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US20130142823A1 - Cmv glycoproteins and recombinant vectors - Google Patents

Cmv glycoproteins and recombinant vectors Download PDF

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Publication number
US20130142823A1
US20130142823A1 US13/626,398 US201213626398A US2013142823A1 US 20130142823 A1 US20130142823 A1 US 20130142823A1 US 201213626398 A US201213626398 A US 201213626398A US 2013142823 A1 US2013142823 A1 US 2013142823A1
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Prior art keywords
cmv
vector
rhcmv
cell
infection
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Inventor
Louis Picker
Klaus Frueh
Scott G. Hansen
Colin Powers
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Oregon Health and Science University
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Oregon Health and Science University
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Priority to US13/626,398 priority Critical patent/US20130142823A1/en
Publication of US20130142823A1 publication Critical patent/US20130142823A1/en
Priority to US14/179,152 priority patent/US9541553B2/en
Assigned to OREGON HEALTH & SCIENCE UNIVERSITY reassignment OREGON HEALTH & SCIENCE UNIVERSITY ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: POWERS, COLIN
Assigned to OREGON HEALTH & SCIENCE UNIVERSITY reassignment OREGON HEALTH & SCIENCE UNIVERSITY ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: FRUEH, KLAUS, PICKER, LOUIS, HANSEN, SCOTT
Priority to US15/374,938 priority patent/US10101329B2/en
Abandoned legal-status Critical Current

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Definitions

  • This invention relates to recombinant cytomegalovirus vectors, methods of making them, uses for them, expression products from them, and uses for the expression products.
  • This invention also relates to cytomegalovirus glycoproteins US2, US3, US6 and US11, in particular recombinant cytomegalovirus vectors lacking one or more of the glycoproteins US2, US3, US6 and US11.
  • This invention also relates to recombinant vectors expressing one or more of the glycoproteins US2, US3, US6 and US11 of HCMV and the homologous proteins Rh182, Rh184, Rh185 and Rh189 of RhCMV.
  • HCMV is an ubiquitous virus that is present in over 60% of the population depending on socioeconomic status. Following primary infection, HCMV persists for the life span of the host. Although HCMV is generally benign in healthy individuals, the virus may cause devastating disease in immunocompromised populations resulting in high morbidity and mortality (for review, see (Pass, R. F. 2001. Cytomegalovirus, p. 2675-2705. In P. M. H. David M. Knipe, Diane E. Griffin, Robert A. Lamb Malcolm A. Martin, Bernard Roizman and Stephen E. Straus (ed.), Fields Virology, 4th ed. Lippincott Williams & Wilkins, Philadelphia)).
  • HCMV ulcerative colitis .
  • WT wild-type
  • HCMV-specific T cell immunity has also been shown to afford protection against CMV disease in transplant patients, presenting another population wherein the ability to safely induce an immunity comparable to that acquired by natural CMV infection would have a clinical impact on CMV disease (Leen, A. M., and H. E. Heslop. 2008. Br J Haematol 143:169-79, Riddell, S. R., and P. D. Greenberg. 2000. J Antimicrob Chemother 45 Suppl T3:35-43 and Riddell, S. R. et al. 1994. Bone Marrow Transplantation 14:78-84). Cytomegalovirus is highly immunogenic, but has evolved immune evasion mechanisms to enable virus persistence and re-infection of the sero-positive host:
  • a large proportion of the host T cell repertoire is also directed against CMV antigens, with 5-10 fold higher median CD4 + T cell response frequencies to HCMV than to acute viruses (measles, mumps, influenza, adenovirus) or even other persistent viruses such as herpes simplex and varicella-zoster viruses (Sylwester, A. W. et al. 2005. J Exp Med 202:673-85).
  • a high frequency of CD8 + responses to defined HCMV epitopes or proteins is also commonly observed (Gillespie, G. M. et al. 2000. J Virol 74:8140-50, Kern, F. et al. 2002. J Infect Dis 185:1709-16, Kern, F. et al. 1999.
  • the high levels of CMV-specific immunity are unable to either eradicate the virus from the healthy infected individual, or confer protection of the CMV sero-positive individual against re-infection.
  • This ability of CMV to escape eradication by the immune system, and to re-infect the sero-positive host has long been believed to be linked to the multiple viral immunomodulators encoded by the virus (for review, see (Mocarski, E. S., Jr. 2002. Trends Microbiol 10:332-9)).
  • the HCMV US6 family of proteins (RhCMV homologues: Rh182-Rh189) are the most extensively studied of these immunomodulators (Loenen, W. A. et al. 2001. Semin Immunol 13:41-9).
  • CMV Consistent with persistent replication/chronic reactivation within the host, CMV also induces and maintains a characteristic and unique T cell immune response.
  • Memory T cells induced by vaccination or infection may be broadly characterized into either effector (T EM ) or central (T CM ) memory, which follow from the distinct functions of these two memory populations (Cheroutre, H., and L. Madakamutil. 2005. Cell Mol Life Sci 62:2853-66, Mackay, C. R. et al. 1990. J Exp Med 171:801-17, Masopust, D. et al. 2001. Science 291:2413-7, Sallusto, F. et al. 1999. Nature 401:708-12 and Wherry, E. J. et al.
  • T EM are designed for immediate function against the invading pathogen, being highly enriched at epithelial mucosal surfaces, are polyfunctional expressing high levels of multiple effector cytokines (expressing TNF ⁇ , IFN ⁇ , MIP-1 ⁇ effector molecules), and have high cytotoxic potential (CD8 + ).
  • T EM and T CM may also be easily distinguished on the basis of cell surface markers, with T EM being CCR7 ⁇ , CD28 +/ ⁇ and T CM being CCR7 + , CD28 + .
  • Multiple studies indicate that persistently replicating viruses such as CMV maintain a T cell response that is heavily biased toward the T EM phenotype (Amyes, E. et al. 2003.
  • the present invention relates to relates to recombinant vectors, advantageously viral vectors that either express human cytomegalovirus (HCMV) glycoproteins US2, US3, US6 and US11 or rhesus cytomegalovirus (RCMV) glycoproteins Rh182, Rh184, Rh185 and Rh189.
  • HCMV human cytomegalovirus
  • RCMV rhesus cytomegalovirus
  • the invention also related to HCMV vectors that have HCMV glycoproteins US2, US3, US6 and US11 deleted therefrom.
  • Further objects of the invention include any or all of: to provide expression products from such recombinants, methods for expressing products from such recombinants, compositions containing the recombinants or the expression products, methods for using the expression products, methods for using the compositions, DNA from the recombinants, and methods for replicating DNA from the recombinants.
  • One embodiment of the invention relates to a method of superinfecting or repeatedly an animal (including human) which may comprise (a) constructing a vector containing and expressing at least one human cytomegalovirus (HCMV) glycoprotein, wherein the glycoprotein is US2, US3, US6 or US11 (or the corresponding RhCMV homologues), and (b) administering the vector into the animal, wherein the animal might have already been infected with the same vector.
  • HCMV human cytomegalovirus
  • the vector may be an adenovirus vector, adeno-associated virus (AAV) vector, alphavirus vector, herpesvirus vector (including HCMV), retrovirus vector and poxvirus vector.
  • AAV adeno-associated virus
  • the vector may contain and express US2, US3, US6 and US11 or Rh182, Rh184, Rh185 and Rh189 or the vector may contain and express all of the glycoproteins within the US2 to US11 region of HCMV or the Rh182-189 region of RhCMV
  • Another embodiment of the present invention relates to a method of determining efficacy of a HCMV vaccine, which may comprise (a) administering a HCMV vaccine to a test subject, (b) challenging the test subject with a HCMV vector, wherein glycoproteins within the US2 to US11 region of HCMV are deleted from the HCMV vector, and (c) measuring a protective CD8+ T cell response, wherein the HCMV vaccine is efficacious if a CD8+ T cell response protects againstchallenge with the HCMV vector with the glycoproteins within the US2 to US11 region of CMV deleted.
  • the US2-11 deleted HCMV vector may be an HIV vaccine.
  • the HIV antigen may be a HIVprotein.
  • the US2-11 deleted HCMV vector may be a HCMV vaccine.
  • a further embodiment of the present invention relates to a method of inducing a different CD8+ T cell response in an animal or human, which may comprise (a) administering a HCMV vector with at least one cytomegalovirus (CMV) glycoprotein deleted from the CMV vector, wherein the glycoprotein is US2, US3, US6 or US11, and wherein the CMV vector contains and expresses at least one immunogen, and (b) administering the vector to the animal or human, wherein the CD8+ T cell response in the animal or human differs as compared to a a CMV vector that contains and expresses the same at least one immunogen and wherein a CMV glycoprotein is not deleted from the CMV vector.
  • CMV cytomegalovirus
  • the vector may have CMV glycoproteins US2, US3, US6 and US11 deleted individually from the CMV vector.
  • the vector vector may also have all of the glycoproteins within the US2 to US11 region of CMV deleted from the CMV vector.
  • FIGS. 1A-1C CMV-infected rhesus macaques are not protected against super-infection with RhCMV and superinfection of RhCMV-positive animals is independent of viral dose.
  • A At day 0, two cohorts of four RhCMV + animals each were infected subcutaneously with 10 2 or 10 4 PFU of RhCMV(gagL). The SIVgag-specific T cell responses in PBMC or in bronchoalveolar lavage (BAL) were monitored by flow cytometric analysis of intrcellular cytokine staining (ICCS) for CD69 and tumor necrosis factor- ⁇ (TNF- ⁇ ) (S. G. Hansen et al. Science 328, 5974 (2010)) (see FIGS.
  • ICCS intrcellular cytokine staining
  • TNF- ⁇ tumor necrosis factor- ⁇
  • Urine was isolated at the indicated days post-infection (PID) from each of the RhCMV(gagL)-infected RM, and SIVgag expression was detected from cocultured virus by immunoblot.
  • PID days post-infection
  • SIVgag expression was detected from cocultured virus by immunoblot.
  • a RhCMV-positive animal that did not receive RhCMV(gagL) was included.
  • FIGS. 2A-2C Interference with MHC-I assembly is not required for primary infection of CMV-na ⁇ ve animals.
  • ⁇ US2-11(gag) lacks the RhCMV gene region Rh182—Rh189 encoding the homologues of HCMV US2-11 (N. T. Pande et al.
  • ⁇ VIHCE ⁇ US2-11(gag) additionally lacks the RhCMV gene Rh178 encoding the viral inhibitor of heavy chain expression (VIHCE)
  • VIHCE viral inhibitor of heavy chain expression
  • A The RhCMV-specific T cell response in PBMC and the SIVgag-specific T cell response in PBMC and BAL were determined at the indicated days post-infection using overlapping peptides to RhCMV immediate early genes IE1 and IE2 or SIVgag by flow cytometric analysis of ICCS for CD69, TNF- ⁇ , and interferon- ⁇ (IFN- ⁇ ) (S. G. Hansen et al.
  • FIGS. 3A-3D US2-11-deleted RhCMV is unable to superinfect RhCMV + rhesus macaques.
  • a cohort of four RhCMV + RM was inoculated subcutaneously with 10 7 PFU of ⁇ VIHCE ⁇ US2-11(gag) ( ⁇ V ⁇ U) at days 0 and 91.
  • the CD4 + and CD8 + T cell response to SIVgag or RhCMV-IE was monitored by flow cytometric analysis of ICCS for CD69, TNF- ⁇ , and IFN- ⁇ in PBMC. The percentage of the responding, specific T cells within the overall memory subset is shown for each time point.
  • the same cohort was inoculated with 10 7 PFU of ⁇ US2-11(gag) (AU), and RhCMV-IE and SIVgag-specific T cell responses were monitored bi-weekly.
  • the cohort was inoculated with ⁇ VIHCE(gag) ( ⁇ V), and the T cell response was monitored as before.
  • the cohort was inoculated with ⁇ Rh186-8(retanef) ( ⁇ R).
  • a T cell response to SIVrev/nef/tat was detected by ICCS in all four animals (black lines) using corresponding overlapping peptides.
  • Virus was isolated at the indicated days by coculture with telomerized rhesus fibroblasts (TRFs), and cell lysates were probed for expression of SIVgag by immunoblot.
  • TRFs telomerized rhesus fibroblasts
  • D Expression of RhCMV-IE2, SIVgag, and SIVretanef by virus secreted in urine collected at the indicated days. Note that all animals were IE2-positive at the onset of the experiment, confirming their RhCMV-positive T cell status (Table 1D).
  • FIGS. 4A-4D CD8 + T cells protect rhesus macaques from infection by RhCMV lacking MHC-I inhibitors.
  • A Four CMV-positive RM were treated at the indicated days with CM-T807, an antibody to CD8, before and after inoculation with 10 7 PFU of ⁇ VIHCE ⁇ US2-11(gag) (two animals, black lines) or ⁇ US2-11(gag) (two animals, red lines). The absolute counts of CD8 + T cells in the blood of each animal are shown over time.
  • B The presence of CD4 + and CD8 + T cell populations in PBMC of one representative animal is shown for the indicated days.
  • FIG. 5 Diagram of viruses used in Example 1. The deletion strategy is described in (S. G. Hansen et al. Science 328, 5974 (2010)). Regions of the genome that were altered to create mutant viruses are shown in detail. All RhCMV ORFs are depicted as arrows that correspond to the direction of the ORF within the genome. Blue arrows represent genes that down-regulate MHC class I. The RhCMV nomenclature is used for all ORFs (S. G. Hansen et al. J Virol 77, 6620 (2003)). For ORFs with homology to HCMV genes the name of the corresponding HCMV homologue is shown in brackets.
  • FIG. 6 Response frequency gating strategy. Lymphocytes originating from PBMC and BAL were stimulated with Ag, stained and collected on a flow cytometer as described in Example 1. Data was analyzed using a hierarchical gating strategy to delineate Ag-responding subsets. Gates are depicted here in pink, with corresponding subset names numbered and displayed above the cytometric plots. For FIG. 1 , response frequencies were determined using the CD69 + /TNF ⁇ + subset (CD4 + , cytometric plot 6a; CD8 + , cytometric plot 8a).
  • FIG. 7 Memory correction gating hierarchy.
  • Cell preparations were stained and the data collected as described (C. J. Pitcher et al., J Immunol 168, 29 (2002)), followed by hierarchical analysis shown here.
  • the pink boxes in cytometric plot 2 and 3 indicate the overall T cell and T cell subset gates, respectively.
  • the memory correction values used for PBMC response calculations reflect the percentage of the events within the memory gate of CD4 + or CD8 + T cell-gated profiles (cytometric plots 4 and 5, respectively).
  • FIGS. 8A-8C Characterization of recombinant RhCMVs in vitro.
  • A) RT-PCR. TRFs were infected at MOI 1 with the indicated virus and total RNA was harvested at 24 hpi. cDNA was synthesized by random hexamer priming, and transcripts were amplified with primers specific for the ORFs indicated on the left. Genes flanking the deleted regions were included to detect possible changes in transcription due to the deletions.
  • WT BACderived wild type RhCMV.
  • RT reverse transcriptase.
  • FIG. 9 Comparative genome sequencing of recombinant RhCMV.
  • the top panel shows the probe signal intensities for labeled genomic DNA fragments obtained from the co-hybridization of ⁇ VIHCE ⁇ US2-11(gag) ( ⁇ V ⁇ U, Cy5 channel, green) and BACderived RhCMV (WT, Cy3 channel, blue) to the RhCMV-DNA-microarray of overlapping oligonucleotides. Differences in hybridization signals between the reference and test genomes are shown in red as the ratio of probe intensities for WT versus ⁇ VIHCE ⁇ US2-11(gag).
  • the second and third panels show the ratios in probe intensities for WT versus ⁇ US2-11(gag) ( ⁇ U) and WT versus ⁇ VIHCE(gag) ( ⁇ V).
  • the bottom panel shows the nucleotide numbers of the RhCMV genome, depicted in 20 kbp increments. Also indicated are the positions of the VIHCE and US2-11 deletions. Positive red spikes represent signals that are present in the reference, but absent in the deletion viruses. These spikes correspond to the expected location of the deletions. Note that significant differences outside the deleted regions were not observed, indicating that the genomes of the deletion viruses are identical to that of the parental BAC in all but the deleted regions.
  • FIG. 10 Outcome of repeated, limiting dose, intra-rectal SIVmac239 challenge of RM vaccinated with A) RhCMV vectors alone (encoding gag, retanef, pol and env; given at wks 0,14); Group B) the same RhCMV/SIV vectors (wk 0) followed by pan-proteome Ad5 vectors (wk 14); Group C) pan-proteome DNA (wks, 0,4,8), followed by pan-proteome Ad5 vectors (wk 14); and Group D) unvaccinated controls (with challenge initiated at wk 59).
  • FIG. 11 No effector site CD4+ T cell depletion in protected RhCMV vector-vaccinated RM (“Controllers”). Analysis of the extent and kinetics of CD4+ memory T cell depletion in BAL following infection of controllers (red) vs. non-controllers (black) within Groups A-D, with the significance of differences in average depletion from days 21-70 pi of Group A and B controllers vs. Group C determined by the Wilcoxon rank sum test.
  • FIG. 12 Neutralizing Ab titres against lab-adapted SIVmac251 are induced or boosted with the onset of systemic infection in unvaccinated control RM, DNA/AD5-vaccinated RM and non-controllers in the RhCMV vector-vaccinated groups; however, controllers (red) in the latter groups show little to no such induction or boosting, consistent with limited Ag exposure and thus, stringent virologic control.
