid residues with amino acid analogs is possible if they do not have a significant impact on the properties of the encoded polypeptide. Conservative amino acid substitutions are glycine/alanine; valine/isoleucine/leucine; asparagine/glutamine; aspartic acid/glutamic acid; serine/threonine/methionine; lysine/arginine; and phenylalanine/tyrosine/tryptophan. One embodiment has variants that contain at minimum 50%, 55%, 60%, and at most 75%.

“Sequence homology or sequence identity is determined by comparing sequences aligned in such a way as to maximize overlap and identify while minimising sequence gaps. A variety of mathematical algorithms can be used to determine sequence identity. The algorithm of Karlin & Altschul (Proc.) is a non-limiting example of a mathematical algorithm that can be used to compare two sequences. Natl. Acad. Sci. USA 1990; 87, 2264-2268. Modified as in Karlin & Altschul. Proc. Natl. Acad. Sci. USA 1993; 90: 5873-5877.

“Another mathematical algorithm that is used to compare sequences is the one of Myers & Miller. CABIOS 1988, 4: 11-17. This algorithm is included in the ALIGN program (version 2.0), which is part the GCG sequence alignment package. A PAM120 weight residue table can be used to compare amino acid sequences. There is a 12 and 4 gap penalties, respectively, when using the ALIGN program. Another useful algorithm to identify regions of alignment and sequence similarity is the FASTA algorithm, as described by Pearson & Lipman in Proc. Natl. Acad. Sci. USA 1988; 85, 2444-2448

The WU-BLAST version 2.0 software is a preferred option for use according the invention. Download WU-BLAST 2.0 executable programs on several UNIX platforms. This program is based upon WU-BLAST 1.4. It can also be downloaded from the public domain NCBI BLAST version 1.4. Natl. Acad. Sci. Sci.

“The invention’s various recombinant sequences, antibodies and/or an antigens are created using standard recombinant and cloning methods. These techniques are well-known to those skilled in the art. For example, see?Molecular Cloning? A Laboratory Manual? second edition (Sambrook and al. 1989).”

“The nucleotide sequences described in the present invention can be used to insert into?vectors. The term “vector” is used interchangeably with the term “vector”. The term “vector” is widely used by skilled artists. As such, the term “vector” herein will be used. It is used in accordance with its meaning for those skilled in the art. The term “vector” is an example of this. “Vector” is a term that is used commonly by those skilled in art to describe a vehicle that permits or facilitates the transfer nucleic acids molecules from one environment or allows or facilitates manipulation of a nucleic Acid molecule.

The present invention allows the use of any vector that permits expression of the viruses. The viruses of the invention can be used in vitro, such as by using cell-free expression systems and/or cultured cells that have been grown in vitro to generate the HIV-antigens or antibodies. These may then be used for various purposes, including the production of proteinaceous vaccinations. Any vector that permits expression of the virus in vitro or in cultured cells can be used for such applications.

“The heterologous antibodies of the invention must be expressed if the heterologous sequence’s protein coding sequence should be?operable linked? to regulate or nucleic acids control sequences that direct transcription/translation of the protein. A coding sequence and a promoter or nucleic acid control sequence are referred to as ‘operably linked’. When they are covalently linked in a manner that places the expression, transcription, and/or translation from the coding sequence under control of the nucleic acids control sequence. What is the ‘nucleic acids control sequence? Any nucleic acids element that controls the expression of a sequence or code sequence can be considered. The term “promoter” is used herein. The term?promoter? will be used herein to describe a group transcriptional control modules clustered around RNA polymerase II’s initiation site. These modules, when operationally linked with the protein coding sequences described in the invention, lead to the expression the encoded protein. A constitutive or inducible promoter can control the expression of the transgenes according to the invention. This induces transcription when the promoter is exposed to a particular stimulus such as antibiotics like tetracycline or hormones like ecdysone or heavy metals. A promoter may also be specific for a particular type of cell, tissue, or organ. There are many suitable enhancers and promoters that can be used to express the transgenes. You can find suitable enhancers and promoters in the Eukaryotic Promoter Database, EPDB.

