Biomedical subjects

S L Wechsler

Publications and source records attributed to S L Wechsler.

At least 37 records · Page 2Linked to original sources

PubMed1999

Herpes simplex virus type 1 serum neutralizing antibody titers increase during latency in rabbits latently infected with latency-associated transcript (LAT)-positive but not LAT-negative viruses.

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) gene is essential for efficient spontaneous reactivation in the rabbit ocular model of HSV-1 latency and reactivation. LAT is also the only viral gene abundantly expressed during latency. Rabbits were ocularly infected with the wild-type HSV-1 strain McKrae or the McKrae-derived LAT null mutant dLAT2903. Serum neutralizing antibody titers were determined at various times during acute and latent infection. The neutralizing antibody titers induced by both viruses increased and were similar throughout the first 45 days after infection (P > 0.05). However, by day 59 postinfection (approximately 31 to 45 days after latency had been established), the neutralizing antibody titers induced by wild-type virus and dLAT2903 diverged significantly (P = 0.0005). The dLAT2903-induced neutralizing antibody titers decreased, while the wild-type virus-induced neutralizing antibody titers continued to increase. A rescuant of dLAT2903, in which spontaneous reactivation was fully restored, induced wild-type neutralizing antibody levels on day 59 postinfection. A second LAT mutant with impaired spontaneous reactivation had neutralizing antibody levels comparable to those of dLAT2903. In contrast to the results obtained in rabbits, in mice, neutralizing antibody titers did not increase over time during latency with any of the viruses. Since LAT is expressed in both rabbits and mice during latency, the difference in neutralizing antibody titers between these animals is unlikely to be due to expression of a LAT protein during latency. In contrast, LAT-positive (LAT(+)), but not LAT-negative (LAT(-)), viruses undergo efficient spontaneous reactivation in rabbits, while neither LAT(+) nor LAT(-) viruses undergo efficient spontaneous reactivation in mice. Thus, the increase in neutralizing antibody titers in rabbits latently infected with LAT(+) viruses may have been due to continued restimulation of the immune system by spontaneously reactivating virus.

Animals↗

PubMed1999

A herpes simplex virus type 1 latency-associated transcript mutant with increased virulence and reduced spontaneous reactivation.

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) gene is essential for efficient spontaneous reactivation of HSV-1 from latency. We previously reported that insertion of the LAT promoter and just the first 1.5 kb of the 8. 3-kb LAT gene into an ectopic location in the virus restored wild-type spontaneous reactivation to a LAT null mutant. This mutant, LAT3.3A (previously designated LAT1.5a), thus showed that the expression of just the first 1.5 kb of LAT is sufficient for wild-type spontaneous reactivation. We also showed that in the context of the entire LAT gene, deletion of LAT nucleotides 76 to 447 (LAT mutant dLAT371) had no effect on spontaneous reactivation or virulence. We report here on a LAT mutant designated LAT2.9A. This mutant is similar to LAT3.3A, except that the ectopic LAT insert contains the same 371-nucleotide deletion found in dLAT371. We found that LAT2.9A had a significantly reduced rate of spontaneous reactivation compared to marker-rescued and wild-type viruses. This was unexpected, since the combined results of dLAT371 and LAT3.3A predicted that spontaneous reactivation of LAT2.9A would be wild type. We also found that LAT2.9A was more virulent than wild-type or marker-rescued viruses after ocular infection of rabbits. This was unexpected, since LAT null mutants and LAT3.3A have wild-type virulence. These results suggest for the first time (i) that regions past the first 1.5 kb of LAT can compensate for deletions in the first 1.5kb of LAT and may therefore play a role in LAT dependent spontaneous reactivation and (ii) that regions of LAT affect viral virulence.

Animals↗

PubMed1998

Therapeutic periocular vaccination with a subunit vaccine induces higher levels of herpes simplex virus-specific tear secretory immunoglobulin A than systemic vaccination and provides protection against recurrent spontaneous ocular shedding of virus in latently infected rabbits.