  • RP rapid progressor
  • FIG. 13 Total SIV (gag, env, rev/nef/tat, and pol)-specific CD8+ memory T cell responses in blood during the vaccine phase of Groups A and B with subsequent controllers shown in red and noncontrollers in black. Note that in both groups the peak response postboost, but not the response at challenge, correlated with outcome.
  • FIGS. 14A-14B Peak and post-peak control and boosting responses in study RM with progressive infection (protected RM in the CMV/CMV and CMV/Ad5 groups are not included in this figure). Note that peak and post-peak viral suppression correlates with the ability to manifest an anamnestic CD8+ T cell response boost to infection. Quantitative real-time RT-PCR and PCR assays targeting a highly conserved sequence in Gag were used for standard measurements of plasma SIV RNA and cell-associated SIV RNA and DNA within peripheral blood and lymph node mononuclear cells, as previously described (Cline, A. N. et al. J Med Primatol 34, 303-312, (2005); Venneti, S. et al. Am J Pathol 172, 1603-1616, (2008)).
  • FIG. 15 CD4+ T Cell-associated SIV in a protected RM.
  • FIG. 16 The “CMV/SIV Vector Shield”: CMV vectors elicit and maintain high frequency SIV-specific T cells in effector sites—sites that contain high SIV target cell densities and comprise the likely sites of early SIV amplification after mucosal inoculation. [quadrant % s shown; background was negligible for all assays.] Data are shown from the necropsy of one animal seven years after inoculation with RhCMV(gagL).
  • FIG. 17 RhCMV/SIV vector-elicited SIV-specific CD8+ T cell responses do not include the typical immunodominant responses that are targeted in SIV infection itself or after vaccination with DNA and/or conventional viral vectors.
  • the figure shows peripheral blood CD8+ T cell responses to a total SIVgag 15 mer peptide mix (blue) or to the Mamu A*01-restricted SIVgag CM9 epitope (red) in 2 representative Mamu A*01+ RM, one that received RhCMV/gag twice (week 0, 14) and one that received RhCMV/gag at week 0 and Ad5/gag at week 14. Note that CM9 responses do not arise after RhCMV/gag vaccination, but do develop after subsequent Ad5/gag vaccination.
  • FIG. 18 Comparison of the ability of RhCMV(gag) (wt) vs. ⁇ US2-11(gag) (US2-11 KO) vectors to infect RhCMV seronegative (CMV-na ⁇ ve) RM (left panel), RhCMV seropositive RM (middle panel), and RhCMV seropositive RM that were depleted of CD8+ lymphocytes with mAb cM-T807 at the time of inoculation (right panel; 100% CD8+ T cell depletion in blood for 14 days).
  • RhCMV vectors may infect RhCMV-na ⁇ ve RM, but not RhCMV+RM, unless CD8+ T cells are depleted during the first 2 weeks. Infection or lack or infection was confirmed by isolation or failure of isolation, respectively, of the designated vector after co-culture of urine in all cases.
  • FIG. 19 Comparison of CD8+ T cell epitope targeting of SIVgag-specific responses arising after vaccination of Mamu A*01+, CMV-na ⁇ ve RM with wt RhCMV(gag) vs. ⁇ US2-11(gag) (US2-11 KO RhCMV(gag)) vectors.
  • FIG. 20 Recognition of individual, consecutive gag 15 mer peptides by 3 each Mamu A*01+, CMV-na ⁇ ve RM vaccinated with RhCMV(gag) (WT) vs. ⁇ US2-11(gag) (KO) vectors. Note that whereas both wt and KO vectors elicit diverse CD8+ T cell recognition of gag epitopes, only the KO vector-elicited responses include recognition of peptides containing conventional immunodominant epitopes (yellow rectangles; epitopes designated at top).
  • FIG. 21 Antigen-specific response assays: routine staining panel.
  • FIG. 22 Deletion of the pp71-homologue Rh110 attenuates RhCMV in vitro. Reduced growth of RhCMV ⁇ Rh110 and ⁇ Rh110(retanef), but not control RhCMV WT virus on telomerized rhesus fibroblasts (tRF). Growth is rescued by growth in pp71-expressing complementing cells (tRFs+pp71 tet). Fibroblasts were infected with the indicated viruses at a multiplicity of infection (MOI) of 0.01. Culture supernatant was collected at the indicated days and the viral titer was determined on pp71-expressing complementing cells.
  • MOI multiplicity of infection
  • Multi-step growth curves show replication deficiency of only ⁇ Rh110 and ⁇ Rh110(retanef), but not WT RhCMV on normal tRFs.
  • Rescue of normal growth of ⁇ Rh110 and ⁇ Rh110(retanef) on pp71 complementing cells (cTRF/pp71) shows that growth deficiency is due to lack of pp71 expression.
  • FIG. 23A-23C RhCMV ⁇ Rh110 is attenuated in vivo and protects against challenge with ⁇ US2-11(gag).
  • the ⁇ Rh110-infected animals were challenged with 10 7 PFU of RhCMV ⁇ US2-11(gag) ( ⁇ Ugag) and the T cell response against RhCMV lysate was measured at the indicated intervals.
  • RhCMV RhCMV in urine collected at the indicated days from two RM infected with RhCMV(gag) or two RM infected with ⁇ Rh110.
  • Expression of SIVgag, RhCMV IE or the cellular protein GAPDH (included as loading control) was determined from viral cocultures by immunoblot using specific antibodies (S. G. Hansen et al. Science 328, 5974 (2010)).
  • the two animals infected with RhCMV(gag) secreted RhCMV (as shown by IE expression) because they were CMV-positive at the onset of the experiment.
  • FIG. 24 RhCMV lacking the tegument proteins pp65a and pp65b ( ⁇ Rh111-112) encoded by the genes Rh111 and Rh112, respectively, was created.
  • Upper panel: ⁇ Rh111-112 grows like WT RhCMV in tissue culture. Telomerized rhesus fibroblasts (tRFs) were infected with the indicated viruses at a multiplicity of infection (MOI) of 0.01. Culture supernatant was collected at the indicated days and the viral titer was determined.
  • RhCMV IE or RhCMV pp65 were determined from viral cocultures by immunoblot using specific antibodies. All animals infected with WT and ⁇ Rh111-112 secreted RhCMV as shown by IE expression. While the virus secreted from WT-infected animals also expressed pp65, this was not observed for ⁇ Rh111-112 because this virus lacks the genes encoding pp65a and pp65b. This demonstrates that the secreted virus corresponds to ⁇ Rh111-112 and that this virus is not attenuated in vivo.
  • FIG. 25A-25B RhCMV lacking pp65 protects against challenge with ⁇ US2-11(gag).
  • Two animals were infected with 5 ⁇ 10 6 pfu ⁇ Rh111-112 and the T-cell response to SIV gag (overlapping 15 mers; 4 amino acid overlap) and T-cell response to RhCMV lysate was determined by ICCS at the indicated days.
  • animals were challenged by sub-cutaneous inoculation of 10 7 PFU of ⁇ US2-11(gag) and the T cell response to RhCMV and SIVgag was measured.
  • the invention relates to a method of a vector capable of repeatedly infecting an organism which may comprise (a) constructing a vector containing and expressing at least one cytomegalovirus (CMV) glycoprotein, wherein the glycoprotein is US2, US3, US6 or US11, and (b) administering the vector repeatedly into the animal or human.
  • CMV cytomegalovirus
  • any vector advantageously a viral vector, may express one or more of the HCMV glycoproteins US2, US3, US6 and US11 (or the RhCMV homologues Rh182, Rh184, Rh185, Rh189).
  • Viral expression vectors are well known to those skilled in the art and include, for example, viruses such as adenoviruses, adeno-associated viruses (AAV), alphaviruses, herpesviruses (including cytomegalovirus itself), retroviruses and poxviruses, including avipox viruses, attenuated poxviruses, vaccinia viruses, and particularly, the modified vaccinia Ankara virus (MVA; ATCC Accession No. VR-1566).
  • viruses when used as expression vectors are innately non-pathogenic in the selected subjects such as humans or have been modified to render them non-pathogenic in the selected subjects.
  • replication-defective adenoviruses and alphaviruses are well known and may be used as gene delivery vectors. Without US2-11 all of these vectors (except for CMV which contains US2-11 naturally) elicit vector-specific immunity which prohibits their repeated use.
  • any vector may express one or more of the glycoproteins US2, US3, US6 and US11.
  • the vector expresses glycoproteins US2, US3, US6 and US11. More advantageously, the vector contains and expresses all of the glycoproteins within the US2 to US11 region of CMV.
  • the one or more of the glycoproteins US2, US3, US6 and US11 may include, but not limited to, the glycoproteins of U.S. Pat. Nos.
  • any vector may express one or more of the glycoproteins RhCMV homologues Rh182, Rh184, Rh185, Rh189.
  • the vector expresses glycoproteins RhCMV homologues Rh182, Rh184, Rh185 and Rh189.
  • the one or more of the glycoproteins Rh182, Rh184, Rh185 and Rh189 may include, but not limited to, the glycoproteins of U.S. Pat. Nos. 7,635,485; 7,323,619; 6,964,762; 6,712,612; 6,544,780; 6,426,196; 6,391,632; 5,858,740; 5,834,256; 5,767,250 and 5,750,106.
  • the present invention also encompasses a method of determining efficacy of a CMV vaccine.
  • efficacy of CMV vaccines are difficult to measure because CMV easily superinfects CMV-immune individuals.
  • the invention may comprise (a) administering a CMV vaccine to a test subject, (b) challenging the test subject with a CMV vector, wherein glycoproteins within the US2 to US11 region of CMV are deleted from the CMV vector, and wherein the CMV vector contains and expresses at least one immunogen of the CMV vaccine, and (c) measuring a CD8+ T cell response, wherein the CMV vaccine is efficacious if a CD8+ T cell response is able to prevent infection with the CMV vector lacking the glycoproteins within the US2 to US11 region of CMV and wherein the CMV vector contains and expresses at least one immunogen of the CMV vaccine.
  • RhCMV ⁇ Rh110 is growth-deficient in vitro and is attenuated in vitro since it is not secreted from infected monkeys (see FIG. 22 ).
  • RhCMV ⁇ Rh1110 thus represents an example for an attenuated CMV vaccine.
  • monkeys infected with wildtype-virus typically show a boost of the CMV-specific T cell response and develop a de novo response to SIIVgag (see FIG. 43 ).
  • This result indicates that spread-deficient, attenuated CMV is capable of inducing a T cell response that protects against challenge with US2-11 deleted virus.
  • This result also indicates that a US2-11 deleted virus may be used to monitor the efficacy of the T cell response.
  • a CMV-vector lacking pp71 may be used as a vaccine against CMV.
  • Applicants further believe that a vaccine against HCMV may be validated by challenge with HCMV lacking US2-11.
  • RhCMV lacking the tegument proteins pp65a and pp65b encoded by the genes Rh111 and Rh112, respectively (see FIG. 24 ). These proteins are not required for viral growth in vitro or in vivo since Applicants observed that RhCMV ⁇ Rh111-112 is secreted from infected animals
  • pp65 is an immunodominant protein that is included in current formulations of subunit vaccines for CMV developed by various investigators. To examine whether pp65-specific T cells are required for protection against challenge with ⁇ US2-11, Applicants infected rhesus macaques with RhCMV ⁇ Rh111-112. As expected Applicants observed an immune response against the IE-proteins of CMV, but not against pp65.
  • a pp65-specific T cell response was readily detected in animals infected with RhCMV (blue line).
  • monkeys infected with wildtype-virus typically show a boost of the CMV-specific T cell response and develop a de novo response to SIVgag (see FIG. 25 ).
  • the present invention also relates to a method of inducing a different CD8+ T cell response in an animal, which may comprise (a) administering a CMV vector with at least one cytomegalovirus (CMV) glycoprotein deleted from the CMV vector, wherein the glycoprotein is US2, US3, US6 or US11, and wherein the CMV vector contains and expresses at least one immunogen, and (b) administering the vector to the animal or human, wherein the CD8+ T cell response in the animal or human differs as compared to a CMV vector that contains and expresses the same immunogen and wherein a CMV glycoprotein is not deleted from the CMV vector.
  • CMV cytomegalovirus
  • the present invention also relates to a method of inducing a different pathogen-specific CD8+ T cell response in an animal, which may comprise (a) administering a CMV vector with at least one cytomegalovirus (CMV) glycoprotein deleted from the CMV vector, wherein the glycoprotein is US2, US3, US6 or US11, and wherein the CMV vector contains and expresses at least one pathogen-derived immunogen, and (b) administering the vector to the animal, wherein the CD8+ T cell response in the animal differs as compared to a CMV vaccine with a CMV vector that contains and expresses the same immunogen and wherein a CMV glycoprotein is not deleted from the CMV vector.
  • CMV cytomegalovirus
  • the animal is a human.
  • the pathogen may be a viral pathogen and the immunogen may be a protein derived from the viral pathogen.
  • Viruses include, but are not limited to Adenovirus, coxsackievirus, hepatitis A virus, poliovirus, rhinovirus, Herpes simplex, type 1, Herpes simplex, type 2, Varicella-zoster virus, Epstein-barr virus, Human cytomegalovirus, Human herpesvirus, type 8, Hepatitis B virus, Hepatitis C virus, yellow fever virus, dengue virus, West Nile virus, Human immunodeficiency virus (HIV), Influenza virus, Measles virus, Mumps virus, Parainfluenza virus, Respiratory syncytial virus, Human metapneumovirus, Human papillomavirus, Rabies virus, Rubella virus, Human bocavirus and Parvovirus B19.
  • the pathogen may be a bacterial pathogen and the immunogen may be a protein derived from the bacterial pathogen.
  • the pathogenic bacteria include, but are not limited to, Bordetella pertussis , Borrelia burgdorferi, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Legionella pneumophila, Leptospira interrogans
  • the pathogen may be a parasite and the immunogen may be a protein derived from the parasite pathogen.
  • the parasite may be a protozoan organism or disease caused by a protozoan organism such as, but not limited to, Acanthamoeba, Babesiosis, Balantidiasis, Blastocystosis, Coccidia, Dientamoebiasis, Amoebiasis, Giardia, Isosporiasis, Leishmaniasis, Primary amoebic meningoencephalitis (PAM), Malaria, Rhinosporidiosis, Toxoplasmosis—Parasitic pneumonia, Trichomoniasis, Sleeping sickness and Chagas disease.
  • Acanthamoeba Babesiosis
  • Balantidiasis Balantidiasis
  • Blastocystosis Coccidia
  • Dientamoebiasis Amoebiasis
  • Giardia Iso
  • the parasite may be a helminth organism or worm or a disease caused by a helminth organism such as, but not limted to, Ancylostomiasis/Hookworm, Anisakiasis, Roundworm—Parasitic pneumonia, Roundworm—Baylisascariasis, Tapeworm—Tapeworm infection, Clonorchiasis, Dioctophyme renalis infection, Diphyllobothriasis—tapeworm, Guinea worm—Dracunculiasis, Echinococcosis—tapeworm, Pinworm—Enterobiasis, Liver fluke—Fasciolosis, Fasciolopsiasis—intestinal fluke, Gnathostomiasis, Hymenolepiasis, Loa boa filariasis, Calabar swellings, Mansonelliasis, Filariasis, Metagonimiasis—intestinal fluke, River blindness, Chinese Liver Fluke
  • the parasite may be an organism or disease caused by an organism such as, but not limited to, parasitic worm, Halzoun Syndrome, Myiasis, Chigoe flea, Human Botfly and Candiru.
  • the parasite may be an ectoparasite or disease caused by an ectoparasite such as, but not limited to, Bedbug, Head louse—Pediculosis, Body louse—Pediculosis, Crab louse—Pediculosis, Demodex—Demodicosis, Scabies, Screwworm and Cochliomyia.
  • the pathogen may be a cancer and the immunogen may be a protein derived from the cancer.
  • the cancers include, but are not limited to, Acute lymphoblastic leukemia; Acute myeloid leukemia; Adrenocortical carcinoma; AIDS-related cancers; AIDS-related lymphoma; Anal cancer; Appendix cancer; Astrocytoma, childhood cerebellar or cerebral; Basal cell carcinoma; Bile duct cancer, extrahepatic; Bladder cancer; Bone cancer, Osteosarcoma/Malignant fibrous histiocytoma; Brainstem glioma; Brain tumor; Brain tumor, cerebellar astrocytoma; Brain tumor, cerebral astrocytoma/malignant glioma; Brain tumor, ependymoma; Brain tumor, medulloblastoma; Brain tumor, supratentorial primitive neuroectodermal tumors; Brain tumor, visual pathway and hypothalamic glioma; Breast cancer; Bronchial
  • US2-11 deleted vector may induce a qualitatively different immune response to a heterologous antigen as compared to a recombinant wildtype virus (see FIGS. 19 and 20 ).
  • Applicants inoculate animals carrying different US2-11 deleted vectors expressing antigens from SIV and examining T cell responses SIV antigens such as gag, retanef, env and pol, which are used to vaccinate CMV-na ⁇ ve rhesus macaques (RMs) against SIV.
  • the T cell response is determined as indicated below.
  • CMV cytomegalovirus
  • Further objects of the invention include any or all of: to provide expression products from such recombinants, methods for expressing products from such recombinants, compositions containing the recombinants or the expression products, methods for using the expression products, methods for using the compositions, DNA from the recombinants, and methods for replicating DNA from the recombinants.
  • the invention provides a CMV synthetically modified to contain therein exogenous DNA.
  • the CMV advantageously has had one or more glycoproteins US2, US3, US6 and US11, deleted therefrom.
  • the invention also pertains to any viral vector that contains and expresses one or more CMV glycoproteins US2, US3, US6 and US11.
