Objective: The kinetics of immediate early (IE) and early (E) herpes simplex virus 1 (HSV-1) mRNA transcription was followed in explanted trigeminal ganglia from rabbits with established latency. Methods: The expression of IE and E mRNAs was first assessed in infected Vero cells by RT-PCR and then in explanted trigeminal ganglia by nested RT-PCR. Results: In infected Vero cells, IE mRNAs [for infected cell protein (ICP) 0, ICP4 and ICP27] were first detected 1–2 h post-inoculation (p.i.), peaking at 3 h p.i. The transcription of E mRNAs [for thymidine kinase (TK), RR1 and UL9], which were first detected from 3 h p.i., peaked between 5 and 10 h p.i. In explanted ganglia, the ICP0, ICP4 and ICP27 mRNAs were first detected after 4 h in culture. This was followed by the appearance of TK mRNA at 8 h and then by the UL9 mRNA, detected from 12 h post-explantation. A further E mRNA (RR1), as well as the late gC mRNA, were first observed after 24 h in culture. Moreover, ICP4 mRNA could be found in non-cultured ganglia. Conclusions: During reactivation of latent HSV-1 in explanted ganglia, the onset of ICP0 and ICP27 transcription at 4 h in culture was followed by TK transcription (at 8 h). Thus, in the rabbit reactivation model, ICP0 gene transcription rather than ICP4 transcription represents the relevant indicator of latency reactivation.
To compare the immunogenity of the herpes simplex virus 1 (HSV-1/HHV-1) recombinant glycoprotein D (gD1), as a potential protective vaccine, Balb/c mice were immunized with either gD1/313 (the ectodomain of the gD1 fusion protein consisting of 313 amino acid residues), or the plasmid pcDNA3.1-gD (coding for a full length gD1 protein, FLgD1). A live attenuated HSV-1 (deleted in the gE gene), and a HSV-1 (strain HSZP)-infected cell extract served as positive controls, and three non-structural recombinant HSV-1 fusion proteins (ICP27, UL9/OBP and thymidine kinase--TK) were used as presumed non-protective (negative) controls. Protection tests showed that the LD50 value of the challenging infectious virus increased 90-fold in mice immunized with ICP27, but remained unchanged in other control mice immunized with TK and OBP polypeptides. A significant protection (the LD50 value of challenging virus increased 800-fold) was noted following immunization with gD1/313, while immunization with the gE-del virus and/or the gD1 DNA vaccine resulted in a more than 4,000-fold increase of the challenging virus dose killing 50% of the animals. Using ELISA, elevated antibody titers were detected following immunizations with gD1/313, gE-del virus, and/or HSV-1-infected-cell extract. In addition, all of the three non-structural proteins elicited a good humoral response (with titres ranging from 1:16,000 to 1:128,000). The lowest IgG response (1:8,000) was noted after immunization with the gD1 DNA vaccine. Peripheral blood leukocytes (PBLs) as well as splenocytes from mice immunized with gD1/313, gE-del virus, and gD1-plasmid responded in lymphocyte transformation test (LTT) to the presence of purified gD1/313 antigen. For PBLs, the most significant stimulation of thymidine incorporation was registered at a gD1/313 concentration of 5 microg/100 microl, while the splenocytes from DNA vaccine-immunized mice responded already at a concentration of 1 microg/100 microl.
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