INTRODUCTION

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Infection with human cytomegalovirus (HCMV), a betaherpesvirus, continues to be a significant cause of sequelae in infants infected in utero following maternal infection. Combined annual rates of disease and death caused by congenital HCMV infection have been estimated to be between 8,000 and 9,000 cases in the United States and Europe (49). In addition, HCMV is a major infectious complication in immunosuppressed individuals, such as transplant recipients and patients suffering from AIDS (12).

A key determinant for the outcome of an HCMV infection in these clinical settings is preexisting immunity. The presence of seroimmunity to HCMV prior to conception reduces the frequency of mother-to-fetus viral transmission and, more importantly, decreases the risk of damage in the infected fetus (23). In solid-organ allograft recipients, the lack of HCMV-specific immunity correlates with more severe clinical manifestations and increased mortality rates in patients infected with HCMV in the posttransplant period (17, 18, 66, 74). Posttransplant immunity against HCMV has also been demonstrated to influence the outcome of infection in patients receiving allogeneic bone marrow transplantation. Reconstitution of major histocompatibility complex (MHC) class I-restricted, HCMV-specific cytotoxic T cells (CTL) in the immediate posttransplantation period has been inversely correlated with severe manifestations of HCMV infections (53, 59, 60).

Cytomegalovirus-specific CD8⁺ CTL have been identified as major immunologic effectors that limit virus replication in vivo (57). The adoptive transfer of HCMV-specific CTL has been shown to prevent severe disease in allogeneic bone marrow transplant recipients (63, 85). Yet efficient reconstitution of CD8⁺ CTL was dependent on the presence of CD4⁺ helper T cells, documenting the importance of both CD4⁺ and CD8⁺ T cells for the control of HCMV infection (60, 85). The presence of transplacentally acquired antiviral antibodies has been demonstrated to modify the severity of HCMV disease in transfusion-associated HCMV infection in newborn infants (90). Passive transfer of antibodies has been shown to be effective in preventing disease in premature newborns and in solid-organ recipients (19, 75, 86). In addition, the presence of HCMV-specific antibodies prior to conception has been shown to correlate with decreased viral transmission and a reduction in the incidence of clinical manifestations in the child (23). Finally, recent studies have emphasized the importance of neutralizing antibodies in bone marrow transplant recipients, as their presence correlated with the lack of severe HCMV disease in these patients (71). Together these results suggest that both cellular and humoral functions contribute to protective immunity against HCMV infection.

Limiting the severity of the HCMV disease that occurs in the nonimmune host after prenatal infection or under conditions of immunosuppression will require the development of an effective vaccine strategy. The potential benefit from vaccine-induced immunity has been estimated to be 40-fold with respect to intrauterine transmission and 25- to 30-fold with respect to decrease in central nervous system damage in congenitally infected infants (11). Several vaccine strategies have been employed in the past. Live attenuated HCMV strains have been tested both in healthy volunteers and in transplant recipients (15, 49, 78). However, protection was less effective than that seen after natural infection. More recently, a subunit vaccine using the viral surface glycoprotein gB (gpUL55) as the antigen has been tested in human populations. gB is a major target of neutralizing antibodies against HCMV (6, 26, 44, 83). Recombinant gB, either produced in CHO cells or expressed by recombinant canarypox viruses, proved to be immunogenic both in laboratory animals and in clinical trials (10, 25, 49). However, with the vaccination protocols used thus far, synthesis of neutralizing antibodies was limited in either quantity or duration or both. An alternative strategy recently tested is to prime with a recombinant canarypox virus which expresses gB and to boost with attenuated Towne virus (2). A lymphoproliferative response could not be detected by priming with gB alone. This could be explained by the fact that although gB is the major target of neutralizing antibodies against HCMV, viral tegument proteins and at least one nonstructural regulatory protein have been shown to be dominant antigens for the generation of a cellular immune response against HCMV (7, 8, 34, 43, 87). Of these, phosphoprotein pp65 has been shown to play a central role both in the induction of CTL and in the stimulation of CD4⁺ Th lymphocytes. Thus, a combination of vectors expressing both gB and pp65 may be required to stimulate both cellular and humoral immune responses against HCMV (16, 49).

Based on these previous results, we have asked whether the combination of both humoral and cellular antigens of HCMV in one virus-like particle could be effective in stimulating both arms of the immune system. We took advantage of the fact that HCMV-infected human fibroblast culture cells release, in addition to virions, noninfectious defective particles termed dense bodies (DB) and noninfectious enveloped particles (NIEPs) (13, 21, 31, 68). The structure and protein composition of noninfectious enveloped particles is comparable to that of virions except for the presence of an additional polypeptide, termed the assembly protein, and the lack of DNA (31). DB are enveloped spherical structures that lack viral capsids and DNA. They consist mainly of viral tegument proteins and glycoproteins, with pp65 and gB being major constituents (5, 21, 24, 31, 65, 68, 79). Human immune sera have been shown to react with DB antigen (22). In addition, most commercially available test kits for the detection of antibodies against HCMV contain large amounts of DB in their antigen preparations, demonstrating the antigenicity of such particles.

As has been shown previously, DB enter cells efficiently and deliver their protein components into the cell (70, 81). Since HCMV virions enter cells by interaction of envelope glycoproteins with cell surface receptors and subsequent membrane fusion, we hypothesized that DB enter cells by the same route and thereby would mimic infection. Using the mouse model, we demonstrated that DB induced a neutralizing antibody response, and although they are noninfectious and did not lead to the synthesis of viral proteins within the cell, they also induced a significant anti-HCMV CTL and T-helper lymphocyte response. Thus, we propose that DB, by virtue of their unique properties, could serve as the basis for the development of a nonreplicating recombinant vaccine against HCMV.

