Systemic Priming-Boosting Immunization with a Triv

http://www.100md.com 病菌学杂志 2005年第1期

     Section of Molecular Biology and Center for Molecular Genetics, University of California, San Diego, La Jolla, California

    ABSTRACT

    We previously demonstrated that vaccination of BALB/c mice with a pool of 13 plasmid DNAs (pDNAs) expressing murine cytomegalovirus (MCMV) genes followed by formalin-inactivated MCMV (FI-MCMV) resulted in complete protection against viral replication in the spleen and salivary glands following sublethal intraperitoneal (i.p.) challenge. Here, we found that following intranasal (i.n.) challenge, titers of virus in the lungs of the immunized mice were reduced approximately 1,000-fold relative to those for mock-immunized controls. We next sought to extend these results and to determine whether similar protection levels could be achieved by priming with a pool of three pDNAs containing three key plasmids (IE1, M84, and gB). We found that the three-pDNA priming elicited IE1- and M84-p65-specific CD8+ T lymphocytes and, following FI-MCMV boost, high levels of virion-specific immunoglobulin G (IgG) and virus-neutralizing antibodies. When mice were i.n. challenged 4 months after the last boost, titers of virus in the lungs of immunized mice were reduced 1,000- to 2,000-fold from those for controls during the peak of viral replication. Additionally, titers of virus were either at or below the detection limits for the salivary glands, liver, and spleen of the majority of the immunized mice. Following sublethal i.p. challenge, virus was undetectable in all of the above target organs of the immunized mice. Virion-specific IgA in the lungs was consistently detected by day 6 post-i.n. challenge for the immunized mice and by day 14 for controls. These results demonstrate the immunity and high levels of protection of the priming-boosting vaccination against both systemic and mucosal challenge.

    INTRODUCTION

    Human cytomegalovirus (HCMV), an opportunistic herpesvirus, continues to be a highly prevalent causative agent of birth defects (for a review, see reference 47). Between 0.5 and 2.5% of all newborns are infected with HCMV, and of the 5 to 10% that are symptomatic at birth, most develop sequelae, such as microcephaly, sensorineural hearing loss, optic atrophy and chorioretinitis, and motor disabilities. While the serological status of the mother positively correlates with protection of the newborn from disease, recent evidence strongly suggests that prior maternal immunity is not completely protective against neonatal disease from recurrent infection or infection with a different HCMV strain (3, 4, 17). The ubiquity of HCMV and devastating lifelong sequelae associated with infection make it important to understand the protective mechanisms of host immunity and to develop a safe and effective vaccine.

    Findings from studies of HCMV immunity and disease in transplant recipients have highlighted the importance of cell-mediated immunity in protecting against HCMV disease (2, 56), and work with animal models, such as the murine cytomegalovirus (MCMV) model, has allowed elucidation of the protective roles of specific leukocyte subsets (53). The immediate effect that NK cells have on viral control has been well demonstrated by the impact on MCMV replication and disease that results from depletion of NK cells or NK function (10, 36, 58, 60). The necessity for the adaptive component of cell-mediated immunity, the CD8+ and CD4+ T lymphocytes, to limit the acute, persistent, latent, and reactivating infections has been documented through depletion and adoptive transfer studies (reviewed in reference 52). Immune reconstitution of gamma-irradiated mice with MCMV-specific CD8+ T lymphocytes has been shown to reduce the viral load in the spleen, lungs, liver, and adrenals, while long-term depletion of CD4+ T lymphocytes in infected mice results in persistent infection in the salivary glands (31). The identity of the specificities of the antiviral CD8+ T cells has long been a subject of interest, since the findings have strong implications for choosing viral antigens to use in antiviral cytoimmunotherapies and vaccines. While the identity of the immunodominant peptide of the immediate-early 1 (IE1) gene product and the protective ability of IE1-specific CD8+ T cells in BALB/c mice have long been known (53), there had also been strong evidence pointing to the existence of CD8+ T cells that were generated against unidentified viral early (E) and late (L) gene products (54). With the advent of more-reliable methods for the detection and quantification of specific CD8+ T cells, the identities of additional CD8+-T-cell specificities have been revealed. These include the HCMV UL83-pp65 homologs M83-pp105 and M84-p65, the antiapoptotic gene product M45, the MCMV immunoevasin gene product m04 (gp34), and two additional genes unique to MCMV, m164 and m18 (18, 21, 22, 24-28). One common feature of these MCMV genes is their expression at either E or E/L times of infection.

    The identification of these E and E/L gene products as CD8+-T-cell targets was initially somewhat paradoxical due to the known expression of the immunoevasin E genes that encode glycoproteins that block the cell surface presentation or recognition of virus-derived antigenic peptides on MHC class I complexes (52). The m152 gene product, gp37/40, retains peptide-loaded class I complexes in the endoplasmic reticulum-cis-Golgi intermediate compartment, while m06-gp48 reroutes these complexes to the lysosome for degradation (18, 34, 39, 55, 67). The m04 gene product, gp34, binds to MHC class I complexes without hindering their transport to the cell surface but appears to prevent recognition of the complex by CD8+ T cells (33). Mutational analysis of the MCMV genome has demonstrated the relative roles of the known immunoevasins in MHC class I downregulation as well as some of the cooperative and competitive interactions among the immunoevasins (32, 59). In addition, the m152 deletion mutant was demonstrated to be attenuated in T-cell-competent mice (34), and cells infected with wild-type, but not m152 deletion, MCMV are not recognized by M45-specific CD8+ T lymphocytes (18, 25). This is a particularly striking result, since M45 has been shown to be a dominant antigen during the acute and memory responses in C57BL/6 mice. This finding has important ramifications for vaccine design, since it was found that cytoimmunotherapy using a specific cytotoxic-T-lymphocyte (CTL) line for this dominant antigen was not effective in limiting viral replication (25).

    An efficacious vaccine against HCMV disease has been an elusive goal for many years, even though many of the antigenic targets of the neutralizing antibody and CD8+-T-cell responses have been identified (for reviews, see references 5 and 19). Clinical trials using the tissue culture-passaged Towne strain, which conceivably could induce protective responses against the full complement of viral antigens, was indeed found to induce both neutralizing antibodies and CTLs and provided limited protection against severe disease in transplant recipients and in volunteers given a low-dose HCMV challenge but failed to prevent infection in women exposed to young children shedding HCMV. The envelope glycoprotein B (gB) has been the basis for virus-neutralizing antibody-inducing vaccines, both as a subunit vaccine (with MF59 as an adjuvant) and as a recombinant replication-deficient canarypox vector, ALVAC-CMV(gB). Both vaccines were found in clinical trials to be well tolerated, and although the subunit gB vaccine was found to elicit high levels of HCMV-neutralizing antibodies in seronegative volunteers, ALVAC-CMV(gB) was able to elicit neutralizing antibodies only after subsequent boosting with Towne. Encouraging preliminary results have been obtained after vaccination of seronegative subjects with the pp65-expressing ALVAC-CMV(pp65) vector, since strong pp65-specific CTL levels were elicited, as well as CTL precursor frequencies similar to those found in HCMV-seropositive subjects. Other vaccination approaches to date that have undergone preclinical testing with mice include plasmid DNA (pDNA) encoding gB or pp65, a peptide of the conserved CD8+-T-cell epitope of pp65, dense bodies, and more recently a recombinant vaccinia virus Ankara that expresses gB (1, 12, 13, 35, 48, 66).

    Because the species specificity of HCMV limits the evaluation of the protective efficacies of these vaccines for mice, we have used the MCMV model to develop and test cytomegalovirus vaccines for their immunogenicity and protective efficacy. We found that intradermal (i.d.) immunization of BALB/c mice with a pDNA expressing the IE1 gene of MCMV elicited CTLs against the defined immunodominant peptide and was able to protect mice against subsequent lethal MCMV challenge and reduce the viral load in the spleen after sublethal intraperitoneal (i.p.) challenge (20). We subsequently demonstrated that i.d. pDNA immunization with an MCMV homolog of HCMV UL83-pp65, M84, encoding the nonstructural E protein M84-p65 (9, 44), was similarly protective against splenic viral load and that coimmunization of mice with IE1 and M84 resulted in a synergistic level of protection (45). i.d. pDNA immunization with the m04 gene, which had been found to encode a Dd-restricted CD8+-T-cell epitope in strain Smith (26), conferred protection against a range of challenge doses, while a pool of the individually nonprotective putative tegument and capsid genes tested (M32, M48, M56, M69, M82, M83, M85, M86, and M99) together with the nonstructural M112/113-e1 was able to reduce the splenic viral load following low to intermediate challenge doses (46). The highest level of protection was observed with mice coimmunized with a pool of IE1, M84, and the matrix and capsid genes above, with splenic titer reductions of 104 relative to those for mock-immunized controls following short-term challenge (46). Because even the most efficacious pDNA vaccine was found to reduce the viral load in the salivary glands only by approximately 10-fold, we developed a priming-boosting strategy entailing i.p. boosting the pDNA-immunized mice with formalin-inactivated MCMV (FI-MCMV). We found that i.p. immunization with FI-MCMV elicited high levels of neutralizing antibodies as well as CD8+ T cells specific for the virion-associated antigens and, most importantly, that the mice that were i.d. primed with a cocktail of 13 pDNAs and i.p. boosted with FI-MCMV (with alum as an adjuvant) had undetectable levels of virus in the spleen and salivary glands following i.p. challenge (46).

    In this report, we sought to extend these findings by testing the efficacy of the pDNA/FI-MCMV parenteral vaccine in a mucosal challenge model, since this infection route is important in the horizontal spread of the virus through infected saliva and other bodily fluids (47). In view of the role of gB in the generation of neutralizing antibodies to the cytomegaloviruses (6), we also evaluated whether inclusion of a gB-expressing pDNA into a simplified pDNA pool consisting only of the synergistically protective IE1 and M84 pDNAs could prime a protective neutralizing antibody response that could be boosted by subsequent immunization with FI-MCMV. Most importantly, we examined whether priming with the IE1, M84, and gB pDNA pool and boosting with FI-MCMV provided protective immunity against both mucosal and systemic challenge.

    MATERIALS AND METHODS

    Mice, cells, viruses, and viral purification. Three- to four-week-old pathogen-free female BALB/c mice were purchased from Harlan Sprague Dawley, Inc., and housed in microisolator covered cages in the Pacific Hall vivarium, University of California, San Diego. Mice were allowed to acclimate for 1 week prior to immunization.

