WO2006110728A2 - Immunogenic cmv tegument aggregates - Google Patents

Abstract

The present invention provides for cytomegalovirus protein tegument aggregates, pharmaceutical compositions, and the use thereof. The present invention provides for methods for anti-viral drug screening, production of CMV antigenic proteins, recombinant protein expression, immunogenic compounds such as CMV tegument aggregates with or without additional haptens, and vaccines useful for raising an immune response in an animal.

Description

IMMUNOGENIC TEGUMENT AGGREGATES

CROSS-REFERENCE TO RELATED APPLICAΉON

The present application is related to, and claims benefit under 35 U.S.C. § 119(e), of U.S. Provisional Patent Application No. 60/670,262, entitled "Immunogenic Tegument Aggregates," filed 12 April 2005, which is incorporated entirely herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was produced in part using funds obtained through grants from the National Institutes of Health. Consequently, the federal government may have certain rights in this invention.

FIELD OF THE INVENTION

The present invention is in the fields of molecular virology, anti- viral drug screening, recombinant protein expression, immunogenic compounds, and vaccines useful for raising an immune response in an animal. In particular, the invention relates to cytomegalovirus protein tegument aggregates, pharmaceutical compositions, and the use thereof.

BACKGROUND OF THE INVENTION Cytomegalovirus, or CMV, is a member of the herpesvirus group, which includes herpes simplex virus types 1 and 2, varicella-zoster virus (which causes chickenpox), and Epstein-Barr virus (which causes infectious mononucleosis). These viruses share a characteristic ability to remain dormant within the body over a long period. Initial CMV infection, which may have few symptoms, is always followed by a prolonged, unapparent infection during which the virus resides in cells without causing detectable damage or clinical illness. Severe impairment of the body's immune system by medication or disease consistently reactivates the virus from the latent or dormant state.

Infectious CMV may be shed in the bodily fluids of any previously infected person, and thus may be found in urine, saliva, blood, tears, semen, and breast milk. The shedding of virus may take place intermittently, without any detectable signs, and without causing symptoms. CMV is found universally throughout all geographic locations and socioeconomic groups, and infects between 50% and 85% of adults in the United States by 40 years of age. CMV is also the virus most frequently transmitted to a developing child before birth. CMV infection is more widespread in developing countries and in areas of lower socioeconomic conditions.

CMV infection is important to certain high-risk groups. Major areas of concern are (1) the risk of infection to the unborn baby during pregnancy, (2) the risk of infection to people who work with children, and (3) the risk of infection to immunocompromised people such as organ transplant recipients and persons infected with HIV. CMV remains the most important cause of congenital viral infection in the United States. During a pregnancy when a woman who has never had CMV infection becomes infected with CMV, there is a potential risk that after birth the infant may experience CMV-related complications, the most common of which are associated with hearing loss, visual impairment, or diminished mental and motor capabilities. Some of these complications can be permanent or even fatal.

Currently, no treatment exists for CMV infection in otherwise healthy adults. Antiviral drug therapy is being evaluated in infants. Ganciclovir treatment is used for patients with depressed immunity, having either sight-related or life-threatening illnesses. Vaccines, however, are still in the research and development stage.

Hence, there remains a need for a vaccine that will protect, particularly women of child-bearing age and immunocompromised individuals, from CMV infection. There is also a need, in general, for improved vaccine protein carriers and adjuvants.

SUMMARY OF THE INVENTION

An object of the present invention provides for immunogenic pp65 tegument aggregates suitable for a vaccine for the prevention of CMV disease, particularly congenital CMV disease.

Another object of the invention provides for a vector system facilitating the production of vaccine antigens for other diseases by fusing other haptens of interest to pp65 and recovering the tegument aggregates. In particular, the invention relates to the development of vaccines and specifically to the development of an improved method for antigen presentation. The invention further relates to the delivery of haptens and/or nucleic acids to cells via a particle. In one aspect, the invention features use of the pp65 antigen as a human cytomegalovirus vaccine. The vaccine of the invention is useful in conferring protective immunity in human subjects at risk for a CMV-mediated disease or as a therapeutic vaccine. In another aspect of the invention, a different or non-CMV hapten is incorporated (either genetically or via chemical conjugation) within the tegument aggregate particles.

Another embodiment of the invention provides for a method of providing an immune response and protective immunity to a patient against cytomegalovirus-mediated diseases. The method includes administering the tegument antigen of the invention to an animal or human.

Another aspect of the present invention provides a recombinant virus useful for screening the effectiveness of inhibitors of CMV viral kinase activity. Tegument aggregates of pp65 accumulate as large intracellular structures in cytomegalovirus- infected cells in the absence of the viral UL97 protein kinase activity. The formation of these structures occurs when cells infected with virus are treated with an inhibitor of UL97 kinase activity, such as maribavir (MBV). Screening can also be conducted by transiently expressing pp65 and the UL97 kinase in COS7 cells, or other cells, and identifying compounds that induce the production of pp65 aggregates through the inhibition of the protein kinase. Another objective of the present invention provides for a process of obtaining quantities of CMV pp65 by infecting a cell culture with CMV, treating the infected cells with an agent such as maribavir such that tegument aggregates for within the infected cells, then harvesting the pp65-containing tegument aggregates. Another aspect of the invention provides for the utilization of a UL97 deletion virus for over-production viral antigens or tegument aggregates.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Photographs of inclusions in infected human foreskin fibroblast (HF) cells and the effect of antiviral drugs on their formation. All images were obtained with a IOOX objective and are shown merged with images of DAPI staining. Phase contrast image of HF cells infected with the UL97 deletion virus (RCΔ97.08) (A) or wt virus (AD169) with the addition of 15 μM MBV (B) are shown at 72 hours post-infection (hpi). The formation of aggregates at 96 hpi was visualized with monoclonal antibody to pp65 in AD 169 infected cells without the addition of drug (C), with 15 μM MBV (D), with 15 μM 2-bromo-5,6- dichloro-1-beta-D-ribofuranosyl benzimidazole (BDCRB) (E), or with cidofovir (F). Localization of aggregates in cells infected with RCΔ97.08 was visualized with a pp65 monoclonal antibody at 48 hpi (G), and 72 hpi (H). Figure 2. Phase contrast images of isolated tegument aggregates from the nuclear fraction (A) and the cytoplasmic fraction (B) of HF cells infected with RCΔ97.08. Phase contrast images are shown from a 4OX objective.

Figure 3. Most abundant proteins in tegument aggregates. Aggregates were isolated the nuclear and cytoplasmic fractions of HF cells infected with RCΔ97.08 and are shown with the crude lysates from which they were derived (A). Viral proteins identified by mass spectrometry from tegument aggregates isolated from the nuclear fraction are indicated by arrows. The isolation procedure was used to separate aggregates from uninfected HF cells, and HF cells infected with RCΔ97.08, AD169, and AD169 and treated with MBV (B). Figure 4. Tegument aggregates do not form in that absence of pp65. HF cells were infected with AD169 (A) or RCΔ97.08 (B) and incubated for 96 h in the presence of 30 μM MBV. Cells infected with recombinant virus RVAd65 containing a deletion of pp65 were incubated for 96 h with the addition of 60 μM MBV (C). Tegument aggregates were stained with a monoclonal antibody to ppl50 and images shown are merged with DAPI stained images.

Figure 5. Aggregation of pp65-GFP in transfected COS7 cells is controlled by UL97 kinase activity. Transient expression of a pp65 GFP fusion protein in COS7 cells results in the formation of large nuclear aggregates (A) 24 h post transfection. Resulting aggregates fluoresce so brightly that they are detected with the filter used for DAPI staining (B). Transiently expressed ppUL97 with a V5 epitope tag localizes to the nucleus (C), whereas the same ppUL97-V5 fusion protein with a K355M point mutation to abolish the enzymatic activity localizes both to the nucleus and the cytoplasm (D) as shown in the images merged with DAPI staining. Coexpression of pp65-GFP (E), and ppUL97-V5 (F) eliminates the formation of nuclear pp65-GFP aggregates and instead, results in the diffuse nuclear localization of both proteins as shown in the merged image with DAPI (H). The inhibition of pp65 aggregation by ppUL97 is antagonized by the addition of MBV and pp65-GFP are formed (I-L). The inhibition of UL97 kinase activity by MBV allows both the formation of pp65-GFP aggregates (I), as well as the recruitment of ppUL97 to the aggregates (J). Similarly, the coexpression of ppUL97-V5-K355M (N), is unable to stop the aggregation pp65-GFP (M), and the kinase deficient ppUL97 fusion protein is recruited to the nuclear inclusions as shown in the merged image (P). DETAILED DESCRIPTION OF THE INVENTION

It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

As used herein and in the claims, the singular forms "a," "an," and "the" include the plural reference unless the context clearly indicates otherwise. Thus, for example, the reference to a protein is a reference to one or more such proteins, including equivalents thereof known to those skilled in the art. Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term "about." The term "about" when used in connection with percentages may mean ±1%.

All patents and other publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those commonly understood to one of ordinary skill in the art to which this invention pertains. Although any known methods, devices, and materials may be used in the practice or testing of the invention, the preferred methods, devices, and materials in this regard are described here.

The present invention relates to molecular virology, drug screening, immunogenic compounds, and vaccines useful for raising an immune response in an animal. In particular, the invention relates to cytomegalovirus protein pp65 tegument aggregates, assays that screen for anti-UL97 agents, pharmaceutical compositions, and the uses thereof. The pp65 tegument aggregates of the present invention are large stable spherical aggregates composed substantially of viral pp65 proteins and possess a number of interesting and potentially useful properties: (a) they are readily isolated from infected cells; (b) they can represent more than 6% of the total cellular protein; (c) they can be mass produced in a substrate cell that is acceptable to the FDA for the manufacture of vaccines; and (d) they are noninfectious but appear to elicit a robust immune response.

The tegument of CMV is a proteinaceous layer surrounding the viral nucleocapsid consisting of as many as twenty proteins, many of which are phosphorylated. Phosphoprotein pp65 (also known as tegument protein UL83 because it is expressed from the UL83 viral gene), also known as lower matrix protein, is the primary target of phosphorylation by protein kinases and makes up approximately 95% of total tegument mass.

The pp65 protein has been identified as a target antigen for CMV-specific class I MHC restricted CTL derived from the peripheral blood of most asymptomatic CMV seropositive individuals. Mclaughlin-Taylor et al, 43 J. MED. VlROL. 103-10 (1994). Importantly, CD8⁺ class I MHC restricted CTL specific for pp65 will recognize autologous HCMV-infected cells without the requirement for viral gene expression, presumably as a result of processing of the internal depot of pp65 that is transferred into the cell during infection. Gilbert et al, 67 J. ViROL. 3461-69 (1993).

