INTRODUCTION

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Human cytomegalovirus (HCMV) is a serious opportunistic pathogen for both newborn and immunocompromised individuals. It is the leading viral cause of birth defects, affecting 6,000 to 7,000 infants yearly in the United States alone (16). In addition, nearly 10% of deaths of AIDS patients in 1992 were attributed to HCMV disease (45), while in bone marrow and solid organ transplant recipients, HCMV infection continues to lead to a high incidence of morbidity and even mortality (4). The severe medical problems associated with HCMV infection in these particularly vulnerable populations underline the necessity of developing a safe and effective vaccine. Studies of HCMV immunity and the testing of candidate vaccines have highlighted the importance of cell-mediated immunity for control of the infection and prevention of disease.

Because of the strict species specificity of the betaherpesviruses, animal models of CMV immunity have used murine cytomegalovirus (MCMV), which is similar to HCMV with regard to virion structure, genome organization, gene expression, tissue tropism, and latency. As with HCMV, the cellular immune response to MCMV has been shown to be critical for control of the acute infection (28). In the susceptible BALB/c strain, a single nonstructural proteinimmediate-early protein 1-pp89 (IE1-pp89) has been shown to be immunodominant for the generation of protective cytotoxic T lymphocytes (CTLs), and a single, nonpeptide epitope was determined to be targeted during natural infection (28). Delivery of this single epitope to BALB/c mice by vaccinia virus (14) or synthetic peptide (42) resulted in protective immunity. Similarly, immunization of BALB/c mice with a pp89-expressing plasmid generated similar levels of pp89-nonapeptide-specific CTL activity as did immunization with tissue culture-derived MCMV (17). However, this plasmid-mediated protection against MCMV was incomplete (17) compared to that generated by the attenuated virus. This suggests that, to be fully protective, the MCMV-specific CTL response may need to be directed against multiple viral antigens.

The number of MCMV antigens that contribute to the generation of protective CTL immunity is likely limited to those viral antigens that not only contain the appropriate epitopes for efficient binding to major histocompatibility complex (MHC) class I glycoprotein complexes and subsequent recognition by / T-cell receptors (TCRs) but are also capable of escaping the MCMV-mediated shutdown of antigen presentation. Similar to HCMV, immune evasion mechanisms affecting the MHC class I presentation pathway have evolved in MCMV to downregulate the presentation of MCMV-derived peptides on the cell surface (19). Seminal experiments characterizing MCMV immunity showed that, beginning in the early phase of viral replication, CTLs specific for an IE antigen could no longer recognize and lyse MCMV-infected cells (13). Levels of surface expression of MHC class I molecules did not appear to be affected at this time, and CTLs specific for an early antigen were able to lyse infected target cells, indicating the specific inhibition of pp89 presentation by early genes. However, later in the early phase, surface expression of MHC class I molecules is severely reduced as a result of MCMV early gene products blocking transport of peptide-loaded MHC class I complexes into the medial-Golgi compartment (12). This block appears to be mediated, at least in part, by the m152 early gene product (46, 50). This retention of class I complexes and the resultant prevention of antigen presentation were found to be abolished in infected cells treated with gamma interferon (IFN-) in vitro (20). Another MCMV gene, m06, was found to encode the glycoprotein gp48, which strongly associates with MHC class I molecules in the endoplasmic reticulum and reroutes them into the endosomal-lysosomal compartment for rapid proteolysis, thus preventing class I surface expression by an independent mechanism. Therefore, although CTLs directed against several viral epitopes may be generated during infection, there may exist only a limited number of epitopes that are sufficiently presented by the infected cell to trigger lysis of the infected cell at times early enough to be protective for the host.

Several lines of evidence point to the existence of other viral targets of CTLs during infection, although their identities have remained elusive. Reddehase and colleagues showed that target cells incubated with increasing levels of UV-inactivated virus became more susceptible to CTL-mediated cytolysis by splenocytes from MCMV-immune mice (37). Thus, target cells presenting virion proteins were specifically lysed by T cells generated during the natural infection. MHC-restricted T-cell clones have also been isolated and characterized as being specific for virion (36) and early-phase (13) antigens. Particularly noteworthy is a recent report in which pulmonary infiltrate cells in BALB/c bone marrow recipients were examined for relative CTL activities specific for the well-characterized IE1 nonapeptide versus the total CD8⁺ cytolytic activity. The total activity was measured by CD3-redirected lysis in order to bypass differences in TCR affinities for MHC-viral peptide complexes (21). A critical finding from these studies was that pulmonary CTLs recognized infected cells in all phases of the viral replicative cycle and that IE1-specific lysis accounted for only a fraction of the total CTL-mediated lysis in BALB/c mice. In addition, it was shown that target cells expressing early antigens were the most susceptible to lysis by pulmonary CTLs harvested during the peak of lytic activity. In view of the apparent shutdown of antigen presentation beginning during the early phase of viral gene expression, a requisite property of protective MCMV antigens may be that they either are expressed very early in the infection or are virion proteins introduced into the cytoplasm upon cell membrane-viral envelope fusion. However, the lack of knowledge regarding the specific identities of CTL targets of MCMV other than pp89 prevents this question from being rigorously addressed.

