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  • C12N2710/10011—Adenoviridae
  • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
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  • C12N2740/16334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

• Adenoviruses are nonenveloped, icosahedral viruses that have been identified in several avian and mammalian hosts; Home et al., 1959 J. MoI. Biol. 1:84-86; Horwitz, 1990 In Virology, eds. B.N. Fields and D.M. Knipe, pps. 1679-1721.
• the first human adenoviruses (Ads) were isolated over four decades ago. Since then, over 100 distinct adenoviral serotypes have been isolated which infect various mammalian species, 51 of which are of human origin; Straus, 1984, In The Adenoviruses, ed. H. Ginsberg, pps.
• the human serotypes have been categorized into six subgenera (A-F) based on a number of biological, chemical, immunological and structural criteria which include hemagglutination properties of rat and rhesus monkey erythrocytes, DNA homology, restriction enzyme cleavage patterns, percentage G+C ⁇ ; content and oncogenicity; Straus, supra; Horwitz, supra.
• Adenoviruses are attractive targets for the delivery and expression of heterologous genes.
• Adenoviruses are able to infect a wide variety of cells (dividing and non-dividing), and are extremely efficient in introducing their DNA into infected host cells. Adenoviruses have not been found to be associated with severe human pathology in immuno-competent individuals. The viruses can be produced at high virus titers in large quantities.
• the adenovirus genome is very well characterized, consisting of a linear double-stranded DNA molecule of approximately 30,000-45,000 base pairs (Adenovirus serotype 5 ("Ad5"), for instance, is -36,000 base pairs). Furthermore, despite the existence of several distinct serotypes, there is some general conservation found amongst the various serotypes.
• adenoviruses as gene delivery vehicles
• the safety of adenoviruses as gene delivery vehicles is enhanced by rendering the viruses replication-defective through deletion/modification of the essential early-region 1 ("El") of the viral genomes, rendering the viruses devoid (or essentially devoid) of El activity and, thus, incapable of replication in the intended host/vaccinee; see, e.g., Brody et al, 1994 Ann N Y Acad ScL, 716:90-101.
• Deletion of adenoviral genes other than El e.g., in E2, E3 and/or E4
• E4 furthermore, creates adenoviral vectors with greater capacity for heterologous gene inclusion.
• two well-characterized adenovirus serotypes of subgroup C, serotypes 5 ("Ad5") and 2 form the basis of the most widely used gene delivery vectors.
• adenovectors relates to any cellular and humoral immune response elicited by the virus (Chirmule et al, 1999 Gene Titer. 6:1574-1583). Although an immune response associated with the initial administration of a vector may be advantageous (Zhang et al, 2001 MoI. Ther. 3:697-707), the generation of systemic levels of adenovirus-specific neutralizing antibody may cause poor transduction when the vectors are readministered (booster immunizations; Kass-Eisler et al, 1996 Gene Ther. 3: 154-162; Chirmule et al, 1999 J. Immunol. 163:448-455).
• the instant invention offers vector compositions and methods for evading such host immunity.
• the present invention relates to novel methods and compositions for improving the efficiency of adenoviral vectors in the delivery and expression of heterologous polypeptides.
• Adenoviral infection is relatively common in the general population, and a large percentage of people have neutralizing antibodies to the more prevalent adenoviral serotypes largely found in group C.
• Such pre ⁇ existing anti-adenoviral immunity can dampen or possibly abrogate the effectiveness of these viruses for the delivery and expression of heterologous proteins or antigens.
• the methods taught herein function to offset pre-existing immunity through the delivery and expression of heterologous polypeptides by a cocktail of at least two adenoviral serotypes.
• adenoviral serotypes Utilizing at least two adenoviral serotypes in accordance with the methods and compositions disclosed herein has been found to increase the effectiveness of adenoviral administration.
• Adenoviral vectors of utility in the elicitation of an immune response against Human Immunodeficiency Virus ("HTV") are also disclosed herein.
• Figure 1 illustrates the nucleotide sequence of a codon optimized version of full-length p55 gag (SEQ ID NO: 2).
• Figures 2A-1 through 2A-2 illustrate a codon optimized wt-pol sequence, wherein sequences encoding protease (PR) activity are deleted, leaving codon optimized "wild type" sequences which encode RT (reverse transcriptase and RNase H activity) and IN integrase activity (SEQ HD NO: 3).
• the open reading frame starts at an initiating Met residue at nucleotides 10-12 at ends at a termination codon at nucleotides 2560-2562.
• Figures 3A-1 through 3A-2 illustrate the open reading frame (SEQ ID NO: 4) of the wild type pol construct disclosed as SEQ ID NO: 3.
• Figures 4A-1 through 4A-3 illustrate the nucleotide (SEQ ID NO: 5) and amino acid sequence (SEQ ID NO: 6) of IA-PoI. Underlined codons and amino acids denote mutations, as listed in Table 1 herein.
• Figure 5 illustrates a codon optimized version of HTV-I jrfl nef (SEQ ID NO: 7).
• the open reading frame starts at an initiating methionine residue at nucleotides 12-14 and ends at a "TAA" stop codon at nucleotides 660-662.
• Figure 6 illustrates the open reading frame (SEQ ID NO: 8) of codon optimized HTV jrfl Nef.
• Figures 7A- 1 through 7A-2 illustrate a nucleotide sequence comparison between wild type nef (jrfl) and codon-optimized nef.
• the wild type nef gene from the jrfl isolate consists of 648 nucleotides capable of encoding a 216 amino acid polypeptide.
• WT wild type sequence (SEQ ID NO: 11); opt, codon-optimized sequence (contained within SEQ ID NO: 7).
• the Nef amino acid sequence is shown in one-letter code (SEQ ID NO: 8).
• Figure 8 illustrates nucleic acid (herein, "opt nef (G2A, LLAA)"; SEQ ID NO: 9) which encodes optimized HTV-I Nef wherein the open reading frame encodes for modifications at the amino terminal myristylation site (Gly-2 to Ala-2) and substitution of the Leu-174-Leu-175 dileucine motif to Ala-174-Ala-175.
• the open reading frame starts at an initiating methionine residue at nucleotides 12-14 and ends at a "TAA" stop codon at nucleotides 660-662.
• Figure 9 illustrates the open reading frame (SEQ TD NO: 10) of opt nef (G2A, LLAA).
• Figure 10 illustrates nucleic acid (herein, "opt nef (G2A)"; SEQ TD NO: 12) which encodes optimized HTV-I Nef wherein the open reading frame encodes for modifications at the amino terminal myristylation site (Gly-2 to Ala-2).
• the open reading frame starts at an initiating methionine residue at nucleotides 12-14 and ends at a "TAA" stop codon at nucleotides 660-662.
• Figure 11 illustrates the open reading frame (SEQ JD NO: 13) of opt nef (G2A).
• Figure 12 illustrates a schematic presentation of nef and nef derivatives. Amino acid residues involved in Nef derivatives are presented. Glycine 2 and Leucine 174 and 175 are the sites involved in myristylation and dileucine motif, respectively.
• Figure 13 illustrates, in tabular format, the seroprevalence of Adenovirus subtypes 5 and 6.
• Brazilian and Thai subjects were selected for high risk behavior for HTV infection.
• * Thai subjects were primarily high risk for HTV infection.
• Figure 14 illustrates, diagrammatically, the construction of the pre-adenovirus plasmid construct, MRKAd5Pol.
• Figure 15 illustrates, diagrammatically, the construction of the pre-adenovirus plasmid construct, MRKAd5Nef.
• Figure 16 illustrates the homologous recombination protocol utilized to recover pMRKAd ⁇ El-.
• Figure 17 illustrates MRKAd5gagnef, a modification of a prototype Group C Adenovirus serotype 5 vector in which the El region (nucleotides 451-3510) is deleted and replaced by nef and gag expression cassettes.
• Figures 18 A-I through 18A- 12 illustrate a nucleic acid sequence (SEQ ID NO: 16) for MRKAd5gagnef.
• Figure 19 illustrates key steps involved in the construction of adenovirus vector MRKAd5gagnef.
• Figure 20 illustrates MRKAd ⁇ gagnef, a modification of a prototype Group C Adenovirus serotype 6 vector in which the El region (nucleotides 451-3507) was deleted and replaced by nef and gag expression cassettes.
• Figures 21A-1 through 21A-12 illustrate a nucleic acid sequence (SEQ ID NO: 17) for MRKAd ⁇ gagnef.
• Figure 22 illustrates key steps involved in the construction of adenovirus vector MRKAd ⁇ gagnef.
• Figure 23 illustrates MRKAd5gagpol, a modification of a prototype Group C Adenovirus serotype 5 vector in which the El region (nucleotides 451-3510) is deleted and replaced by a gagpol fusion expression cassette.
• Figures 24A- 1 through 24A- 11 illustrate a nucleic acid sequence (SEQ ID NO: 18) for MRKAd5gagpol.
• Figure 25 illustrates key steps involved in the construction of adenovirus vector
• Figure 26 illustrates the PCR strategy for generating the gagpol fusion fragment for use in MRKAd5 gagpol.
• Figure 27 illustrates MRKAd5nef-gagpol, a modification of a prototype Group C Adenovirus serotype 5 vector in which the El region (nucleotides 451-3510) is deleted and replaced by nef and gagpol expression cassettes.
• Figures 28A-1 through 28A-12 illustrate a nucleic acid sequence (SEQ ID NO: 19) for MRKAd5nef-gagpol.
• Figure 29 illustrates key steps involved in the construction of adenovirus vector MRKAd5nef-gagpol.
• Figure 30 illustrates MRKAd5gagpolnef, a modification of a prototype Group C Adenovirus serotype 5 vector in which the El region (nucleotides 451-3510) is deleted and replaced by a gagpolnef expression cassette.
• Figures 3 IA-I through 31A-12 illustrate a nucleic acid sequence (SEQ ID NO: 20) for MRKAd5gagpolnef.
• Figure 32 illustrates key steps involved in the construction of adenovirus shuttle plasmid pMRKAd5gagpolnef.
• Figure 33 illustrates the PCR strategy for generating the polnef fusion fragment for use in MRKAd5gagpolnef.
• Figure 34 illustrates key steps involved in the construction of adenovirus vector MRKAd5gagpolnef.
• Figure 35 illustrates MRKAd ⁇ nef-gagpol, a modification of a prototype Group C
• Figures 36A-1 through 36A-12 illustrate a nucleic acid sequence (SEQ ID NO: 21) for MRKAd ⁇ nef-gagpol.
• Figure 37 illustrates key steps involved in the construction of adenovirus vector
• Figure 38 illustrates MRKAd ⁇ gagpolnef, a modification of a prototype Group C
• FIGS 39A-1 through 39A-11 illustrate a nucleic acid sequence (SEQ ID NO: 22) for
• Figure 40 illustrates key steps involved in the construction of adenovirus vector MRKAd ⁇ gagpolnef.
• Figure 41 illustrates, in tabular format, the levels of Nef-specific T cells during the course of immunization. Values reflect the mock-subtracted numbers of IFN- ⁇ secreting cells per million PBMC; wk, week. The bold numbers (the final row of each group) are the cohort geometric means in SFC/10 ⁇ 6 PBMC.
• Figure 42 illustrates, in tabular format, the effect of pre-existing Ad5-specific immunity on the efficacy of MRKAd5gag and a cocktail of MRKAd5gag +MRKAd6gag.
• the first two cohorts have Ad5-specific neutralization titers averaging 1300-1400 prior to immunization with the gag- expressing vectors.
• the third cohort had no detectable pre-existing neutralization titers. Shown are the SFC/106 PBMC values for each animal at week 4 and week 8 against the entire gag peptide pool and mock control. In bold are the cohort geometric means for the T cell responses.
• Figure 43 illustrates, in tabular format, the levels of Gag, Pol, and Nef-specific T cells in rhesus macaques immunized with lOlO vp/vector of one of the following vaccines: (1) MRKAd5gag + MRKAd5pol + MRKAd5nef; (2) MRKAd5hCMVnefmCMVgag + MRKAd5pol; (3) MRKAd5hCMVnefMCMVgagpol; and (4) MRKAd5hCMVgagpolnef.
• Cytokine secretion was induced using entire nef, gag, and pol peptide pools consisting of 15-aa peptides with 11-aa overlaps. Shown are the mock-corrected SFC/106 PBMC values for each animal at week 4 and week 8. In bold are the cohort geometric means for the T cell responses to each of the antigens.
• Figure 44 illustrates, in tabular format, the levels of Gag, Pol, and Nef-specific T cells in rhesus macaques immunized with I ⁇ 8 vp/vector of one of the following vaccines: (1) MRKAd5gag + MRKAd5pol + MRKAd5nef; (2) MRKAd5hCMVnefmCMVgag + MRKAd5pol; (3) MRKAd5hCMVnefmCMVgagpol; and (4) MRKAd5hCMVgagpolnef.
• Cytokine secretion was induced using entire nef, gag, and pol peptide pools consisting of 15-aa peptides with 11-aa overlaps. Shown are the mock-corrected SFC/1()6 PBMC values for each animal at week 4 and week 8. In bold are the cohort geometric means for the T cell responses to each of the antigens.
• Figure 45 illustrates, in tabular format, the levels of Gag, Pol, and Nef-specific T cells in rhesus macaques immunized with IOIO vp /vector of one of the following vaccines: (1) MRKAd5nefgagpol; (2) MRKAd ⁇ nefgagpol; (3) MRKAd5nefgagpol + MRKAd ⁇ nefgagpol. Cytokine secretion was induced using entire nef, gag and pol peptide pools consisting of 15-aa peptides with 11-aa overlaps. Shown are the mock-corrected SFC/106 PBMC values for each animal at week 4 and week 8. In bold are the cohort geometric means for the T cell responses to each of the antigens.
• Figure 46 illustrates, in tabular format, the levels of Gag, Pol, and Nef-specific T cells in rhesus macaques immunized with IO ⁇ vp /vector of one of the following vaccines: (1) MRKAd5nefgagpol; (2) MRKAd ⁇ nefgagpol; (3) MRKAd5nefgagpol + MRKAd ⁇ nefgagpol. Cytokine secretion was induced using entire nef, gag and pol peptide pools consisting of 15-aa peptides with 11-aa overlaps. Shown are the mock-corrected SFC/ I ⁇ 6 PBMC values for each animal at week 4 and week 8. In bold are the cohort geometric means for the T cell responses to each of the antigens.
