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The rational design and development of genetically attenuated HSV-2 mutant viruses represent an attractive approach for developing both prophylactic and therapeutic vaccines for genital herpes. Previously, HSV-2 UL24 was shown to be a virulence determinant in both murine and guinea pig vaginal infection models. An UL24-ßgluc insertion mutant produced syncytial plaques and replicated to nearly wild type levels in tissue culture, but induced little or no pathological effects in recipient mice or guinea pigs following vaginal infection. Here we report that immunization of mice or guinea pigs with high or low doses of UL24-ßgluc elicited a highly protective immune response. UL24-ßgluc immunization via the vaginal or intramuscular routes was demonstrated to protect mice from a lethal vaginal challenge with wild type HSV-2. Moreover, antigen re-stimulated splenic lymphocytes harvested from immunized mice exhibited both HSV-2 specific CTL activity and IFN-γ expression. Humoral anti-HSV-2 responses in serum were Th1-polarized (IgG2a>IgG1) and contained high-titer anti-HSV-2 neutralizing activity. Guinea pigs vaccinated subcutaneously with UL24-ßgluc or the more virulent parental strain (186) were challenged with a heterologous HSV-2 strain (MS). Acute disease scores were nearly indistinguishable in guinea pigs immunized with either virus. Recurrent disease scores were reduced in UL24-ßgluc immunized animals but not to the same extent as those immunized with strain 186. In addition, challenge virus was not detected in 75% of guinea pigs subcutaneously immunized with UL24-ßgluc. In conclusion, disruption of the UL24 gene is a prime target for the development of a genetically attenuated live HSV-2 vaccine.

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BACKGROUND: A recent review found standardised patient (SP) methodology to be the gold standard methodology for evaluating clinical care. We used this to investigate the content of optometric eye care for a young myopic patient with headaches suggestive of migraine. METHODS: We recruited 100 community optometrists who consented to be visited by an unannounced actor for an eye examination and to have that eye examination recorded. The actor received extensive training to enable accurate reporting of the content of the eye examinations, via an audio recording and a checklist completed for each clinical encounter. The actor presented as a 20-year-old student seeking a private eye examination and complaining of symptoms suggestive of migraine headaches. The results of each clinical encounter were recorded on a pre-designed checklist based on evidence-based reviews on headaches, clinical guidelines and the views of an expert panel of optometrists. RESULTS: The presence of headache was detected in 98% of cases. Eight standard headache questions were considered to be the gold standard for primary care headache investigation. Although none of the optometrists asked all of these questions, 22% asked at least four of the eight questions. Sixty-nine per cent of practitioners asked the patient to seek a medical opinion regarding the headaches. The proportion of the tests recommended by the expert panel that was carried out varied from 33% to 89% and the durations of the eye examination varied from 5 to 50 min. CONCLUSION: SP encounters are an effective way of measuring clinical care within optometry and should be considered for further comparative measurements of quality of care. As in research using SPs in other healthcare disciplines, our study has highlighted substantial differences between different practitioners in the duration and depth of their clinical investigations. This highlights the fact that not all eye examinations are the same and that there is no such thing as a 'standard sight test'. We recommend that future optometric continuing education could usefully focus on migraine diagnosis and assessment.

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A herpes simplex virus type 2 (HSV-2) UL24 beta-glucuronidase (UL24-betagluc) insertion mutant was derived from HSV-2 strain 186 via standard marker transfer techniques. Cell monolayers infected with UL24-betagluc yielded cytopathic effect with syncytium formation. UL24-betagluc replicated to wild-type viral titers in three different cell lines. UL24-betagluc was not virulent after intravaginal inoculation of BALB/c mice in that all inoculated animals survived doses up to 400 times the 50% lethal dose (LD50) of the parental virus. Furthermore, few UL24-betagluc-inoculated mice developed any vaginal lesions. Intravaginal inoculation of guinea pigs with UL24-betagluc at a dose equivalent to the LD50 of parental virus (approximately 5 x 10(3) PFU) was not lethal (10/10 animals survived). Although genital lesions developed in some UL24-betagluc-inoculated guinea pigs, both the overall number of lesions and the severity of disease were far less than that observed for animals infected with parental strain 186.

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Expression of Norwalk virus nonstructural polyprotein precursor in vitro resulted in rapid cotranslational cleavage at specific sites. The cleavage products were similar to those previously identified for Southampton virus, a highly related virus. We inactivated the virally encoded proteinase responsible for cleavage of the nonstructural polyprotein by mutation of the putative catalytic cysteine residue, which resulted in production of full-length polyprotein precursor. NV proteinase was expressed in Escherichia coli as a glutathione S-transferase fusion and purified by GST-affinity chromatography. Activity of the purified proteinase was demonstrated by incubation with the full-length precursor protein. trans cleavage of the nonstructural protein precursor resulted in cleavage products similar to those observed during cotranslational cleavage, however, at lesser efficiency. NV proteinase displayed sensitivities to cysteine and serine protease inhibitors similar to poliovirus 3C proteinase, suggesting that NV proteinase is a member of the viral cysteine proteinase family. We propose that the proteinase may play a regulatory role in viral replication.

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In this study, we evaluated the potential of a genetically modified cholera toxin, CT-E29H as an adjuvant for recombinant Norwalk virus like particle (NV-VLP) vaccine. This detoxified mutant, containing E to H substitution at amino acid 29 of the CT-A1 subunit, was administered with a recombinant Norwalk virus like particle vaccine to Balb/c mice by mucosal routes to monitor the induction of mucosal, humoral and cellular responses. We observed that a low dose of NV-VLP (5 microg) with the adjuvant delivered by the intranasal route (IN) was more effective than the highest dose (200 microg) delivered by oral route at inducing both cellular and NV-VLP specific IgG and IgA responses. Higher counts of antigen specific IgA secreting cells were observed in the Peyer's Patches (PP) following delivery of the vaccine with CT-E29H as compared to delivery of vaccine by mucosal routes without CT-E29H. Furthermore, there was an increase in antigen specific cells producing IL-4 from animals that received the vaccine with the adjuvant. Delivery of the vaccine by the oral route results in antigen specific CD4(+) and CD8(+) T cells in PP and spleen. Addition of CT-E29H results in an increase of antigen specific CD4(+) cell population in PP and both CD4(+) and CD8(+) populations in the spleen. These cellular and cytokine responses suggest that combining the vaccine with CT-E29H results in a stronger Th2 type response. Collectively, these results indicate that immune responses to NV-VLP vaccine are qualitatively and quantitatively improved when the vaccine is delivered along with CT-E29H, and thus merits its further consideration as a mucosal adjuvant.

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