    NIH 3T3 (ATCC CRL 1658), COS-7 (ATCC CRL 1651), J774A.1 (ATCC CRL TIB-67) macrophages (H-2d), and BALB/c mouse embryonic cells were propagated as previously described (7, 46).

    MCMV strain K181 was used for all experiments. Salivary gland-derived MCMV (SG-MCMV) and tissue culture-derived MCMV (TC-MCMV) were prepared as previously described (45). TC-MCMV was partially purified for preparation of FI-MCMV vaccine as described previously (46) and stored at –80°C, and its final titer was approximately 5 x 108 PFU equivalents per ml. Virion for enzyme-linked immunosorbent assay (ELISA) was similarly prepared by pelleting TC-MCMV through a 25% sorbitol cushion in Tris-buffered saline, pH 7.4 (TBS), washing the pellets in Dulbecco's phosphate-buffered saline (DPBS) (Invitrogen/Life Technologies, Inc.), repelleting through a fresh cushion, and resuspending the final pellets in DPBS. The protein concentration of the resultant virion preparation was measured by a Bio-Rad (Bradford) protein assay using bovine serum albumin (Pierce) as a standard, and the titer of the virion was measured by plaque assay on NIH 3T3 cells. The virion remained infectious through the purification (<20% loss in infectivity after purification) and had a specific infectivity of approximately 107 PFU per μg of protein. Glycerol was added to the ELISA virion to a 50% (vol/vol) final concentration, and the preparation was stored at –20°C.

    Plasmid construction and expression. The expression vectors pc3neo and its derivatives pc3-pp89 (IE1) and pc3-M84, as well as the amino-terminal ubiquitin fusion constructs pc3-U-pp89 (U-IE1) and pc3-U-M84, were described previously (65). The other plasmids comprising the All-U pDNA pool, encoding M32, M48, M56, M69, M82, M83, M99, M85, M86, M112-113 (e1), and m04-gp34, have also been described previously (45, 46). The M55 open reading frame (ORF) encoding gB of MCMV K181 was subcloned from the pACYC184-derived subgenomic constructs (41) into the expression vector pCMV-int-BL (a gift from Eyal Raz, University of California, San Diego). Expression from this vector is driven by the HCMV major IE promoter-enhancer and contains a 5' HCMV-derived intron, a 3' simian virus 40-derived intron, and a simian virus 40-derived polyadenylation signal. Of note, we had not been successful subcloning the MCMV gB ORF into any mammalian expression vector without the inclusion of at least a 5' intron. The full-length gB ORF was subcloned as a BamHI fragment, replacing the vector's tissue plasminogen activator signal sequence from the 5' PstI site (blunted and ligated with a BamHI linker) to the 3' BamHI site. The DNA sequence of the entire gB ORF in the final vector, designated pCMV-int-BL-gB, was sequenced (University of California, San Diego Cancer Center Core Facility) and found to be identical to our previously published sequence (11). Plasmids were propagated in E. coli DH5, purified by using a QIAGEN EndoFree Mega or Giga column, and resuspended in endotoxin-free 10 mM Tris-HCl (pH 8).

    Expression of gB was tested by Western blot following transient transfection of COS-7 cells. Cells were transfected with Effectene (QIAGEN) following the manufacturer's recommendations, and 48 h posttransfection, cell lysates were made in reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) buffer, resolved on SDS-PAGE gels, and electrophoretically transferred to nitrocellulose. As controls, similar lysates of either uninfected NIH 3T3 cells or NIH 3T3 cells infected with MCMV at a multiplicity of infection of 3.5 for 48 h were analyzed on the same gel. To detect gB, blocked blots were incubated with the monoclonal antibody 2E8.12A (a gift from Lambert Loh, University of Saskatchewan, Saskatoon, Canada), and bound antibody was detected using a goat-anti-mouse immunoglobulin G (IgG) horseradish peroxidase conjugate (Calbiochem) and enhanced chemiluminescence (Supersignal West Pico; Pierce).

    To detect gB-specific antibodies resulting from the in vivo expression of the gB plasmid following i.d. immunization of mice, the preparative lane of an SDS-10% PAGE gel was loaded with a lysate of MCMV-infected NIH 3T3 cells made 48 h p.i., and resolved lysate proteins were transferred to nitrocellulose as described above. Using a Mini Protean II multiscreen apparatus (Bio-Rad Laboratories), the blot was simultaneously incubated with sera from individual BALB/c mice that were collected either 5 or 10 weeks post-i.d. immunization with either vector or gB pDNA. Bound antibodies were detected by enhanced chemiluminescence as described above.

    Immunization and virus challenge. In the first (All-U pDNA pool) experiment, plasmids were diluted in endotoxin-free 10 mM Tris-HCl (pH 8)-buffered saline such that mice were immunized with either 26 μg of either empty vector DNA (pc3-Ua) or 26 μg of the All-U pDNA cocktail (46) consisting of 2 μg each of m04-gp34, M32, M48, M56, M69, M82, M83, M85, M86, M99, M112-113 (e1), U-IE1, and U-M84 DNAs, with U indicating amino-terminal ubiquitinated IE1 and M84, respectively (46, 65). Mice were i.d. immunized into the shaved back near the base of the tail three times within 2 weeks. At 4 and 7 weeks following the last i.d. pDNA immunization, the pc3-Ua-immunized control mice were i.p. boosted with phosphate-buffered saline (PBS) plus alum, while the All-U-immunized mice were boosted with 107 PFU equivalents of FI-MCMV plus alum (FI+alum). Boosts (0.2 ml) contained Imject Alum (Pierce) freshly mixed with either PBS or FI-MCMV (diluted in PBS) at a ratio of 1:1 (vol:vol) as recommended by the manufacturer. At 13 or 14 weeks following the last boost, mice were challenged either i.p. or i.n., respectively. For i.n. challenge, mice were lightly anesthetized by inhalation of isoflurane (Isoflo; Abbott Laboratories) prior to instilling 50 μl of DPBS containing 5 x 105 PFU of TC-MCMV into both nares. For i.p. challenge, mice were i.p. injected with 0.5 ml of DPBS containing 4 x 105 PFU (0.5x 50% lethal dose [LD50]) of SG-MCMV.

    In the second (three-pDNA pool) experiment, mice were immunized three times within 2 weeks with 30 μl containing either 15 μg of pc3neo or a cocktail containing 5 μg each of pc3-pp89, pc3-M84, and pCMV-int-BL-gB. At 4 and 9 weeks after the last i.d. pDNA immunization, the pc3neo-immunized mice were i.p. boosted with 0.2 ml containing PBS plus alum, and the three-pDNA immunized mice were i.p. boosted with FI-MCMV plus alum as above. At 19 or 20 weeks after the last i.p. boost, half of the mice from each immunization group were i.n. or i.p. challenged, respectively, as described above.

    Virus titration. On days 6, 10, 14, 18, 24, and 32 post-i.n. challenge or days 6, 10, 14, and 18 post-i.p. challenge, four to six mice per immunization group were sacrificed and the spleen, lungs, liver, and salivary glands were aseptically removed and washed with DPBS. Homogenates were made of each organ (10% [wt/vol] for spleen, salivary glands and lungs; 20% for liver) in Dulbecco's modified Eagle medium plus 10% bovine calf serum (Life Technologies) plus 10% dimethyl sulfoxide and stored at –80°C in three separate aliquots. The titer of infectious MCMV in each organ was determined by plaque assay, using NIH 3T3 cells in 24-well dishes as previously described (20). If the level of virus in a homogenate was at or below five times the limit of detection, another aliquot of the homogenate was subjected to a more sensitive plaque assay (46). NIH 3T3 monolayers in 10-cm dishes were infected with either 100 μl of spleen, liver, or lung homogenate or 20 μl of salivary gland homogenate. By using exogenously added virus in this assay, these volumes were found to be the maximal for detection of virus without the target cells being inhibited by the toxicity of the organ homogenate. The limits of sensitivity for this assay, therefore, are 10 PFU per spleen or lungs, 30 PFU for liver, and 50 PFU for the salivary glands. The log10 values of the individual viral titers in each group were determined, and the mean of the log10 values was calculated. If virus was undetectable in a given organ in all of the assays, the individual titer of virus for that organ was arbitrarily set to the log10 of one-half the respective detection limit for display purposes and mean calculation.

    Intracellular cytokine staining (ICS) assay. Ten days after the last pDNA immunization, four mice from each immunization group were sacrificed and the splenocytes were prepared for the measurement of pp89- and M84-p65-specific CD8+ T lymphocytes by ICS assay as described previously (64, 65). Briefly, following the removal of erythrocytes, splenocytes were incubated with brefeldin A (GolgiPlug; PharMingen) and either a 1 μM concentration of the dominant nonapeptide epitope of IE1 (168YPHFMPTNL176) or J774A.1 macrophages infected for 10 h with an M84-expressing recombinant vaccinia virus, M84-vacc (45), at a 1:6 stimulator-to-responder ratio. After incubation, splenocytes were stained with anti-CD8 and anti-gamma interferon (IFN-) fluorescent monoclonal antibody conjugates as described previously. The lymphocytes were gated, and the dually stained splenocytes were enumerated by Epics Elite flow cytometer (Beckman Coulter) at the Flow Cytometry Core, VA Medical Center, La Jolla, Calif.

    Quantification of virion-specific IgG, IgA, and virus-neutralizing antibodies. Blood samples were collected retroorbitally from the immunized mice on weeks 14, 17, and 20 in the first (All-U pDNA pool) experiment and on weeks 4, 10, 24, and 30 in the second (three-pDNA pool) experiment, and sera were prepared and stored at –20°C until analysis. Virion-specific serum IgG levels were measured by indirect ELISA as follows. The partially purified, intact TC-MCMV as prepared above was diluted in PBS plus 0.05% (wt/vol) sodium azide and adsorbed to Nunc Maxisorp F96 plates (0.1 μg of virion protein in 50 μl of PBS plus 0.05% [wt/vol] sodium azide per well) overnight at 4°C. Virion-coated plates were subsequently washed with TBS using a Wellwash II Mk4 plate washer (Thermo Labsystems) and incubated with 100 μl of blocking buffer (TBS plus 5% [vol/vol] bovine calf serum) for 1 to 2 h, with all incubations at room temperature. Serial dilutions of sera in blocking buffer (1:16 initial dilution and 1:4 subsequent dilutions) were incubated in the blocked wells for 1 h with shaking at 220 rpm. Plates were washed six times with TBS, 50 μl of goat antimouse IgG (whole molecule)-alkaline phosphatase conjugate (Sigma A-3562) diluted 1:10,000 in blocking buffer was added, and the plates were incubated for 1 h with shaking. Following TBS washes as described above, 75 μl of a freshly prepared p-nitrophenyl phosphate (p-NPP) solution (Sigma FAST, N-2770 and N-1891) was added and the plates were incubated for 1 h. Absorbance at 405 nm (A405) was measured in a Bio-Rad model 405 microplate reader, and the ELISA titer of each serum was defined as the highest reciprocal dilution that resulted in an A405 of 0.195 (twice the background level.)