The goals of the work presented here were (i) to identify other MCMV antigens that generate protective cellular immunity in BALB/c mice and (ii) to determine whether this protection is limited to the H-2^d haplotype. We previously showed that intradermal (i.d.) DNA immunization with a pp89-expressing plasmid elicits a protective CTL response in BALB/c mice. In this study, we extended these findings by constructing plasmid DNA vaccines encoding candidate MCMV antigens and testing their protective efficacies against subsequent MCMV challenge. Because MCMV-specific CTLs against early antigens as well as virion proteins are likely generated during the infection of BALB/c mice, we focused this study on an early antigen, M84, and the MCMV homologs of HCMV virion proteins. The M84 gene was chosen based on our previous work showing that the protein encoded by this gene was an early, nonstructural protein with strong homology to the HCMV CTL target pp65 (34). Other genes included in this study were the homologs of seven tegument and putative tegument antigens and two putative capsid antigens. Two inbred mouse strains of different H-2 haplotypes were immunized by injecting these plasmids either singly or in cocktails of multiple plasmids. We found that in BALB/c mice only plasmids expressing either the nonstructural pp89 or M84 protein could limit viral replication in the spleen following subsequent challenge with a wide range of sublethal MCMV doses. None of the other plasmids elicited consistent protection. Moreover, these genes were not protective even when delivered by immunizing the mice with recombinant vaccinia viruses expressing the same MCMV open reading frames (ORFs). The results also showed that coimmunization of BALB/c mice with the protective pp89 and M84 plasmids provided synergistic protection against viral replication in the spleen. The response to M84 to was found to be specific for M84 epitopes, as mice immunized with the M84-expressing plasmid were not protected against an MCMV deletion mutant lacking M84. The reductions in spleen titers were accompanied by smaller reductions in salivary gland titers, indicating that spread of virus to secondary organs of replication was only partially limited by these vaccines. Lastly, we found that the protective responses generated by our vaccines were haplotype dependent, as pp89 was the only antigen that provided detectable protection in C3H/HeN mice.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Mice. Only female, specific-pathogen-free inbred mice were used in these experiments. Mice were at least 6 weeks old upon arrival and were not injected with DNA or MCMV until at least 1 week later. BALB/c (H-2^d) mice were obtained from Harlan-Sprague-Dawley, Inc., or Simonsen Laboratories, Inc., at 5 to 6 weeks of age. C3H/HeN (H-2^k) mice were obtained from Simonsen Laboratories at 5 to 6 weeks of age. Mice were housed in microisolator-covered cages in a vivarium (University of California, San Diego) and given food and water ad libitum.

Cells and virus. NIH 3T3 cells (ATCC CRL 1658), CV-1 cells (ATCC CCL 70), and HeLa S3 cells (ATCC CCL 2.2) were maintained in Dulbecco's modified Eagle's medium containing 10% (vol/vol) heat-inactivated bovine calf serum and 200 U of penicillin, 0.2 mg of streptomycin, 0.05 mg of gentamicin, 1.5 µg of amphotericin B, and 0.29 mg of L-glutamine per ml. MCMV strain K181 and the M84 deletion mutant M84 (34) were prepared as salivary gland homogenates following intraperitoneal (i.p.) injection of female BALB/c mice as previously described (15). The titers of these viruses were determined on NIH 3T3 cells as described below.

Plasmid constructs. Restriction endonucleases, T4 DNA ligase, T4 DNA polymerase, Klenow fragment of DNA polymerase, and competent Escherichia coli cells (DH5 MAX) were obtained from Bethesda Research Laboratories, Inc., unless otherwise noted. DNA restriction fragments contained in agarose gels were purified using the Geneclean Kit (Bio 101) or by centrifugation of gel slices in 0.45-µm-pore-size Ultrafree-MC filter columns (Millipore) according to the manufacturer's recommendations. Unless otherwise specified, phosphorylated linker DNAs were obtained from Stratagene, Inc., and linker ligations and digestions were performed as described previously (40). When appropriate, orientations of MCMV sequences in recombinant clones were confirmed by restriction analysis. Construction and characterization of pACYC184-based plasmids containing the HindIII fragments of the MCMV genome (i.e., H3B, H3D, H3H, etc.) were described previously (33). To allow easier cloning of a number of ORFs into the vaccinia virus vector plasmid pSC11 (6), this plasmid was modified to place either a NotI, NheI, or KpnI restriction site at a position allowing expression of the cloned gene in recombinant vaccinia viruses under the control of the vaccinia virus p7.5 promoter. The pSC11 vector was digested with SmaI, then phosphorylated NotI (Promega), NheI, or KpnI linkers were ligated to the cut plasmid, and the plasmid was then digested with an excess of NotI, NheI, or KpnI, respectively. The linker-ligated plasmid was then isolated and recircularized, yielding pSC11(Not), pSC11(Nhe), or pSC11(Kpn), respectively. Restriction analysis in all three cases demonstrated placement of the introduced site at a position corresponding to the SmaI site of the parent plasmid.

Expression of MCMV ORFs. Expression of full ORFs from the pcDNA3-based plasmids was assessed prior to immunization. Antigens were expressed and [³⁵S]methionine-labeled by TNT (Promega) coupled in vitro transcription-translation using the T7 promoter in the vector and following the manufacturer's recommendations. Labeled translation products were added to Laemmli sodium dodecyl sulfate (SDS) sample buffer, heated to 100°C (or 42°C for M83 and M84) for 2 min, separated by SDS-polyacrylamide gel electrophoresis on gels with various acrylamide concentrations ranging from 5 to 15%, and visualized by fluorography or autoradiography.

