Fernando J. Bravo

Protection against Congenital Cytomegalovirus Infection and Disease in Guinea Pigs, Conferred by a Purified Recombinant Glycoprotein B Vaccine

Glycoprotein B (gB) has emerged as a subunit-vaccine candidate for congenital cytomegalovirus (CMV) infection, a major public health problem. The present study evaluated a cloned, recombinant gB vaccine in the guinea pig cytomegalovirus (GPCMV) model of congenital infection. Guinea pigs were immunized with gB, which was coadministered with either Freunds adjuvant or alum. All gB-immunized dams had enzyme-linked immunosorbent-assay and neutralizing-antibody responses, with significantly higher titers in the gB/Freunds group. Pregnant dams were challenged with GPCMV subcutaneously during the 3rd trimester. Maternal DNAemia on day 10 after infection trended lower in gB-immunized dams than in control animals, with significant reductions in the gB/Freunds group. Vaccination resulted in a highly significant reduction in pup mortality. For the gB-vaccine groups, pup mortality was significantly lower, and reduced rates of GPCMV transmission were noted, for dams immunized with gB and Freunds adjuvant, compared with dams immunized with gB and alum. These are the first data indicating that a recombinant gB vaccine protects against congenital CMV infection and disease.

Preconception Immunization with a Cytomegalovirus (CMV) Glycoprotein Vaccine Improves Pregnancy Outcome in a Guinea Pig Model of Congenital CMV Infection

The guinea pig (gp) model of congenital cytomegalovirus (CMV) infection was used to evaluate a gpCMV glycoprotein vaccine. Hartley guinea pigs were immunized 3 times with 50 microg of lectin column-purified glycoproteins prepared from gpCMV-infected or -uninfected tissue culture. Immunization with the gpCMV vaccine produced seroconversion in all animals. Animals then were placed with gpCMV-seronegative male animals and were challenged late in pregnancy with virulent salivary gland-passaged gpCMV. Immunization with gpCMV glycoproteins significantly improved pregnancy outcome, with 54 of 63 pups live-born in immunized animals, compared with 21 of 48 in the controls (P<.001). In addition, virus was isolated from 24 of 54 live-born pups born to immunized mothers, compared with 16 of 20 live-born pups born to controls, indicating that immunization significantly reduced in utero transmission in surviving animals (P<.01).

Inhibition of LSD1 reduces herpesvirus infection, shedding, and recurrence by promoting epigenetic suppression of viral genomes

Epigenetic suppression of herpes simplex virus demonstrates an approach to control persistent viruses. Epigenetic Control of Herpes Epigenetic modifications are a sort of genetic metadata—they alter gene expression without changing the underlying DNA. Hill et al. hypothesized that epigenetic modification could be a therapeutic approach for treating herpesvirus infection. Indeed, herpesvirus infection and reactivation from latency previously has been shown to depend on the histone demethylases LSD1 and JMJD2. The authors found that blocking the activity of LSD1 could inhibit viral infection in animal models that represent three different stages of herpes simplex virus infection: suppression of primary infection, a block to subclinical shedding, and reduction in recurrent lesions. If these data are confirmed in humans, epi-pharmaceuticals may provide a promising therapeutic avenue to treat this prevalent, lifelong disease. Herpesviruses are highly prevalent and maintain lifelong latent reservoirs, thus posing challenges to the control of herpetic disease despite the availability of antiviral pharmaceuticals that target viral DNA replication. The initiation of herpes simplex virus infection and reactivation from latency is dependent on a transcriptional coactivator complex that contains two required histone demethylases, LSD1 (lysine-specific demethylase 1) and a member of the JMJD2 family (Jumonji C domain–containing protein 2). Inhibition of either of these enzymes results in heterochromatic suppression of the viral genome and blocks infection and reactivation in vitro. We demonstrate that viral infection can be epigenetically suppressed in three animal models of herpes simplex virus infection and disease. Treating animals with the monoamine oxidase inhibitor tranylcypromine to inhibit LSD1 suppressed viral lytic infection, subclinical shedding, and reactivation from latency in vivo. This phenotypic suppression was correlated with enhanced epigenetic suppression of the viral genome and suggests that, even during latency, the chromatin state of the virus is dynamic. Therefore, epi-pharmaceuticals may represent a promising approach to treat herpetic diseases.

The Adjuvant CLDC Increases Protection of a Herpes Simplex Type 2 Glycoprotein D Vaccine in Guinea Pigs

Herpes simplex virus (HSV) infections are common but there is no vaccine available. We evaluated cationic liposome-DNA complexes (CLDC) as an adjuvant for an HSV gD2 vaccine and compared it to an MPL/Alum adjuvant in a guinea pig model of genital herpes. The addition of CLDC to the gD2 vaccine significantly decreased acute and recurrent disease and most importantly the number of days with recurrent virus shedding compared to gD2 alone. Reductions in these outcomes were also detected when gD2+CLDC was compared to gD2+MPL/Alum. When the vaccine and adjuvants were evaluated as therapeutic vaccines, they were ineffective. CLDC enhanced protection compared to MPL/Alum and is the first vaccine to reduce recurrent virus shedding, a key to decreasing the spread of HSV-2.

