Dimethyl sulfoxide blocks herpes simplex virus-1 productive infection in vitro acting at different stages with positive cooperativity. Application of micro-array analysis

BACKGROUND: Dimethyl sulfoxide (DMSO) is frequently used at a concentration of up to 95% in the formulation of antiherpetic agents because of its properties as a skin penetration enhancer. Here, we have analyzed the effect of DMSO on several parameters of Herpes Simplex Virus replication. METHODS: Productive infection levels of HSV-1 were determined by plaque assay or by reporter gene activity, and its DNA replication was estimated by PCR. Transcript levels were evaluated with HSV-specific DNA micro-arrays. RESULTS: DMSO blocks productive infection in vitro in different cell types with a 50% inhibitory concentration (IC(50)) from 0.7 to 2% depending upon the multiplicity of infection. The concentration dependence exhibits a Hill coefficient greater than 1, indicating that DMSO blocks productive infection by acting at multiple different points (mechanisms of action) with positive cooperativity. Consistently, we identified at least three distinct temporal target mechanisms for inhibition of virus growth by DMSO. At late stages of infection, DMSO reduces virion infectivity, and markedly inhibits viral DNA replication. A third mode of action was revealed using an oligonucleotide-based DNA microarray system for HSV. These experiments showed that DMSO reduced the transcript levels of many HSV-1 genes; including several genes coding for proteins involved in forming and assembling the virion. Also, DMSO markedly inhibited some but not all early transcripts indicating a previously unknown mode for inhibiting the early phase of HSV transcription-replication cycle. CONCLUSION: These observations suggest that DMSO itself may have a role in the anti-herpetic activity of formulations utilizing it as a dispersant

A Probiotic Adjuvant Lactobacillus rhamnosus Enhances Specific Immune Responses after Ocular Mucosal Immunization with Chlamydial Polymorphic Membrane Protein C

Recent advances in the development of chlamydia vaccines, using live-attenuated or ultraviolet light-inactivated chlamydia, are paving the way for new possibilities to oppose the societal challenges posed by chlamydia-related diseases, such as blinding trachoma. An effective subunit vaccine would mitigate the risks associated with the use of a whole-cell vaccine. Our rationale for the design of an efficient subunit vaccine against Chlamydia trachomatis (Ct) is based on the membrane proteins involved in the initial Ct-host cell contact and on the route of immunization that mimics the natural infection process (i.e., via the ocular mucosa). The first aim of our study was to characterize the specific conjunctival and vaginal immune responses following eye drop immunization in BALB/c mice, using the N-terminal portion of the Ct serovar E polymorphic membrane protein C (N-PmpC) as the subunit vaccine antigen. Second, we aimed to examine the adjuvant properties of the probiotic Lactobacillus rhamnosus (LB) when formulated with N-PmpC. N-PmpC applied alone stimulated the production of N-PmpC-and Ct serovar B-specific antibodies in serum, tears and vaginal washes, whereas the combination with LB significantly enhanced these responses. The N-PmpC/LB combination initiated a T cell response characterized by an elevated percentage of CD25+ T cells and CD8+ effector T cells, enhanced CD4+ T-helper 1 skewing, and increased regulatory T cell responses. Together, these results show that eye drop vaccination with combined use of N-PmpC and a live probiotic LB stimulates specific cellular and humoral immune responses, not only locally in the conjunctiva but also in the vaginal mucosa, which could be a promising approach in Ct vaccine development

HSV-1 latent rabbits shed viral DNA into their saliva

BACKGROUND: Rabbits latent with HSV-1 strain McKrae spontaneously shed infectious virus and viral DNA into their tears and develop recurrent herpetic-specific corneal lesions. The rabbit eye model has been used for many years to assess acute ocular infections and pathogenesis, antiviral efficacy, as well as latency, reactivation, and recurrent eye diseases. This study used real-time PCR to quantify HSV-1 DNA in the saliva and tears of rabbits latent with HSV-1 McKrae. METHODS: New Zealand white rabbits used were latent with HSV-1 strain McKrae and had no ocular or oral pathology. Scarified corneas were topically inoculated with HSV-1. Eye swabs and saliva were taken from post inoculation (PI) days 28 through 49 (22 consecutive days). Saliva samples were taken four times each day from each rabbit and the DNA extracted was pooled for each rabbit for each day; one swab was taken daily from each eye and DNA extracted. Real-time PCR was done on the purified DNA samples for quantification of HSV-1 DNA copy numbers. Data are presented as copy numbers for each individual sample, plus all the copy numbers designated as positive, for comparison between left eye (OS), right eye (OD), and saliva. RESULTS: The saliva and tears were taken from 9 rabbits and from 18 eyes and all tested positive at least once. Saliva was positive for HSV-1 DNA at 43.4% (86/198) and tears were positive at 28.0% (111/396). The saliva positives had 48 episodes and the tears had 75 episodes. The mean copy numbers ± the SEM for HSV-1 DNA in saliva were 3773 ± 2019 and 2294 ± 869 for tears (no statistical difference). CONCLUSION: Rabbits latent with strain McKrae shed HSV-1 DNA into their saliva and tears. HSV-1 DNA shedding into the saliva was similar to humans. This is the first evidence that documents HSV-1 DNA in the saliva of latent rabbits

Decreased reactivation of a herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) mutant using the in vivo mouse UV-B model of induced reactivation

Blinding ocular herpetic disease in humans is due to herpes simplex virus type 1 (HSV-1) reactivations from latency, rather than to primary acute infection. The cellular and molecular mechanisms that control the HSV-1 latency-reactivation cycle remain to be fully elucidated. The aim of this study was to determine if reactivation of the HSV-1 latency associated transcript (LAT) deletion mutant (dLAT2903) was impaired in this model, as it is in the rabbit model of induced and spontaneous reactivation and in the explant TG induced reactivation model in mice. The eyes of mice latently infected with wild type HSV-1 strain McKrae (LAT((+)) virus) or dLAT2903 (LAT((−)) virus) were irradiated with UV-B and reactivation was determined. We found that compared to LAT((−)) virus, LAT((+)) virus reactivated at a higher rate as determined by shedding of virus in tears on days 3 to 7 after UV-B treatment. Thus, the UV-B induced reactivation model of HSV-1 appears to be a useful small animal model for studying the mechanisms involved in how LAT enhances the HSV-1 reactivation phenotype. The utility of the model for investigating the immune evasion mechanisms regulating the HSV-1 latency/reactivation cycle and for testing the protective efficacy of candidate therapeutic vaccines and drugs are discussed