  • the invention further provides a vector for cloning or expression of heterologous DNA which may comprise the recombinant CMV.
  • the heterologous DNA encodes an expression product which may comprise: an epitope of interest, a biological response modulator, a growth factor, a recognition sequence, a therapeutic gene, or a fusion protein.
  • An epitope of interest is an antigen or immunogen or immunologically active fragment thereof from a pathogen or toxin of veterinary or human interest.
  • An epitope of interest may be an antigen of pathogen or toxin, or from an antigen of a pathogen or toxin, or another antigen or toxin which elicits a response with respect to the pathogen, of from another antigen or toxin which elicits a response with respect to the pathogen.
  • An epitope of interest may be an antigen of a human pathogen or toxin, or from an antigen of a human pathogen or toxin, or another antigen or toxin which elicits a response with respect to the pathogen, or from another antigen or toxin which elicits a response with respect to the pathogen, such as, for instance: a Morbillivirus antigen, e.g., a measles virus antigen such as HA or F; a rabies glycoprotein, e.g., rabies virus glycoprotein G; an influenza antigen, e.g., influenza virus HA or N; a Herpesvirus antigen, e.g., a glycoprotein of a herpes simplex virus (HSV), a human cytomegalovirus (HCMV), Epstein-Barr; a flavivirus antigen, a JEV, Yellow Fever virus or Dengue virus antigen; a Hepatitis virus antigen, e.g.,
  • tetani antigen a mumps antigen; a pneumococcal antigen, e.g., PspA; a Borrelia antigen, e.g., OspA, OspB, OspC of Borrelia associated with Lyme disease such as Borrelia burgdoreferi, Borrelia atzelli and Borrelia garinii ; a chicken pox (varicella zoster) antigen; or a Plasmodium antigen.
  • PspA pneumococcal antigen
  • a Borrelia antigen e.g., OspA, OspB, OspC of Borrelia associated with Lyme disease such as Borrelia burgdoreferi, Borrelia atzelli and Borrelia garinii
  • a chicken pox (varicella zoster) antigen a Plasmodium antigen.
  • the epitope of interest is an immunodeficiency antigen, advantageously HIV or SIV.
  • epitope of interest may be an antigen of any veterinary or human pathogen or from any antigen of any veterinary or human pathogen.
  • the heterologous DNA may be a growth factor or therapeutic gene
  • the recombinant CMV may be used in gene therapy.
  • Gene therapy involves transferring genetic information; and, with respect to gene therapy and immunotherapy, reference is made to U.S. Pat. No. 5,252,479, which is incorporated herein by reference, together with the documents cited in it and on its face, and to WO 94/16716 and U.S. application Ser. No. 08/184,009, filed Jan. 19, 1994, each of which is also incorporated herein by reference, together with the documents cited therein.
  • the growth factor or therapeutic gene may encode a disease-fighting protein, a molecule for treating cancer, a tumor suppressor, a cytokine, a tumor associated antigen, or interferon; and, the growth factor or therapeutic gene may, for example, be selected from the group consisting of a gene encoding alpha-globin, beta-globin, gamma-globin, granulocyte macrophage-colony stimulating factor, tumor necrosis factor, an interleukin, macrophage colony stimulating factor, granulocyte colony stimulating factor, erythropoietin, mast cell growth factor, tumor suppressor p53, retinoblastoma, interferon, melanoma associated antigen or B7.
  • the invention still further provides an immunogenic, immunological or vaccine composition containing the recombinant CMVvirus or vector, and a pharmaceutically acceptable carrier or diluent.
  • An immunological composition containing the recombinant CMV virus or vector (or an expression product thereof) elicits an immunological response—local or systemic. The response may, but need not be, protective.
  • An immunogenic composition containing the recombinant CMVvirus or vector (or an expression product thereof) likewise elicits a local or systemic immunological response which may, but need not be, protective.
  • a vaccine composition elicits a local or systemic protective response.
  • the terms “immunological composition” and “immunogenic composition” include a “vaccine composition” (as the two former terms may be protective compositions).
  • the invention therefore also provides a method of inducing an immunological response in a host vertebrate which may comprise administering to the host an immunogenic, immunological or vaccine composition which may comprise the recombinant CMVvirus or vector and a pharmaceutically acceptable carrier or diluent.
  • an immunogenic, immunological or vaccine composition which may comprise the recombinant CMVvirus or vector and a pharmaceutically acceptable carrier or diluent.
  • animal includes all vertebrate species, except humans; and “vertebrate” includes all vertebrates, including animals (as “animal” is used herein) and humans.
  • a subset of “animal” is “mammal”, which for purposes of this specification includes all mammals, except humans.
  • the invention even further provides a therapeutic composition containing the recombinant CMVvirus or vector and a pharmaceutically acceptable carrier or diluent.
  • the therapeutic composition is useful in the gene therapy and immunotherapy embodiments of the invention, e.g., in a method for transferring genetic information to an animal or human in need of such which may comprise administering to the host the composition; and, the invention accordingly includes methods for transferring genetic information.
  • the invention provides a method of expressing a protein or gene product or an expression product which may comprise infecting or transfecting a cell in vitro with a recombinant CMVvirus or vector of the invention and optionally extracting, purifying or isolating the protein, gene product or expression product or DNA from the cell.
  • the invention provides a method for cloning or replicating a heterologous DNA sequence which may comprise infecting or transfecting a cell in vitro or in vivo with a recombinant CMV virus or vector of the invention and optionally extracting, purifying or isolating the DNA from the cell or progeny virus
  • the invention in another aspect provides a method for preparing the recombinant CMV virus or vector of the invention which may comprise inserting the exogenous DNA into a non-essential region of the CMVgenome.
  • the method may further comprise deleting a non-essential region from the CMVgenome, preferably prior to inserting the exogenous DNA.
  • the method may comprise in vivo recombination.
  • the method may comprise transfecting a cell with CMV DNA in a cell-compatible medium in the presence of donor DNA which may comprise the exogenous DNA flanked by DNA sequences homologous with portions of the CMV genome, whereby the exogenous DNA is introduced into the genome of the CMV, and optionally then recovering CMV modified by the in vivo recombination.
  • the method may also comprise cleaving CMV DNA to obtain cleaved CMV DNA, ligating the exogenous DNA to the cleaved CMV DNA to obtain hybrid CMV-exogenous DNA, tranfecting a cell with the hybrid CMV-exogenous DNA, and optionally then recovering CMVmodified by the presence of the exogenous DNA.
  • the invention accordingly also provides a plasmid which may comprise donor DNA not naturally occurring in CMVencoding a polypeptide foreign to CMV, the donor DNA is within a segment of CMV DNA which would otherwise be co-linear with a non-essential region of the CMVgenome such that DNA from a non-essential region of CMVis flanking the donor DNA.
  • the exogenous DNA may be inserted into CMV to generate the recombinant CMV in any orientation which yields stable integration of that DNA, and expression thereof, when desired.
  • the exogenous DNA in the recombinant CMVvirus or vector of the invention may include a promoter.
  • the promoter may be from a herpesvirus.
  • the promoter may be a cytomegalovirus (CMV) promoter, such as a human CMV (HCMV) or murine CMVpromoter.
  • CMV cytomegalovirus
  • the promoter may be a truncated transcriptionally active promoter which may comprise a region transactivated with a transactivating protein provided by the virus and the minimal promoter region of the full-length promoter from which the truncated transcriptionally active promoter is derived.
  • a “promoter” is composed of an association of DNA sequences corresponding to the minimal promoter and upstream regulatory sequences;
  • a “minimal promoter” is composed of the CAP site plus TATA box (minimum sequences for basic level of transcription; unregulated level of transcription); and, “upstream regulatory sequences” are composed of the upstream element(s) and enhancer sequence(s).
  • the term “truncated” indicates that the full-length promoter is not completely present, i.e., that some portion of the full-length promoter has been removed.
  • the truncated promoter may be derived from a herpesvirus such as MCMVor HCMV, e.g., HCMV-IE or MCMV-IE.
  • the inventive promoter is preferably a herpesvirus, e.g., a MCMVor HCMVsuch as MCMV-IE or HCMV-IE promoter; and, there may be up to a 40% and even up to a 90% reduction in size, from a full-length promoter, based upon base pairs.
  • a herpesvirus e.g., a MCMVor HCMVsuch as MCMV-IE or HCMV-IE promoter
  • the invention thus also provides an expression cassette for insertion into a recombinant virus or plasmid which may comprise the truncated transcriptionally active promoter.
  • the expression cassette may further include a functional truncated polyadenylation signal; for instance an SV40 polyadenylation signal which is truncated, yet functional. Considering that nature provided a larger signal, it is indeed surprising that a truncated polyadenylation signal is functional; and, a truncated polyadenylation signal addresses the insert size limit problems of recombinant viruses such as CMV.
  • the expression cassette may also include exogenous or heterologous DNA with respect to the virus or system into which it is inserted; and that DNA may be exogenous or heterologous DNA as described herein.
  • the present invention encompasses CMV, recombinants which may comprise the HCMV-IE or MCMV-IE promoter, preferably a truncated promoter therefrom.
  • CMV CMV
  • recombinants which may comprise the HCMV-IE or MCMV-IE promoter, preferably a truncated promoter therefrom.
  • the HCMV-IE or MCMV-IE promoter or a truncated promoter therefrom is transactivated by CMV-induced gene products.
  • the invention further comprehends antibodies elicited by the inventive compositions and/or recombinants and uses for such antibodies.
  • the antibodies, or the product (epitopes of interest) which elicited them, or monoclonal antibodies from the antibodies, may be used in binding assays, tests or kits to determine the presence or absence of an antigen or antibody.
  • Flanking DNA used in the invention may be from the site of insertion or a portion of the genome adjacent thereto (wherein “adjacent” includes contiguous sequences, e.g., codon or codons, as well as up to as many sequences, e.g., codon or codons, before there is an intervening insertion site).
  • the exogenous or heterologous DNA may be DNA encoding any of the aforementioned epitopes of interest, as listed above.
  • the exogenous DNA may include a marker, e.g., a color or light marker.
  • the exogenous DNA may also code for a product which would be detrimental to an insect host such that the expression product may be a pesticide or insecticide.
  • the exogenous DNA may also code for an anti-fungal polypeptide; and, for information on such a polypeptide and DNA therefor, reference is made to U.S. Pat. No. 5,421,839 and the documents cited therein, incorporated herein by reference.
  • the heterologous or exogenous DNA in recombinants of the invention preferably encodes an expression product which may comprise: an epitope of interest, a biological response modulator, a growth factor, a recognition sequence, a therapeutic gene, or a fusion protein.
  • an expression product which may comprise: an epitope of interest, a biological response modulator, a growth factor, a recognition sequence, a therapeutic gene, or a fusion protein.
  • antigens for use in vaccine or immunological compositions see also Stedman's Medical Dictionary (24th edition, 1982, e.g., definition of vaccine (for a list of antigens used in vaccine formulations; such antigens or epitopes of interest from those antigens may be used in the invention, as either an expression product of the inventive recombinant virus, or in a multivalent composition containing an inventive recombinant virus or an expression product therefrom).
  • epitopes of interest one skilled in the art may determine an epitope or immunodominant region of a peptide or polypeptide and ergo the coding DNA therefor from the knowledge of the amino acid and corresponding DNA sequences of the peptide or polypeptide, as well as from the nature of particular amino acids (e.g., size, charge, etc.) and the codon dictionary, without undue experimentation.
  • a general method for determining which portions of a protein to use in an immunological composition focuses on the size and sequence of the antigen of interest. “In general, large proteins, because they have more potential determinants are better antigens than small ones. The more foreign an antigen, that is the less similar to self configurations which induce tolerance, the more effective it is in provoking an immune response.” Ivan Roitt, Essential Immunology, 1988.
  • the skilled artisan may maximize the size of the protein encoded by the DNA sequence to be inserted into the viral vector (keeping in mind the packaging limitations of the vector).
  • the DNA sequence may exclude introns (regions of a gene which are transcribed but which are subsequently excised from the primary RNA transcript).
  • the DNA sequence may code for a peptide at least 8 or 9 amino acids long. This is the minimum length that a peptide needs to be in order to stimulate a CD4+ T cell response (which recognizes virus infected cells or cancerous cells). A minimum peptide length of 13 to 25 amino acids is useful to stimulate a CD8+ T cell response (which recognizes special antigen presenting cells which have engulfed the pathogen). See Kendrew, supra. However, as these are minimum lengths, these peptides are likely to generate an immunological response, i.e., an antibody or T cell response; but, for a protective response (as from a vaccine composition), a longer peptide is preferred.
  • the DNA sequence preferably encodes at least regions of the peptide that generate an antibody response or a T cell response.
  • One method to determine T and B cell epitopes involves epitope mapping.
  • the protein of interest “is fragmented into overlapping peptides with proteolytic enzymes.
  • the individual peptides are then tested for their ability to bind to an antibody elicited by the native protein or to induce T cell or B cell activation. This approach has been particularly useful in mapping T-cell epitopes since the T cell recognizes short linear peptides complexed with MHC molecules (see FIG. 20 ).
  • the method is less effective for determining B-cell epitopes” since B cell epitopes are often not linear amino acid sequence but rather result from the tertiary structure of the folded three dimensional protein. Janis Kuby, Immunology, (1992) pp. 79-80.
  • Another method for determining an epitope of interest is to choose the regions of the protein that are hydrophilic. Hydrophilic residues are often on the surface of the protein and are therefore often the regions of the protein which are accessible to the antibody. Janis Kuby, Immunology, (1992) p. 81.
  • Yet another method for determining an epitope of interest is to perform an X-ray crystallographic analysis of the antigen (full length)-antibody complex. Janis Kuby, Immunology, (1992) p. 80.
  • Still another method for choosing an epitope of interest which may generate a T cell response is to identify from the protein sequence potential HLA anchor binding motifs which are peptide sequences which are known to be likely to bind to the MHC molecule.
  • the peptide which is a putative epitope of interest, to generate a T cell response should be presented in a MHC complex.
  • the peptide preferably contains appropriate anchor motifs for binding to the MHC molecules, and should bind with high enough affinity to generate an immune response.
  • Factors which may be considered are: the HLA type of the patient (vertebrate, animal or human) expected to be immunized, the sequence of the protein, the presence of appropriate anchor motifs and the occurrence of the peptide sequence in other vital cells.
  • T cells recognize proteins only when the protein has been cleaved into smaller peptides and is presented in a complex called the “major histocompatability complex MHC” located on another cell's surface.
  • MHC complexes There are two classes of MHC complexes—class I and class II, and each class is made up of many different alleles. Different patients have different types of MHC complex alleles; they are said to have a different HLA type.
  • Class I MHC complexes are found on virtually every cell and present peptides from proteins produced inside the cell. Thus, Class I MHC complexes are useful for killing cells which when infected by viruses or which have become cancerous and as the result of expression of an oncogene.
  • T cells which have a protein called CD8 on their surface, bind to the MHC class I cells and secrete lymphokines. The lymphokines stimulate a response; cells arrive and kill the viral infected cell.
  • Class II MHC complexes are found only on antigen-presenting cells and are used to present peptides from circulating pathogens which have been endocytosed by the antigen-presenting cells.
  • T cells which have a protein called CD4 bind to the MHC class II cells and kill the cell by exocytosis of lytic granules.
  • Peptide length the peptide should be at least 8 or 9 amino acids long to fit into the MHC class I complex and at least 13-25 amino acids long to fit into a class II MCH complex. This length is a minimum for the peptide to bind to the MHC complex. It is preferred for the peptides to be longer than these lengths because cells may cut the expressed peptides.
  • the peptide should contain an appropriate anchor motif which will enable it to bind to the various class I or class II molecules with high enough specificity to generate an immune response (See Bocchia, M.
  • an epitope of interest by comparing the protein sequence with sequences listed in the protein data base. Regions of the protein which share little or no homology are better choices for being an epitope of that protein and are therefore useful in a vaccine or immunological composition. Regions which share great homology with widely found sequences present in vital cells should be avoided.
  • Another method is simply to generate or express portions of a protein of interest, generate monoclonal antibodies to those portions of the protein of interest, and then ascertain whether those antibodies inhibit growth in vitro of the pathogen from which the from which the protein was derived.
  • the skilled artisan may use the other guidelines set forth in this disclosure and in the art for generating or expressing portions of a protein of interest for analysis as to whether antibodies thereto inhibit growth in vitro.
  • portions of a protein of interest may be generated by: selecting 8 to 9 or 13 to 25 amino acid length portions of the protein, selecting hydrophilic regions, selecting portions shown to bind from X-ray data of the antigen (full length)-antibody complex, selecting regions which differ in sequence from other proteins, selecting potential HLA anchor binding motifs, or any combination of these methods or other methods known in the art.
  • Epitopes recognized by antibodies are expressed on the surface of a protein. To determine the regions of a protein most likely to stimulate an antibody response one skilled in the art may preferably perform an epitope map, using the general methods described above, or other mapping methods known in the art.
  • a biological response modulator modulates biological activity; for instance, a biological response modulator is a modulatory component such as a high molecular weight protein associated with non-NMDA excitatory amino acid receptors and which allosterically regulates affinity of AMPA binding (See Kendrew, supra).
  • the recombinant of the present invnention may express such a high molecular weight protein.
  • Modulation of activity may be carried out through mechanisms as complicated and intricate as allosteric induced quaternary change to simple presence/absence, e.g., expression/degradation, systems. Indeed, the repression/activation of expression of many biological molecules is itself mediated by molecules whose activities are capable of being modulated through a variety of mechanisms.
