| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Center for Comparative Medicine, University of California, Davis, County Road 98 and Hutchison Drive, Davis, CA 95616, USA
2 Department of Immunology, New England Primate Research Center, Harvard Medical School, Southborough, MA 02129, USA
3 Department of Pathology and Laboratory Medicine, University of California, Davis, County Road 98 and Hutchison Drive, Davis, CA 95616, USA
4 California National Primate Research Center, University of California, Davis, County Road 98 and Hutchison Drive, Davis, CA 95616, USA
Correspondence
Yujuan Yue
yyue{at}ucdavis.edu
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). There is no licensed vaccine for HCMV, although there is a clear clinical need for one that could protect those most at risk for HCMV infection and disease. Natural history studies have identified immunological correlates of protection, particularly the role of both neutralizing (NT) antibodies and cytotoxic T lymphocytes (CTLs) in limiting transplacental transmission of HCMV (Boppana & Britt, 1995) and protecting from disease in bone marrow- and solid organ-transplant recipients (Li et al., 1994; Reusser et al., 1991, 1997, 1999; Walter et al., 1995), respectively. Dominant viral targets for NT antibodies and CTL activity include the proteins encoded by the UL55 (glycoprotein B; gB) and UL83 (phosphoprotein 65; pp65) open reading frames (ORFs). As such, both HCMV proteins represent logical components of HCMV vaccine strategies. Likewise, other studies have indicated that the immediate-early 1 protein (IE1) should be included as a vaccine target (Bunde et al., 2005), a conclusion bolstered by vaccine studies involving the IE1 protein of murine CMV (MCMV) (Del Val et al., 1991; González Armas et al., 1996; Jonji
et al., 1988; Ye et al., 2002).
Although the immune responses that control HCMV are not known precisely, the quality and magnitude of humoral responses (e.g. NT antibodies or high-avidity antibodies) appear to be critical for protection against primary infection and limiting virus dissemination (Boppana & Britt, 1995; Fowler et al., 1992; Nigro et al., 2005) and the severity of disease during recurrence (Campbell & Herold, 2004), whereas cell-mediated immune responses probably play the dominant role in protection against HCMV sequelae and the recovery from fulminant HCMV infection (Li et al., 1994; Reusser et al., 1991, 1997, 1999; Walter et al., 1995). Thus, induction of protective antibody and CTL responses should be considered for vaccine design. As an abundant structural protein, HCMV pp65 is the immunodominant target for CTL and lymphoproliferative responses (Beninga et al., 1995; Boppana & Britt, 1996; Gyulai et al., 2000; McLaughlin-Taylor et al., 1994; Wills et al., 1996) and, as such, has been studied as a vaccine candidate. A recombinant canarypox vector expressing HCMV pp65 has been shown to induce CD8+ cytotoxic T cells that persist for at least 2 years following immunization in healthy, HCMV-seronegative humans (Berencsi et al., 2001). Similarly, a modified vaccinia virus Ankara expressing pp65 elicits pp65-specific CTLs in mice (Wang et al., 2004). However, there has not been an evaluation of their ability to protect against either infection or disease. DNA immunization against the MCMV homologue (M84) of HCMV pp65 stimulates cellular responses that reduce viral titres significantly following challenge (Morello et al., 2000, 2005). Together, the natural history and vaccination studies point to the need to investigate pp65-based immunization strategies further.
Towards that end, we have characterized a rhesus CMV (RhCMV) homologue of HCMV pp65 as a prelude to evaluating its utility as a model system for testing pp65 strategies in a non-human primate host. RhCMV encodes two ORFs, Rh111 and Rh112, that are both positional and sequence homologues of HCMV pp65 (Hansen et al., 2003). Of the two, the predicted protein encoded by Rh112 (pp65-2) has slightly higher amino acid identity (35 %) to HCMV pp65 than does the protein encoded by Rh111 (32 %). Immunologically, pp65-2 elicits antibody and cellular immune responses stronger than those generated by pp65-1 (Hansen et al., 2003; unpublished data). This report characterizes the expression of pp65-2 in tissue-culture cells and host immune responses following natural exposure to RhCMV. Like its HCMV pp65 counterpart, RhCMV pp65-2 is expressed with late-gene kinetics, localizes to the nucleus and stimulates humoral and cellular immune responses in infected rhesus macaques (Fowler et al., 1992). The results demonstrate that RhCMV pp65-2 is a target of host immune responses and, thus, represents a valid surrogate for investigating pp65 as a vaccine component.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Kirchoff et al., 2002) were cultured in complete DMEM containing 10 % (v/v) FCS, 100 µg G418 ml–1 (Invitrogen) and Pen/Strep. 293T cells (DuBridge et al., 1987) were grown in complete DMEM containing 10 % (v/v) FCS and Pen/Strep. All of the cell lines were tested and found to be free of mycoplasma contamination. Cell-free stocks of RhCMV strain 68-1 (Lockridge et al., 1999) and total virus antigens for ELISA were prepared in RhDFs as described previously (Yue et al., 2003) and stored at –80 °C. Telo-RFs were used for the kinetics of protein expression and localization of pp65-2 following RhCMV infection. 293T cells were used for the transfection of pND/pp65-2 and the preparation of antigens for pp65-2-specific ELISA.
Sequence analysis.
Alignment of RhCMV pp65-1, pp65-2 and HCMV pp65 was performed with the Vector NTI 7 software package from Invitrogen. Identification of potential nuclear-localization signals (NLSs) was performed with PSORT (http://psort.nibb.ac.jp) (Nakai & Horton, 1999; Nakai & Kanehisa, 1992).
Expression plasmids for RhCMV pp65-2 gene.
