The efficacy of 2-amino-7-[(1,3-dihydroxy-2-propoxy)methyl]purine (S2242) was evaluated in several animal models for herpesvirus infections. Compound S2242 was more effective than acyclovir (i) when administered subcutaneously in a model for herpes simplex virus type 1 (HSV-1)-induced mortality in immunocompetent mice and (ii) when applied topically to hairless (hr/hr) mice that had been infected intracutaneously with HSV-2. In SCID (severe combined immune deficient) mice that had been infected with a thymidine kinase-deficient HSV-1 strain, S2242 (administered subcutaneously at a dosage of 50 mg/kg/day) completely protected against virus-induced mortality whereas foscarnet was less effective and acyclovir had no or little protective effect. Compound S2242 was far more effective than ganciclovir in preventing or delaying murine cytomegalovirus-induced mortality in immunocompetent and SCID mice. The compound was more effective when a given dose was fractionated and administered on subsequent days than when this dose was administered in one single injection. A 5-day treatment course with S2242 (10 and 50 mg/kg/day) for newborn mice that had been infected with a lethal dose of murine cytomegalovirus suppressed virus-induced mortality. Compound S2242 had no inhibitory effect on the growth of weanling (at 50 mg/kg for 5 days) and 3- to 4-week-old mice (at doses of 50 to 200 mg/kg for 6 weeks). However, akin to ganciclovir, compound S2242 significantly reduced testicle weight, testicle morphology, and spermatogenesis.
2-Amino-7-[(1,3-dihydroxy-2-propoxy)methyl]purine (compound S2242) represents the first antivirally active nucleoside analog with the side chain attached to the N-7 position of the purine ring. Compound S2242 strongly inhibits the in vitro replication of both herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) (50% effective concentration [EC50], 0.1 to 0.2 microgram/ml), varicella-zoster virus (EC50, 0.01 to 0.02 microgram/ml) and thymidine kinase (TK)-deficient strains of HSV (EC50, 0.4 microgram/ml) and varicella-zoster virus (EC50, 0.2 to 0.5 microgram/ml). Potent activity was also observed against murine cytomegalovirus (EC50, 1 microgram/ml), human cytomegalovirus (HCMV) (EC50, 0.04 to 0.1 microgram/ml), and human herpesvirus 6 (EC50, 0.0005 microgram/ml). Compound S2242 (i) was not cytotoxic to confluent Vero, HeLa, or human fibroblast cells at concentrations of > 100 micrograms/ml, (ii) proved somewhat more cytostatic to Vero, HEL, HeLa, and C127I cells than ganciclovir, and (iii) was markedly more cytostatic than ganciclovir to the growth of the human lymphocytic cell lines HSB-2 and CEM degrees. In contrast to ganciclovir, (i) compound S2242 proved not to be cytocidal to murine mammary carcinoma (FM3A) cells transfected with the HSV-1 or HSV-2 TK gene, (ii) exogenously added thymidine had only a limited effect on its anti-HSV-1 activity, and (iii) the compound was not phosphorylated by HSV-1-encoded TK derived from HSV-1 TK-transfected FM3A cells, indicating that the compound is not activated by a virally encoded TK. Compound S2242 inhibited (i) the expression of late HCHV antigens at an EC50 of 0.07 microgram/ml (0.6 microgram/ml for ganciclovir) and (ii) HCMV DNA synthesis at an EC50 of 0.1 microgram/ml (0.32 microgram/ml for ganciclovir), i.e., values that are close to the EC50S for inhibition of HCMV-induced cytopathogenicity. Neither ganciclovir nor S2242 had any effect on the expression of immediate-early HCMV antigens, which occurs before viral DNA synthesis. In time-of-addition experiments, S2242 behaved like ganciclovir and acyclovir; i.e., the addition of the drugs could be delayed until the onset of viral DNA synthesis.