Vaccinia virus construction. Recombinant vaccinia viruses expressing the MCMV M32, M82, M84, M85, M86, and e1 ORFs or -galactosidase were constructed using the vaccinia virus vector plasmids pSC11-M32, pSC11-M82, pSC11-M84, pSC11-M85, pSC11-M86, pGS-e1, and pSC11, respectively, and the WR strain of vaccinia virus as the parent virus (ATCC VR-119) using methods previously described (6, 23, 31, 47). The resulting recombinant viruses were designated M32-vacc, M82-vacc, M84-vacc, M85-vacc, M86-vacc, e1-vacc, and pSC11-vacc, respectively. Construction of the vaccinia virus recombinants expressing M83 (M83-vacc), M99 (M99-vacc, formerly UL99-vacc), and pp89 (pp89-vacc) was described previously (10, 11, 47). Recombinant viruses were grown and plaque purified on the 143B cell line (ATCC CRL 8303) under selection with 50 µg of 5-bromo-2'-deoxyuridine per ml. Southern blot analysis using probes to either the thymidine kinase region of the vaccinia virus genome or the MCMV gene to be inserted showed the expected hybridization pattern consistent with the appropriate insert DNA and also confirmed the homogeneity of the plaque-purified stock (data not shown). Large-scale viral stocks were prepared by growth of the plaque-purified virus on monolayers of HeLa S3 cells. Virus stocks were prepared by freeze-thawing infected cell pellets, prior to storage in aliquots at 70°C. Viral stock titers were determined by plaque assay on monolayers of CV-1 cells after treatment of the viral stock for 30 min at 37°C with 0.25% (vol/vol) trypsin. Production of immunoreactive products of the expected sizes from M99-vacc, M83-vacc, M84-vacc, M32-vacc, pp89-vacc, and e1-vacc was confirmed by Western blot analysis of infected cell extracts (data not shown).

Immunizations. Plasmid DNAs were prepared from standard Luria-Bertani cultures using Qiagen Maxi and Mega columns according to the manufacturer's instructions. Plasmids were resuspended in Tris-EDTA (pH 8.0) made with endotoxin-free ddH₂O (Life Technologies, Inc.). The endotoxin content of the DNA was reduced to 0.5 to 5 ng per mg of DNA, as measured by the Limulus amoebocyte lysate assay (Associates of Cape Cod, Inc., Woods Hole, Mass.), by four extractions with Triton X-114 essentially as described previously (35). Following ethanol precipitation, DNA pellets were washed with 70% ethanol (in endotoxin-free ddH₂O) and resuspended in endotoxin-free 10 mM Tris-HCl (pH 8.0) to a concentration of approximately 2 mg per ml. DNA concentrations were measured by A₂₆₀, and the plasmid DNA was verified by agarose gel electrophoresis to be >95% supercoiled, undegraded, and free of chromosomal DNA or RNA contamination. For injection, DNA was diluted in 10 mM Tris-buffered, endotoxin-free saline (pH 8.0).

Virus challenge, tissue harvest, and plaque assay. Two to four weeks after the last immunization, mice were challenged by i.p. injection of virulent MCMV strain K181 in 0.5 ml of sterile PBS. At various times postchallenge, mice were sacrificed by CO₂ asphyxiation, spleens or salivary glands were aseptically removed, and 10% (wt/vol) homogenates were made in Dulbecco's modified Eagle's medium-10% bovine calf serum-10% dimethyl sulfoxide using sterile Pyrex homogenizers. Homogenates were stored at 70°C. All organ homogenates from a single experiment were diluted, and the titers were determined simultaneously by plaque assay on NIH 3T3 cells as previously described (17). Plaques were counted on day 4 or 5 postinfection (p.i.). Unless otherwise indicated, organ titers presented are the means of the log₁₀ PFU per organ for four mice and error bars representing either the standard deviation or, for better visibility of data points in most line graphs, the standard error of the mean.

Statistical analysis. MCMV organ titers were compared by one-factor analysis of variance (ANOVA) and Fisher's protected least significant difference post hoc test for pairwise comparisons between groups.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Expression of MCMV antigens from plasmid DNA vaccine vectors. The genome of MCMV contains approximately 170 ORFs, and of all of the virally encoded antigens, only one IE antigenIE1-pp89has been identified as eliciting a protective CTL response in infected BALB/c mice. Based upon work by Koszinowski and Reddehase documenting the presence of other specific CTL populations, it appears that other CTL epitopes may be contained within viral early-phase and structural proteins (13, 36). In order to begin the identification of antigens that generate the protective CTL response against MCMV, we first isolated the coding sequences for several candidate MCMV antigens for subsequent testing in a plasmid DNA immunization-based protection assay. The virion proteins that we tested were the MCMV homologs of known HCMV tegument and capsid proteins (see Table 1 for list). The homology between the published DNA sequences of HCMV strain AD169 (7) and MCMV strain Smith (35) facilitated our subcloning of these MCMV homologs from the K181 genome into the mammalian expression vector pcDNA3 for strong HCMV major IE promoter-enhancer-driven expression of the ORFs in vivo. The tegument and putative tegument antigens of HCMV include UL32-pp150, UL48-p212 and UL56-p130 (3), pUL69 (49), UL82-pp71, UL83-pp65, and UL99-pp28, and their respective FASTA amino acid homologies are shown in Table 1. The major and minor capsid antigens of HCMV encoded by UL86 and UL85, respectively, share approximately 50% FASTA amino acid homology with their MCMV counterparts, M86 and M85, and both of these MCMV genes were subcloned for immunization trials.