Rabbits latently infected with herpes simplex virus type 1 (HSV-1) were vaccinated either periocularly or systemically with a subunit vaccine (gB2 + gD2) plus adjuvant or adjuvant alone. Tear films were collected daily to measure recurrent infectious HSV-1 shedding. After systemic vaccination, the latently infected rabbits were not protected against recurrent ocular viral shedding (HSV-1-positive tear film cultures/total cultures) compared with either the systemic or periocular adjuvant controls (systemic vaccination = 49 of 972, 5.0%; systemic control = 46 of 972, 4.7%; periocular control = 43 of 930, 4.6%; P > 0.8). In contrast, latently infected rabbits vaccinated periocularly with the same vaccine had significantly reduced recurrent shedding (20 of 1026, 2.0%) compared with controls (P < 0.001) or systemic vaccination (P = 0.0002). Thus, recurrent HSV-1 shedding was significantly reduced by therapeutic local periocular subunit vaccination but not by therapeutic systemic subunit vaccination. Neutralizing antibody titers in the serum of systemically and ocularly vaccinated rabbits was similar. In contrast, HSV-specific tear secretory immunoglobulin A was significantly higher in the ocularly vaccinated group (P < 0.01). These results strongly suggest that in the rabbit, and presumably in humans, the local ocular (mucosal) immune response is much more important than the systemic immune response for therapeutic protection against recurrent ocular HSV-1. Thus development of a therapeutic vaccine against recurrent ocular HSV-1 should be directed at enhancing the local ocular (mucosal) immune response.

Animals↗

PubMed1998

The region of the herpes simplex virus type 1 LAT gene involved in spontaneous reactivation does not encode a functional protein.

We previously showed that the LAT function required for efficient spontaneous reactivation of herpes simplex virus type 1 (HSV-1) from neuronal latency in the rabbit maps within the first 1.5 kb of the 8.3-kb primary LAT transcript. This demonstrated that LAT does not function via an antisense mechanism, since the first 1.5 kb of LAT does not overlap any other known HSV-1 gene. Furthermore, if LAT encodes a protein essential for efficient spontaneous reactivation, it must map within the functional first 1.5 kb of LAT. Thus, the absence of a well-conserved LAT open reading frame in this region among all HSV-1 LAT genes capable of supporting high levels of spontaneous reactivation would demonstrate that LAT does not encode a protein essential for efficient spontaneous reactivation. In this report, we sequenced the first 1.5 kb of LAT from HSV-1 McKrae, a strain with a very high spontaneous reactivation rate. Of the HSV-1 LAT sequences available for comparison (17syn+, KOS, and F), only strain 17syn+ has a high spontaneous reactivation rate. However, as shown in this report, a chimeric virus containing the KOS LAT gene on an HSV-1 McKrae genetic background had a spontaneous reactivation rate indistinguishable from McKrae (15 versus 13.6%; P > 0.05). Thus, the spontaneous reactivation competency of the LAT gene from HSV-1 KOS was similar to that of the McKrae LAT gene. Comparative sequence analysis of the LAT genes from McKrae, 17syn+, and KOS revealed that none of the eight potential McKrae LAT ORFs were well conserved. Additional types of sequence analyses further confirmed that none of the potential ORFs were likely to encode a functional LAT protein. These results strongly support the notion that the LAT function involved in spontaneous reactivation is mediated by a direct DNA or RNA mechanism rather than a protein.

Animals↗

PubMed1998

The persistent elevated cytokine mRNA levels in trigeminal ganglia of mice latently infected with HSV-1 are not due to the presence of latency associated transcript (LAT) RNAs.

Trigeminal ganglia (TG) from mice latently infected with wild type HSV-1 contain detectable levels of cytokine transcripts that are not present in TG from uninfected mice. This suggests that during HSV-1 neuronal latency, the immune system is stimulated by the production of one or more viral proteins. Since the LAT (latency associated transcript) gene is essential for wild type levels of spontaneous reactivation and is the only highly active viral gene during latency, the stimulation of cytokines may indicate the presence of a LAT encoded latency protein. We therefore compared the cytokine transcript profiles in the TG of mice latently infected with wild type and LAT negative viruses. Mice were latently infected with either: (1) the LAT null mutant dLAT2903; (2) its marker rescued virus dLAT2903R; or (3) the parental wild type HSV-1 strain McKrae. As expected, reactivation following explant cultivation of TG from latently infected mice was significantly decreased with dLAT2903 (P < 0.05)(40 +/- 8%, n = 24) compared with dLAT2903R (85 +/- 7.6%, n = 36) or the parental virus (70 +/- 10.0%, n = 36). The relative levels of various cytokines was determined by RT-PCR analysis of TG extracts. None of the cytokine transcripts detected in mice latently infected with the wild type or marker rescued viruses were missing or decreased in mice latently infected with the LAT null mutant 30 or 60 days post infection. There were also no differences in the HSV-1 antibody titers induced by the LAT negative virus compared to the LAT positive viruses. Thus, although LAT facilitated reactivation of HSV-1 from explanted mouse TG, expression of LAT during latency did not appear to be involved in persistent cytokine expression in TG. This suggests that during latency, HSV-1 does not produce a highly antigenic abundant LAT encoded protein.