  • modulation of biological functions may be mediated simply through the proper/improper localization of a molecule.
  • Molecules may function to provide a growth advantage or disadvantage only if they are targeted to a particular location.
  • a molecule may be typically not taken up or used by a cell, as a function of that molecule being first degredaded by the cell by secretion of an enzyme for that degradation.
  • production of the enzyme by a recombinant may regulate use or uptake of the molecule by a cell.
  • the recombinant may express a molecule which binds to the enzyme necessary for uptake or use of a molecule, thereby similarly regulating its uptake or use.
  • Localization targeting of proteins carried out through cleavage of signal peptides another type of modulation or regulation.
  • a specific endoprotease catalytic activity may be expressed by the recombinant.
  • RNA virus poly-proteins Other examples of mechanisms through which modulation of function may occur are RNA virus poly-proteins, allosteric effects, and general covalent and non-covalent steric hindrance.
  • HIV is a well studied example of an RNA virus which expresses non-functional poly-protein constructs.
  • the gag, pol, and env poly-proteins are processed to yield, respectively, the viral structural proteins p17, p24, and p15-reverse transcriptase and integrase—and the two envelope proteins gp41 and gp120” (Kohl et al., PNAS USA 85:4686-90 (1988)).
  • the functional usefulness of enzymes may also be modulated by altering their capability of catalyzing a reaction.
  • modulated molecules are zymogens, formation/disassociation of multi-subunit functional complexes, RNA virus poly-protein chains, allosteric interactions, general steric hindrance (covalent and non-covalent) and a variety of chemical modifications such as phosphorylation, methylation, acetylation, adenylation, and uridenylation (see Table 1 of Neidhardt, supra, at page 315 and Table 2 at page 73).
  • Zymogens are examples of naturally occurring protein fusions which cause modulation of enzymatic activity.
  • Zymogens are one class of proteins which are converted into their active state through limited proteolysis. See Table 3 of Reich, Proteases and Biological Control, Vol. 2, (1975) at page 54). Nature has developed a mechanism of down-modulating the activity of certain enzymes, such as trypsin, by expressing these enzymes with additional “leader” peptide sequences at their amino termini. With the extra peptide sequence the enzyme is in the inactive zymogen state. Upon cleavage of this sequence the zymogen is converted to its enzymatically active state. The overall reaction rates of the zymogen are “about 10.sup.5-10.sup.6 times lower than those of the corresponding enzyme” (See Table 3 of Reich, supra at page 54).
  • a recombinant may express peptide sequences containing additional amino acids at one or both terminii.
  • the formation or disassociation of multi-subunit enzymes is another way through which modulation may occur. Different mechanisms may be responsible for the modulation of activity upon formation or disassociation of multi-subunit enzymes.
  • the recombinant of the invention may express a molecule which sterically hinders a naturally occurring enzyme or enzyme complex, so as to modulate biological functions.
  • Suicide substrates which irreversibly bind to the active site of an enzyme at a catalytically important amino acid in the active site are examples of covalent modifications which sterically block the enzymatic active site.
  • An example of a suicide substrate is TPCK for chymotrypsin (Fritsch, Enzyme Structure and Mechanism, 2d ed; Freeman & Co. Publishers, 1984)). This type of modulation is possible by the recombinant expressing a suitable suicide substrate, to thereby modulate biological responses (e.g., by limiting enzyme activity).
  • non-covalent steric hindrance including many repressor molecules.
  • the recombinant may express repressor molecules which are capable of sterically hindering and thus down-modulating the function of a DNA sequence by preventing particular DNA-RNA polymerase interactions.
  • Aspartate transcarbamoylase is a well characterized allosteric enzyme. Interacting with the catalytic subunits are regulatory domains. Upon binding to CTP or UTP the regulatory subunits are capable of inducing a quaternary structural change in the holoenzyme causing down-modulation of catalytic activity. In contrast, binding of ATP to the regulatory subunits is capable of causing up-modulation of catalytic activity (Fritsch, supra). Using methods of the invention, molecules may be expressed which are capable of binding and causing modulatory quaternary or tertiary changes.
  • a growth factor may be defined as multifunctional, locally acting intercellular signalling peptides which control both ontogeny and maintenance of tissue and function (see Kendrew, especially at page 455 et seq.).
  • the growth factor or therapeutic gene may encode a disease-fighting protein, a molecule for treating cancer, a tumor suppressor, a cytokine, a tumor associated antigen, or interferon; and, the growth factor or therapeutic gene may, for example, be selected from the group consisting of a gene encoding alpha-globin, beta-globin, gamma-globin, granulocyte macrophage-colony stimulating factor, tumor necrosis factor, an interleukin (e.g., an interleukin selected from interleukins 1 to 14, or 1 to 11, or any combination thereof), macrophage colony stimulating factor, granulocyte colony stimulating factor, erythropoietin, mast cell growth factor, tumor suppressor p53, retinoblastoma, interferon, melanoma associated antigen or B7.
  • a gene encoding alpha-globin, beta-globin, gamma-globin, granulocyte macrophage-colony stimulating factor,
  • U.S. Pat. No. 5,252,479 provides a list of proteins which may be expressed in an adenovirus system for gene therapy, and the skilled artisan is directed to that disclosure.
  • WO 94/16716 and U.S. application Ser. No. 08/184,009, filed Jan. 19, 1994, provide genes for cytokines and tumor associated antigens and immunotherapy methods, including ex vivo methods, and the skilled artisan is directed to those disclosures.
  • the exogenous or heterologous DNA may itself include a promoter for driving expression in the recombinant CMV, or the exogenous DNA may simply be coding DNA and appropriately placed downstream from an endogenous promoter to drive expression. Further, multiple copies of coding DNA or use of a strong or early promoter or early and late promoter, or any combination thereof, may be done so as to amplify or increase expression.
  • the exogenous or heterologous DNA may be suitably positioned with respect to an endogenous promoter like the E3 or the MLP promoters, or those promoters may be translocated to be inserted at another location, with the exogenous or heterologous DNA.
  • the coding DNA may be DNA coding for more than one protein so as to have expression of more than one product from the recombinant CMV.
  • the expression products may be antigens, immunogens or epitopes of interest; and therefore, the invention further relates to immunological, antigenic or vaccine compositions containing the expression products. Further, since the CMVvector, in certain instances, may be administered directly to a suitable host, the invention relates to compositions containing the CMV vector.
  • the invention relates to methods for expressing a product, e.g., which may comprise inserting the exogenous DNA into a CMVas a vector, e.g., by restriction/ligation or by recombination followed by infection or transfection of suitable cells in vitro with a recombinant CMV, and optionally extracting, purifying or isolating the expression product from the cells. Any suitable extraction, purification or isolation techniques may be employed.
  • the protein(s) from the expression of the exogenous DNA are collected by known techniques such as chromatography (see Robbins, EPA 0162738A1; Panicali, EPA 0261940A2); Richardson, supra; Smith et al., supra; Pennock et al., supra; EP Patent Publication No. 0265785).
  • the collected protein(s) may then be employed in a vaccine, antigenic or immunological composition which also contains a suitable carrier.
  • the recombinant CMV may be used to prepare proteins such as antigens, immunogens, epitopes of interest, etc. which may be further used in immunological, antigenic or vaccine compositions.
  • a recombinant CMV expressing a product detrimental to growth or development of insects may be used to prepare an insecticide
  • a recombinant CMV expressing a product detrimental to growth of plants may be used to prepare a herbicide (by isolating the expression product and admixing it with an insecticidally or herbicidally acceptable carrier or diluent)
  • a recombinant CMV expressing an anti-fungal polypeptide may be used to prepare an anti-fungal preparation (by isolating the expression product and admixing it with a suitable carrier or diluent).
  • the invention further relates to products therefrom; namely, antibodies and uses thereof. More in particular, the expression products may elicit antibodies.
  • the antibodies may be formed into monoclonal antibodies; and, the antibodies or expression products may be used in kits, assays, tests, and the like involving binding, so that the invention relates to these uses too.
  • the invention since the recombinants of the invention may be used to replicate DNA, the invention relates to recombinant CMVas a vector and methods for replicating DNA by infecting or transfecting cells with the recombinant and harvesting DNA therefrom. The resultant DNA may be used as probes or primers or for amplification.
  • compositions of the invention such as immunological, antigenic or vaccine compositions or therapeutic compositions may be via a parenteral route (intradermal, intramuscular or subcutaneous). Such an administration enables a systemic immune response.
  • the administration may be via a mucosal route, e.g., oral, nasal, genital, etc. Such an administration enables a local immune response.
  • compositions containing the CMV recombinants of the invention or expression products may be prepared in accordance with standard techniques well known to those skilled in the pharmaceutical arts. Such compositions may be administered in dosages and by techniques well known to those skilled in the medical arts taking into consideration such factors as the breed or species, age, sex, weight, and condition of the particular patient, and the route of administration.
  • the compositions may be administered alone, or may be co-administered or sequentially administered with other compositions of the invention or with other immunological, antigenic or vaccine or therapeutic compositions.
  • Such other compositions may include purified native antigens or epitopes or antigens or epitopes from the expression by a recombinant CMV or another vector system; and are administered taking into account the aforementioned factors.
  • compositions of the invention include liquid preparations for orifice, e.g., oral, nasal, anal, genital, e.g., vaginal, etc., administration such as suspensions, syrups or elixirs; and, preparations for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration (e.g., injectable administration) such as sterile suspensions or emulsions.
  • the recombinant may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose or the like.
  • Antigenic, immunological or vaccine compositions typically may contain an adjuvant and an amount of the recombinant CMVor expression product to elicit the desired response.
  • alum aluminum phosphate or aluminum hydroxide
  • Saponin and its purified component Quil A, Freund's complete adjuvant and other adjuvants used in research and veterinary applications have toxicities which limit their potential use in human vaccines.
  • Chemically defined preparations such as muramyl dipeptide, monophosphoryl lipid A, phospholipid conjugates such as those described by Goodman-Snitkoff et al. J. Immunol.
  • encapsulation of the protein within a proteoliposome as described by Miller et al., J. Exp. Med. 176:1739-1744 (1992) and incorporated by reference herein, and encapsulation of the protein in lipid vesicles such as Novasome lipid vesicles (Micro Vescular Systems, Inc., Nashua, N.H.) may also be used.
  • lipid vesicles such as Novasome lipid vesicles (Micro Vescular Systems, Inc., Nashua, N.H.) may also be used.
  • the composition may be packaged in a single dosage form for immunization by parenteral (i.e., intramuscular, intradermal or subcutaneous) administration or orifice administration, e.g., perlingual (i.e., oral), intragastric, mucosal including intraoral, intraanal, intravaginal, and the like administration.
  • parenteral i.e., intramuscular, intradermal or subcutaneous
  • orifice administration e.g., perlingual (i.e., oral), intragastric, mucosal including intraoral, intraanal, intravaginal, and the like administration.
  • the effective dosage and route of administration are determined by the nature of the composition, by the nature of the expression product, by expression level if recombinant CMV is directly used, and by known factors, such as breed or species, age, sex, weight, condition and nature of host, as well as LD 50 and other screening procedures which are known and do not require undue experimentation.
  • Dosages of expressed product may range from a few to a few hundred micrograms, e.g., 5 to 500 ⁇ g.
  • the inventive recombinant may be administered in any suitable amount to achieve expression at these dosage levels.
  • the vaccinal CMV is administered in an amount of about 10 3.5 pfu; thus, the inventive recombinant is preferably administered in at least this amount; more preferably about 10 4 pfu to about 10 6 pfu.
  • Other suitable carriers or diluents may be water or a buffered saline, with or without a preservative.
  • the expression product or recombinant CMV may be lyophilized for resuspension at the time of administration or may be in solution.
  • the carrier may also be a polymeric delayed release system.
  • Synthetic polymers are particularly useful in the formulation of a composition having controlled release. An early example of this was the polymerization of methyl methacrylate into spheres having diameters less than one micron to form so-called nano particles, reported by Kreuter, J., Microcapsules and Nanoparticles in Medicine and Pharmacology, M. Donbrow (Ed). CRC Press, p. 125-148.
  • Microencapsulation has been applied to the injection of microencapsulated pharmaceuticals to give a controlled release.
  • a number of factors contribute to the selection of a particular polymer for microencapsulation.
  • the reproducibility of polymer synthesis and the microencapsulation process, the cost of the microencapsulation materials and process, the toxicological profile, the requirements for variable release kinetics and the physicochemical compatibility of the polymer and the antigens are all factors that must be considered.
  • useful polymers are polycarbonates, polyesters, polyurethanes, polyorthoesters and polyamides, particularly those that are biodegradable.
  • PLGA poly (d,1-lactide-co-glycolide)
  • PLGA poly (d,1-lactide-co-glycolide)
  • the entrapment of antigens in PLGA microspheres of 1 to 10 microns in diameter has been shown to have a remarkable adjuvant effect when administered orally.
  • the PLGA microencapsulation process uses a phase separation of a water-in-oil emulsion.
  • the compound of interest is prepared as an aqueous solution and the PLGA is dissolved in a suitable organic solvents such as methylene chloride and ethyl acetate. These two immiscible solutions are co-emulsified by high-speed stirring.
  • a non-solvent for the polymer is then added, causing precipitation of the polymer around the aqueous droplets to form embryonic microcapsules.
  • microcapsules are collected, and stabilized with one of an assortment of agents (polyvinyl alcohol (PVA), gelatin, alginates, polyvinylpyrrolidone (PVP), methyl cellulose) and the solvent removed by either drying in vacuo or solvent extraction.
  • agents polyvinyl alcohol (PVA), gelatin, alginates, polyvinylpyrrolidone (PVP), methyl cellulose
  • solid, including solid-containing-liquid, liquid, and gel including “gel caps” compositions are envisioned.
  • inventive vectors e.g., recombinant CMV, and the expression products therefrom may stimulate an immune or antibody response in animals.
  • monoclonal antibodies may be prepared and, those monoclonal antibodies, may be employed in well known antibody binding assays, diagnostic kits or tests to determine the presence or absence of antigen(s) and therefrom the presence or absence of the natural causative agent of the antigen or, to determine whether an immune response to that agent or to the antigen(s) has simply been stimulated.
  • Monoclonal antibodies are immunoglobulin produced by hybridoma cells.
  • a monoclonal antibody reacts with a single antigenic determinant and provides greater specificity than a conventional, serum-derived antibody. Furthermore, screening a large number of monoclonal antibodies makes it possible to select an individual antibody with desired specificity, avidity and isotype.
  • Hybridoma cell lines provide a constant, inexpensive source of chemically identical antibodies and preparations of such antibodies may be easily standardized. Methods for producing monoclonal antibodies are well known to those of ordinary skill in the art, e.g., Koprowski, H. et al., U.S. Pat. No. 4,196,265, issued Apr. 1, 1989, incorporated herein by reference.
  • Monoclonal antibodies have also been used to recover materials by immunoadsorption chromatography, e.g. Milstein, C., 1980, Scientific American 243:66, 70, incorporated herein by reference.
  • inventive recombinant CMVor expression products therefrom may be used to stimulate a response in cells in vitro or ex vivo for subsequent reinfusion into a patient.
  • the reinfusion is to stimulate an immune response, e.g., an immunological or antigenic response such as active immunization.
  • the reinfusion is to stimulate or boost the immune system against a pathogen.
  • the recombinant CMV of the invention are also useful for generating DNA for probes or for PCR primers which may be used to detect the presence or absence of hybridizable DNA or to amplify DNA, e.g., to detect a pathogen in a sample or for amplifying DNA.
  • the invention comprehends promoters and expression cassettes which are useful in adenovirus systems, as well as in any viral or cell system which provides a transactivating protein.
  • the expression cassette of the invention may further include a functional truncated polyadenylation signal; for instance an SV40 polyadenylation signal which is truncated, yet functional.
  • the expression cassette may contain exogenous or heterologous DNA (with respect to the virus or system into which the promoter or expression cassette is being inserted); for instance exogenous or heterologous coding DNA as herein described above, and in the Examples. This DNA may be suitably positioned and operably linked to the promoter for expression.
  • the expression cassette may be inserted in any orientation; preferably the orientation which obtains maximum expression from the system or virus into which the expression cassette is inserted.
  • promoter and expression cassette are specifically exemplified with reference to adenoviruses, the skilled artisan may adapt these embodiments of the invention to other viruses and to plasmids for cells such as eukaryotic cells, without undue experimentation, by simply ascertaining whether the virus, plasmid, cell or system provides the transactivating protein.
  • HCMV promoters reference is made to U.S. Pat. Nos. 5,168,062 and 5,385,839, incorporated herein by reference.
  • transfecting cells with plasmid DNA for expression therefrom reference is made to Felgner et al. (1994), J. Biol. Chem. 269, 2550-2561, incorporated herein by reference.
  • direct injection of plasmid DNA as a simple and effective method of vaccination against a variety of infectious diseases reference is made to Science, 259:1745-49, 1993, incorporated herein by reference. It is therefore within the scope of this invention that the inventive promoter and expression cassette be used in systems other than adenovirus; for example, in plasmids for the direct injection of plasmid DNA.
  • the protein fragments of the present invention form a further aspect of the invention; and, such compounds may be used in methods of medical treatments, such as for diagnosis, preventing or treating HIV or for eliciting antibodies for diagnosis of HIV, including use in vaccines. Further, such compounds may be used in the preparation of medicaments for such treatments or prevention, or compositions for diagnostic purposes. The compounds may be employed alone or in combination with other treatments, vaccines or preventatives; and, the compounds may be used in the preparation of combination medicaments for such treatments or prevention, or in kits containing the compound and the other treatment or preventative.
