Cytomegalovirus antivirals and development of improved animal models

Abstract

Introduction: Cytomegalovirus (CMV) is a ubiquitous pathogen that establishes a lifelong asymptomatic infection in healthy individuals. Infection of immunesuppressed individuals causes serious illness. Transplant and AIDS patients are highly susceptible to CMV leading to life-threatening end-organ disease. Another vulnerable population is the developing fetus in utero, where congenital infection can result in surviving newborns with long-term developmental problems. There is no vaccine licensed for CMV and current antivirals suffer from complications associated with prolonged treatment. These include drug toxicity and emergence of resistant strains. There is an obvious need for new antivirals. Candidate intervention strategies are tested in controlled preclinical animal models but species specificity of human CMV precludes the direct study of the virus in an animal model.

Areas covered: This review explores the current status of CMV antivirals and development of new drugs. This includes the use of animal models and the development of new improved models such as humanized animal CMV and bioluminescent imaging of virus in animals in real time.

Expert opinion: Various new CMV antivirals are in development, some with greater spectrum of activity against other viruses. Although the greatest need is in the setting of transplant patients, there remains an unmet need for a safe antiviral strategy against congenital CMV. This is especially important as an effective CMV vaccine remains an elusive goal. In this regard, greater emphasis should be placed on suitable preclinical animal models and greater collaboration between industry and academia.

Figure 1. Diseases caused by cytomegalovirus

Solid line arrows indicate disease directly attributed to viral infection. Dotted lines indicate disease linked to CMV in association with other factors. The link between CMV and certain cancers remains controversial but growing epidemiological data supports a link to cardiovascular disease.

Figure 2. Antivirals against cytomegalovirus and modes of action

Currently there are 4 antivirals that are used in the clinic for the treatment of CMV (GCV, VGCV, FOS and CDV). GCV is the most commonly used drug and VGCV is a prodrug that can be given orally. Various CMV antiviral under development or currently in use target different aspects of viral DNA synthesis/ packaging in virus infected cells (see text for more details). Mutations in HCMV genes result in resistance to specific antivirals. The antiviral targets are UL97 (viral kinase), viral DNA polymerase (UL54 and UL44 complex) and viral terminase (UL56, UL89 and UL104). Antiviral targets where codon mutations lead to specific drug resistance are indicated as UL97, UL54, UL89, UL44 and UL27. Antivirals under development include MBV, BAY compound (38-4766) and CPV (CDV prodrug). Prodrugs have similar mechanisms of action to their parent drugs once activated but have the advantage of better bioavailability. Other antiviral strategies are mentioned in the text but are not shown (see sections 2 & 3). Figure based on Michel and Mertens (198).

Figure 3. Viral genome structure of human and animal cytomegalovirus

(i) Comparative genome structures of HCMV, mouse (MCMV) and guinea pig (GPCMV) cytomegalovirus. (ii) Comparative % G+C content along the length of HCMV, MCMV and GPCMV.

Figure 4. Generation of UL97 chimeric GPCMV encoding wild type or mutant versions of HCMV UL97

An infectious GPCMV BAC was modified in bacteria to delete the UL97 homolog gene (GP97) and introduce in place the HCMV UL97 ORF under GP97 promoter control. (i) Mutagenesis of the GPCMV BAC and generation of infectious virus. 1. A GP97 locus shuttle vector was modified by the insertion of the UL97 coding sequence from HCMV Towne strain. Bacteria (DH10B) carrying the GPCMV BAC were modified by introduction of a plasmid encoding red ET recombination to temporally induce recombination positive conditions in bacteria (2 and 3). Linearized UL97 chimeric plasmid shuttle vector is introduced into bacteria (4) and bacterial colonies carrying modified BAC are selected by insertion of kanamycin (Km) drug marker co-inserted into the GP97 locus along with the UL97 coding sequences. Selection is made at a higher temperature (31°C switched 39°C) to remove the ts ET plasmid (5). Chimeric UL97 GPCMV BAC DNA is characterized and subsequently transfected onto guinea pig fibroblast cells (GPL) to generate virus (6). (ii) Left, viral spread across cell monolayer detected by GFP reporter gene expression. Right, western blot analysis of chimeric virus infected cell lysate (UL97GPCMV) or control uninfected cell lysate (mi) vs wild type GPCMV (wt GPCMV) or GP97 deletion mutant (GP97 del) demonstrated a band corresponding to UL97 protein in the chimeric virus infected cell lysate corresponding to a similar sized band in HCMV infected cell lysate. (iii). GCV antiviral plaque reduction assays of wild type GPCMV and UL97 chimeric GPCMV. Antiviral plaque reduction assay demonstrates that the UL97 chimeric GPCMV has modified susceptibility to antiviral GCV in comparison to wild type GPCMV. Assay was carried out as described in McGregor et al. (2008). The IC₅₀ values of both viruses are indicated. (iv) GCV antiviral plaque reduction assays of wild type and mutant UL97 chimeric GPCMV. A chimeric UL97 GPCMV was generated that contained a common UL97 codon mutation commonly found in GCV resistant HCMV strains. Original chimeric virus (UL97 GPCMV) is sensitive to GCV in contrast the mutant UL97 (L595S GPCMV) chimera (codon 595 change L to S) is resistant to GCV with modified IC₅₀ value compared to wild type chimera.