Effects of herpes simplex virus type 2 glycoprotein vaccines and CLDC adjuvant on genital herpes infection in the guinea pig.

Genital herpes simplex virus (HSV) infections are common but results from vaccine trials with HSV-2 glycoprotein D (gD) have been disappointing. We therefore compared a similar HSV gD2 vaccine, to a further truncated gD2 vaccine, to a vaccine with gD2 plus gB2 and gH2/gL2 and to a vaccine with only gB2 and gH2/gL2 in a guinea pig model of genital herpes. All vaccines were administered with cationic liposome-DNA complexes (CLDC) as an adjuvant. All vaccines significantly decreased the severity of acute genital disease and vaginal virus replication compared to the placebo group. The majority of animals in all groups developed at least one episode of recurrent disease but the frequency of recurrent disease was significantly reduced by each vaccine compared to placebo. No vaccine was significantly more protective than gD2 alone for any of the parameters described above. No vaccine decreased recurrent virus shedding. When protection against acute infection of dorsal root ganglia and the spinal cord was evaluated all vaccines decreased the per cent of animal with detectable virus and the quantity of virus but again no vaccine was significantly more protective than another. Improvements in HSV-2 vaccines may require inclusion of more T cell targets, more potent adjuvants or live virus vaccines.

Amphipathic DNA polymers exhibit antiviral activity against systemic Murine Cytomegalovirus infection

BackgroundPhosphorothioated oligonucleotides (PS-ONs) have a sequence-independent, broad spectrum antiviral activity as amphipathic polymers (APs) and exhibit potent in vitro antiviral activity against a broad spectrum of herpesviruses: HSV-1, HSV-2, HCMV, VZV, EBV, and HHV-6A/B, and in vivo activity in a murine microbiocide model of genital HSV-2 infection. The activity of these agents against animal cytomegalovirus (CMV) infections in vitro and in vivo was therefore investigated.ResultsIn vitro, a 40 mer degenerate AP (REP 9) inhibited both murine CMV (MCMV) and guinea pig CMV (GPCMV) with an IC50 of 0.045 μM and 0.16 μM, respectively, and a 40 mer poly C AP (REP 9C) inhibited MCMV with an IC50 of 0.05 μM. Addition of REP 9 to plaque assays during the first two hours of infection inhibited 78% of plaque formation whereas addition of REP 9 after 10 hours of infection did not significantly reduce the number of plaques, indicating that REP 9 antiviral activity against MCMV occurs at early times after infection. In a murine model of CMV infection, systemic treatment for 5 days significantly reduced virus replication in the spleens and livers of infected mice compared to saline-treated control mice. REP 9 and REP 9C were administered intraperitoneally for 5 consecutive days at 10 mg/kg, starting 2 days prior to MCMV infection. Splenomegaly was observed in infected mice treated with REP 9 but not in control mice or in REP 9 treated, uninfected mice, consistent with mild CpG-like activity. When REP 9C (which lacks CpG motifs) was compared to REP 9, it exhibited comparable antiviral activity as REP 9 but was not associated with splenomegaly. This suggests that the direct antiviral activity of APs is the predominant therapeutic mechanism in vivo. Moreover, REP 9C, which is acid stable, was effective when administered orally in combination with known permeation enhancers.ConclusionThese studies indicate that APs exhibit potent, well tolerated antiviral activity against CMV infection in vivo and represent a new class of broad spectrum anti-herpetic agents.

Potent Adjuvant Activity of Cationic Liposome-DNA Complexes for Genital Herpes Vaccines

ABSTRACT Development of a herpes simplex virus (HSV) vaccine is a priority because these infections are common. It appears that potent adjuvants will be required to augment the immune response to subunit HSV vaccines. Therefore, we evaluated cationic liposome-DNA complexes (CLDC) as an adjuvant in a mouse model of genital herpes. Using a whole-virus vaccine (HVAC), we showed that the addition of CLDC improved antibody responses compared to vaccine alone. Most important, CLDC increased survival, reduced symptoms, and decreased vaginal virus replication compared to vaccine alone or vaccine administered with monophosphoryl lipid A (MPL) plus trehalose dicorynomycolate (TDM) following intravaginal challenge of mice. When CLDC was added to an HSV gD2 vaccine, it increased the amount of gamma interferon that was produced from splenocytes stimulated with gD2 compared to the amount produced with gD2 alone or with MPL-alum. The addition of CLDC to the gD2 vaccine also improved the outcome following vaginal HSV type 2 challenge compared to vaccine alone and was equivalent to vaccination with an MPL-alum adjuvant. CLDC appears to be a potent adjuvant for HSV vaccines and should be evaluated further.