  • the present invention also encompassed the use of the protein fragments of the present invention described herein as immunogens, advantageously as HIV-1 vaccine components.
  • protein protein
  • peptide polypeptide
  • amino acid sequence amino acid sequence
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling or bioactive component.
  • the terms “antigen” or “immunogen” are used interchangeably to refer to a substance, typically a protein, which is capable of inducing an immune response in a subject.
  • the term also refers to proteins that are immunologically active in the sense that once administered to a subject (either directly or by administering to the subject a nucleotide sequence or vector that encodes the protein) is able to evoke an immune response of the humoral and/or cellular type directed against that protein.
  • antibody includes intact molecules as well as fragments thereof, such as Fab, F(ab′) 2 , Fv and scFv which are capable of binding the epitope determinant. These antibody fragments retain some ability to selectively bind with its antigen or receptor and include, for example:
  • a “neutralizing antibody” may inhibit the entry of HIV-1 virus for example SF162 and/or JRCSF with a neutralization index >1.5 or >2.0.
  • Broad and potent neutralizing antibodies may neutralize greater than about 50% of HIV-1 viruses (from diverse clades and different strains within a Glade) in a neutralization assay.
  • the inhibitory concentration of the monoclonal antibody may be less than about 25 mg/ml to neutralize about 50% of the input virus in the neutralization assay.
  • proteins including the antibodies and/or antigens of the invention may differ from the exact sequences illustrated and described herein.
  • the invention contemplates deletions, additions and substitutions to the sequences shown, so long as the sequences function in accordance with the methods of the invention.
  • particularly preferred substitutions will generally be conservative in nature, i.e., those substitutions that take place within a family of amino acids.
  • amino acids are generally divided into four families: (1) acidic—aspartate and glutamate; (2) basic—lysine, arginine, histidine; (3) non-polar—alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar—glycine, asparagine, glutamine, cysteine, serine threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids.
  • nucleotide sequences and “nucleic acid sequences” refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequences, including, without limitation, messenger RNA (mRNA), DNA/RNA hybrids, or synthetic nucleic acids.
  • the nucleic acid may be single-stranded, or partially or completely double-stranded (duplex).
  • Duplex nucleic acids may be homoduplex or heteroduplex.
  • transgene may used to refer to “recombinant” nucleotide sequences that may be derived from any of the nucleotide sequences encoding the proteins of the present invention.
  • the term “recombinant” means a nucleotide sequence that has been manipulated “by man” and which does not occur in nature, or is linked to another nucleotide sequence or found in a different arrangement in nature. It is understood that manipulated “by man” means manipulated by some artificial means, including by use of machines, codon optimization, restriction enzymes, etc.
  • nucleotide sequences may be mutated such that the activity of the encoded proteins in vivo is abrogated.
  • nucleotide sequences may be codon optimized, for example the codons may be optimized for human use.
  • nucleotide sequences of the invention are both mutated to abrogate the normal in vivo function of the encoded proteins, and codon optimized for human use. For example, each of the Gag, Pol, Env, Nef, RT, and Int sequences of the invention may be altered in these ways.
  • the nucleic acid molecules of the invention have a nucleotide sequence that encodes the antigens of the invention and may be designed to employ codons that are used in the genes of the subject in which the antigen is to be produced.
  • codons that are used in the genes of the subject in which the antigen is to be produced.
  • Many viruses including HIV and other lentiviruses, use a large number of rare codons and, by altering these codons to correspond to codons commonly used in the desired subject, enhanced expression of the antigens may be achieved.
  • the codons used are “humanized” codons, i.e., the codons are those that appear frequently in highly expressed human genes (Andre et al., J. Virol.
  • codon usage provides for efficient expression of the transgenic HIV proteins in human cells. Any suitable method of codon optimization may be used. Such methods, and the selection of such methods, are well known to those of skill in the art. In addition, there are several companies that will optimize codons of sequences, such as Geneart (geneart.com). Thus, the nucleotide sequences of the invention may readily be codon optimized.
  • the invention further encompasses nucleotide sequences encoding functionally and/or antigenically equivalent variants and derivatives of the antigens of the invention and functionally equivalent fragments thereof.
  • These functionally equivalent variants, derivatives, and fragments display the ability to retain antigenic activity. For instance, changes in a DNA sequence that do not change the encoded amino acid sequence, as well as those that result in conservative substitutions of amino acid residues, one or a few amino acid deletions or additions, and substitution of amino acid residues by amino acid analogs are those which will not significantly affect properties of the encoded polypeptide.
  • Conservative amino acid substitutions are glycine/alanine; valine/isoleucine/leucine; asparagine/glutamine; aspartic acid/glutamic acid; serine/threonine/methionine; lysine/arginine; and phenylalanine/tyrosine/tryptophan.
  • the variants have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology or identity to the antigen, epitope, immunogen, peptide or polypeptide of interest.
  • sequence identity or homology is determined by comparing the sequences when aligned so as to maximize overlap and identity while minimizing sequence gaps.
  • sequence identity may be determined using any of a number of mathematical algorithms.
  • a nonlimiting example of a mathematical algorithm used for comparison of two sequences is the algorithm of Karlin & Altschul, Proc. Natl. Acad. Sci. USA 1990; 87: 2264-2268, modified as in Karlin & Altschul, Proc. Natl. Acad. Sci. USA 1993; 90: 5873-5877.
  • Another example of a mathematical algorithm used for comparison of sequences is the algorithm of Myers & Miller, CABIOS1988; 4: 11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 may be used. Yet another useful algorithm for identifying regions of local sequence similarity and alignment is the FASTA algorithm as described in Pearson & Lipman, Proc. Natl. Acad. Sci. USA 1988; 85: 2444-2448.
  • WU-BLAST Woodington University BLAST
  • WU-BLAST version 2.0 executable programs for several UNIX platforms may be downloaded from ftp://blast.wust1.edu/blast/executables.
  • the nucleotide sequences of the present invention may be inserted into “vectors.”
  • vehicle is widely used and understood by those of skill in the art, and as used herein the term “vector” is used consistent with its meaning to those of skill in the art.
  • vector is commonly used by those skilled in the art to refer to a vehicle that allows or facilitates the transfer of nucleic acid molecules from one environment to another or that allows or facilitates the manipulation of a nucleic acid molecule.
  • any vector that allows expression of the antibodies and/or antigens of the present invention may be used in accordance with the present invention.
  • the antigens and/or antibodies of the present invention may be used in vitro (such as using cell-free expression systems) and/or in cultured cells grown in vitro in order to produce the encoded HIV-antigens and/or antibodies which may then be used for various applications such as in the production of proteinaceous vaccines.
  • any vector that allows expression of the antigens and/or antibodies in vitro and/or in cultured cells may be used.
  • any vector that allows for the expression of the antibodies and/or antigens of the present invention and is safe for use in vivo may be used.
  • the vectors used are safe for use in humans, mammals and/or laboratory animals.
  • the protein coding sequence should be “operably linked” to regulatory or nucleic acid control sequences that direct transcription and translation of the protein.
  • a coding sequence and a nucleic acid control sequence or promoter are said to be “operably linked” when they are covalently linked in such a way as to place the expression or transcription and/or translation of the coding sequence under the influence or control of the nucleic acid control sequence.
  • nucleic acid control sequence may be any nucleic acid element, such as, but not limited to promoters, enhancers, IRES, introns, and other elements described herein that direct the expression of a nucleic acid sequence or coding sequence that is operably linked thereto.
  • promoter will be used herein to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II and that when operationally linked to the protein coding sequences of the invention lead to the expression of the encoded protein.
  • the expression of the transgenes of the present invention may be under the control of a constitutive promoter or of an inducible promoter, which initiates transcription only when exposed to some particular external stimulus, such as, without limitation, antibiotics such as tetracycline, hormones such as ecdysone, or heavy metals.
  • the promoter may also be specific to a particular cell-type, tissue or organ.
  • suitable promoters and enhancers are known in the art, and any such suitable promoter or enhancer may be used for expression of the transgenes of the invention.
  • suitable promoters and/or enhancers may be selected from the Eukaryotic Promoter Database (EPDB).
  • the present invention relates to a recombinant vector expressing a foreign epitope.
  • the epitope is an HIV epitope.
  • the HIV epitope is a protein fragment of the present invention, however, the present invention may encompass additional HIV antigens, epitopes or immunogens.
  • the HIV epitope is an HIV antigen, HIV epitope or an HIV immunogen, such as, but not limited to, the HIV antigens, HIV epitopes or HIV immunogens of U.S. Pat. Nos.
  • HIV, or immunogenic fragments thereof may be utilized as the HIV epitope.
  • any epitope recognized by an HIV antibody may be used in the present invention.
  • the anti-HIV antibodies of U.S. Pat. Nos. 6,949,337, 6,900,010, 6,821,744, 6,768,004, 6,613,743, 6,534,312, 6,511,830, 6,489,131, 6,242,197, 6,114,143, 6,074,646, 6,063,564, 6,060,254, 5,919,457, 5,916,806, 5,871,732, 5,824,304, 5,773,247, 5,736,320, 5,637,455, 5,587,285, 5,514,541, 5,317,009, 4,983,529, 4,886,742, 4,870,003 and 4,795,739 are useful for the present invention.
  • the present invention relates to a recombinant vector expressing a foreign epitope.
  • the epitope is a SIV epitope. It is understood by one of skill in the art that anything referring to HIV in the specification also applies to SIV.
  • the SIV epitope is a protein fragment of the present invention, however, the present invention may encompass additional SIV antigens, epitopes or immunogens.
  • the SIV epitope is an SIV antigen, SIV epitope or an SIV immunogen, such as, but not limited to, the SIV antigens, SIV epitopes or SIV immunogens of U.S. Pat. Nos.
  • the vectors used in accordance with the present invention should typically be chosen such that they contain a suitable gene regulatory region, such as a promoter or enhancer, such that the antigens and/or antibodies of the invention may be expressed.
  • expression vectors that are suitable for expression on that subject, and that are safe for use in vivo, should be chosen.
  • any vectors that are suitable for such uses may be employed, and it is well within the capabilities of the skilled artisan to select a suitable vector.
  • the vectors used for these in vivo applications are attenuated to vector from amplifying in the subject.
  • plasmid vectors preferably they will lack an origin of replication that functions in the subject so as to enhance safety for in vivo use in the subject.
  • viral vectors preferably they are attenuated or replication-defective in the subject, again, so as to enhance safety for in vivo use in the subject.
  • viral vectors are used.
  • the vector is a CMV vector, preferably lacking one or more of the glycoproteins US2, US3, US6 and US11.
  • all of the genes between US2 and US11 region of the CMV genome are deleted.
  • any vector advantageously a viral vector, may express one or more of the glycoproteins US2, US3, US6 and US11.
  • Viral expression vectors are well known to those skilled in the art and include, for example, viruses such as adenoviruses, adeno-associated viruses (AAV), alphaviruses, herpesviruses, retroviruses and poxviruses, including avipox viruses, attenuated poxviruses, vaccinia viruses, and particularly, the modified vaccinia Ankara virus (MVA; ATCC Accession No. VR-1566).
  • viruses when used as expression vectors are innately non-pathogenic in the selected subjects such as humans or have been modified to render them non-pathogenic in the selected subjects.
  • replication-defective adenoviruses and alphaviruses are well known and may be used as gene delivery vectors. However, these vectors are immunogenic and induce immunity against the vector which prohibits their repeated use unless they express US2-11.
  • any vector may express one or more of the glycoproteins US2, US3, US6 and US11.
  • the vector expresses glycoproteins US2, US3, US6 and US11. More advantageously, the vector contains and expresses all of the glycoproteins within the US2 to US11 region of CMV.
  • the one or more of the glycoproteins US2, US3, US6 and US11 may include, but not limited to, the glycoproteins of U.S. Pat. Nos.
  • the nucleotide sequences and vectors of the invention may be delivered to cells, for example if aim is to express and the HIV-1 antigens in cells in order to produce and isolate the expressed proteins, such as from cells grown in culture.
  • any suitable transfection, transformation, or gene delivery methods may be used. Such methods are well known by those skilled in the art, and one of skill in the art would readily be able to select a suitable method depending on the nature of the nucleotide sequences, vectors, and cell types used. For example, transfection, transformation, microinjection, infection, electroporation, lipofection, or liposome-mediated delivery could be used.
  • antibodies and/or antigens may be carried out in any suitable type of host cells, such as bacterial cells, yeast, insect cells, and mammalian cells.
  • the antibodies and/or antigens of the invention may also be expressed using including in vitro transcription/translation systems. All of such methods are well known by those skilled in the art, and one of skill in the art would readily be able to select a suitable method depending on the nature of the nucleotide sequences, vectors, and cell types used.
  • the nucleotide sequences, antibodies and/or antigens of the invention are administered in vivo, for example where the aim is to produce an immunogenic response in a subject.
  • a “subject” in the context of the present invention may be any animal.
  • the subject is a human, for example a human that is infected with, or is at risk of infection with, HIV-1.
  • the nucleotide sequences, antibodies and/or antigens of the invention are preferably administered as a component of an immunogenic composition which may comprise the nucleotide sequences and/or antigens of the invention in admixture with a pharmaceutically acceptable carrier.
  • the immunogenic compositions of the invention are useful to stimulate an immune response against HIV-1 and may be used as one or more components of a prophylactic or therapeutic vaccine against HIV-1 for the prevention, amelioration or treatment of AIDS.
  • the nucleic acids and vectors of the invention are particularly useful for providing genetic vaccines, i.e. vaccines for delivering the nucleic acids encoding the antibodies and/or antigens of the invention to a subject, such as a human, such that the antibodies and/or antigens are then expressed in the subject to elicit an immune response.
  • compositions of the invention may be injectable suspensions, solutions, sprays, lyophilized powders, syrups, elixirs and the like. Any suitable form of composition may be used.
  • a nucleic acid or vector of the invention having the desired degree of purity, is mixed with one or more pharmaceutically acceptable carriers and/or excipients.
  • the carriers and excipients must be “acceptable” in the sense of being compatible with the other ingredients of the composition.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, or combinations thereof, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobul
  • An immunogenic or immunological composition may also be formulated in the form of an oil-in-water emulsion.
  • the oil-in-water emulsion may be based, for example, on light liquid paraffin oil (European Pharmacopea type); isoprenoid oil such as squalane, squalene, EICOSANETM or tetratetracontane; oil resulting from the oligomerization of alkene(s), e.g., isobutene or decene; esters of acids or of alcohols containing a linear alkyl group, such as plant oils, ethyl oleate, propylene glycol di(caprylate/caprate), glyceryl tri(caprylate/caprate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, e.g., isostearic acid esters.
  • the oil advantageously is used in combination with emulsifiers to form the emulsion.
  • the emulsifiers may be nonionic surfactants, such as esters of sorbitan, mannide (e.g., anhydromannitol oleate), glycerol, polyglycerol, propylene glycol, and oleic, isostearic, ricinoleic, or hydroxystearic acid, which are optionally ethoxylated, and polyoxypropylene-polyoxyethylene copolymer blocks, such as the Pluronic® products, e.g., L121.
  • the adjuvant may be a mixture of emulsifier(s), micelle-forming agent, and oil such as that which is commercially available under the name Provax® (IDEC Pharmaceuticals, San Diego, Calif.).
  • the immunogenic compositions of the invention may contain additional substances, such as wetting or emulsifying agents, buffering agents, or adjuvants to enhance the effectiveness of the vaccines (Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, (ed.) 1980).
  • Adjuvants may also be included.
  • Adjuvants include, but are not limited to, mineral salts (e.g., AlK(SO 4 ) 2 , AlNa(SO 4 ) 2 , AlNH(SO 4 ) 2 , silica, alum, Al(OH) 3 , Ca 3 (PO 4 ) 2 , kaolin, or carbon), polynucleotides with or without immune stimulating complexes (ISCOMs) (e.g., CpG oligonucleotides, such as those described in Chuang, T. H. et al, (2002) J. Leuk. Biol. 71(3): 538-44; Ahmad-Nejad, P. et al (2002) Eur. J. Immunol.
  • mineral salts e.g., AlK(SO 4 ) 2 , AlNa(SO 4 ) 2 , AlNH(SO 4 ) 2 , silica, alum, Al(OH) 3 , Ca 3 (PO
  • monophosphoryl lipid A in particular, 3-de-O-acylated monophosphoryl lipid A (3D-MPL), imiquimod (also known in the art as IQM and commercially available as Aldara®; U.S. Pat. Nos. 4,689,338; 5,238,944; Zuber, A. K. et al (2004) 22(13-14): 1791-8), and the CCR5 inhibitor CMPD167 (see Veazey, R. S. et al (2003) J. Exp. Med. 198: 1551-1562).
  • 3D-MPL 3-de-O-acylated monophosphoryl lipid A
  • imiquimod also known in the art as IQM and commercially available as Aldara®; U.S. Pat. Nos. 4,689,338; 5,238,944; Zuber, A. K. et al (2004) 22(13-14): 1791-8
  • CMPD167 see Veazey, R. S. et al (2003) J. Exp. Med
  • Aluminum hydroxide or phosphate (alum) are commonly used at 0.05 to 0.1% solution in phosphate buffered saline.
  • Other adjuvants that may be used, especially with DNA vaccines, are cholera toxin, especially CTA1-DD/ISCOMs (see Mowat, A. M. et al (2001) J. Immunol. 167(6): 3398-405), polyphosphazenes (Allcock, H.R. (1998) App. Organometallic Chem. 12(10-11): 659-666; Payne, L. G. et al (1995) Pharm. Biotechnol.