The importance of human adenovirus infections in immunocompromised patients urges for new and adequate antiadenovirus compounds. Since human adenoviruses are species specific, animal models for systemic adenovirus infections rely on a nonhuman adenovirus. We established mouse adenovirus type 1 (MAV-1) infection of BALB/c SCID mice as a model for the evaluation of antiadenovirus therapy. In vitro studies with mouse embryonic fibroblasts pointed to the acyclic nucleoside phosphonate cidofovir and the N-7-substituted acyclic derivative 2-amino-7-(1,3-dihydroxy-2-propoxymethyl)purine (S-2242) as markedly active compounds against MAV-1. SCID mice, infected intranasally with MAV-1, developed a fatal disseminated infection after approximately 19 days, characterized by hemorrhagic enteritis. Several techniques were optimized to monitor viral, immunological, and pathological aspects of MAV-1 infection. Real-time PCR quantification of viral DNA revealed that after replication in the lungs, virus disseminated to several organs, including the brain, liver, spleen, intestine, heart, and kidneys (resulting in viruria). Immunohistochemical staining showed that MAV-1 was localized in the endothelial cells of the affected organs. Using reverse transcription-PCR, tissue levels of proinflammatory cytokines (i.e., interleukin-1β and tumor necrosis factor alpha) were found to be markedly increased. The MAV-1/SCID model appears to be an appropriate model for in vivo evaluation of antiadenovirus agents. Treatment with cidofovir or S-2242 at a dose of 100 mg per kg of body weight resulted in a significant delay in MAV-1-related death, although these antivirals were unable to completely suppress virus replication despite continued drug treatment. These findings suggest that complete virus clearance during antiviral therapy for disseminated adenovirus infection may require an efficient adaptive immune response from the host.
Compound 2242, also known as 2-amino-7-[(1,3-dihydroxy-2-propoxy)methyl]purine, is the first known antivirally active nucleoside analog with the side chain substituted at the N-7 position of the purine ring system. Our purpose was to evaluate its retinal toxicity and assess the efficacy of its highest nontoxic concentration in a rabbit model of herpes simplex retinitis. Concentrations of the drug from 0.5 to 2,000 microM were injected intravitreally in twelve New Zealand White rabbits. Fundoscopic, histologic, and electrophysiologic data revealed no evidence of toxicity even at the highest dose of the compound. Dutch pigmented rabbits (n = 34) had their left eyes injected with herpes simplex virus type 1 3 days after, concurrently, or 3 days before intravitreal injection of either 2,000 microM compound 2242 or 480 microM ganciclovir (final concentration in the eye). Both compound 2242 and ganciclovir were equally effective compared with saline when administered simultaneously with the virus (P < 0.0001). In the 3-day pretreatment paradigm, compound 2242 was superior to ganciclovir (P < 0.04), but there was no clear difference between the two with regard to their effects on an established infection. The pharmacokinetics of compound 2242 in 10 rabbits injected intravitreally with 30 microM showed an intravitreal half-life of 8 h. This compound, which may be orally active in its pro form, has a very high therapeutic index in the eye and is more efficient than ganciclovir in this animal model of herpes retinitis.
Resistant herpes simplex virus type 1 strains were obtained under the selective pressure of acyclovir, ganciclovir, bromovinyldeoxyuridine, foscarnet, 2-phosphonylmethoxyehtyl (PME) derivatives of adenine and 2,6-diaminopurine, 3-hydroxy-2-phosphonylmethoxypropyl derivatives of adenine and cytosine, and 2-amino-7-(1,3-dihydroxy-2-propoxymethyl)purine (S2242). The drug susceptibility profiles of resistant strains point to differences in the modes of action of PME and 3-hydroxy-2-phosphonylmethoxypropyl derivatives and common mechanisms of action of foscarnet, S2242, and PME derivatives against herpes simplex virus type 1 replication.
Poxviruses are large DNA viruses that replicate in the cytoplasm of infected cells. Myxoma virus is a rabbit poxvirus that belongs to the Leporipoxvirus genus. It causes a lethal disease called myxomatosis in European rabbits but cannot sustain any detectable infection in nonlagomorphs. Vaccinia virus is a prototypal orthopoxvirus that was used as a vaccine to eradicate smallpox. Myxoma virus is nonpathogenic in mice, whereas systemic infection with vaccinia virus can be lethal even in immunocompetent mice. Plasmacytoid dendritic cells (pDCs) are potent type I interferon (IFN)-producing cells that play important roles in antiviral innate immunity. How poxviruses are sensed by pDCs to induce type I IFN production is not well understood. Here we report that infection of primary murine pDCs with myxoma virus, but not with vaccinia virus, induces IFN-α, IFN-β, tumor necrosis factor (TNF), and interleukin-12p70 (IL-12p70) production. Using pDCs derived from genetic knockout mice, we show that the myxoma virus-induced innate immune response requires the endosomal DNA sensor TLR9 and its adaptor MyD88, transcription factors IRF5 and IRF7, and the type I IFN positive-feedback loop mediated by IFNAR1. It is independent of the cytoplasmic RNA sensing pathway mediated by the mitochondrial adaptor molecule MAVS, the TLR3 adaptor TRIF, or the transcription factor IRF3. Using pharmacological inhibitors, we demonstrate that myxoma virus-induced type I IFN and IL-12p70 production in murine pDCs is also dependent on phosphatidylinositol 3-kinase (PI3K) and Akt. Furthermore, our results reveal that the N-terminal Z-DNA/RNA binding domain of vaccinia virulence factor E3, which is missing in the orthologous M029 protein expressed by myxoma virus, plays an inhibitory role in poxvirus sensing and innate cytokine production by murine pDCs.