The RhCMV pp65-2 gene was amplified from the genome of RhCMV 68-1 by using primers that overlapped the start and stop codons of Rh112 (Hansen et al., 2003) and contained XhoI and EcoRI restriction-endonuclease sites, respectively, for cloning into the SalI and EcoRI sites of the pND expression vector (Loomis-Huff et al., 2001) to create the vector pND/pp65-2. The fidelity of the amplification was confirmed by sequence analysis after cloning the amplicon into pND. The plasmid pECFP-pp65-2 was constructed by inserting the pp65-2 coding sequence downstream of and in frame with that of the enhanced cyan fluorescent protein (ECFP) by using the BglII and EcoRI sites of the pECFP-c1 expression vector (BD Biosciences). This resulted in the expression of a chimeric ECFP–pp65-2 fusion protein under the transcriptional control of the HCMV immediate-early promoter.
Transient-transfection assay.
For the preparation of antigen for a pp65-2-specific ELISA, 293T cells were transiently transfected with pND/pp65-2 according to our previously published protocol (Yue et al., 2003). Cells were harvested at 48 h post-transfection and resuspended in 0·1 M glycerin/saline buffer (Yue et al., 2003). Untransfected 293T-cell extracts prepared similarly served as a source of negative-control antigen.
To investigate the subcellular localization of pp65-2, the plasmids pECFP-pp65-2 and pECFP-c1 vector were transfected individually into Telo-RFs with FuGENE 6, a lipid-based transfection reagent (Roche Diagnostics) at a ratio of 3 µl FuGENE : 0·5 µg DNA in the absence of serum (Chang et al., 2002). Complete DMEM was replaced with 2 ml plain DMEM prior to transfection. After a 4 h incubation at 37 °C in 5 % CO2, the medium containing the DNA–lipid complex was removed and fresh DMEM/serum was added. Cells were either fixed with 3 % paraformaldehyde at different times post-transfection (6, 12, 24, 48 and 78 h) or harvested into lysis buffer (Vogel et al., 1994) at 48 h post-transfection for Western blot analysis. The fixed cells were observed under a fluorescence microscope for the subcellular distribution of the ECFP–pp65-2 fusion protein.
Immunofluorescence assay.
Telo-RFs grown on chamber slides were infected with RhCMV strain 68-1 (m.o.i. of 2). At 24 h post-infection (p.i.), the cells were fixed in glacial acetic acid : ethanol (5 : 95) for 10 min and permeabilized with CSK buffer (50 mM NaCl, 300 mM sucrose, 10 mM PIPES, 3 mM MgCl2, 0·5 % Triton X-100) for 5 min. After rinsing in PBS, the cells were blocked with 20 % (v/v) normal goat serum in PBS for 30 min at 37 °C and then were incubated in either pp65-2-specific or non-immune mouse sera (both diluted 1 : 500 in 1 % goat serum/PBS) for 1 h at 37 °C. The cells were washed three times in PBS (5 min per wash) and incubated with Alexa Fluor 594-conjugated goat anti-mouse IgG (diluted 1 : 3000 in 1 % goat serum/PBS) (Molecular Probes Invitrogen Detection Techologies) for 1 h at 37 °C. After rinsing three times with PBS, the slides were mounted with SlowFade gold anti-fade reagent with DAPI (4,6-diamidino-2-phenylindole; Molecular Probes Invitrogen Detection Techologies) and viewed on a Zeiss fluorescence microscope.
Protein-expression analysis.
Telo-RFs were infected with RhCMV strain 68-1 at an m.o.i. of 1 and harvested at 6, 24, 48 and 72 h p.i. in the presence or absence of either cycloheximide (CHX, 50 µg ml–1, 6 h) or ganciclovir (GCV, 20 µg ml–1, 24, 48 and 72 h). Cell lysates were harvested at the different time points p.i. for Western blot analysis (Vogel et al., 1994). Mock-infected Telo-RF cells were also prepared as a negative control. pp65-2 protein expression was detected by using serum from mice immunized with the pND/pp65-2 expression plasmid. The protocol for genetic immunization in mice has been described previously (Loomis-Huff et al., 2001).
Animals and sample collection.
To study the humoral and cellular responses to RhCMV pp65-2 after infection, blood samples were collected from three groups of healthy, immunocompetent rhesus macaques at the California National Primate Research Center (CNPRC) (University of California, Davis, CA, USA). Group I monkeys (n=7, <1 year of age) were part of an earlier study that evaluated genetic immunization against herpes B virus (BV) (Loomis-Huff et al., 2001). These juveniles were housed indoors and were seronegative for RhCMV at the beginning of the BV immunization study. Their RhCMV serostatus changed from negative to positive during the course of the original study following an unknown exposure to RhCMV unrelated to the immunization procedure. Blood samples were collected every 2–4 weeks over a period of 8 months. Group II consisted of 14 RhCMV-seronegative juveniles (n=14, 1–2 years) that were relocated to a new housing arrangement into which a lone RhCMV-seropositive adult monkey was introduced. The seronegative juveniles seroconverted to RhCMV over the course of 36 weeks following initial horizontal transmission of RhCMV from the sentinel adult (L. Strelow, Y. Yue, A. Kaur, S. S. Zhou & P. A. Barry, unpublished data). Blood samples were collected every 1–3 months over a period of 1 year. Group III consisted of 79 randomly selected animals housed either in outdoor natal groups or indoors. Of these, 74 animals had pre-existing RhCMV antibodies at the start of the investigation and five animals seroconverted to RhCMV during the period of observation. Blood samples were collected at least twice from each animal over a period of 4–7 months. Blood samples were processed for either plasma/peripheral blood mononuclear cells (PBMCs) or, in some cases, just serum.