  • cytokines such as, but not limited to, IL-2, IL-4, GM-CSF, IL-12, IL-15 IGF-1, IFN- ⁇ , IFN- ⁇ , and IFN- ⁇
  • immunoregulatory proteins such as CD40L (ADX40; see, for example, WO03/063899)
  • CD1a ligand of natural killer cells also known as CRONY or ⁇ -galactosyl ceramide; see Green, T. D. et al, (2003) J. Virol.
  • immunostimulatory fusion proteins such as IL-2 fused to the Fc fragment of immunoglobulins (Barouch et al., Science 290:486-492, 2000) and co-stimulatory molecules B7.1 and B7.2 (Boyer), all of which may be administered either as proteins or in the form of DNA, on the same expression vectors as those encoding the antigens of the invention or on separate expression vectors.
  • the immunogenic compositions may be designed to introduce the nucleic acids or expression vectors to a desired site of action and release it at an appropriate and controllable rate.
  • Methods of preparing controlled-release formulations are known in the art.
  • controlled release preparations may be produced by the use of polymers to complex or absorb the immunogen and/or immunogenic composition.
  • a controlled-release formulations may be prepared using appropriate macromolecules (for example, polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine sulfate) known to provide the desired controlled release characteristics or release profile.
  • Another possible method to control the duration of action by a controlled-release preparation is to incorporate the active ingredients into particles of a polymeric material such as, for example, polyesters, polyamino acids, hydrogels, polylactic acid, polyglycolic acid, copolymers of these acids, or ethylene vinylacetate copolymers.
  • a polymeric material such as, for example, polyesters, polyamino acids, hydrogels, polylactic acid, polyglycolic acid, copolymers of these acids, or ethylene vinylacetate copolymers.
  • microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacrylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • Suitable dosages of the nucleic acids and expression vectors of the invention in the immunogenic composition of the invention may be readily determined by those of skill in the art.
  • the dosage of the immunogens may vary depending on the route of administration and the size of the subject.
  • Suitable doses may be determined by those of skill in the art, for example by measuring the immune response of a subject, such as a laboratory animal, using conventional immunological techniques, and adjusting the dosages as appropriate.
  • Such techniques for measuring the immune response of the subject include but are not limited to, chromium release assays, tetramer binding assays, IFN- ⁇ ELISPOT assays, IL-2 ELISPOT assays, intracellular cytokine assays, and other immunological detection assays, e.g., as detailed in the text “Antibodies: A Laboratory Manual” by Ed Harlow and David Lane.
  • the immunogenic compositions of the invention are ideally administered to a subject in advance of HIV infection, or evidence of HIV infection, or in advance of any symptom due to AIDS, especially in high-risk subjects.
  • the prophylactic administration of the immunogenic compositions may serve to provide protective immunity of a subject against HIV-1 infection or to prevent or attenuate the progression of AIDS in a subject already infected with HIV-1.
  • the immunogenic compositions may serve to ameliorate and treat AIDS symptoms and are advantageously used as soon after infection as possible, preferably before appearance of any symptoms of AIDS but may also be used at (or after) the onset of the disease symptoms.
  • the immunogenic compositions may be administered using any suitable delivery method including, but not limited to, intramuscular, intravenous, intradermal, mucosal, and topical delivery. Such techniques are well known to those of skill in the art. More specific examples of delivery methods are intramuscular injection, intradermal injection, and subcutaneous injection. However, delivery need not be limited to injection methods. Further, delivery of DNA to animal tissue has been achieved by cationic liposomes (Watanabe et al., (1994) Mol. Reprod. Dev.
  • delivery routes may be oral, intranasal or by any other suitable route. Delivery also be accomplished via a mucosal surface such as the anal, vaginal or oral mucosa.
  • Immunization schedules are well known for animals (including humans) and may be readily determined for the particular subject and immunogenic composition. Hence, the immunogens may be administered one or more times to the subject. Preferably, there is a set time interval between separate administrations of the immunogenic composition. While this interval varies for every subject, typically it ranges from 10 days to several weeks, and is often 2, 4, 6 or 8 weeks. For humans, the interval is typically from 2 to 6 weeks.
  • the interval is longer, advantageously about 10 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 22 weeks, 24 weeks, 26 weeks, 28 weeks, 30 weeks, 32 weeks, 34 weeks, 36 weeks, 38 weeks, 40 weeks, 42 weeks, 44 weeks, 46 weeks, 48 weeks, 50 weeks, 52 weeks, 54 weeks, 56 weeks, 58 weeks, 60 weeks, 62 weeks, 64 weeks, 66 weeks, 68 weeks or 70 weeks.
  • the interval is about 16 weeks or about 53 weeks.
  • the immunization regimes typically have from 1 to 6 administrations of the immunogenic composition, but may have as few as one or two or four.
  • the methods of inducing an immune response may also include administration of an adjuvant with the immunogens.
  • annual, biannual or other long interval (5-10 years) booster immunization may supplement the initial immunization protocol.
  • the present methods also include a variety of prime-boost regimens, for example DNA prime-Adenovirus boost regimens.
  • one or more priming immunizations are followed by one or more boosting immunizations.
  • the actual immunogenic composition may be the same or different for each immunization and the type of immunogenic composition (e.g., containing protein or expression vector), the route, and formulation of the immunogens may also be varied.
  • an expression vector is used for the priming and boosting steps, it may either be of the same or different type (e.g., DNA or bacterial or viral expression vector).
  • Prime-boost regimen provides for two priming immunizations, four weeks apart, followed by two boosting immunizations at 4 and 8 weeks after the last priming immunization. It should also be readily apparent to one of skill in the art that there are several permutations and combinations that are encompassed using the DNA, bacterial and viral expression vectors of the invention to provide priming and boosting regimens. In the event that the viral vectors express US2-11 they may be used repeatedly while expressing different antigens derived from different pathogens.
  • a specific embodiment of the invention provides methods of inducing an immune response against HIV in a subject by administering an immunogenic composition of the invention, preferably which may comprise an US2-11 expressing adenovirus vector containing DNA encoding one or more of the epitopes of the invention, one or more times to a subject wherein the epitopes are expressed at a level sufficient to induce a specific immune response in the subject.
  • an immunogenic composition of the invention preferably which may comprise an US2-11 expressing adenovirus vector containing DNA encoding one or more of the epitopes of the invention, one or more times to a subject wherein the epitopes are expressed at a level sufficient to induce a specific immune response in the subject.
  • Such immunizations may be repeated multiple times at time intervals of at least 2, 4 or 6 weeks (or more) in accordance with a desired immunization regime.
  • the immunogenic compositions of the invention may be administered alone, or may be co-administered, or sequentially administered, with other HIV immunogens and/or HIV immunogenic compositions, e.g., with “other” immunological, antigenic or vaccine or therapeutic compositions thereby providing multivalent or “cocktail” or combination compositions of the invention and methods of employing them.
  • the ingredients and manner (sequential or co-administration) of administration, as well as dosages may be determined taking into consideration such factors as the age, sex, weight, species and condition of the particular subject, and the route of administration.
  • the other HIV immunogens may be administered at the same time or at different times as part of an overall immunization regime, e.g., as part of a prime-boost regimen or other immunization protocol.
  • the other HIV immunogen is env, preferably the HIV env trimer.
  • HIVA (described in WO 01/47955), which may be administered as a protein, on a plasmid (e.g., pTHr.HIVA) or in a viral vector (e.g., MVA.HIVA).
  • RENTA (described in PCT/US2004/037699), which may also be administered as a protein, on a plasmid (e.g., pTHr.RENTA) or in a viral vector (e.g., MV ⁇ RENTA).
  • one method of inducing an immune response against HIV in a human subject may comprise administering at least one priming dose of an HIV immunogen and at least one boosting dose of an HIV immunogen, wherein the immunogen in each dose may be the same or different, provided that at least one of the immunogens is an epitope of the present invention, a nucleic acid encoding an epitope of the invention or an expression vector, preferably a VSV vector, encoding an epitope of the invention, and wherein the immunogens are administered in an amount or expressed at a level sufficient to induce an HIV-specific immune response in the subject.
  • the HIV-specific immune response may include an HIV-specific T-cell immune response or an HIV-specific B-cell immune response.
  • Such immunizations may be done at intervals, preferably of at least 2-6 or more weeks.
  • Cytomegalovirus may superinfect persistently infected hosts despite CMV-specific humoral and cellular immunity; however, how it does so remains undefined.
  • Applicants have demonstrated that superinfection of rhesus CMV-infected rhesus macaques (RM) requires evasion of CD8 + T cell immunity by virally encoded inhibitors of major histocompatibility complex class I (MHC-I) antigen presentation, particularly the homologs of human CMV US2, 3, 6, and 11.
  • MHC-I interference was dispensable for primary infection of RM, or for the establishment of a persistent secondary infection in CMV-infected RM transiently depleted of CD8 + lymphocytes.
  • CMV herpesvirus family member cytomegalovirus
  • RhCMV RhCMV-specific T cell responses
  • the SIVgag-specific T cell responses in peripheral blood mononuclear cells (PBMC) or in broncho-alveolar lavage lymphocytes (BAL) were monitored by flow cytometric analysis of intracellular cytokine staining (ICCS) ( FIGS. 6 and 7 ) after stimulation with consecutive overlapping 15-amino acid peptides corresponding to SIVgag. Reduction of the inoculating dose had minimal impact on superinfection dynamics: All animals developed SIVgag-specific T cell responses within 2 weeks ( FIG. 1A ), and secretion of SIVgag-expressing virus in urine or buccal swabs was observed within 4 to 10 weeks of infection in both cohorts ( FIG. 1B ).
  • PBMC peripheral blood mononuclear cells
  • BAL broncho-alveolar lavage lymphocytes
  • RhCMV RhCMV + animals infected with 10 7 PFU of RhCMV(gagL)
  • SIVgag-specific T cell responses and RhCMV(gagL) secretion were stable for more than 3 years regardless of initial dose ( FIG. 1 , A and C).
  • FIG. 1 10 4 PFU 21985 1.70 0.68 RhCMV 22046 1.29 0.37 (gagL) 22463 1.27 0.36 22499 1.71 0.30 10 2 PFU 22052 2.04 0.12 RhCMV 22063 2.37 0.43 (gagL) 22511 3.16 0.55 22559 1.05 0.42 avg ⁇ sd 1.82 ⁇ 0.69 0.41 ⁇ 0.16 B FIG.
  • FIG. 3 ⁇ VIHCE (gag) 23101 2.112 0.576 ⁇ Rh186-8 23126 2.242 0.809 (retanef) 23132 2.273 1.343 23244 3.295 0.779 avg ⁇ sd 2.48 ⁇ 0.55 0.88 ⁇ 0.33 D FIG.
  • CTL cytotoxic T cells
  • MHC-I major histocompatibility complex class I
  • HCMV encodes at least four related glycoproteins, each with a unique mechanism to prevent antigen presentation: US2 and US11 mediate the retrograde translocation of MHC-I into the cytosol for proteasomal destruction (F. J. van der Wal et al. Curr. Top. Microbiol. Immunol. 269, 37 (2002)), US3 retains MHC-I in the endoplasmic reticulum by interfering with chaperone-controlled peptide loading (Z. Liu et al. Int. J. Biochem. Cell Biol.
  • RhCMV encodes sequence and functional homologs of these genes in a genomic region spanning Rh182 (US2) to Rh189 (US11) ( FIG. 5 ) (N. T. Pande et al. J. Virol. 79, 5786 (2005)). Furthermore, the Rh178 gene encodes the RhCMV-specific viral inhibitor of heavy chain expression (VIHCE), which prevents signal-sequence-dependent translation/translocation of MHC-I (C. J. Powers, K. ceremonies, PLoS Pathog. 4, e1000150 (2008)).
  • VIHCE RhCMV-specific viral inhibitor of heavy chain expression
  • RhCMV open reading frames ORFs
  • FIGS. 8 and 9 Applicants examined whether these viruses were able to infect animals that were CMV-na ⁇ ve as shown by a lack of CMV-specific T cell responses (Table 1B).
  • T cell responses against both CMV and SIVgag in PBMC and against SIVgag in BAL were comparable between animals infected with the deletion mutants and the wild-type RhCMV(gag) control ( FIG. 2A ).
  • all animals secreted SIVgag-expressing virus from day 56 onward for the duration of the experiment (>700 days) FIG. 2B .
  • Polymerase chain reaction (PCR) analysis of DNA isolated from urine cocultured virus at day 428 confirmed that the secreted viruses lacked the respective gene regions and were able to persist in the host ( FIG. 2C ). Together these results show that viral MHC-I interference is dispensable for primary infection and the establishment and maintenance of persistent infection, despite the development of a substantial CMV-specific T cell response.
  • FIG. 3C SIVgag-expressing virus
  • Applicants did not detect SIVgag-specific T cell responses in PBMC or BAL in RM inoculated with ⁇ VIHCE ⁇ US2-11(gag), even after repeated inoculation ( FIG. 3 , A and B), and SIVgag-expressing virus was not detected in secretions ( FIG. 3C ).
  • MHC-I interference was essential for superinfection. Inoculation of the same animals with ⁇ US2-11(gag) and, later, ⁇ VIHCE(gag) demonstrated that superinfection required the conserved US2-11 region but not the VIHCE region.
  • Rh186 to Rh188 genes within the US2-11 region are essential for superinfection, which is consistent with the known function of US2, US3, US6, and US11 as inhibitors of MHC-I antigen presentation.
  • Rh186 and Rh187 are most closely related to the HCMV glycoproteins US8 and US10, respectively (N. T. Pande et al. J. Virol. 79, 5786 (2005)), whereas Rh188 is an uncharacterized RhCMV-specific ORF.
  • Rh186-8(retanef) Applicants inoculated the same cohort with ⁇ Rh186-8(retanef) and monitored the T cell response to this fusion protein as well as to RhCMV-IE and SIVgag using corresponding peptides.
  • FIG. 3 A and B, all four RM developed a SIVretanef-specific T cell response within 2 weeks post-challenge, indicating successful superinfection.
  • virus expressing SIVretanef was shed in the secretions of infected animals together with SIVgag-expressing ⁇ VIHCE(gag) ( FIG. 3D ). Applicants thus conclude that the Rh186-8 region is dispensable for superinfection.
  • RhCMV + RM Table 1D
  • cM-T807 a humanized monoclonal antibody to CD8
  • ⁇ US2-11(gag) or ⁇ VIHCE ⁇ US2-11(gag) Flow cytometric analysis of total CD8 + T cells revealed that depletion was extensive, but transient, with detectable CD8 + T cell recovery beginning on day 21 after challenge ( FIG. 4 , A and B).
  • SIVgag-specific CD4 + T cell responses were recorded as early as day 7 post-challenge, showing the ability of the deletion viruses to superinfect these animals ( FIG. 4C ).
  • SIVgag-specific CD8 + T cells were observed within the rebounding CD8 + T cells in blood and BAL at day 21 in two RM and at day 28 in a third; in the fourth RM, such responses were only observed in BAL after day 56.
  • CD8 + lymphocytes most likely CD8 + T cells, were essential for preventing superinfection by ⁇ US2-11 virus, strongly indicating that the MHC-I inhibitory function of these molecules is necessary for superinfection of the CMV-positive host.
  • CMV-specific CD8 + T cells were unable to eliminate RhCMV lacking MHC-I inhibitors once persistent infection had been established ( FIG. 4D ), providing additional evidence that persistent infection is insensitive to CD8 + T cell immunity, even when the ability of the virus to prevent MHC-I presentation is compromised.
  • T cell evasion is not required for establishment of primary CMV infection or once the sites of persistence (e.g., kidney and salivary gland epithelial cells) have been occupied, but rather it is essential to enable CMV to reach these sites of persistence from the peripheral site of inoculation in the CMV-immune host.
  • sites of persistence e.g., kidney and salivary gland epithelial cells
  • One possible scenario is that viral infection of circulating cells, for example, monocytes, may succeed only if the virus prevents elimination of these cells by virus-specific CTLs. More work, however, will be required to identify the cell type supporting superinfection.
  • Anti-RhCMV-IE1 was described previously (S. G. Hansen et al., Nat Med 15, 293 (2009)).
  • the following antibodies used in flow cytometry were from BD Bioscience: L200 (CD4; AmCyan); SP34-2 (CD3; Alex700, PacBlu); SK1 (CD8alpha; TruRed); DX2 (CD95; PE); 25723.11 (IFN- ⁇ ; APC); 6.7 (TNF; FITC).
  • L200 CD4; AmCyan
  • SP34-2 CD3; Alex700, PacBlu
  • SK1 CD8alpha; TruRed
  • DX2 CD95; PE
  • 25723.11 IFN- ⁇ ; APC
  • 6.7 TNF; FITC
  • the following antibodies were obtained from Beckman Coulter: CD28.2 (CD28; PE-Texas Red); L78 (CD69; PE).
  • RhCMV(gagL) was generated by replacing the loxP-flanked enhanced green-fluorescent protein (EGFP) in RhCMV-EGFP (W. L. Chang et al. J Virol 76, 9493 (2002)) with a loxP-flanked expression cassette for SIVmac239-gag under control of the EF1 ⁇ -promoter by in vivo recombination in tissue culture.
  • EGFP enhanced green-fluorescent protein
  • RhCMV-BAC RhCMV bacterial artificial chromosome
  • FIG. 5 The BAC-cassette was inserted between the RhCMV homologs of US1 and US2 and self-excises via Cre-recombinase (W. L. Chang, P. A. Barry, J Virol 77, 5073 (2003)).