• Applicants disclose herein novel methods and compositions for circumventing pre ⁇ existing anti-adenoviral immunity through administration of desired nucleic acid encoding a polypeptide(s) of interest via at least two adenoviral serotypes. This method is based on results of experiments conducted by Applicants employing serotypes of high homology and same group classification, contemporaneously, in the delivery and expression of nucleic acid of interest, and the favorable comparison of such delivery methodology to single serotype administrations utilizing the individual serotypes of the contemporaneous administration.
• a nucleic acid of interest by at least two adenoviral serotypes proved effective in both evading pre-existing host immunity and effectuating the delivery and expression of a polypeptide of interest.
• the expression effected was sufficient to elicit a host immune response to the expressed polypeptide that was comparable to that effectuated by single serotype administration where pre-existing immunity did not present a challenge.
• Pre-existing immunity did not have any apparent detrimental effect on the induced immunity.
• pre-existing immunity had a measurable impact on single serotype administration in situations where the serotype utilized was that to which pre-existing immunity was directed towards.
• the cellular immune response was found to be comparable to that of the individual serotype administration that was not challenged by pre-existing immunity.
• Applicants submit that the disclosed methods and vector compositions should improve the breadth of patient coverage in gene therapy and/or vaccination protocols by overcoming potential pre-existing immunity to single serotype delivery. Consequently, the disclosed methods and compositions form a viable prospect for mass administration in the face of pre-existing immunity, even to the more prevalent (group C) adenoviral serotypes.
• the present invention therefore, relates to methods for effecting the delivery and expression of heterologous nucleic acid encoding a polypeptide(s) of interest, which comprises contemporaneously administering purified replication-defective adenovirus particles of at least two different serotypes, wherein said replication-defective adenovirus particles comprise heterologous nucleic acid encoding at least one common polypeptide.
• the polypeptide can be any protein or antigen which one desires to have expressed in a particular cell, tissue, or subject of interest.
• Administration can be either within the same composition or in separate formulations administered contemporaneously; "contemporaneous" as defined herein meaning within the same period of time.
• contemporaneous administration refers to the administration of viral particles of alternative serotypes either simultaneously (whether in the same or separate formulations) or with some period of time between the administrations of the two or more different serotypes.
• This period of time can be of any duration, generally extending from simultaneous administration to a period of eighteen ("18") weeks between the administrations.
• the period of time between the administrations does not exceed a period of more than 18 weeks. More preferably, the period of time between administrations is significantly less than 18 weeks.
• the period of time between administrations is, in an increasing order of preference, less than four weeks, less than two weeks, less than one week, less than two days, less than one day, less than one hour, within five minutes (“simultaneous" administration).
• the result sought by contemporaneous administration is not that of a "prime-boost" effect but rather the effect of a single administration (albeit alternative administrations can be present), whether that administration be in the form of a prime (or primes employing the at least two serotypes), in the form of a boost (employing the at least two serotypes), or involving prime and boost administrations (the administrations of which independently both comprise the at least two serotypes).
• the present invention contemplates as well the contemporaneous administration of at least two adenoviral serotypes encoding at least one common polypeptide in a sole administration not dependent on a prime/boost regimen.
• the present invention also relates to compositions comprising the at least two adenoviral serotypes; said at least two adenoviral serotypes comprising heterologous nucleic acid encoding at least one common polypeptide.
• the methods in accordance with the present invention utilize (and compositions in accordance with the present invention comprise) purified replication-defective adenovirus particles of at least two different serotypes.
• adenoviral serotypes including, but not limited to, (1) the numerous serotypes of subgenera A-F discussed above, (2) unclassified adenovirus serotypes, (3) non-human serotypes (including but not limited to primate adenoviruses (see, e.g., Fitzgerald et al., 2003 J. Immunol 170(3)1416-1422; Xiang et al, 2002 J. Virol 76(6):2667-2675)), and equivalents, modifications, or derivatives of the foregoing.
• Adenoviruses can readily be obtained from the American Type Culture Collection ("ATCC”) or other publicly available/private source; and adenoviral sequences can be discerned from both the published literature and widely accessible public databases, where not obtained elsewhere.
• ATCC American Type Culture Collection
• the specific combination of serotypes suitable for use in the methods and compositions disclosed herein is limitless. There are numerous means by which one can choose a candidate combination of serotypes.
• One means by which to evaluate a candidate pairing of serotypes is to evaluate the seroprevalence of the vectors in combination (i.e., determine whether the population tends to be more/less/equally infected by all of the serotypes of the combination).
• the effective neutralizing antisera titer to the combination of serotype components is lower than that exhibited to an individual serotype (particularly to a serotype(s) of real interest) or, in the alternative, the percentage of individuals with serotype-specific neutralizing antisera titers to all the serotype components is less than that with titers to an individual serotype tested (again, particularly to the serotype(s) of real interest).
• the effective neutralizing antisera titer against a candidate composition (Le., the combination of serotype components) is the lower of the titers tested since that component of the vector will therefore be more potent.
• ranges can, but need not be, established as a qualitative reference for the potency of a determined serum towards specific serotypes (for example, ranges used herein for Ad5 were as follows: very low or undetectable [ ⁇ 18], low [18-200], medium [201-1000], and high [>1000]).
• adenovirus (Ad) serotypes several methods are available for determining type-specific antibodies to adenovirus (Ad) serotypes.
• Assay formats can be used such as, for example, end point dilution assays, or any available assays designed to evaluate gene expression.
• the basic principle behind such assays is to ascertain the specificity/existence of any preexisting antisera in the subject population.
• serum neutralization studies were utilized to evaluate the preexisting antisera of the candidate population; see Example 1.
• Serum neutralization assays generally involve incubating serum (from a candidate(s)) along with virus of the serotype of interest and cells to ascertain whether the serum contains antibodies specific for the virus sufficient to inhibit infection of the cells. Infection can be detected by a number of methods, the most frequently utilized being cell viability or transgene expression; Sprangers et al., supra.
• serotypes can be utilized with one or more that is a bit more prevalent to support the administration in the event that neutralizing antisera to the prevalent adenovirus poses a threat/challenge.
• rare serotypes can be administered contemporaneously.
• two or more relatively prevalent serotypes can be administered contemporaneously, particularly where the effective neutralizing antisera titer to the combination of serotype components is lower than that exhibited to an individual serotype (particularly to a serotype(s) of real interest) or, in the alternative, the percentage of individuals with serotype-specific neutralizing antisera titers to the combination of serotype components is less than that with titers to an individual serotype tested (again, particularly to the serotype(s) of real interest).
• the present invention encompasses and is exemplified herein by contemporaneous administration of adenovirus serotypes 5 and 6, both encoding at least one common polypeptide of interest.
• Adenovirus serotypes 5 and 6 are well known in the art (American Type Culture Collection (“ATCC”) Deposit Nos. VR-5 and VR-6, respectively, and sequences therefore have been published; see Chroboczek et al, 1992 J. Virol. 186:280, and PCT/US02/32512, published April 17, 2003, respectively).
• ATCC American Type Culture Collection
• pre-existing immunity did not have any apparent detrimental impact on their contemporaneous delivery.
• pre-existing immunity had a measurable impact on administration using one of the serotypes for which pre-existing immunity was present.
• the cocktail was, furthermore, effectively able to express sufficient amounts of the polypeptide to elicit a cellular immune response which was comparable to that of the individual serotype of the cocktail that was not effected by pre-existing immunity.
• Another embodiment of the present invention involves the combination/contemporaneous administration of human serotypes of adenovirus with serotypes that naturally infect other species.
• this could entail administering, contemporaneously, a human adenovirus and an adenovirus that naturally infects primates, including but not limited to chimpanzees.
• adenoviruses of alternative and distinct serotype e.g., the various serotypes found in subgenera A-F discussed above; including but not limited to those on deposit with widely accessible public depositories such as the American Type Culture
• any combination of these adenoviral serotypes is suitable for use in the present invention, provided that neutralizing antisera does not present a hindrance to administration of a desired combination of serotypes.
• this can be determined very readily by one of skill in the pertinent art from published literature concerning the relative prevalence of the various serotypes in specific populations, from actual experiments conducted, or from the various assays discussed above which are available to identify the existence of/quantify immunity to the serotype/classification group of interest.
• Adenoviral serotypes administered via the methods and compositions of the present invention should be replication-impaired in the intended host; unless the replication thereof in the intended host is determined not to pose a safety issue.
• the vectors are at least partially deleted/mutated in El such that any resultant virus is devoid (or essentially devoid) of El activity, rendering the vector incapable of replication in the intended host.
• the El region is completely deleted or inactivated.
• Specific embodiments of the present invention employ adenoviral vectors as described in PCT/US01/28861, published March 21, 2002.
• Said vectors are at least partially deleted in El and comprise several adenoviral packaging repeats (Le., the El deletion does not start until approximately base pairs 450-458, with base pair numbers assigned corresponding to a wildtype Ad5 sequence).
• the adenoviruses may contain additional deletions in E3, and other early regions, albeit in certain situations where E2 and/or E4 is deleted, E2 and/or E4 complementing cell lines may be required to generate recombinant, replication-defective adenoviral vectors.
• Vectors devoid of adenoviral protein- coding regions (“gutted vectors") are also feasible for use herein. Such vectors typically require the presence of helper virus for the propagation and development thereof.
• Adenoviral vectors can be constructed using well known techniques, such as those reviewed in Graham & Prevec, 1991 In Methods in Molecular Biology: Gene Transfer and Expression Protocols, (Ed. Murray, EJ.), p. 109; and Hitt et al, 1997 "Human Adenovirus Vectors for Gene Transfer into Mammalian Cells" Advances in Pharmacology 40: 137-206.
• Example 2 details the construction of several adenoviral vector constructs suitable for use herein.
• El -complementing cell lines used for the propagation and rescue of recombinant adenovirus should provide elements essential for the viruses to replicate, whether the elements are encoded in the cell's genetic material or provided in trans.
• the El- complementing cell line and the vector not contain overlapping elements which could enable homologous recombination between the nucleic acid of the vector and the nucleic acid of the cell line potentially leading to replication competent virus (or replication competent adenovirus "RCA").
• propagation cells are human cells derived from the retina or kidney, although any cell line capable of expressing the appropriate El and any other critical deleted region(s) can be utilized to generate adenovirus suitable for use in the methods of the present invention.
• Embryonal cells such as amniocytes have been shown to be particularly suited for the generation of El complementing cell lines.
• PER.C6® ECACC deposit number 96022940
• PER.C6® cell lines are described in WO 97/00326 (published January 3, 1997) and issued U.S. Patent No. 6,033,908.
• PER.C6® is a primary human retinoblast cell line transduced with an El gene segment that complements the production of replication deficient (FG) adenovirus, but is designed to prevent generation of replication competent adenovirus by homologous recombination. 293 cells are described in Graham et ah, 1977 J. Gen. Virol. 36:59-72.
• a cell line expressing an El region which is complementary to the El region deleted in the virus being propagated can be utilized.
• a cell line expressing regions of El and E4 derived from the same serotype can be employed; see, e.g., U.S. Patent No. 6,270,996.
• Another alternative would be to propagate non-group C adenovirus in available El -expressing cell lines (e.g., PER.C6®, A549 or 293). This latter method involves the incorporation of a critical E4 region into the adenovirus to be propagated.
• the critical E4 region is native to a virus of the same or highly similar serotype as that of the El gene product(s) (particularly the ElB 55K region) of the complementing cell line, and comprises typically, at a minimum, E4 open reading frame 6 ("ORF6")); see, PCT/US2003/026145, published March 4, 2004.
• ORF6 E4 open reading frame 6
• One of skill in the art can readily appreciate and carry out numerous other methods suitable for the production of recombinant, replication-defective adenoviruses suitable for use in the methods of the present invention. Following viral production in whatever means employed, viruses may be purified, formulated and stored prior to host administration.
• compositions described herein are well suited to effectuate the expression of heterologous polypeptides, especially in situations where pre-existing immunity prevents administration or readministration of at least one of the adenoviral serotypes employed.
• specific embodiments of the present invention comprise methods for effecting the delivery and expression of heterologous nucleic acid encoding a polypeptide(s) of interest, which comprises contemporaneously administering purified replication-defective adenovirus particles of at least two different serotypes, wherein said replication-defective adenovirus particles comprise heterologous nucleic acid encoding at least one common polypeptide.
• compositions comprising purified replication-defective adenovirus particles of at least two different serotypes, wherein said replication-defective adenovirus particles comprise heterologous nucleic acid encoding at least one common polypeptide.
• the expressed nucleic acid can be DNA and/or RNA, and can be double or single stranded.
• the nucleic acid can be inserted in an El parallel (transcribed 5' to 3' relative to the vector backbone) or anti-parallel (transcribed 3' to 5' relative to the vector backbone) orientation.
• the nucleic acid can be codon-optimized for expression in the desired host (e.g., a mammalian host).
• the heterologous nucleic acid can be in the form of an expression cassette.
• a gene expression cassette can contain (a) nucleic acid encoding a protein or antigen of interest; (b) a heterologous promoter operatively linked to the nucleic acid encoding the protein/antigen; and (c) a transcription termination signal.
• the heterologous promoter is recognized by a eukaryotic RNA polymerase.
• a promoter suitable for use in the present invention is the immediate early human cytomegalovirus promoter (Chapman et al., 1991 Nucl. Acids Res. 19:3979-3986).
• promoters that can be used in the present invention are the strong immunoglobulin promoter, the EFl alpha promoter, the murine CMV promoter, the Rous Sarcoma Virus promoter, the SV40 early/late promoters and the beta actin promoter, albeit those of skill in the art can appreciate that any promoter capable of effecting expression of the heterologous nucleic acid in the intended host can be used in accordance with the methods of the present invention.
• the promoter may comprise a regulatable sequence such as the Tet operator sequence. Sequences such as these that offer the potential for regulation of transcription and expression are useful in circumstances where repression/modulation of gene transcription is sought.
• the adenoviral gene expression cassette may comprise a transcription termination sequence; specific embodiments of which are the bovine growth hormone termination/polyadenylation signal (bGHpA) or the short synthetic polyA signal (SPA) of 50 nucleotides in length defined as follows: AATAAAAGATCTTTATTTTCATTAGATCTGTGTGTTGGTTTTTTGTGTG (SEQ ID NO: 1).
• a leader or signal peptide may also be incorporated into the transgene.
• the leader is derived from the tissue-specific plasminogen activator protein, tPA.