The pp65 protein has been sequenced and may be expressed in E. coli. Bankier et al, 2(1) DNA SEQ. 1-12 (1991); Pande et al, 182(1) ViROL. 220-28 (1991). CMV-pp65 contains immunodominant regions within amino acids 297-510. See, e.g., Mocarski, Cytomegalovirus Biology and Replication, in THE HUMAN HERPESVIRUSES (Roizman et al, eds. Raven Press, 1993). Additionally, the amino acid sequences of several pp65 epitopes have been identified. For example, pp65 residues 495-503 (NLVPMATV) provide a target for CD8⁺ T-cells. Kahm et al, 185 J. INFECT. DIS. 1025-34 (2002); U.S. Patents No. 6,727,093; No. 6,843,992. Full-length pp65 as well of portions (peptides) of pp65 have been shown to generate a T-cell response when expressed in recombinant Vaccinia virus, pp65 has been immunogenic when expressed in recombinant adenovirus, retrovirus, and canarypox virus. See, e.g., Wang et al, 104(3) BLOOD 847-56 (2004).

The inventors have discovered that the aggregation of pp65 tegument proteins is inversely related to the activity of the CMV protein kinase UL97. Protein kinases are encoded by diverse virus families including herpesviruses, poxviruses, and rotaviruses. Blackhall et al, 71 J. VIROL. 138-44 (1997); Purves et al, 89 PROC. NATL. ACAD. SCI. USA 7310-4 (1992); Rempel et al, 66 J. VIROL. 4413-26 (1992). All herpesviruses encode at least one conserved herpesvirus protein kinase that may mimic aspects of cellular protein kinase cdc2, and play a role in viral replication. Kawaguchi et al, 11 J. VIROL. 2359-68 (2003). Each of these viral enzymes contain conserved subdomains found in cellular serine/threonine (ser/thr) kinases and possess both autophosphorylating and transphosphorylating activity. Kawaguchi et aL, 13 REV. MED. VIROL. 331-40 (2003). These conserved kinases likely share some common functions among all the herpesviruses since the human cytomegalovirus (HCMV) UL97 kinase can substitute to some degree for the UL13 kinase in herpes simplex virus. Ng et aL, 225 VlROL. 347-58 (1996).

The UL97 protein kinase shares homology with this family of kinases and appears to be closely related to human ser/thr kinases. Michel et ah, 79(9) J. GEN. VIROL. 2105-12 (1998). This enzyme exhibits an unusual substrate specificity in that it can activate both ganciclovir (GCV) and acyclovir through the selective phosphorylation of these nucleoside analogs (Littler et ah, 358 NATURE 160-2 (1992); Sullivan et aL, 359 NATURE 85 (1992); Talarico et aL, 43 ANΉMICROB. AGENTS CHEMOTHER. 1941-6 (1999)), and it can autophosphorylate and transphosphorylate protein and peptide substrates on serine and threonine residues (Baek et aL, 277 J. BiOL. CHEM. 29593-9 (2002); He et al., 11 J. VIROL. 405-11 (1997)). The high frequency of UL97 mutations in drug resistant clinical isolates reflects its central role in mechanism of action of GCV as well as its importance in the management of CMV infections in the clinic. Chou et aL, 171 J. INFECT. DIS. 576-83 (1995). More recently, this kinase has become an antiviral target in its own right, as MBV was shown to be a selective inhibitor of this enzyme and exhibits potent antiviral activity Biron et aL, 46 ANTIMICROB. AGENTS CHEMOTHER. 2365-72 (2002). This and other inhibitors of this enzyme are being developed as new therapies for the treatment of CMV infections. Herget et aL, 48 ANTIMICROB. AGENTS CHEMOTHER. 4154-62 (2004); Koszalka et aL, 46 ANTIMICROB. AGENTS CHEMOTHER. 2373-80 (2002). Thus, defining the function of this kinase in viral replication is vital to understanding the emergence of drug resistance in the clinic, as well as the development of better antiviral therapies.

The UL97 open reading frame is contained in a complex transcriptional unit that produces a number of structurally polycistronic 3' coterminal transcripts, of which at least five contain this gene. Wing et aL, 69 J. VIROL. 1521-31 (1995). Only the 4.7 kb transcript is thought to be translated, but mutations in this open reading frame may also disrupt the transcription of other genes that are thought to be essential. UL97 kinase is expressed with early/late kinetics, localizes to the nucleus and has an apparent migration rate of approximately 80 kilodaltons. Michel et aL, 70 J. VlROL. 6340-6 (1996). The kinase is also a constituent of virions and is post-translationally modified by the phosphorylation of serines and threonines. Michel et al. (1996). The nuclear localization signal lies in the amino terminal domain, and this region is not required for GCV phosphorylation. Michel et al, 79(9) J. GEN. VIROL. 2105-12 (1998). Point mutations in the conserved kinase domains including amino acids 340, 442, 446, and 523, result in the loss of both autophosphorylation and GCV phosphorylation activity. Michel et al, 73 J. VIROL. 8898-901 (1999).

Although an intact UL97 open reading frame is not essential for viral replication, a deficiency of the gene product reduces virus yield by more than two orders of magnitude. Prichard et al, 73 J. ViROL. 5663-70 (1999). The first observable defect during the replication of a UL97 null virus is a modest 2-6 fold decrease in the accumulation of viral DNA replication, but the magnitude of this defect is insufficient to explain the poor replication characteristics of this virus. Krosky et al, 11 J. VIROL. 905-14 (2003); Wolf et al, 98 PROC. NATL. ACAD. SCI. USA 1895-900 (2001). The first striking defect is a marked decrease in the number of mature capsids in the cytoplasm of cells infected with the UL97 deletion virus, suggesting that mature virions never exited the nucleus. Krosky et al, 11 J. VIROL. 905-14 (2003); Wolf et al, 98 PROC. NATL. ACAD. SCI. USA 1895-900 (2001). The underlying molecular defects that occur in the absence of UL97 remain undefined, but could be related to the poorly understood events related to the cleavage and packaging of DNA, virion morphogenesis in the nucleus, or the process of nuclear egress where the nuclear lamina is degraded to facilitate the exit of mature capsids. The first report of recombinant viruses with large deletions in UL97 described a distinctive plaque morphology characterized by the appearance of highly refractile bodies in the nucleus of infected cells. Prichard et al, 13 J. VIROL. 5663-70 (1999). These structures were not observed in cells infected with any wild type (wt) virus, so their appearance in two independently constructed recombinant viruses suggested that they were a result of the engineered mutations rather than other mutations in the genome. Their appearance was also markedly diminished in a population of cells expressing ppUL97, and suggested that their appearance was due to a deficiency in this viral protein. An investigation into the nature of these nuclear inclusions revealed that these molecular defects were associated with a deficiency in UL97 kinase activity. These studies demonstrated that the inclusions were composed predominantly of tegument and structural proteins and that their formation was a result of inappropriate aggregation by pp65 in the absence of UL97 kinase activity. The spontaneous aggregation of pp65 was reproduced in uninfected cells and was inhibited specifically by the enzymatic activity of the kinase. These results suggest that the UL97 kinase is required for the proper function of tegument proteins in the nuclei of infected cells. The appearance of the pp65 aggregates are also observed in infected cells treated with the UL97 kinase inhibitor, MBV. Nuclear inclusions may be purified to near homogeneity and the constituent proteins identified by MALDI-TOF mass spectrometry. This analysis demonstrates that the principle components of the inclusions are the tegument proteins pp65 and ppUL25 and also contained major capsid protein. Immunoblotting experiments also identify a number of additional viral proteins present in the purified tegument aggregates. Interestingly, the formation of these structures appear to be dependent on pp65, because they do not occur in MBV-treated cells infected with a recombinant virus that does not express this protein. Similar aggregates could be reproduced in nuclei of uninfected cells by over expressing pp65, and aggregate formation may be prevented by co-expressing the UL97 kinase. Inhibition of UL97 kinase activity with MBV, or mutation of an essential amino acid in the kinase, abolishes its ability to prevent aggregate formation. These data suggest that the UL97 regulates the aggregation of pp65 in the nuclei of infected cells and that the misregulation of pp65 results in the formation of tegument aggregates. Hence, control of pp65 aggregation by the kinase may be required for the normal acquisition of tegument by capsids in the nucleus and may be an important step in virion morphogenesis.

Hence, the present invention provides for a system useful to screen drug libraries to identify anti-viral drugs, particularly those which inhibit the protein kinase activity of UL97, without the need for producing purified enzyme. One embodiment of the present invention provides for techniques useful for screening the effectiveness of inhibitors of CMV viral kinase activity. Briefly, in this approach, cells infected with CMV are treated with the antiviral agent and then later observed for the presence of inclusion bodies. See Example 2, below. For example, the formation of tegument aggregates of pp65 protein accumulate in CMV-infected cells when ppUL97 protein kinase activity is inhibited with the drug MBV. The effectiveness of MBV and drugs like it may be determined by observing the presence of inclusion bodies within CMV-infected cells. Another approach to identifying inhibitors of the UL97 kinase expresses both the pp65 and the kinase transiently in COS7 cells: any compound that inhibits the kinase would induce the formation of large aggregates in the cells. Such inhibitors of the protein kinase exhibit potent antiviral activity and some have been investigated clinically for the treatment of HCMV infections. The present invention also provides for a process of preparing useful quantities of pp65 from CMV-infected cells by treating infected cells with a ppUL97-inhibitor. In these cells, tegument aggregates including pp65 may constitute six percent of cell protein, and may be isolated and purified readily from cells by methods well-known in the art. For example, wild type virus such as AD 169 may be grown in suitable human cells such as human foreskin fibroblasts or fetal diploid lung cells cells in which UL97 activity is inhibited by the addition of MBV. After growing the infected cells from about 48 to 120 hours or more in a suitable medium, the cells may be separated from the medium, washed, and lysed in a suitable buffer and the large, spherical aggregates purified by known methods. Alternatively, any appropriate cell line may be transfected with a vector that expresses pp65, and grown for a suitable length of time. For example, prokaryotes such as E. coli may be used to express recombinant pp65. It may be desirable to alter the pp65 genetic sequence to improve solubility in bacteria. The pp65 aggregates may then be separated from the lysate using known methods such as gel exclusion or immunoaffinity column chromatography, or as described in the examples below. Tegument aggregates have also been produced by the inventors in recombinant baculovirus-infected sf9 cells and in MRC-5 cells, the latter representing a desirable cell substrate for vaccines.