ST-246 (Tecovirimat) is a small synthetic antiviral compound being developed to treat pathogenic orthopoxvirus infections of humans. The compound was discovered as part of a high throughput screen designed to identify inhibitors of vaccinia virus-induced cytopathic effects. The antiviral activity is specific for orthopoxviruses and the compound does not inhibit the replication of other RNA- and DNA-containing viruses or inhibit cell proliferation at concentrations of compound that are antiviral. ST-246 targets vaccinia virus p37, a viral protein required for envelopment and secretion of extracellular forms of virus. The compound is orally bioavailable and protects multiple animal species from lethal orthopoxvirus challenge. Preclinical safety pharmacology studies in mice and non-human primates indicate that ST-246 is readily absorbed by the oral route and well tolerated with the no observable adverse effect level (NOAEL) in mice measured at 2000 mg/kg and the no observable effect level (NOEL) in non-human primates measured at 300 mg/kg. Drug substance and drug product processes have been developed and commercial scale batches have been produced using Good Manufacturing Processes (GMP). Human phase I clinical trials have shown that ST-246 is safe and well tolerated in healthy human volunteers. Based on the results of the clinical evaluation, once a day dosing should provide plasma drug exposure in the range predicted to be antiviral based on data from efficacy studies in animal models of orthopoxvirus disease. These data support the use of ST-246 as a therapeutic to treat pathogenic orthopoxvirus infections of humans.
Smallpox; ST-246; Tecovirimat; orthopoxvirus; p37; egress inhibitor; antiviral drug
A novel class of acyclic nucleoside phosphonates has been discovered in which the base consists of a pyrimidine preferably containing an amino group at C-2 and C-4 and a 2-(phosphonomethoxy)ethoxy (PMEO) or a 2-(phosphonomethoxy)propoxy (PMPO) group at C-6. The 6-PMEO 2,4-diaminopyrimidine (compound 1) and 6-PMPO 2,4-diaminopyrimidine (compound 11) derivatives showed potent activity against human immunodeficiency virus (HIV) in the laboratory (i.e., CEM and MT-4 cells) and in primary (i.e., peripheral blood lymphocyte and monocyte/macrophage) cell cultures and pronounced activity against Moloney murine sarcoma virus in newborn NMRI mice. Their in vitro and in vivo antiretroviral activity was comparable to that of reference compounds 9-[(2-phosphonomethoxy)ethyl]adenine (adefovir) and (R)-9-[(2-phosphonomethoxy)-propyl]adenine (tenofovir), and the enantiospecificity of (R)- and (S)-PMPO pyrimidine derivatives as regards their antiretroviral activity was identical to that of the classical (R)- and (S)-9-(2-phosphonomethoxy)propyl purine derivatives. The prototype PMEO and PMPO pyrimidine analogues were relatively nontoxic in cell culture and did not markedly interfere with host cell macromolecular (i.e., DNA, RNA, or protein) synthesis. Compounds 1 and 11 should be considered attractive novel pyrimidine nucleotide phosphonate analogues to be further pursued for their potential as antiretroviral agents in the clinical setting.
The acyclic nucleoside 9-(1,3-dihydroxy-2- propoxymethyl )guanine (DHPG) and natural mouse interferon beta ( MuIFN -beta) were evaluated for their efficacy alone and in combination against herpes simplex virus type 2 systemic infections in mice. Intraperitoneally infected animals were treated once a day with the drugs at various concentrations for 5 days starting 24 h after inoculation. DHPG was injected subcutaneously at doses of 0.7 to 6 mg/kg. MuIFN -beta was given intraperitoneally at doses ranging from 3 X 10(3) to 3 X 10(4) IU per mouse. For DHPG alone, the effective dose at which 50% of the mice survived (ED50) was greater than 6 mg/kg. However, when given in combination with an ineffective dose of MuIFN -beta (10(4) IU per mouse), the ED50 for DHPG was 0.8 mg/kg. In addition, at the highest dose tested, MuIFN -beta alone had no protective activity against herpes simplex virus type 2 (ED50, greater than 3 X 10(4) IU per mouse). However, when given in combination with a marginally effective dose of DHPG (2 mg/kg), the ED50 for MuIFN -beta was less than 3 X 10(3) IU per mouse. Calculation of the fractional protective dose index (less than 0.23 where values of less than or equal to 0.5 are considered synergistic) indicates an enhanced protective interaction by the combination of the two drugs. These results represent the first time that potentiation of the antiviral activity of an acyclic nucleoside by interferon has been demonstrated in animal studies.