  • Recombinant virus RhCMV(gag) contains a codon-optimized, FLAG-tagged SIVmac239-gag sequence under control of the EF1 ⁇ -promoter inserted between ORFs R213 and R214 (S. G.
  • the mutant FRT-flanked KanR cassette was obtained from plasmid pOri6K-F5 (E. M. Borst, M. Messerle, J Virol 79, 3615 (2005)) using primers 5′-TAAAAGTGTCGGATGAATGTGCGGCGCCAACACGCAGACCGAAAAGTGCCACCTGC AGAT-3′ and 5′-GCCTGACTGATGACTAGTCATCGCACGCCTCTTCCCGCCCCAGGAACACTTAACGGC TGA-3′.
  • ⁇ VIHCE was created by replacing base pairs 181320-182060 with the SIVgag expression cassette using primers 5′-TTTGTTCGTATAAAAGTGTCGGATGAATGTGCGG CGCCAACACGCAGACCGTAAAACGACGGCCAGT-3′ and 5′-CGCTCCCTCG GCCTGACTGATGACTAGTCATCGCACGCCTCTTCCCGCCCGTATGTTGTGTGGAATT GTGAG-3′.
  • ⁇ Rh186-8(retanef) was created from previously described V5-tagged RhCMV(retanef) (S. G.
  • RhCMV virus was reconstituted by electroporation of telomerized rhesus fibroblasts (TRFs) (V. Kirchoff et al. Arch Virol 147, 321 (2002)).
  • RNAse I Applied Biosystems
  • DNAse I Applied Biosystems
  • 1 ⁇ g of DNAse-treated RNA was used in a 20 ⁇ l reverse transcription reaction containing 50 ng random hexamers, 0.5 mM dNTPs, 10 mM DTT, and 1 ⁇ l superscript III RT in 1 ⁇ RT buffer (Invitrogen) for 1 hour at 37° C.
  • 1 ⁇ l of the RT reaction was used for semi-quantitative PCR with Platinum taq polymerase (Invitrogen) under the following conditions: 1 ⁇ platinum taq buffer, 1.5 mM MgCl 2 , 0.2 mM dNTPs, 0.504 each primer, and 1.5 U polymerase. 35 cycles of amplification was performed under the following conditions: 94° C. for 30 sec, 55° C. for 30 sec, and 72° C. for 15 sec.
  • RhA186-8(retanef) virus RT-PCR was performed as described above with the following primer pairs: Rh185 5′-AGCGTAGCTCCTCCATACCG CT-3′ and 5′-ATCCGCGGACTGTTTGGGTGT-3′; Rh 186 5′-GCTTCTTCCAGAAGTTGCA TAGGATGA-3′ and 5′-CGACTTTCCGGATCCTACGTGGC-3′; Rh187 5′-CCATAGCCATGCAATGGTCGCA-3′ and 5′-GCGCCATCCCGTGTTACCCC-3′; Rh188 5′-AGAGCTCTGGTCGTCGGCGT-3′ and 5′-TGGCTGGCCACCAGATGGATGT-3′; Rh189 5′-AACCAGTAGGAGCGCCCGGT-3′ and 5′-CGACTCCTGCATGCTTACTGGGGA-3′; ⁇ -actin 5′-TCACCCACACTGTGCCCATCTACGA-3′ and 5′-CAGCGGAACCGCTCATTGCCAATGG-3′.
  • CGS Comparative Genome Sequencing
  • Oligonucleotides that comprised this array were designed to be between 29 and 32 bp, with overlapping sequences of at least 7 bp, with coverage of both strands of the RhCMV 68.1 genome.
  • Viral DNA was isolated using standard methods from a) parental RhCMV-BAC (W. L. Chang, P. A. Barry, J Virol 77, 5073 (2003)), b) ⁇ VIHCE ⁇ US2-11(gag), c) ⁇ US2-11(gag), or d) ⁇ VIHCE(gag).
  • telomerized rhesus fibroblasts TRFs
  • supernatants were collected and, after proteinase K treatment, DNA was isolated by cesium chloride gradient centrifugation.
  • the resulting viral DNA was ethanol precipitated and brought to a final concentration of 1 ⁇ g/ ⁇ l.
  • Viral DNA was fragmented and labeled with Cy3 (RhCMV-BAC as reference) or Cy5 (deletion viruses). Labeled reference and test viral DNA probes were co-hybridized to the tiling arrays and the Cy3 and Cy5 signals were scanned.
  • SignalMap software (NimbleGen Systems, Inc.) was used to analyze all CGS data.
  • RM purpose-bred juvenile and adult male rhesus macaques
  • SPF pathogen-free
  • All other animals used in the study acquired RhCMV naturally while in the colony.
  • RhCMV-specific T cell responses was confirmed by intracellular cytokine staining of RhCMV Ag-stimulated PBMC (Table 1).
  • All RM were free of cercopithicine herpesvirus 1, D-type simian retrovirus, simian T-lymphotrophic virus type 1 and SIV infection.
  • virus was concentrated from cleared urine and co-cultured with rhesus fibroblasts and cell lysates were collected after cytopathic effects were observed or after 28 days.
  • Debris was pelleted at 16000 ⁇ g for 15 min, supernatant removed, and DNA precipitated with 450 ⁇ l isopropanol. 50 ng of DNA was used for PCR analysis under the following conditions: 1 ⁇ platinum taq buffer, 1.5 mM MgCl 2 , 0.2 mM dNTPs, 0.5 ⁇ M each primer, and 1.5 U platinum taq polymerase. 35 cycles of amplification was performed under the following conditions: 94° C. for 30 sec, 55° C. for 30 sec, and 72° C. for 80 sec.
  • Brefeldin A 10 ⁇ g/ml; Sigma Aldrich
  • this background-subtracted value was divided by the fraction of total memory cells (determined as described below) to achieve the reported “memory corrected” response frequency (C. J. Pitcher et al., J Immunol 168, 29 (2002)).
  • BAL the reported responses were background response (no antigen) subtracted only, as BAL T cells are entirely memory cells.
  • To determine the memory fraction of circulating T cells memory and na ⁇ ve T cell subset populations were delineated based on CD28 and CD95 expression patterns, as described in (C. J. Pitcher et al., J Immunol 168, 29 (2002))(see FIG. 7 ).
  • Applicants develop a number of attenuated RhCMV-vaccines to examine the highest level of attenuation that may still achieve protection against ⁇ US2-11-Gag.
  • a limitation of Applicants' preliminary data was that Applicants had only shown that natural infection with RhCMV was protective against re-infection with ⁇ US2-11, but Applicants had yet to demonstrate that experimental infection with recombinant RhCMV would be protective.
  • Applicants now demonstrate that a recombinant virus lacking the major tegument proteins pp65a and pp65b or pp71 protects against re-infection by ⁇ US2-11-Gag.
  • PP65 is one of the most abundant proteins in HCMV particles and the most abundant component of the viral tegument, an amorphous protein structure layered between the capsid and the envelope.
  • pp65 is one of the most immunogenic proteins encoded by HCMV and it is therefore included in most experimental vaccines and pp65-specific T cells are routinely included in adoptive transfer protocols.
  • the role of pp65 for acute and persistent infection in vivo has never been examined.
  • RhCMV encodes two homologues of HCMV pp65 (UL83), pp65a (Rh111) and pp65b (Rh112).
  • BAC-mutagenesis Applicants deleted Rh111 and Rh112 from the RhCMV genome.
  • ⁇ Rh111-2 does not show a growth defect in fibroblast cultures (data not shown).
  • WT-RhCMV a representative animal is shown in FIG.
  • pp65 does not seem to be required for primary infection of CMV-na ⁇ ve animals by RhCMV.
  • RhCMV/SIV vectors may 1) establish persistent infection in RhCMV-seropositive rhesus macaques (RM), 2) elicit potent, long-lasting SIV-specific CD4+ and CD8+ T cell responses with a strong “effector memory” (T EM ) bias (see FIG. 16 ), and 3) protect ⁇ 50% of vaccinated RM from progressive SIV infection after limiting dose, intra-rectal challenge with the highly pathogenic SIVmac239 virus (see FIG. 10 ).
  • RM RhCMV-seropositive rhesus macaques
  • T EM effector memory
  • RhCMV/SIV vaccinated RM The protection manifested in RhCMV/SIV vaccinated RM is distinct from previous T cell SIV vaccines in its abruptness and extent, with protected RM manifesting a viral burst in plasma of varying size upon initial infection, followed by immediate control to undetectable levels. While these RM may subsequently show periodic, low level “blips” of viremia, CD4+ memory depletion is not observed and SIV-specific antibody (Ab) responses do not boost, indicating a very high level of control. Moreover, to date, this stringent control has been stable for >30 weeks in 16/17 protected RM. Protection correlates with peak total SIV-specific CD8+ T cell responses in blood during the vaccine phase, which likely reflects the degree to which these cells are seeded into effector tissues.
  • RhCMV-vectored SW-specific CD8+ T cell responses are distinct from responses elicited by conventional viral vectors or SIV itself:
  • RhCMV-vectored CD8+ T cells target a broad array of (likely cross-presented) epitopes that exclude the typical immunodominant epitopes (e.g., CM9 or TL8 in Mamu A*01+ RM) that are internally processed in and presented by virally infected cells (see FIG. 20 ).
  • RhCMV/SIV vector-mediated protection including the mechanisms, timing and location of protection, and the impact of differential CD8+ T epitope targeting on the efficiency of SIV control, in particular asking whether broadening RhCMV/SIV-vectored CD8+T cell responses to include typical dominant epitopes may improve response quality and enhance efficacy.
  • the goals of this Example are:
  • HIV/SIV adaptations that provide for efficient immune evasion include 1) massive replication, high mutation rates, genetic malleability and functional plasticity leading to rapid evolution, 2) specific genetic mechanisms to thwart innate and adaptive immune mechanisms (e.g., countering tetherin, APOBEC, Trim5 ⁇ innate anti-viral mechanisms and cytotoxic T cells by class 1 MHC down-regulation), 3) env adaptations to avoid antibody (Ab) neutralization, 4) latency, and 5) dysregulated immune function (Evans, D. T., and Desrosiers, R. C. 2001. Immunol Rev 183:141-158, Johnson, W. E., and Desrosiers, R. C. 2002. Annu Rev Med 53:499-518, Goulder, P.
  • protection is 1) uneven within identically vaccinated RM cohorts (and often correlated with protective MHC alleles), 2) seemingly limited to ⁇ 1.5-2 log mean reduction in peak and plateauphase plasma viral loads with SIVmac challenge, and 3) subject to reversion over time (18-29).
  • This pattern of protection is similar for intravenous, high (single) and low (repeated) dose mucosal challenge, and appears to derive from a massive anamnestic CD8+ T cell response after infection that “intercepts” viral replication fairly late during systemic spread, with anti-viral activity first manifested by a blunting of peak SIV replication at day 10 to 14. As illustrated in FIG.
  • the CD8+ memory T cells that result from prime-boost vaccines with non-replicating vectors are dramatically expandable upon infection, but the proliferation, differentiation and effector cell delivery to viral replication sites is quite delayed relative to viral kinetics, a temporal relationship that clearly limits both the extent and durability of protection.
  • HVTN 502 phase 2b Merck STEP trial
  • the STEP regimen may, in retrospect, have been insufficiently potent (or unable to elicit a sufficiently broad HIV-specific CD8+ T cell response) to achieve significant protection, but the results clearly illustrate the difficulty in attaining meaningful efficacy with a vaccine designed to elicit conventional CD8+ memory T cells.
  • T cell memory memory responses that, upon initial pathogen encounter, require effector expansion, differentiation and migration to mediate anti-viral activity
  • T EM effector memory T cells
  • T EM lack robust expansion capacity, but are localized in effector sites and poised for immediate effector function (Bannard, O. et al. 2009. Eur J Immunol 39:2083-2087, Hansen, S. G. et al. 2009. Nat Med 15:293-299, Sallusto, F. et al. 2004. Annu Rev Immunol 22:745-763, Picker, L. J. et al. 2006.
  • CD4+ T EM are the primary targets of HIV/SIV (Grossman, Z. et al. 2006. Nat Med 12:289-295), and as CD4+ and CD8+ T EM cohabit the same sites, a vaccine-generated CD8+ T EM response would theoretically be ideally positioned to intercept initial/early viral replication in primary infection, providing anti-viral effector activity during the most vulnerable phase of infection.
  • T EM bias characterizes T cell responses to chronic/persistent agents, in particular CMV (Hansen, S. G. et al. 2009. Nat Med 15:293-299, 35, 38-40).
  • Applicants therefore initiated a RhCMV vector development program to assess the ability of T EM to intervene early in primary SIV infection.
  • RhCMV vector development program to assess the ability of T EM to intervene early in primary SIV infection.
  • RhCMV may be modified to highly express SIV proteins, without disruption of other RhCMV genes, and with preservation of wildtype growth characteristics (in vitro and in vivo).
  • These vectors may re-infect RhCMV-seropositive RM in a clinically silent manner, and in the process of re-infection elicit indefinitely persistent, high frequency CD4+ and CD8+ T cell responses against the SIV gene products.
  • RhCMV-vector elicited SIV-specific T cell responses manifest a polyfunctional, highly T EM -biased phenotype, and in keeping with this phenotype were highly enriched in effector sites [(Hansen, S. G. et al. 2009. Nat Med 15:293-299); and see FIG. 16 ].
  • RhCMV/SIV vectors do not elicit significant SIV-specific Ab responses, nor do they appear even to prime for such responses (Hansen, S. G. et al. 2009. Nat Med 15:293-299).
  • RM immunized with RhCMV/gag, /rev/nef/tat, and /env were challenged with repeated, limiting dose intra-rectal SIVmac239 at 486-615 days after the last vaccination.
  • This challenge protocol was designed to infect RM via mucosal exposure with viral doses more akin to sexual HIV transmission in humans (24).
  • 4/12 vaccinees vs. 0/16 controls
  • 4/12 vaccinees were demonstrably infected with SIV but completely controlled infection, to the extent that CD8+ cell depletion failed to elicit viral recrudescence (Hansen, S. G. et al. 2009. Nat Med 15:293-299).
  • RhCMV/SIV-vaccinated RM have been highly protected from progressive SIV infection after intra-rectal challenge with highly pathogenic, CCR5-tropic SIV.
  • This Example addresses these priorities by providing detailed analysis of 1) the distribution and functional characteristics of RhCMV vector-elicited, SIV-specific T cells and the relationship between these characteristics and efficacy, 2) where and how these responses “intercept” and suppress mucosally administered SIV in primary infection, and control SIV replication over the long-term, and 3) the differential CD8+ T cell epitope targeting mediated by US2-11 gene function in wt vs. mutant CMV/SIV vectors, and the impact of this differential targeting on efficacy.
  • RhCMV vectors expressing SIVgag, rev/nef/tat, env and pol extensive assessment of vector biology and immunogenicity, and most importantly, efficacy assessment and delineation of immune correlates.
  • RhCMV vectors and the T EM responses they elicit may be efficacious against highly pathogenic SIV has been confirmed, and a new pattern of early protection has been discovered.
  • the details of these accomplishments are described in Hansen, et al. (Hansen, S. G. et al. 2009. Nat Med 15:293-299).
  • RhCMV/SIV vectors Hansen, S. G. et al. 2009. Nat Med 15:293-299
  • RhCMV/pol-1 RhCMV/pol-1
  • RhCMV pol-2 wks 0, 14
  • C) pan-proteome DNA pan-proteome DNA (wks, 0, 4, 8) followed by pan-proteome Ad5 vectors (wk 14), and D) additional unvaccinated controls.
  • RhCMV/SIV-vaccinated RM manifested variable numbers of low level viral blips at subsequent time points ( FIG. 10 ), but overall viral control was sufficiently early and stringent to preclude any CD4+ target cell depletion ( FIG. 11 ), as well as prevent induction (Group A) or boosting (Group B) of the anti-SIVenv antibody response ( FIG. 12 ).
  • protection in both RhCMV vector-vaccinated groups significantly correlated with the magnitude of the peak total SIV-specific CD8+ T cell response in blood during the vaccine phase ( FIG. 13 ), not the responses present immediately pre-challenge (and not SIV-specific CD4+ T cell responses, not shown).
  • RhCMV-vectored, SIV-specific T cell responses were fundamentally different from the responses elicited by DNA/Ad5. The latter did not provide the immediate protection seen in the RhCMV/SIV vector-vaccinated RM, but did demonstrate significant mean reductions in peak and early plateau-phase viral loads, and were associated with strong boosting upon infection ( FIG. 14 ). In contrast, the protection associated with RhCMV/SIV vectors alone was binary, providing either immediate stringent control, or no control, and in keeping with this, these responses did not significantly boost in the unprotected (progressively infected) RM ( FIG. 14 ).
  • RhCMV/SIV and Ad5/SIV vectors showed both the immediate “T EM ” protection, an element of peak and post-peak (“T CM ”) control, and a post-infection response boost, similar to, but less than, the DNA/Ad5-vaccinated group ( FIG. 14 ).
  • T EM immediate “T EM ” protection
  • T CM element of peak and post-peak
  • post-infection response boost similar to, but less than, the DNA/Ad5-vaccinated group ( FIG. 14 ).
  • 12/13 of RhCMV vector-protected RM maintained stringent protection for >30 weeks, whereas 7/9 DNA/Ad5-vaccinated RM were indistinguishable from controls by 26 weeks post-infection.
  • RhCMV-vectored SIV-specific T cell responses intercept mucosally administered SIV infection very early in primary infection, prior to extensive systemic replication and may maintain stringent control after this early “intercept”.