pp65-2 ELISA.
pp65-2-specific antibodies were analysed by following previously described protocols (Yue et al., 2003). For the detection of pp65-2-specific antibodies, lysates of 293T cells transiently transfected with pND/pp65-2 were used as coating antigen. Lysates of untransfected 293T cells served as a source of negative-control antigen. Microtitre plates (Immunlon 4 HBX; Fisher) were coated with 0·5 µg cell extract per well in 100 µl 0·05 M carbonate/bicarbonate buffer (Lockridge et al., 1999) and incubated overnight at 4 °C. All subsequent steps were carried out at room temperature. The plates were blocked with 300 µl per well of PBS containing 1 % BSA for 2 h. After washing the plate six times in PBS/0·05 % Tween 20 (wash buffer), 100 µl plasma (diluted 1 : 100 in PBS/1 % BSA/0·05 % Tween 20 dilution buffer) was added to each well for 2 h. Each plasma sample was assayed in duplicate. After washing, 100 µl peroxidase-conjugated goat anti-monkey IgG (KPL Inc.) (1 : 150 000 in dilution buffer) was added to each well for 1 h. One hundred microlitres of tetramethylbenzidine liquid substrate (Sigma) was added to each well for 30 min following a six-time washing. The reaction was terminated by the addition of 50 µl 0·5 M H2SO4 per well. A450 was recorded spectrophotometrically. For each sample, the net A450 was determined by subtracting the mean A450 values obtained in the duplicate control antigen-coated wells from the mean A450 obtained in the pp65-2 antigen-coated wells. Samples were considered positive if the net absorbance values were >0·1.
Gamma interferon (IFN-
) ELISPOT assay.
pp65-2-specific T-cell responses were quantified by an IFN- ELISPOT assay (U-CyTech) using a previously published protocol (Kaur et al., 2002). Briefly, cryopreserved PBMCs were stimulated for 16 h with either a recombinant vaccinia virus expressing pp65-2 (rVV-pp65-2) (Kaur et al., 2003) or an overlapping peptide pool (15mers overlapping by 11 aa – 134 peptides in total) representing the entire amino acid sequence of pp65-2 (BioSource). Each peptide was resuspended in 100 % DMSO at a concentration of 100 mg ml–1 and equal volumes of each peptide were mixed to form the pool (final concentration of each peptide in the pool, 0·75 mg ml–1). The peptide pool was diluted so that the final concentration of individual peptides used for stimulation was 1 µg ml–1 and the concentration of DMSO did not exceed 0·5 %. Spots were counted on a KS ELISPOT Automated Reader System (Carl Zeiss) using KS ELISPOT 4.2 software (performed by Zellnet Consulting). Frequencies of pp65-2-specific cells obtained after subtracting background spots in negative-control wells (control vaccinia NYCBH strain for rVV-pp65-2 and medium for pp65-2 peptides) were expressed as the number of spot-forming cells (SFCs) per million PBMCs. pp65-2-specific T-cell frequencies 
50 SFCs per 106 PBMCs were considered positive.
Statistical analysis.
Statistical analysis was carried out by using Prism (GraphPad Software). The relationship between antibodies to RhCMV and antibodies to pp65-2 was analysed by a linear-regression test.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). The two pp65 proteins predicted from the nucleotide sequences are 539 (pp65-1) and 542 (pp65-2) aa in length. They have 32 % (pp65-1) and 35 % (pp65-2) identity to HCMV pp65 and 41 % identity to each other. Alignment of the predicted amino acid sequences derived from the Rh111, Rh112 and HCMV UL83 genes indicates that conserved amino acids amongst all three ORFs were dispersed throughout the proteins, with minimal stretches of contiguous conservation (Fig. 1). There were only three regions where five consecutive amino acids were absolutely conserved. In contrast, the pp65 proteins of HCMV and chimpanzee CMV (Dolan et al., 2004) are 81 % identical (data not shown). Amino acids conserved between the two RhCMV ORFs were found throughout the proteins, including the regions with minimal identity to HCMV pp65.
|
; Schmolke et al., 1995). Nuclear localization is due in part to the presence of multiple NLSs, although there are other domains involved (Gallina et al., 1996; Schmolke et al., 1995). PSORT analysis of RhCMV pp65-1 and pp65-2 (Nakai & Horton, 1999; Nakai & Kanehisa, 1992) revealed that, of four potential NLSs, two have been conserved in RhCMV pp65-1 and three in pp65-2 (Fig. 1
). Only one NLS was common to both RhCMV pp65 homologues.
RhCMV pp65-2 expression kinetics and virion association
The kinetics of pp65-2 protein expression following RhCMV infection of Telo-RFs were analysed by Western blot (Fig. 2a). There was a large increase in the intensity of pp65-2 between 24 and 48 h p.i., with abundant steady-state levels present also at 72 h p.i. The approximate size of pp65-2 detected by Western blot (63 kDa) was consistent with the predicted size of 61·7 kDa (Hansen et al., 2003). Expression of pp65-2 was abrogated almost completely in the presence of the replication inhibitor GCV, consistent with true late-expression kinetics. There was faint staining observed at 6 h p.i. in both the presence and absence of the protein-synthesis inhibitor CHX. This was probably due to the uptake of the protein from the infecting virion following viral attachment and entry. This interpretation was substantiated by Western blot analysis demonstrating the presence of an immunoreactive protein of the expected size within sucrose gradient-purified RhCMV virions (Fig. 2b).
|
a). The latter pattern was characterized by relatively uniform nuclear staining together with a few punctate nodes of intense staining. At 78 h post-transfection, staining was confined to numerous intense foci of variable size within the nucleus (Fig. 3c). In contrast, the control ECFP protein remained distributed uniformly throughout the cell post-transfection (Fig. 3b, d).
|
–g).