Within the scope of the present invention are derivatives of the pp65 tegument aggregates. "Derivative" is intended to include modifications of the native pp65 protein that retain the immunizing activity of the native pp65 aggregate. The term is intended to include, without limitation, fragments, oligomers or complexes of the protein, polypeptides or fusion proteins made by recombinant DNA techniques whose amino acid sequences are identical or substantially identical (i.e., differ in a manner that does not affect immunizing activity adversely) to that of the protein or that of an active fragment thereof, or that lack or have different substituents (e.g., lack glycosylation or differ in glycosylation), and conjugates of the protein or such fragments, oligomers, polypeptides, fusion proteins, and carrier proteins.

Additionally, the pp65 protein or a derivative thereof may be used to detect the presence of CMV antibodies in samples (e.g., sera) of human Ig-containing body fluids. This procedure might be used, for instance, to identify potential donors of anti-CMV sera for use in passive immunization therapy. The basic procedure involves incubating the sample with the protein or derivative under conditions that permit antigen-antibody binding and detecting resulting immune complexes that include the protein. The immune complexes may be detected by incorporating a detectable label (e.g., radionuclide, fluorochrome, enzyme) into the complex. A standard solid phase immunoassay procedure is preferred. In such a procedure the protein is immobilized on a solid phase and the immobilized protein is incubated with the sample. The solid phase is then separated from the sample and washed to remove residual, unbound sample. The solid phase is next incubated with a labeled anti- human Ig antibody. Following the second incubation the solid phase separated from the labeled reagent and washed to remove residual unbound labeled reagent. Immune complexes containing anti-CMV antibodies are detected via the label. The complexes may be detected on the surface of the solid phase or eluted from the solid phase. In the case of radionuclides, the immune complexes are normally detected by scintillation scanning. Fluorescent labels are detected by exposing the analyte to excitation energy and detecting the resulting fluorescence. Enzyme labels are detected by incubating the solid phase with an appropriate substrate solution and detecting enzyme activity by spectrophotometric analysis of the substrate solution.

The present invention also provides for novel monoclonal antibodies against tegument aggregate protein epitopes. In particular, a monoclonal antibody against the viral ppUL50 was generated as described below in Example 7. A monoclonal antibody to HCMV ppUL50 has not been reported previously. Antibodies within the scope of this invention include portions or fragments of antibodies that are capable of binding to an antigenic hapten. A hapten, as used herein, refers to a disease-specific antigenic determinant identified by biochemical, genetic, or computational means.

Moreover, by manipulating or removing the expression of UL97 kinase or expressing a cloned pp65 gene, the present invention provides for pp65 tegument structures that are immunogenic. These structures not only raise an immune response against CMV, but may also serve as a scaffold for the delivery of other antigens in a vaccine. Vaccination with inactivated or attenuated organisms or their products has been shown to be an effective method for increasing host resistance and ultimately has led to the eradication of certain common and serious infectious diseases. The use of vaccines is based on the stimulation of specific immune responses within a host.

It has been reported previously that "dense bodies" including CMV viral envelopes, pp65 and gB were produced in fibroblasts infected with CMV. These dense bodies were able to raise an immune response in mice. Pepperl et al, 25 J. VIROL. S75-S85 (2002). It has also been reported that, in the context of dense bodies, phosphoprotein pp65 plays a central role both in the induction of CTL and in the stimulation of CD4⁺ Th lymphocytes. Pepperl et ah, 74(13) J. VIROL. 6132-46 (2000). Additionally, ρp65 fused on its carboxyl end to both green fluorescent protein and neomycin phosphotransferase (pp65-GFP-Neo) was packaged into dense bodies which subsequently transported the fusion protein into target cells. Pepperl et al, 10 GENE THERAPY 178-84 (2003). In contrast to dense bodies, the spherical tegument aggregates of the present invention are as much as 100 times larger than CMV dense bodies, and lack the viral envelope and other possible protein contaminants associated with dense bodies. Importantly, the tegument aggregates of the present invention are highly immunogenic. Indeed, tegument aggregates of the present invention comprising HCMVpp65 were immunogenic without the use of an adjuvant. The tegument antigen described in this invention generates an immune response. The term "immune response" refers to a cytotoxic T-cell response or increased serum levels of antibodies specific to an antigen, or to the presence of neutralizing antibodies to an antigen. The immune response may be sufficient to make the tegument antigen of the invention useful as a vaccine for protecting human subjects from CMV infection. Additionally, antibodies generated by the tegument antigen of the invention can be extracted and used to detect a virus in a body fluid sample. The term "protection" or "protective immunity" refers herein to the ability of the serum antibodies and/or cytotoxic T-cell response induced during immunization to protect (partially or totally) against a disease caused by an infectious agent, e.g., human cytomegalovirus. The use of the combined antigen as a vaccine is expected to provide protective immunity to humans against severe CMV infection by inducing antibodies against CMV which are known to prevent severe clinical symptoms.

In another embodiment of the present invention, the tegument aggregate is conjugated to another antigen (hapten), thus acting as an effective protein carrier or adjuvant for that hapten. Hapten refers to a disease specific antigenic determinant identified by biochemical, genetic or computational means. The haptens may be associated with a disease condition caused by an agent selected from the group consisting of: single stranded DNA viruses, double stranded DNA viruses, single stranded RNA viruses, double stranded RNA viruses, intracellular parasites, fungi, bacteria, and cancer. The hapten may be a protein, peptide, or polysaccharide. The protein or peptide may also be a mimetic of a polysaccharide epitope. Conjugation may be accomplished by known methods, including genetic manipulation of recombinant pp65 proteins and chemical conjugation to the tegument aggregate proteins. An example of such a hapten, provided for in the present invention, is a HIV ρ24 epitope as shown in Example 9 and Example 10. This hapten was expressed recombinantly with the pp65 tegument aggregate and proved antigenic, raising an immune response to the p24 epitope.

Haptens of the group B streptococci are another example of haptens that could be presented with the tegument aggregate of the present invention: women of child bearing age are one of the populations in need of an effective CMV vaccine because of the risk to fetuses and infants following CMV infection. These women would benefit from a vaccine against group B streptococcal disease, also implicated in serious and life-threatening sequelae for infants. In an aspect of the invention, GBD capsular polysaccharides are coupled to the pp65 tegument aggregate. See, e.g., Paoletti et al., 20(3-4) VACCINE 370-76 (2001). This vaccine approach may prove highly immunogenic.

Additionally, any appropriate immune stimulating nucleic acid may be associated with purified tegument aggregates, with or without additional haptens, and used as a vaccine in the practice of the present invention. See, e.g., U.S. Pat. Appl. Pub. No. 2004/0219164. The invention includes a method of providing an immune response and protective immunity to a patient against cytomegalovirus-mediated diseases. The method includes administering the tegument antigen of the invention to an animal or human. The tegument antigen of the invention is preferably administered as a formulation comprising an effective amount of the tegument antigen. A variety of physiologically acceptable carriers are known in the art, including, for example, saline. Routes of administration, amounts, and frequency of administration are known to those skilled in the art for providing protective immunity to a recipient subject. Routes of administration include any method which confers protective immunity to the recipient, including, but not limited to, inhalation, intravenous, intramuscular, intraperitoneal, intradermal, intranasal, and subcutaneous. Preferably the tegument antigen of the invention is provided to a human subject by subcutaneous or intramuscular injection. A range of amounts and frequency of administration is acceptable so long as protective immunity of the recipient is achieved. For example, 5 μg to 20 μg can be administered by intramuscular injection between one and four times over a three- month period.

Inoculation with the vaccine of the present invention is particularly suitable for individuals about to undergo immunosuppression, such as those undergoing skin grafting or organ transplantation. Such individuals, if they lack immunity (antibodies), are at great risk for CMV infection. The vaccine is also of great benefit to women of childbearing years, perhaps at or before the early stages of pregnancy. The primary threat from CMV infection arises from transmission or reactivation of CMV during the gestation period. Thus, if the infective cycle of CMV can be inhibited by stimulation of the mother's immune system, the likelihood of infection of the infant can be reduced or eliminated.

The approaches to determine the suitability of a vaccine candidate are well known. For example immunogenicity studies are often conducted before implementation of a randomized, placebo controlled, double-blind efficacy trial. Once a protective immune mechanism is defined, e.g., from an efficacy trial, vaccine performance can then be judged by serologic parameters. See, e.g., Boome, Vaccine Efficacy Determination, 14(2) NEPH ANN. 219-21 (1991). Thereafter, national surveillance and, for example, indirect cohort analysis may determine protective efficacy. See, e.g., Butler et al, 270(15) JAMA 1826-31 (1993). '

EXAMPLES Example 1. Expression of pp65 in UL97 wt and UL97 defective CMV-infected cells Tegument aggregate formation as it relates to the expression of CMV UL 97 was studied as described in Prichard et al, 79(24) J. ViROL. 15494-502 (2005), incorporated fully herein by reference. Cells, viruses, and drugs: Primary or low-passage human foreskin fibroblast cells were routinely propagated in monolayer cultures in minimum Eagle's medium with Earle's salts supplemented with 10% fetal bovine serum, 2mM L-glutamine, 100 μg/ml penicillin G and 25 μg/ml gentamicin. HCMV strain AD 169 was obtained from the American Type Culture Collection (Manassas, VA) and virus stocks were prepared and titered as described previously. Spaete et al, 56 J. VIROL. 135-43 (1985). The UL97 null mutant, RCΔ97.08 was described previously (Prichard et al, 13 J. VlROL. 5663-70 (1999)), and the pp65 deficient virus was obtained from Dr. Bodo Plachter (University of Mainz, Mainz, Germany) and its constructions details were described previously (Schmolke et al, 69 J. VIROL. 5959-68 (1995)). Maribavir (MBV), 2-bromo-5,6-dichloro-l-beta-D-ribofuranosyl benzimidazole (BDCRB), and cidofovir (CDV) were obtained through the Antiviral Substances Program of the National Institute for Allergy and Infectious Diseases.