    Virion-specific IgA was measured in the lung and salivary gland homogenates post-i.n. challenge by ELISA as described above with the following modifications. Virion-coated plates were blocked with TBS plus 2% (wt/vol) bovine serum albumin (United States Biochemical no. 10857), and homogenates that were serially diluted in this blocking buffer (1:4 initial dilution and then either 1:2 subsequent dilutions for lungs or 1:3 subsequent dilutions from salivary glands) were incubated in the blocked plates for 2 h with shaking. After washes in TBS as described above, 50 μl of goat-antimouse IgA (-chain specific)-alkaline phosphatase conjugate (Sigma A-4937) diluted 1:1,000 in blocking buffer was added to each well and plates were incubated for 1 h as described above.

    SG-MCMV neutralizing-antibody titers were measured by plaque reduction assay as previously described (46). Neutralization titers were defined as the highest reciprocal serum dilution that resulted in a 50% reduction of the number of input PFU (ca. 50 PFU).

    RESULTS

    Protection of mice primed with All-U pDNA and boosted with FI-MCMV against viral replication after sublethal i.p. or i.n. challenge. Our previous work evaluating the efficacy of i.d. pDNA immunization against MCMV showed that optimal protection against viral replication in the spleen was observed following immunization with a pDNA cocktail containing equal masses of pDNAs expressing the 13 MCMV genes m04, M32, M48, M56, M69, M82, M83, M85, M86, M99, e1, U-IE1, and U-M84, with U representing amino-terminal ubiquitin fusion with the MCMV gene. Vaccination with this pDNA vaccine, designated All-U, resulted in four log10 reductions in the levels of infectious virus in the spleen relative to levels for vector-immunized controls following short-term i.p. challenge (46; also data not shown). When All-U pDNA-primed mice were subsequently i.p. boosted with 107 PFU equivalents of FI-MCMV adsorbed to alum adjuvant, no infectious virus was detectable in the spleen (<10 PFU per spleen) or salivary glands (<100 PFU per salivary glands) following i.p. challenge (46). We next sought to examine the efficacy of i.d. All-U pDNA priming and i.p. FI-MCMV boosting in conferring long-term protection against both i.p. and mucosal i.n. MCMV challenge.

    To test for protection, two groups of BALB/c mice were i.d. primed with either empty vector pDNA (pc3-Ua) or the All-U pDNA cocktail (see Fig. 1 for immunization groups and timeline). Starting at 3 weeks after the last pDNA priming, the pc3-U vector-immunized mice received two i.p. boosts with PBS plus alum, while the All-U pDNA-primed mice received 107 PFU equivalents of FI-MCMV plus alum. Blood was collected retroorbitally three times in the subsequent 12 weeks leading up to the challenge, and sera were prepared for antibody analyses. As antibody controls, two additional groups of mice were immunized with 2.5 x104 PFU of TC-MCMV given either i.p. or i.n. on week 1 of the experiment (Fig. 1) and bled with the mice above. Thirteen weeks after the last i.p. boost with either PBS plus alum or FI-MCMV plus alum, one-half of the immunized mice were i.p. challenged with 4 x 105 PFU (0.5x LD50) of SG-MCMV, while at 14 weeks the other half were i.n. challenged with 5 x 105 PFU of TC-MCMV. Four to five mice from each immunization and challenge group were sacrificed on days 6, 10, 14, and 18 postchallenge to determine levels of infectious virus in key target organs: the lungs, salivary glands, spleen, and liver.

    In order to measure the levels of virus-specific IgG in the serum after immunization with the MCMV pDNAs or FI-MCMV, we developed and optimized an ELISA for MCMV antibodies (see Materials and Methods). Plates were coated with intact, partially purified TC-MCMV in order to detect antibodies to only virus structural antigens, since these levels would likely correlate more closely with neutralization than would nonstructural antigens, such as IE1 and m04-gp34, that elicit antibody responses from MCMV infection (46; also data not shown). When the virion-specific IgG levels elicited by All-U pDNA priming and FI-MCMV boosting (All-U plus FI+alum) were measured, we found that at 6 weeks following the last boost (week 14), six of six mice tested had seroconverted with a mean ELISA titer 2.5 logs above the background levels in the vehicle-only (pc3-U plus PBS plus alum) control mice (Fig. 2A). These levels remained high over the next 6 weeks leading up to challenge, with the sera of all six mice reaching a plateau of approximately 3 logs above background. As seen in Fig. 2A, this level is approximately 1 log higher than the plateau levels observed following i.n. or i.p. infection with 2.5 x 104 PFU of live TC-MCMV.

    We previously demonstrated that i.p. boosting with FI-MCMV resulted in virus-neutralizing antibodies in the serum. In the experiment shown in Fig. 2B, we found that 6 weeks after the last FI-MCMV boost, six of six mice had detectable virus-neutralizing antibodies, with a mean titer of 2.7 and a range of 1 to 4 (Fig. 2B). The mean neutralization titer increased to a plateau by 9 weeks after the final FI-MCMV boost (mean, 4.3; range, 3 to 7). These levels are consistent with those observed in the previous experiment (46) and remained stable until 12 weeks after the final FI-MCMV boost. As expected, no virus-neutralizing activity was seen with the vector-immunized controls (pc3-U plus PBS plus alum). Mice that had been i.n. infected with live TC-MCMV had higher levels of neutralizing antibodies after infection than the All-U- and FI-MCMV-immunized mice (Fig. 2B), with a mean level of 3.5 (range, 2 to 4) starting on week 14 (13 weeks p.i.) and reaching a plateau level of 6 (range, 5 to 7) on week 17. Mice i.p. infected with TC-MCMV had mean neutralization titers of approximately 4 to 5 (range, 3 to 6) on the weeks tested.

    To determine the protective efficacy of the All-U pDNA prime and FI-MCMV boost, half of the immunized mice were i.p. challenged with 4 x 105 PFU (0.5x LD50) of SG-MCMV 13 weeks after the last boost with FI-MCMV. We subsequently sacrificed four to five mice per group on days 6, 10, 14, and 18 postchallenge. Spleen, liver, lungs, and salivary glands were harvested, and infectious virus was quantified by a sensitive plaque assay on NIH 3T3 cells. Following i.p. challenge, MCMV replicates first in organs such as the spleen, liver, and lungs and subsequently in the salivary glands. In the spleens of the pc3-Ua-primed and PBS plus alum-boosted controls, there was a mean MCMV titer of 105.3 PFU/spleen on day 6 postchallenge and no detectable virus on day 10 (<10 PFU/spleen), followed by spurious low levels of virus thereafter (Fig. 3A), indicating that most of the vector-immunized control mice were able to control viral replication by day 10 postchallenge. Although a minor, secondary peak in viral replication has previously been documented in the spleen following i.p. infection (42), virus was only spuriously detected in this immunization experiment. In contrast, no virus was detectable in the spleens of all of the All-U pDNA-primed and FI-MCMV-boosted mice on all four days examined. Virus persisted in the livers of controls at a level of 103 PFU per liver, with a decrease to 102 PFU per liver seen on day 10 postchallenge (Fig. 3B). In the lungs of the control mice, approximately 103 PFU per lungs was detected on day 6 postchallenge, with virus detectable in 11 of 15 mice through day 18 postchallenge (Fig. 3C). In contrast, none of the All-U pDNA-primed and FI-MCMV-boosted mice had detectable virus in the liver (<40 PFU per liver) (Fig. 3B) or in the lungs (<10 PFU per lungs) (Fig. 3C). In the salivary glands after i.p. challenge, 13 of 19 of the All-U pDNA- and FI-MCMV-immunized mice had undetectable levels of virus (<50 PFU per salivary glands) and 4 had viral titers near the level of detection (Fig. 3D). In contrast, the control mice had mean viral titers of approximately 106 PFU per salivary glands from day 10 to day 18 postchallenge (Fig. 3D). Of note, the variance of viral titers was high in many of the mouse groups in this experiment. We have observed that in immunization experiments in which mice are housed for periods of 6 months or more prior to challenge, the variability of challenge virus levels in the target organs increases substantially, even in nonimmune mice (46, 65). Therefore, each titer of virus is individually plotted so that the overall trends of protection within an experiment can be assessed with each target organ over all of the time points examined.

    Fourteen weeks after the last FI-MCMV boost, the other half of the immunized mice were i.n. challenged with TC-MCMV. Pilot i.n. infections with TC-MCMV and pelleted and resuspended SG-MCMV demonstrated that TC-MCMV administration resulted in higher levels of infectious virus in the lungs (data not shown). We found that following i.n. challenge with TC-MCMV, the lungs of the control-immunized mice had mean titers of virus of 105.2 PFU on day 6 postchallenge, and this level decreased to 103.3 PFU over the next 12 days postchallenge (Fig. 4A). The lungs of mice immunized with All-U pDNA and FI-MCMV had a mean viral titer of 101.8 PFU on day 6, a level nearly 3,000-fold below that for controls. Thereafter, challenge titers of virus in the lungs of the All-U pDNA- and FI-MCMV-immunized mice decreased on days 14 and 18 to levels such that 7 of the 10 mice tested had undetectable virus in the lungs, with the remaining 3 mice having titers at or below 102 PFU (Fig. 4A). As seen in Fig. 4B, the salivary glands of the control mice had peak levels of challenge virus of 105.5 PFU on days 10 to 14, while on these days, 7 of 10 of the All-U pDNA- and FI-MCMV-immunized mice had undetectable levels of virus, with the other 3 having levels of 102.5 PFU or below. On day 18 post-i.n. challenge, the salivary glands of the All-U pDNA- and FI-MCMV-immunized mice had a slightly increased mean titer of virus of 102.7, with four of five mice showing detectable levels of virus. Following i.n. challenge, virus was undetectable in the spleens of 14 of 20 of the All-U pDNA- and FI-MCMV-immunized mice and in the livers of all 20 of these mice on the 4 days examined (Fig. 4C and 4D). In the controls, virus was detectable in the spleen and liver on day 6 and then in the livers of 4 of 14 mice thereafter. It should be noted that virus in the spleens of the All-U pDNA- and FI-MCMV-immunized mice from days 10 to 18 was detectable in only one of the three aliquots of spleen homogenate, since the initial low-sensitivity plaque assay and the final highly sensitive assay yielded no detectable virus. Because these results differed, the data showing the possibility of viral breakthrough on these days are presented (Fig. 4C, black arrows). In addition, while one of five control mice on day 10 had a low level of virus (log10 titer of 1.70) in the final plaque assay (Fig. 4C, arrowhead), the titers for the rest of the samples were consistent for all of the assays. Of the four All-U pDNA- and FI-MCMV-immunized mice with detectable virus in the salivary glands on day 18, three of them also had infectious MCMV in the spleen (Fig. 4C), while two had virus in the lungs (Fig. 4A). Together, these results demonstrate that following i.n. challenge, titers of virus in the lungs of the All-U pDNA- and FI-MCMV-immunized mice were reduced approximately 103-fold below those in controls, while titers in the salivary glands were reduced approximately 104-fold at the times corresponding to the peak of replication in the mock-immunized controls.

