CD8-positive T lymphocytes specific for murine cytomegalovirus immediate-early antigens mediate protective immunity.

We have shown in a murine model system for acute, lethal cytomegalovirus (CMV) disease in the immunocompromised natural host that control of virus multiplication in tissues, protection from virus-caused tissue destruction, and survival are mediated by virus-specific CD8+ CD4-T lymphocytes. Protection from a lethal course of disease did not result in a rapid establishment of virus latency, but led to a long-lasting, persistent state of infection. The CD8- CD4+ subset of T lymphocytes was not effective by itself in controlling murine CMV (MCMV) multiplication in tissue or essential for the protective function of the CD8+ CD4- effector cells. The antiviral efficacy of the purified CD8+ CD4- subset was not impaired by preincubation with fibroblasts that presented viral structural antigens, but was significantly reduced after depletion of effector cells specific for the nonstructural immediate-early antigens of MCMV, which are specified by the first among a multitude of viral genes expressed during MCMV replication in permissive cells. Thus, MCMV disease provides the first example of a role for nonstructural herpesvirus immediate-early antigens in protective immunity.     

Mucosal and Parenteral Vaccination against Acute and Latent Murine Cytomegalovirus (MCMV) Infection by Using an Attenuated MCMV Mutant

We used a live attenuated murine cytomegalovirus (MCMV) mutant to analyze mechanisms of vaccination against acute and latent CMV infection. We selected MCMV mutant RV7 as a vaccine candidate since this virus grows well in tissue culture but is profoundly attenuated for growth in normal and severe combined immunodeficient (SCID) mice (V. J. Cavanaugh et al., J. Virol. 70:1365–1374, 1996). BALB/c mice were immunized twice (0 and 14 days) subcutaneously (s.c.) with tissue culture-passaged RV7 and then challenged with salivary gland-passaged wild-type MCMV (sgMCMV) intraperitoneally (i.p.) on day 28. RV7 vaccination protected mice against challenge with 10⁵ PFU of sgMCMV, a dose that killed 100% of mock-vaccinated mice. RV7 vaccination reduced MCMV replication 100- to 500-fold in the spleen between 1 and 8 days after challenge. We used the capacity to control replication of MCMV in the spleen 4 days after challenge as a surrogate for protection. Protection was antigen specific and required both live RV7 and antigen-specific lymphocytes. Interestingly, RV7 was effective when administered s.c., i.p., perorally, intranasally, and intragastrically, demonstrating that attenuated CMV applied to mucosal surfaces can elicit protection against parenteral virus challenge. B cells and immunoglobulin G were not essential for RV7-induced immunity since B-cell-deficient mice were effectively vaccinated by RV7. CD8 T cells, but not CD4 T cells, were critical for RV7-induced protection. Depletion of CD8 T cells by passive transfer of monoclonal anti-CD8 (but not anti-CD4) antibody abrogated RV7-mediated protection, and RV7 vaccination was less efficient in CD8 T-cell-deficient mice with a targeted mutation in the β2-microglobulin gene. Although gamma interferon is important for innate resistance to MCMV, it was not essential for RV7 vaccination since gamma interferon receptor-deficient mice were protected by RV7 vaccination. Establishment of and/or reactivation from latency by sgMCMV was decreased by RV7 vaccination, as measured by diminished reactivation of MCMV from splenic explants. We found no evidence for establishment of splenic latency by RV7 after s.c. vaccination. We conclude that RV7 administered through both systemic and mucosal routes is an effective vaccine against MCMV infection. It may be possible to design human CMV vaccines with similar properties.     