Plasmids: Two PCR reactions were used to construct a K355M mutation in the UL97 open reading frame (ORF) using UL97 forward primer 5'-CAC CAT GTC CTC CGC ACT TCG GTC T-3' (SEQ ID NO: 1) and UL97^*B reverse primer 5'-CTA TCG CGT GGT CAT GGT GGC GCG TAA G-3' (SEQ ID NO: 2) for the first reaction and UL97^*B forward primer 5'-CTT ACG CGC CAC CAT GAC CAC GCG ATA G-3' (SEQ ID NO: 3) and the UL97 reverse primer 5'-TTA CTC GGG GAA CAG TTG G-3' (SEQ ID NO: 4) for the second reaction. The fragments were amplified with Taq PCR Master Mix (Qiagen, Valencia, CA) and the resulting PCR products were gel purified using a Qiagen Gel Purification Kit. PCR products were combined in an equal ratio and used as templates for the final PCR product using UL97 forward and reverse primers, and PFU Turbo polymerase (Stratagene, La Jolla, CA). The resulting PCR product was TOPO cloned into pENTR (Invitrogen, Carlsbad, CA). The UL97 ORF was also amplified with the UL97 forward and UL97 reverse primers and cloned into pENTR, and the open reading frames were sequenced. Both the wt AD 169 UL97 ORF and the K355M mutated ORF were recombined into pcDNA3.1/nV5-DEST vector using LR clonase (Invitrogen) expression vector to provide amino terminal epitope tags and were designated pMP93 and pMP92, respectively. Both plasmids expressed immunoreactive proteins of the predicted molecular weight, and the K355M mutant did not appear to be phosphorylated. A pp65-green fluorescent protein-expressing plasmid was constructed by ligating the coding sequence of AD 169 UL83 (pp65) into the enhanced green fluorescent protein (EGFP) Cl plasmid (Clontech/BD, Palo Alto, CA), resulting in the expression of an EGPF protein having pp65 fused to the carboxyl terminus of EGFP.

Transformation of a host cell with vectors containing DNA encoding the recombinant virus of the invention may be carried out by conventional techniques as are well known to those skilled in the art. Such transformed host cells are capable of expressing the pp65 tegument aggregate antigen. Isolation and purification of the expressed antigen may be carried out by conventional means well known in the art, or as described further below. Polyacrylamide gels and blotting: Protein samples were disrupted in 2 X Laemlli buffer (Sigma, St. Louis, MO) and separated on 10% polyacrylamide gels (BioRad, Hercules, CA). For Western blots, proteins were transferred to polyvinylidene difluoride membranes (Roche Applied Science, Indianapolis, IN) in a buffer containing 28 mM Tris, 39 mM glycine, 0.0375 % SDS and 20% methanol in a semi dry transfer cell (BioRad, Hercules, CA). Blots were blocked in 1% blocking buffer (Roche Applied Science, Indianapolis, ESf), incubated with primary antibodies overnight at 4°C and washed in PBS with the addition of 0.02% tween 20. An alkaline phosphatase conjugated goat anti-mouse secondary antibody (Southern Biotechnology Associates, Birmingham, AL) was used to detect the primary antibody and the blots were developed with CDP* (Roche Applied Science, Indianapolis, IN). Immunofluorescence microscopy: Immunofluorescence staining was performed in a manner that is similar to that previously described by Sanchez et al, 74 J. VlROL. 975-86 (2000). Briefly, monolayer cultures of HF cells were grown on 13 mm diameter coverslips in 24- well plates. Infected coverslips were fixed for 15 minutes with freshly prepared 1% formaldehyde in phosphate buffered saline (PBS), washed two times with PBS, and permeabilized with 0.2 % Triton X-IOO in PBS for 15 minutes. Monoclonal antibodies to pρ65 (28-19), ρpl50 (36-14), ppUL44 (28-21) and major capsid protein (28-4) were used as culture supernatants with goat anti-mouse secondary antibodies conjugated to fluorescein isothiocyanate or Texas Red (Southern Biotechnology Associates, Birmingham, AL). Mass spectrometry: Proteins were excised from polyacrylamide gels stained with coomassie brilliant blue and were analyzed at the UAB Comprehensive Cancer Center/Department of Pharmacology and Toxicology shared mass spectrometry facility. Matrix assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometers (Applied Biosystems, Foster City, CA) were used to identify proteins present in the inclusions.

Isolation of nuclear and cytoplasmic tegument aggregates: Low-passage HFF cells were infected with RCΔ97.08 at an MOI of 0.01 PFU/cell in growth media in 175 cm² flasks. Monolayers of infected cells were passaged at 7 days postinfection (dpi) as plaques started to form as well as 12 dpi and 16 dpi until 100% cytopathic effect was observed. Infected cells were rinsed once in PBS, dislodged with 0.25% trypisn-EDTA (Gibco), and resuspended in a volume of 10 ml growth medium. The cells were collected at 1000 x g for 5 minutes, and the residual media decanted. The cell pellet was resuspended in PBS with the addition of 0.6 % NP-40 and nuclei were collected by centrifugation through a cushion of Histopaque 1077 (Sigma Chemical Company, St. Louis, MO) at 1000 x g for 5 minutes. Nuclei were resuspended in PBS, and an equal volume of 5 M NaCl was added to lyse the nuclei. Cellular DNA was degraded by diluting the nuclear lysate five-fold in distilled water, adding 10,000 units of DNase I, and incubating for 15 min at 37°C. An equal volume of 5 M urea was added to the nuclear lysate and the nuclear bodies were collected by centrifugation at 3500 x g for 10 minutes through a Histopaque cushion. Nuclear bodies were resuspended in PBS supplemented with 0.5% NP-40 and frozen at -80⁰C. Aggregates were isolated from the cytoplasmic fraction using a similar strategy. Viral inclusions from the cytoplasmic fraction of the NP-40 lysate were collected by sedimentation at 3500 x g. The sedimented material was resuspended in a PBS buffer containing 5M urea and incubated for 5 min at ambient temperature. Viral inclusions were then isolated by sedimentation through a Histopaque cushion, resuspended in PBS with 0.5% NP-40, and frozen at -80⁰C.

Example 2. Characteristic inclusions are formed in infected cells in the absence of ρpUL97 activity

The first report of recombinant viruses with large deletions in UL97 described a distinctive plaque morphology characterized by the appearance of highly retractile bodies in the nuclei of infected cells. Prichard et al., 73 J. VIROL. 5663-70 (1999). In subsequent experiments, it was noted that these mutants did not induce the formation of these structures in cells that expressed ppUL97 kinase in trans, suggesting that a deficiency of this gene product results in the formation of unusual inclusions in infected cells. The recent description of a specific inhibitor of ppUL97 kinase activity, MBV, presented an opportunity to confirm these observations and to test the hypothesis that a deficiency in the enzymatic activity of this protein was sufficient to induce the production of these inclusions. Biron et al, 46 ANTIMICROB. AGENTS CHEMOTHER. 2365-72 (2002). Cells infected with the UL97 deletion mutant developed the characteristic nuclear inclusions observed previously by 72 hours postinfection (hpi) (Fig. IA). Cells infected with AD169 and treated with 15 μM MBV also formed large retractile nuclear inclusions that were microscopically indistinguishable from those observed with the deletion mutant (Fig. IB). The formation of inclusions was also observed in The Towne or Toledo strain of HCMV. Thus it appeared that their formation might be related to the absence of UL97 kinase activity.

To investigate whether the tegument aggregate structures resulted from infected nuclei becoming saturated with viral structural proteins during the late block in viral replication induced by MBV, or whether the physical properties of some viral proteins are controlled by phosphorylation and that they aggregate inappropriately in an underphosphorylated state, cells infected with AD 169 were treated with MBV, CDV, or BDCRB. BDCRB is an analog of MBV that blocks viral replication late in infection through the inhibition of DNA packaging and does not inhibit UL97 kinase activity. Underwood et al, 72 J. VIROL. 717-25 (1998). A monoclonal antibody specific for the pp65 tegument phosphoprotein bound specifically these structures and was used to monitor their formation in subsequent experiments. Cells infected with AD 169 exhibited punctate cytoplasmic and perinuclear staining by 96 hpi, as observed previously (Fig. 1C). Sanchez et al, 74 J. VIROL. 975-86 (2000). In contrast, infected cells treated with MBV induced the development of very large cytoplasmic inclusions at this period of time (Fig. ID). In the presence of BDCRB (Fig. IE), the localization pattern of pp65 resembled that in untreated cells infected with the wt virus and no large inclusions were observed. This observation suggested that the formation of these structures was not simply a consequence of a late block in the replication cycle, but was more closely related to the specific mechanism of action of MBV, presumably its inhibition of UL97 kinase activity. An early block in viral replication by CDV greatly reduced the synthesis of pp65 and eliminated its export to the cytoplasm, as expected for an inhibitor of DNA synthesis (Fig. IF). Thus, both the genetic data as well as the pharmacologic data suggest that a deficiency of UL97 kinase activity leads to the formation of these aberrant structures in infected cells.

Example 3. Aggregates of structural proteins form in the nucleus and move to the cytoplasm The composition of the aggregate protein structures was investigated further to help understand the molecular defects that result from a deficiency of UL97 kinase activity. Aggregates produced in infected HF monolayers were stained with a panel of monoclonal antibodies specific for viral proteins and monoclonal antibodies to the tegument phosphoproteins, pp65 and ppl50, as well as the major capsid protein and ppUL44 specifically labeled these structures, suggesting that they contained a number of viral proteins. In cells infected with RCΔ97.08, small inclusions that stained with a monoclonal antibody to pp65 were observed in the nuclei starting approximately 48 hpi, while none were observed in the cells infected with AD 169. By 72 hpi, the nuclear aggregations had enlarged and a few cytoplasmic inclusions were observed (Fig. IG). By 96 hpi, the aggregations had attained considerable mass and were predominantly cytoplasmic although a few remained in the nuclei (Fig. IH). Aggregates formed in cells infected with the UL97 null mutant, or induced with MBV differ from the small inclusions observed in the wt virus in that they are very large and typically reach a characteristic size of 5-10 microns. The existence of aggregates in both compartments was also confirmed by electron microscopy. The formation and apparent translocation to the cytoplasm occurred with the same kinetics in cells infected with AD 169 and treated with MBV. The quantitative translocation of pp65 from the nuclei to the cytoplasmic perinuclear compartments described previously (Sanchez et ah, 72 J. VlROL. 3321-9 (1998)), also occurred very late in cells infected with RCΔ97 (Fig. 1C, H). The temporal changes in the relative frequency of nuclear and cytoplasmic inclusions suggests that the aggregates of viral proteins form in the nuclei of infected cells in the absence of UL97 kinase activity, and that they are subsequently translocated to the cytoplasm late in infection. These data do not exclude the possibility that the aggregates first form in the nucleus, then disaggregate and reform in the cytoplasm late in infection, but this is unlikely given the remarkable stability of these structures. It is clear that aggregates formed in cells infected with the mutant virus and those induced with MBV were observed in both the nucleus and the cytoplasm of infected cells.