    Immunization of mice with a gB, IE1, and M84 pDNA cocktail with or without subsequent boosts with FI-MCMV. Since the above-described experiments showed that the protection was not complete following i.n. challenge and long-term i.p. challenge, we proceeded to determine whether the vaccine could be improved by using for the priming step a DNA cocktail that consisted of the synergistically protective IE1 and M84 genes plus a pDNA that expressed the envelope glycoprotein gB. We chose gB because it is a target of neutralizing antibodies and can confer some protection against a subsequent MCMV challenge when mice are immunized with this gene delivered by vaccinia virus (51) or replication-deficient adenovirus vector (57).

    We subcloned the M55 gene encoding gB from genomic constructs of strain K181 (41) into a mammalian expression vector for pDNA-mediated immunization. Analysis of the putative gB amino acid sequence in the gB plasmid showed it to be identical to the K181 sequence of the previously published M54-M55 region (11) and to have a 1-amino-acid divergence from the gB gene of the K181 SG-MCMV used for challenge (R-to-W mutation at position 811), a mutation that likely occurred during PCR cloning of the M55 gene from the challenge virus (data not shown). To test expression of the gB plasmid, COS-7 cells were transiently transfected with either the gB plasmid or the empty vector. Cell lysates were prepared 48 h posttransfection and subjected to SDS-PAGE and Western blot analysis using the gB-specific monoclonal antibody 2E8.12A, which specifically binds to a single epitope located in the uncleaved polypeptides of gB as well as the carboxy-terminal cleavage product, gp52 (37). As shown in Fig. 5A, a single 128-kDa immunoreactive band was detected in the cells transfected with the gB-expressing plasmid. For migration comparison, lysates from MCMV-infected NIH 3T3 cells were resolved on the same gel. The corresponding 128-kDa gB precursor band was specifically detected in the infected NIH 3T3 cells, as well as the 52-kDa cleavage product of fully glycosylated gB. These results indicate that the gB plasmid expresses the appropriately sized gB-reactive protein species that comigrates with the 128-kDa gB precursor. It has been seen previously that this is the predominant form of MCMV gB that accumulates in cells infected with a gB-expressing recombinant vaccinia virus (50). The 150-kDa fully processed form of gB could be detected in the MCMV-infected NIH 3T3 lane upon longer exposure (data not shown).

    The immunogenicity of the encoded gB gene product was subsequently tested by i.d. injection of mice with the gB expression plasmid. BALB/c mice were i.d. immunized with 30 μg of either vector DNA or the gB pDNA on weeks 0, 1, 2, 6, and 8. Sera collected on weeks 5 and 10 were tested by Western blotting for seroreactivity with MCMV-infected NIH 3T3 cell lysates. As shown in Fig. 5B, sera from the mice immunized with the vector alone did not seroreact with the infected NIH 3T3 proteins. In contrast, two of the three mice tested at week 5 had weakly seroconverted against the 128- and 52-kDa forms of gB, and at week 10, all three mice tested had seroconverted. In addition, two of the three gB pDNA-immunized mice at week 10 produced antisera that could also detect the fully glycosylated 150-kDa product of gB in the infected cells. Taken together, these results show that the gB pDNA expresses the glycosylated 128-kDa gB precursor in infected NIH 3T3 cells and after immunization elicits antibodies that specifically bind to gB species from MCMV-infected cells.

    To determine the protective efficacy of a vaccine consisting of IE1, M84, and gB pDNA plus or minus a FI-MCMV boost, BALB/c mice were immunized in three groups as diagrammed in Fig. 6. The vector group received immunizations with the vaccine vehicles only: three i.d. immunizations with empty vector DNA (pc3neo) followed by two boosts with PBS plus alum i.p. The pDNA-only-immunized group was injected i.d. with the IE1, M84, and gB pDNAs and then boosted with PBS plus alum. Finally, the pDNA-plus-FI-MCMV group received both the IE1, M84, and gB pDNA cocktail i.d. and two i.p. boosts with 107 PFU equivalents of FI-MCMV in alum. Eight days after the last i.d. pDNA immunization, mice were sacrificed for quantification of the pDNA-induced CD8+-T-lymphocyte responses by ICS assay. Sera were taken prior to each boost with FI-MCMV or PBS, at 13 weeks following the second FI-MCMV boost, and last, 1 week prior to challenge. Finally, mice were either i.n. or i.p. challenged 19 or 20 weeks, respectively, after the last FI-MCMV boost, and target organs were harvested and homogenized for MCMV titer determination as described above.

    CD8+ T responses to IE1 and M84-p65. The three-pDNA cocktail in this experiment was formulated in order to elicit both protective CD8+-T-cell responses against IE1 and M84-p65 and a neutralizing-antibody response against gB. To document the levels of IE1- and M84-p65-specific CD8+ T lymphocytes elicited by the three-pDNA cocktail, groups of mice were sacrificed 8 days after the last i.d. immunization with either vector (pc3-Ua) or the IE1, M84, or gB pDNA cocktail. Splenocytes were harvested and subjected to ICS assay to quantify the number of CD8+ T lymphocytes that produce IFN- following 7 h of stimulation in vitro. Splenocytes were stimulated with either the IE1-derived nonapeptide 168YPHFMPTNL176 or J774A.1 cells infected for 10 h with an M84-expressing vaccinia virus. We have recently shown that stimulation of splenocytes with the M84-expressing J774A.1 macrophages as stimulators results in a higher sensitivity level for detection of M84-specific CD8+ T cells with the ICS assay due to multiple epitopes in M84-p65 contributing to the CD8+-T-cell response following pDNA immunization or MCMV infection (64). When we measured the IE1-specific CD8+-T-cell responses in the mice immunized with the three-pDNA cocktail, we found that a mean of 1.6% (range, 0.66 to 2.64%) of the splenic CD8+ T lymphocytes were directed against the IE1 epitope, while the background in the vector control group was 0.21% (range, 0.18 to 0.29%) (Fig. 7). When CD8+-T-cell responses against M84-p65 were measured with the mice primed with the IE1, M84, and gB pDNAs, we found 6.1% (range, 3.89 to 7.32%) of CD8+ T cells were IFN- positive after stimulation with the M84-p65-expressing antigen-presenting cells, while the background level was 0.32% (range, 0.21 to 0.41%) in the splenocytes from vector-only-immunized mice (Fig. 7). These levels are comparable to those seen previously following coimmunization with IE1 and M84, when IE1-specific CD8+-T-cell responses of 2 to 4% (65) and M84-p65-specific responses of 4.5 to 5.5% (64) have been demonstrated. In addition, we have recently demonstrated the antigen specificity of J774 cell-based stimulation, since the percentage of IFN--positive splenocytes from mice immunized with M84 pDNA is at background levels (0.12 to 0.20%) after stimulation with J774 cells infected with the parental vaccinia virus (64). Together, these results confirm that immunization with the gB plasmid included in the IE1 and M84 pDNA cocktail results in the expected levels of CD8+ T responses to the latter two gene products.

    Virus-specific serum antibody responses following IE1, M84, and gB pDNA and FI-MCMV immunization. In this experiment we were particularly interested in virus-specific IgG elicited by the gB pDNA prime and its effect on the response to the FI-MCMV boost. In a pilot experiment, BALB/c mice were immunized with the gB plasmid, and gB-specific antibodies were detected using immunoblots of MCMV-infected NIH 3T3 lysates. However, following subsequent i.p. challenge with either of two sublethal doses of SG-MCMV, no significant protection was observed in the spleen or salivary glands (data not shown). In order to quantify the MCMV-specific IgG elicited by the pDNA prime alone or following FI-MCMV boost, sera from the mice immunized with the IE1, M84, and gB pDNAs were collected throughout the experiment and subjected to virion-specific ELISA as described above. Sera collected from the mice 2 weeks after the last injection with the IE1, M84, and gB pDNA cocktail (week 4) had ELISA titers either at (9 of 12 mice) or just above (3 of 12 mice) the detection limit (Fig. 8A). Six weeks later, mice immunized with the 3 pDNA cocktail and boosted with PBS plus alum had slightly increased virion-specific IgG titers of 102.2 (range, 102.1 to 103.0). The IgG that the assay detected is likely gB specific only, since both IE1-pp89 and M84-p65 are nonstructural antigens and we have found that pp89-specific sera does not yield detectable virion-specific ELISA using this assay (data not shown). Compared with the mice immunized with the three pDNAs alone, mice boosted once with FI-MCMV plus alum (Fig. 8A) had mean serum titers of IgG that were higher by 1.5 logs (mean, 103.7; range, 103.6 to 104.4). Mean titers of IgG in the FI-MCMV boosted group remained stable at 103.8 for the next 20 weeks prior to challenge, while the titers in the pDNA-only group remained stable at mean levels of 101.8 to 101.9. This experiment demonstrated that both the IE1, M84, and gB pDNA prime and FI-MCMV boosts elicited virion-specific IgG but that the maintenance levels of IgG in the pDNA-primed and FI-MCMV-boosted mice were 100-fold higher than the IgG levels in the pDNA primed-only mice.