Complete DNA sequence of the rat cytomegalovirus genome

We have determined the complete genome sequence of the Maastricht strain of rat cytomegalovirus (RCMV). The RCMV genome has a length of 229,896 bp and is arranged as a single unique sequence flanked by 504-bp terminal direct repeats. RCMV was found to have counterparts of all but one of the open reading frames (ORFs) that are conserved between murine CMV (MCMV) and human CMV (HCMV). Like HCMV, RCMV lacks homologs of the genes belonging to the MCMV m02 glycoprotein gene family. However, RCMV contains 15 ORFs with homology to members of the MCMV m145 glycoprotein gene family. Four ORFs are predicted to encode homologs of host proteins; R33 and R78 both putatively encode G protein-coupled receptors, whereas r144 and r131 encode homologs of major histocompatibility class I heavy chains and CC chemokines, respectively. An intriguing feature of the RCMV genome is the presence of an ORF, r127, with similarity to the rep gene of parvoviruses as well as ORF U94 of human herpesvirus 6A (HHV-6A) and HHV-6B. Counterparts of these ORFs have not been found in the other sequenced herpesviruses.     

Vaccination of mice with bacteria carrying a cloned herpesvirus genome reconstituted in vivo

Bacterial delivery systems are gaining increasing interest as potential vaccination vectors to deliver either proteins or nucleic acids for gene expression in the recipient. Bacterial delivery systems for gene expression in vivo usually contain small multicopy plasmids. We have shown before that bacteria containing a herpesvirus bacterial artificial chromosome (BAC) can reconstitute the virus replication cycle after cocultivation with fibroblasts in vitro. In this study we addressed the question of whether bacteria containing a single plasmid with a complete viral genome can also reconstitute the viral replication process in vivo. We used a natural mouse pathogen, the murine cytomegalovirus (MCMV), whose genome has previously been cloned as a BAC in Escherichia coli. In this study, we tested a new application for BAC-cloned herpesvirus genomes. We show that the MCMV BAC can be stably maintained in certain strains of Salmonella enterica serovar Typhimurium as well and that both serovar Typhimurium and E. coli harboring the single-copy MCMV BAC can reconstitute a virus infection upon injection into mice. By this procedure, a productive virus infection is regenerated only in immunocompromised mice. Virus reconstitution in vivo causes elevated titers of specific anti-MCMV antibodies, protection against lethal MCMV challenge, and strong expression of additional genes introduced into the viral genome. Thus, the reconstitution of infectious virus from live attenuated bacteria presents a novel concept for multivalent virus vaccines launched from bacterial vectors.     

Immunization with cytomegalovirus envelope glycoprotein M and glycoprotein N DNA vaccines can provide mice with complete protection against a lethal murine cytomegalovirus challenge

Human cytomegalovirus virions contain three major glycoprotein complexes (gC I, II, III), all of which are required for CMV infectivity. These complexes also represent major antigenic targets for anti-viral immune responses. The gC II complex consists of two glycoproteins, gM and gN. In the current study, DNA vaccines expressing the murine cytomegalovirus (MCMV) homologs of the gM and gN proteins were evaluated for protection against lethal MCMV infection in a mouse model. Humoral and cellular immune responses, spleen viral titers, and mice survival and body-weight changes were examined. The results showed that immunization with gM or gN DNA vaccine alone was not able to offer good protection, whereas co-immunization with both gM and gN induced an effective neutralizing antibody response and cellular immune response, and provided mice with complete protection against a lethal MCMV challenge. This study provides the first in vivo evidence that the gC II (gM-gN) complex may be able to serve as a protective subunit antigen for future HCMV vaccine development.     

Murine model for immunoprophylaxis of cytomegalovirus infection. I. Efficacy of immunization.

Murine cytomegalovirus was utilized as a model for human cytomegalovirus, which had no experimental animal, to study immunoprophylaxis of the cytomegalovirus infections. (1) Murine cytomegalovirus (MCMV) serially propagated in mouse embryonic fibroblasts had lost pathogenicity for weanling mice including neonatally thymectomized mice. (2) The cell culture-adapted MCMV was effective as a "live, attenuated virus vaccine" against challenge by virulent, mouse-passaged MCMV. (3) The immunization via intraperitoneal route protected mice from every parameter of MCMV infection. These included clinical signs, virus replication, histopathology and mortality. (4) The protective immunity was active against the virulent MCMV which was not neutralized by the rabbit anti-attenuated MCMV serum.     