Effect of Maternal Treatment with Cyclic HPMPC in the Guinea Pig Model of Congenital Cytomegalovirus Infection

BACKGROUND Cytomegalovirus (CMV) infection of the fetus is the leading cause of congenital infection. Using the guinea pig model of congenital CMV infection, we sought to determine whether antiviral treatment of a CMV-infected dam could improve the outcome of offspring. METHODS Pregnant Hartley guinea pigs were inoculated with guinea pig CMV (GPCMV) during the late second/early third trimester of gestation. Guinea pigs received either 1 dose of cyclic 1-[((s)-2-hydroxy-2-oxo-1,4,2,-dioxaphosphorinan-5-yl)methyl]cytosine dihydrate (cHPMPC; 35 mg/kg) or placebo 24 h after GPCMV infection. Guinea pigs were monitored until delivery or were killed 10 days after infection, for the evaluation of the effect of cHPMPC on viral replication by polymerase chain reaction analysis in various dam and pup tissues. RESULTS cHPMPC treatment of infected dams improved pup survival from 28.2% (11/39) to 83.7% (36/43) (P < .001) and extended the duration of pregnancy. cHPMPC treatment did not prevent infection of the placenta or disseminated infection of the dam and pup but significantly decreased the amount of GPCMV in tissues. GPCMV DNA levels in the placenta were reduced from 3.54 to 2.12 log(10) genome copies/ mu g of DNA (P < .0001). CONCLUSIONS Treatment of the GPCMV-infected pregnant dam with 1 dose of cHPMPC improves the outcome of congenital infection and decreases viral replication in a guinea pig model.

Oral Hexadecyloxypropyl-Cidofovir Therapy in Pregnant Guinea Pigs Improves Outcome in the Congenital Model of Cytomegalovirus Infection

ABSTRACT Cytomegalovirus (CMV) infection is the leading cause of congenital infection, producing both sensorineural hearing loss and mental retardation. We evaluated the in vivo efficacy of an orally bioavailable analog of cidofovir, hexadecyloxypropyl-cidofovir (HDP-CDV), against guinea pig CMV (GPCMV) in a guinea pig model of congenital CMV infection. HDP-CDV exhibited antiviral activity against GPCMV with a 50% effective concentration (EC50) of 0.004 μM ± 0.001 μM. To evaluate in vivo efficacy, pregnant Hartley guinea pigs were inoculated with GPCMV during the late second/early third trimester of gestation. Animals were administered 20 mg HDP-CDV/kg body weight orally at 24 h postinfection (hpi) and again at 7 days postinfection (dpi) or administered 4 mg/kg HDP-CDV orally each day for 5 days or 9 days. Virus levels in dam and pup tissues were evaluated following delivery, or levels from dam, placenta, and fetal tissues were evaluated following sacrifice of dams at 10 dpi. All HDP-CDV regimens significantly improved pup survival, from 50 to 60% in control animals to 93 to 100% in treated animals (P ≤ 0.019). Treatment with 20 mg/kg HDP-CDV significantly reduced the viral load in pup spleen (P = 0.017) and liver (P = 0.029). Virus levels in the placenta were significantly reduced at 10 dpi following daily treatment with 4 mg/kg HDP-CDV for 5 or 9 days. The 9-day treatment also significantly reduced the viral levels in the dam spleen and liver. Although the 4-mg/kg treatment improved pup survival, virus levels in the fetal tissues were similar to those in control tissues. Taken together, HDP-CDV shows potential as a well-tolerated antiviral candidate for treatment of congenital human CMV (HCMV) infection.

Quantitative-competitive PCR monitoring of viral load following experimental guinea pig cytomegalovirus infection.

Human cytomegalovirus (HCMV) is the most common cause of congenital viral infection in the developed world, and can lead to significant morbidity. Animal models of HCMV infection are required for study of pathogenesis, because of the strict species-specificity of cytomegalovirus (CMV). Among the small animal CMV models, the guinea pig CMV (GPCMV) has unique advantages, in particular its propensity to cross the placenta, causing disease in utero. In order to develop quantitative endpoints for vaccine and antiviral therapeutic studies in the GPCMV model, a quantitative-competitive PCR (qcPCR) assay was developed, based on the GPCMV homolog of the HCMV UL83 gene, GP83. Optimal amplification of GPCMV DNA was observed using primers spanning a 248 base pair (bp) region of this gene. A 91 bp deletion of this cloned fragment was generated for use as an internal standard (IS) for PCR amplification. Standard curves based upon the fluorescent intensity of full-length external target to IS were compared with signal intensity of DNA extracted from blood and organs of experimentally infected guinea pigs in order to quantify viral load. Viral load in newborn guinea pigs infected transplacentally was determined and compared with that of pups infected with GPCMV as neonates. Viral loads were highest in pups infected as neonates. The most consistent isolation and highest quantities of viral DNA were observed in liver and spleen, although viral genome could be readily identified in brain, lung, and salivary gland. Viral load determination should be useful for monitoring outcomes following vaccine studies, as well as responses to experimental antiviral agents.