• Heterologous nucleic acids of interest typically encode immunogenic and/or therapeutic proteins.
• Preferred therapeutic proteins are those which elicit some measurable therapeutic benefit in the individual host upon administration.
• Preferred immunogenic proteins are those proteins which are capable of eliciting a protective and/or beneficial immune response in an individual.
• a specific embodiment of the instant invention, illustrated herein, is the delivery of nucleic acid encoding representative immunogenic proteins (HTV Gag, Nef and/or Pol) by the methods and compositions disclosed, albeit any gene encoding a therapeutic or immunogenic protein can be used in accordance with the methods disclosed herein and form important embodiments hereof.
• the methods and compositions disclosed in the present invention do not hinge upon any specific heterologous nucleic acid.
• the methods and compositions of the instant invention can be used to effectuate the delivery of any polypeptide whose presence/function brings about a desired effect in a given host, particularly a therapeutic/immunogenic effect useful in the treatment/alteration/modification of various conditions associated with, caused by, effected by (positively or negatively), exacerbated by, or modified by the presence or absence of a particular nucleic acid, protein, antigen, fragment, or activity associated with any of the foregoing.
• One aspect of the present invention as indicated above, relates to methods and compositions employing adenoviral vectors carrying heterologous nucleic acid encoding an HTV antigen(s)/protein(s).
• HTV Human Immunodeficiency Virus
• AIDS acquired human immune deficiency syndrome
• HTV is an RNA virus of the Retroviridae family and exhibits the 5'LTR-g ⁇ g-poZ-env-LTR 3' organization of all retroviruses.
• the integrated form of HTV, known as the provirus, is approximately 9.8 Kb in length.
• Each end of the viral genome contains flanking sequences known as long terminal repeats (LTRs).
• Heterologous nucleic acid encoding an HTV antigen/protein may be derived from any HIV strain, including but not limited to HIV-I and HIV-2, strains A, B, C, D, E, F, G, H, I, O, TUB, LAV, SF2, CM235, and US4; see, e.g., Myers et al, eds. "Human Retroviruses and AIDS: 1995 (Los Alamos National Laboratory, Los. Alamos NM 97545).
• Another HTV strain suitable for use in the methods disclosed herein is HTV-I strain CAM-I; Myers et al, eds. "Human Retroviruses and AIDS”: 1995, HA3- IIA19.
• HTV gene sequence(s) may be based on various clades Of HTV-I; specific examples of which are Clades A, B, and C. Sequences for genes of many HTV strains are publicly available from GenBank and primary, field isolates of HTV are available from the National Institute of Allergy and Infectious Diseases (NIAID) which has contracted with Quality Biological (Gaithersburg, MD) to make these strains available. Strains are also available from the World Health Organization (WHO), Geneva Switzerland.
• HTV genes encode at least nine proteins and are divided into three classes; the major structural proteins (Gag, Pol, and Env), the regulatory proteins (Tat and Rev); and the accessory proteins (Vpu, Vpr, Vif and Nef).
• the gag gene encodes a 55-kilodalton (kDa) precursor protein (p55) which is expressed from the unspliced viral mRNA and is proteolytically processed by the HTV protease, a product of the pol gene.
• the mature p55 protein products are pl7 (matrix), p24 (capsid), p9 (nucleocapsid) and p6.
• the pol gene encodes proteins necessary for virus replication - protease (Pro, PlO), reverse transcriptase (RT, P50), integrase (TN, p31) and RNAse H (RNAse, pl5) activities. These viral proteins are expressed as a Gag or Gag-Pol fusion protein which is generated by a ribosomal frame shift. The 55 kDa gag and 160 kDa gagpol precursor proteins are then proteolytically processed by the virally encoded protease into their mature products.
• the nef gene encodes an early accessory HTV protein (Nef) which has been shown to possess several activities such as down regulating CD4 expression, disturbing T-cell activation and stimulating HTV infectivity.
• the env gene encodes the viral envelope glycoprotein that is translated as a 160-kilodalton (kDa) precursor (gpl ⁇ O) and then cleaved by a cellular protease to yield the external 120-kDa envelope glycoprotein (gpl20) and the transmembrane 41-kDa envelope glycoprotein (gp41). Gpl20 and gp41 remain associated and are displayed on the viral particles and the surface of HTV-mfected cells.
• the tat gene encodes a long form and a short form of the Tat protein, a RNA binding protein which is a transcriptional transactivator essential for HTV replication.
• the rev gene encodes the 13 kDa Rev protein, a RNA binding protein.
• the Rev protein binds to a region of the viral RNA termed the Rev response element (RRE).
• RRE Rev response element
• the Rev protein promotes transfer of unspliced viral RNA from the nucleus to the cytoplasm.
• the Rev protein is required for HTV late gene expression and in turn, HTV replication.
• Nucleic acid encoding any HTV antigen may be utilized in the methods and compositions of the present invention (specific examples of which include but are not limited to the aforementioned genes, nucleic acid encoding active and/or immunogenic fragments thereof, and/or modifications/derivatives of any of the foregoing).
• the present invention contemplates as well the various codon-optimized forms of nucleic acid encoding HIV antigens, including codon-optimized HTV gag (including but by no means limited to p55 versions of codon-optimized full length ("EL") Gag and tPA-Gag fusion proteins), HTV pol, HTV nef, HTV env, HTV tat, HTV rev, and modifications/derivatives of immunological relevance.
• Embodiments exemplified herein employ nucleic acid encoding codon- optimized Nef antigens; codon-optimized p55 Gag antigens; and codon-optimized Pol antigens.
• Codon- optimized HIV-I gag genes are disclosed in PCT International Application PCT/USOO/18332, published January 11, 2001 (WO 01/02607). Codon-optimized HTV-I env genes are disclosed in PCT International Applications PCT/US97/02294 and PCT/US97/10517, published August 28, 1997 (WO 97/31115) and December 24, 1997 (WO 97/48370), respectively. Codon-optimized HIV-I pol genes are disclosed in U.S. Application Serial No. 09/745,221, filed December 21, 2000 and PCT International Application PCT/USOO/34724, also filed December 21, 2000. Codon-optimized HIV-I nef genes are disclosed in U.S. Application Serial No.
• Immunologically relevant or “antigenic” as defined herein means (1) with regard to a viral antigen, that the protein is capable, upon administration, of eliciting a measurable immune response within an individual sufficient to retard the propagation and/or spread of the virus and/or to reduce/contain viral load within the individual; or (2) with regards to a nucleotide sequence, that the sequence is capable of encoding for a protein capable of the above.
• a nucleotide sequence that the sequence is capable of encoding for a protein capable of the above.
• a codon-optimized gag gene that can be utilized in the methods and compositions of the present invention is that disclosed in PCT/USOO/18332, published January 11, 2001 (see Figure 1; SEQ ID NO: 2).
• the sequence is derived from HTV-I strain CAM-I and encodes full-length p55 gag.
• the gag gene of HtV-I strain CAM-I was selected as it closely resembles the consensus amino acid sequence for the clade B (North American/European) sequence (Los Alamos HTV database).
• the sequence was designed to incorporate human preferred ("humanized") codons in order to maximize in vivo mammalian expression (Lathe, 1985, J. MoI. Biol. 183:1-12).
• Open reading frames for various synthetic pol genes contemplated herein and disclosed in PCT/USOO/34724 comprise coding sequences for reverse transcriptase (or RT which consists of a polymerase and RNase H activity) and integrase (IN).
• the protein sequence is based on that of Hxb2r, a clonal isolate of ITJDB; this sequence has been shown to be closest to the consensus clade B sequence with only 16 nonidentical residues out of 848 (Korber, et al., 1998, Human retroviruses and AIDS, Los Alamos National Laboratory, Los Alamos, New Mexico).
• a particular embodiment of this portion of the invention comprises methods and compositions comprising codon optimized nucleotide sequences which encode wt-pol constructs (herein, "wt-pol” or “wt-pol (codon optimized))" wherein sequences encoding the protease (PR) activity are deleted, leaving codon optimized "wild type” sequences which encode RT (reverse transcriptase and RNase H activity) and DSf integrase activity.
• wt-pol or wt-pol (codon optimized)
• the open reading frame of the wild type pol construct contains 850 amino acids.
• Alternative specific embodiments relate to methods and compositions utilizing adenoviral vector constructs which comprise codon optimized HIV-I pol wherein, in addition to deletion of the portion of the wild type sequence encoding the protease activity, a combination of active site residue mutations are introduced which are deleterious to HTV-I pol (RT-RH-TN) activity of the expressed protein.
• the present invention relates to methods and compositions employing an adenoviral construct comprising HTV-I pol wherein the construct is devoid of sequences encoding any PR activity, as well as containing a mutation(s) which at least partially, and preferably substantially, abolishes RT, RNase and/or TN activity.
• HTV-I pol mutant which is part and parcel of an adenoviral vector construct of use in the methods and compositions disclosed herein may include but is not limited to a mutated nucleic acid molecule comprising at least one nucleotide substitution which results in a point mutation which effectively alters an active site within the RT, RNase and/or TN regions of the expressed protein, resulting in at least substantially decreased enzymatic activity for the RT, RNase H and/or TN functions of HTV-I Pol.
• a HTV-I DNA pol construct contains a mutation (or mutations) within the Pol coding region which effectively abolishes RT, RNase H and TN activity.
• a specific HTV-I pol-containing construct contains at least one point mutation which alters the active site of the RT, RNase H and TN domains of Pol, such that each activity is at least substantially abolished.
• Such a HTV-I Pol mutant will most likely comprise at least one point mutation in or around each catalytic domain responsible for RT, RNase H and TN activity, respectfully.
• encoding nucleic acid comprises nine codon substitution mutations which result in an inactivated Pol protein (IA Pol: SEQ ID NO: 6, Figures 4A-1 to 4A-3) which has no PR, RT, RNase or IN activity, wherein three such point mutations reside within each of the RT, RNase and IN catalytic domains. Therefore, one exemplification contemplated employs an adenoviral vector construct which comprises, in an appropriate fashion, a nucleic acid molecule which encodes IA-PoI, which contains all nine mutations as shown below in Table 1. An additional amino acid residue for substitution is Asp551, localized within the RNase domain of Pol.
• any combination of the mutations disclosed herein may be suitable and therefore may be utilized in the vectors, methods and compositions of the present invention. While addition and deletion mutations are contemplated and within the scope of the invention, the preferred mutation is a point mutation resulting in a substitution of the wild type amino acid with an alternative amino acid residue.
• SEQ ID NO: 5 discloses the nucleotide sequence which codes for a codon optimized pol in addition to the nine mutations shown in Table 1 and referred to herein as "IApol".
• adenoviral constructs comprising IA-pol for use in the vectors, methods and compositions of the present invention
• inactivation of the enzymatic functions was achieved by replacing a total of nine active site residues from the enzyme subunits with alanine side-chains.
• Table 1 all residues that comprise the catalytic triad of the polymerase, namely Aspll2, Aspl87, and Aspl88, were substituted with alanine (Ala) residues (Larder, et al., Nature 1987, 327: 716-717; Larder, et al., 1989, Proc. Natl. Acad. Sci. 1989, 86: 4803-4807).
• any combination of the mutations disclosed above may be suitable and therefore be utilized in adenoviral HIV constructs, methods and compositions of the present invention, either when administered alone, with other heterologous genes, in a combined modality regime and/or as part of a prime-boost regimen.
• adenoviral vector constructs comprising codon optimized HIV-I Pol comprising a eukaryotic trafficking signal peptide or a leader peptide such as is found in highly expressed mammalian proteins such as immunoglobulin leader peptides. Any functional leader peptide may be tested for efficacy.
• the respective DNA may be modified by known recombinant DNA methodology.
• a nucleotide sequence which encodes a leader/signal peptide may be inserted into a DNA vector housing the open reading frame for the Pol protein of interest.
• the end result is a vector construct which comprises vector components for effective gene expression in conjunction with nucleotide sequences which encode a modified HIV-I Pol protein of interest, including but not limited to a HTV-I Pol protein which contains a leader peptide.
• codon usage for mammalian optimization is preferred: Met (ATG), GIy (GGC), Lys (AAG), Trp (TGG), Ser (TCC), Arg (AGG), VaI (GTG), Pro (CCC), Thr (ACC), GIu (GAG); Leu (CTG), His (CAC), He (ATC), Asn (AAC), Cys (TGC), Ala (GCC), GIn (CAG), Phe (TTC) and Tyr (TAC).
• the present invention also relates to vectors, methods and compositions comprising/utilizing non-codon optimized or partially codon optimized versions of nucleic acid molecules and associated recombinant adenoviral HTV constructs which encode the various wild type and modified forms of the HTV proteins.
• codon optimization of these constructs constitutes a preferred embodiment of this invention. Codon optimized versions of HTV-I nef and HIV-I nef modifications of use in specific embodiments of the present invention can be found in U.S. Application Serial No.
• nef and nef modifications relate to nucleic acid encoding HIV-I Nef from the HTV-I jrfl isolate wherein the codons are optimized for expression in a mammalian system such as a human.
• a DNA molecule which encodes this protein is disclosed herein as SEQ TD NO: 7 ( Figure 5), while the expressed open reading frame is disclosed herein as SEQ TD NO: 8.
• Figures 7A-1 to 7A-2 illustrate a comparison of wild type vs. codon optimized nucleotides comprising the open reading frame of HTV-nef.
• the open reading frame for SEQ TD NO: 7 comprises an initiating methionine residue at nucleotides 12-14 and a "TAA" stop codon from nucleotides 660-662.
• the open reading frame of SEQ TD NO: 7 provides for a 216 amino acid HTV-I Nef protein expressed through utilization of a codon optimized DNA vaccine vector.
• the 216 amino acid HTV-I Nef (jrfl) protein is disclosed herein as SEQ TD NO: 8; Figure 6.
• Another modified nef optimized coding region relates to a nucleic acid molecule encoding optimized HTV-I Nef wherein the open reading frame codes for modifications at the amino terminal myristylation site (Gly-2 to Ala-2) and substitution of the Leu-174-Leu-175 dileucine motif to Ala-174-Ala-175, herein described as opt nef (G2A, LLAA).
• a DNA molecule which encodes this protein is disclosed herein as SEQ TD NO: 9, while the expressed open reading frame is disclosed herein as SEQ TD NO: 10.
• Yet another modified nef optimized coding region relates to a nucleic acid molecule encoding optimized HTV-I Nef wherein the open reading frame codes for modifications at the amino terminal myristylation site (Gly-2 to Ala-2), herein described as opt nef (G2A).