All is fair in virus-host interactions: NK cells and cytomegalovirus

The infection of mice with mouse cytomegalovirus (MCMV) as a model of human cytomegalovirus (HCMV) infection has been particularly informative in elucidating the role of innate and adaptive immune response mechanisms during infection. Millions of years of co-evolution between cytomegaloviruses (CMV) and their hosts has resulted in numerous attempts to overwhelm each other. CMVs devote many genes to modulating the host natural killer (NK) cell response and NK cells employ many strategies to cope with CMV infection. While focusing on these attack-counterattack measures, this review will discuss several novel mechanisms of immune evasion by MCMV, the role of Ly49 receptors in mediating resistance to MCMV, and the impact of the initial NK cell response on the shaping of adaptive immunity.     

Discrete clusters of virus-encoded micrornas are associated with complementary strands of the genome and the 7.2-kilobase stable intron in murine cytomegalovirus

The prevalence and importance of microRNAs (miRNAs) in viral infection are increasingly relevant. Eleven miRNAs were previously identified in human cytomegalovirus (HCMV); however, miRNA content in murine CMV (MCMV), which serves as an important in vivo model for CMV infection, has not previously been examined. We have cloned and characterized 17 novel miRNAs that originate from at least 12 precursor miRNAs in MCMV and are not homologous to HCMV miRNAs. In parallel, we applied a computational analysis, using a support vector machine approach, to identify potential precursor miRNAs in MCMV. Four of the top 10 predicted precursor sequences were cloned in this study, and the combination of computational and cloning analysis demonstrates that MCMV has the capacity to encode miRNAs clustered throughout the genome. On the basis of drug sensitivity experiments for resolving the kinetic class of expression, we show that the MCMV miRNAs are both early and late gene products. Notably, the MCMV miRNAs occur on complementary strands of the genome in specific regions, a feature which has not previously been observed for viral miRNAs. One cluster of miRNAs occurs in close proximity to the 5' splice site of the previously identified 7.2-kb stable intron, implying a variety of potential regulatory mechanisms for MCMV miRNAs.     

Suppression of murine cytomegalovirus (MCMV) replication with a DNA vaccine encoding MCMV M84 (a homolog of human cytomegalovirus pp65)

The cytotoxic T-lymphocyte (CTL) response against the murine cytomegalovirus (MCMV) immediate-early gene 1 (IE1) 89-kDa phosphoprotein pp89 plays a major role in protecting BALB/c mice against the lethal effects of the viral infection. CTL populations specific to MCMV early-phase and structural antigens are also generated during infection, but the identities of these antigens and their relative contributions to overall immunity against MCMV are not known. We previously demonstrated that DNA vaccination with a pp89-expressing plasmid effectively generated a CTL response and conferred protection against infection (J. C. Gonzalez Armas, C. S. Morello, L. D. Cranmer, and D. H. Spector, J. Virol. 70:7921-7928, 1996). In this report, we have sought (i) to identify other viral antigens that contribute to immunity against MCMV and (ii) to determine whether the protective response is haplotype specific. DNA immunization was used to test the protective efficacies of plasmids encoding MCMV homologs of human cytomegalovirus (HCMV) tegument (M32, M48, M56, M82, M83, M69, and M99), capsid (M85 and M86), and nonstructural antigens (IE1-pp89 and M84). BALB/c (H-2(d)) and C3H/HeN (H-2(k)) mice were immunized by intradermal injection of either single plasmids or cocktails of up to four expression plasmids and then challenged with sublethal doses of virulent MCMV administered intraperitoneally. In this way, we identified a new viral gene product, M84, that conferred protection against viral replication in the spleens of BALB/c mice. M84 is expressed early in the infection and encodes a nonstructural protein that shares significant amino acid homology with the HCMV UL83-pp65 tegument protein, a major target of protective CTLs in humans. Specificity of the immune response to the M84 protein was confirmed by showing that immunization with pp89 DNA, but not M84 DNA, protected mice against subsequent infection with an MCMV deletion mutant lacking the M84 gene. The other MCMV genes tested did not generate a protective response even when mice were immunized with vaccinia viruses expressing the viral proteins. However, the M84 plasmid was protective when injected in combination with nonprotective plasmids, and coimmunization of BALB/c mice with pp89 and M84 provided a synergistic level of protection in the spleen. Viral titers in the salivary glands were also reduced, but not to the same extent as observed in the spleen, and the decrease was seen only when the BALB/c mice were immunized with pp89 plus M84 or with pp89 alone. The experiments with the C3H/HeN mice showed that the immunity conferred by DNA vaccination was haplotype dependent. In this strain of mice, only pp89 elicited a protective response as measured by a reduction in spleen titer. These results suggest that DNA immunization with the appropriate combination of CMV genes may provide a strategy for improving vaccine efficacy.     