Humoral immune responses to pp65-2
RhCMV is ubiquitous in captive rhesus macaques, with an infection rate of almost 100 % by the first year of age (Vogel et al., 1994). To determine whether pp65-2 was immunogenic in RhCMV-seropositive macaques, a pp65-2-specific ELISA was developed. Antibody (IgG) responses to pp65-2 were characterized in two groups of seronegative juvenile macaques (groups I and II, n=7 and 14, respectively) that underwent a primary infection following natural exposure to RhCMV and a third group of animals of mixed ages and RhCMV serostatus (group III, n=79). The difference between groups I and II was that the source(s) of infectious virus for group I was not known, whereas the source of infectious virus for group II was a single seropositive adult macaque that was co-housed with the seronegative juveniles.
A total of 20 monkeys of the 21 animals comprising groups I and II (95 %) developed IgG to pp65-2 during primary infection (Fig. 4a; data from only four animals are presented). Generally, binding antibodies to pp65-2 developed contemporaneously with antibodies to a total viral-antigen preparation consisting of structural and non-structural viral proteins. Antibody responses to pp65-2 reached maximal levels prior to the time of peak responses to total viral antigens. In addition, pp65-2 antibody responses declined over time and became undetectable in approximately 50 % of the animals over a period of 2–6·5 months since the first time point of detectable pp65-2-binding antibodies. The levels of pp65-2-binding antibodies were quite variable between the infected monkeys. Taken together, these data suggested that the humoral responses to RhCMV pp65-2 were elicited early, varied considerably among individuals and were not always sustained after production.
|
). Of these, 27 (59 % of those with pp65-2 antibodies) either had declining or fluctuating responses to pp65-2 over a period of time when contemporaneous responses to total RhCMV antigens remained relatively stable. Only five of the pp65-2-positive animals (11 %) exhibited stable antibody responses over time. As RhCMV DNA was detected periodically in the saliva and genital secretion, the fluctuating pp65-2-specific antibody responses may indicate the reactivation of RhCMV or reinfection (Huff et al., 2003). Statistical analysis showed that the antibody responses to pp65 and those directed against total RhCMV antigens were related (P<0·001; r=0·256). This result was consistent with the interpretation that antibodies to pp65-2 were not dominant during chronic infection.
|
; Reusser et al., 1991, 1997, 1999; Walter et al., 1995). The kinetics of cellular responses to pp65-2 during primary infection were analysed in four animals. Three of the four animals developed detectable pp65-2 IgG during the period of observation, whereas all four were positive for T cells secreting IFN- following stimulation with pp65-2 antigen (Fig. 4). The cellular responses to pp65-2 varied between animals. The peak responses ranged from 66 to 466 SFCs per million PBMCs following stimulation with an overlapping peptide pool of pp65-2 and from 94 to 938 SFCs per million PBMCs following stimulation with rVV-pp65-2. The kinetics of the pp65-2-specific T cells were generally concordant with seroconversion to RhCMV. It was noted that one animal (MMU31039; Fig. 4b) that was negative for antibody against pp65-2 exhibited robust T-cell responses (280–390 SFCs per 106 PBMCs) following stimulation with either rVV-pp65-2 or an overlapping peptide pool comprising the entire protein. It was also noted that the cellular responses declined over time in this animal.
To determine whether cellular responses persisted over time, a cross-sectional study was performed by using PBMCs from 20 long-term RhCMV-seropositive monkeys, ten of which were positive for IgG to pp65-2 and ten that were negative. Unlike long-term antibody responses to pp65-2, cellular responses were mostly retained in chronically infected animals (80 %, 16/20). Eight monkeys in each of the pp65-2 antibody-negative and -positive groups had detectable T-cell responses to either or both sources of pp65-2 antigen (Table 2). Conversely, two monkeys were IFN- ELISPOT-negative/IgG-positive and two were IFN- ELISPOT-negative/IgG-negative with respect to pp65-2. Although the frequencies of IFN--positive T cells in some of the pp65-2 antibody-positive animals were higher than the highest frequencies observed in pp65-2 antibody-negative animals, there was no statistical difference between the two groups. The ages of the animals at which seroconversion occurred for this group were unknown. However, the dates of birth and times of the PBMC isolation were consistent with an interval of up to 2 years between primary infection and the time of sampling, based on previous seroepidemiological studies of colony-reared macaques (Vogel et al., 1994). Thus, T-cell responses to pp65-2 appeared to persist for years.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
), pp65-2, is expressed late in the replication cycle, localizes to the nucleus after expression, is present within the mature virion and elicits both humoral and cellular immune responses. As such, RhCMV pp65-2 bears strong similarity to the pp65 protein (UL83) of HCMV in terms of genomic collinearity, sequence homology, expression kinetics, localization within the cell and virion, and immunogenic profile (Boppana & Britt, 1996; Chee et al., 1990; Depto & Stenberg, 1989; Gallina et al., 1996; Geballe et al., 1986; Gyulai et al., 2000; Jin et al., 2000; McLaughlin-Taylor et al., 1994; Ohlin et al., 1995; Sanchez et al., 1998; Schmolke et al., 1995; van Zanten et al., 1995; Wills et al., 1996).