  • RhCMV/SIV-vectored T EM responses are unable to “chase” and do not provide peak or post-peak protection.
  • RhCMV/SIV vectors indefinitely maintain strikingly high frequencies of SIV-specific T cells in target cell-rich effector sites that are likely candidates for initial viral infection and amplification, including both rectal mucosa and sites of potential hematogenous viral spread—spleen, liver, bone marrow ( FIG. 16 ).
  • RhCMV/SIV vector immunogenicity assessed the degree to which the CD8+ T cell responses elicited by these vectors recognized epitopes that had been previously shown to represent dominant targets in SIV-infected or DNA/Adenovirus/pox vector-vaccinated RM with the appropriate restricting MHC class I allele.
  • These epitopes included Mamu A*01-restricted gag-CM9, -LW9, -VL8, -QI9, -VT10, -LF8, -LA9, env-TL9, and tat-SL8 (12 RM); Mamu A*02-restricted gag-GY9, env-RY8, and nef-YY9 (4 RM); Mamu B*08-restricted nef-RL10 (1 RM), and Mamu B*17-nef-IW9, -MW9, and env-FW9 (7 RM) (41-44).
  • RhCMV/SIV-vectored responses differ from DNA and conventional viral vectors (e.g., Ad5) not only in their T EM -biased phenotype, function, distribution and longevity, but also in their CD8+ T cell recognition patterns.
  • Ad5 conventional viral vectors
  • HCMV encodes 4 related glycoproteins that act together to prevent presentation of MHC class I-restricted epitopes by infected cells: US2 and US11 mediate the retrograde translocation of MHC-I molecules into the cytosol for proteosomal degradation; US3 retains MHC-I molecules in the endoplasmic reticulum (ER); and US6 inhibits translocation of viral and host peptides across the ER membrane by the peptide transporter TAT (Powers, C. et al. 2008. Curr Top Microbiol Immunol 325:333-359. Liu, Z. et al. 2009. Int J Biochem Cell Biol 41:503-506, van der Wal, F. J. et al. 2002.
  • RhCMV encodes sequence and functional homologues of these 4 proteins in a genomic region spanning Rh182 (US2) to Rh189 (US11) (Powers, C., and Fruh, K. 2008. Microbiol Immunol 197:109-115 and Pande, N. T. et al. 2005. J Virol 79:5786-5798).
  • RhCMV vector cross-presentation mechanism is notable for its apparent complete exclusion of CD8+ T cell responses to multiple, conventional immunodominant epitopes, suggesting the very efficient inhibition of direct presentation and a distinct epitope processing mechanism for indirect presentation.
  • the second implication of these data, which is more speculative, but potentially more significant, is the suggestion that cytotoxicity (direct killing of SIV-infected cells) may not play the primary role in the protection afforded by wt RhCMV/SIV vector-elicited CD8+ T cell responses. It is not that these cross-presentation-derived responses would lack intrinsic cytotoxic function [the cytotoxic apparatus of CD8+ T EM is present (Hansen, S. G. et al. 2009.
  • RhCMV/SIV vector-elicited responses would most likely be due to a more indirect effector function (by T cells stimulated by APC in the neighborhood of infected cells), such as, for example, elaboration of CCR5 binding chemokines (Cocchi, F. et al. 1995. Science 270:1811-1815 and Arenzana-Seisdedos, F., and Parmentier, M. 2006. Semin Immunol 18:387-403).
  • SIV-specific T cell responses elicited by the US2-11 KO RhCMV/SIV vectors clearly retain the broad epitope recognition that arises from cross-presentation, and maintain the same phenotypic and functional capabilities of wt vector-elicited responses (not shown), but in addition, include recognition of the epitopes that are efficiently processed by SIV-infected cells, and therefore, would include epitopes, like gag CM9 or tat SL8, that may mediate direct cytotoxicity (Lrissado, J. T. et al. 2007. J Virol 81:2624-2634).
  • the responses elicited by US2-11 KO RhCMV/SIV vectors may have enhanced anti-viral function, either by more efficient cytotoxicity or direct viral suppression, or simply greater CD8+ T cell response breadth, or both.
  • CD8+ responses elicited by US2-11 deletant vectors might therefore more efficiently protect RM from progressive SIV infection, increasing the fraction of protected RM above the current level, a important goal of Applicants' efforts to optimize T EM vaccine approaches.
  • Routine T cell response quantification is accomplished by cytokine flow cytometry (CFC; see FIG. 16 ) using mixes of overlapping 15 mer peptides for SIVgag, env, pol, and rev/nef/tat, and RhCMV IE, compared to control peptides (mTB Ag85b and ESAT6) and co-stimulation alone (Hansen, S. G. et al. 2009. Nat Med 15:293-299).
  • CFC cytokine flow cytometry
  • CFC tube #1 is used on fresh cell preparations from all individual tissue samples for all SIV proteins to comprehensively establish the systemic distribution of the SIV-specific T cell responses.
  • CFC tube #s 2 and 3 retrospectively provide further functional characterization and phenotypic assessment (expression of CD25, HLA-DR, and PD-1) of the degree to which the Ag-specific T cells being measured were subject to activation in vivo. These additional “tubes” are applied in more limited fashion, focusing on one (cryopreserved) cell preparation from each tissue and on the dominant responses identified with the fresh tube #1 analysis.
  • Cryopreserved splenic cells (which are not limiting) are analyzed with single peptide ⁇ -IFN ELISPOT to “deconvolute” overall SIV protein-specific responses into individual 15 mer peptide responses, and then these single peptide responses are confirmed, lineage typed and functionally characterized with CFC tube #1 from the same cryopreserved splenic cell preps.
  • Responses to the 5 highest frequency (CD8) epitopes are analyzed by CFC tube #1 (and selectively tube #s 2 and 3) in all tissues to define the distribution of these individual epitope responses.
  • Proliferative potential to whole protein peptide mixes and to the 5 selected individual peptide responses per RM are determined in each tissue by 6 day CFSE dilution cultures of PBMC and selected tissue cell preps (Onlamoon, N. et al. 2007. J Med Primatol 36:206-2), and supernatants from these cultures are sampled after 48 hrs and analyzed for cytokine secretion patterns by Luminex analysis (IL-2, -4, -5, -13, -17, IFN- ⁇ , GM-CSF, TNF, MCP-1, MIP-1 ⁇ / ⁇ , RANTES) (Giavedoni, L. D. 2005. J Immunol Methods 301:89-101).
  • Viral suppression assays on SIVmac239-infected autologous CD4+ T cells are performed on selected cell preparations to compare the anti-viral activity of responses in different tissues and arising from different vaccination routes.
  • the CD8+ T cell responses elicited by wt RhCMV/SIV vectors do not include the typical immunodominant epitopes defined for SIV or other viral vectors, precluding the use of existing tetramers to analyze wt RhCMV/SIV vector-elicited responses.
  • Applicants may identify and determine the restricting MHC allele on common, dominant CMV/SIV vector-elicited epitopes and have tetramers constructed for the most common of these epitopes. As they become available, these tetramers (as well as existing tetramers for “typical” epitopes in RM likely to have such responses—DNA/Ad5-vaccinated and/or SW-infected), are applied to this Example in appropriate RM (e.g., correct MHC types), both to directly quantify and phenotypically characterize tetramer defined responses in PBMC and tissues by flow cytometry, but more importantly, for sorting of defined epitope-specific populations by microarray analysis (see below).
  • appropriate RM e.g., correct MHC types
  • Wildtype (wt) RhCMV/SIV vectors elicit high frequency and broadly targeted CD8+ T EM responses to SIV epitopes, yet do not include responses to the typical immunodominant epitopes targeted in SIV infection itself or after vaccination with DNA or conventional viral vectors.
  • this “hole” in the wt RhCMV/SIV vector-elicited CD8+ T cell targeting is a direct result of the action of CMV genes in the US2-11 region that prevent MHC class I-restricted presentation by infected cells, a mechanism that might serve CMV biology by directing CD8+ T cell responses away from epitopes most likely to allow for efficient direct CD8+ T cell recognition of CMV-infected cells, and therefore, efficient CD8+ T cell-mediated cytolysis.
  • HCMV human cytomegalovirus
  • rhesus macaque Using the rhesus macaque (RM) model, Applicants' team demonstrated that rhesus CMV (RhCMV) may repeatedly re-infect sero-positive animals even when the same strain of CMV is used and an high level of antibody and T cell immunity is present (Hansen, S. G. et al. 2009. Nat Med 15:293-9 and Price, D. A. et al. 2008. J Immunol 180:269-). In preliminary experiments Applicants further show that such re-infection occurs with as little as 100 plaque-forming units (PFU) given subcutaneously (s.c.).
  • PFU plaque-forming units
  • RhCMV recombinant designated ⁇ Rh182-9
  • MHC-I major histocompatibility complex class I
  • Depletion of CD8 + T cells restores the ability of ⁇ Rh182-9 to re-infect sero-positive RMs, showing that inhibition of antigen presentation is one of the underlying reasons for re-infection.
  • the capacity of immunity induced by natural CMV infection to protect against the US2-11 deleted virus ⁇ Rh182-9 is thus an excellent measure for the quality of the CD8 + T cell response against CMV induced naturally by prior infection or artificially by vaccination.
  • This measure far surpasses all other means currently available to monitor CMV vaccine efficacy, since it is effectively ‘all-or-none’: once the sites of persistence have been reached by ⁇ Rh182-9 (even by a few viruses) long-term infection and easily detectable levels of viral shedding occur.
  • other measures such as viremia and clinical signs of infection are notoriously variable, subjective, and are especially problematic where vaccine effects are rather subtle.
  • Applicants therefore propose to use protection against ⁇ Rh182-9 to systematically re-evaluate some of the basic assumptions regarding vaccine approaches that have been made over the years regarding CMV vaccines. This allows Applicants to develop empirically based recommendations for the best strategy to develop a vaccine against HCMV. To accelerate future development of HCMV-based vaccines Applicants also generate and characterize in vitro recombinant HCMVs containing the same attenuating genetic disruptions present in the attenuated RhCMV vaccines.
  • ⁇ Rh182-9 Applicants' recent findings using a RhCMV virus deleted for the US2-11 genes ( ⁇ Rh182-9) show that this family of immune evasion genes is responsible for the ability of CMV to re-infect the healthy sero-positive host.
  • CD8′ depletion prior to challenge overcomes the block to ⁇ Rh182-9 infection in CMV sero-positive animals shows that the US6 family proteins function by their effect on the CMV-specific CD8 + T cell response.
  • ⁇ Rh182-9 infection may serve as an all-or-none read-out for whether a CMV-specific CD8 + T cell immune response is functionally comparable to that induced by natural WT CMV infection.
  • CMV possesses the remarkable ability to re-infect and establish a persistent infection regardless of host CMV immunity (Boppana, S. B. et al. 1999. Pediatrics 104:55-60, Farroway, L. N. et al. 2005. Epidemiol Infect 133:701-10 and Hansen, S. G. et al. 2009. Nat Med 15:293-9) ( FIG. 31 ).
  • CMV is shed for years from epithelial surfaces into body fluids (saliva, tears, urine, genital secretions and breast milk), and transmission generally involves mucosal exposure to such fluids, most commonly in early childhood or adolescence (Boppana, S. B. et al. 1999.
  • Virus was also detected in throat swabs and urine from one symptomatically infected individual, and in the blood from a second asymptomatic individual.
  • Toledo-1 was administered via a parenteral (subcutaneous) route, and it is possible that protection against natural infection via mucosal routes may by impacted by HCMV immunity (Adler, S. P. et al. 1995. J Infect Dis 171:26-32).
  • HCMV immunity Adler, S. P. et al. 1995. J Infect Dis 171:26-312.
  • HCMV sero-positive pregnant women using CMV strain-specific antibodies as an indicator of re-infection suggests that natural HCMV re-infection of the sero-positive healthy adult is a common occurrence (Boppana, S. B. et al. 2001. N Engl J Med 344:1366-71).
  • Gag-expressing virus was shed by infected animals, Applicants sampled saliva and urine on a weekly basis. Upon co-culturing virus pellets with RM fibroblasts (RFs), Applicants monitored Gag expression in immunoblots. In animals that received 10 7 PFU of RhCMV-Gag, Applicants detected Gag-positive virus in the urine of some animals within 7-14 days p.i., and in all animals by 42 days p.i. Similarly, buccal swabs of all animals were positive by 70 days p.i. Animals inoculated with lower doses had a trend toward longer time p.i. before detection of RhCMV-Gag in saliva and urine, but all animals were eventually positive.
  • RFs RM fibroblasts
  • RhCMV overcomes a substantial, pre-existing anti-CMV immune response during re-infection suggests that CMV has evolved mechanisms to evade host immune surveillance.
  • the adaptive cellular immune response is known to be particularly important for controlling CMV.
  • CMV-specific T cells comprise on average approximately 10% of both the CD4 + and CD8 + memory compartments (Sylwester, A. W. et al. 2005. J Exp Med 202:673-85) suggesting that enormous resources are constantly devoted to controlling this virus.
  • RhCMV genomic region Rh182—Rh189 encodes functional homologues of the US2, US3, US6 and US11 immunevasins of HCMV (Pande, N. T. et al. 2005. J Virol 79:5786-98).
  • Applicants replaced the Rh182-189 region with an expression cassette for the SIV Gag antigen, which allowed Applicants to monitor Gag-specific immune responses in infected animals (virus designated ⁇ Rh182-9Gag). Deletion of the Rh182-9 region was confirmed by PCR and Southern Blot. Applicants further monitored in vitro growth in primary RFs and observed no difference to WT, BAC-derived RhCMV (data not shown). Initially, Applicants determined whether ⁇ Rh182-9Gag would be able to establish persistent infection in CMV-negative animals.
  • ⁇ Rh182-9Gag (confirmed by immunoblot) was detected in the secretions of the infected animals even >1 year p.i. (data not shown). Therefore, Applicants conclude that ⁇ Rh182-9Gag is competent to establish persistent infection in CMV-na ⁇ ve animals.
  • CMV-encoded immunevasins enable re-infection of the sero-positive host due to their ability to evade the host CMV-specific CD8 + T response.
  • immunity induced by natural CMV infection is able to protect against CMV re-infection in the absence of these virally encoded immunevasins.
  • challenge with ⁇ Rh182-9Gag may be used to determine whether any type of vaccination effort has successfully generated a CD8 + T cell response that is as protective as that induced by natural infection.
  • this assay is far superior to any other measure of CMV-vaccine efficacy, since this all-or-nothing read-out allows candidate vaccine efficacy to be determined using relatively small groups of animals.
  • ⁇ Rh182-9Gag challenge Applicants re-examine whether non-replicating, partially replicating or non-replicating heterologous ‘prime-boost’ vaccines are able to induce a protective response.
  • RhCMV/RM infection model a model that closely mimics HCMV infection in humans, but that permits empirical ‘fine-tuning’ of the level of CMV attenuation.
  • Applicants introduce an on/off switch into the RhCMV genome which allow Applicants to inhibit or re-start viral replication at any time after infection thus addressing the roles of initial virus dissemination to sites of latent/persistent infection in the host, as well as of acute versus persistent replication in inducing a CMV-specific immune response.
  • Applicants propose to use the ability of a vaccine to protect against the US2-11 deleted recombinant virus ⁇ Rh1829Gag.
  • Applicants also evaluate the ability of ‘Prime-Boost’ vaccines (IE-1, pp65b, gB) to prevent ⁇ Rh182-9Gag re-infection using a DNA prime/adenovirus boost strategy.
  • Vaccinated animals that were protected against ⁇ Rh182-9Gag are further challenged with WT-RhCMV followed by monitoring of viremia.
  • RhCMV model Protection against ⁇ Rh182-9Gag thus indicate that a given vaccine was able to generate a CMV-specific immune response, particularly a CD8 + T cell response, similar to that induced during natural infection.
  • One of the great advantages of the RhCMV model is the close evolutionary relationship between the human and rhesus CMVs, and their respective hosts. Therefore, Applicants in parallel generate and characterize HCMV-derived constructs containing identical genetic deletions thereby ensuring that any of the attenuated vaccines that shows promise in the RhCMV system is functionally comparable to its HCMV counterpart. Applicants anticipate that results obtained in this project are directly transferable to development of an HCMV-based vaccine candidate.
  • RhCMV ⁇ Rh110 is growth-deficient in vitro but is not secreted from infected monkeys. Applicants have tested whether monkeys infected with RhCMV ⁇ Rh110 are protected against challenge with RhCMV ⁇ US2-11 expressing the SIV antigen Gag. Protection was demonstrated by the absence of a boost in RhCMV-specific T cell responses. In contrast, monkeys infected with wildtype-virus show a boost of the CMV-specific T cell response (see FIG. 23 ). This result indicates that spread-deficient CMV is capable of inducing a T cell response that protects against challenge with US2-11 deleted virus. This result also indicates that a US2-11 deleted virus may be used to monitor the efficacy of the T cell response.
  • RhCMV lacking the tegument proteins pp65a and pp65b encoded by the genes Rh111 and Rh112, respectively (see FIG. 24 ). These proteins are not required for viral growth in vitro.
  • pp65 is an immunodominant protein that is included in current formulations of subunit vaccines for CMV developed by various investigators.
  • pp65-specific T cells are required for protection against challenge with ⁇ US2-11, Applicants infected rhesus macaques with RhCMV ⁇ Rh111-112.
  • RhCMV ⁇ Rh111-112 was readily detected in animals infected with RhCMV (blue line). Applicants also observed that RhCMV ⁇ Rh111-112 is secreted from infected animals.

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