View larger version (39K): [in this window] [in a new window]   FIG. 1.   Coupled in vitro transcription-translation of pcDNA3-based vaccine plasmids. All antigens encoded by the plasmids used for immunization of mice were expressed in vitro from the T7 promoter of the plasmid vector using the TNT (Promega) expression system. [35S]methionine-labeled translation products were denatured in reducing SDS-polyacrylamide gel electrophoresis buffer, electrophoresed on gels of various acrylamide concentrations ranging from 5 to 15%, and visualized by fluorography or autoradiography. Each panel represents a different expression experiment and gel with the migration of the molecular mass standards (in kilodaltons) in each gel shown at the left.

The relative protective effect elicited by pcDNA3-pp89 immunization increases with increased MCMV challenge dose. After demonstrating that immunization with pp89-expressing plasmid pcDNA-89 can protect BALB/c mice against both lethal and sublethal MCMV challenges (17), we proceeded to optimize the experimental conditions to increase our probability of identifying other potentially protective antigens. Although pcDNA-89 provides protection against lethal challenge with virulent virus, we found that this protection could be overwhelmed by increases of the lethal challenge dose occurring from normal experimental variation in virus administration. We therefore sought to develop a protection assay which used a sublethal challenge in order to prevent the small variations in challenge dose from acutely affecting the measure of protection. To this end, we titrated the level of challenge dose to find a sublethal dose that would not overwhelm the protective response from plasmid DNA immunization. We measured the replication of MCMV in the spleens of vaccinated mice on day 6 postchallenge, as these replication levels correlate well with mortality of BALB/c mice from MCMV infection. In addition, control of the acute MCMV infection in the spleens of BALB/c mice has been found to be CD8⁺ T cell mediated (24), and we previously demonstrated that a specific CTL response can be elicited by plasmid DNA immunization with a pp89-expressing plasmid (17).

View larger version (15K): [in this window] [in a new window]   FIG. 2.   The relative protective effect elicited by pcDNA3-pp89 immunization, as measured by replication in the spleen, increases with increasing viral challenge dose. Naive BALB/c mice were immunized three times in 10 days by i.d. injections of 50 µg of either pcDNA3 or pcDNA3-pp89 as described in Materials and Methods. Two weeks after the last immunization, mice were i.p. challenged with one of four serial dilutions of salivary gland-derived MCMV corresponding to 21 × 103, 47 × 103, 95 × 103, and 127 × 103 PFU in 0.5 ml of PBS per mouse. Six days postchallenge, spleens were removed and the viral titers were determined. Spleen titer values presented are the means of the log10 PFU per spleen for four mice with the standard deviation (SD) indicated by error bars. Values above each challenge dose indicate the fold reductions of the nonlogarithmic mean titers (i.e., PFU per organ) of pcDNA3-pp89-immunized mice relative to the corresponding pcDNA3-immunized controls.

Plasmid DNA immunization with M84, either alone or in combination with other expression plasmids, protects BALB/c mice against challenge with increasing sublethal doses of MCMV. Since we were able to elicit consistent protection against a wide range of sublethal challenge doses following plasmid immunization with pp89, we used this challenge strategy as a measure of the protective abilities of the other plasmids encoding MCMV antigens. In order to efficiently screen a dozen additional antigens in mice of various MHC backgrounds, we chose to immunize groups of mice with mixtures of the different plasmids rather than immunize with each plasmid alone. This method is roughly analogous to that used by Barry and coworkers (1), who screened pools of expression clones from sublibraries made from the genome of Mycoplasma pulmonis, although their immunization pools contained large numbers of uncharacterized plasmids. If we found a particular plasmid pool to be protective in the initial screen, then its constituent plasmids would be used singly in a later immunization experiment to determine which antigen(s) provided the protective effect. Because the results of a pilot immunization experiment using a single sublethal challenge dose suggested that pcDNA3-M84 may provide protection (data not shown), the M84-expressing plasmid was singled out to be used alone as well as in combination with other expression plasmids. The pcDNA3-based vaccine plasmids were divided into three groups of plasmid cocktails. pcDNA3-M84 was included in group I (Gp. I) with M32 and M82two antigens which did not confer protection in pilot immunization or recombinant vaccinia virus immunization experiments (see below). Gp. I consisted of equal masses of pcDNA3-M32, -M82, and -M84, as well as pcDNA3 vector alone added to normalize the injected DNA mass with that of Gp. II. Gp. II contained pcDNA3-M83, -M85, -M86, and -M99, and Gp. III contained pcDNA3-M48, -M56, and -M69.

View larger version (21K): [in this window] [in a new window]   FIG. 3.   Plasmid DNA immunization of BALB/c mice with pcDNA3-based vectors used alone or in combination and the reductions in spleen titers following increasing sublethal challenge doses of virulent MCMV. BALB/c mice were i.d. immunized three times over 2 weeks with 40 µg of DNA consisting of either pcDNA3 or a cocktail of 10 µg of each antigen-expressing plasmid to be tested and the balance of 40 µg consisting of pcDNA3. Two weeks after the last immunization, mice were i.p. challenged with either 20 × 103, 40 × 103, 80 × 103, or 120 × 103 PFU, and spleens were harvested 6 days postchallenge for MCMV titer determination. Spleen titer values presented are the means of the log10 PFU per spleen for four mice with the standard error (SE) of the mean indicated by error bars. (A) Spleen titers of mice immunized with either pcDNA3, pcDNA3-pp89, pcDNA3-M84, or Gp. I. (B) Spleen titers from the same pcDNA3- or pcDNA3-pp89-immunized mice as in panel A as well as the Gp. II-immunized mice. (C) Spleen titers from an independent experiment in which mice were immunized with 30 µg of pcDNA3, 10 µg of pcDNA3-pp89 plus 20 µg of pcDNA3 to normalize masses), or 30 µg of Gp. III (10 µg of each plasmid shown).