• G2A opt nef
• a DNA molecule which encodes this protein is disclosed herein as SEQ TD NO: 12, while the expressed open reading frame is disclosed herein as SEQ TD NO: 13.
• HTV-I Nef is a 216 amino acid cytosolic protein which associates with the inner surface of the host cell plasma membrane through myristylation of Gly-2 (Franchini et al., 1986, Virology 155: 593-599). While not all possible Nef functions have been elucidated, it has become clear that correct trafficking of Nef to the inner plasma membrane promotes viral replication by altering the host intracellular environment to facilitate the early phase of the HTV-I life cycle and by increasing the infectivity of progeny viral particles.
• the methods, vectors and compositions of the present invention employ an adenoviral vector(s) comprising codon-optimized nef sequence modified to contain a nucleotide sequence which encodes a heterologous leader peptide such that the amino terminal region of the expressed protein will contain the leader peptide.
• adenoviral vector(s) comprising codon-optimized nef sequence modified to contain a nucleotide sequence which encodes a heterologous leader peptide such that the amino terminal region of the expressed protein will contain the leader peptide.
• Sorting decisions for most proteins need to be made only once as they traverse their biosynthetic pathways since their final destination, the cellular location at which they perform their function, becomes their permanent residence. Maintenance of intracellular integrity depends in part on the selective sorting and accurate transport of proteins to their correct destinations. Defined sequence motifs exist in proteins which can act as 'address labels'. A number of sorting signals have been found associated with the cytoplasmic domains of membrane proteins. An effective induction of CTL responses often required sustained, high level endogenous expression of an antigen.
• mutants lacking myristylation, by glycine-to-alanine change, change of the dileucine motif and/or by substitution with a leader sequence will be functionally defective, and therefore will have improved safety profile compared to wild-type Nef for use as an HIV-I vaccine component.
• the nucleotide sequence is modified to include a leader or signal peptide of interest. This may be accomplished by known recombinant DNA methodology.
• insertion of a nucleotide sequence may be inserted into a DNA vector housing the open reading frame for the Nef protein of interest. It has been shown that myristylation of Gly-2 in conjunction with a dileucine motif in the carboxy region of the protein is essential for Nef-induced down regulation of CD4 (Aiken et al., 1994, Cell 76: 853-864) via endocytosis.
• Nef expression promotes down regulation of MHCI (Schwartz et al., 1996, Nature Medicine 2(3): 338-342) via endocytosis.
• the present invention contemplates adenoviral vectors which comprise sequence encoding a modified Nef protein altered in trafficking and/or functional properties and the use thereof in the methods and compositions of the present invention.
• modifications introduced into the adenoviral vector HTV constructs of the present invention include but are not limited to additions, deletions or substitutions to the nef open reading frame which results in the expression of a modified Nef protein which includes an amino terminal leader peptide, modification or deletion of the amino terminal myristylation site, and modification or deletion of the dileucine motif within the Nef protein and which alter function within the infected host cell.
• a recombinant adenoviral construct of use in accordance with the methods and compositions disclosed herein can comprise sequence encoding optimized HIV-I Nef with modifications at the amino terminal myristylation site (Gly-2 to Ala-2) and substitution of the Leu-174-Leu-175 dileucine motif to Ala-174-Ala-175.
• This open reading frame is herein described as opt nef (G2A,LLAA) and is disclosed as SEQ ID NO: 9, which comprises an initiating methionine residue at nucleotides 12-14 and a "TAA" stop codon from nucleotides 660-662.
• SEQ ID NO: 9 The nucleotide sequence of this codon optimized version of HIV-I jrfl nef gene with the above mentioned modifications is disclosed herein as SEQ ID NO: 9; Figure 8.
• the open reading frame of SEQ ID NO: 9 encodes Nef (G2AJLLAA), disclosed herein as SEQ ID NO: 10; Figure 9.
• Another recombinant adenoviral construct of use in accordance with the methods and compositions disclosed herein can comprise sequence encoding optimized HIV-I Nef with modifications at the amino terminal myristylation site (Gly-2 to Ala-2).
• This open reading frame is herein described as opt nef (G2A) and is disclosed as SEQ ID NO: 13, which comprises an initiating methionine residue at nucleotides 12-14 and a "TAA" stop codon from nucleotides 660-662.
• SEQ ID NO: 12 The nucleotide sequence of this codon optimized version of HIV-I jrfl nef gene with the above mentioned modification is disclosed herein as SEQ ID NO: 12; Figure 10.
• the open reading frame of SEQ ID NO: 12 encodes Nef (G2A), disclosed herein as SEQ ID NO: 13 ; Figure 11.
• Figure 12 shows a schematic presentation of nef and nef derivatives. Amino acid residues involved in Nef derivatives are presented. Glycine 2 and Leucine 174 and 175 are the sites involved in myristylation and dileucine motif, respectively.
• Adenoviral vectors of use in the methods and compositions of the present invention may comprise one or more HTV genes/encoding nucleic acid.
• the administration of at least one (preferably, at least two) recombinant adenoviral vector(s) comprising two or more HTV genes, their derivatives, or modifications are anticipated as well as exemplified herein.
• Two or more HTV genes can be expressed on at least one of the recombinant adenoviral vector constructs and/or two or more HIV genes can be expressed across two or more constructs.
• the present invention encompasses those situations where, while only one antigen is in common amongst at least two of the vectors of different serotype, the vectors may have additional HTV genes that (1) differ, (2) are the same, (3) while not in common with that vector, are in common with another vector utilized in the disclosed methods or compositions, or (4) are derived from the same common antigen.
• the present invention offers the possibility of using the methods and compositions of the present invention to evade/bypass host immunity and effectuate a multi-valent HTV gene administration, specific examples, but not limitations of which, include the administration of adenoviral vectors comprising nucleic acid sequence encoding (1) Gag and Nef polypeptides, (2) Gag and Pol polypeptides, (3) Pol and Nef polypeptides, and (4) Gag, Pol and Nef polypeptides.
• Open reading frames for the multiple genes/encoding nucleic acid can be operatively linked to distinct promoters and transcription termination sequences.
• the open reading frames may be operatively linked to a single promoter, with the open reading frames operatively linked by an internal ribosome entry sequence (IRES; as disclosed in WO 95/24485), or suitable alternative allowing for transcription of the multiple open reading frames to run off of a single promoter.
• the open reading frames may be fused together by stepwise PCR or suitable alternative methodology for fusing together two open reading frames.
• Various combined modality administration regimens suitable for use in the present invention are disclosed in PCT/USOl/28861, published March 21, 2002.
• Said vectors comprise nucleic acid encoding at least two antigens selected from the group consisting of gag, nef and/or pol antigens.
• the nucleic acid can be as disclosed herein or can be any modification, derivative or functional equivalent of same.
• the nucleic acid sequences are codon-optimized or partially codon-optimized.
• Specific embodiments of the present invention are such constructs which are di/tri-cistronic (i.e., the individual antigens are under the control of distinct promoters).
• adenoviral vectors comprising nucleic acid encoding (1) gag and nef; (2) gag and pol; and (3) gag, pol and nef.
• the adenoviral serotypes are of adenoviral serotype 5 or 6.
• the adenoviral vectors are deleted in El and E3 to accommodate the heterologous nucleic acid.
• the adenoviral vectors disclosed herein have the heterologous nucleic acid present in an El deletion of a region which corresponds to that of nucleotides 451-3510 of adenovirus serotype 5 or nucleotides 451-3507 of adenovirus serotype 6.
• the adenoviral vectors comprise the nucleic acid encoding the at least two antigens under the control of at least two promoters, one driving expression of nucleic acid encoding at least one of the antigens and at least one other driving the expression of nucleic acid encoding at least one other antigen.
• adenoviral vectors comprising nucleic acid encoding: (1) nef and gag under the control of two distinct promoters; (2) nef and gag under the control of the hCMV and mCMV promoters (see, e.g., Examples 2H and 21 and Figures 17 and 20); (3) gagpol (a fusion of coding sequences of gag and pol); (4) nef and gagpol; (5) nef and gagpol under the control of hCMV and mCMV promoters (see, e.g., Examples 2K and 2M and Figures 27 and 35); and (6) gagpolnef ( a fusion of coding sequence of gag, pol and nef).
• HTV-I Env protein e.g., gpl20
• nucleic acid encoding Env may be added to the constructs described herein, the constructs absent such nucleic acid have proven sufficient to elicit a significant immune response in treated subjects. It is well within the purview of one of skill in the art to arrive at and effectively utilize various fusion/multi-valent constructs.
• inventions of the present invention relate to the contemporaneous administration of more than one vector administered by the at least two serotypes.
• two or more serotypes both comprising nucleic acid A can be co-administered with two or more serotypes both comprising nucleic acid B.
• the properties of the instant administration strategies can be exploited to administer nucleic acid that one may want, for one reason or another, across more than one vector.
• contemporaneous administration of recombinant adenoviruses in accordance with the methods of the present invention may be the subject of a single administration or form part of a broader prime/boost-type administration regimen.
• Prime-boost regimens can employ different viruses (including but not limited to different viral serotypes and viruses of different origin), viral vector/protein combinations, and combinations of viral and polynucleotide administrations.
• an individual is first administered a priming dose of a protein/antigen/derivative/modification utilizing a certain vehicle (be that a viral vehicle, purified and/or recombinant protein, or encoding nucleic acid).
• Multiple primings typically 1-4, are usually employed, although more may be used.
• the priming dose(s) effectively primes the immune response so that, upon subsequent identification of the protein/antigen(s) in the circulating immune system, the immune response is capable of immediately recognizing and responding to the protein/antigen(s) within the host.
• the individual is administered a boosting dose of at least one of the previously delivered protein(s)/antigen(s), derivatives or modifications thereof (administered by viral vehicle/protein/nucleic acid).
• the length of time between priming and boost may typically vary from about four months to a year, albeit other time frames may be used as one of ordinary skill in the art will appreciate.
• the follow-up or boosting administration may also be repeated at selected time intervals.
• contemporaneous administration in accordance herewith can be employed for both the prime and boost administrations.
• a mixed modality prime and boost inoculation scheme should result in an enhanced immune response, specifically where there is pre-existing anti-vector immunity.
• Selection of the alternate administration vehicle (be it viral/nucleic acid/protein) to be employed in conjunction with the methods and compositions disclosed herein in a prime-boost administration regimen is not critical to the successful practice hereof. Any vehicle capable of delivering the antigen (or effectuating expression of the antigen) to sufficient levels such that a cellular and/or humoral-mediated response is elicited should be sufficient to prime or boost the presently disclosed administration.
• Suitable viral vehicles include but are not limited to distinct serotypes of adenovirus, including but not limited to adenovirus serotypes 6, 24, 34 and 35 (see, e.g., PCT/US02/32512, published April 17, 2003 (Ad6); PCT/US2003/026145, published March 4, 2004 (Ad24, Ad34); PCT/NLOO/00325, published November 23, 2000 (Ad35)).
• the adenoviral administration can be followed or preceded by a viral vehicle of diverse origin.
• examples of different viral vehicles include but are not limited to adeno-associated virus ("AAV"; see, e.g., Samulski et al, 1987 J. Virol. 61:3096-3101;
• Potential hosts/vaccinees/individuals that can be administered the recombinant adenoviral vectors of the present invention include but are not limited to primates and especially humans and non-human primates, and include any non-human mammal of commercial or domestic veterinary importance.
• compositions of adenoviral vectors whether of single or multiple serotype, including but not limited to vaccine compositions, administered in accordance with the methods and compositions of the present invention may be administered alone or in combination with other viral- or non-viral-based DNA/protein vaccines. They also may be administered as part of a broader treatment regimen.
• the present invention thus, encompasses those situations where the disclosed adenoviral cocktails are administered in conjunction with other therapies; including but not limited to other antimicrobial (e.g., antiviral, antibacterial) agent treatment therapies.
• a specific antimicrobial agent(s) selected is not critical to successful practice of the methods disclosed herein.
• the antimicrobial agent can, for example, be based on/derived from an antibody, a polynucleotide, a polypeptide, a peptide, or a small molecule. Any antimicrobial agent that effectively reduces microbial replication/spread/load within an individual is sufficient for the uses described herein. Antiviral agents antagonize the functioning/life cycle of a virus, and target a protein/function essential to the proper life cycle of the virus; an effect that can be readily determined by an in vivo or in vitro assay.
• Some representative antiviral agents which target specific viral proteins are protease inhibitors, reverse transcriptase inhibitors (including nucleoside analogs; non-nucleoside reverse transcriptase inhibitors; and nucleotide analogs), and integrase inhibitors.
• Protease inhibitors include, for example, indinavir/CRIXIVAN®; ritonavir/NORVIR®; saquinavir/FORTOVASE®; nelfinavir/VIRACEPT®; amprenavir/AGENERASE®; lopinavir and ritonavir/KALETRA®.
• Reverse transcriptase inhibitors include, for example, (1) nucleoside analogs, e.g.,zidovudine/RETROVIR® (AZT); didanosine/VIDEX® (ddl); zalcitabine/HTVID® (ddC); stavudine/ZERTT® (d4T); lamivudine/EPrVIR® (3TC); abacavir/ZIAGEN® (ABC); (2) non-nucleoside reverse transcriptase inhibitors, e.g., nevirapme/VIRAMUNE® (NVP); delavirdine/RESCRIPTOR® (DLV); efavirenz/SUSTIVA® (EFV); and (3) nucleotide analogs, e.g., tenofovir DF/VIREAD® (TDF).
• nucleoside analogs e.g.,zidovudine/RETROVIR® (AZT); didanosine/VIDEX® (d
• Integrase inhibitors include, for example, the molecules disclosed in U.S. Application Publication No. US2003/0055071, published March 20, 2003; and International Application WO 03/035077.
• the antiviral agents can target as well a function of the virus/viral proteins, such as, for instance the interaction of regulatory proteins tat or rev with the trans-activation response region ("TAR") or the rev-responsive element ("RRE"), respectively.
• An antiviral agent is, preferably, selected from the class of compounds consisting of: a protease inhibitor, an inhibitor of reverse transcriptase, and an integrase inhibitor.
• the antiviral agent administered to an individual is some combination of effective antiviral therapeutics such as that present in highly active anti-retroviral therapy ("HAART"), a term generally used in the art to refer to a cocktail of inhibitors of viral protease and reverse transcriptase.