The acyclic nucleoside DHPG [9-(1,3-dihydroxy-2-propoxymethyl)guanine] and recombinant human alpha-interferon of clones A/D potentiate each other's antiviral activity against a systemic infection with herpes simplex virus type 2. The effective dose at which 50% of the mice survived was lowered approximately 10-fold for DHPG when it was given in combination with a marginally effective dose of alpha-interferon and greater than 10-fold for alpha-interferon when it was given in combination with a nontherapeutic dose of DHPG.
The antiherpetic effects of a novel purine acyclic nucleoside, 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG), were compared with those of acyclovir in cell cultures and in mice. The modes of action of DHPG and acyclovir were similar in that herpes thymidine kinase phosphorylated each compound, and both agents selectively inhibited viral over host cell DNA synthesis. In 50% plaque reduction assays in Vero cells, the drugs inhibited herpes simplex virus types 1 and 2 thymidine kinase-positive strains at 0.2 to 2.4 microM. DHPG was markedly more active than acyclovir against human cytomegalovirus (50% inhibitory doses were 7 and 95 microM, respectively). Each nucleoside inhibited uninfected cell macromolecule synthesis and cell proliferation at concentrations far above those required to inhibit herpes simplex virus replication. Although the two compounds had many similarities in their behavior in vitro, the important difference was the superior performance of DHPG against herpesvirus-induced encephalitis and vaginitis in vivo. Thus, mortality in mice infected with herpesvirus type 2 was reduced 50% by daily doses of 7 to 10 mg of DHPG/kg, whereas an equally effective daily dose of acyclovir was approximately 500 mg/kg. DHPG at a daily dose of 50 mg/kg was also superior to acyclovir at 100 mg/kg per day in its inhibition of herpetic vaginal lesions in mice.
The activities of the purine acyclic nucleoside 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) against two human and five animal strains of cytomegalovirus were compared with those of acyclovir. DHPG was significantly more active than acyclovir against all but one (mouse cytomegalovirus) of the strains tested, with 50% effective doses ranging from 5 to 13 microM, as determined by plaque reduction assays in human embryonic lung (MRC-5) and human embryonic tonsil cells. Both DHPG and acyclovir inhibited virus replication at concentrations considerably lower than those necessary to inhibit cell proliferation. In mode-of-action studies, the triphosphates of DHPG and acyclovir inhibited human cytomegalovirus DNA polymerase. DHPG phosphorylation to the active triphosphate was enhanced in infected cells; however, this enzymatic activity was unrelated to thymidine kinase. In animal studies, DHPG was slightly more effective than acyclovir in reducing mouse cytomegalovirus-induced mortality.
The acyclic nucleoside 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) was evaluated for its efficacy in protecting guinea pigs from primary and recrudescent infections of herpes simplex virus type 2. Vaginally infected guinea pigs were treated twice a day with DHPG at 25 mg/kg per dose for 3 weeks. Subcutaneous doses were started 3 h, 24 h, or 5 weeks after virus inoculation. Treatment starting at 3 or 24 h reduced the severity of the primary infection by greater than 70% when lesions were graded for 3 weeks; lesion duration was lessened by greater than 55%. For 6 weeks after treatment, the number of recrudescent lesions was reduced by greater than 60%, and the duration of the recrudescences declined by greater than 40%. When dosing was started at 3 h postinfection, 33% of the animals did not develop any sign of primary or recrudescent infection throughout the 9-week test. By comparison, all the animals treated with DHPG starting at 24 h or with saline became infected. A 3-week DHPG regimen starting 5 weeks postinfection reduced the number of animals that developed recrudescent lesions by 70%. When treatment ended, however, recrudescent episodes in the animals increased to the level of saline-treated controls.These results suggest that (i) DHPG is highly effective in reducing the severity of both primary and recrudescent lesions of herpes simplex virus type 2, (ii) early treatment of a primary infection or treatment of recrudescences reduces the incidence of recrudescences, and (iii) the drug appears to have no effect on the latent form of the virus, as the incidence of recrudescences increases when DHPG treatment is ended.