View larger version (32K): [in this window] [in a new window]   FIG. 4.   Immunization of BALB/c mice with recombinant vaccinia viruses expressing the MCMV ORFs. BALB/c mice were i.p. immunized with the recombinant vaccinia viruses and challenged with virulent MCMV, and spleens were harvested 6 days postchallenge for MCMV titer determination. Spleen titer values presented are the means of the log10 PFU per spleen for each group with the standard deviation (SD) indicated by error bars. Values above each titer bar indicate the fold reductions of the nonlogarithmic mean titers (i.e., PFU per spleen) of that immunized mouse group relative to the corresponding pSC11-vacc-immunized controls, with statistically significant titer reductions denoted as superscripts (*, P < 0.05; , P < 0.01; , P < 0.001; one-factor ANOVA and Fisher's protected least significant difference test). (A) Three mice per group were i.p. immunized with 107 PFU of one of the recombinant vaccinia viruses expressing the MCMV antigens shown or the -galactosidase-expressing pSC11-vacc. A booster injection was given 31 days postimmunization, and 12 days following the boost, mice were i.p. challenged with 40 × 103 PFU of MCMV. (B) Four mice per group were given a single i.p. immunization with 107 PFU of the vaccinia virus recombinants shown and, 21 days postimmunization, were challenged with 30 × 103 PFU of MCMV. (C) Twelve mice per group were immunized as described for panel B and challenged with 80 × 103 PFU of MCMV.

Coimmunization of BALB/c mice with plasmids expressing pp89 and M84 results in synergistic reduction in spleen replication following challenge with a high sublethal dose. As described above, screening of 11 antigen-expressing plasmids in two inbred mouse strains revealed only two antigens which could protect BALB/c mice from challenge, the major IE1 protein pp89 and the pp65 homolog M84. Protection elicited by either plasmid against MCMV replication was incomplete as evidenced by the presence of detectable virus in the spleen. We therefore sought to determine whether a combination of these two MCMV genes could augment the antiviral response. BALB/c mice were i.d. immunized three times over 2 weeks with either 30 µg of pcDNA3; 15 µg of pp89, M83, or M84 (each combined with 15 µg of pcDNA3 to normalize DNA masses); or a cocktail of 15 µg each of pp89 and M83 or pp89 and M84. Three weeks after the last immunization, vaccinated mice were i.p. challenged with 60 × 10³ or 120 × 10³ PFU of virulent MCMV. On day 6 postchallenge, spleens were homogenized for MCMV titer determination.

View larger version (31K): [in this window] [in a new window]   FIG. 5.   Plasmid DNA immunization of BALB/c mice with protective and nonprotective plasmids, either alone or in combination, and the resulting viral spleen titers following sublethal MCMV challenge. BALB/c mice were i.d. immunized three times with either 30 µg of pcDNA3 or a cocktail of 15 µg of each antigen-expressing pcDNA3-based plasmid to be tested and the remainder of 30 µg consisting of pcDNA3. Two weeks after the last immunization, mice were i.p. challenged with either 60 × 103 (A) or 120 × 103 (B) PFU of MCMV. On day 6 postchallenge, spleens were harvested, and the resultant spleen titers are shown as the means of the log10 PFU per spleen for four mice with the standard deviation (SD) indicated by error bars. Values above each titer bar indicate the fold reductions of the nonlogarithmic mean titers (i.e., PFU per organ) of that group relative to the corresponding pcDNA3-immunized controls. The subscript 0 indicates that three of the four mice in this group had MCMV titers below the detection limit of 102 PFU/spleen and that the titer values of each of these three mice were arbitrarily set to the detection limit for mean titer calculation. Note also that the standard deviation of this group was too low to depict with an error bar.

The immune response against pcDNA3-M84 does not protect against challenge with an MCMV mutant lacking M84 expression. The above results showed that prior immunity against M84 is protective against subsequent MCMV challenge and that coimmunization with both M84 and pp89 results in a synergistic level of protection against a high challenge dose. We previously demonstrated that plasmid DNA immunization with a pp89-expressing plasmid induces CTL responses against the dominant pp89 nonapeptide epitope as strong as those generated during infection with tissue culture-derived MCMV (17). Because M84 is also a nonstructural protein (34), we would expect that the protection generated by the M84-expressing plasmid is also a cell-mediated response. From our experiments above, however, we could not exclude the possibility that protection mediated by pcDNA3-M84 (or M84-vacc) was due to a cytokine response that could result in a more rapid nonspecific priming of naive CTLs or activation of natural killer (NK) cells or, in the case of pp89-M84-coimmunized mice, a more rapid induction of pp89 memory CTLs into cytolytic effectors. We therefore tested whether the protection mediated by pcDNA3-M84 required that the infected cells express the M84 protein. We previously found that M84 was dispensable for viral replication in tissue culture and that a K181 deletion mutant, M84, was able to replicate with slightly attenuated growth levels in the target organs of BALB/c and other strains of mice (34). We therefore tested whether this M84 deletion mutant could escape pcDNA3-M84-based immunity in plasmid DNA-immunized mice.