Deficient major histocompatibility complex-linked innate murine cytomegalovirus immunity in MA/My.L-H2b mice and viral downregulation of H-2k class I proteins

NK cells are key effectors of innate immunity and host survival during cytomegalovirus (CMV) infection. Innate murine CMV (MCMV) resistance in MA/My mice requires Ly49H/m157-independent H-2k-linked NK cell control. Here we show that replacement of MA/My H-2k with C57L H-2b susceptibility genes led to a remarkable loss of innate virus immunity, though NK gamma interferon was induced in H-2b and H-2k strains shortly after infection. Thus, H-2b genes expressed in C57L or MA/My.L-H2b are sufficient in alerting NK cells to intrusion but fail to support NK restraint of viral infection. In addition, novel H-2 recombinant strains were produced and utilized in a further refinement of a critical genetic interval controlling innate H-2k-linked MCMV resistance. Importantly, this analysis excluded the gene interval from Kk class I through class II. The responsible gene(s) therefore resides in an interval spanning Dk class Ia and more-distal major histocompatibility complex (MHC) nonclassical class Ib genes. Recently, the NK activation receptor Ly49P and MHC class I Dk proteins were genetically implicated in MCMV resistance, in part because Ly49P-expressing reporter T cells could specifically bind Dk molecules on MCMV-infected mouse embryonic fibroblasts (MEFs). However, as we found that H-2k innate resistance differs in the C57L or MA/My backgrounds and because MCMV very efficiently downregulates H-2k class I proteins in L929 cells and primary MEFs shortly after infection, a Ly49P/Dk model should not fully explain H-2k-linked MCMV resistance.     

Complete Protection of Mice against Lethal Murine Cytomegalovirus Challenge by Immunization with DNA Vaccines Encoding Envelope Glycoprotein Complex III Antigens gH,gL and gO

Human cytomegalovirus infects the majority of humanity which may lead to severe morbidity and mortality in newborns and immunocompromised adults. Humoral and cellular immunity are critical for controlling CMV infection. HCMV envelope glycoprotein complexes (gC I, II, III) represent major antigenic targets of antiviral immune responses. The gCIII complex is comprised of three glycoproteins, gH, gL, and gO. In the present study, DNA vaccines expressing the murine cytomegalovirus homologs of the gH, gL, and gO proteins were evaluated for protection against lethal MCMV infection in the mouse model. The results demonstrated that gH, gL, or gO single gene immunization could not yet offer good protection, whereas co-vaccination strategy apparently showed effects superior to separate immunization. Twice immunization with gH/gL/gO pDNAs could provide mice complete protection against lethal salivary gland-derived MCMV (SG-MCMV) challenge, while thrice immunization with pgH/pgL, pgH/pgO or pgL/pgO could not provide full protection. Co-vaccination with gH, gL and gO pDNAs elicited robust neutralizing antibody and cellular immune responses. Moreover, full protection was also achieved by simply passive immunization with anti-gH/gL/gO sera. These data demonstrated that gCIII complex antigens had fine immunogenicity and might be a promising candidate for the development of HCMV vaccines.     