Example 4. Isolation of aggregates from infected cells

That aggregates observed in both RCΔ97 infected cells and AD 169 infected cells treated with MBV represented similar structures was confirmed by isolating them from infected cells and examining the constituent proteins. A large scale purification scheme was developed based on the remarkable stability, density, and relatively large size of these structures. Briefly, nuclei from cells infected with RCΔ97 were lysed and treated with DNase I to eliminate DNA from the preparation. Aggregates in the crude nuclear lysates were then resuspended in 2.5 M urea and harvested by sedimentation through a ficoll cushion (Fig. 2A). Aggregates were also isolated from the cytoplasmic fraction using a similar procedure (Fig. 2B). Final preparations of aggregates retained their highly refractile appearance and appeared to be largely free of cellular debris. The final yield of isolates derived from nuclear and cytoplasmic fractions represented approximately 7% and 3%, respectively, of the total cellular protein in infected cells. This high yield is consistent with the numerous large inclusions observed in infected cells (Fig. 1). The fact that these aggregates withstand dissolution in the high urea concentration suggests that they are highly stable structures and that the constituent proteins aggregate very high affinity interactions.

Aggregates are formed predominantly from viral structural proteins. Aggregates were isolated from the nuclei of cells infected with RCΔ97.08. Constituent proteins were separated by ID and 2D polyacrylamide gel electrophoresis, and protein species were excised and identified by MALDI-TOF mass spectrometry (Fig. 3, Table 1). This analysis confirmed that the major constituents of the aggregates were the tegument phosphoproteins pp65 and pρUL25 as well as the major capsid protein encoded by UL86. Proteins with migration rates similar to MCP, ppUL25, and pp65 were also observed from aggregates derived from the cytoplasmic fraction (Fig. 3B). These findings were confirmed by western blots of isolated aggregates and immunofluorescence of infected cells. These experiments confirmed the presence of pp65, and MCP in these aggregates and also suggested that the tegument protein ppl50 and ppUL44 were present in the aggregates (Table 1). The constituent proteins in the tegument aggregates described here likely represent a subset of the viral gene products in these structures. The fact that two of the most abundant proteins in these aggregates are pp65 and ppUL25, suggests that they are largely composed of tegument proteins and are similar in composition to dense bodies. Varnum et al, 78 J. VIROL. 10960-66 (2004).

Table 1. Proteins identified in nuclear tegument aggregates isolated from cells infected with RCΔ97

Protein No. Peptides Confirmed by Confirmed by

Observed by MS^a Immunoblotting^b Immunofluorescence⁰

MCP 16 X X pp65 18 X X pρUL25 15 ppl50 X X ppUL44 X X ^a Number of peptides identified by MS following trypsin digestion of excised protein species. ^b Proteins from tegument aggregates were separated by SDS-PAGE and stained with monoclonal antibodies. ^c Monolayers of HF cells were infected with RCΔ97 and stained with monoclonal antibodies.

An additional mass spectrometric study compared the composition of nuclear and cytoplasmic tegument aggregates with the compositions of virions and dense bodies. Results from this analysis were similar to those reported above. Some thirty viral proteins and fifty- one host proteins were identified in tegument aggregates. Like dense bodies, both nuclear and cytoplasmic tegument aggregates contain large quantities of both pp65 and ppUL, but capsid proteins pUL86, pUL85, and pUL46 are under represented, as are the tegument proteins ppUL32 and pUL35 (Table 2).

Table 2. Composition of tegument aggregates is similar to that of dense bodies

Nuclear Cytoplasmic

Dense Tegument Tegument

Gene Product Function Virions'¹ bodies^a Aggregates'³ Aggregates⁰ ppUL83

(pp65) tegument 123^d 40 82 60 ppUL25 tegument 60 17 27 11 pUL86 (MCP) capsid 149 22 21 23 pUL85 capsid 21 4 3 2 pUL46 capsid 20 1 3 0.9 ppUL32

(ppl50) tegument 135 11 4 5 pUL35 tegument 42 5 3 0.9 a. Varnum et al, 78(20) J. VlROL. 10960-66 (2004). b. Tegument aggregates isolated from the nuclear fraction of cells. c. Tegument aggregates isolated from the cytoplasmic fraction of cells. d. Number of peptides observed by MS.

Aggregates isolated from cells infected with RCΔ97 or induced with MBV are similar in composition. To compare the inclusions isolated from cells infected with RCΔ97 to those isolated from cells infected with AD 169 and treated with MBV, aggregates from the nuclear fraction were isolated by methods described above, although on a smaller scale. Untreated AD169 infected and uninfected cells were also subjected to the same isolation scheme and served as additional controls. Few proteins remained in samples derived from either uninfected cells or cells infected with the wt virus (Fig. 3B). Aggregates isolated from the UL97 deletion mutant as well as the MBV treated sample appeared to be similar in composition and contained at least four proteins that were over represented, or not observed in the control samples. Differences were observed in the relative abundance of some constituent proteins in MBV induced aggregates and those produced by the mutant virus (Fig. 3B). These differences may be related either to the mechanism of action of MBV on viral replication, or the higher MOI for cells infected with the wt virus. Nevertheless, the protein profiles are similar and support the notion that the aggregates produced under these different circumstances are similar in composition.

Example 5. A pp65 knockout virus does not form tegument aggregates

To confirm that the expression of pp65 was required for the formation of tegument aggregates in infected cells, a recombinant virus that does not express pp65 Schmolke et al, 69 J. VlROL. 5959-68 (1995)), was evaluated for its ability of make tegument aggregates in the presence of MBV. Monolayers of infected HF cells were infected with RVAd65, treated with increasing concentrations of MBV, and a monoclonal antibody to ppl50 was used to detect the formation of tegument aggregates. Cells infected with RCΔ97.08 and cells infected with the wt virus and treated with 30 μM MBV formed tegument aggregates by 96 hpi (Fig. 4A, B). In contrast, cells infected with RVAd65 did not produce these aggregates, even when the MBV concentration was increased 60 μM (Fig. 4C). This result suggests strongly that pp65 is required for the formation of tegument aggregates in the absence of UL97 kinase activity and is consistent with the observation that this protein is the predominant constituent of these structures Example 6. Expression of pp65 is sufficient to induce aggregates in uninfected cells and their formation is inhibited by UL97 kinase activity

In CMV-infected cells, the absence of UL97 kinase activity leads to the formation of tegument aggregates in which pp65 is a principle and essential component. The phosphorylation of pp65 by the kinase might alter its physical characteristics and lead to inappropriate aggregation in an underphosphorylated state - resulting in the formation of tegument aggregates. The inventors took advantage of an unreported observation that pp65- EGFP fusion proteins transiently expressed in COS7 cells formed large nuclear aggregates. Indeed, COS7 cells produce pp65 tegument aggregates very efficiently. Initially, this result was presumed to be an artifact related to the insoluable nature of the fusion protein, but could also be interpreted as aggregation of underphosphorylated forms of this protein. To distinguish between these possibilities, plasmids expressing EGFP-pp65 fusion proteins were transfected into COS7 cells either alone or with UL97 expression plasmids containing amino terminal V5 epitope tags. Cells transfected with an expression plasmid for the pp65-EGFP fusion protein exhibited the very bright nuclear aggregations observed previously (Fig. 5 A, B). Transiently expressed ppUL97 fusion proteins exhibit diffuse nuclear staining (Fig. 5 C), whereas the transient expression of ppUL97 fusion proteins containing a K355M point mutation are expressed both in the nucleus and the cytoplasm (Fig. 5 D). The coexpression of the UL97 kinase with pp65-EGFP fusion protein eliminated the formation of nuclear aggregations even though both proteins were abundantly expressed in the nucleus (Fig. 5 E, H). These results suggested that the expression of pp65 in uninfected cells was sufficient to form nuclear aggregates that were similar in appearance to those induced in infected cells treated with MBV, or observed in the UL97 deletion mutant. The inhibition of pp65 aggregation by ppUL97 might be mediated by phosphorylation because the inhibition of kinase activity in infected cells leads to the formation aggregates. To determine if UL97 kinase activity was required to prevent aggregation, the experiment was repeated and transfected cells were treated with MBV to specifically inhibit UL97 kinase activity. When ppUL97 and pp65 were co-expressed in COS7 cells without MBV, both proteins were distributed evenly throughout the nucleus (Fig. 5 E, H). In contrast, when UL97 kinase activity was inhibited with MBV, nuclear aggregation was restored in cells expressing both tegument proteins (Fig. 5 I, L), suggesting that UL97 kinase activity was required to inhibit aggregation. Also of interest, was the localization of ppUL97 to the aggregates in the presence of MBV, suggesting that pp65 can recruit the kinase to these structures, presumably through a physical interaction. To confirm these data, the experiment was repeated using the ppUL97 expression construct containing the K355M mutation that abrogates its kinase activity. The ppUL97 K355M mutant was unable to prevent the formation of pp65 aggregates in cells expressing both proteins (Fig. 5 M, P), confirming that UL97 kinase activity was required to control the aggregation of pp65. The kinase negative form of ppUL97 was also recruited to the aggregates, as observed with native ppUL97 in the presence of MBV. Although, the ppUL97 mutant was recruited to the pp65 aggregates in the nucleus, its nuclear localization was reduced compared to the native ppUL97 fusion protein and cytoplasmic localization was also observed. These data taken together suggest that: (a) the expression of pp65 is sufficient to form aggregates in uninfected cells; (b) coexpression of the viral kinase can prevent this aggregation; (c) UL97 kinase activity is specifically required to prevent the aggregation of pp65; and (d) pp65 can recruit ppUL97 to the aggregates in the absence of kinase activity. A recombinant virus that does not express pp65 is resistant to MBV. Data presented here suggest that the UL97 kinase, as well as pp65 play a central role in the formation of tegument aggregates in the nuclei of infected cells. If this interpretation is correct, then the deletion of either UL97 or UL83 might be expected to disrupt the normal nuclear tegumentation process and force the virus to rely on an alternative, albeit inefficient, mechanism to produce infectious virus. One prediction of this hypothesis is that MBV should be less effective against recombinant viruses with deletions in either of these genes since the normal pathway would be inoperative in either case. To test this hypothesis, the sensitivity of AD169, RCΔ97.08 and RVAd65 to the antiviral effects of MBV were determined. Each of these isogenic strains exhibited the same sensitivity to the cidofovir and yielded EC₅₀ values that were indistinguishable (Table 3). As reported previously, RCΔ97.08 was highly resistant to the antiviral effects of MBV. Williams et al, 47 ANΉMICROB. AGENTS CHEMOTHER. 2186-92 (2003). Interestingly, RVAd65 was also significantly resistant to this MBV and is consistent with the notion that both these tegument proteins function in a common pathway in infected cells. It is also suggests that pp65 is involved in the mechanism of action of MBV. The pp65 null mutant did not exhibit the high level of resistance seen with the UL97 null mutant. This was not unexpected given that MBV should inhibit the phosphorylation of all UL97 kinase substrates, rather than just that of pp65. Resistance to MBV has been ascribed to mutations that map to either JJL97 or UL27. To confirm that RVAd65 did not have mutations at these loci, both genes were sequenced and no mutations were observed. These data taken together suggest that both the UL97 kinase and pp65 are involved in the formation of tegument aggregates and that they likely function in a common pathway related to the mechanism of action of MBV.