The localization of RhCMV pp65-2 in discrete areas within the nucleus is consistent with the patterns identified for the pp65 proteins of HCMV and guinea pig CMV (GPCMV) (Gallina et al., 1996; McGregor et al., 2004; Schleiss et al., 1999; Schmolke et al., 1995). The punctate staining pattern in our study is consistent with co-localization in the nucleoli, although this remains to be proved formally. If substantiated, this would be similar to that of HCMV pp65, which has been shown to traffic rapidly to nucleoli after infection (Arcangeletti et al., 2003). The early distribution of virion-associated pp65 to the nucleus, recapitulated by pp65 expressed from a plasmid, suggests a possible regulatory role in the growth of CMV-infected cells, virus replication and transcription, and subviral particle assembly (Arcangeletti et al., 2003; Sanchez et al., 1998; Sindre et al., 2000). Whatever functions pp65 performs, the distribution of the protein within the cell nucleus further suggests that it may be conserved amongst HCMV, RhCMV and GPCMV.
HCMV pp65 represents a critical vaccine candidate because it is an immunodominant target of host cell-mediated immunity, and HCMV-specific CTLs are vital for protection from HCMV pathogenesis associated with transplantation (Riddell et al., 1992; Walter et al., 1995). In the present study, pp65-2-specific IFN--secreting T lymphocytes were detected in the peripheral blood of all animals with primary infection and roughly two-thirds of RhCMV-seropositive macaques with long-standing RhCMV infection following stimulation of PBMCs with either rVV-pp65-2 or an overlapping pp65-2 peptide pool. It has been reported that recombinant vaccinia virus-delivered antigen mainly stimulates specific CD8+ T cells and that 15mer overlapping peptide pool stimulates both specific CD4+ and CD8+ T cells. The disparate results between rVV-pp65-2 and pp65-2 peptide stimulation indicate that pp65-2 is likely to elicit both CD4+ and CD8+ T-cell responses. As the macaque colony at the CNPRC is an outbred population with multiple MHC haplotypes (Penedo et al., 2005), the results indicate that pp65-2 contains epitopes recognized in the context of diverse HLA backgrounds. The results of this study, however, do not attest to either the relative cellular immunodominance of pp65-2 or the protective efficacy of these responses in limiting the extent of RhCMV replication. The ontogeny of RhCMV pp65-2-specific cell-mediated immunity observed in this study was coincident with the clearance of viral DNA from the blood following either experimental inoculation with or natural exposure to RhCMV observed in previous studies (Lockridge et al., 1999; Sequar et al., 2002; L. Strelow, Y. Yue, A. Kaur, S. S. Zhou & P. A. Barry, unpublished data). CD8+ CTLs against MHC-matched target cells either pulsed with an overlapping pp65-2 peptide pool or infected with rVV-pp65-2 have been observed in the peripheral blood of naturally infected macaques by using pp65-2 antigen-stimulated PBMCs (A. Kaur, unpublished data). However, a previous report has noted that an early RhCMV protein(s) was primarily responsible for stimulating cytolytic activity of bulk peripheral CTLs in seropositive macaques (Kaur et al., 1996). This observation, together with our characterization of pp65-2 as a protein expressed with late-gene expression kinetics, would suggest that RhCMV pp65-2 may not have as prominent a role in generating CTL activity as HCMV pp65 does in infected humans. This conclusion should be tempered by recent studies evaluating HCMV-specific CTL activity in the context of HCMV-mediated immune modulation. The breadth and specificity of CTL activity to HCMV proteins are greater when target cells are infected with a variant of HCMV lacking the US2, 3, 6 and 11 ORFs, which downmodulate antigen presentation in the context of MHC class I (Manley et al., 2004). Moreover, studies examining T-cell responses to CD8+ epitopes of 14 HCMV antigens predicted by HLA-binding algorithms or overlapping peptide pools comprising 213 HCMV ORFs also demonstrated that T-cell recognition of HCMV is very broad, including functionally and kinetically diverse ORFs (Elkington et al., 2003; Sylwester et al., 2005). As RhCMV expresses functional homologues of these immunomodulatory ORFs of HCMV (Pande et al., 2005), the relative dominance of RhCMV proteins in stimulating CTL activity should be examined with a modified variant of RhCMV lacking MHC class I-modulating activity or, alternatively, overlapping peptide pools.
The homology between pp65-1 and pp65-2 raises the question of antibody and T-cell cross-reactivity of these two proteins. pp65-2-specific antibodies, generated in pp65-2 expression plasmid-vaccinated mice, do not cross-react by Western blot with an epitope (FLAG)-tagged pp65-1 expressed in transfected mammalian cells (data not shown). Analysis of plasma from 12 monkeys infected naturally with RhCMV (group II) and four experimentally infected animals indicates that only a minority of infected animals generated antibodies to pp65-1 (data not shown). Of the 16 infected monkeys, two animals had weak responses and only two had robust reactivity (data not shown). The antibody status of pp65-1 in these animals was not correlated to the levels of pp65-2 antibodies, suggesting that there is little cross-reactivity of pp65-1 antibodies to pp65-2. Similar findings have been noted for a lack of cross-reactivity of cellular responses. The cellular immune responses to pp65-1 and pp65-2 are largely independent of each other (L. Picker, personal communication), consistent with the interpretation that our results reflect pp65-2-specific immune responses.
Infection of rhesus macaques with RhCMV strongly recapitulates HCMV persistence and pathogenesis in humans (Barry & Chang, 2005), thereby offering a system to address HCMV vaccine strategies in a non-human primate host that complements other animal models (Schleiss & Heineman, 2005). Together with previous reports demonstrating the conservation of sequence and immunogenic determinants of the glycoprotein B (gB) proteins of primate CMV representatives, the results presented herein enable the macaque model to be used for evaluating the pp65- and gB-based vaccine strategies proposed for humans (Gonczol & Plotkin, 2001).