Requisite H2k role in NK cell-mediated resistance in acute murine cytomegalovirus-infected MA/My mice

Human CMV infections are a major health risk in patients with dysfunctional or compromised immunity, especially in patients with NK cell deficiencies, as these are frequently associated with high morbidity and mortality. In experimental murine CMV (MCMV) infections, Ly49H activation receptors on C57BL/6 (B6) NK cells engage m157 viral ligands on MCMV-infected cells and initiate dominant virus control. In this study, we report that MCMV resistance in MA/My relies on Ly49H-independent NK cell-mediated control of MCMV infection as NK cells in these mice do not bind anti-Ly49H mAb or soluble m157 viral ligands. We genetically compared MA/My resistance with MCMV susceptibility in genealogically and NK gene complex-Ly49 haplotype-related C57L mice. We found that MCMV resistance strongly associated with polymorphic H2k-linked genes, including MHC and non-MHC locations by analysis of backcross and intercross progeny. The H2b haplotype most frequently, but not absolutely, correlated with MCMV susceptibility, thus confirming a role for non-MHC genes in MCMV control. We also demonstrate a definite role for NK cells in H2k-type MCMV resistance because their removal from C57L.M-H2k mice before MCMV infection diminished immunity. NK gene complex-linked polymorphisms, however, did not significantly influence MCMV control. Taken together, effective NK cell-mediated MCMV control in this genetic system required polymorphic H2k genes without need of Ly49H-m157 interactions.     

Cytomegalovirus Replicon-Based Regulation of Gene Expression In Vitro and In Vivo

There is increasing evidence for a connection between DNA replication and the expression of adjacent genes. Therefore, this study addressed the question of whether a herpesvirus origin of replication can be used to activate or increase the expression of adjacent genes. Cell lines carrying an episomal vector, in which reporter genes are linked to the murine cytomegalovirus (MCMV) origin of lytic replication (oriLyt), were constructed. Reporter gene expression was silenced by a histone-deacetylase-dependent mechanism, but was resolved upon lytic infection with MCMV. Replication of the episome was observed subsequent to infection, leading to the induction of gene expression by more than 1000-fold. oriLyt-based regulation thus provided a unique opportunity for virus-induced conditional gene expression without the need for an additional induction mechanism. This principle was exploited to show effective late trans-complementation of the toxic viral protein M50 and the glycoprotein gO of MCMV. Moreover, the application of this principle for intracellular immunization against herpesvirus infection was demonstrated. The results of the present study show that viral infection specifically activated the expression of a dominant-negative transgene, which inhibited viral growth. This conditional system was operative in explant cultures of transgenic mice, but not in vivo. Several applications are discussed.     

A replicating cytomegalovirus-based vaccine encoding a single Ebola virus nucleoprotein CTL epitope confers protection against Ebola virus

Background

Human outbreaks of Ebola virus (EBOV) are a serious human health concern in Central Africa. Great apes (gorillas/chimpanzees) are an important source of EBOV transmission to humans due to increased hunting of wildlife including the ‘bush-meat’ trade. Cytomegalovirus (CMV) is an highly immunogenic virus that has shown recent utility as a vaccine platform. CMV-based vaccines also have the unique potential to re-infect and disseminate through target populations regardless of prior CMV immunity, which may be ideal for achieving high vaccine coverage in inaccessible populations such as great apes.

Methodology/Principal Findings

We hypothesize that a vaccine strategy using CMV-based vectors expressing EBOV antigens may be ideally suited for use in inaccessible wildlife populations. To establish a ‘proof-of-concept’ for CMV-based vaccines against EBOV, we constructed a mouse CMV (MCMV) vector expressing a CD8⁺ T cell epitope from the nucleoprotein (NP) of Zaire ebolavirus (ZEBOV) (MCMV/ZEBOV-NP_CTL). MCMV/ZEBOV-NP_CTL induced high levels of long-lasting (>8 months) CD8⁺ T cells against ZEBOV NP in mice. Importantly, all vaccinated animals were protected against lethal ZEBOV challenge. Low levels of anti-ZEBOV antibodies were only sporadically detected in vaccinated animals prior to ZEBOV challenge suggesting a role, at least in part, for T cells in protection.