Animals↗

PubMed1998

The role of neutralizing antibody and T-helper subtypes in protection and pathogenesis of vaccinated mice following ocular HSV-1 challenge.

In order to determine the possible correlation of specific immune responses with protection against mortality and ocular disease following ocular herpes simplex virus type 1 (HSV-1) challenge, BALB/c mice were vaccinated with different doses and regimens of baculovirus-expressed gD. Neutralizing antibody, virus titres in the eyes, corneal scarring, and survival were measured. In addition, infiltration into the cornea of CD4+ T cells and cells containing the lymphokines interleukin-2 (IL-2), IL-4, IL-6 and tumour necrosis factor-alpha (TNF-alpha) were monitored on days 3, 7, 10, 14 and 21 post-challenge by immunocytochemistry. The vaccination regimens used induced varying degrees of immune responses and protection upon ocular challenge with HSV-1. Our results suggest that neutralizing antibody was the most important immune response in protecting mice against lethal ocular challenge and corneal scarring. TNF-alpha and IL-2 were not crucial in terms of survival and corneal scarring, since gD1 (one vaccination with 1 microg of gD) and gD0.1 (one vaccination with 0.1 microg of gD), both of which provided high levels of protection, showed no TNF-alpha or IL-2 expression. However, TNF-alpha and IL-2 were crucial in terms of virus clearance from the eyes, since gD3 (three vaccinations with 1 microg of gD), which had less virus in their eyes, had high numbers of TNF-alpha and IL-2 infiltrates. Finally, mock-vaccinated mice were not protected from death and corneal disease following HSV-1 challenge. Eyes of mock-vaccinated mice had little or no TNF-alpha or IL-2 responses and the strongest IL-4 and IL-6 responses.

Animals↗

PubMed1998

Local periocular vaccination protects against eye disease more effectively than systemic vaccination following primary ocular herpes simplex virus infection in rabbits.

Vaccination of experimental animals can provide efficient protection against ocular herpes simplex virus type 1 (HSV-1) challenge. Although it is suspected that local immune responses are important in protection against ocular HSV-1 infection, no definitive studies have been done to determine if local ocular vaccination would produce more efficacious protection against HSV-1 ocular challenge than systemic vaccination. To address this question, we vaccinated groups of rabbits either systemically or periocularly with recombinant HSV-2 glycoproteins B (gB2) and D (gD2) in MF59 emulsion or with live KOS (a nonneurovirulent strain of HSV-1). Three weeks after the final vaccination, all eyes were challenged with McKrae (a virulent, eye disease-producing strain of HSV-1). Systemic vaccination with either HSV-1 KOS or gB2/gD2 in MF59 did not provide significant protection against any of the four eye disease parameters measured (conjunctivitis, iritis, epithelial keratitis, and corneal clouding). In contrast, periocular vaccination with gB2/gD2 in MF59 provided significant protection against conjunctivitis and iritis, while ocular vaccination with live HSV-1 KOS provided significant protection against all four parameters. Thus, local ocular vaccination provided better protection than systemic vaccination against eye disease following ocular HSV-1 infection. Since local vaccination should produce a stronger local immune response than systemic vaccination, these results suggest that the local ocular immune response is very important in protecting against eye disease due to primary HSV-1 infection. Thus, for clinical protection against primary HSV-1-induced corneal disease, a local ocular vaccine may prove more effective than systemic vaccination.

Adjuvants, Immunologic↗

PubMed1998

The US5 open reading frame of herpes simplex virus type 1 does encode a glycoprotein (gJ).