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Arcangeletti, M. C., De Conto, F., Ferraglia, F. & 11 other authors (2003). Human cytomegalovirus proteins PP65 and IEP72 are targeted to distinct compartments in nuclei and nuclear matrices of infected human embryo fibroblasts. J Cell Biochem 90, 1056–1067.[CrossRef][Medline]
Barry, P. A. & Chang, W.-L. W. (2005). Primate betaherpesviruses. In Human Herpesviruses: Biology, Therapy and Immunoprophylaxis. Edited by A. Arvin, G. Campadielli-Fiume, P. S. Moore, E. Mocarski, B. Roizman, R. Whitley & K. Yamanishi. Cambridge: Cambridge University Press.
Beninga, J., Kropff, B. & Mach, M. (1995). Comparative analysis of fourteen individual human cytomegalovirus proteins for helper T cell response. J Gen Virol 76, 153–160.
Berencsi, K., Gyulai, Z., Gönczöl, E. & 9 other authors (2001). A canarypox vector-expressing cytomegalovirus (CMV) phosphoprotein 65 induces long-lasting cytotoxic T cell responses in human CMV-seronegative subjects. J Infect Dis 183, 1171–1179.[CrossRef][Medline]
Boppana, S. B. & Britt, W. J. (1995). Antiviral antibody responses and intrauterine transmission after primary maternal cytomegalovirus infection. J Infect Dis 171, 1115–1121.[Medline]
Boppana, S. B. & Britt, W. J. (1996). Recognition of human cytomegalovirus gene products by HCMV-specific cytotoxic T cells. Virology 222, 293–296.[CrossRef][Medline]
Bunde, T., Kirchner, A., Hoffmeister, B. & 8 other authors (2005). Protection from cytomegalovirus after transplantation is correlated with immediate early 1-specific CD8 T cells. J Exp Med 201, 1031–1036.
Campbell, A. L. & Herold, B. C. (2004). Strategies for the prevention of cytomegalovirus infection and disease in pediatric liver transplantation recipients. Pediatr Transplant 8, 619–627.[CrossRef][Medline]
Chang, W. L. W., Kirchoff, V., Pari, G. S. & Barry, P. A. (2002). Replication of rhesus cytomegalovirus in life-expanded rhesus fibroblasts expressing human telomerase. J Virol Methods 104, 135–146.[CrossRef][Medline]
Chee, M. S., Bankier, A. T., Beck, S. & 12 other authors (1990). Analysis of the protein-coding content of the sequence of human cytomegalovirus strain AD169. Curr Top Microbiol Immunol 154, 125–169.[Medline]
Del Val, M., Schlicht, H.-J., Volkmer, H., Messerle, M., Reddehase, M. J. & Koszinowski, U. H. (1991). Protection against lethal cytomegalovirus infection by a recombinant vaccine containing a single nonameric T-cell epitope. J Virol 65, 3641–3646.
Depto, A. S. & Stenberg, R. M. (1989). Regulated expression of the human cytomegalovirus pp65 gene: octamer sequence in the promoter is required for activation by viral gene products. J Virol 63, 1232–1238.
Dolan, A., Cunningham, C., Hector, R. D. & 12 other authors (2004). Genetic content of wild-type human cytomegalovirus. J Gen Virol 85, 1301–1312.
DuBridge, R. B., Tang, P., Hsia, H. C., Leong, P.-M., Miller, J. H. & Calos, M. P. (1987). Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system. Mol Cell Biol 7, 379–387.
Elkington, R., Walker, S., Crough, T., Menzies, M., Tellam, J., Bharadwaj, M. & Khanna, R. (2003). Ex vivo profiling of CD8+-T-cell responses to human cytomegalovirus reveals broad and multispecific reactivities in healthy virus carriers. J Virol 77, 5226–5240.
Fowler, K. B., Stagno, S., Pass, R. F., Britt, W. J., Boll, T. J. & Alford, C. A. (1992). The outcome of congenital cytomegalovirus infection in relation to maternal antibody status. N Engl J Med 326, 663–667.[Abstract]
Gallina, A., Percivalle, E., Simoncini, L., Revello, M. G., Gerna, G. & Milanesi, G. (1996). Human cytomegalovirus pp65 lower matrix phosphoprotein harbours two transplantable nuclear localization signals. J Gen Virol 77, 1151–1157.
Geballe, A. P., Leach, F. S. & Mocarski, E. S. (1986). Regulation of cytomegalovirus late gene expression: genes are controlled by posttranscriptional events. J Virol 57, 864–874.
Gonczol, E. & Plotkin, S. (2001). Development of a cytomegalovirus vaccine: lessons from recent clinical trials. Expert Opin Biol Ther 1, 401–412.[CrossRef][Medline]
González Armas, J. C., Morello, C. S., Cranmer, L. D. & Spector, D. H. (1996). DNA immunization confers protection against murine cytomegalovirus infection. J Virol 70, 7921–7928.[Abstract]
Gyulai, Z., Endresz, V., Burian, K. & 7 other authors (2000). Cytotoxic T lymphocyte (CTL) responses to human cytomegalovirus pp65, IE1-Exon4, gB, pp150, and pp28 in healthy individuals: reevaluation of prevalence of IE1-specific CTLs. J Infect Dis 181, 1537–1546.[CrossRef][Medline]
Hansen, S. G., Strelow, L. I., Franchi, D. C., Anders, D. G. & Wong, S. W. (2003). Complete sequence and genomic analysis of rhesus cytomegalovirus. J Virol 77, 6620–6636.
Huff, J. L., Eberle, R., Capitanio, J., Zhou, S.-S. & Barry, P. A. (2003). Differential detection of B virus and rhesus cytomegalovirus in rhesus macaques. J Gen Virol 84, 83–92.