• HAART highly active anti-retroviral therapy
• the present invention can be employed in conjunction with any pharmaceutical composition useful for the treatment of microbial infections.
• Antimicrobial agents are typically administered in their conventional dosage ranges and regimens as reported in the art, including the dosages described in the Physicians' Desk Reference, 54 th edition, Medical Economics Company, 2000.
• compositions comprising the recombinant viral vectors may contain physiologically acceptable components, such as buffer, normal saline or phosphate buffered saline, sucrose, other salts and polysorbate.
• the viral particles are formulated in A195 formulation buffer.
• the formulation has: 2.5-10 mM TRIS buffer, preferably about 5 mM TRIS buffer; 25-100 mM NaCl, preferably about 75 mM NaCl; 2.5-10% sucrose, preferably about 5% sucrose; 0.01 -2 mM MgCl 2 ; and 0.001%-0.01% polysorbate 80 (plant derived).
• the pH should range from about 7.0-9.0, preferably about 8.0.
• the formulation contains 5mM TRIS, 75 mM NaCl, 5% sucrose, ImM MgCl 2 , 0.005% polysorbate 80 at pH 8.0. This has a pH and divalent cation composition which is near the optimum for virus stability and minimizes the potential for adsorption of virus to glass surface. It does not cause tissue irritation upon intramuscular injection. It is preferably frozen until use.
• the amount of viral particles in the vaccine composition(s) to be introduced into a vaccine recipient will depend on the strength of the transcriptional and translational promoters used and on the immunogenicity of the expressed gene product(s).
• an immunologically or prophylactically effective dose of IxIO 7 to IxIO 12 particles and preferably about IxIO 10 to IxIO 11 particles per adenoviral vector is administered directly into muscle tissue.
• Subcutaneous injection, intradermal introduction, impression through the skin, and other modes of administration such as intraperitoneal, intravenous, or inhalation delivery are also contemplated.
• modes of administration can be employed to administer the different viruses of the methods and compositions taught herein.
• one serotype can feasibly be administered via one injection route and another serotype via another route and still maintain contemporaneous delivery.
• the total dose of adenoviral particles administered does not exceed 1 X I ⁇ l2.
• Administration of additional agents able to potentiate or broaden the immune response e.g., the various cytokines, interleukins
• concurrently with or subsequent to parenteral introduction of the viral vectors of this invention is appreciated herein as well and can be advantageous.
• the benefits of administration as described herein should be (1) a comparable or broader population of individuals successfully immunized/treated with recombinant adenoviral vectors, and (2) in situations of immunization, a lower transmission rate to (or occurrence rate in) previously uninfected individuals (i.e., prophylactic applications) and/or a reduction in/control of the levels of virus/bacteria/foreign agent within an infected individual (i.e., therapeutic applications).
• Serum samples were collected from HIV-infected patients from six countries - North America, Brazil, Thailand, Malawi, South Africa, and Cameroon. The samples were complement- inactivated at 56 0 C for 90 mins before use.
• FIG. 1 illustrates the nucleotide sequence of the exemplified optimized codon version of full-length p55 gag; SEQ H) NO: 2.
• the gag gene of HTV-I strain CAM-I was selected as it closely resembles the consensus amino acid sequence for the clade B (North American/European) sequence (Los Alamos HTV database).
• PVlJnsHTVgag is a plasmid comprising the CMV immediate-early (IE) promoter and intron A, a full-length codon-optimized HTV gag gene, a bovine growth hormone- derived polyadenylation and transcriptional termination sequence, and a minimal pUC backbone; see Montgomery et al. , 1993 DNA Cell Biol. 12:777-783, for a description of the plasmid backbone.
• IE immediate-early
• GMP grade pVUnsHTVgag was used as the starting material to amplify the hCMV promoter.
• the amplification was performed with primers suitably positioned to flank the hCMV promoter.
• a 5' primer was placed upstream of the Mscl site of the hCMV promoter and a 3' primer
• This ligation reaction resulted in the construction of a hCMV promoter (minus intron A) + bGHpA expression cassette within the original pVlJnsHTVgag vector backbone.
• This vector is designated pVUnsCMV(no intron).
• the FLgag gene was excised from pVlJnsHTVgag using BgITL digestion and the 1,526 bp gene was gel purified and cloned into ⁇ VUnsCMV(no intron) at the SgZII site. Colonies were screened using Smal restriction enzymes to identify clones that carried the FLgag gene in the correct orientation. This plasmid, designated pVUnsCMV(no intxon)-FLgag-bGHpA, was fully sequenced to confirm sequence integrity.
• Ad5 shuttle vector pdelElsplA; a vector comprising Ad5 sequences from base pairs 1-341 and 3524-5798, with a multiple cloning region between nucleotides 341 and 3524 of Ad5, included the following three manipulations carried out in sequential cloning steps as follows:
• the left ITR region was extended to include the Pad site at the junction between the vector backbone and the adenovirus left ITR sequences. This allowed for easier manipulations using the bacterial homologous recombination system.
• the packaging region was extended to include sequences of the wild-type (WT) adenovirus from 342 bp to 450 bp inclusive.
• An original adenovector pADHVE3 (comprising all Ad5 sequences except those nucleotides encompassing the El region) was reconstructed so that it would contain the modifications to the El region. This was accomplished by digesting the newly modified shuttle vector (MRKpdelEl shuttle) with Pad and BstZl 101 and isolating the 2,734 bp fragment which corresponds to the adenovirus sequence. This fragment was co-transformed with DNA from Clal linearized pAdHVE3 (E3+adenovector) into E. coli BJ5183 competent cells. At least two colonies from the transformation were selected and grown in TerrificTM broth for 6-8 hours until turbidity was reached. DNA was extracted from each cell pellet and then transformed into E.
• coli XLl competent cells One colony from the transformation was selected and grown for plasmid DNA purification. The plasmid was analyzed by restriction digestions to identify correct clones. The modified adenovector was designated MRKpAdHVE3 (E3+ plasmid). Virus from the new adenovector (MRKHVE3) as well as the old version were generated in the PER.C6 ® cell lines.
• the multiple cloning site of the original shuttle vector contained CIaI , Bamffl, Xho I, EcoRV, Hindm, Sal I, and BgI ⁇ sites.
• This MCS was replaced with a new MCS containing Not I, CIa I, EcoRV and Asc I sites.
• This new MCS has been transferred to the MRKpAdHVE3 pre-plasmid along with the modification made to the packaging region and pIX gene. 4. Construction of the new shuttle vector containing modified sag trans gene - " MRKpdelEl - CMV(no intron)-FLsas-bGHpA"
• the modified plasmid pVUnsCMV(no intron)-FLgag-bGHpA was digested with Mscl overnight and then digested with Sfil for 2 hours at 50 0 C.
• the DNA was then treated with Mungbean nuclease for 30 minutes at 30 0 C.
• the DNA mixture was desalted using the Qiaex II kit and then Klenow treated for 30 minutes at 37°C to fully blunt the ends of the transgene fragment.
• the 2,559 bp transgene fragment was then gel purified.
• the modified shuttle vector (MRKpdelEl shuttle) was linearized by digestion with EcoRV, treated with calf intestinal phosphatase and the resulting 6,479 bp fragment was then gel purified. The two purified fragments were then ligated together and several dozen clones were screened to check for insertion of the transgene within the shuttle vector. Diagnostic restriction digestion was performed to identify those clones carrying the transgene in the El parallel orientation.
• the shuttle vector containing the HTV-I gag transgene in the El parallel orientation, MRKpdelEl -CMV(no intron)-FLgag-bGHpA was digested with Pad.
• the reaction mixture was digested with Bs ⁇ l 1.
• the 5,291 bp fragment was purified by gel extraction.
• the MRKpAdHVE3 plasmid was digested with Clal overnight at 37 0 C and gel purified. About 100 ng of the 5,290 bp shuttle +transgene fragment and -100 ng of linearized MRKpAdHVE3 DNA were co-transformed into E. coli BJ5183 chemically competent cells.
• the pre-plasmid clone is designated MRKpAdHVE3+CMV(no intron)-FLgag-bGHpA and is 37,498 bp in size.
• MRKpAdHVE3+CMV(no intron)-FLgag-bGHpA is 37,498 bp in size.
• a nucleotide sequence for pMRKAd5HTV-lgag adenoviral vector and details of its construction are disclosed in PCT/US01/28861, published March 21, 2002.
• MRK Ad5 HIV- 1 gag contains the hCMV(no intron)-FLgag-bGHpA transgene inserted into the new E3+ adenovector backbone, MRKpAdHVE3, in the El parallel orientation.
• MRKpAdHVE3 new E3+ adenovector backbone
• the pre-plasmid MRKpAdHVE3+CMV(no intron)-FLgag-bGHpA was digested with Pad to release the vector backbone and 3.3 ⁇ g was transfected by the calcium phosphate method (Amersham Pharmacia Biotech.) in a 6 cm dish containing PER.C6 ® cells at ⁇ 60% confluence. Once CPE was reached (7-10 days), the culture was freeze/thawed three times and the cell debris pelleted. 1 ml of this cell lysate was used to infect into a 6 cm dish containing PER.C6 ® cells at 80-90% confluence. Once CPE was reached, the culture was freeze/thawed three times and the cell debris pelleted.
• the cell lysate was then used to infect a 15 cm dish containing PER.C6 ® cells at 80-90% confluence. This infection procedure was continued and expanded at passage 6.
• the virus was then extracted from the cell pellet by CsCl method. Two bandings were performed (3-gradient CsCl followed by a continuous CsCl gradient). Following the second banding, the virus was dialyzed in A105 buffer. Viral DNA was extracted using pronase treatment followed by phenol chloroform. The viral DNA was then digested with HincHB. and radioactively labeled with [33p]dATP. Following gel electrophoresis to separate the digestion products the gel was dried down on Whatman paper and then subjected to autoradiography. The digestion products were compared with the digestion products from the pre-plasmid (that had been digested with Pacl/HindJR prior to labeling). The expected sizes were observed, indicating that the virus had been successfully rescued.
• HTV Pol from HTV-I was constructed using codons frequently used in humans; see Korber et al., 1998 Human Retroviruses and AIDS, Los Alamos Nat'l Lab., Los Alamos, New Mexico; and Lathe, R., 1985 J. MoI. Biol. 183:1-12.
• the protein sequence is based on that of Hxb2r, a clonal isolate of JLJULB; this sequence has been shown to be closest to the consensus clade B sequence with only 16 nonidentical residues out of 848 (Korber et al, 1998 Human Retroviruses and AIDS, Los Alamos National Laboratory, Los Alamos, New Mexico).
• FIGS. 4A- 1 to 4A-3 illustrate the nucleotide sequence of an exemplified codon optimized version of HTV-I pol.
• the pol gene encodes optimized HTV-I Pol wherein the open reading frame of a recombinant adenoviral HTV vaccine encodes for nine codon substitution mutations which result in an inactivated Pol protein (IA Pol: SEQ JD NO: 6; Figures 4A-1 to 4A-3) which has no protease, reverse transcriptase, RNase or integrase activity, with three point mutations residing within each of the RT, RNase and In catalytic domains.
• IA Pol SEQ JD NO: 6; Figures 4A-1 to 4A-3
• adenoviral shuttle vector for the full- length inactivated HTV-I pol gene is as follows.
• MRKpdelEl(Pac/pIX/pack450)+CMVmin+BGHpA(str.) is a derivative of the shuttle vector used in the construction of the MRKAd5gag adenoviral pre-plasmid.
• the vector contains an expression cassette with the hCMV promoter (no intronA) and the bovine growth hormone polyadenylation signal.
• the expression unit has been inserted into the shuttle vector such that insertion of the gene of choice at a unique BgIK site will ensure the direction of transcription of the transgene will be Ad5 El parallel when inserted into the MRKpAd5(El-/E3+)Clal (or MRKpAdHVE3) pre-plasmid.
• the vector similar to the original shuttle vector contains the Pad site, extension to the packaging signal region, and extension to the pIX gene.
• the synthetic full-length codon-optimized HIV-I pol gene was isolated directly from the plasmid pVlJns-HTV-pol-inact(opt). Digestion of this plasmid with BgI II releases the pol gene intact (comprising a codon optimized IA pol sequence as disclosed in SEQ ID NO: 5).
• the pol fragment was gel purified and ligated into the MRKpdelEl(Pac/pIX/pack450)+CMVmin+BGHpA(str.) shuttle vector at the BgIE site.
• the clones were checked for the correct orientation of the gene by using restriction enzymes DrdSUNotl.
• a positive clone was isolated and named MRKpdel+hCMVmin+FL- pol+bGHpA(s).
• the genetic structure of this plasmid was verified by PCR, restriction enzyme and DNA sequencing.
• the pre-adenovirus plasmid was constructed as follows: Shuttle plasmid MRKpdel+hCMVmin+FL-pol+bGHpA(S) was digested with restriction enzymes Pad and BstllOl I (or its isoschizomer, BstZlOl I) and then co-transformed into E.
• pMRKAd5pol linearized (Clal digested) adenoviral backbone plasmid, MRKpAd(E l-/E3+)Clal.
• the resulting pre-plasmid originally named MRKpAd+hCMVmin+FL-pol+bGHpA(S)E3+ is now referred to as "pMRKAd5pol".
• the genetic structure of the resulting pMRKAd5pol was verified by PCR, restriction enzyme and DNA sequence analysis.
• the vectors were transformed into competent E. coli XL-I Blue for preparative production.
• the recovered plasmid was verified by restriction enzyme digestion and DNA sequence analysis, and by expression of the pol transgene in transient transfection cell culture.
• a nucleotide sequence for pMRKAd5HTV-lpol adenoviral vector and details of its construction are disclosed in PCT/USOl/28861, published March 21, 2002.
• PER.C6 ® adherent monolayer cell culture.
• 12 ⁇ g of pMRKAd5pol was digested with restriction enzyme Pad (New England Biolabs) and 3.3 ⁇ g was transfected per 6 cm dish of PER.C6 ® cells using the calcium phosphate co-precipitation technique (Cell Phect Transfection Kit, Amersham Pharmacia Biotech Inc.). Pad digestion releases the viral genome from plasmid sequences allowing viral replication to occur after entry into PER.C6 ® cells.
• Infected cells and media were harvested 6 -10 days post-transfection, after complete viral cytopathic effect (CPE) was observed. Infected cells and media were stored at ⁇ -6O 0 C.