	MATERIALS AND METHODS

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Mice. BALB/cJ (H-2^d) mice were obtained from the breeding colony of the Institute for Virology of the University of Mainz. HLA-A2 transgenic mice C57BL6-A2.K^b were provided by Linda Sherman, La Jolla, Calif. Eight- to 12-week-old mice were immunized subcutaneously in the left hind footpad with antigen resuspended in phosphate-buffered saline (PBS) in a volume of 25 to 50 µl or intraperitoneally with antigen resuspended in PBS in a volume of 200 to 250 µl.

Cells and viruses. Human foreskin fibroblasts (HFF) were grown in minimal essential medium (MEM; Gibco-BRL, Glasgow, Scotland) supplemented with 5% fetal calf serum (FCS), L-glutamine (100 mg/liter), and gentamicin (50 mg/liter). HFF were used between passages 6 and 16 and infected with HCMV laboratory strain Ad169 at a multiplicity of infection of 1 to 10 while subconfluent.

Immunoblotting, immunofluorescence, and antibodies. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and immunoblot analyses were carried out as described before (70). For immunoreactions, specific monoclonal antibodies (MAbs) directed against pp65 (MAb 65-33), against gB (MAb 27-287), against pp28 (MAb 41-18) (all kindly provided by W. Britt, University of Alabama, Birmingham), against gH (MAb SA4) (82), and against pp150 (MAb XP1) (32) were used. For MAbs 65-33, 27-287, 41-18, and SA4, hybridoma supernatant was diluted 1:50 to 1:100; purified MAb XP1 was used at a concentration of 1 to 10 µg/ml.

DB preparations. DB were isolated by glycerol-tartrate gradient centrifugation from the supernatants of HCMV strain Ad169-infected HFF (31), resuspended in PBS, and stored at 70°C until further use. DB preparations were quantitated by standardization according to their pp65 content. For this, serial DB dilutions were run on an SDS-10% polyacrylamide gel adjacent to serial dilutions of a bovine serum albumin stock of 10 mg/ml (68 kDa) and stained with Coomassie brilliant blue. By comparing band intensity using the TINA software (Raytest, Straubenhardt, Germany), the pp65 content per microliter of DB preparation was calculated. Deduced from that, the amount of DB in each preparation was determined. Part of the DB preparations was sonicated for 10 min in short intervals on ice to avoid heating in a Branson sonifier using a horn tip in pulsed mode (Branson, Danburry, United Kingdom). Subsequent to that, DB were further subjected to 10 subsequent cycles of freeze-thawing using liquid nitrogen. The resulting preparation was called sonicated freeze-thawed DB (sfDB). To obtain soluble proteins, DB were treated with 2% SDS-2% -mercaptoethanol for 2 h at 37°C.

Sucrose gradient centrifugation. DB material (4 µg) was diluted in 1.4 ml of PBS, layered onto a 10 to 66% (wt/wt) sucrose gradient, and centrifuged in a TH641 rotor for 2.5 h at 29,000 rpm and 20°C. Seventeen 0.6-ml fractions were collected from the top and trichloroacetic acid (TCA) precipitated for 2.5 h on ice. Protein pellets were resuspended in Laemmli buffer and separated on a 10% SDS gel. After transfer to nitrocellulose membranes, the blots were probed with anti-pp65 MAb p65-33 and horseradish peroxidase (HRP)-labeled anti-mouse IgG (diluted 1:10,000; Dako, Hamburg, Germany), with subsequent chemiluminescence detection (ECL Plus; Amersham Corp., Arlington Heights, Ill.).

Negative-staining electron microscopy. DB and sfDB were diluted in PBS to a final concentration of 1 µg/µl to 200 ng/µl, respectively. Negative staining of the specimen was done by the single-droplet procedure (28, 29), with carbon support films that were glow discharged for 20 s. After adsorption of the sample to the carbon film, the samples were washed five times with distilled water; the negative stain, consisting of 5% (wt/vol) ammonium heptamolybdate (pH 7.0) containing 1% (wt/vol) trehalose, was then added. Transmission electron microscopy was performed with a Zeiss EM 900 at 80 kV, and images were recorded on Kodak EM film, type 4489, at magnifications of ×12,000 to ×50,000.