We studied the effect of a novel purine acyclic nucleoside, 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG), on human cytomegalovirus (HCMV) replication. The susceptibility of HCMV to this drug was monitored in cell culture by plaque reduction assay. HCMV replication of various strains was inhibited to the extent of 50% by 1 to 5 microM DHPG. DHPG was highly specific in its anti-HCMV activity, since at concentrations as high as 100 microM it did not exert any detectable inhibitory effect on uninfected cell macromolecular synthesis and cell growth. At concentrations of 2 to 4 microM, the drug inhibited the synthesis of six virus-specific polypeptides with molecular weights of 200,000 (VP200), 150,000 (VP150), 67,000 (VP67), 54,000 (VP54), 32,000 (VP32), and 27,000 (VP27) up to 96 h after infection. HCMV DNA synthesis was also considerably suppressed at concentrations of 2 to 4 microM DHPG. Upon removal of the inhibitor, however, viral DNA synthesis resumed and infectious virus reappeared, indicating that this inhibition was a virostatic reversible-type inhibition.
An in vitro tissue culture system consisting of reaggregated embryonic brain cells was used to evaluate the inhibition of herpes simplex type 1 (HSV-1) by several antiviral compounds. The efficacy of acyclovir, vidarabine, bromovinyldeoxyuridine, and 9-(1,3-dihydroxy-2-propoxymethyl) guanine in HSV-1-infected Vero cell monolayer cultures was compared with that seen with brain cell aggregates. At a mean 50% inhibitory dose with Vero cells, acyclovir showed a 99% reduction of virus titer in brain cell aggregates. Vidarabine and 9-(1,3-dihydroxy-2-propoxymethyl) guanine gave a dose-dependent reduction in virus titer with Vero cells; however, in aggregate cultures treated with the same drugs a dose-dependent decrease at 24 h was followed by an increase to a point of no inhibition at 72 h postinfection. Pretreatment of brain cell aggregates with a hybrid human leukocyte interferon (Le IF-AD) reduced virus titers at 48 h postinfection but did not maintain this reduction at 72 h. In contrast, infected Vero cell monolayer cultures demonstrated a dose-dependent reduction in virus titers with Le IF-AD. Postinfection treatment with Le IF-AD did not reduce plaque formation in Vero cells but was effective in reducing virus titer in HSV-1-infected brain cell aggregates at 48 h postinfection. Antiviral concentrations of up to 200 micrograms or 200,000 IU/ml for interferon did not appear morphologically toxic to brain cells. Antiviral therapy of HSV-1-infected brain cell aggregates may more closely mimic in vivo responses than monolayer cultures.
The emergence of zoonotic orthopoxvirus infections and the threat of possible intentional release of pathogenic orthopoxviruses have stimulated renewed interest in understanding orthopoxvirus infections and the resulting diseases. Ectromelia virus (ECTV), the causative agent of mousepox, offers an excellent model system to study an orthopoxvirus infection in its natural host. Here, we investigated the role of the vaccinia virus ortholog N1L in ECTV infection. Respiratory infection of mice with an N1L deletion mutant virus (ECTVΔN1L) demonstrated profound attenuation of the mutant virus, confirming N1 as an orthopoxvirus virulence factor. Upon analysis of virus dissemination in vivo, we observed a striking deficiency of ECTVΔN1L spreading from the lungs to the livers or spleens of infected mice. Investigating the immunological mechanism controlling ECTVΔN1L infection, we found the attenuated phenotype to be unaltered in mice deficient in Toll-like receptor (TLR) or RIG-I-like RNA helicase (RLH) signaling as well as in those missing the type I interferon receptor or lacking B cells. However, in RAG-1−/− mice lacking mature B and T cells, ECTVΔN1L regained virulence, as shown by increasing morbidity and virus spread to the liver and spleen. Moreover, T cell depletion experiments revealed that ECTVΔN1L attenuation was reversed only by removing both CD4+ and CD8+ T cells, so the presence of either cell subset was still sufficient to control the infection. Thus, the orthopoxvirus virulence factor N1 may allow efficient ECTV infection in mice by interfering with host T cell function.
We studied the susceptibility of human herpesvirus 8 (HHV-8) to a number of antiherpesvirus agents. The acyclic nucleoside phosphonate (ANP) analogs cidofovir and HPMPA [(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)adenine] effected potent inhibition of HHV-8 DNA synthesis, with 50% effective concentrations (EC50) of 6.3 and 0.6 microM, respectively. Adefovir, an ANP with both antiretrovirus and antiherpesvirus activity, blocked HHV-8 DNA replication at a fourfold-lower concentration than did foscarnet (EC50 of 39 and 177 microM, respectively). The most potent inhibitory effect was obtained with the N-7-substituted nucleoside analog S2242 (EC50, 0.11 microM). The nucleoside analogs acyclovir, penciclovir, H2G ((R)-9-[4-hydroxy-2-(hydroxymethyl) butyl]guanine), and brivudine had weak to moderate effects (EC50 of > or =75, 43, 42, and 24 microM, respectively, and EC90 of > or =75 microM), whereas ganciclovir elicited pronounced anti-HHV-8 activity (EC50, 8.9 microM).