• This pol containing recombinant adenovirus is referred to herein as "MRKAd5pol”.
• This recombinant adenovirus expresses an inactivated HTV-I Pol protein as shown in SEQ ID NO: 6.
• HTV Nef from HTV-I was constructed using codons frequently used in humans; see Korber et al., 1998 Human Retroviruses and AIDS, Los Alamos Nat'l Lab., Los Alamos, New Mexico; and Lathe, R., 1985 J. MoI. Biol. 183:1-12.
• Figure 8 illustrates the nucleotide sequence of an exemplified codon optimized version of HTV- 1 jrfl nef gene.
• the nef gene encodes optimized HTV-I Nef wherein the open reading frame of a recombinant adenoviral HTV vaccine encodes for modifications at the amino terminal myristylation site (Gly-2 to Ala-2) and substitution of the Leu-174-Leu-175 dileucine motif to Ala-174-Ala-175.
• the open reading frame is herein described as opt nef (G2A,LLAA), and is disclosed as SEQ ID NO: 10, which comprises an initiating methionine residue at nucleotides 12-14 and a "TAA" stop codon from nucleotides 660-662.
• Figure 10 illustrated the nucleotide sequence of an exemplified codon optimized version of HTV-I jrfl nef gene.
• the nef gene encodes optimized HIV-I Nef wherein the open reading frame of a recombinant adenoviral HTV vaccine encodes for modifications at the amino terminal myristylation site (Gly-2 to Ala-2).
• the open reading frame is herein described as opt nef (G2A) and is disclosed as SEQ ID NO: 12, which comprises an initiating methionine residue at nucleotides 12-14 and a "TAA" stop codon from nucleotides 660-662.
• G2A opt nef
• SEQ ID NO: 12 comprises an initiating methionine residue at nucleotides 12-14 and a "TAA" stop codon from nucleotides 660-662.
• the vector MRKpdelEl(Pac/pIX/pack450)+CMVmin+BGHpA(str.) is the shuttle vector used in the construction of the MRKAd5gag adenoviral pre-plasmid. It has been modified to contain the Pad site, extension to the packaging signal region, and extension to the pIX gene. It contains an expression cassette with the hCMV promoter (no intronA) and the bovine growth hormone polyadenylation signal.
• the expression unit has been inserted into the shuttle vector such that insertion of the gene of choice at a unique BgIl 1 site will ensure the direction of transcription of the transgene will be Ad5 El parallel when inserted into the MRKpAd5(El-/E3+)Clal pre-plasmid.
• the synthetic full-length codon-optimized HIV-I nef gene was isolated directly from the plasmid pVl Jns/nef (G2A,LLAA). Digestion of this plasmid with BgIl 1 releases the pol gene intact, which comprises the nucleotide sequence as disclosed in SEQ ID NO: 9.
• the nef fragment was gel purified and ligated into the MRKpdelEl+CMVmin+BGHpA(str.) shuttle vector at the BgIl 1 site.
• the clones were checked for correct orientation of the gene by using restriction enzyme Seal.
• a positive clone was isolated and named MRKpdelElhCMVminFL-nefBGHpA(s).
• the genetic structure of this plasmid was verified by PCR, restriction enzyme and DNA sequencing.
• the pre- adenovirus plasmid was constructed as follows.
• pMRKAd5nef MRKpdelElhCMVminFL-nefBGHpA(s) is now referred to as "pMRKAd5nef '.
• the genetic structure of the resulting pMRKAdSnef was verified by PCR, restriction enzyme and DNA sequence analysis.
• the vectors were transformed into competent E. coli XL-I Blue for preparative production.
• the recovered plasmid was verified by restriction enzyme digestion and DNA sequence analysis, and by expression of the nef transgene in transient transfection cell culture.
• a nucleotide sequence for pMRKAd5H3V-lnef adenoviral vector and details of its construction are disclosed in PCT/USOl/28861, published March 21, 2002. 2. Generation of research-grade recombinant adenovirus
• Infected cells and media were harvested 6 -10 days post-transfection, after complete viral cytopathic effect (CPE) was observed. Infected cells and media were stored at ⁇ -6O 0 C. This nef containing recombinant adenovirus is now referred to as "MRKAd5nef '.
• the general strategy used to recover a pMRKAd ⁇ El- bacterial plasmid is illustrated in Figure 16.
• cotransformation of BJ 5183 bacteria with purified wt Ad6 viral DNA and a second DNA fragment termed the Ad6 ITR cassette effectuates circularization of the viral genome by homologous recombination.
• the ITR cassette contains sequences from the right (bp 35460 to 35759) and left (bp 1 to 450 and bp 3508 to 3807) end of the Ad6 genome separated by plasmid sequences containing a bacterial origin of replication and an ampicillin resistance gene.
• pNEB193 a commonly used commercially available cloning plasmid (New England Biolabs cat# N305 IS) containing a bacterial origin of replication, an ampicillin resistance gene, and a multiple cloning site into which the PCR products are introduced
• pNEBAd6-3 the ITR cassette
• the ITR cassette contains a deletion of El sequences from Ad6 sequence from 451 to 3507.
• the Ad6 sequences in the ITR cassette provide regions of homology with the purified Ad6 viral DNA in which recombination can occur.
• pMRKAd6El- can then be used to generate first generation Ad6 vectors containing transgenes in El.
• a synthetic full-length codon-optimized HTV-I gag gene was inserted into a universal shuttle vector comprising adenovirus serotype 6 ("Ad6") sequences from bpl to bp450 and bp bp3508 to bp3807 (basepairs 451 to 3507 are deleted), a CMV promoter (minus Intron A) and bGHpA. Direction of transcription was El parallel.
• the synthetic full-length codon-optimized HTV-I gag gene was obtained from plasmid pVl Jns-HTV-FLgag-opt by BgIR digestion, gel purified and ligated into the BgIQ. restriction endonuclease site in the shuttle vector. The genetic structure of the resultant shuttle vector comprising full length gag was verified by PCR, restriction enzyme and DNA sequence analyses.
• the shuttle vector was digested with restriction enzymes Pad and Bstl 1071 and then co- transformed into E. coli strain BJ5183 with linearized (C/ ⁇ l-digested) adenoviral backbone plasmid, pAd6El-E3+.
• the genetic structure of the resulting pMRKAd ⁇ gag was verified by restriction enzyme and DNA sequence analysis.
• the vectors were transformed into competent E. coli XL-I Blue for large- scale production.
• the recovered plasmid was verified by restriction enzyme digestion and DNA sequence analysis, and by expression of the gag transgene in transient transfection cell culture.
• pMRKAd ⁇ gag contains Ad6 bps 1 to 450 and bps 3508 to 35759 (bp numbers refer correspond to that of an Ad6 sequence; see, e.g., PCT/US02/32512, published April 17, 2003).
• the viral ITRs are joined by plasmid sequences that contain the bacterial origin of replication and an ampicillin resistance gene.
• the pre-adenovirus plasmid pMRKAd ⁇ gag was rescued as infectious virions in PER.C6 ® adherent monolayer cell culture.
• 10 ⁇ g of pMRKAd ⁇ gag was digested with restriction enzyme Pad (New England
• PER.C6 ® cells transfected into a 6 cm dish of PER.C6 ® cells using the calcium phosphate co-precipitation technique (Cell Phect Transfection Kit, Amersham Pharmacia Biotech Inc.). Pad digestion releases the viral genome from plasmid sequences allowing viral replication to occur after entry into PER.C6 ® cells. Infected cells and media were harvested after complete viral cytopathic effect (CPE) was observed. The virus stock was amplified by multiple passages in PER.C6 ® cells. At the final passage virus was purified from the cell pellet by CsCl ultracentrifugation.
• CPE viral cytopathic effect
• the identity and purity of the purified virus was confirmed by restriction endonuclease analysis of purified viral DNA and by Gag ELISA of culture supernatants from virus infected mammalian cells grown in vitro.
• digested viral DNA was end-labeled with P 33 -dATP, size-fractionated by agarose gel electrophoresis, and visualized by autoradiography.
• a synthetic full-length codon-optimized HTV-I nef gene (opt nef G2A, LLAA) was inserted into a universal shuttle vector comprising adenovirus serotype 6 ("Ad6") sequences from bpl to bp450 and bp bp3508 to bp3807 (basepairs 451 to 3507 are deleted), a CMV promoter (minus Intron A) and bGHpA.
• Ad6 adenovirus serotype 6
• the synthetic full-length codon-optimized HTV- 1 nef gene was obtained from plasmid pVl Jns-HTV-FLnef-opt by BgIR digestion, gel purified and ligated into the .BgZII restriction endonuclease site in the shuttle vector.
• the genetic structure of the resultant shuttle vector comprising full length /ze/was verified by PCR, restriction enzyme and DNA sequence analyses.
• the shuttle vector was digested with restriction enzymes Pad and Bstl 1071 and then co- transformed into E. coli strain BJ5183 with linearized (C/ ⁇ l-digested) adenoviral backbone plasmid, pAd6El-E3+.
• the genetic structure of the resulting pMRKAd ⁇ nef was verified by restriction enzyme and DNA sequence analysis.
• the vectors were transformed into competent E. coli XL-I Blue for large- scale production.
• the recovered plasmid was verified by restriction enzyme digestion and DNA sequence analysis, and by expression of the nef transgene in transient transfection cell culture.
• pMRKAd ⁇ nef contains Ad6 bps 1 to 450 and bps 3508 to 35759 (bp numbers refer correspond to that of an Ad6 sequence; see, e.g., PCT/US02/32512, published April 17, 2003).
• the viral ITRs are joined by plasmid sequences that contain the bacterial origin of replication and an ampicillin resistance gene.
• the pre-adenovirus plasmid pMRKAd ⁇ nef was rescued as infectious virions in PER.C6 ® adherent monolayer cell culture.
• 10 ⁇ g of pMRKAd ⁇ nef was digested with restriction enzyme Pad (New England
• PER.C6 ® cells transfected into a 6 cm dish of PER.C6 ® cells using the calcium phosphate co-precipitation technique (Cell Phect Transfection Kit, Amersham Pharmacia Biotech Inc.). Pad digestion releases the viral genome from plasmid sequences allowing viral replication to occur after entry into PER.C6 ® cells. Infected cells and media were harvested after complete viral cytopathic effect (CPE) was observed. The virus stock was amplified by multiple passages in PER.C6 ® cells. At the final passage virus was purified from the cell pellet by CsCl ultracentrifugation.
• CPE viral cytopathic effect
• the identity and purity of the purified virus was confirmed by restriction endonuclease analysis of purified viral DNA and by nef ELISA of culture supernatants from virus infected mammalian cells grown in vitro.
• digested viral DNA was end-labeled with P 33 -dATP, size-fractionated by agarose gel electrophoresis, and visualized by autoradiography.
• MRKAd5gagnef is depicted in Figure 17, with a sequence of such character being illustrated in Figure 18 (SEQ ID NO: 16).
• the vector is a modification of a prototype Group C Adenovirus serotype 5 whose genetic sequence has been described previously; Chroboczek et at, 1992 /. Virol. 186:280-285.
• the El region of the wild-type Ad5 (nt 451-3510) was deleted and replaced by nef and gag expression cassettes.
• the nef expression cassette consists of: 1) the immediate early gene promoter from the human cytomegalovirus (Chapman et ah, 1991 Nucl. Acids Res.
• the amino acid sequence of the Nef and Gag proteins closely resembles the Clade B consensus amino acid sequence (G.
• gag open reading frame encodes the matrix, capsid, and nucleocapsid proteins.
• An otherwise identical version of this construct was also generated that contains the nef open reading frame with only the mutated myristylation site.
• the shuttle plasmid P MRKAd5-HCMV-nef-BGHpA-MCMV36gagSV40-S was constructed by inserting the gag expression cassette into the Ascl site in pMRKAd5-hCMV-nef-BGHpA.
• gag expression cassette was obtained by PCR using S-MRKAd5MCMV36gagSV40pA as template.
• the PCR primers were designed to introduce Ascl sites at each end of the transgene.
• the Ascl digested PCR fragment was ligated with pMRKAd5-hCMV-nef-BGHpA, also digested with Ascl, generating pMRKAd5-hCMV-nef-BGHpA-mCMV36gagSV40-S.
• BGHpA-mCMV36gagSV40-S was verified by restriction enzyme analyses and sequencing.
• the transgene containing fragment was liberated from shuttle plasmid pMRKAd5-hCMV-nef-BGHpA-mCMV36gagSV40-S by digestion with restriction enzymes BstT ⁇ .11 + SgrAI and gel purified.
• the purified transgene fragment was then co- transformed into E. coli strain BJ5183 with linearized (C/ ⁇ l-digested) adenoviral backbone plasmid, pHVE3. Plasmid DNA isolated from BJ5183 transformants was then transformed into competent E. coli Sabl2TM for screening by restriction analysis.
• the desired plasmid pMRKAd5gagnef also referred to as pMRKAd5-hCMV-nef-BGH-mCMV36gagSV40-S
• the pre-adenovirus plasmid pMRKAd5gagnef was rescued as infectious virions in PER.C6TM adherent monolayer cell culture.
• 10 ⁇ g of pMRKAd5gagnef was digested with restriction enzyme Pad (New England Biolabs) and then transfected into one T25 flask of PER.C6TMcells using the calcium phosphate co-precipitation technique. Pad digestion releases the viral genome from plasmid sequences, allowing viral replication to occur after entry into PER.C6TM cells. Infected cells and media were harvested 7 days post-transfection, after complete viral cytopathic effect (CPE) was observed.
• Pad restriction enzyme
• the virus stock was amplified by 2 passages in PER.C6TM cells. At passage 2, virus was purified on CsCl density gradients. To verify that the rescued virus had the correct genetic structure, viral DNA was isolated and analyzed by restriction enzyme (HincflR) analysis. The expression of Gag and Nef was also verified by ELISA and Western blot. The rescued virus was referred to as MRKAd5gagnef (also referred to as MRK-Ad5-hCMVnefbGH- MCMV36gagSV40-S).
• MRKAd5gagnef also referred to as MRK-Ad5-hCMVnefbGH- MCMV36gagSV40-S.
• MRKAd ⁇ gagnef is depicted in Figure 20, with a sequence of such character being illustrated in Figure 21 (SEQ ID NO: 17).
• the vector is a modification of a prototype Group C
• the El region of the wild type Ad6 (nt 451-3507) was deleted and replaced by nef and gag expression cassettes.