“In one instance, the epitope refers to an SIV epitope. One of ordinary skill in the art will know that any reference to HIV in the specification applies to SIV. The SIV epitope in an advantageous embodiment is a protein fragment according to the present invention. However, the invention may also include additional SIV antigens and epitopes. The SIV epitope, which is an SIV-antigen, includes but not limited, to the U.S. Pat. SIV antigens. Nos. Nos.

“The vectors according to the present invention should be chosen so that they contain a suitable gene regulation region, such as a enhancer or promoter, so that the antigens can be expressed.”

When the goal is to express the antigens of an invention in vivo in a subject (e.g. to generate an immune response to an HIV-1 antigen or protective immunity against HIV-1), expression vectors that can be used in vivo and are suitable for that subject should be chosen. In some embodiments, it might be desirable to express the antigens and antibodies of the invention in a laboratory animal for preclinical testing of the HIV-1 vaccines and immunogenic compositions. It may be possible to express the antigens in human subjects in other embodiments. This is for clinical trials or actual clinical use. It is possible to use any vector that is suitable for such purposes. The skilled artisan can easily choose a suitable vector. It may be preferable that vectors used in in vivo applications be attenuated so as to prevent amplifying in the subject. If plasmid vectors were used, it would be preferable that they do not have an origin of replication that functions within the subject. This will increase safety for in-vivo use. If viral vectors are used, preferably they are attenuated or replication-defective in the subject, again, so as to enhance safety for in vivo use in the subject.”

“Viral vectors are preferred in the preferred embodiments. Advantageously, the vector can be a CMV Vector, which is lacking at most the glycoprotein UUL128 or CMV Vector lacking at minimum the glycoprotein UUL130. Every CMV vector also contains the glycoprotein Ul131.

The CMV vectors disclosed can be administered in vivo. This is possible, for example, when the goal is to induce an immunogenic response. In some cases, it might be desirable to use the CMV vectors in laboratory animals, such as rhesus monkeys, for pre-clinical testing and development of vaccines using RhCMV. Other embodiments will allow the use of the CMV vectors in humans for clinical trials or clinical use of immunogenic compositions with HCMV.

“The CMV vectors disclosed are used in such in vivo applications as a component of an immunogenic mixture further comprising a pharmaceutically accepted carrier. The immunogenic compositions described in the invention can be used to induce an immune response against heterologous antigens, including pathogen-specific antigens. They may also be used as components of a prophylactic, therapeutic, or diagnostic vaccine against HIV-1. The invention’s vectors and nucleic acid are especially useful in providing genetic vaccines. The invention provides vaccines that deliver the nucleic acid encoding antigens to a subject such as a person to incite an immune response.

“The immunogenic compositions can include additional substances such as buffering agents or wetting agents to increase the effectiveness of vaccines (Remington?s Pharmaceutical Sciences, 18th Edition, Mack Publishing Company). 1980).”

“Immunogenic compositions can be used to deliver the CMV vectors at a desired location of action and then release them at a controlled rate. The art has many methods for preparing controlled-release formulations. Controlled release formulations can be made by using polymers that absorb or complex the immunogen. A controlled-release formulation can be prepared using appropriate macromolecules (for example, polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine sulfate) known to provide the desired controlled release characteristics or release profile. A controlled-release formulation can also be controlled by the incorporation of active ingredients in particles of polymeric materials such as polyesters, polyamino acid, hydrogels or polylactic acid, copolymers, ethylene vinylacetate copolymers, and copolymers. Alternatively, instead of incorporating these active ingredients into polymeric particles, it is possible to entrap these materials into microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacrylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. These techniques are described in New Trends and Developments in Vaccines by Voller et al. (eds. (eds.