Table 3. RVAd65 is resistant to the antiviral effects of maribavir. Drug AD169 RCΔ97.08 RVAd65

CDV 0.22 ± 0.17a 0.09 ± 0.04 (0.4)b 0.09 ± 0 (0.4)b MBV 0.82 ± 0.8 33 ± 26 (40)b 3.6 ± 0.4 (4.4)b ^a EC₅₀ values are the average of three of more experiments with the standard deviations shown. ^b Fold resistance compared to the parent virus AD 169.

Although the biochemical activity of the UL97 kinase has been well-characterized and its central role in the emergence of ganciclovir resistance in the clinic has been established, the functions that this tegument protein performs during viral replication remain somewhat unclear. Studies reported herein suggest that tegument aggregates are produced in the absence of UL97 kinase activity, and can be induced either through the deletion of UL97, or through the inhibition of its enzymatic activity with MBV. These structures were shown to be composed predominantly of the tegument proteins pp65 and ppUL25, but also contain other viral proteins. Significantly, we demonstrated that pp65 is essential for the formation of these structures in infected cells, that its transient expression is sufficient to produce similar nuclear aggregates in uninfected cells, and that this aggregation can be inhibited by UL97 kinase activity. The fact that the regulation of pp65 by ppUL97 can be reproduced in uninfected cells reflects the close relationship between these proteins and suggests strongly that they function together in infected cells.

Results described herein are intriguing in light of results from previous studies regarding the phenotype of RCΔ97 and its severely impaired ability to produce infectious virions. Previous reports regarding the phenotype of pUL97 null mutants as well as those describing the mechanism of action specific inhibitors of this enzyme have concluded that the activity of this enzyme is critical for viral replication. See Biron et al, 46 ANTEMICROB. AGENTS CHEMOTHER. 2365-72 (2002); Herget et al, 48 ANTDVΠCROB. AGENTS CHEMOTHER. 4154-62 (2004); Krosky et al, 11 J. VIROL. 905-14 (2003); Marschall et al, 83 J. GEN. VIROL. 1013-23 (2002); Prichard et al, 73 J. VIROL. 5663-70 (1999); Wolf et al, 98 PROC. NATL. ACAD. SCI. USA 1895-900 (2001). Defining precisely the critical functions of the kinase has been difficult due to variation in the nature of the phenotype within and among laboratories.

Similarly, drugs that inhibit this kinase also induce effects that vary significantly within laboratories and also suggest that the virus utilizes the kinase to a greater or lesser extent depending on variables likely associated with the primary cell substrate. Pleiotropic effects are expected when a broadly active kinase is deleted and phenotypes are complicated further by differentially regulated cellular factors that can complement the mutant virus to some degree, as evidenced by improved replication of the null mutant in dividing cells. Nevertheless, it is clear that replication of the RCΔ97 proceeds normally and expresses appropriate levels of α, β, and γ gene products and DNA synthesis proceeds, although a modest 3 to 6 fold reduction in accumulated DNA is observed. Krosky et al, 77 J. VIROL. 905-14 (2003); Prichard et al, 73 J. VIROL. 5663-70 (1999); Wolf et al, 98 PROC. NATL. ACAD. SCI. USA 1895-900 (2001). In one report, modest defects in viral DNA synthesis were noted as well as more pronounced defects in the packaging of viral DNA and an accumulation of immature capsids in the nucleus and an absence of mature virions in the cytoplasm. Wolf et al, 98 PROC. NATL. ACAD. SCI. USA 1895-900 (2001). The authors concluded that the kinase played a role both in DNA synthesis and encapsidation of nascent genomes.

A subsequent report did not observe either a defect in DNA packaging or an overabundance of immature particles in the nucleus, although mature capsids did not appear in the cytoplasm. Krosky et al, 11 J. VIROL. 905-14 (2003). The authors concluded that the virus was defective in nuclear egress, defined broadly as events following the packaging of genomic DNA but prior to exit from the nucleus of intact mature virions. The authors also noted that a single cell was observed with an overabundance of immature capsids, and it is possible that the differences observed between these studies may reflect the variation associated with differences in the condition of the primary cell substrate.

Mechanism of action studies with specific inhibitors of UL97 kinase activity suggest that they exert their antiviral effects through the inhibition of viral DNA synthesis, although the polymerase is not inhibited directly. Biron et al, 46 ANTMICROB. AGENTS CHEMOTHER. 2365-72 (2002); Marschall et al, 83 J. GEN. VIROL. 1013-23 (2002). Observed differences between these results and those obtained with the UL97 null mutant are likely the result of inhibiting UL97 kinase activity rather than eliminating its expression from a viral infection. It is also likely that ppUL97 has functions that are unrelated to its kinase activity and the kinase inhibitors also likely affect one or more other cellular homologs to some degree. Each of the reported defects attributed to either UL97 kinase inhibition or the deletion of UL97, occur in the nucleus of infected cells and both mature cytoplasmic virions and the production of infectious virus are severely reduced. In the absence of ppUL97, viral infection appears to progress rather normally through the expression of late gene products and the assembly of immature capsids in the nucleus occurs. Viral DNA can be packaged to some degree but mature capsids are not observed in the cytoplasm. This suggests that a defect occurs in virion morphogenesis, the transit of mature capsids to the nuclear membrane, or the process of nuclear egress including the degradation of the nuclear lamina and exit from the nucleus. The assembly of mature CMV particles within the nuclei of infected cells is a highly ordered progression of complex and concerted molecular events. Procapsid assembly appears to be relatively simple, but subsequent steps of DNA cleavage/packaging, capsid maturation, acquisition of tegument, transit to the nuclear membrane and process of nuclear egress remain ill defined and incompletely understood. Mettenleiter, 76 J. VIROL. 1537-47 (2002). The recombinant viruses described here, as well as a host of new antiviral drugs that inhibit distinct facets of this process in CMV promise to improve our understanding of virion morphogenesis in the betaherpesviruses. See, e.g., Biron et al, 46 ANΉMICROB. AGENTS CHEMOTHER. 2365-72 (2002); Buerger et al, 75 J. ViROL. 9077-86 (2001); Marschall et al, 82 J. GEN. VIROL. 1439 50 (2001); Underwood et al, 48 ANTIMICROB. AGENTS CHEMOTHER. 1647-51 (2004); Underwood et al, 72 J. VlROL. 717-25 (1998).

The investigation of the formation of tegument aggregates sheds light on the molecular defects resulting from a deletion in the viral protein kinase as well as the mechanism of action of UL97 kinase inhibitors. Results presented herein have defined and characterized a new facet of the phenotype associated with a deficiency of ppUL97. In the absence of the kinase, the aggregation of pp65 is unregulated and leads to the formation of very large aberrant inclusions composed largely of this protein as well as other viral proteins. The observed defects in pp65 function and the gross morphological changes in the organization of tegument and structural proteins in the nucleus likely reflect a deficiency of virion morphogenesis at a late stage when the initial acquisition of tegument proteins by capsids normally occurs. We propose that the UL97 kinase is required for normal virion morphogenesis and that in the absence of its enzymatic activity, capsids do not acquire tegument proteins normally and do not mature fully. It is possible that these final maturational events are required to target mature capsids for a subsequent step nuclear egress and could account for the failure of capsids to exit the nucleus. It is also possible that the multifunctional kinase is required both for virion morphogenesis and the process of nuclear egress. The fact that the pp65 knockout virus is resistant to MBV confirms that this protein is important in the functions performed by this kinase and is consistent with proposed role in morphogenesis described here, but it is also possible that pp65 could be involved in the process of nuclear egress as well. Clearly these two proteins are closely connected and function together in viral infection.

Neither UL97 nor UL83 are required for the replication of HCMV, but replication is severely impaired when either of these genes is deleted. The observed recruitment of ppUL97 to pp65 aggregates in uninfected cells is intriguing and is consistent with a direct interaction between these two proteins. These data are consistent with a previous observation that the kinase directly phosphorylates pp65. Sethna et al, 23rd Int'l Herpesvirus Workshop, York, UK (1998). This suggests that they both perform an important function in viral replication and is also consistent with the data presented here suggesting that they function together. The formation of tegument aggregates is interesting because of the similarity of these structures to both virions and dense bodies. The principle constituent of both dense bodies and tegument aggregates is pp65 and both require this tegument protein for their formation (Fig. 4) Schmolke et al, 69 J. VIROL. 5959-68 (1995). Further research regarding the interactions of these tegument proteins may aid in developing new antiviral therapies as well as understanding the complex stages in virion morphogenesis in the betaherpesviruses.

Example 7. Immunogenicity of HCMV tegument aggregates

The immunogenicity of the HCMV tegument aggregates was confirmed in the generation of monoclonal antibodies against tegument aggregate proteins using standard techniques well-known in the art. Briefly, three adult Balb/c mice were immunized with 50ug of tegument aggregates emulsified in Freunds adjuvant, and two weeks later boosted with 50ug of tegument aggregates emulsified in incomplete Freunds adjuvant. Animals were sacrificed, a sample of blood was obtained for further analysis, and spleen cells fused with murine myeloma cells, P63Ag8-, using polyethylene glycol and cell fusions plated in 48 well tissue culture plates. Hybridoma cell lines were screened for monoclonal antibody production using an immunofluorescence assays in which HCMV infected human fibroblast cells served as screening template. Greater than 30% of wells contained HCMV specific monoclonal antibodies as determined by reactivity for HCMV-infected cells but not for non-infected cells. Subsequent clones were obtained that reacted with several different HCMV encoded proteins. Some of the antibodies were specific for proteins for which antibody reagents are not available previously, such as ppUL50. In addition, serum obtained at the time of sacrifice of the immunized mice exhibited strong reactivity for HCMV-infected cells but minimal to no reactivity for non-infected cells.