Neutralization assays. Neutralization assays were carried out as described by Andreoni and colleagues (4). Briefly, serum samples were collected from anesthetized mice by cardiocentesis and allowed to clot at 37°C for 2 h and then stored at 4°C overnight. The following day, samples were centrifuged at 4°C for 10 min at 8,000 × g. The clarified sera were transferred to sterile Eppendorf tubes. All sera were prediluted 1:3 with PBS and then further diluted in 10 serial twofold dilutions. In the last sample, no serum was added to generate a nonimmune control. For analysis of neutralizing antibodies to HCMV strain Ad169, equal volumes of virus supernatant were added to the diluted sera and mixed. This supernatant had been generated by infecting HFF with strain Ad169 until complete late cytopathic effect developed. Supernatants were stored at 70°C in aliquots until further use. The 50% tissue culture infective dose (TCID₅₀) (41) was determined to be 6.5 TCID₅₀ for the virus supernatant used for our analyses. For the neutralization assays, frozen supernatant was thawed and vortexed for 20 s and prediluted 1:200 in MEM. This dilution of virus resulted in 100 to 200 IE1-stained cells per microscopic field in 96-well microtiter plates in the absence of specific antiserum (positive control). Generally, 250 µl of diluted serum and 250 µl of virus suspension were mixed and incubated at 37°C for 4 h. In the meantime 1.5 × 10⁴ HFF per well were seeded into 96-well plates in a volume of 25 µl in four rows for each serum to be tested. After 4 h of incubation, 100 µl of each serum-virus mix was added to the cells in four replicates. The plates were incubated at 37°C and 5% CO₂ for 24 h. The following day, the cells were fixed in 96% ethanol for 15 min at 20°C, washed in PBS, and stained with the anti-IE1 MAb p63-27 for 1 h at 37°C; 50 µl of hybridoma supernatant was used. Subsequently, cells were washed again, and IE1 detection was carried out by incubating with HRP-conjugated anti-mouse IgG antibodies for 45 min at 37°C (Dako), diluted 1:500 in PBS. Following that, AEC staining was performed with 4 mg of AEC (aminoethylcarbazole) resolved in 1 ml of DMF (dimethylformamide; both from Sigma, Deisenhofen, Germany) and diluted 1:20 in AEC buffer containing 50 mM sodium acetate and 50 mM acetic acid (pH 4.9). This solution was filtered twice, and 1/1,000 volume of H₂O₂ (30%) was added. Then 100 µl of this substrate was added to each well and incubated for 10 to 30 min at 37°C until the nuclei in the positive control (without serum) stained dark brown. The wells were washed again in PBS, and the number of IE1-positive nuclei per well was counted. Percent neutralization was calculated as the serum dilution that resulted in 100% (V/V₀ = 1) and 50% (V/V₀ = 0.5) reduction in the number of infected cells 24 h after infection compared to a negative control without serum. Results are given as averages for quadruplicate wells.

Cytotoxicity assays. Mice were immunized or infected subcutaneously in the left hind footpad with different amounts of antigen or 10⁵ PFU of purified murine CMV, strain Smith. All antigens were resuspended in PBS and administered in a volume of 25 µl. Popliteal lymph nodes were collected 8 days after immunization or infection and pooled for each group of mice. Lymphocytes were isolated, washed several times, counted, and seeded in macrocultures in MEM-alpha supplemented with 10% FCS, HEPES buffer (0.01 M), L-glutamine (200 mg/liter), 28 µl of -mercaptoethanol, gentamicin (50 mg/liter), and 100 to 200 U of recombinant human interleukin-2 (IL-2) (Pan Systems, Aidenbach, Germany) mediating T-cell growth. After a cultivation period of 8 days in IL-2-containing medium, the cytolytic activity of CTL was measured in a standard 4-h ⁵¹Cr release assay using the indicated effector-to-target cell (E:T) ratios with a constant number (10³) of ⁵¹Cr-labeled target cells and graded numbers of effector cells in 0.2-ml round-bottomed 96-well plates. Throughout, reported cytolytic activities represent the mean percent specific ⁵¹Cr release from three replicate microcultures.

(i) Redirected lysis. The strategy of redirected lysis (30, 38) was used to measure the total cytolytic activity of CTL populations. For this, Fc receptor-expressing P815 cells were labeled with ⁵¹Cr for 75 min and then armed with antibodies by incubation for 15 min at 25°C with an optimized dose of hamster MAb (IgG1) specific for mouse CD3 (clone 145-2C11; Southern Biotechnology Associates, Inc., Birmingham, Ala.). After washing twice in RPMI 1640, these cells were used as targets. The CTL response was considered positive if lysis of CD3 target cells was >5% above the level of lysis obtained with untreated control P815 target cells.

(ii) Peptide-specific lysis. CTL activity directed against the peptide pp65 (NLVPMVATV; amino acids [aa] 495 to 503) presented in the context of HLA-A2 was monitored with HLA-A2.k^b-positive T2 and Jurkat cells labeled with peptide using concentrations of peptide ranging from 10⁶ to 10¹⁰ M. Synthetic peptides were purchased from Jerini Biotools GmbH, Berlin, Germany, and diluted to 10³ M in 30% acetonitrile. Further dilutions were carried out in PBS. Peptide labeling was performed on 10⁶ target cells for 1 h at 37°C after ⁵¹Cr labeling for 90 min. Excess peptides were removed by washing before labeled cells were used as targets. Values for specific percent lysis were obtained by subtraction of the lysis values obtained with an irrelevant control peptide from tumor suppressor protein p53, known to be presented by HLA-A2 (STPPPGTRV; aa 149 to 157).

Cytokine ELISAs. For determining the type of T-helper-cell response, BALB/cJ mice were immunized subcutaneously in the left hind footpad with the different antigen preparations. Eight days postimmunization, the draining lymph nodes of each group were pooled and the lymphocytes were isolated. Cells were seeded in 48-well plates in RPMI 1640 supplemented with 5% FCS, L-glutamine (100 mg/liter), gentamicin (50 mg/liter), and 28 µl of -mercaptoethanol (99%) at a density of 10⁶ cells/ml. Cell suspension (0.5 ml) was seeded into each well and restimulated as indicated. Each restimulation was done in four parallel wells. The supernatants were harvested at 24 h and 36 h after restimulation from two wells each. Cell debris was removed by centrifugation for 5 min at 8,000 × g, and the resulting supernatants were frozen at 70°C. For determining the amount of gamma interferon (IFN-) and interleukin-5 (IL-5), all supernatants were thawed once and analyzed using commercially available IFN- and IL-5 enzyme-linked immunosorbent assays (ELISAs) (Endogen, Woburn, Mass.).