Figure 4. Generation of UL97 chimeric GPCMV encoding wild type or mutant versions of HCMV UL97

An infectious GPCMV BAC was modified in bacteria to delete the UL97 homolog gene (GP97) and introduce in place the HCMV UL97 ORF under GP97 promoter control. (i) Mutagenesis of the GPCMV BAC and generation of infectious virus. 1. A GP97 locus shuttle vector was modified by the insertion of the UL97 coding sequence from HCMV Towne strain. Bacteria (DH10B) carrying the GPCMV BAC were modified by introduction of a plasmid encoding red ET recombination to temporally induce recombination positive conditions in bacteria (2 and 3). Linearized UL97 chimeric plasmid shuttle vector is introduced into bacteria (4) and bacterial colonies carrying modified BAC are selected by insertion of kanamycin (Km) drug marker co-inserted into the GP97 locus along with the UL97 coding sequences. Selection is made at a higher temperature (31°C switched 39°C) to remove the ts ET plasmid (5). Chimeric UL97 GPCMV BAC DNA is characterized and subsequently transfected onto guinea pig fibroblast cells (GPL) to generate virus (6). (ii) Left, viral spread across cell monolayer detected by GFP reporter gene expression. Right, western blot analysis of chimeric virus infected cell lysate (UL97GPCMV) or control uninfected cell lysate (mi) vs wild type GPCMV (wt GPCMV) or GP97 deletion mutant (GP97 del) demonstrated a band corresponding to UL97 protein in the chimeric virus infected cell lysate corresponding to a similar sized band in HCMV infected cell lysate. (iii). GCV antiviral plaque reduction assays of wild type GPCMV and UL97 chimeric GPCMV. Antiviral plaque reduction assay demonstrates that the UL97 chimeric GPCMV has modified susceptibility to antiviral GCV in comparison to wild type GPCMV. Assay was carried out as described in McGregor et al. (2008). The IC₅₀ values of both viruses are indicated. (iv) GCV antiviral plaque reduction assays of wild type and mutant UL97 chimeric GPCMV. A chimeric UL97 GPCMV was generated that contained a common UL97 codon mutation commonly found in GCV resistant HCMV strains. Original chimeric virus (UL97 GPCMV) is sensitive to GCV in contrast the mutant UL97 (L595S GPCMV) chimera (codon 595 change L to S) is resistant to GCV with modified IC₅₀ value compared to wild type chimera.

Figure 5. Strategy for bioluminescence imaging of virus infected animals

(i) Bioluminescent signal from cells. Firefly luciferase enzyme expressed in cells acts on specific substrate (D-luciferin) to release light photons which can be detected by a CCD camera above the imaging chamber of the Xenogen/Kalipar imaging apparatus and software enables overlay of detected photon intensity over a still black and white image of the subject. (ii) Imaging procedure.1. Bioluminescence imaging station (Xenogen IVIS 50). 2. Sedated animal is placed in imaging chamber and imaging acquired over a set period of time (usually 5 min.). 3. Photons emitted from the animal is detected and software converts results into intensity of light emission over a black and white image of the animal. Bioluminescence measured in photons per second per centimeter square per steradian. (iii) BLI of GPCMV dissemination in the guinea pig. Neonatal guinea pigs infected with recombinant luciferase tagged GPCMV (1× 10⁶ pfu via intraperitoneal route). Virus infection that starts in the spleen progresses to the liver, lungs and eventually to the brain. Bioluminescence image A and B of the same animal from above. A. upper body and head. B. lower torso. C. Two animals side image upper body and head. All images taken at 7 days post infection with 5 min. imaging. Animals injected with 0.2 ml D-luciferin (30 mg/ml) and sedated prior to imaging. Animals were handled following University of Minnesota IACUC guidelines. Highest levels of bioluminescence appear in red as indicated in side bar. Bioluminescence measured in photons per second per centimeter square per steradian.

Figure 5. Strategy for bioluminescence imaging of virus infected animals

(i) Bioluminescent signal from cells. Firefly luciferase enzyme expressed in cells acts on specific substrate (D-luciferin) to release light photons which can be detected by a CCD camera above the imaging chamber of the Xenogen/Kalipar imaging apparatus and software enables overlay of detected photon intensity over a still black and white image of the subject. (ii) Imaging procedure.1. Bioluminescence imaging station (Xenogen IVIS 50). 2. Sedated animal is placed in imaging chamber and imaging acquired over a set period of time (usually 5 min.). 3. Photons emitted from the animal is detected and software converts results into intensity of light emission over a black and white image of the animal. Bioluminescence measured in photons per second per centimeter square per steradian. (iii) BLI of GPCMV dissemination in the guinea pig. Neonatal guinea pigs infected with recombinant luciferase tagged GPCMV (1× 10⁶ pfu via intraperitoneal route). Virus infection that starts in the spleen progresses to the liver, lungs and eventually to the brain. Bioluminescence image A and B of the same animal from above. A. upper body and head. B. lower torso. C. Two animals side image upper body and head. All images taken at 7 days post infection with 5 min. imaging. Animals injected with 0.2 ml D-luciferin (30 mg/ml) and sedated prior to imaging. Animals were handled following University of Minnesota IACUC guidelines. Highest levels of bioluminescence appear in red as indicated in side bar. Bioluminescence measured in photons per second per centimeter square per steradian.