“Those skilled in the art can easily determine the appropriate dosage of CMV vectors in immunogenic compositions. The route of administration and size of the subject can affect the dosage of CMV vectors. The art of measuring the immune response of subjects, such as laboratory animals, can help determine the appropriate doses. This can be done using standard immunological techniques and then adjusting the dosages accordingly. These techniques include, but are not limited, chromium release, tetramer binding, and IFN-? ELISPOT and IL-2 ELISPOT tests, intracellular cytokine (IFN-) assays and other immunological detection methods assays are all available. Ed Harlow and David Lane.

The immunogenic compositions may be administered by any method, including intramuscular, intravenous and mucosal. These techniques are well-known to those who have mastered the art. You can also use intramuscular injections, subcutaneous injections, or intradermal injections to deliver the drugs. Delivery does not have to be restricted to injection methods.

“Immunization regimens or schedules are well-known for animals, including humans. They can be easily determined for each subject and their immunogenic composition. The immunogens may be administered to the subject one or more times. It is preferable that there is a time limit between administrations of the immunogenic mixture. Although the interval will vary for each subject, it is usually between 10 days and several weeks. It can be 2, 4, 6, or 8 weeks. The interval for humans is usually between 2 and 6 weeks. The interval can be extended in a particularly beneficial embodiment of the invention. It is typically between 2 and 6 weeks.

“The immunization regimens usually have between 1 and 6 administrations of immunogenic composition. However, it is possible to have only one, two, or even four. Inducing an immune response may also involve the administration of an adjuvant. Sometimes, booster vaccination can be administered at intervals of 5-10 years, or biannually, to supplement the initial protocol.

The present methods include a variety prime-boost regimens such as DNA prime-Adenovirus booster regimens. These methods include one or more priming vaccines, followed by one or several boosting vaccinations. You can vary the composition of the actual immunogenic composition, such as the type and amount of immunogenic composition (e.g. containing protein or expression vector), and also the route and formulation of the immunogens. An expression vector used in the priming or boosting steps can be either the same type or a different one (e.g. DNA, bacterial, or viral). A prime-boost program provides two immunizations four weeks apart followed by two booster immunizations 4 and 8 weeks later. One skilled in the art will also know that the invention includes many combinations and permutations of viral, bacterial, and DNA expression vectors that provide priming or boosting regimens. If the viral vectors contain US2-11, or any of the genes encoded within the US2-11 region, they can be used repeatedly to express different antigens from different pathogens.

“A specific embodiment provides methods for inducing an immune reaction against a pathogen within a subject by administering immunogenic compositions one or more times. The epitopes are sufficiently expressed to induce a specific immune response. These immunizations may be repeated at intervals of at most 2, 4, or 6 weeks (or longer) according to a desired immunization regimen.

The immunogenic compositions can be administered by themselves, or co-administered or sequentially administered with other antigens (e.g., with?other?). Multivalent or ‘cocktail? immunological, antigenic, vaccine or therapeutic compositions are available. The invention may also include combinations of compositions and methods for using them. The ingredients and methods (sequential and co-administration), as well as dosages, can be determined by taking into account such factors as age, gender, weight, species, and condition of the subject and the route of administration.

“When administered in combination, antigens may be administered simultaneously or at different times. This is part of an overall immunization regimen, such as a prime-boost regimen, or any other immunization protocol.

“Although this invention has been described in great detail, it is important to understand that many modifications, substitutions, and alterations are possible without departing from its spirit and scope as set forth in the appended claims.”

“EXAMPLES”

“The following are examples of disclosed methods. Those skilled in the art will be able to see that there are variations of these and other methods disclosed. This disclosure is intended to show that it is possible to use the method without any undue experimentation.

“Example 1: Immunization with RhCMV vectors and a deletion of UL128 or UL130 results in an immune response characterized by a wide variety of CD8+ T cell epitopes against an SIV antigen.”