IgG subclass analysis. To determine the IgG subclasses induced after immunization with DB, serum samples were collected from anesthetized mice by cardiocentesis, allowed to clot at 37°C for 2 h, and then stored at 4°C overnight. The following day, samples were centrifuged at 4°C for 10 min at 8,000 × g. The clarified sera were transferred to sterile Eppendorf tubes in several aliquots. Half of them were heat inactivated by incubation at 56°C for 30 min. Native and heat-inactivated sera were serially diluted in dilution buffer (PBS supplemented with 2% Tween 20 and 3% FCS) and added to ELISA plates coated with HCMV particles as well as with a control antigen isolated from noninfected cells (Biotest). Plates were incubated at 37°C for 2 h in a humidifier and washed four times. The bound antibodies were detected with peroxidase-conjugated IgG1-specific and IgG2a-specific antibodies (both from Pharmingen, San Diego, Calif.) and IgG-specific anti-mouse antibodies for 90 min at 37°C. After four washing steps, 100 ml of substrate (o-phenylenediamine; 2 mg/ml) was added for 20 min. The reaction was stopped by the addition of 100 µl of 12% H₂SO₄, and the optical density at 492 nm was determined. The endpoint dilution was defined as the serum dilution at which the absorbance at 492 nm equaled 2.5 times the absorbance of a preimmune serum.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

HCMV DB efficiently deliver viral proteins into human and mouse cells. Incubation with HCMV DB leads to the rapid translocation of viral proteins into human fibroblast cells (70). Others have shown that uptake is mediated by the envelope surrounding these nonreplicative particles (81). We hypothesized that this entry into cells, which resembled viral infection, rendered such particles an efficient delivery system for antigenic proteins for processing by and presentation to the immune system. In the first set of experiments, we determined whether DB could also deliver proteins into professional antigen-presenting cells and whether this process of protein transfer was also effective in mouse cells. DB were purified from the culture supernatant of infected HFF cells using glycerol-tartrate gradient centrifugation (31, 80). About 0.4 µg of purified DB was used to inoculate human lymphoblastoid cells (LCL) and murine mastocytoma cells (P815) as well as murine embryonic fibroblasts and BALB/c-3T3 cells.

View larger version (51K): [in this window] [in a new window]   FIG. 1.   Localization of pp65 (UL83) after incubation of various human and murine cell types with DB and sfDB. Adherent HFF, BALB/c-3T3 (3T3), and MEF were grown on glass coverslips and incubated with DB or sfDB overnight. The panel on the right shows PBS-treated (mock) controls. Nonadherent P815 and LCL cells were incubated overnight with DB, sfDB, or PBS in 24-well plates. Subsequent to that, cells were cytocentrifuged onto glass coverslips and fixed. The localization of pp65 in adherent cells was determined by immunofluorescence using MAb 65-33 as the primary antibody, followed by a murine IgG-specific, FITC-conjugated secondary antibody. Cytospin preparations were stained with a commercially available kit, using APAAP technology and a pp65-specific MAb as the primary antibody.

View larger version (61K): [in this window] [in a new window]   FIG. 2.   Analysis of the reduction of infectivity of virions by physical treatment and determination of the purity of DB preparations. (A and B) Reduction of infectivity of virion preparations by sonication and freeze-thawing. HFF were grown on glass coverslips and incubated with virions (A) and sf-virions (B) overnight. Following fixation, the expression of IE1 was determined by using MAb p63-27 followed by a murine IgG-specific, FITC-conjugated secondary antibody. (C and D) Analysis of the purity of DB preparations. HFF were grown on glass coverslips and incubated with DB (C) and sfDB (D) overnight. Following fixation, the expression of the nonstructural protein IE1 was determined as described for panels A and B to provide a measure of residual virion contamination in the DB preparations.

Mechanical disruption does not impair the particulate nature of DB. One major issue of this work was to analyze the potential of DB to stimulate immune responses in a small-animal model and to investigate whether the delivery of antigens by DB could induce specific immunological effector functions. Initially, we determined whether sonication and freeze-thawing altered the structure of DB so as to prevent their cellular entry but without altering their particulate nature or protein composition. We first determined whether sonication and freeze-thawing led to significant degradation of selected protein constituents of DB. DB and sfDB were subjected to immunoblot analysis using MAbs directed against the tegument proteins pp65 and pp150, as well as against glycoprotein B (gB) (Fig. 3). Protein degradation attributable to sonication and freeze-thawing was not found, and the reporter proteins pp65, pp150, and gB were still present in equal amounts in DB and sfDB (Fig. 3). With the pp65-specific MAb, additional bands besides the major band at 65 kDa were detected. We do not know the origin of these bands. Since they were comparable in DB and sfDB, we argued that they were the result of overloading and not of increased pp65 degradation in one of the preparations. It should also be noted that we consistently found the viral tegument protein pp150 in our DB preparations.

View larger version (59K): [in this window] [in a new window]   FIG. 3.   Comparative immunoblot analysis of DB and sfDB. DB and sfDB were separated on SDS-10% polyacrylamide gels and blotted onto nitrocellulose membranes. Triplicate filters were probed with MAbs directed against the tegument proteins pp65 (MAb 65-33) and pp150 (MAb XP1) as well as against glycoprotein gB (MAb 27-287) of HCMV. The positions of the proteins are indicated by arrows. The positions of molecular size standards are indicated on the left.