Smallpox preparedness research has led to development of antiviral therapies for treatment of serious orthopoxvirus infections. Monkeypox virus is an emerging, zoonotic orthopoxvirus which can cause severe and transmissible disease in humans, generating concerns for public health. Monkeypox virus infection results in a systemic, febrile-rash illness closely resembling smallpox. Currently, there are no small-molecule antiviral therapeutics approved to treat orthopoxvirus infections of humans. The prairie dog, using monkeypox virus as a challenge virus, has provided a valuable nonhuman animal model in which monkeypox virus infection closely resembles human systemic orthopoxvirus illness. Here, we assess the efficacy of the antiorthopoxvirus compound ST-246 in prairie dogs against a monkeypox virus challenge of 65 times the 50% lethal dose (LD50). Animals were infected intranasally and administered ST-246 for 14 days, beginning on days 0, 3, or after rash onset. Swab and blood samples were collected every 2 days and analyzed for presence of viral DNA by real-time PCR and for viable virus by tissue culture. Seventy-five percent of infected animals that received vehicle alone succumbed to infection. One hundred percent of animals that received ST-246 survived challenge, and animals that received treatment before symptom onset remained largely asymptomatic. Viable virus and viral DNA were undetected or at greatly reduced levels in animals that began treatment on 0 or 3 days postinfection, compared to control animals or animals treated post-rash onset. Animals treated after rash onset manifested illness, but all recovered. Our results indicate that ST-246 can be used therapeutically, following onset of rash illness, to treat systemic orthopoxvirus infections.
Camelpox virus (CMLV) is the closest known orthopoxvirus genetically related to variola virus. So far, CMLV was restricted to camelids but, recently, three human cases of camelpox have been described in India, highlighting the need to pursue research on its pathogenesis, which has been hampered by the lack of small animal models. Here, we confirm that NMRI immunocompetent mice are resistant to intranasal (i.n.) CMLV infection. However, we demonstrate that CMLV induced a severe disease following i.n. challenge of athymic nude mice, which was accompanied with a failure in gaining weight, leading to euthanasia of the animals. On the other hand, intracutaneous (i.c.) infection resulted in disease development without impacting the body weight evolution. CMLV replication in tissues and body fluids was confirmed in the two models. We further analyzed innate immune and B cell responses induced in the spleen and draining lymph nodes after exposure to CMLV. In both models, strong increases in CD11b+F4/80+ macrophages were seen in the spleen, while neutrophils, NK and B cell responses varied between the routes of infection. In the lymph nodes, the magnitude of CD11c+CD8α+ lymphoid and CD11c+CD11b+ myeloid dendritic cell responses increased in i.n. challenged animals. Analysis of cytokine profiles revealed significant increases of interleukin (IL)-6 and IL-18 in the sera of infected animals, while those of other cytokines were similar to uninfected controls. The efficacy of two antivirals (cidofovir or HPMPC, and its 2, 6-diaminopurine analog) was evaluated in both models. HPMPC was the most effective molecule affording 100% protection from morbidity. It appeared that both treatments did not affect immune cell responses or cytokine expression. In conclusion, we demonstrated that immunodeficient mice are permissive for CMLV propagation. These results provide a basis for studying the pathogenesis of CMLV, as well as for evaluating potential antiviral therapies in an immunodeficiency context.
The metabolisms of 9-(1,3-dihydroxy-2-propoxymethyl)guanine (2'NDG) and its cyclic phosphate, 9-[(2-hydroxy-1,3,2-dioxophosphorinan-5-yl) oxymethyl]guanine P-oxide (2'-nor-cGMP), were compared in cultures of primary rabbit kidney cells infected with herpes simplex virus type 1 (HSV-1). 2'-Nor-cGMP was taken up by the cells essentially intact, after which it was opened to the acyclic monophosphate and phosphorylated further, ultimately to the triphosphate. Formation of the triphosphate was independent of HSV thymidine kinase expression, unlike what is observed with 2'NDG. In addition, there was a direct correlation between the antiviral activity of 2'NDG and the level of triphosphate formed in HSV-1-infected cells, whereas such a correlation was absent with 2'-nor-cGMP. In vivo experiments indicated that only a small percentage of free 2'NDG was formed in the bloodstream of mice after oral administration of 2'-nor-cGMP. Incubation of 2'-nor-cGMP with crude extracts of HSV-1-infected or uninfected HeLa cells resulted in the direct production of 2'NDG triphosphate. The possibility that the triphosphate of 2'NDG produced from 2'-nor-cGMP was the enantiomer of the triphosphate made from 2'NDG by viral and cellular kinases was investigated and disproved. Taken together, these data indicate that (i) 2'-nor-cGMP does not act simply as a prodrug of 2'NDG, (ii) 2'-nor-cGMP does not require viral thymidine kinase for its activity, and (iii) 2'-nor-cGMP may have an additional, triphosphate-independent mode of action.