    The virus-neutralizing activity of the sera of these mice was also tested by plaque reduction assay as described above. No neutralization activity was observed in the pc3-U primed and PBS plus alum-boosted mice (Fig. 8B). Similarly, neutralization activity was undetectable in the pDNA-primed and PBS plus alum-boosted mice on weeks 4 through 24, and two of eight mice on week 30 had a titer of 21 (the assay detection limit). Boosting of the IE1, M84, and gB pDNA-primed mice with FI-MCMV elicited virus-neutralizing antibodies that were detectable 4 weeks after the first boost (week 10), with a mean titer of approximately 24 (range, 23 to 25) (Fig. 8B). The neutralization titers in this group continued to increase from weeks 10 to 30 to a mean of 26 (range, 25 to 27) at week 30, even though the sera maintained a constant level of virion-specific IgG (Fig. 8B). Taken together, these data show that while the IE1, M84, and gB pDNA cocktail can elicit virus-specific IgG that maintains a stable level for at least 28 weeks after the last immunization, this response is not significantly neutralizing in vitro compared with the sera from mice subsequently i.p. boosted with FI-MCMV.

    Protection from i.n. challenge following IE1, M84, and gB pDNA priming and FI-MCMV boosting. To determine the efficacy of the three-pDNA cocktail prime and FI-MCMV boost in protecting against challenge virus replication, half of the immunized mice were i.n. challenged with 5 x 105 PFU of TC-MCMV on week 30 of the experiment, 19 weeks following the last boost with PBS plus alum or FI-MCMV plus alum. The levels of infectious virus were quantified by a sensitive plaque assay on homogenates from the lungs and salivary glands on days 6, 10, 14, 18, 24, and 32 postchallenge and from the liver and spleen on days 6 and 10 postchallenge. In the lungs of the control (pc3neo; PBS plus alum) mice following i.n. challenge, a mean of approximately 106 PFU of challenge virus per lungs was detected on day 6 postchallenge, and this level decreased over the next 26 days after challenge to 102.2 PFU per lungs on day 32 (Fig. 9A). The levels of challenge virus in the lungs of the IE1, M84, and gB pDNA-only-immunized mice were decreased from day 6 to day 18 by 4- to 20-fold compared with controls, with the 20-fold decrease on day 18 resulting from the lungs of one mouse with undetectable virus, and then an 8-fold decrease on day 32. In contrast, the mean titer of virus in the lungs of the FI-MCMV-boosted mice on day 6 was 102.8, an approximately 2,000-fold decrease relative to the control group. Titers in the pDNA- and FI-MCMV-immunized group decreased until day 14, when virus was detectable in one of five mice. The titers then slightly increased to a secondary peak of 101.5 PFU per lungs on day 24 and were below detection limits in four of four mice on day 32. In the salivary glands following i.n. challenge, viral titers peaked (106 PFU per salivary glands) in the control mice on day 14 postchallenge and then decreased to undetectable levels on day 32 (Fig. 9C). Titers of virus in the salivary glands of the pDNA-only-immunized mice were comparable with those for the control group, with the mean titer decreases of five- and sixfold on days 14 and 18 being within the variability of the pDNA immunization and challenge assay. In the pDNA-primed and FI-MCMV-boosted mice, challenge virus was undetectable in the salivary glands of 23 of 29 mice examined, with a single mouse per group having a detectable titer of virus on each of days 10, 14, 18, and 32 and 2 mice having detectable virus on day 24. Similarly, one of five of the pDNA-primed and FI-MCMV-boosted mice had virus in the spleen on day 6 with a titer at the detection limit of 10 PFU per spleen, while the pDNA-only-immunized mice had variable protection of up to 10-fold in the spleen (Fig. 9D). Finally, while the titers of virus in the livers of the i.n.-challenged mice in the pDNA-only-immunized group were slightly reduced on days 6 and 10 postchallenge relative to control levels, virus was undetectable (<30 PFU per liver) in the FI-MCMV-boosted mice on both days of examination (Fig. 9B).

    Protection from i.p. challenge after IE1, M84, and gB pDNA priming and FI-MCMV boosting. The other half of the IE1, M84, and gB pDNA-primed and FI-MCMV-boosted mice were sublethally i.p. challenged with 4 x 105 PFU of SG-MCMV on week 31 of the experiment, 20 weeks after the last PBS or FI-MCMV boost. The resulting levels of protection against viral replication in the lungs, liver, salivary glands, and spleen are shown in Fig. 10. In the lungs of the control mice, a mean of approximately 103 PFU per lungs of challenge virus was observed on days 6 and 10 postchallenge, and this level decreased to approximately 102 PFU per lungs on day 18 (Fig. 10A). Titers of virus in the pDNA-only-immunized mice were reduced by 4-fold (day 6) to 40-fold (day 14), with 7 of 10 mice on days 14 and 18 postchallenge having undetectable virus. Similarly, in the liver, while challenge virus persisted in the control group at mean levels of approximately 103 to 103.7 PFU per liver through day 18 postchallenge, for mice in the pDNA-only group, titers of virus were reduced by only 3- and 5.9-fold on days 6 and 10, respectively, and then 14- and more than 47-fold on days 14 and 18, respectively (Fig. 10B). In the spleen, while a 200-fold reduction in titers of virus was observed for the pDNA-only-immunized mice on day 6 postchallenge, no virus was detectable in the spleen of five of five mice on day 10 (Fig. 10D). Finally, in the salivary glands of the mice of the pDNA-only-immunized mice, while titers of virus were initially similar on day 6 to those in the control group, thereafter there was a trend of three- to sevenfold-reduced mean titers, with the titers on day 18 being highly variable in the pDNA-only group (Fig. 10C). The most striking result was that virus was undetectable in the lungs, liver, spleen, and salivary glands of all 20 of the 20 mice immunized with both the IE1, M84, and gB pDNA and FI-MCMV (Fig. 10). Taken together, these results demonstrate that for mice i.d. primed with a pDNA cocktail consisting of IE1, M84, and gB DNAs and i.p. boosted with FI-MCMV, the replication of challenge virus administered i.n. was reduced by 2.5 to 3 logs in the lungs during the peak times of viral replication relative that for the control mice. Additionally, in the liver, spleen, and salivary glands, viral replication was reduced to below detectable levels in the majority of the mice tested. Following i.p. challenge, all of the mice that were primed with the three MCMV pDNAs and boosted with FI-MCMV were completely protected from detectable virus in all four organs tested.

    IgA responses in the lungs and salivary glands following i.n. challenge. Because of the importance of IgA in providing a first line of defense against the invasion of pathogens through mucosal surfaces, we sought to examine the IgA responses in two IgA-producing mucosal organs, the lungs and salivary glands, in the immunized mice following i.n. MCMV challenge. Specific IgA has been shown to be detectable in bronchoalveolar lavage fluid in mouse models of vaccination and infection, and B cells in the salivary glands secrete IgA that is detectable in whole or parotid saliva (29, 30, 43, 57, 62). To this end, the postchallenge lung and salivary gland homogenates that were subjected to titer determination for infectious challenge virus were subjected to ELISA, using the partially purified virion as the target antigen and an IgA -chain-specific secondary antibody conjugate. Two- or threefold dilutions of individual homogenates were adsorbed to virion bound to ELISA plates, and the titer of IgA was defined as the reciprocal dilution that produced an A405 greater than or equal to twice the background absorbance (see Materials and Methods). Control experiments demonstrated that virion-specific serum IgG was not detected in this assay (data not shown). In addition, the lung homogenates from naive mice yielded no detectable virion-specific IgA in this assay (Fig. 11A). Beginning on day 6 post-i.n. challenge, three of four of the control mice had levels of virus-specific IgA at the detection limit (Fig. 11A). By day 14 postchallenge, five of five mice tested had detectable virus-specific IgA with a mean titer of 101.7. This level was stably maintained at levels from 101.6 to 102.1 through day 32 postchallenge. In the IE1, M84, and gB pDNA-only-immunized mice, while IgA titers were similar to those for the controls on day 6 postchallenge, titers rapidly increased to a mean of 101.6 on day 10 and 102.5 on day 14 postchallenge, with peak titers of 103 observed on day 14 (Fig. 11A). The IgA titers in the pDNA-only group remained at 102.0 to 102.4 through day 24 before decreasing to 101.7 on day 32. IgA titers were detectable in five of five mice that were primed with the IE1, M84, and gB pDNAs and boosted with FI-MCMV as early as day 6 postchallenge (mean, 101.6), a time in which IgA titers in the majority of mice in the control and pDNA-only groups were at or below the detection limit. By day 10 postchallenge, titers of IgA in the pDNA-primed and FI-MCMV-boosted mice slightly increased to a mean of 102.1 and remained stable thereafter at levels between 102.0 and 102.4. These results show that following i.n. MCMV challenge, virion-specific IgA was detectable by ELISA in all of the vector control mice by day 14, in the pDNA-only-immunized mice by day 10, and in the pDNA and FI-MCMV-immunized mice as early as day 6. While the kinetics of IgA induction were more rapid in the mice immunized with pDNA with or without FI-MCMV, peak levels were only slightly increased relative to those for the control mice.

    The virion-specific IgA levels in the salivary gland homogenates were also quantified by ELISA. In the salivary glands of the control group mice, virion-specific IgA titers above background levels were observed in four of four mice by day 18 post-i.n. challenge; the mean titer was approximately 10, with the titer of IgA for one mouse at the detection limit (Fig. 11B). The mean titer of IgA in the control group increased sharply on day 24 postchallenge to 102.5, and this level remained stable through day 32. The salivary glands of the pDNA-only-immunized mice had titers of IgA similar to those for the controls through the time course, with a trend towards higher titers in the pDNA-only-immunized mice on days 14 and 18 postchallenge. While a mean IgA titer of 101.1 was observed in the salivary glands of the pDNA-primed and FI-MCMV-boosted mice as early as day 6 postchallenge, the mean titer of IgA in this group decreased to a level at or below the detection limit from day 14 to day 24, with only one mouse per group having significant IgA levels of 10 (Fig. 11B). On day 32 postchallenge, variable titers of virion-specific IgA were observed, with a mean of 100.9 (one mouse was at and one mouse was below the detection limit). Taken together, these results show that the virion-specific IgA titers increase in the salivary glands of vector alone-immunized and pDNA alone-immunized mice to a peak by day 24 post-i.n. MCMV challenge. In contrast, in the salivary glands of the mice that were primed with the pDNA and boosted with FI-MCMV, there are increased levels of IgA soon after challenge, but that IgA is detectable only spuriously thereafter at levels that are consistently lower than in the vector and pDNA-only groups.