• the nef expression cassette consists of: 1) the immediate early gene promoter from the human cytomegalovirus (Chapman et al, 1991 Nucl. Acids Res. 19:3979-3986), 2) the coding sequence of the human immunodeficiency virus type 1 (HTV-I) nef (strain JR-FL) gene, and 3) the bovine growth hormone polyadenylation signal sequence; Goodwin & Rottman, 1992 J. Biol. Chem. 267:16330-16334.
• the nef expression cassette is directly followed by the gag expression cassette which consists of: 1) the immediate early gene promoter from the mouse cytomegalovirus (Keil et al, 1987 J. Virol. 61:1901- 1908), 2) the coding sequence of the human immunodeficiency virus type 1 (HTV-I) gag (strain CAM-I) gene, and 3) the simian virus 40 polyadenylation signal sequence.
• HTV-I gag human immunodeficiency virus type 1 gag gag gene
• simian virus 40 polyadenylation signal sequence The amino acid sequence of the Nef and Gag proteins closely resembles the Clade B consensus amino acid sequence (G. Myers et al., eds., Human Retroviruses and ATDS, 1995: Tl-A-I to TI-A-22) and the codon usage was optimized for expression in human cells; R.
• the nef open reading frame was altered by mutating the myristylation site located at Gly-2 to an alanine (opt nef G2A). This mutation prevents attachment of Nef to cytoplasmic membranes, thereby functionally inactivating Nef; Pandori et al, 1996 J. Virol. 70:4283-4290; Bresnahan et al., 1998 Curr. Biol. 8:1235-1238.
• the gag open reading frame encodes the matrix, capsid, and nucleocapsid proteins.
• the shuttle plasmid pMRKAd6-hCMV-nefG2A-BGHpA-mCMV36gagSV40-S was constructed by inserting the gag expression cassette into the Ascl site in pMRKAd6-hCMV-nefG2A ⁇ BGHpA.
• the gag expression cassette was obtained by PCR using S-MRKAd5-mCMV36gagSV40 as template.
• the PCR primers were designed to introduce Ascl sites at each end of the transgene.
• the Ascl digested PCR fragment was ligated with pMRKAd6-hCMV-nefG2A-BGHpA, also digested with Ascl, generating pMRKAd6-hCMV-nefG2A-BGHpA-mCMV36gagSV40-S.
• the genetic structure of pMRKAd6-hCMV-nefG2A-BGHpA-mCMV36gagSV40-S was verified by restriction enzyme analyses and sequencing.
• the transgene containing fragment was liberated from shuttle plasmid pMRKAd6-hCMV-nefG2A-BGHpA-mCMV36gagSV40-S by digestion with restriction enzymes Pad and Pmel and gel purified.
• the purified transgene fragment was then co- transformed into E. coli strain BJ5183 with linearized (CZ ⁇ l-digested) adenoviral backbone plasmid, pMRKAd ⁇ El-. Plasmid DNA isolated from BJ5183 transformants was then transformed into competent E. coli XL-I Blue for screening by restriction analysis.
• the desired plasmid pMRKAd ⁇ gagnef (also referred to as pMRKAd6-hCMV-nefG2A-BGH-mCMV36gagSV40-S) was verified by restriction enzyme digestion and DNA sequence analysis.
• pMRKAd ⁇ gagnef was rescued as infectious virions in PER.C6TM adherent monolayer cell culture.
• 10 ⁇ g of pMRKAd ⁇ gagnef was partially digested with restriction enzyme Pad (New England Biolabs) and then transfected into one T25 flask of PER.C6TM cells using the calcium phosphate co-precipitation technique.
• pMRKAd ⁇ gagnef contains three Pad restriction sites. One at each ITR and one located in early region 3.
• Digestion conditions were used which favored the linearization of pMRKAd ⁇ gagnef (digestion at only one of the three Pad sites) since the release of only one ITR is required to allow the initiation of viral DNA replication after entry into PER.C6 ® cells.
• Infected cells and media were harvested 7 days post-transfection, after complete viral cytopathic effect (CPE) was observed.
• the virus stock was amplified by 2 passages in PER.C6TM cells. At passage 2 virus was purified on CsCl density gradients. To verify that the rescued virus had the correct genetic structure, viral DNA was isolated and analyzed by restriction enzyme (HindTH) analysis. The expression of Gag and Nef was also verified by ELISA.
• HindTH restriction enzyme
• the rescued virus was referred to as MRKAd ⁇ gagnef (also referred to as Ad ⁇ -hCMVnefG2AbGH- MCMV36gagSV40-S).
• MRKAd ⁇ gagnef also referred to as Ad ⁇ -hCMVnefG2AbGH- MCMV36gagSV40-S.
• MRKAd5gagpol is depicted in Figure 23, with a sequence of such character being illustrated in Figure 24 (SEQ ID NO: 18).
• the vector is a modification of a prototype Group C Ad5 whose genetic sequence has been reported previously; Chroboczek et al., 1992 J. Virol. 186:280-285.
• the El region of the wild-type Ad5 (nt 451-3510) is deleted and replaced with the transgene.
• the transgene contains the gagpol expression cassette consisting of: 1) the immediate early gene promoter from the human cytomegalovirus (Chapman et al, 1991 Nucl. Acids Res.
• the pol open reading frame encodes the reverse transcriptase, RNAse H, and integrase proteins, each of which was completely inactivated by substitution of alanine residues for each amino acid residue that was part of the enzymatic active sites (reverse transcriptase Asp-112, Asp-187 and Asp-188; RNase H Asp-445, Glu-480, and Asp-500; integrase Asp-626, Asp-678, and Glu-714) for a total of nine site mutations; Larder et al, 1987 Nature 327:716-717; Larder et al., 1989 Proc. Natl. Acad. Sci.
• the vector has an E3 deletion (nt 28138 to 30818) in order to accommodate the transgene.
• the shuttle plasmid pMRKAd5gagpol was constructed by inserting a synthetic full- length codon-optimized HIV-I gagpol fusion gene into
• the genetic structure of pMRKAd5gagpol was verified by restriction enzyme and DNA sequence analyses.
• transgene containing fragment was liberated from shuttle plasmid pMRKAd5gagpol by digestion with restriction enzymes Pad and BstZlll and gel purified.
• the purified transgene fragment was then co-transformed into E. coli strain BJ5183 with linearized (CM-digested) adenoviral backbone plasmid, pAd5HVO (also referred to as pAd5 E1-E3-).
• Plasmid DNA isolated from BJ5183 transformants was then transformed into competent E. coli XL-I Blue for screening by restriction analysis.
• the desired plasmid pMRKAd5DElHCMVgagpolBGHpADE3 (also referred to as pAd5HVOMRKgagpol) was verified by restriction enzyme digestion and DNA sequence analysis.
• pMRKAd5DElHCMVgagpolBGHpADE3 was rescued as infectious virions in PER.C6TM adherent monolayer cell culture.
• 10 ⁇ g of pMRKAd5DElHCMVgagpolBGHpADE3 was digested with restriction enzyme Pad (New England Biolabs) and then transfected into one T25 flask of PER.C6 ® cells using the calcium phosphate co-precipitation technique. Pad digestion releases the viral genome from plasmid sequences, allowing viral replication to occur after entry into PER.C6TM cells.
• the virus stock was amplified by 2 passages in PER.C6TM cells. At passage 2, virus was purified on CsCl density gradients.
• PCR primer GP-I was designed to contain a BgIR site (underlined) for cloning .
• PCR primer GP-2 was designed to define the desired junction region between gag and pol, one half of the primer consists of 3' end of gag (bold) and the other the 5 'end of pol (italics)
• GP-4 5' CAGCAGATCTGCCCGGGCTTTAGTC (SEQ ID NO: 24)).
• PCR primer GP-3 was designed to be complementary to primer GP-2 thus defining the desired junction region between gag and pol.
• Primer GP-4 was designed to contain a BgI II site (underlined) for cloning. In PCR reaction three the products of PCR reactions one and two were mixed with PCR primers GP-I and GP-4. The homologous sequences in PCR product 1 and product 2 allow them to prime the amplification of the full gagpol fusion product. K. Construction of an Ad5 vector containing HIV gagpol and nef transgenes
• MRKAd5nef-gagpol is depicted in Figure 27, with a sequence of such character being illustrated in Figure 28 (SEQ ID NO: 19).
• the vector is a modification of a prototype Group C Ad5 whose genetic sequence has been reported previously; Chroboczek et al, 1992 J. Virol. 186:280-285.
• the El region of the wild-type Ad5 (nt 451-3510) is deleted and replaced with the transgene.
• the tri- antigen transgene contains the nef expression cassette consisting of: 1) the immediate early gene promoter from the human cytomegalovirus (Chapman et al, 1991 Nucl. Acids Res.
• HTV-I human immunodeficiency virus type 1
• nef strain JR-FL
• bovine growth hormone polyadenylation signal sequence Goodwin & Rottman, 1992 J. Biol. Chem. 267:16330-16334.
• the nef cassette is directly followed by the gagpol expression cassette consisting of: 1) the immediate early gene promoter from the mouse cytomegalovirus (Keil et al., 1987 J. Virol.
• HTV-I gag strain CAM-I
• HTV-I pol strain TTlB
• simian virus 40 polyadenylation signal sequence The amino acid sequence of the Nef, Gag and Pol proteins closely resembles the Clade B consensus amino acid sequence (G. Myers et al, eds., Human Retroviruses and ADDS, 1995: II-A-1 to II-A-22) and the codon usage was optimized for expression in human cells; R. Lathe, 1985 J. Molec. Biol. 183:1-12.
• the nef open reading frame was altered by mutating the myristylation site located at Gly-2 to an alanine. This mutation prevents attachment of Nef to the cytoplasmic membrane and retrotrafficking into endosomes, thereby functionally inactivating Nef; Pandori et al, 1996 J. Virol. 70:4283-4290; Bresnahan et al, 1998 Curr. Biol. 8:1235-1238.
• the gag open reading frame encodes the matrix, capsid, and nucleocapsid proteins.
• the pol open reading frame encodes the reverse transcriptase, RNAse H, and integrase proteins, each of which was completely inactivated by substitution of alanine residues for each amino acid residue that was part of the enzymatic active sites (reverse transcriptase Asp- 112, Asp- 187 and Asp-188; RNase H Asp-445, Glu-480, and Asp-500; integrase Asp-626, Asp-678, and Glu-714) for a total of nine site mutations; Larder et al, 1987 Nature 327:716-717; Larder et al, 1989 Proc. Natl. Acad.
• the vector has an E3 deletion (nt 28138 to 30818) in order to accommodate the transgene.
• Shuttle plasmid pMRKAd5HCMVnefMCMVgagpol was constructed in two steps. First the gagpol fusion open reading frame was obtained from pMRKAd5gagpol (described in Example 2J) by BgIH digestion and inserted into the BgIE site in S-MRKAd5-mCMV36-SV40, generating MRKAd5MCMVgagpolSV40.
• MRKAd5MCMVgagpolSV40 was then digested with Mfel and Xhol to generate a gagpol transgene containing fragment that was cloned into the Mfel and Xhol sites in MRKAd5-hCMVnefG2ABGH-mCMV36gagSv40-S, generating pMRKAd5HCMVnefMCMVgagpol.
• the genetic structure of pMRKAd5HCMVnefMCMVgagpol was verified by restriction enzyme analysis and sequencing.
• transgene containing fragment was liberated from shuttle plasmid pMRKAd5HCMVnefMCMVgagpol by digestion with restriction enzymes Pad and BstTAll and gel purified.
• the purified transgene fragment was then co-transformed into E. coli strain BJ5183 with linearized (C/ ⁇ l-digested) adenoviral backbone plasmid pAd5HVO, (also referred to as pAd5El-E3-). Plasmid DNA isolated from BJ5183 transformants was then transformed into competent E.
• pAd5MRKDElHCMVnefG2ABGHMCMV36gagpolSV40DE3 was rescued as infectious virions in PER.C6TM adherent monolayer cell culture.
• 10 ⁇ g of pAd5MRKDElHCMVnefG2ABGHMCMV36gagpolSV40DE3 was digested with restriction enzyme Pad (New England Biolabs) and then transfected into one T25 flask of PER.C6TM cells using the calcium phosphate co-precipitation technique. Pad digestion releases the viral genome from plasmid sequences, allowing viral replication to occur after entry into PER.C6TM cells.
• the virus stock was amplified by 2 passages in PER.C6TM cells. At passage 2, virus was purified on CsCl density gradients.
• MRKAd5gagpolnef is depicted in Figure 30, with a sequence of such character being illustrated in Figure 31 (SEQ ID NO: 20).
• the vector is a modification of a prototype Group C Ad5 whose genetic sequence has been reported previously; Chroboczek et al, 1992 J. Virol. 186:280-285.
• the El region of the wild-type Ad5 (nt 451-3510) is deleted and replaced with the transgene.
• the transgene contains the gagpolnef expression cassette consisting of: 1) the immediate early gene promoter from the human cytomegalovirus (Chapman et al, 1991 Nucl. Acids Res.
• the pol open reading frame encodes the reverse transcriptase, RNAse H, and integrase proteins, each of which was completely inactivated by substitution of alanine residues for each amino acid residue that was part of the enzymatic active sites (reverse transcriptase Asp-112, Asp-187 and Asp- 188; RNase H Asp-445, Glu-480, and Asp-500; integrase Asp-626, Asp-678, and Glu-714) for a total of nine site mutations; Larder et al., 1987 Nature 327:716-717; Larder et al, 1989 Proc. Natl. Acad. ScL 86:4803-4807; Davies et al, 1991 Science 252:88-95; Schatz et al, 1989 FEBS Lett. 257:311-314;
• the shuttle plasmid pMRKAd5gagpolnef was constructed in three steps ( Figure 32).
• First shuttle plasmid pMRKAd5gagpol (described in Example 2J) was digested with BamHI to remove part of the gagpol transgene, generating pMRKAd5gagpolBamHIcollapse.
• the BamHI fragment containing the partial gagpol transgene was. gel purified and used in step three.
• the polnef fusion gene obtained by overlap PCR as depicted in Figure 33, was ligated into the BamHI and BgIJl sites in pMRKAd5gagpolBamHIcollapse, generating pMRKAd5gagpolBamHIcollapsenef.
• the BamHI fragment containing the partial gagpol transgene obtained in step one was inserted into the BamHL site in pMRKAd5gagpolBamHIcollapsenef, generating pMRKAd5gagpolnef.