Although a highly effective vaccine against smallpox, vaccinia virus (VV) is not without adverse events, some of which can be life-threatening, particularly in immunocompromised individuals. We have recently demonstrated that the immunogenicity and protective efficacy of Dryvax® in immunocompetent mice is preserved even when co-administered with ST-246, an orally bioavailable small-molecule inhibitor of orthopoxvirus egress and dissemination. In addition, ST-246 markedly reduced the reactogenicity of the smallpox vaccine ACAM2000 and the highly neurovirulent VV strain Western Reserve (VV-WR). Here, we evaluated the impact of ST-246 co-administration on ACAM2000 reactogenicity, immunogenicity, and protective efficacy in seven murine models of varying degrees of humoral and cellular immunodeficiency: BALB/c and B-cell deficient (JH-KO) mice depleted of CD4+ or CD8+ or both subsets of T cells. We observed that ST-246 reduced vaccine lesion severity and time to complete resolution in all of the immunodeficient models examined, except in those lacking both CD4+ and CD8+ T cells. Although VV-specific humoral responses were moderately reduced by ST-246 treatment, cellular responses were generally comparable or slightly enhanced at both 1 and 6 months post-vaccination. Most importantly, in those models in which vaccination given alone conferred protection against lethal VV challenge, similar levels of protection were observed at both time points when vaccination was given with ST-246. These data suggest that, with the exception of individuals with irreversible, combined CD4+ and CD8+ T-cell deficiency, ST-246 co-administered at the time of vaccination may help reduce vaccine reactogenicity –even in those lacking humoral immunity– without impeding the induction of protective immunity.
ST-246; ACAM2000; immunodeficient mice
The absence of any formally licensed antiadenovirus drugs and the increasing incidence of life-threatening adenovirus infections in immunosuppressed patients warrant the development of effective antiadenovirus compounds. A detailed study was performed on the antiadenovirus activities of several classes of nucleoside and nucleotide analogues in human embryonic lung fibroblast cells. The antiadenovirus activities were evaluated by three methods, viz., evaluating the adenoviral cytopathic effect, monitoring cell viability by a colorimetric assay, and real-time PCR quantitation of viral DNA as a direct parameter for virus replication. The most active and selective compounds were the acyclic nucleoside phosphonate analogues cidofovir, its adenine analogue (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [(S)-HPMPA], and the new derivative (S)-2,4-diamino-6-[3-hydroxy-2-(phosphonomethoxy)propoxy]pyrimidine [(S)-HPMPO-DAPy]; the N7-substituted acyclic derivative 2-amino-7-(1,3-dihydroxy-2-propoxymethyl)purine (S-2242); and the 2′,3′-dideoxynucleoside analogues zalcitabine and alovudine. No antiadenovirus activity was observed for the antiviral drugs ribavirin, foscarnet, acyclovir, penciclovir, and brivudin, while ganciclovir displayed modest activity. However, in human osteosarcoma cells transfected with herpes simplex virus thymidine kinase, ganciclovir demonstrated highly potent antiadenovirus activity, suggesting that the efficacy of ganciclovir against adenovirus is limited by inefficient phosphorylation in adenovirus-infected cells, rather than by insufficient inhibition at the viral DNA polymerase level. Collectively, our antiviral data show that the adenovirus DNA polymerase exhibits sensitivity to a relatively broad spectrum of inhibitors and should be studied further as an antiviral target in antiadenovirus drug development programs.