View larger version (41K): [in this window] [in a new window]   FIG. 6.   Protection of plasmid DNA-immunized mice against either wild-type K181 or the M84 deletion mutant M84. Four BALB/c mice per group were i.d. immunized as described for Fig. 5, and 2 weeks following the last immunization, mice were i.p. challenged with 120 × 103 PFU of MCMV from salivary gland-derived stocks of either wild-type K181 or M84. Titer values and error bars are as described for Fig. 5. Values above each titer bar indicate the fold reductions of the nonlogarithmic mean titers (i.e., PFU per organ) of that group relative to the corresponding pcDNA3-immunized controls challenged with the same virus. SD, standard deviation.

Immunization with pcDNA3-pp89 alone or in combination with pcDNA3-M84, but not immunization with pcDNA3-M84 alone, results in consistent reductions in salivary gland titer. Having observed augmented protection from viral replication in the spleens of BALB/c mice coimmunized with the plasmids expressing pp89 and M84, we sought to determine if the limited replication in the spleen (and possibly other abdominal organs) could substantially reduce viral spread to the salivary glands. We found previously that immunization with a pp89 plasmid vaccine resulted in reduced salivary gland titers relative to those observed in mice immunized with vector backbone alone (17), but titers were still approximately 10⁶ PFU/organ. In an independent coimmunization experiment, BALB/c mice were i.d. immunized with the same DNAs as described above. The range of challenge doses chosen (2 × 10³, 10 × 10³, 30 × 10³, and 60 × 10³ PFU) was lower than that used when measuring spleen titer reductions because salivary gland titers are maximal following injection of lower input viral doses. Day 10 postchallenge salivary gland titers for the mice immunized with pcDNA3, -pp89, or -84 or pp89-M84 can be seen in Fig. 7. Salivary gland titers of pcDNA3-alone-immunized mice increased from 10^6.5 to 10^7.2 PFU/organ over the range of challenge doses and remained maximal at approximately 10⁷ PFU/organ. In mice immunized with pp89 and challenged with the three lowest viral doses, there were approximately twofold (P < 0.05), sixfold (P < 0.001), and eightfold (P < 0.01) reductions in salivary gland titers relative to those for pcDNA3-immunized controls, respectively, while at the highest dose the reduction was threefold (P < 0.05). Thus, the protective effect of pcDNA3-pp89 in the salivary gland increased with increasing challenge dose, but unlike the protection found in the spleen, the titer reductions approached only a single order of magnitude. In contrast to pp89, salivary gland titers in the M84 group were not reduced relative to those for pcDNA3-immunized mice over the range of challenge doses given. When pp89 and M84 were used for coimmunization, salivary gland titer reductions of 3-fold (P < 0.05), 9-fold (P < 0.001), and 11-fold (P < 0.01) were observed following the lower challenge doses and a reduction of 5-fold (P < 0.01) was observed with the highest challenge dose: reductions at least as great as or just measurably greater than those with pp89 immunization alone. However, coimmunization with pp89 and M84 still allowed the virus to replicate to titers of 10⁶ PFU in salivary glands. Thus, while protection in the salivary glands mediated by immunization with pp89 alone or in combination with M84 was statistically significant following the range of the challenge doses tested, the magnitude was less than that observed in the spleen.

View larger version (18K): [in this window] [in a new window]   FIG. 7.   Plasmid DNA immunization of BALB/c mice with protective plasmids, used alone or in combination, and the resulting salivary gland viral titers following sublethal MCMV challenge. BALB/c mice were i.d. immunized as described for Fig. 5 and 2 weeks after the last immunization were i.p. challenged with either 2 × 103, 10 × 103, 30 × 103, or 60 × 103 PFU of MCMV. Day 10 postchallenge salivary gland titers are shown as the means of the log10 PFU per salivary gland for four mice with the standard error (SE) of the mean indicated by error bars. Statistically significant titer reductions relative to the pcDNA3-immunized mice are indicated as follows: *, P < 0.05; , P < 0.01; and , P < 0.001 (one-factor ANOVA and Fisher's protected least significant difference test).

Plasmid DNA immunization with pcDNA3-pp89, but not pcDNA3-M84, provides detectable protection in C3H/HeN mice. An optimal vaccine should include antigen(s) that is presented by MHC molecules derived from wide immunogenetic backgrounds. In the MCMV model of immunity, the BALB/c inbred strain has been the primary animal used for determining which antigens were immunodominant for CTL recognition and protection (28). However, it has since been found that the BALB/c strain may rely upon CD8⁺ T lymphocytes for the control of MCMV to a greater extent than do other inbred mouse strains (29). Other inbred strains possess higher resistance to MCMV due to both H-2 haplotype and non-H-2-linked genes that are responsible for activating NK cells early in the infection before CTLs are fully active. Because the C3H/HeN inbred strain is highly resistant to MCMV due to both its H-2^k haplotype (18) and its rapid activation of NK cells by secretion of high levels of IFN-, we chose to test the efficacy of the plasmid DNA immunization cocktails in these mice.