Conclusions/Significance

This study demonstrates the ability of a CMV-based vaccine approach to protect against an highly virulent human pathogen, and supports the potential for ‘disseminating’ CMV-based EBOV vaccines to prevent EBOV transmission in wildlife populations.     

Cross-Presentation of a Spread-Defective MCMV Is Sufficient to Prime the Majority of Virus-Specific CD8+ T Cells

CD8+ T cells can be primed by peptides derived from endogenous proteins (direct presentation), or exogenously acquired protein (cross-presentation). However, the relative ability of these two pathways to prime CD8+ T cells during a viral infection remains controversial. Cytomegaloviruses (CMVs) can infect professional antigen presenting cells (APCs), including dendritic cells, thus providing peptides for direct presentation. However, the viral immune evasion genes profoundly impair recognition of infected cells by CD8+ T cells. Nevertheless, CMV infection elicits a very strong CD8+ T cell response, prompting its recent use as a vaccine vector. We have shown previously that deleting the immune evasion genes from murine cytomegalovirus (MCMV) that target class I MHC presentation, has no impact on the size or breadth of the CD8+ T cell response elicited by infection, suggesting that the majority of MCMV-specific CD8+ T cells in vivo are not directly primed by infected professional APCs. Here we use a novel spread-defective mutant of MCMV, lacking the essential glycoprotein gL, to show that cross-presentation alone can account for the majority of MCMV-specific CD8+ T cell responses to the virus. Our data support the conclusion that cross-presentation is the primary mode of antigen presentation by which CD8+ T cells are primed during MCMV infection.     

Inhibition of the TRAIL Death Receptor by CMV Reveals Its Importance in NK Cell-Mediated Antiviral Defense

TNF-related apoptosis inducing ligand (TRAIL) death receptors (DR) regulate apoptosis and inflammation, but their role in antiviral defense is poorly understood. Cytomegaloviruses (CMV) encode many immune-modulatory genes that shape host immunity, and they utilize multiple strategies to target the TNF-family cytokines. Here we show that the m166 open reading frame (orf) of mouse CMV (MCMV) is strictly required to inhibit expression of TRAIL-DR in infected cells. An MCMV mutant lacking m166 expression (m166^stop) is severely compromised for replication in vivo, most notably in the liver, and depleting natural killer (NK) cells, or infecting TRAIL-DR^−/− mice, restored MCMV-m166^stop replication completely. These results highlight the critical importance for CMV to have evolved a strategy to inhibit TRAIL-DR signaling to thwart NK-mediated defenses.     

Rapid identification of essential and nonessential herpesvirus genes by direct transposon mutagenesis

Herpesviruses are important pathogens in animals and humans. The large DNA genomes of several herpesviruses have been sequenced, but the function of the majority of putative genes is elusive. Determining which genes are essential for their replication is important for identifying potential chemotherapy targets, designing herpesvirus vectors, and generating attenuated vaccines. For this purpose, we recently reported that herpesvirus genomes can be maintained as infectious bacterial artificial chromosomes (BAC) in Escherichia coli. Here we describe a one-step procedure for random-insertion mutagenesis of a herpesvirus BAC using a Tn1721-based transposon system. Transposon insertion sites were determined by direct sequencing, and infectious virus was recovered by transfecting cultured cells with the mutant genomes. Lethal mutations were rescued by cotransfecting cells containing noninfectious genomes with the corresponding wild-type subgenomic fragments. We also constructed revertant genomes by allelic exchange in bacteria. These methods, which are generally applicable to any cloned herpesvirus genome, will facilitate analysis of gene function for this virus family.