View larger version (86K): [in this window] [in a new window]   FIG. 4.   (A) Immunoblot analysis of TCA-precipitated fractions from a sucrose density gradient centrifugation of DB, sfDB, and detergent-treated DB. DB, sfDB, and DB treated with 2% SDS-2% -mercaptoethanol were resuspended in PBS and layered onto a 0 to 66% (wt/wt) sucrose gradient. Centrifugation was carried out as described in Materials and Methods. The fractions were collected from the top, TCA precipitated, and analyzed by SDS-PAGE and immunoblotting using the pp65-specific MAb 65-33. The numbers below each blot indicate the refractive index of the respective sample. (B) Negative-stained electron micrographs of DB and sfDB preparations. Negative staining of specimens was performed by the single-droplet procedure as described in Materials and Methods. The negative stain consisted of 5% (wt/vol) ammonium heptamolybdate (pH 7.0) containing 1% (wt/vol) trehalose. The magnifications used for photodocumentation are indicated.

Immunization with DB induces a durable neutralizing antibody response. Next we determined whether immunization with DB would induce significant levels of neutralizing antibody and if the capacity for cell entry would influence the development of the antiviral antibody response. BALB/cJ mice were immunized subcutaneously with various amounts of DB or sfDB in a single dose without adjuvant. After 55 days, blood was obtained by cardiocentesis. Using 20 and 2 µg of DB as the antigen source, 50% neutralization titers were found to range between 1:384 and 1:768 (Table 1). No significant differences were seen in sera obtained after immunization with 2 or 20 µg of DB (Table 1). A 10-fold increase in the amount of antigen administered did not lead to higher antibody titers in the sera, suggesting that a plateau in the response had been reached. Even very small amounts of immunogen (0.2 and 0.02 µg) induced a significant neutralizing antibody response. In contrast, although neutralizing antibodies were also induced after immunization with sfDB, levels were consistently lower than with DB (Table 1).

View larger version (18K): [in this window] [in a new window]   FIG. 5.   Analysis of the neutralizing capacity of murine immune sera obtained at different time points after initial immunization. BALB/c mice were immunized with 20 µg of DB intraperitoneally three times at 4-week intervals. Blood was collected by cardiocentesis at the indicated time points after initial immunization. The sera were analyzed by microneutralization assays as described in Materials and Methods. Neutralization is expressed as percent surviving infectivity (V/V0) and plotted as a function of the serum dilution. Results represent the means of four experiments. Dotted line, V/V0 = 0.5.

Serum antibodies induced by DB immunization are directed against HCMV tegument proteins and glycoproteins. To analyze the specificity of antibodies induced by DB immunizations, immunoblot analyses were carried out. Gradient-purified virions were subjected to electrophoresis, blotted, and probed with sera obtained from immunized mice (Fig. 6). Several bands in the range between 35 and 150 kDa became detectable. By comparison with the reactivity of MAbs, the tegument proteins pp150 and pp65 and the glycoproteins gB and gH were identified as being reactive with murine sera. No antibodies against the tegument protein pp28 were detectable. In addition to the proteins that could be identified by comparison using available reagents, immunization with DB led to the induction of antibody specificities directed against proteins of approximately 35, 45, and 80 kDa. It remains unclear which of the viral proteins correlate to these bands.

View larger version (48K): [in this window] [in a new window]   FIG. 6.   Comparative immunoblot analysis of antibody specificities in a murine immune serum collected 55 days after a single subcutaneous immunization with 20 µg of DB. Purified virion particles were used as the antigen. They were separated by SDS-PAGE and blotted onto nitrocellulose membranes. The filters were incubated either with a 1:2,000 dilution of murine serum, a 1:100 dilution of hybridoma supernatant of MAb 65-33 (pp65), 41-18 (pp28), 27-287 (gB), or SA4 (gH), or a 1:5,000 dilution of purified MAb XP1 (pp150). Bound antibodies were detected by HRP-conjugated murine IgG-specific secondary antibodies and visualized by enhanced chemiluminescence. The positions of molecular size standards are indicated on the left.

DB deliver pp65 into the MHC class I presentation pathway to prime HCMV-specific CTL responses. CMV-specific MHC class I-restricted CTL have been shown to be primed and expanded when viral proteins are synthesized de novo in infected cells and subsequently processed to peptides by the proteasome complex. These peptides are introduced into the MHC class I presentation pathway and stimulate virus-specific CTL responses. Although this process is generally accepted as being the major mechanism for the efficient induction of CTL responses, stimulation of CTL has also been shown to occur by exogenous introduction of HCMV-encoded proteins from infecting particles into the MHC class I presentation pathway (55, 62). It thus appeared reasonable to assume that the exogenous loading of large amounts of the dominant T-cell antigen pp65 by DB would induce antiviral CTL.

6

8

View larger version (20K): [in this window] [in a new window]   FIG. 7.   Analysis of HCMV-specific CTL induced after subcutaneous immunization of C57BL6-HLA-A2.Kb transgenic mice with DB or sfDB in chromium release assays. Effector lymphocytes were prepared from C57BL6-HLA-A2Kb transgenic mice 8 days after a single subcutaneous immunization with the antigens indicated. Lymphocytes were cultured for another 8 days in medium containing IL-2 without further restimulation. Chromium release assays were carried out at the indicated E:T ratios. Mean values of triplicate cultures are shown. (A) Chromium release assay (4 h) using T2-A2.Kb target cells loaded with 108 M peptide 495-503 (NLVPMVATV) for 1 h after 51Cr labeling. (B) Chromium release assay (4 h) using JA2.Kb target cells loaded with 106 M peptide 495-503 for 1 h after 51Cr labeling. Lysis values were obtained by subtracting the lysis of equivalent cells loaded with the HLA-A2-restricted control peptide 149 to 157 from p53 (STPPPGTRV).