View larger version (18K): [in this window] [in a new window]   FIG. 8.   Plasmid DNA immunization of C3H/HeN mice with pcDNA3-based vectors used either alone or in combination and the reductions in spleen titers following increasing sublethal challenge doses of virulent MCMV. C3H/HeN mice were immunized as described for Fig. 3 and then i.p. challenged 2 weeks following the last immunization with either approximately 40 × 103, 80 × 103, 160 × 103, or 320 × 103 PFU of MCMV. Day 3 postchallenge spleen titer values presented are the means of the log10 PFU per spleen for four mice with the standard error (SE) of the mean indicated by error bars. Statistically significant titer reductions relative to the pcDNA3-immunized mice are indicated as in Fig. 7. Spleen titers are those of mice immunized with either pcDNA3, pcDNA3-pp89, pcDNA3-M84, Gp. I, or Gp. II.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

The goal of this study was to identify the other potential targets of protective CTLs in BALB/c mice and to determine whether similar protective responses would be generated in a strain with a different H-2 haplotype. Our approach was to use plasmid DNA immunization, an effective and straightforward method for generating CTL responses, to elicit cell-mediated immunity specific for an MCMV early antigen and several MCMV homologs of HCMV virion-associated proteins. These antigens were chosen by virtue of their ability to be presented on the cell surface in association with MHC class I complexes before the early-phase MHC class I shutdown. CTLs directed against these antigens may theoretically lyse the infected cell prior to release and spread of progeny virus. Although CTLs are likely generated to an array of MCMV antigens during the course of infection, our assay was designed specifically to identify those antigens that not only generate CTL responses in vivo but also are protective against challenge with virulent virus.

The results of the experiments presented here showed that of the two MCMV homologs of HCMV UL83-pp65, the M84 protein was protective while the positional homolog M83 was not. Protection was observed in BALB/c, but not C3H/HeN, mice. We previously reported the homologies and possible evolutionary relationships of the M83 and M84 proteins with their HCMV homologs UL83 and UL84 (11). The positional homologs M83 and UL83 are both late proteins that are virion associated. However, UL84 is an early, nonstructural protein, and the M84 protein is also expressed early and is not detectable in the virion (34). In addition, both M83 and UL83 proteins are targets of humoral responses during infection of their respective hosts. Whereas pp65 has been documented to be targeted by the HCMV-specific CTLs across various HLA types in healthy seropositive individuals (2, 26, 32, 48), only the M84 homolog conferred protection to mice following DNA immunization. Because the M84 protein is nonstructural, the protective immunity elicited by the M84 plasmid is almost certainly cell mediated. This is further supported by the demonstration that the M84-mediated protection from challenge required M84 expression in the infected cell. Therefore, the M84 protein likely contains one or more epitopes that may be presented on class I molecules of the H-2^d BALB/c strain. The consistently weaker protection levels afforded by M84 relative to pp89 could be due to several factors including in vivo protein expression levels from the vaccine plasmids, relative efficiencies of peptide processing and loading, TCR-MHC affinities, or the relative availability of M84 protein during infection for proteolysis and presentation. Interestingly, recent experiments reevaluating the relative frequencies of CTL responses against HCMV IE1-pp72 and UL83-pp65 in healthy HCMV-seropositive blood donors showed that, in 12 donors tested, all 12 had CTLs specific for either pp72 or pp65, but only 4 had CTLs for both antigens (26).

Although at least some virion proteins elicit specific CTLs during MCMV infection, none of the plasmids or recombinant vaccinia viruses encoding structural or putative structural MCMV antigens was protective in vivo. It could be reasoned that either none of these proteins contained epitopes that could be presented on cells of either of the H-2 haplotypes tested or the immunization methods failed to elicit CTL responses strong enough to alter the course of infection. It is noteworthy that only the two plasmids coding for nonstructural proteins, pp89 and M84, were protective in BALB/c mice. In contrast to the late expression patterns generally observed for the structural proteins, peak expression of pp89 and M84 is at IE and early times, respectively. DNA immunization may have been able to elicit responses too weak to detect the relatively small amounts of virion-associated protein entering the cytoplasm upon viral penetration but strong enough to detect the presentation of the de novo-synthesized antigens at the peak of their expression. Although CTLs are thought to be exquisitely sensitive to peptide-loaded class I molecules (9), adequate levels of these complexes may be required for the migration and activation of memory cells from the immunization site to the infected target organs. Even in measuring the activity of CTLs from MCMV-immune mice using target cells presenting only virion-associated antigens, high doses of UV-inactivated virus particles (200 PFU equivalents per cell) and an excess of in vitro restimulated effectors (5:1 to 25:1 effector/target ratios) are required to achieve levels of specific lysis similar to those using target cells undergoing de novo expression of viral antigens (36, 37). The additional need for the migration of DNA-primed effector cells may be reflected in the augmented protection reported from priming mice with plasmid DNA and then boosting with the appropriate antigen-expressing recombinant vaccinia virus (43, 44). The vaccinia virus infection may draw the DNA-primed CTLs into tissues where they can encounter their specific antigen, proliferate, and provide increased immunosurveillance in the organs targeted by the challenge virus. Experiments examining the protection levels following immunization and challenge of mice in proximal or distal sites may help determine if these spatial relationships apply to this system.