“Epitope targeting profiles for SIVgag specific CD8+ T cells responses elicited from RhCMV/gag Vectors derived RhCMV 68-1 strain. These vectors, which are devoid of active UL128 or UL130 (?UL128-135) but contain an active UL131 (Hansen S G et. al., J Virol 77. 6620 (2003); incorporated herein by reference) were compared with those elicites such as well as well as well as well as well as well as well as well as well as well as well as well as well as well as well as ed vectors. Flow cytometric intracellular staining was used for individual CD8+ T cells responses to each of the 125 consecutive 15mer-peptides (with an 11-amino acid overlap). This covered the entire SIVgag proteins. Twenty-nine rhesus monkeys (RM) were used. Fourteen were vaccinated using the?UL128-13 RhCMV/gag virus. Fourteen were vaccinated using electroporated DNA/gag+interleukin (IL-12). Three animals were vaccinated using adenovirus (Ad5/gag) and three others with vaccinia virus(MVA/gag). Five additional animals had been previously infected with SIV (SIVmac239) and were treated with spontaneous viral control.

“Peripheral blood CD8+T cells from?UL128-13 RhCMV/gag vector vaccined RM showed an average response to 46 of the 125 15mer sIVgag peptides. This corresponded with an average of 35 distinct epitopes. (FIG. 1A). SIV-infected controllers and RM vaccinated using electroporated DNA/gag+IL-12, Ad5/gag, and MVA/gag each responded to an average 10-19 peptides. This corresponds to an average 9-15 distinct epitopes. The range of responses from the?UL128-13 RhCMV/gag vector-vaccinated RM was such that many of these SIVgag 15mer peptides were reacted to by CD8+ cells in all or most of the 14 outbred animals (FIG. 1A).”

“To determine if this finding is indicative of promiscuous recognition a single common epitope? T cell recognition of?hotspots? The response to a series truncated propeptides was analysed. These truncated peptides corresponded with 7 of 15mers that were recognized in 3 RM per reaction. These peptides were used to identify core epitopes within each RM (FIG. 1B).”

Two distinct responses were observed with the truncated proteomes. Type 1 is the first. This pattern is characterized by a drop in response frequencies and loss of an essential amino residue. These truncations often resulted in a 9mer epitope (e.g. Gag259-267 or Gag276-284), and Gag482-490. The second type of response, Type 2, is a pattern where response frequencies slowly decline as the optimal sequence is truncated. These truncations often resulted in a 12mer-core epitope (Gag41-52, Gag211-222, Gag290-301 and Gag495-506). These truncation responses patterns and core peptides were identical in each RM. In all cases, core peptides showed superior stimulation (higher frequency response) than the parent 15mer (FIG. 1C).”

These data strongly suggest that many SIVgag epitopes that CD8+ T cell receptors in?UL128/130 RhCMV/gag vector vaccined RM target are specific determinants that can be recognized by MHC haplotypes of different types. A detectable CD8+ response to the core (optimal peptide) for five of these truncated15mers was observed in 100% of 42 RhCMV/gag-vaccinated RM. Responses to six other peptides, two optimal peptides, and four 15mers, were also found in >60% (FIG. 1D). These epitopes were not recognized by CD8+ cells in conventionally SIV infected RM. These CD8+ T cells that are stimulated by?UL128-13 RhCMV/gag vector are three times as wide as those that are infected with SIVgag-specific CD8+ cell responses. They are also unique because they are often targeted at’supertopes’.

