Nigel Bourne

Dendrimers, a New Class of Candidate Topical Microbicides with Activity against Herpes Simplex Virus Infection

ABSTRACT Dendrimers are large highly branched macromolecules synthesized from a polyfunctional core. They have shown a variety of biological properties, including, in some instances, antiviral activity. In this study, five dendrimers were evaluated for in vitro activity against herpes simplex virus (HSV) types 1 and 2 by cytopathic effect (CPE) inhibition and plaque reduction (PR) assay in human foreskin fibroblast cells. All of the compounds were active against both virus types in the CPE inhibition assay, in which drug was added to the cells prior to the addition of virus. Antiviral activity was reduced or lost in the PR assays, in which the cells were incubated with the virus before the drug was added. The prophylactic efficacy suggested that the dendrimers might have potential as topical microbicides, products intended to be applied to the vaginal or rectal mucosa to protect against sexually transmitted infections. Three dendrimers were evaluated for this application against genital HSV infection in mice. Two of the compounds, BRI-2999 and BRI-6741, significantly reduced infection rates when 15 μl of a 100-mg/ml solution was administered immediately prior to intravaginal challenge, and the most effective compound, BRI-2999, provided significant protection even when applied 30 min before challenge. This is the first report of microbicidal activity by dendrimers in vivo.

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.

Poly(Sodium 4-Styrene Sulfonate): An Effective Candidate Topical Antimicrobial for the Prevention of Sexually Transmitted Diseases

Presently marketed vaginal barrier agents are cytotoxic and damage the vaginal epithelium and natural vaginal flora with frequent use. Novel noncytotoxic agents are needed to protect women from sexually transmitted diseases. One candidate compound is a high-molecular-mass form of soluble poly(sodium 4-styrene sulfonate) (T-PSS). The antimicrobial activity of T-PSS was evaluated in primary culture systems and in a genital herpes murine model. Results obtained indicate that T-PSS is highly effective against herpes simplex viruses, Neisseria gonorrhoeae, and Chlamydia trachomatis in vitro. A 5% T-PSS gel protected 15 of 16 mice from vaginal herpes, compared with 2 of 16 mice treated with a placebo gel. Moreover, T-PSS exhibited little or no cytotoxicity and has an excellent selectivity index. T-PSS is an excellent candidate topical antimicrobial that blocks adherence of herpes simplex virus at low concentrations, inactivates virus at higher concentrations, and exhibits a broad spectrum of antimicrobial activity.

Preconception Vaccination with a Glycoprotein B (gB) DNA Vaccine Protects against Cytomegalovirus (CMV) Transmission in the Guinea Pig Model of Congenital CMV Infection

DNA vaccines expressing the guinea pig cytomegalovirus (GPCMV) homologs of the glycoprotein B (gB) and UL83 proteins were evaluated for protection against congenital GPCMV infection. After 4 doses of DNA administered by epidermal (gene gun) route, all guinea pigs developed enzyme-linked immunosorbent assay (ELISA) antibody and, for gB-vaccine recipients, neutralizing antibody. Dams were challenged with 1 x 10(4) plaque-forming units of GPCMV in the third trimester. Preconception vaccination with gB did not decrease overall pup mortality, although, within the gB-vaccine group, pup mortality was lower among dams with high ELISA responses. Preconception maternal vaccination with gB vaccine significantly reduced congenital transmission in liveborn pups. In contrast, UL83 vaccine had no significant effect on pup mortality or vertical transmission of GPCMV. Virus load was significantly lower in infected pups born to gB- and UL83-vaccinated dams than in infected pups born to control dams. These data support the concept that subunit gB vaccination may be useful in protecting against CMV-induced 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).

Effect of indomethacin on ultraviolet radiation-induced recurrent herpes simplex virus disease in guinea-pigs

Exposure to u.v. radiation increases the local level of prostaglandins which may play a role in u.v. radiation-induced herpes simplex virus (HSV) recurrences. We used the guinea-pig model of u.v. radiation-induced recurrent genital HSV-2 disease for examining the effects of indomethacin, a prostaglandin inhibitor, on u.v.-induced recurrences. In the first experiment, performed 100 days after HSV-2 inoculation, treatment with indomethacin for 5 days begun 24 h before u.v.-irradiation decreased the proportion of animals developing HSV disease recurrences from 11/13 (84.6%) to 2/13 (15.4%) (P < 0.001). In the second experiment, performed 135 days after HSV-2 inoculation, treatment with indomethacin for 5 days begun 24 h before u.v.-irradiation decreased the number of animals developing recurrences from 12/21 (57.1%) to 5/21 (23.8%) (P < 0.05). Five days of indomethacin treatment begun 4 h after u.v.-irradiation, however, did not reduce the percentage of animals developing disease recurrences but did decrease the mean number of days with recurrent lesions in animals that developed recurrences. Our data suggest that indomethacin may modify u.v. radiation-induced recurrent lesions by decreasing viral reactivation when given before u.v. radiation exposure or by reducing prostaglandin-induced immunosuppression when given before or after exposure. Future studies are needed for evaluating indomethacin prophylaxis for recurrent HSV disease when prolonged u.v. radiation exposure is anticipated.

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.

An animal model of neonatal cytomegalovirus infection

Cytomegalovirus (CMV) is the most common cause of congenital infection in the developed world and can lead to a life-threatening disease. We therefore developed an animal model to evaluate candidate anti-CMV drugs and to further define the pathogenesis of CMV infections. Newborn guinea pigs were infected by intraperitoneal administration of 10(6) pfu of a virulent salivary gland (SG) passaged guinea pig CMV (gpCMV) within 48 h of birth. Inoculation of animals produced 50% overall mortality. A lack of weight gain was also a hallmark of infection. By day 14 after inoculation the weight of gpCMV-infected animals was significantly less than controls (152.9+/-45 g versus 254.7+/-38.5 g, P<0.0001). The most consistent isolation and highest titers of virus were found in the liver and spleen early while lung titers were maximal at day 10. A quantitative competitive PCR (qcPCR) assay confirmed the presence of a high CMV viral load in infected organs. Antiviral treatment with cyclic HPMPC (cHPMPC) for 7 days significantly reduced mortality (1/20 versus 14/20, P<0.001) and viral replication but did not improve weight gain. This model should be useful for further evaluations of the pathogenesis of CMV infections and for evaluation of antiviral drugs and vaccines.