The l-nucleoside analog β-l-2′,3′-dideoxy-2′,3′-didehydro-5-fluorocytidine (β-l-Fd4C) was first shown to exhibit potent activity against hepatitis B virus (HBV) in tissue culture and then to significantly inhibit viral spread during acute infection in the duck HBV model (F. Le Guerhier et al., Antimicrob. Agents Chemother. 44:111–122, 2000). We have therefore examined its antiviral activity in a mammalian model of chronic HBV infection, the woodchuck chronically infected with woodchuck hepatitis virus (WHV). Side-by-side comparison of β-l-Fd4C and lamivudine administered intraperitoneally during short-term and long-term protocols demonstrated a more profound inhibition of viremia in β-l-Fd4C-treated groups. Moreover, β-l-Fd4C induced a marked inhibition of intrahepatic viral DNA synthesis compared with that induced by lamivudine. Nevertheless, covalently closed circular (CCC) DNA persistence explained the lack of clearance of infected hepatocytes expressing viral antigens and the relapse of WHV replication after drug withdrawal. Liver histology showed a decrease in the inflammatory activity of chronic hepatitis in woodchucks receiving β-l-Fd4C. An electron microscopy study showed the absence of ultrastructural changes of hepatic mitochondria, biliary canaliculi, and bile ducts. However, a loss of weight was observed in all animals, whatever the treatment, as was a transient skin pigmentation in all woodchucks during β-l-Fd4C treatment. There was no evidence that lamivudine or β-l-Fd4C could prevent the development of hepatocellular carcinoma with the protocols used. These results indicate that β-l-Fd4C exhibits a more potent antiviral effect than lamivudine in the WHV model but was not able to eradicate CCC DNA and infected cells from the liver at the dosage and with the protocol used.
Plasmacytoid dendritic cells (pDCs) play important roles in antiviral innate immunity by producing type I interferon (IFN). In this study, we assess the immune responses of primary human pDCs to two poxviruses, vaccinia and myxoma virus. Vaccinia, an orthopoxvirus, was used for immunization against smallpox, a contagious human disease with high mortality. Myxoma virus, a Leporipoxvirus, causes lethal disease in rabbits, but is non-pathogenic in humans. We report that myxoma virus infection of human pDCs induces IFN-α and TNF production, whereas vaccinia infection does not. Co-infection of pDCs with myxoma virus plus vaccinia blocks myxoma induction effects. We find that heat-inactivated vaccinia (Heat-VAC; by incubating the virus at 55°C for 1 h) gains the ability to induce IFN-α and TNF in primary human pDCs. Induction of IFN-α in pDCs by myxoma virus or Heat-VAC is blocked by chloroquine, which inhibits endosomal acidification required for TLR7/9 signaling, and by inhibitors of cellular kinases PI3K and Akt. Using purified pDCs from genetic knockout mice, we demonstrate that Heat-VAC-induced type I IFN production in pDCs requires the endosomal RNA sensor TLR7 and its adaptor MyD88, transcription factor IRF7 and the type I IFN feedback loop mediated by IFNAR1. These results indicate that (i) vaccinia virus, but not myxoma virus, expresses inhibitor(s) of the poxvirus sensing pathway(s) in pDCs; and (ii) Heat-VAC infection fails to produce inhibitor(s) but rather produces novel activator(s), likely viral RNA transcripts that are sensed by the TLR7/MyD88 pathway. Using vaccinia gene deletion mutants, we show that the Z-DNA/RNA binding domain at the N-terminus of the vaccinia immunomodulatory E3 protein is an antagonist of the innate immune response of human pDCs to poxvirus infection and TLR agonists. The myxoma virus ortholog of vaccinia E3 (M029) lacks the N-terminal Z-DNA/RNA binding domain, which might contribute to the immunostimulating properties of myxoma virus.
The nucleotide phosphonates cidofovir (CDV) and cyclic cidofovir (cCDV) are potent antiviral compounds when administered parenterally but are not well absorbed orally. These compounds have been reported to have activity against orthopoxvirus replication in vitro and in animal models when administered parenterally or by aerosol. To obtain better oral activity, we synthesized a novel series of analogs of CDV and cCDV by esterification with two long-chain alkoxyalkanols, 3-hexadecyloxy-1-propanol (HDP-CDV; HDP-cCDV) or 3-octadecyloxy-1-ethanol (ODE-CDV; ODE-cCDV). Their activities were evaluated and compared with those of CDV and cCDV in human foreskin fibroblast (HFF) cells infected with vaccinia virus (VV) or cowpox virus (CV) using a plaque reduction assay. The 50% effective concentrations (EC50s) against VV in HFF cells for CDV and cCDV were 46.2 and 50.6 μM compared with 0.84 and 3.8 μM for HDP-CDV and HDP-cCDV, respectively. The EC50s for ODE-CDV and ODE-cCDV were 0.20 and 1.1 μM, respectively. The HDP analogs were 57- and 13-fold more active than the parent nucleotides, whereas the ODE analogs were 231- and 46-fold more active than the unmodified CDV and cCDV. Similar results were obtained using CV. Cytotoxicity studies indicated that although the analogs were more toxic than the parent nucleotides, the selective index was increased by 4- to 13-fold. These results indicate that the alkoxyalkyl esters of CDV and cCDV have enhanced activity in vitro and need to be evaluated for their oral absorption and efficacy in animal models.