“Example 2” Type 1 CD8+ responses are MHC-1 Restricted, Type 2-CD8+ responses are MHC-2 Restricted

MHC-I-restricted epitopes typically have 8-10 amino acid lengths and contain position-specific amino acids which engage binding pockets (anchor residues), so that they can fit in a closed end. The MHC-1 binding groove (Rammensee G et al., Ann Rev Immunol 11 213 (1993); incorporated herein) has characteristics that are consistent with the Type 1 pattern. The Type 2 truncation is more typical for MHC II-restricted epitopes. They are usually longer, have a >12mer core and are more tolerant to length heterogeneity (Southwood S et al., J Immunol 160 (1998) and Chelvanayagam GL, Hum Immunol 58 (1997), both of which are incorporated herein). It was suggested that CD8+ T cells that recognize Type 2 SIVgag epitopes within the RhCMV/gag-vaccinated RM may be MHC-II-restricted. This suggests that class II-restricted CD8 T cells responses might be unusual. However, such responses were previously reported in mice (Mizuochi T et Al, J Exp Med 168.437 (1988); Suzuki E et Al, J Immunol 153.4496 (1994); Matechak E et A, Immunity 4, 337 (1996), Shimizu T and Takeda T, Eur J Immunol 27,500 (1997); Rist M et, Blood 102, 11244 (2002); and J ets (1997), Eur J eta eta, 21.

“To determine if?UL128-135, RhCMVgag might be eliciting an MHCII-restricted CD8+ cell response to gag, we tested the ability of?blocking? Monoclonal antibodies (mAbs), specific for MHC-128 and MHC-1II, as well as the invariant-chain-derived, MHC-2II-specific binding peptide CLIP were tested to block Type 1 and Type 2 epitopes-specific CD8+ T cells responses in RM immunized by?UL130 RhCMVgag. 2A). These reagents inhibited the 5 universal supertope-specific CD8+ cell responses. This corresponded exactly to the Type 1 and 2 truncation patterns, with T cells recognizing the three Type 2 epitopes Gag211-222-301, Gag290-306 and Gag495-506 being blocked by anti?MHCII and CLIP but not anti?MHCII. The reverse was true for the 2 Type 1 epitopes Gag276-284 and Gag482-490

“The epitope-specific responses shown in FIG. 2A in relation to MHC-1 vs MHC-2 blockade (FIG. 2B, FIG. 2C). 2C).

“To confirm that the MHC-II-blocked CD8+ T cell responses were MHC-II-restricted?defined as the epitope in question being recognized in the context of MHC-II?and to investigate the basis of the promiscuity of these responses across MHC-disparate RM, cell lines expressing single rhesus MHC-II allomorphs were constructed. Four RhCMV/gag-vaccinated RM were used to express the MHC-II alleles. They had characterized SIVgag epitope recognition profiles. Flow cytometric ICS tests showed that pulsing the MHCII allomorph transfectants but not the parent MHCII negative line with individual peptides resulted a robust CD8+ T-cell stimulation of only those responses classed as MHCII-associated (FIG. These responses can be blocked by anti-MHCII mAbs or CLIP peptide but not anti-MHCII mAbs. Importantly, MHC-II Allomorphs often presented multiple peptides. Individual peptides were also frequently presented by multiple MHC?II allomorphs (FIG. 3A). Individual allomorphs can present multiple gag peptides, which helps to explain the range of MHC-II-restricted reactions. Multiple MHCII allomorphs can present individual peptides. This suggests that all RM expressing at most one MHCII allomorph are likely to explain the widespread recognition of these peptides across RhCMV/gag vector elicited CD8+ cells.

“As previously reported for MHCII-restricted CD4+ cell responses (Corradin A and Lanzaveccia, Int Rev Immunl 7, 139 (1991); incorporated herein), MHCII-restricted SIVgag specific CD8+ T cells elicited with RhCMV/gag Vectors can respond to the peptide epitope within the context of peptide binding MHCII allomorphs not expressed by the donor (FIGS). 3A and 3B, respectively, indicate that these T cells recognize the bound protein either as a single molecule or in combination with other non-polymorphic structures of the MHC-II molecules.