    DISCUSSION

    We previously demonstrated that parenteral priming of mice with a pDNA cocktail and boosting with FI-MCMV plus alum could provide complete protection against detectable MCMV replication in the spleen and salivary glands when mice were sublethally i.p. challenged 8 weeks after the last FI-MCMV boost (46). In this report, we confirmed this result with two independent experiments as well as extending our findings to a mucosal challenge model. While the mice immunized with the All-U pDNA pool and FI-MCMV were completely protected in the spleen, liver, and lungs following i.p. challenge (Fig. 3), there was low-level replication in the salivary glands of four of the i.p. challenged mice, and there were titers above 102.5 PFU for two other mice. In contrast, no infectious virus was detected in 20 of 20 of the mice that were primed with the IE1, M84, and gB pDNAs and boosted with FI-MCMV (Fig. 10). It is possible that the minor differences in immunization and challenge schedules may have contributed to the presence of challenge virus in some of the mice. In both experiments, it appears that although the virion-specific IgG and neutralizing-antibody titers were close to the peak levels the week before each of the challenges, the mean neutralization titer for the All-U pDNA primed/FI-MCMV boosted mice was twofold lower than that for the IE1, M84, and gB pDNA-primed/FI-MCMV-boosted mice at the time of challenge. Although it is possible that the priming with gB pDNA provided slightly higher levels of antibody prior to challenge, the twofold difference was observed between different immunization experiments and is within the mouse-to-mouse variance. Experiments are in progress to better define the priming effect of the gB pDNA. Differences in the actual dose of challenge virus delivered may also affect whether challenge virus is detectable in the target organs. Because virus was detectable in the salivary glands but not in the other organs of the All-U/FI-MCMV-immunized mice, it is possible that either low levels of viral replication were occurring at another site before seeding of the salivary gland or that some of the initial input virus was able to disseminate to the salivary glands. Figure 3D shows that titers of virus in the salivary glands of the All-U pDNA and FI-MCMV-immunized mice peaked on day 14 compared with day 10 for the controls. In any case, the strong potential of the virus to replicate in the salivary glands makes this organ a key indicator for detecting a very low level of challenge virus that is able to elude the vaccine-induced immunity and reach an important organ for horizontal dissemination. Finally, we cannot exclude the presence of infectious virus in these target organs at levels below the detection limit.

    The fate of the challenge virus administered i.n. into the pDNA-primed/FI-MCMV-boosted mice is perhaps more readily observable in these experiments. While titers of virus in the lungs of these mice are greatly reduced compared with those for the mock-immunized controls, it is apparent that the challenge virus is still able to replicate at the initial portal of entry. Of note, pilot i.n. infection experiments showed that virus was not detectable in the lungs of mice 1 day following the i.n. administration of the same dose of virus showed here (data not shown), suggesting that the virus detected in the lungs of the pDNA/FI-MCMV-immunized mice resulted from newly replicated virus. While the initial reduction of titers of virus to levels at or below the detection limits in the lungs of the pDNA/FI-MCMV-immunized mice was observed on day 14 for both immunization experiments, there appeared to be an increase in titers of virus thereafter. However, as shown in the second experiment in which the time course was extended, the secondary peak of viral replication occurred on day 24 post-i.n. challenge, and virus was then undetectable by day 32. These results indicate that the virus did not likely continue to replicate in the lungs of the immunized mice, as was the case with the controls (Fig. 9A). While peak titers of approximately 103 PFU were observed in the lungs of the IE1, M84, and gB pDNA/FI-MCMV-immunized mice on day 6 post-i.n. challenge, virus in the liver, spleen, and salivary glands of these mice was undetectable in the majority of mice, with the levels of virus in the salivary glands only at the detection limit in many of the mice with breakthrough virus. This trend suggests that the viral replication occurring in the lungs of these mice did not result in high levels of spread to or poorly controlled replication in the secondary organs following i.n. challenge. This observation may reflect the presence of a suboptimal level of mucosal immunity at the portal of entry but high levels of systemic immunity induced by the parenterally administered vaccine. While it is possible that there was significant immunity in the lungs of the pDNA/FI-MCMV-immunized mice that was overwhelmed by such a high-challenge dose, we found after i.n. challenge of these mice with a 50-fold-lower challenge dose that viral replication still resulted in detectable virus in the lungs on day 6 to day 14 postchallenge (data not shown). Experiments in progress are aimed at improving the levels of protection in the lungs through the mucosal administration of the pDNAs and/or the FI-MCMV.

    A caveat that is always relevant in experiments measuring protection and immunity is the correlation of the types and levels of immune responses with the protection observed. In the second pDNA priming/FI-MCMV boosting experiment presented here, we immunized a group of mice only with the three-pDNA cocktail to assess the levels of protection if the pDNA alone is given. Following i.p. challenge, the clearance of infectious virus from the lungs of these mice was more rapid than in controls, with no detectable virus in 7 of 10 pDNA-only-immunized mice on days 14 and 18 postchallenge. Holtappels et al. showed that the clearance of MCMV in the lungs following subcutaneous infection correlated with the infiltration of CD8+ T cells. Moreover, these CD8+ T cells not only were cytolytically active ex vivo without the need for secondary restimulation in vitro but also lysed syngeneic target cells that were either pulsed with the IE1-pp89 peptide or MCMV infected under conditions in which either IE, E, or L proteins were expressed (23). Thus, it may be possible that the IE1- or M84-p65-specific CD8+ T cells elicited by the i.d. pDNA immunization provided some or all of the protection observed in the three-pDNA-only-immunized mice, as well as some in the three-pDNA-primed/FI-MCMV-boosted mice, following i.p. challenge. In addition, although MCMV gB has not been demonstrated to contain CD8+-T-cell epitopes, it is possible that the gB pDNA elicited a gB-specific CD8+-T-cell response. Similarly, titers of virus in the lungs of the three-pDNA-immunized mice were reduced approximately 10-fold relative to controls on each of the days examined after i.n. challenge. We showed previously that pDNA immunization with IE1 and M84 is highly protective in the spleen but only nominally protective in the salivary glands, and we found in this experiment that the pDNA alone has a modest protective effect in the liver, particularly at day 14 postchallenge and later (Fig. 9 and 10). Adoptive transfer of CTL lines specific for M83, M84, or IE1 has been shown to reduce MCMV titers in the liver (as well as lungs and spleen) of gamma-irradiated BALB/c recipients on day 12 postinfection (24, 27), demonstrating the ability of stimulated MCMV-specific CD8+ T cells to confer protection in these organs.

    In the second immunization experiment, we found that immunization with the gB pDNA as part of the three-pDNA cocktail elicited stable levels of virion-specific serum IgG but did not generate detectable neutralizing antibodies. This was despite the construct encoding the gB ORF of strain K181 expressing the appropriately sized precursor protein following transfection of COS7 cells and eliciting IgG specific for the various gB species in MCMV-infected NIH 3T3 cells (Fig. 5). In addition, this same gB gene delivered to mice via recombinant vaccinia virus (data not shown) and replication-deficient adenovirus (57) was found to elicit protection against subsequent MCMV challenge. The simplest explanation for the lack of neutralizing antibodies after gB pDNA immunization is that the expression levels following i.d. injection were insufficient to elicit high enough levels of antibodies to show neutralization activity in vitro. Of note, the mean levels of virion-specific IgG were consistently 100-fold lower for the mice that were primed only with the IE1, M84, and gB pDNAs than for their FI-MCMV-boosted counterparts for the 7 to 8 weeks prior to challenge (Fig. 8A). However, in the latter case, antibodies against neutralizing-antibody targets of the virion other than gB, such as gH (38, 49), and seroreactive, nonenvelope antigens, such as M83, were likely present in the serum as well. Work is in progress that uses a gB-specific ELISA to compare the relative levels of gB-specific IgG following immunization with either gB pDNA or FI-MCMV. Alternatively, gB protein expressed by the gB pDNA in vivo may not undergo the posttranslational modifications, such as glycosylation, cleavage, or disulfide linkage, necessary for the generation of the neutralizing epitope(s) of gB. We have found that sera from mice immunized with the gB pDNA often react poorly if at all to the gp105 species of the cleaved, mature gB (Fig. 5B and data not shown) that appears on the surface of the mature virion (37). It has been reported that this species may contain at least one neutralizing-antibody epitope as assayed with sera from mice immunized with bacterially expressed gB fragments (63).

    Surprisingly, we found that parenteral administration of the IE1, M84, and gB pDNAs and FI-MCMV resulted in the priming of B cells to secrete IgA in the lungs. Virion-specific IgA was found in the lungs by day 6 post-i.n. challenge in the pDNA-primed/FI-MCMV-boosted mice and in the pDNA-primed mice by day 10 in four of five mice. Although the organs used for IgA analysis, the lungs and salivary glands, were not perfused prior to homogenization, IgA-specific ELISAs of the sera of these mice showed that the blood did not contain detectable virion-specific IgA (data not shown). Thus, we conclude that the IgA was present in the parenchyma and not the circulating blood. In the salivary glands, while virion-specific IgA was present in the pDNA-primed/FI-MCMV-boosted mice on day 6 post-i.n. challenge, significant IgA levels were only spuriously observed throughout the rest of the course of challenge (Fig. 11B). This is in contrast to the IgA induction kinetics observed in the pDNA primed-only mice and in the mock-immunized controls. In these cases, similar levels of virion-specific IgA were induced by day 18 postchallenge, and a stable peak was observed by day 24. The immunological basis for the discrepancy between the induction of IgA in the salivary glands of the vector and pDNA-only groups versus the pDNA/FI-MCMV group is not yet known. It may be that the absence of sustained viral antigen in the salivary glands of the pDNA/FI-MCMV group prevented the adequate stimulation of IgA-secreting B cells. As seen from the viral titer data in Fig. 9, infectious virus was consistently present in the lungs of the pDNA/FI-MCMV mice through day 10 postchallenge but only spuriously in the salivary glands of this group throughout the 32 days postchallenge examined. Another explanation for the reduced IgA levels detected in the salivary glands of the pDNA-primed/FI-MCMV-boosted mice may be that IgA bound to viral antigens was rapidly cleared from this organ by macrophage or dendritic cell phagocytosis and subsequent endosomal proteolysis. The kinetics of this clearance may be different from that in the lungs, since both infectious virus and virus-specific IgA were simultaneously detected in the lungs following i.n. challenge (Fig. 9A and 11A), and the levels of unbound IgA in the salivary glands may not reflect the role, if any, that IgA plays in the control of virus in this organ. Quantification of the IgA-secreting B cells in the lungs and salivary glands before and after challenge may be helpful in determining the relative rates of IgA secretion in these organs. In progress are studies to measure the levels of lung and salivary gland IgA that are elicited by the priming-boosting vaccination alone and to determine whether restimulation by challenge virus is necessary for the detection of antiviral IgA.