Our studies also showed that in the H-2^k strain C3H/HeN, immunization with the pp89-expressing plasmid provided some protection against splenic viral replication. Although the reductions in spleen titer were within 10-fold compared with vector-alone-immunized controls, our results suggest that one or more pp89 epitopes are presented in association with the MHC H-2^k heavy chain. C3H mice are among the most resistant to MCMV infection, partially due to their H-2^k haplotype (18). Early activation of NK cells through IFN- production also helps to rapidly control viral replication in visceral organs. Results from experiments utilizing monoclonal antibody-mediated depletion of T-lymphocyte subsets in such resistant strains suggest that CD8⁺ T-cell populations may play less of a role in viral clearance during the acute infection than innate responses (29). Thus, even moderate CTL responses generated by the pp89 plasmid may have been overshadowed by the strong innate response. However, it has been shown for mutant and wild-type 129 strains that previous vaccination with an attenuated MCMV deletion mutant significantly limits the splenic replication upon subsequent virulent MCMV challenge regardless of the IFN- receptor-mediated mechanisms of viral control (30). The efficacy of the vaccine was compromised, however, in ₂ microglobulin null 129 mice. Taken together, these data suggest that effective vaccination with an attenuated MCMV should provide T-cell-mediated protection in mice with intact IFN- responses. In addition, the efficient control of splenic viral replication in C3H forced us to measure viral replication at day 3 postchallenge, a time that was perhaps too early for CTLs primed by DNA immunization to become fully active effectors. Previous work by Del Val et. al demonstrated that CTLs generated in C57BL/6 (H-2^b) mice following infection with MCMV or a pp89-expressing vaccinia virus specifically lyse syngeneic target cells expressing IE-phase genes (13). Thus, it appears as though at least one pp89-derived CTL epitope may be presented in association with MHC class I complexes from H-2^d, H-2^b, and H-2^k strains. Levels of specific lysis in vitro of target cells expressing IE or IE plus early antigens by C57BL/6 haplotype-derived CTLs, however, were reduced approximately two- to threefold relative to those obtained from the BALB/c mice (13). Thus, the protective ability of pp89 in the C3H/HeN strain may be less pronounced than that in the BALB/c strain due to a lower relative affinity of pp89-derived epitopes for H-2^k class I complexes and/or non-CD8⁺ T-lymphocyte-mediated effectors that dominate the antiviral response in this strain.

When BALB/c mice were coimmunized with pp89 and M84 DNAs, a synergistic reduction in spleen titers was observed relative to those after immunization with either plasmid alone. However, this synergistic effect was observed only when the mice were challenged with the highest doses of virus. This suggests that, upon more stringent challenge conditions, the observed interaction between the two CTL epitopes may have been synergistic. Since these two CTL epitopes are located on proteins from different phases of viral gene expression, it is possible that inclusion of MCMV genes encoding both IE and early proteins in a DNA vaccine accounts for such a substantial increase in protection.

In a recent study, the breadth of the CTL response against MCMV was analyzed using a bone marrow transplantation model in BALB/c mice (21). The authors found that, when pulmonary CTL activity peaked at 4 weeks p.i., CTLs displayed the highest CD3-redirected cytolytic activity against target cells presenting early (12 h p.i.) antigens, while target cells presenting only virion proteins were not significantly lysed. Pulmonary CTLs isolated at 3 weeks p.i., 1 week before activities were optimal, however, displayed the highest levels of activity against IE and late proteins. Thus, in order for complete CTL-mediated protection to occur in the lung, the CTL repertoire may need to encompass multiple epitopes in order to adapt to changing conditions in the infected tissue such as the relative availability of viral antigens for MHC class I presentation. Although in our experiments the antigens in Gp. II did not provide protection against the highest MCMV challenge dose, the inclusion of one or more of them in the pp89-M84 DNA vaccine may help diversify the resulting CTL responses and provide more complete protection against viral replication.

In contrast to the reduction of spleen titers to nearly undetectable levels in pp89-and-M84-coimmunized BALB/c mice, viral replication in the salivary glands was reduced only up to 10-fold below that of control mice. Previously, we reported salivary gland titer reductions of approximately 50-fold following pp89 DNA immunization (17). However, pp89 was expressed from pcDNA-I/Amp, and the use of the pcDNA3 vector in this report may account for differences in immunity. Through depletion studies of immune T lymphocytes, it was previously found that CD4⁺ cells are the effectors of viral clearance from this organ (24, 25). Although the protective immunity provided by CD8⁺ lymphocytes may suppress viral replication in the abdominal organs, virus that seeds the salivary glands replicates to high levels until CD4⁺ lymphocyte-mediated clearance can occur. Therefore, it appears likely that, although our plasmid DNA immunization-mediated protection may dramatically reduce MCMV replication in the spleen to nearly undetectable levels, the ability of the virus to disseminate to and amplify in the salivary gland was not significantly impeded in the immunized animals. This is not surprising, since plasmid DNA immunization favors the generation of CD8-mediated T-cell responses. Taken together, these data suggest that, in order for vaccination to significantly reduce salivary gland titers, adequate CD4⁺ antiviral activity in the salivary gland must be established prior to or coinciding with viral seeding of that organ.

DNA-mediated immunization has become a valuable research tool and holds great promise for future use in preventing infectious diseases. The field is moving rapidly, and with continued innovations in augmenting immune responses against plasmid-encoded antigens by coadministration of cytokines or cytokine-expressing plasmids (8, 22, 27), utilizing immunostimulatory DNA sequences (41), improving chemical or physical delivery systems, and delivering antigens as immune-response-enhancing ubiquitin fusion proteins (38, 39), it seems likely that it will be possible to vigorously prime both arms of the acquired cellular immune response to MCMV and provide complete protection against acute infection and the establishment of latency. The identification of MCMV antigens that elicit these types of protective responses will be crucial to directing this prophylactic immunity. The insights provided from animal models can then be used for the development of a safe and effective vaccine against HCMV.