“Example 3?” Phenotype and Function of the?UL128-133/SIV Vector-Elicited T Cell Responses to CD8+”

“The epitope-specificity of SIV-specific CD8-specific T cells generated and maintained with RhCMV/SIV vector vaccine raises questions about their functional potential, particularly the unusual MHC-II-restricted population that dominates these responses. This is because supertope-specific CD8T cell responses are not due to high peptide levels used in standard ICS assays. Responses to optimal peptides (both Type 1 and 2) can be demonstrated at peptide dilutions greater than 1:105 (FIG. 4A). Second, supertope-specific Type 1 and 2 responses appear immediately following vaccination (FIG. 4B and coordinately distribute throughout the body in the same pattern as previously reported for RhCMV/SIV vector vaccined RM (Hansen, S G et. al., Nature 473/523 (2011); incorporated herein by reference (FIGS. 4C and 4D. Third, as previously reported in RhCMV-specific CD8+ and RhCMV/SIV vector elicited SIV?specific T cells (Hansen S. G. et al., Nat Med 15, 293 (2008); incorporated herein), both Type 1 supertope-specific and Type 2 T cells exhibit an identical phenotype that is indicative of effector-memory T cell differentiation (CCR7, CD28?). This effector-memory profile is consistent with the identical polyfunctional profile (high TNF, IFN, and MIP-1). Production, high CD107 externalization (degranulation), and low IL-2 production. 4E and 4F. These data indicate that CD8+ T cells in vaccinated RM receive the same in vivo exposures to Type 1 and 2 epitopes, since effector memory differentiation is believed to be Ag-driven.

“Example 4?” UL128 and/or UL130 Control Targeting CMV-Elicited CD8+ Cell Responses

“To identify candidate CMV gene associations with this unusual CD8+ immune reaction, it was first questioned whether CD8+ T cells responses to an endogenous CMV instant early (IE protein) also target unorthodox epitopes (in particular supertopes that are restricted by MHC?II). This was done by comparing RM infected naturally with wildtype RhCMV (colony circulating viruses) and RM vaccinated using the exemplary?UL128-13 deficient strain 68.1 RhCMV/SIV virus vector. It was not surprising that RM vaccinated using the?UL128-13 vector showed IE-specific CD8+ cell responses with identical targeting characteristics to the SIVgag specific CD8+ cell responses in the same RM. There were >30 distinct IE epitopes/RM. The majority of these responses were blocked by anti-MHCII and a few blocked with anti?MHCII.

“In striking contrast, however, the IE-specific CD8+ responses in naturally RhCMV infected RM were more targeted (?8 epitopes/RM) and did not show evidence of MHC-2 restriction or epitope promiscuity. These results are consistent with the conventional immunodominance hierarchies. These results likely explain why CMV-specific CD8+ cell responses that are not targeted by CMV have not been reported in naturally exposed CMV+ RM or humans (despite extensive analysis). They also implicate genetic differences in the mechanisms responsible for generating these unconventionally targeted CD8+ cell responses between wildtype RhCMV and the?UL128-130-deficient strain 68.1-based RhCMV vectors.

“To evaluate the role of these genes during the targeting of CD8+ cells during priming, a RhCMV/gag was created in which expressions of the UL128, UL130 orthologs were re-established (Lilja A E et al., Proc Natl Aca Sci USA 105, 19950 (2008). The question was then posed whether this “repair”? The epitope targeting profiles for vector-elicited gag specific CD8+ T cells responses were affected by the UL128 or UL130 ortholog expression. The UL128 and UL130-repaired RhCMV/gag vector-elicited SIVgag specific CD8+ T cells responses, which did not include recognition or any of the MHC?II supertopes, were more targeted than the response elicited from the unrepaired 68.1 strain vector. They were also entirely MHC?I-associated (FIGS). 5D, 5E and 5F.

“Example 5:CMV Vectors with Single Delitions of UL128 and UL130 Display CD8 Responds Characterized By Class II Restriction. CMV Vectors with one deletion of Ul131 are Incapable of Suprainfection.”

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