DB immunization leads to induction of cytolytic cells comparable to standard immunization with replication-competent murine CMV. Having demonstrated that DB induce pp65-specific CTL responses, we next wanted to know how the intensity of the cytolytic activity induced by immunization with DB compared to immunization with a replicating virus. Therefore, one group of six BALB/c mice was subcutaneously infected with 10⁵ PFU of purified murine CMV in the left hind footpad, a standard method for the induction of CMV-specific cytolytic lymphocytes in mice (54, 56). Four other groups of mice were immunized the same way with 20 µg of DB, 2 µg of DB, 20 µg of sfDB, and PBS as a control.

View larger version (23K): [in this window] [in a new window]   FIG. 8.   CD3-redirected lysis analysis of lymphocytes from BALB/c mice immunized with either DB, sfDB, murine CMV (MCMV), or PBS, using a 4-h chromium release assay. Lymphocytes were prepared from BALB/c mice 8 days after a single subcutaneous immunization in the left hind footpad with the amounts of DB, virus, or PBS indicated. Lymphocytes were cultured for 8 days in IL-2-containing medium without further restimulation. Subsequent to a 51Cr-labeling period of 90 min, P815 mastocytoma cells were charged with an MAb against murine CD3 for 25 min. Lymphocytes and charged P815 cells were cocultivated at the indicated E:T ratios. Chromium release is shown as the mean values obtained from triplicate cultures. Numbers were corrected for nonspecific lysis by subtracting the values obtained from analyses of uncharged P815 cells exposed to murine lymphocytes.

Immunization with DB leads to a Th1-type T-helper-cell response. From adoptive transfer experiments of HCMV-specific CTL in bone marrow transplant recipients, it has become clear that significant levels of cytolytic cells against HCMV could be sustained only when a helper T-lymphocyte response was present (85). As pp65 had been identified as one of the major HCMV antigens to induce helper T cells (7), we investigated whether immunization of mice with DB led to the generation of a Th lymphocyte response. Functionally distinct Th lymphocyte subsets, known as Th1 and Th2, are characterized by their cytokine secretion patterns and by the antibody isotypes which are induced. In general, Th1 immune responses promote the production of IgG2a subclass antibodies, whereas Th2 immune responses promote the production of IgG1 antibodies. Th1-dominated responses are thought to be associated with protective responses to infectious agents. Thus, it was of particular interest whether DB could induce a Th1-like immune response. We approached this question in two ways. First, the production of two signature lymphokines, IFN- (Th1-like) and IL-5 (Th2-like), by lymphocytes isolated from popliteal lymph nodes after subcutaneous immunization in the left hind footpad was analyzed. For this, six BALB/c mice per group were immunized with 2 or 20 µg of DB, 20 µg of sfDB, or PBS. Lymph node cells were isolated from popliteal lymph nodes 8 days after immunization, seeded at densities of 10⁶ cells/ml, and restimulated in vitro with either 20 µg of DB, 20 µg of sfDB, 20 µg (2 × 10⁵ M) of the synthetic peptide 495-503, representing a CTL epitope, or PBS. After 24 and 36 h of restimulation, the culture supernatant was analyzed for the presence of IFN- and IL-5 using commercially available ELISAs. As shown in Fig. 9, lymphocytes from mice immunized with 20 µg of DB secreted IFN- up to 15 ng/ml upon restimulation with DB and slightly less upon restimulation with sfDB (Fig. 9A). Lymph node cells from animals immunized with 2 µg of DB also secreted large amounts of IFN-, with a peak of 10 ng/ml at 36 h upon restimulation with DB and about half the amount upon restimulation with sfDB (Fig. 9C). IFN- was released to a significantly lesser extent (peak of 3 ng/ml 36 h postrestimulation with DB) and delayed after immunization of mice with 20 µg of sfDB (Fig. 9B). Restimulation with DB induced IFN- secretion most effectively. sfDB were less effective. Neither the pp65-derived CTL epitope nor PBS induced IFN- production. We could not detect IL-5 secretion from cells obtained from any group (data not shown). Taken together, these results indicated that immunization with DB led to a Th1-like immune response with respect to the cytokine secretion of restimulated lymph node cells. Furthermore, the data suggest that immunization with DB resulted in higher levels of IL-5 secretion than immunization with sfDB.

View larger version (31K): [in this window] [in a new window]   FIG. 9.   Quantitative ELISA analysis of IFN- secretion by murine lymphocytes isolated after subcutaneous immunization with DB or sfDB and subsequent stimulation in culture using different antigens. Assays were performed on supernatants of 106 lymphocytes per ml isolated 8 days postimmunization with the antigen indicated above each plot. Lymphocytes were restimulated with the indicated antigens. At 24 and 36 h after restimulation, supernatants were analyzed for IFN- levels. Values represent the mean results of four independently performed experiments. Error bars indicate maximal deviations from the means.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

DB have been well recognized by HCMV researchers primarily as an obstacle to the purification of virions. These noninfectious particles are released in large amounts after infection of primary human fibroblasts with laboratory-adapted strains of HCMV, contaminating virion preparations. One hallmark of DB is their ability to enter eukaryotic cells, and this is reflected by the translocation of their proteinaceous content to the cytoplasm and eventually into the nucleus. As shown in this communication, DB can also deliver antigen into cell lines that belong in the group of professional antigen-presenting cells. In early studies, DB had been proposed as ideal candidates for the development of an anti-HCMV vaccine (24, 68, 69, 79). However, no detailed analysis of their usefulness as immunogens has been reported to date. Recent studies have shown that only a few of the more than 200 proteins of HCMV are dominant antigens for the induction of humoral and cellular immune responses. Most of these antigens are constituents of DB. Thus, elucidation of the immunogenic properties of these particles could be important for the future development of a noninfectious HCMV vaccine.