HCMV tegument aggregates are immunogenic and can be used as source of viral proteins for immunization, antigen in viral screening applications, production of HCMV- specific antibodies, and production of HCMV-specific monoclonal antibodies.

Example 8. Expression of recombinant pp65 in a baculovirus expression system

DNA sequences shown below were isolated from the AD 169 strain of HCMV and BamHI sites were added to each end. The BamHI fragment containing the pp65 open reading frame (in italics) was ligated in a unique BamHI site in a baculovirus transfer vector (a portion of which is shown underlined above). This places the pp65 ORF under the control of the polyhedron promoter. This transfer vector transfected into sf9 cells and a recombinant baculovirus containing this region was selected. Infection of sf9 cells with the recombinant virus expressing pρ65 resulted in large inclusion bodies (tegument aggregates of pp65) within the nuclei of infected cells. The formation of these structures is dependent on the expression of pp65 since they were not observed with other recombinants expressing other HCMV proteins. Thus, the formation of large pp65 aggregates was reproduced in insect cells.

Recombinant pp65 DNA sequence (SEQ ID NO: 5): ctagaggatcgatcccccggatctgatcatggagataattaaaatgataaccatctcgcaaataaataagtattttactgttttcgtaacagt tttgtaataaaaaaacctataaatatcggatccαtggQgtCjgcgCjggfcgccgttgtcccgαααtjgfltotccgtoctjejegtcccfltttcje gggcacgtgctgaaagccgtgtttagtcgcggcgatacgccggtgctgccgcacgagacgcgactcctgcagacgggtatccac gtacgcgtgagccagccctcgctgatcttggtatcgcagtacacgcccgactcgacgccatgccaccgcggcgacaatcagctgc aggtgcagcacacgtactttacgggcagcgaggtggagaacgtgtcggtcaacgtgcacaaccccacgggccgaagcatctgc cccagccaggagcccatgtcgatctatgtgtacgcgctgccgctcaagatgctgaacatccccagcatcaacgtgcaccactacc cgtcggcggccgagcgcaaacaccgacacctgcccgtagctgacgctgtgattcacgcgtcgggcaagcagatgtggcaggcg cgtctcacggtctcgggactggcctggacgcgtcagcagaaccagtggaaagagcccgacgtctactacacgtcagcgttcgtgt ttcccaccaaggacgtggcactgcggcacgtggtgtgcgcgcacgagctggtttgctccatggagaacacgcgcgcaaccaag atgcaggtgataggtgaccagtacgtcaaggtgtacctggagtccttctgcgaggacgtgccctccggcaagctctttatgcacgtc acgctgggctctgacgtggaagaggacctgacgatgacccgcaacccgcaacccttcatgcgcccccacgagcgcaacggcttt acggtgttgtgtcccaaaaatatgataatcaaaccgggcaagatctcgcacatcatgctggatgtggcttttacctcacacgagcat tttgggctgctgtgtcccaagagcatcccgggcctgagcatctcaggtaacctgttgatgaacgggcagcagatcttcctggaggta caagccatacgcgagaccgtggaactgcgtcagtacgatcccgtggctgcgctcttctttttcgatatcgacttgctgctgcagcgcg ggcctcagtacagcgagcaccccaccttcaccagccagtatcgcatccagggcaagcttgagtaccgacacacctgggaccgg cacgacgagggtgccgcccagggcgacgacgacgtctggaccagcggatcggactccgacgaagaactcgtaaccaccgag cgcaagacgccccgcgtcaccggcggcggcgccatggcgggcgcctccacttccgcgggccgcaaacgcaaatcagcatcct cggcgacggcgtgcacgtcgggcgttatgacacgcggccgccttaaggccgagtccaccgtcgcgcccgaagaggacaccga cgaggattccgacaacgaaatccacaatccggccgtgttcacctggccgccctggcaggccggcatcctggcccgcaacctggt gcccatggtggctacggttcagggtcagaatctgaagtaccaggaattcttctgggacgccaacgacatctaccgcatcttcgccg aattggaaggcgtatggcagcccgctgcgcaacccaaacgtcgccgccaccggcaagacgccttgcccgggccatgcatcgcc tcgαcjecccαflαααgcflccgαggttgflggatccgggttattagtacatttattaagcgctagattctgtgcgttgttpatttacagacaa ttgttgtacgtattttaataattcattaaatttataatctttagggtggtatgttagagcgaaaatcaaatgattttcagcgtctttatatctgaattt aaatattaaatcctcaatagatttgtaaaataggtttcgattagtttcaaacaagggttgtttttccgaaccgatggctggactatctaatgga ttttcgctcaacgccacaaaacttgccaaatcttgtagcagcaatctagctttgtcgatattcgtttgtgttttgttttgtaataaaggttcgac gtcgttcaaaatattatgcgcttttgtatttctttcatcactgtcgttagtgtacaattgactcga

Example 9. Molecular determinants of pp65 self aggregation

The self-aggregating properties of pp65 and the regulation of this property by ppUL97 likely contribute to the formation of tegument aggregates and can be reproduced in COS7 cells as reported previously (Prichard et al J. Virol., 2005). Given this property, it might be expected that pp65 expressed transiently could be incorporated in tegument aggregates and a pp65-EGFP fusion protein also colocalized within the tegument aggregates. A study was undertaken to identify the portion of the pp65 required for self aggregation such that fusions of other proteins and haptens could be incorporated within the tegument aggregates. The alteration of the amino terminus pp65 by the addition of an antibody epitope, or a T-cell epitope from the HIV p24 protein precluded self aggregations as did the substitution of the first three serine residues with either aspartic acid, or alanine (Table 4). In contrast, additional amino acids could be readily fused to the carboxyl terminus without affection the ability of the molecule to aggregate when expressed in COS7 cells. Thus, additional haptens or T-cell epitopes can be fused to the carboxyl terminus of ρp65 and transiently expressed in cells for the production of tegument aggregates containing any antigen of interest. Because tegument aggregates are related to highly immunogenic dense bodies, the pp65 tegument aggregates are expected to elicit a strong immune response to the constituent proteins without the use of adjuvant. Table 4. Molecular determinants of pp65 self aggregation

Clone Amino Carboxyl terminus Point mutations Self Aggegation terminus pMP35 yes ρMP136 V5 epitope no pMP165 HIV p24 no epitope ρMP172 HIV p24 V5 epitope no epitope pMP216 V5 epitope yes pMP174 HIV p24 epitope yes pMP175 HIV ρ24,V5 epitope yes pMP212 S3D,S13D,S19D no pMP213 S3A,S13A,S19A no pMP223 S3A yes pMP224 S13A no pMP225 S19A no

The amino acid sequences of pp65 constructs of Table 4 were inferred from the DNA sequences of each construct. The pMP35 construct has 561 amino acids of the native pp65 sequence: pMP35 Native pp65 protein (SEQ ID NO: 6)

MESRGRRCPEMISVLGPISGHVLKAVFSRGDTPVLPHETRLLQTGIHVRVSQPSLILVSQYTPDSTPCHRGDNQL QVQHTYFTGSEVENVSVNVHNPTGRSICPSQEPMSIYVYALPLKMLNIPSINVHHYPSAAERKHRHLPVADAVIH ASGKQMWQARLTVSGLAWTRQQNQWKEPDWYTSAFVFPTKDVALRHWCAHELVCSMENTRATKMQVIGDQYVK VYLESFCEDVPSGKLFMHVTLGSDVEEDLTMTRNPQPFMRPHERNGFTVLCPKNMIIKPGKISHIMLDVAFTSHE HFGLLCPKSIPGLSISGNLLMNGQQIFLEVQAIRETVELRQYDPVAALFFFDIDLLLQRGPQYSEHPTFTSQYRI QGKLEYRHTWDRHDΞGAAQGDDDVWTSGSDSDEELVTTERKTPRVTGGGAMAGASTSAGRKRKSASSATACTSGV MTRGRLKAESTVAPEEDTDEDSDNEIHNPAVFTWPPWQAGILARNLVPMVATVQGQNLKYQEFFWDANDIYRIFA ELEGVWQPAAQPKRRRHRQDALPGPCIASTPKKHRG

The amino acid sequence of pMP216 includes 561 amino acids of the native sequence, with an additional 45 amino acids of the V5 His6 region which were added on the carboxy terminus to enhance solubility: pMP216 (SEQ ID NO: 7)

MESRGRRCPEMISVLGPISGHVLKAVFSRGDTPVLPHETRLLQTGIHVRVSQPSLILVSQYTPDSTPCHRGDNQL QVQHTYFTGSEVENVSVNVHNPTGRSICPSQEPMSIYVYALPLKMLNIPSINVHHYPSAAERKHRHLPVADAVIH ASGKQMWQARLTVSGLAWTRQQNQWKEPDVYYTSAFVFPTKDVALRHWCAHELVCSMENTRATKMQVIGDQYVK VYLESFCEDVPSGKLFMHVTLGSDVEEDLTMTRNPQPFMRPHERNGFTVLCPKNMIIKPGKISHIMLDVAFTSHE HFGLLCPKSIPGLSISGNLLMNGQQIFLEVQAIRETVELRQYDPVAALFFFDIDLLLQRGPQYSEHPTFTSQYRI QGKLEYRHTWDRHDEGAAQGDDDVWTSGSDSDEELVTTERKTPRVTGGGAMAGASTSAGRKRKSASSATACTSGV MTRGRLKAESTVAPEEDTDEDSDNEIHNPAVFTWPPWQAGILARNLVPMVATVQGQNLKYQEFFWDANDIYRIFA ELEGVWQPAAQPKRRRHRQDALPGPCIASTPKKHRGKGNSADIQHSGGRSSLEGPRFEGKPIPNPLLGLDSTRTG HHHHHH

The amino acid sequence of pMP136 includes an additional 45 amino acids, from the

V5 epitope tag, on the amino terminus: pMP136 (SEQ ID NO: 8)