    Western blot analysis of the day 24 post-i.n. challenge IgA in lungs demonstrated that IgAs specific for the pp89 and pp76 forms of IE1 and possibly the gp128 and gp52 forms of gB were primed by the IE1, M84, and gB pDNA cocktail (data not shown). We also noted that lung IgA from the pDNA/FI-MCMV-immunized mice seroreacted with additional antigens in the MCMV-infected NIH 3T3 cells. By day 24 postchallenge, the lungs of the mice in the vector group also had IgA specific for IE1-pp89, although the possible protective ability of the IE1 antibody is not clear. It has been documented that IgA monoclonal antibodies that transcytose through epithelial cells in vitro can bind to intracellular viral antigens, interfering with viral replication, assembly, or egress. In addition, in a mouse model of rotavirus infection, a nonneutralizing IgA monoclonal antibody specific for the inner core protein VP6 was able to prevent primary infection and to resolve a chronic infection (8). It was subsequently found that within epithelial cells, this IgA could bind to VP6 trimer and render it transcriptionally inactive (16). Thus, it is possible that pp89-specific IgA could play a role in the protection against MCMV replication in mucosal epithelia. Notably, passive transfer of MCMV-specific monoclonal antibodies that were nonneutralizing in vitro have been shown to be protective against lethal i.p. challenge (15).

    Heterologous priming-boosting strategies are rapidly emerging as an effective means for the vaccination against such pathogens as human immunodeficiency virus, herpes simplex virus, hepatitis viruses B and C, Mycobacterium tuberculosis, and the malaria-causing Plasmodium parasites, with clinical trials already under way in some cases (for review, see reference 61). One particular strategy, priming with pDNA followed by boosting with a subunit or replication-defective viral vector vaccines, has been shown to elicit synergistic levels of T-cell and antibody immunity against intracellular pathogens, which is not observed with repeated boosts with the same antigen delivery system. While the mechanisms underlying this synergism following priming and boosting are not yet fully understood, increases in T-cell number and avidity have been observed in mice, and both CD4+- and CD8+-T-responses were observed in human subjects after delivery of the antigen by pDNA followed by recombinant vaccinia virus (14, 40). Coupled with their ability to generate high levels of protective antibodies, heterologous priming-boosting vaccination may provide the long-awaited tool for vaccination against pathogens recalcitrant to control by use of the traditional vaccination strategies.

    ACKNOWLEDGMENTS

    This work was supported by research grants 1-FY02-199 from the March of Dimes Birth Defects Foundation and AI51557 from NIH and by NIH training grant T32 AI07036.

    We also thank Lambert Loh and Eyal Raz for contributing reagents.

    Present address: Beckman Coulter, Inc., San Diego, CA 92121.

    REFERENCES

    BenMohamed, L., R. Krishnan, C. Auge, J. F. Primus, and D. J. Diamond. 2002. Intranasal administration of a synthetic lipopeptide without adjuvant induces systemic immune responses. Immunology 106:113-121.

    Boeckh, M., W. G. Nichols, G. Papanicolaou, R. Rubin, J. R. Wingard, and J. Zaia. 2003. Cytomegalovirus in hematopoietic stem cell transplant recipients: current status, known challenges, and future strategies. Biol. Blood Marrow Transplant. 9:543-558.

    Boppana, S. B., K. B. Fowler, W. J. Britt, S. Stagno, and R. F. Pass. 1999. Symptomatic congenital cytomegalovirus infection in infants born to mothers with preexisting immunity to cytomegalovirus. Pediatrics 104:55-60.

    Boppana, S. B., L. B. Rivera, K. B. Fowler, M. Mach, and W. J. Britt. 2001. Intrauterine transmission of cytomegalovirus to infants of women with preconceptional immunity. N. Engl. J. Med. 344:1366-1371.

    Britt, W. J. 1996. Vaccines against human cytomegalovirus: time to test. Trends Microbiol. 4:34-38.

    Britt, W. J., and M. Mach. 1996. Human cytomegalovirus glycoproteins. Intervirology 39:401-412.

    Brune, W., H. Hengel, and U. H. Koszinowski. 1999. A mouse model for cytomegalovirus infection, p. 19.17.11-19.17.13. In J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober (ed.), Current protocols in immunology, vol. 4. John Wiley & Sons, Inc., New York, N.Y.

    Burns, J. W., M. Siadat-Pajouh, A. A. Krishnaney, and H. B. Greenberg. 1996. Protective effect of rotavirus VP6-specific IgA monoclonal antibodies that lack neutralizing activity. Science 272:104-107.

    Cranmer, L. D., C. L. Clark, C. S. Morello, H. E. Farrell, W. D. Rawlinson, and D. H. Spector. 1996. Identification, analysis, and evolutionary relationships of the putative murine cytomegalovirus homologs of the human cytomegalovirus UL82 (pp71) and UL83 (pp65) matrix phosphoproteins. J. Virol. 70:7929-7939.

    Daniels, K. A., G. Devora, W. C. Lai, C. L. O'Donnell, M. Bennett, and R. M. Welsh. 2001. Murine cytomegalovirus is regulated by a discrete subset of natural killer cells reactive with monoclonal antibody to Ly49H. J. Exp. Med. 194:29-44.

    Elliott, R., C. Clark, D. Jaquish, and D. H. Spector. 1991. Transcription analysis and sequence of the putative murine cytomegalovirus DNA polymerase gene. Virology 185:169-186.

    Endresz, V., K. Burian, K. Berencsi, Z. Gyulai, L. Kari, H. Horton, D. Virok, C. Meric, S. A. Plotkin, and E. Gonczol. 2001. Optimization of DNA immunization against human cytomegalovirus. Vaccine 19:3972-3980.

    Endresz, V., L. Kari, K. Berencsi, C. Kari, Z. Gyulai, C. Jeney, S. Pincus, U. Rodeck, C. Meric, S. A. Plotkin, and E. Gonczol. 1999. Induction of human cytomegalovirus (HCMV)-glycoprotein B (gB)-specific neutralizing antibody and phosphoprotein 65 (pp65)-specific cytotoxic T lymphocyte responses by naked DNA immunization. Vaccine 17:50-58.

    Estcourt, M. J., A. J. Ramsay, A. Brooks, S. A. Thomson, C. J. Medveckzy, and I. A. Ramshaw. 2002. Prime-boost immunization generates a high frequency, high-avidity CD8(+) cytotoxic T lymphocyte population. Int. Immunol. 14:31-37.

    Farrell, H. E., and G. R. Shellam. 1991. Protection against murine cytomegalovirus infection by passive transfer of neutralizing and non-neutralizing monoclonal antibodies. J. Gen. Virol. 72:149-156.

    Feng, N., J. A. Lawton, J. Gilbert, N. Kuklin, P. Vo, B. V. Prasad, and H. B. Greenberg. 2002. Inhibition of rotavirus replication by a non-neutralizing, rotavirus VP6-specific IgA mAb. J. Clin. Investig. 109:1203-1213.

    Fowler, K. B., S. Stagno, and R. F. Pass. 2003. Maternal immunity and prevention of congenital cytomegalovirus infection. JAMA 289:1008-1011.

    Gold, M. C., M. W. Munks, M. Wagner, U. H. Koszinowski, A. B. Hill, and S. P. Fling. 2002. The murine cytomegalovirus immunomodulatory gene m152 prevents recognition of infected cells by M45-specific CTL but does not alter the immunodominance of the M45-specific CD8 T cell response in vivo. J. Immunol. 169:359-365.

    Gonczol, E., and S. Plotkin. 2001. Development of a cytomegalovirus vaccine: lessons from recent clinical trials. Expert Opin. Biol. Ther. 1:401-412.

    González Armas, J. C., C. S. Morello, L. D. Cranmer, and D. H. Spector. 1996. DNA immunization confers protection against murine cytomegalovirus infection. J. Virol. 70:7921-7928.

    Holtappels, R., N. K. Grzimek, C. O. Simon, D. Thomas, D. Dreis, and M. J. Reddehase. 2002. Processing and presentation of murine cytomegalovirus pORFm164-derived peptide in fibroblasts in the face of all viral immunosubversive early gene functions. J. Virol. 76:6044-6053.

    Holtappels, R., N. K. Grzimek, D. Thomas, and M. J. Reddehase. 2002. Early gene m18, a novel player in the immune response to murine cytomegalovirus. J. Gen. Virol. 83:311-316.

    Holtappels, R., J. Podlech, G. Geginat, H. P. Steffens, D. Thomas, and M. J. Reddehase. 1998. Control of murine cytomegalovirus in the lungs: relative but not absolute immunodominance of the immediate-early 1 nonapeptide during the antiviral cytolytic T-lymphocyte response in pulmonary infiltrates. J. Virol. 72:7201-7212.

    Holtappels, R., J. Podlech, N. K. Grzimek, D. Thomas, M. F. Pahl-Seibert, and M. J. Reddehase. 2001. Experimental preemptive immunotherapy of murine cytomegalovirus disease with CD8 T-cell lines specific for ppM83 and pM84, the two homologs of human cytomegalovirus tegument protein ppUL83 (pp65). J. Virol. 75:6584-6600.

    Holtappels, R., J. Podlech, M. F. Pahl-Seibert, M. Julch, D. Thomas, C. O. Simon, M. Wagner, and M. J. Reddehase. 2004. Cytomegalovirus misleads its host by priming of CD8 T cells specific for an epitope not presented in infected tissues. J. Exp. Med. 199:131-136.

    Holtappels, R., D. Thomas, J. Podlech, G. Geginat, H. P. Steffens, and M. J. Reddehase. 2000. The putative natural killer decoy early gene m04 (gp34) of murine cytomegalovirus encodes an antigenic peptide recognized by protective antiviral CD8 T cells. J. Virol. 74:1871-1884.

    Holtappels, R., D. Thomas, J. Podlech, and M. J. Reddehase. 2002. Two antigenic peptides from genes m123 and m164 of murine cytomegalovirus quantitatively dominate CD8 T-cell memory in the H-2d haplotype. J. Virol. 76:151-164.

    Holtappels, R., D. Thomas, and M. J. Reddehase. 2000. Identification of a K(d)-restricted antigenic peptide encoded by murine cytomegalovirus early gene M84. J. Gen. Virol. 81:3037-3042.

    Hou, Y., W. G. Hu, T. Hirano, and X. X. Gu. 2002. A new intra-NALT route elicits mucosal and systemic immunity against Moraxella catarrhalis in a mouse challenge model. Vaccine 20:2375-2381.