DB are electron-dense spherical structures surrounded by a lipid bilayer membrane. Inserted into this envelope are an as yet incompletely defined set of viral glycoproteins, including gB and gH (21, 24, 68, 79). gB and gH appear to be essential for adsorption and penetration of viral particles. In addition, these glycoproteins have been shown to be major targets of the neutralizing antibody response against HCMV. As the entry of DB into cells is comparable to that of infectious virions (81), it is likely that gB and gH are present in the DB envelope in a conformation similar to their conformation in virions. For the induction of virus-neutralizing antibodies, this may be desirable, as part of the major neutralizing epitopes on gB and gH are conformation dependent (76, 82). Accordingly, immunization with DB either subcutaneously or intraperitoneally led to the induction of significant levels of HCMV-neutralizing antibodies in mice. This response is analogous to previously reported results obtained after immunization of rabbits with DB (33). DB can enter cell types such as B cells and dendritic cells, which can present antigen in the context of MHC class II molecules (M. Mach, unpublished data; S. Pepperl and B. Plachter, unpublished data). It could be hypothesized that enhanced binding of DB to or entry into such cells improves presentation of viral antigens by MHC class II molecules and stimulates the Th lymphocyte response required for efficient priming and expansion of virus-specific B cells.

One major characteristic of a nonreplicating vaccine is the decay of the immune response over time. Although the issue cannot be addressed directly in an animal model, it appeared remarkable that even after a period of 1 year, neutralizing antibodies could still be detected in sera obtained from animals immunized with DB. A vaccine with immunogenicity similar to that of DB could offer a distinct advantage by providing a persistent neutralizing antibody response compared to the transient response which had been reported to follow a subunit vaccine such as the recombinant gB subunit vaccine (49). However, clinical trials would be necessary to investigate whether DB particles would be superior to purified HCMV glycoproteins for the induction of protective antibodies in humans.

The most abundant protein contained within DB is pp65 (pUL83) (31, 67). This polypeptide is one of the major antigenic determinants for the induction of HCMV-specific CTL during natural infection (8, 43, 87) and has also been demonstrated to induce a Th lymphocyte response to HCMV (7). In addition to pp65, we consistently detected the large phosphorylated tegument protein pp150 in our DB preparations. pp150 has also been shown to be a target of CTL responses against HCMV (42). Using immunoblot analysis with a highly specific MAb, the amount of this protein detected relative to pp65 was reduced in DB compared with virions. This is in agreement with earlier reports by Gibson and coworkers, who found only small amounts of pp150 in DB (31, 65). Besides pp65 and pp150, other tegument proteins have also been shown to be contained within DB, but their immunological properties have not been defined (5, 88, 89).

CTL are usually primed against viral peptides that have been processed by the proteasome from viral proteins synthesized de novo in infected cells. However, in the case of HCMV, introduction of extracellular protein from virus particles into the MHC class I presentation pathway and subsequent recognition of these cells by virus-specific CTL has been described (61). Thus, the relative abundance of pp65 in DB compared to virions and the process of delivery of pp65 to cells by DB made it reasonable to assume that DB could serve as a nonreplicating virus-like particle to induce CTL in the absence of viral infection.

In the HLA-A2.K^b transgenic mouse model, immunization with DB clearly led to the induction of HCMV-specific CTL. Target cells that had been loaded with the synthetic peptide previously shown to induce a response in cells expressing HLA-A2 were efficiently lysed by DB-primed lymphocytes. These results demonstrated that immunization with DB could induce CTL reactive with an epitope of pp65 in the context of HLA-A2. Several possible explanations could account for the continued capacity to induce CTL in the apparent absence of cell entry of sfDB, at least as measured by the lack of pp65 nuclear translocation. The most obvious is the difference in the level of sensitivity of the immunofluorescence assay for detection of pp65 nuclear translocation and the immunological assays which included an in vitro expansion of T lymphocytes. It could also be argued that significant amounts of pp65 continued to enter cells, but because of disruption of other structures in DB, this protein could not translocate to the nucleus. Alternative explanations include the immunological properties of DB being attributable to their particulate nature, regardless of whether an envelope was present. Aggregates of antigen or antigen coupled to insoluble carriers can prime CTL, whereas soluble forms of the antigen fail to induce CTL (37, 48, 77). Macrophages and dendritic cells can present exogenous antigen on MHC class I molecules. Different mechanisms for such alternative MHC class I loading, such as transfer of particulate structures from the lysosomal compartment to the cytoplasm for further antigen degradation by the proteasome, have been described (46). However, professional antigen-presenting cells such as LCL appear to take up DB much more efficiently than sfDB, correlating with the differences in the immunogenicity of these particles. Irrespective of the explanation for the continued immunogenicity of sfDB compared to intact DB, we have shown that physical damage to the envelope structure of DB not only limited the entry of the particle into the cell, as measured by nuclear translocation of pp65, but also significantly reduced the capacity of these particles to induce an immune response characterized by both persistence of neutralizing antibodies and viral protein-specific CTL.