Jin, X., Demoitie, M. A., Donahoe, S. M. & 13 other authors (2000). High frequency of cytomegalovirus-specific cytotoxic T-effector cells in HLA-A*0201-positive subjects during multiple viral coinfections. J Infect Dis 181, 165–175.[CrossRef][Medline]
Jonji, S., del Val, M., Keil, G. M., Reddehase, M. J. & Koszinowski, U. H. (1988). A nonstructural viral protein expressed by a recombinant vaccinia virus protects against lethal cytomegalovirus infection. J Virol 62, 1653–1658.
Kaur, A., Daniel, M. D., Hempel, D., Lee-Parritz, D., Hirsch, M. S. & Johnson, R. P. (1996). Cytotoxic T-lymphocyte responses to cytomegalovirus in normal and simian immunodeficiency virus-infected macaques. J Virol 70, 7725–7733.[Abstract]
Kaur, A., Hale, C. L., Noren, B., Kassis, N., Simon, M. A. & Johnson, R. P. (2002). Decreased frequency of cytomegalovirus (CMV)-specific CD4+ T lymphocytes in simian immunodeficiency virus-infected rhesus macaques: inverse relationship with CMV viremia. J Virol 76, 3646–3658.
Kaur, A., Kassis, N., Hale, C. L. & 9 other authors (2003). Direct relationship between suppression of virus-specific immunity and emergence of cytomegalovirus disease in simian AIDS. J Virol 77, 5749–5758.
Kirchoff, V., Wong, S., St Jeor, S. & Pari, G. S. (2002). Generation of a life-expanded rhesus monkey fibroblast cell line for the growth of rhesus rhadinovirus (RRV). Arch Virol 147, 321–333.[CrossRef][Medline]
Li, C.-R., Greenberg, P. D., Gilbert, M. J., Goodrich, J. M. & Riddell, S. R. (1994). Recovery of HLA-restricted cytomegalovirus (CMV)-specific T-cell responses after allogeneic bone marrow transplant: correlation with CMV disease and effect of ganciclovir prophylaxis. Blood 83, 1971–1979.
Lockridge, K. M., Sequar, G., Zhou, S. S., Yue, Y., Mandell, C. P. & Barry, P. A. (1999). Pathogenesis of experimental rhesus cytomegalovirus infection. J Virol 73, 9576–9583.
Loomis-Huff, J. E., Eberle, R., Lockridge, K. M., Rhodes, G. & Barry, P. A. (2001). Immunogenicity of a DNA vaccine against herpes B virus in mice and rhesus macaques. Vaccine 19, 4865–4873.[CrossRef][Medline]
Manley, T. J., Luy, L., Jones, T., Boeckh, M., Mutimer, H. & Riddell, S. R. (2004). Immune evasion proteins of human cytomegalovirus do not prevent a diverse CD8+ cytotoxic T-cell response in natural infection. Blood 104, 1075–1082.
McGregor, A., Liu, F. & Schleiss, M. R. (2004). Molecular, biological, and in vivo characterization of the guinea pig cytomegalovirus (CMV) homologs of the human CMV matrix proteins pp71 (UL82) and pp65 (UL83). J Virol 78, 9872–9889.
McLaughlin-Taylor, E., Pande, H., Forman, S. J., Tanamachi, B., Li, C. R., Zaia, J. A., Greenberg, P. D. & Riddell, S. R. (1994). Identification of the major late human cytomegalovirus matrix protein pp65 as a target antigen for CD8+ virus-specific cytotoxic T lymphocytes. J Med Virol 43, 103–110.[Medline]
Morello, C. S., Cranmer, L. D. & Spector, D. H. (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., Ye, M., Hung, S., Kelley, L. A. & Spector, D. H. (2005). Systemic priming-boosting immunization with a trivalent plasmid DNA and inactivated murine cytomegalovirus (MCMV) vaccine provides long-term protection against viral replication following systemic or mucosal MCMV challenge. J Virol 79, 159–175.
Nakai, K. & Horton, P. (1999). PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization. Trends Biochem Sci 24, 34–36.[CrossRef][Medline]
Nakai, K. & Kanehisa, M. (1992). A knowledge base for predicting protein localization sites in eukaryotic cells. Genomics 14, 897–911.[CrossRef][Medline]
Nigro, G., Adler, S. P., La Torre, R. & Best, A. M. (2005). Passive immunization during pregnancy for congenital cytomegalovirus infection. N Engl J Med 353, 1350–1362.
Ohlin, M., Plachter, B., Sundqvist, V.-A., Steenbakkers, P. G. A., Middeldorp, J. M. & Borrebaeck, C. A. K. (1995). Human antibody reactivity against the lower matrix protein (pp65) produced by cytomegalovirus. Clin Diagn Lab Immunol 2, 325–329.
Pande, N. T., Powers, C., Ahn, K. & Früh, K. (2005). Rhesus cytomegalovirus contains functional homologues of US2, US3, US6, and US11. J Virol 79, 5786–5798.
Penedo, M. C. T., Bontrop, R. E., Heijmans, C. M. C. & 7 other authors (2005). Microsatellite typing of the rhesus macaque MHC region. Immunogenetics 57, 198–209.[CrossRef][Medline]
Reusser, P., Riddell, S. R., Meyers, J. D. & Greenberg, P. D. (1991). Cytotoxic T-lymphocyte response to cytomegalovirus after human allogeneic bone marrow transplantation: pattern of recovery and correlation with cytomegalovirus infection and disease. Blood 78, 1373–1380.