MGKPIPNPLLGLDSTENLYFQGPDPSTNSADITRYKKAGSAAAPFTMESRGRRCPEMISVLGPISGHVLKAVFSR GDTPVLPHETRLLQTGIHVRVSQPSLILVSQYTPDSTPCHRGDNQLQVQHTYFTGSEVENVSVNVHNPTGRSICP SQEPMSIYVYALPLKMLNIPSINVHHYPSAAERKHRHLPVADAVIHASGKQMWQARLTVSGLAWTRQQNQWKEPD VYYTSAFVFPTIKDVALRHWCAHELVCSMENTRATKMQVIGDQYVKVYLESFCEDVPSGKLFMHVTLGSDVEEDL TMTRNPQPFMRPHERNGFTVLCPKNMIIKPGKISHIMLDVAFTSHEHFGLLCPKSIPGLSISGNLLMNGQQIFLE VQAIRETVELRQYDPVAALFFFDIDLLLQRGPQYSEHPTFTSQYRIQGKLEYRHTWDRHDEGAAQGDDDVWTSGS DSDEELVTTERKTPRVTGGGAMAGASTSAGRKRKSASSATACTSGVMTRGRLKAESTVAPEEDTDEDSDNEIHNP AVFTWPPWQAGILARNLVPMVATVQGQNLKYQEFFWDANDIYRIFAELEGVWQPAAQPKRRRHRQDALPGPCIAS TPKKHRG

The construct pMP165 comprises the native pp65 sequence and an additional amino acid sequence (underlined) of a T-cell epitope from HIV p24: pMP165 (SEQ ID NO: 9) MESLYNTVATSRGRRCPEMISVLGPISGHVLKAVFSRGDTPVLPHETRLLQTGIHVRVSQPSLILVSQYTPDSTP CHRGDNQLQVQHTYFTGSEVENVSVNVHNPTGRSICPSQEPMSIYVYALPLKMLNIPSINVHHYPSAAERKHRHL PVADAVIHASGKQMWQARLTVSGLAWTRQQNQWKEPDVYYTSAFVFPTKDVALRHWCAHELVCSMENTRATKMQ VIGDQYVKVYLESFCEDVPSGKLFMHVTLGSDVEEDLTMTRNPQPFMRPHERNGFTVLCPKNMIIKPGKISHIML DVAFTSHEHFGLLCPKSIPGLSISGNLLMNGQQIFLEVQAIRETVELRQYDPVAALFFFDIDLLLQRGPQYSEHP TFTSQYRIQGKLEYRHTWDRHDEGAAQGDDDVWTSGSDSDEELVTTERKTPRVTGGGAMAGASTSAGRKRKSASS ATACTSGVMTRGRLKAESTVAPEEDTDEDSDNEIHNPAVFTWPPWQAGILARNLVPMVATVQGQNLKYQEFFWDA NDIYRIFAELEGVWQPAAQPKRRRHRQDALPGPCIASTPKKHRG

The construct pMP174 includes the native pp65 amino acid sequence and, at the caraboxy terminus, a T-cell epitope from HIV p24 (underlined): pMP174 (SEQ ID NO: 10)

MESRGRRCPEMISVLGPISGHVLKAVFSRGDTPVLPHETRLLQTGIHVRVSQPSLILVSQYTPDSTPCHRGDNQL QVQHTYFTGSEVENVSVNVHNPTGRSICPSQEPMSIYVYALPLKMLNIPSINVHHYPSAAERKHRHLPVADAVIH ASGKQMWQARLTVSGLAWTRQQNQWKEPDVYYTSAFVFPTKDVALRHWCAHELVCSMENTRATKMQVIGDQYVK VYLESFCEDVPSGKLFMHVTLGSDVEEDLTMTRNPQPFMRPHERNGFTVLCPKNMIIKPGKISHIMLDVAFTSHE HFGLLCPKSIPGLSISGNLLMNGQQIFLEVQAIRETVELRQYDPVAALFFFDIDLLLQRGPQYSEHPTFTSQYRI QGKLEYRHTWDRHDEGAAQGDDDVWTSGSDSDEELVTTERKTPRVTGGGAMAGASTSAGRKRKSASSATACTSGV MTRGRLKAESTVAPEEDTDEDSDNEIHNPAVFTWPPWQAGILARNLVPMVATVQGQNLKYQEFFWDANDIYRIFA ELEGVWQPAAQPKRRRHRQDALPGPCIASTPKKHRGESLYNTVAT

The amino acid sequence of pMP175 includes the native pp65 sequence, a T-cell epitope from HIV p24 (underlined), and the V5 epitope His6 tag on the carboxy terminus: pMP175 (SEQ ID NO: ll)

MESRGRRCPEMISVLGPISGHVLKAVFSRGDTPVLPHETRLLQTGIHVRVSQPSLILVSQYTPDSTPCHRGDNQL QVQHTYFTGSEVENVSVNVHNPTGRSICPSQEPMSIYVYALPLKMLNIPSINVHHYPSAAERKHRHLPVADAVIH

ASGKQMWQARLTVSGLAWTRQQNQWKEPDVYYTSAFVFPTKDVALRHWCAHELVCSMENTRATKMQVIGDQYVK

VYLESFCEDVPSGKLFMHVTLGSDVEEDLTMTRNPQPFMRPHERNGFTVLCPKNMIIKPGKISHIMLDVAFTSHE

HFGLLCPKSIPGLSISGNLLMNGQQIFLEVQAIRETVELRQYDPVAALFFFDIDLLLQRGPQYSEHPTFTSQYRI

QGKLΞYRHTWDRHDEGAAQGDDDVWTSGSDSDEELVTTERKTPRVTGGGAMAGASTSAGRKRKSASSATACTSGV MTRGRLKAESTVAPEEDTDEDSDNEIHNPAVFTWPPWQAGILARNLVPMVATVQGQNLKYQEFFWDANDIYRIFA

ELEGVWQPAAQPKRRRHRQDALPGPCIASTPKKHRGESLYNTVATKGNSADIQHSGGRSSLEGPRFEGKPIPNPL

LGLDSTRTGHHHHHH In the amino acid sequence of the pMP212 construct, polar, hydrophilic serine residues at positions 3, 13, and 19 were replaced with negatively charged, hydrophilic aspartic acid residues. In the amino acid sequence of the pMP213 construct, serine residues at positions 3, 13, and 19 were replaced with nonpolar, hydrophobic alanine residues. In each construct, self aggregation was inhibited by these substitutions. Requirements for self aggregation were refined further and it was shown that the substitution of alanine residues for serine residues at position 13 or 19 were sufficient to eliminate aggregations.

Example 10. Immunogenicty of pp65-hapten constructs The HIV p24 T-cell epitope was fused the carboxyl terminus of pp65 and cloned, using standard techniques, into the bacterial expression vector pET151D to add an additional V5 epitope and HIS6 the terminus of the protein. This construct was designated pl74, and the predicted amino acid sequence is shown in Example 7, above. The protein was expressed in E. coli BL21* cells and the recombinant protein was purified on a Ni-NTA column (Qiagen, Valencia, CA).

Inoculations were conducted in B ALB/c mice by two routes of administration without the use of adjuvant. The first group of mice was vaccinated by the intranasal instillation of 10.25ug of recombinant protein on day 1 and boosting on day 14. The second set of animals received the same quantity of antigen by intramuscular injection on day 1 and boosted by the same route on day 14. Serum samples were collected from both groups and a third group of unvaccinated control animals on day 53.

Antibody response to the vaccine was determined by ELISA using synthetic peptides as antigen. The peptide NLVPMVATV (SEQ ID NO: 12) was use to assess antibodies to pp65 and the peptide SLYNTVATL (SEQ ID NO: 13) was used to measure the immune response to the HIV p24 epitope fused to pp65. ELISA plates were coated with 10 μg per well of the synthetic peptides overnight in a coating buffer. Serum samples were diluted in the plates and the antibodies were allowed to bind overnight at 4°C. Bound antibodies were detected with HRP-labeled goat anti-mouse secondary and developed using the one step turbo TMB (3, 3', 5, 5'-tetra methylbenzidine) ELISA substrate (Pierce Biotechnology, Inc., Rockford, LL).

The geometric mean serum titers were determined for each group of animals against the representative pp65 synthetic peptide and the p24 epitope fused to the pp65 expression construct. Animals vaccinated by intramuscular injection did not develop a detectable response to pp65, although a low but detectable response to the p24 epitope was observed (Table 5). Animals vaccinated by intranasal instillation developed antibody titers to both the pp65 and p24 peptides that were higher than those elicited by intramuscular injection.

Table 5. pp65-hapten immunogenicty data

Average ELISA titer^a Route of pp65 peptide p24 peptide administration

Intramuscular 0.3 ± 0.3 1.05 ± 0.53

Intranasal 1.35 ±0.23 1.66 ± 0.07 a. values are given as the average log 10 serum titers from 4 animals

These data suggest that other antigens can be fused to pp65 and that vaccination with the fusion protein can elicit an immune response to the artificial epitope without the use of adjuvant. Moreover, because the pp65 fusion proteins can be incorporated in tegument aggregates, this method could be used to produce antigens for vaccines to other targets of interest. The highly immunogenic properties of pp65 and will then help induce a strong immune response to the new antigens expressed in the fusion proteins.

Claims

1. A composition comprising isolated CMV tegument aggregates, in which said tegument aggregates comprise a plurality of CMV pp65 proteins and may elicit an immune response in a mammal.

2. The composition of claim 1 wherein said CMV is human CMV.

3. A composition comprising a plurality of CMV tegument proteins, the primary sequence of which is derived from pp65, wherein at least a portion of said proteins has been genetically altered such that at least one hapten has been inserted into its amino acid sequence, and wherein said proteins may be aggregated to form an immunogenic particle.

4. The composition of claim 3 wherein said hapten is associated with a disease condition caused by an agent selected from the group consisting of: single-stranded DNA viruses, double-stranded DNA viruses, single-stranded RNA viruses, double-stranded RNA viruses, intracellular parasites, fungi, bacteria, and cancer.

5. The composition of claim 4 wherein said hapten is a HIV epitope.

6. A method of inducing an immune response in an animal comprising administering the composition of claim 1 to the animal in an amount effective to induce said immune response.

7. A composition comprising pp65 which has been obtained by: infecting cells with CMV; contacting said infected cells with an inhibitor of ppUL97 function; and isolating said pp65 from tegument aggregates formed within said infected cell.

8. The composition of claim 7 in which said inhibitor of ppUL97 is Maribavir.

9. A method of screening agents for CMV antiviral activity comprising the steps of: infecting cells with CMV; contacting said agent with said infected cells; and observing said cells for the formation of tegument aggregates.

10. A method of screening agents for CMV antiviral activity by transiently expressing pp65 and UL97 kinase such that agents that inhibit the kinase induce the formation of pp65 aggregates.