As HCMV does not replicate in mice, we cannot directly compare the intensity of the cytolytic response induced by DB with that induced during natural HCMV infection. However, murine CMV infection has been used in BALB/c mice to study various aspects of CMV immunology (36, 54, 56). We chose to use subcutaneous immunization with a previously defined optimized titer of murine CMV and compared the CD3-redirected cytolysis induced by this with the response induced by DB immunization using the same route of inoculation. Although murine CMV replicated at the site of injection (hind footpad) and was expected to induce a much more vigorous immune response than DB, we found to our surprise that the induction of total cytolytic activity after DB immunization was nearly equivalent to that induced by murine CMV. About twice the number of effector cells were needed to achieve comparable levels of cytolysis. This provides some measure of the immunogenicity of proteinaceous DB to induce cytotoxic cells compared to the established standard of murine CMV infection. However, the mouse model can provide only limited information about the potency of these particles to induce protective HCMV-specific CTL, as processing and presentation of HCMV antigens may be quite different in humans. Thus, detailed analyses of this issue must await future preclinical studies.

Viruses or intracellular microorganisms frequently lead to a Th1-type immune response after infection. It is well established that the presence of Th1-type helper lymphocytes is important for the development of both humoral and cellular immunity. Cell-mediated cytotoxicity, primarily mediated through CD8⁺ CTL, appears to be critical for the control of CMV infections (57, 59, 60). An HCMV-specific CTL response, however, can be sustained only in the presence of T-cell help (85). Thus, a successful vaccine against HCMV will likely require the induction of a Th1-like immune response. Previous work has shown that the differentiation of naive Th cells into Th1 or Th2 cells can be influenced by many factors, the most important of which are cytokines (45, 72). IL-12 and cytokines that modulate the influence of IL-12, such as IFN- and IFN-, are key regulators of Th1 differentiation (9, 58), while IL-4 and IL-5 are key regulators of Th2 differentiation (72). Other factors that can affect Th-cell differentiation include the type of antigen-presenting cells and the dose of antigen. For example, the addition of immunostimulatory substances such as aluminum hydroxide to vaccine preparations as well as gene gun applications often lead to a Th2-like helper response (20), which would be undesirable in the case of HCMV. On the other hand, there are also reports showing that both in vivo and in vitro, DNA vaccines can induce lymphocytes as well as sorted B cells, T cells, and NK cells to produce Th1-inducing cytokines such as IL-12, IFN-, and IFN- (35, 39). For the development of an HCMV vaccine, DB could be a suitable antigen delivery system because they induce a Th1-like immune response. After subcutaneous footpad immunization of mice with DB, lymphocytes isolated from draining lymph nodes secreted high levels of IFN- but no detectable IL-5 upon restimulation with DB. Although immunization with sfDB also resulted in IFN- production, levels were considerably lower and production appeared to be delayed. This was particularly the case when sfDB were used for both immunization and restimulation of the lymphocytes. Thus, the ability of DB to enter cells appears to support the stimulation of a Th response in vivo. One possible explanation for this may be that DB as well as virions are able to enter professional antigen-presenting cells such as B cells and dendritic cells (64; Mach, unpublished; Pepperl and Plachter, unpublished) and may thereby be efficiently introduced into the MHC class II presentation pathway.

It has been shown before that the development of Th1-like or Th2-like immune responses may vary according to the route of immunization. In general, Th1 immune responses promote the production of IgG2a antibodies, whereas Th2 immune responses promote the production of IgG1 antibodies. For DB, the route of administration appeared to be irrelevant for the induction of a Th1-like immune response. Both intraperitoneal and subcutaneous application led to an IgG1/IgG2a ratio of <1. The finding that both intraperitoneal and subcutaneous immunizations resulted in the same type of immune response indicates that the induction of Th1 lymphocytes by DB is attributable to the antigen rather than to the route of immunization.

In the past, several different strategies were used for the development of an HCMV vaccine (11, 49). The primary goal was to induce protective humoral immunity in women of childbearing age in order to prevent congenital HCMV infection. With the advent of the AIDS epidemic and the increasing frequency of iatrogenic immunosuppression, e.g., during transplantation, it became apparent that in addition to antibody synthesis, cellular immunity must be induced by a vaccine which could limit HCMV disease in these patients. Early attempts which used live attenuated strains of HCMV as a vaccine were encouraging and demonstrated that some protective immunity to HCMV could be induced by vaccination (49, 52, 78). The Towne vaccine could modify the outcome of HCMV infection in renal transplant recipients, although infection could not be prevented (50). In a challenge study, limited protection was afforded by vaccination with the Towne strain (51). However, HCMV infection rates were not affected by vaccination in young women (3). Notably, the CTL responses seemed to wane early after Towne vaccination (1).

Other investigators have stressed the need to refine vaccine strategies by combining gB as a target for the humoral immune response with pp65 to induce cellular immunity (16). DB appeared to be an ideal natural source of such an immunogen, combining all relevant antigens which could be delivered by these particles in a manner similar to viral infection. In addition, DB do not contain replicating DNA.

From the data presented, several options for the future development of DB as a vaccine are available. (i) Using recombinant DNA technology, these particles will have to be modified to further optimize antigen composition. This may be critical, as nonstructural viral antigens have been described to be important for the induction of immunity in part of the Caucasian population (34). (ii) DB can be used as a delivery system to include antigens from other viruses in a multivalent vaccine. (iii) Investigating the components necessary to form DB will enable the design of strategies to reconstitute DB-like particles in the absence of infectious virus. In this report, we have shown that DB of HCMV are immunogenic without adjuvant in mice and induce HCMV-neutralizing humoral as well as cellular immune responses. Therefore, these particles appear to be a promising basis for the future development of an nonreplicating recombinant HCMV vaccine.