• the genetic structure of pMRKAd5gagpolnef was verified by restriction enzyme and DNA sequence analyses.
• transgene containing fragment was liberated from shuttle plasmid pMRKAd5gagpolnef by digestion with restriction enzymes Pad and BstZYll and gel purified.
• the purified transgene fragment was then co-transformed into E. coli strain BJ5183 with linearized (Cf ⁇ l-digested) adenoviral backbone plasmid, pAd5HVO (also referred to as pAd5El-E3-).
• Plasmid DNA isolated from BJ5183 transformants was then transformed into competent E. coli XL-I Blue for screening by restriction analysis.
• the desired plasmid pMRKAd5DElHCMVgagpolBGHpADE3 (also referred to as pAd5HVOMRKgagpol) was verified by restriction enzyme digestion and DNA sequence analysis.
• pMRKAd5DElHCMVgagpolnefBGHpADE3 was rescued as infectious virions in PER.C6TM adherent monolayer cell culture.
• 10 ⁇ g of pMRKAd5DElHCMVgagpolnefBGHpADE3 was digested with restriction enzyme Pad (New England Biolabs) and then transfected into one T25 flask of PER.C6TM cells using the calcium phosphate co- precipitation technique. Pad digestion releases the viral genome from plasmid sequences, allowing viral replication to occur after entry into PER.C6TM cells.
• the virus stock was amplified by 2 passages in PER.C6TM cells. At passage 2, virus was purified on CsCl density gradients.
• PCR primer PN-I was chosen to overlap an existing BamHI site (underlined) in the pol sequence that was used for cloning.
• PCR primer PN-2 was designed to define the desired junction region between pol and nef, one half of the primer consists of 3' end of pol (bold) and the other the 5'end of nef (italics).
• PN-3 and PN-4 PN-
• PCR primer PN-3 was designed to be complementary to primer PN-2 thus defining the desired junction region between pol and nef.
• Primer PN-4 was designed to contain a BgIR site for cloning. In PCR reaction three the products of PCR reactions one and two were mixed with PCR primers PN-I and PN-4. The homologous sequences in PCR product 1 and product 2 allow them to prime the amplification of the full gagpol fusion product.
• MRKAd ⁇ nef-gagpol is depicted in Figure 35, with a sequence of such character being illustrated in Figure 36 (SEQ ID NO: 21).
• the vector is a modification of a prototype Group C Adenovirus serotype 6; VR-6; PCT/US02/32512, published April 17, 2003.
• the El region of the wild type Ad6 (nt 451-3507) was deleted and replaced by the transgene.
• the transgene contains the nef expression cassette consisting of: 1) the immediate early gene promoter from the human cytomegalovirus (Chapman et al, 1991 Nucl. Acids Res.
• HTV-I human immunodeficiency virus type 1
• nef strain JR-FL
• bovine growth hormone polyadenylation signal sequence Goodwin & Rottman, 1992 J. Biol. Chem. 267:16330-16334.
• the nef cassette is directly followed by the gagpol expression cassette consisting of: 1) the immediate early gene promoter from the mouse cytomegalovirus (Keil et al., 1987 /. Virol.
• HTV-I gag strain CAM-I
• HTV-I gag strain CAM-I
• HTV-I pol strain TIIB
• simian virus 40 polyadenylation signal sequence The amino acid sequence of the Nef, Gag and Pol proteins closely resembles the Clade B consensus amino acid sequence (G. Myers et al., eds., Human Retroviruses and ATDS, 1995: II-A-1 to II-A-22) and the codon usage was optimized for expression in human cells; R. Lathe, 1985 J. Molec. Biol.
• the nef open reading frame was altered by mutating the myristylation site located at Gly-2 to an alanine. This mutation prevents attachment of Nef to the cytoplasmic membrane and retrotrafficking into endosomes, thereby functionally inactivating Nef; Pandori et al, 1996 J. Virol. 70:4283-4290; Bresnahan et al, 1998 Curr. Biol. 8:1235-1238.
• the gag open reading frame encodes the matrix, capsid, and nucleocapsid proteins.
• the pol open reading frame encodes the reverse transcriptase, RNAse H, and integrase proteins, each of which was completely inactivated by substitution of alanine residues for each amino acid residue that was part of the enzymatic active sites (reverse transcriptase Asp- 112, Asp- 187 and Asp-188; RNase H Asp-445, Glu-480, and Asp- 500; integrase Asp-626, Asp-678, and Glu-714) for a total of nine site mutations; Larder et al, 1987 Nature 327:716-717; Larder et al, 1989 Proc. Natl. Acad. Sd.
• the vector has an E3 deletion (nt 28138 to 30818) in order to accommodate the transgene.
• Shuttle plasmid pNEBAd6-2HCMVnefMCMVgagpol was constructed by inserting the nef-gagpol transgene from pMRKHCMVnefMCMVgagpol (described in Example 2K) into the Ascl and Notl sites in pNEBAd6-2.
• pMRKHCMVnefMCMVgagpol was digested to completion with Notl and Pvul and then partially digested with Ascl. Pvul was used to digest and thus reduce in size the unwanted plasmid fragment so that the desired NoUI Ascl transgene fragment could be more easily gel purified.
• NotUAscl transgene fragment was ligated with pNEBAd6-2 also digested with Not I and Ascl, generating pNEBAd6-2HCMVnefMCMVgagpol.
• the genetic structure of pNEBAd6-2HCMVnefMCMVgagpol was verified by restriction enzyme analysis and sequencing.
• transgene containing fragment was liberated from shuttle plasmid pNEBAd ⁇ - 2HCMVnefMCMVgagpol by digestion with restriction enzymes Pad and Pmel and gel purified.
• the purified transgene fragment was then co-transformed into E. coli strain BJ5183 with linearized (Clal- digested) adenoviral backbone plasmid, pAd6MRKDElDE3. Plasmid DNA isolated from BJ5183 transformants was then transformed into competent E. coli XL-I Blue for screening by restriction analysis.
• the desired plasmid pAd6MRKDElHCMVnefBGHMCMVgagpolSV40DE3 was verified by restriction enzyme digestion and DNA sequence analysis. 3. Generation of recombinant MRKAd ⁇ nef-gaspol To prepare virus the pre-adenovirus plasmid pAd6MRKDElHCMVnefBGHMCMVgagpolSV40DE3 was rescued as infectious virions in PER.C6TM adherent monolayer cell culture.
• pAd6MRKDElHCMVnefflGHMCMVgagpolSV40DE3 was digested with restriction enzyme Pad (New England Biolabs) and then transfected into one T25 flask of PER.C6TM cells using the calcium phosphate co-precipitation technique. Pad digestion releases the viral genome from plasmid sequences, allowing viral replication to occur after entry into PER.C6TM cells. Infected cells and media were harvested 10 days post-transfection, after complete viral cytopathic effect (CPE) was observed.
• the virus stock was amplified by 2 passages in PER.C6TM cells. At passage 2, virus was purified on CsCl density gradients.
• MRKAd ⁇ gagpolnef is depicted in Figure 38, with a sequence of such character being illustrated in Figure 39 (SEQ ID NO: 22).
• the vector is a modification of a prototype Group C Adenovirus serotype 6; VR-6; PCT/US02/32512, published April 17, 2003.
• the El region of the wild type Ad6 was deleted and replaced by the transgene.
• the transgene contains the gagpolnef expression cassette consisting of: 1) the immediate early gene promoter from the human cytomegalovirus (Chapman et al, 1991 Nucl. Acids Res.
• the pol open reading frame encodes the reverse transcriptase, RNAse H, and integrase proteins, each of which was completely inactivated by substitution of alanine residues for each amino acid residue that was part of the enzymatic active sites (reverse transcriptase Asp-112, Asp-187 and Asp-188; RNase H Asp-445, Glu-480, and Asp- 500; integrase Asp-626, Asp-678, and Glu-714) for a total of nine site mutations; Larder et al, 1987 Nature 327:716-717; Larder et al, 1989 Proc. Natl. Acad. Sd.
• Shuttle plasmid pNEBAd6-2gagpolnef was constructed by inserting the gagpolnef transgene from pMRKAd5gagpolnef (described in Example 2K) into the Asc ⁇ and Notl sites in pNEBAd6-2. To obtain the gagpolnef transgene fragment, pMRKAd5gagpolnef was digested with Notl and Asc ⁇ and transgene fragment gel purified. The Notl/Asc ⁇ transgene fragment was then ligated with pNEBAd6-2 also digested with Not I and Ascl, generating pNEBAd6-2HCMVgagpolnef. The genetic structure of pNEB Ad6-2gagpolnef was verified by restriction enzyme analysis and sequencing. 2. Construction of pre-adenovi?'us plasmid
• transgene containing fragment was liberated from shuttle plasmid pNEB Ad6-2gagpolnef by digestion with restriction enzymes Pad and Pmel and gel purified.
• the purified transgene fragment was then co- transformed into E. coli strain BJ5183 with linearized (CZ ⁇ l-digested) adenoviral backbone plasmid, pAd6MRKDElDE3.
• Plasmid DNA isolated from BJ5183 transformants was then transformed into competent E. coli XL-I Blue for screening by restriction analysis.
• the desired plasmid pAd6MRKDElHCMVgagpolnefBGHpADE3 was verified by restriction enzyme digestion. 3. Generation of recombinant MRKAd ⁇ gagpolnef
• pAd6MRKDElHCMVgagpolnefBGHpADE3 was rescued as infectious virions in PER.C6TM adherent monolayer cell culture.
• 10 ⁇ g of pAd6MRKDElHCMVgagpolnefBGHpADE3 was digested with restriction enzyme Pad (New England Biolabs) and then transfected into one T25 flask of PER.C6TM cells using the calcium phosphate co- precipitation technique. Pad digestion releases the viral genome from plasmid sequences, allowing viral replication to occur after entry into PER.C6TM cells.
• the virus stock was amplified by 2 passages in PER.C6TM cells. At passage 2, virus was purified on CsCl density gradients.
• the cells were stimulated in the absence (mock) or presence of a nef peptide pool (4 ⁇ g/mL per peptide).
• the pool consisting of 15 amino acid (“aa”) (15-aa) peptides shifting by 4 aa (Synpep, CA), was constructed from the HIV-I JRFL nef sequence. Cells were then incubated for 20-24 h at 37°C in 5% CO2- Plates were washed six times with
• PBST PBS, 0.05% Tween 20
• U-Cytech-BV anti-IFN- ⁇ polyclonal biotinylated detector antibody solution
• Rhesus macaques were between 3-10 kg in weight. In all cases, the total dose of each vaccine was suspended in 1 mL of buffer. The macaques were anesthetized (ketamine/xylazine) and the vaccines were delivered i.m. in 0.5-mL aliquots into both deltoid muscles using tuberculin syringes (Becton-Dickinson, Franklin Lakes, NJ). Peripheral blood mononuclear cells (PBMC) were prepared from blood samples collected at several time points during the immunization regimen. AU animal care and treatment were in accordance with standards approved by the Institutional Animal Care and Use Committee according to the principles set forth in the Guide for Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Research Council.
• PBMC Peripheral blood mononuclear cells
• the IFN- ⁇ ELISPOT assays for rhesus macaques were conducted following a previously described protocol (Allen et ah, 2001 J. Virol. 75(2):738-749), with some modifications.
• a peptide pool was prepared from 15-aa peptides that encompass the entire HIV-I gag sequence with 11-aa overlaps (Synpep Corp., Dublin, CA).
• 50 ⁇ L of 2-4 x 10 5 were added; the cells were counted using Beckman Coulter Z2 particle analyzer with a lower size cut-off set at 80 fL.
• Rhesus macaques were between 3-10 kg in weight. In all cases, the total dose of each vaccine was suspended in 1 mL of buffer. The macaques were anesthetized (ketamine/xylazine) and the vaccines were delivered i.m. in 0.5-mL aliquots into both deltoid muscles using tuberculin syringes (Becton-Dickmson, Franklin Lakes, NJ). Peripheral blood mononuclear cells (PBMC) were prepared from blood samples collected at several time points during the immunization regimen. All animal care and treatment were in accordance with standards approved by the Institutional Animal Care and Use Committee according to the principles set forth in the Guide for Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Research Council.
• PBMC Peripheral blood mononuclear cells
• the IFN- ⁇ ELISPOT assays for rhesus macaques were conducted following a previously described protocol (Allen et al, 2001 J. Virol. 75(2):738-749), with some modifications.
• peptide pools were prepared from 15-aa peptides that encompass the entire HTV-I nef, gag, and pol sequences with 11-aa overlaps (Synpep Corp., Dublin, CA).
• 50 ⁇ L of 2-4 x 10 5 peripheral blood mononuclear cells (PBMCs) were added; the cells were counted using Beckman Coulter Z2 particle analyzer with a lower size cut-off set at 80 fL.
• Rhesus macaques were between 3-10 kg in weight. In all cases, the total dose of each vaccine was suspended in 1 mL of buffer. The macaques were anesthetized (ketamine/xylazine) and the vaccines delivered i.m. in 0.5-mL aliquots into both deltoid muscles using tuberculin syringes (Becton- Dickinson, Franklin Lakes, NJ). Peripheral blood mononuclear cells (PBMC) were prepared from blood samples collected at several time points during the immunization regimen. AU animal care and treatment were in accordance with standards approved by the Institutional Animal Care and Use Committee according to the principles set forth in the Guide for Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Research Council.
• PBMC Peripheral blood mononuclear cells
• the IFN- ⁇ ELISPOT assays for rhesus macaques were conducted following a previously described protocol (Allen et al., 2001 J. Virol. 75(2):738-749), with some modifications.
• peptide pools were prepared from 15-aa peptides that encompass the entire HTV-I nef, gag and pol sequences with 11-aa overlaps (Synpep Corp., Dublin, CA).
• 50 ⁇ L of 2-4 x 10 3 peripheral blood mononuclear cells (PBMCs) were added; the cells were counted using Beckman Coulter Z2 particle analyzer with a lower size cut-off set at 80 fL.
• the vectors were able to induce specific T cell response to all 3 antigens at 10 ⁇ 10 vp/vector dose.
• the immunogenicity of the Ad5 and Ad6 vectors is comparable when delivered alone or in combination.
• the immunogenicity of the vaccines at 10 ⁇ 8 vp/vector dose is described in Figure 46. Even at a lower 10 ⁇ 8 vp/vector dose, specific T cell response to all three antigens were detected.

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• 2005
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