Reusser, P., Attenhofer, R., Hebart, H., Helg, C., Chapuis, B. & Einsele, H. (1997). Cytomegalovirus-specific T-cell immunity in recipients of autologous peripheral blood stem cell or bone marrow transplants. Blood 89, 3873–3879.
Reusser, P., Cathomas, G., Attenhofer, R., Tamm, M. & Thiel, G. (1999). Cytomegalovirus (CMV)-specific T cell immunity after renal transplantation mediates protection from CMV disease by limiting the systemic virus load. J Infect Dis 180, 247–253.[CrossRef][Medline]
Riddell, S. R., Watanabe, K. S., Goodrich, J. M., Li, C. R., Agha, M. E. & Greenberg, P. D. (1992). Restoration of viral immunity in immunodeficient humans by the adoptive transfer of T cell clones. Science 257, 238–241.
Sanchez, V., Angeletti, P. C., Engler, J. A. & Britt, W. J. (1998). Localization of human cytomegalovirus structural proteins to the nuclear matrix of infected human fibroblasts. J Virol 72, 3321–3329.
Schleiss, M. R. & Heineman, T. C. (2005). Progress toward an elusive goal: current status of cytomegalovirus vaccines. Expert Rev Vaccines 4, 381–406.[CrossRef][Medline]
Schleiss, M. R., McGregor, A., Jensen, N. J., Erdem, G. & Aktan, L. (1999). Molecular characterization of the guinea pig cytomegalovirus UL83 (pp65) protein homolog. Virus Genes 19, 205–221.[CrossRef][Medline]
Schmolke, S., Drescher, P., Jahn, G. & Plachter, B. (1995). Nuclear targeting of the tegument protein pp65 (UL83) of human cytomegalovirus: an unusual bipartite nuclear localization signal functions with other portions of the protein to mediate its efficient nuclear transport. J Virol 69, 1071–1078.[Abstract]
Sequar, G., Britt, W. J., Lakeman, F. D., Lockridge, K. M., Tarara, R. P., Canfield, D. R., Zhou, S.-S., Gardner, M. B. & Barry, P. A. (2002). Experimental coinfection of rhesus macaques with rhesus cytomegalovirus and simian immunodeficiency virus: pathogenesis. J Virol 76, 7661–7671.
Sindre, H., Rollag, H., Degré, M. & Hestdal, K. (2000). Human cytomegalovirus induced inhibition of hematopoietic cell line growth is initiated by events taking place before translation of viral gene products. Arch Virol 145, 99–111.[CrossRef][Medline]
Sylwester, A. W., Mitchell, B. L., Edgar, J. B. & 9 other authors (2005). Broadly targeted human cytomegalovirus-specific CD4+ and CD8+ T cells dominate the memory compartments of exposed subjects. J Exp Med 202, 673–685.
van Zanten, J., Harmsen, M. C., van der Giessen, M., van der Bij, W., Prop, J., de Leij, L. & The, T. H. (1995). Humoral immune response against human cytomegalovirus (HCMV)-specific proteins after HCMV infection in lung transplantation as detected with recombinant and naturally occurring proteins. Clin Diagn Lab Immunol 2, 214–218.
Vogel, P., Weigler, B. J., Kerr, H., Hendrickx, A. & Barry, P. A. (1994). Seroepidemiologic studies of cytomegalovirus infection in a breeding population of rhesus macaques. Lab Anim Sci 44, 25–30.[Medline]
Walter, E. A., Greenberg, P. D., Gilbert, M. J., Finch, R. J., Watanabe, K. S., Thomas, E. D. & Riddell, S. R. (1995). Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor. N Engl J Med 333, 1038–1044.
Wang, Z., La Rosa, C., Mekhoubad, S. & 9 other authors (2004). Attenuated poxviruses generate clinically relevant frequencies of CMV-specific T cells. Blood 104, 847–856.
Wills, M. R., Carmichael, A. J., Mynard, K., Jin, X., Weekes, M. P., Plachter, B. & Sissons, J. G. P. (1996). The human cytotoxic T-lymphocyte (CTL) response to cytomegalovirus is dominated by structural protein pp65: frequency, specificity, and T-cell receptor usage of pp65-specific CTL. J Virol 70, 7569–7579.[Abstract]
Yao, Z.-Q., Gallez-Hawkins, G., Lomeli, N. A., Li, X., Molinder, K. M., Diamond, D. J. & Zaia, J. A. (2001). Site-directed mutation in a conserved kinase domain of human cytomegalovirus-pp65 with preservation of cytotoxic T lymphocyte targeting. Vaccine 19, 1628–1635.[CrossRef][Medline]
Ye, M., Morello, C. S. & Spector, D. H. (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.
Yue, Y., Zhou, S. S. & Barry, P. A. (2003). Antibody responses to rhesus cytomegalovirus glycoprotein B in naturally infected rhesus macaques. J Gen Virol 84, 3371–3379.
Received 9 September 2005;
accepted 29 November 2005.
This article has been cited by other articles:
|
|
 |
|
  A. E. Lilja, W. L. W. Chang, P. A. Barry, S. P. Becerra, and T. E. Shenk Functional Genetic Analysis of Rhesus Cytomegalovirus: Rh01 Is an Epithelial Cell Tropism Factor J. Virol., March 1, 2008; 82(5): 2170 - 2181. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Yue, A. Kaur, M. K. Eberhardt, N. Kassis, S. S. Zhou, A. F. Tarantal, and P. A. Barry Immunogenicity and Protective Efficacy of DNA Vaccines Expressing Rhesus Cytomegalovirus Glycoprotein B, Phosphoprotein 65-2, and Viral Interleukin-10 in Rhesus Macaques J. Virol., February 1, 2007; 81(3): 1095 - 1109. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||