    Hvalbye, B. K., I. S. Aaberge, M. Lovik, and B. Haneberg. 1999. Intranasal immunization with heat-inactivated Streptococcus pneumoniae protects mice against systemic pneumococcal infection. Infect. Immun. 67:4320-4325.

    Jonjic, S., W. Mutter, F. Weiland, M. J. Reddehase, and U. H. Koszinowski. 1989. Site-restricted persistent cytomegalovirus infection after selective long-term depletion of CD4+ T lymphocytes. J. Exp. Med. 169:1199-1212.

    Kavanagh, D. G., M. C. Gold, M. Wagner, U. H. Koszinowski, and A. B. Hill. 2001. The multiple immune-evasion genes of murine cytomegalovirus are not redundant: m4 and m152 inhibit antigen presentation in a complementary and cooperative fashion. J. Exp. Med. 194:967-978.

    Kleijnen, M. F., J. B. Hupp, P. Lucin, S. Mukherjee, H. Farrell, A. E. Campbell, U. H. Koszinowski, A. B. Hill, and H. L. Ploegh. 1997. A mouse cytomegalovirus glycoprotein, gp34, forms a complex with folded class I MHC molecules in the ER which is not retained but is transported to the cell surface. EMBO J. 16:685-694.

    Krmpotic, A., M. Messerle, I. Crnkovic-Mertens, B. Polic, S. Jonjic, and U. H. Koszinowski. 1999. The immunoevasive function encoded by the mouse cytomegalovirus gene m152 protects the virus against T cell control in vivo. J. Exp. Med. 190:1285-1296.

    La Rosa, C., Z. Wang, J. C. Brewer, S. F. Lacey, M. C. Villacres, R. Sharan, R. Krishnan, M. Crooks, S. Markel, R. Maas, and D. J. Diamond. 2002. Preclinical development of an adjuvant-free peptide vaccine with activity against CMV pp65 in HLA transgenic mice. Blood 100:3681-3689.

    Lee, S. H., J. R. Webb, and S. M. Vidal. 2002. Innate immunity to cytomegalovirus: the Cmv1 locus and its role in natural killer cell function. Microbes Infect. 4:1491-1503.

    Loh, L. C., N. Balachandran, and L. F. Qualtiere. 1988. Characterization of a major virion envelope glycoprotein complex of murine cytomegalovirus and its immunological cross-reactivity with human cytomegalovirus. Virology 166:206-216.

    Loh, L. C., and L. F. Qualtiere. 1988. A neutralizing monoclonal antibody recognizes an 87K envelope glycoprotein on the murine cytomegalovirus virion. Virology 162:498-502.

    LoPiccolo, D. M., M. C. Gold, D. G. Kavanagh, M. Wagner, U. H. Koszinowski, and A. B. Hill. 2003. Effective inhibition of Kb- and Db-restricted antigen presentation in primary macrophages by murine cytomegalovirus. J. Virol. 77:301-308.

    McConkey, S. J., W. H. Reece, V. S. Moorthy, D. Webster, S. Dunachie, G. Butcher, J. M. Vuola, T. J. Blanchard, P. Gothard, K. Watkins, C. M. Hannan, S. Everaere, K. Brown, K. E. Kester, J. Cummings, J. Williams, D. G. Heppner, A. Pathan, K. Flanagan, N. Arulanantham, M. T. Roberts, M. Roy, G. L. Smith, J. Schneider, T. Peto, R. E. Sinden, S. C. Gilbert, and A. V. Hill. 2003. Enhanced T-cell immunogenicity of plasmid DNA vaccines boosted by recombinant modified vaccinia virus Ankara in humans. Nat. Med. 9:729-735.

    Mercer, J. A., J. R. Marks, and D. H. Spector. 1983. Molecular cloning and restriction endonuclease mapping of the murine cytomegalovirus genome (Smith strain). Virology 129:94-106.

    Mercer, J. A., and D. H. Spector. 1986. Pathogenesis of acute murine cytomegalovirus infection in resistant and susceptible strains of mice. J. Virol. 57:497-504.

    Moja, P., C. Tranchat, I. Tchou, B. Pozzetto, F. Lucht, C. Desgranges, and C. Genin. 2000. Neutralization of human immunodeficiency virus type 1 (HIV-1) mediated by parotid IgA of HIV-1-infected patients. J. Infect. Dis. 181:1607-1613.

    Morello, C. S., L. D. Cranmer, and D. H. Spector. 1999. In vivo replication, latency, and immunogenicity of murine cytomegalovirus mutants with deletions in the M83 and M84 genes, the putative homologs of human cytomegalovirus pp65 (UL83). J. Virol. 73:7678-7693.

    Morello, C. S., L. D. Cranmer, and D. H. Spector. 2000. Suppression of murine cytomegalovirus (MCMV) replication with a DNA vaccine encoding MCMV M84 (a homolog of human cytomegalovirus pp65). J. Virol. 74:3696-3708.

    Morello, C. S., M. Ye, and D. H. Spector. 2002. Development of a vaccine against murine cytomegalovirus (MCMV), consisting of plasmid DNA and formalin-inactivated MCMV, that provides long-term, complete protection against viral replication. J. Virol. 76:4822-4835.

    Pass, R. F. 2001. Cytomegaloviruses, p. 2675-2706. In D. M. Knipe and P. M. Howley (ed.), Fields virology, 4th ed. Lippincott-Raven, Philadelphia, Pa.

    Pepperl, S., J. Munster, M. Mach, J. R. Harris, and B. Plachter. 2000. Dense bodies of human cytomegalovirus induce both humoral and cellular immune responses in the absence of viral gene expression. J. Virol. 74:6132-6146.

    Rapp, M., P. Lucin, M. Messerle, L. C. Loh, and U. H. Koszinowski. 1994. Expression of the murine cytomegalovirus glycoprotein H by recombinant vaccinia virus. J. Gen. Virol. 75:183-188.

    Rapp, M., M. Messerle, B. Buhler, M. Tannheimer, G. M. Keil, and U. H. Koszinowski. 1992. Identification of the murine cytomegalovirus glycoprotein B gene and its expression by recombinant vaccinia virus. J. Virol. 66:4399-4406.

    Rapp, M., M. Messerle, P. Lucin, and U. H. Koszinowski. 1993. In vivo protection studies with MCMV glycoproteins gB and gH expressed by vaccinia virus, p. 327-332. In P. S. A. Michelsons (ed.), Multidisciplinary approach to understanding cytomegalovirus disease. Excerpta Medica, Amsterdam, The Netherlands.

    Reddehase, M. J. 2002. Antigens and immunoevasins: opponents in cytomegalovirus immune surveillance. Nat. Rev. Immunol. 2:831-844.

    Reddehase, M. J. 2000. The immunogenicity of human and murine cytomegaloviruses. Curr. Opin. Immunol. 12:390-396.

    Reddehase, M. J., G. M. Keil, and U. H. Koszinowski. 1984. The cytolytic T lymphocyte response to the murine cytomegalovirus. II. Detection of virus replication stage-specific antigens by separate populations of in vivo active cytolytic T lymphocyte precursors. Eur. J. Immunol. 14:56-61.

    Reusch, U., W. Muranyi, P. Lucin, H. G. Burgert, H. Hengel, and U. H. Koszinowski. 1999. A cytomegalovirus glycoprotein re-routes MHC class I complexes to lysosomes for degradation. EMBO J. 18:1081-1091.

    Riddell, S. R., and P. D. Greenberg. 2000. T-cell therapy of cytomegalovirus and human immunodeficiency virus infection. J. Antimicrob. Chemother. 45:35-43.

    Shanley, J. D., and C. A. Wu. 2003. Mucosal immunization with a replication-deficient adenovirus vector expressing murine cytomegalovirus glycoprotein B induces mucosal and systemic immunity. Vaccine 21:2632-2642.

    Tay, C. H., and R. M. Welsh. 1997. Distinct organ-dependent mechanisms for the control of murine cytomegalovirus infection by natural killer cells. J. Virol. 71:267-275.

    Wagner, M., A. Gutermann, J. Podlech, M. J. Reddehase, and U. H. Koszinowski. 2002. Major histocompatibility complex class I allele-specific cooperative and competitive interactions between immune evasion proteins of cytomegalovirus. J. Exp. Med. 196:805-816.

    Welsh, R. M., J. O. Brubaker, M. Vargas-Cortes, and C. L. O'Donnell. 1991. Natural killer (NK) cell response to virus infections in mice with severe combined immunodeficiency. The stimulation of NK cells and the NK cell-dependent control of virus infections occur independently of T and B cell function. J. Exp. Med. 173:1053-1063.

    Woodland, D. L. 2004. Jump-starting the immune system: prime-boosting comes of age. Trends Immunol. 25:98-104.

    Wu, X., S. Hall, and S. Jackson. 2003. Tropism-restricted neutralization by secretory IgA from parotid saliva of HIV type 1-infected individuals. AIDS Res. Hum. Retrovir. 19:275-281.

    Xu, J., P. A. Lyons, M. D. Carter, T. W. Booth, N. J. Davis-Poynter, G. R. Shellam, and A. A. Scalzo. 1996. Assessment of antigenicity and genetic variation of glycoprotein B of murine cytomegalovirus. J. Gen. Virol. 77:49-59.

    Ye, M., C. S. Morello, and D. H. Spector. 2004. Multiple epitopes in the murine cytomegalovirus early gene product M84 are efficiently presented in infected primary macrophages and contribute to strong CD8+-T-lymphocyte responses and protection following DNA immunization. J. Virol. 78:11233-11245.

    Ye, M., C. S. Morello, and D. H. Spector. 2002. Strong CD8 T-cell responses following coimmunization with plasmids expressing the dominant pp89 and subdominant M84 antigens of murine cytomegalovirus correlate with long-term protection against subsequent viral challenge. J. Virol. 76:2100-2112.

    Zaia, J. A., G. Gallez-Hawkins, X. Li, Z. Q. Yao, N. Lomeli, K. Molinder, C. La Rosa, and D. J. Diamond. 2001. Infrequent occurrence of natural mutations in the pp65(495-503) epitope sequence presented by the HLA A0201 allele among human cytomegalovirus isolates. J. Virol. 75:2472-2474.

    Ziegler, H., R. Thale, P. Lucin, W. Muranyi, T. Flohr, H. Hengel, H. Farrell, W. Rawlinson, and U. H. Koszinowski. 1997. A mouse cytomegalovirus glycoprotein retains MHC class I complexes in the ERGIC/cis-Golgi compartments. Immunity 6:57-66.(Christopher S. Morello, M)

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