Based on sequence analysis, the protein encoded by the US5 open reading frame (ORF) of herpes simplex virus type 1 (HSV-1) was predicted to contain an N-glycosylation site and was given the designation of glycoprotein J (gJ). However, the US5 gene product has not been identified and the identity of gJ as a glycoprotein has not been confirmed. We have cloned and expressed the DNA encoding the complete sequence of the US5 ORF, using a baculovirus expression system. Western blotting, using polyclonal antibody raised against synthetic US5 peptides, revealed two major baculovirus-US5-expressed protein bands with apparent molecular weights of 16-17 and 10 kD. The recombinant US5 was found on the membrane of Spodoptera frugiperda cells and was susceptible to tunicamycin, endoglycosidase H, glycosidase F and partially resistant to endoglycosidase F. Vaccination of mice with baculovirus-expressed US5 did not induce a neutralizing antibody to HSV-1 or provide protection against lethal HSV-1 challenge. However, serum from these vaccinated mice was able to recognize US5 in purified HSV-1 virions by Western blot analyses and on the surface of HSV-1-infected cells by immunofluorescence. These findings establish that US5 does encode a glycoprotein and confirm the appropriateness of naming the US5 gene product gJ.

Amino Acid Sequence↗

PubMed1998

A therapeutic vaccine that reduces recurrent herpes simplex virus type 1 corneal disease.

PURPOSE: To investigate the therapeutic efficacy of periocular vaccination with herpes simplex virus (HSV) recombinant glycoprotein D from HSV-1 (gD1) or HSV-2 (gD2) in decreasing HSV-induced recurrent dendritic keratitis and HSV-induced recurrent ocular shedding in rabbits latently infected with HSV-1. METHODS: Rabbits latently infected with HSV-1 were vaccinated periocularly (by subconjunctival injection) with gD1 and adjuvant, gD2 and adjuvant, or adjuvant alone. Eyes were examined daily for 49 days for recurrent herpetic keratitis and for recurrent infectious HSV-1 shedding. RESULTS: In both vaccinated groups, a significantly decreased number of eyes exhibited recurrences of herpetic keratitis compared with recurrences in adjuvant-treated control eyes (gD1 group, 27/1372, [2%]; gD2 group, 24/1274, [2%]; and control, 54/1274 [4%]; P < 0.005). Eyes in the gD1-vaccinated group (44/1308 [3.4%]; P = 0.01), but not those in the gD2-vaccinated group (71/1274 [5.6%]; P = 0.93), had significantly decreased viral shedding (positive cultures compared with total cultures) compared with eyes in the adjuvant-treated control group (69 of 1275 [5.4%]). CONCLUSIONS: Recurrent HSV-1 corneal disease was significantly reduced by therapeutic local periocular vaccination. The vaccine may be more efficacious against HSV-1-induced recurrent corneal disease than against recurrent HSV-1 ocular shedding. Its efficacy against corneal disease appeared to be longer lasting than its efficacy against recurrent spontaneous shedding. The heterotypic gD2 vaccine was as efficacious as the homotypic gD1 vaccine against recurrent corneal disease, whereas the homotypic vaccine was much more efficacious than the heterotypic vaccine against recurrent HSV-1 shedding. This is the first report in any animal model of a successful therapeutic vaccine against recurrent HSV-1-induced corneal disease. These results support the concept that development of a therapeutic vaccine for ocular HSV-1 recurrence in humans may be possible.

Acetylmuramyl-Alanyl-Isoglutamine↗

PubMed1997

The importance of MHC-I and MHC-II responses in vaccine efficacy against lethal herpes simplex virus type 1 challenge.

To investigate the importance of major histocompatability complex (MHC) class I- and MHC class II-dependent immune responses in herpes simplex virus-1 (HSV-1) vaccine efficacy, groups of beta 2% (MHC I-) and Ab% (MHC II-) mice were inoculated with various vaccines, and then challenged intraperitoneally with HSV-1. Following vaccination with either live avirulent HSV-1, expressed HSV-1 glycoprotein D (gD), or a mixture of seven expressed HSV-1 glycoproteins (7gPs), Ab% (MHC-II-) mice developed no enzyme-linked immunosorbent assay (ELISA) or neutralizing antibody titres. In contrast, significant ELISA and neutralizing antibody titres were induced in beta 2m% (MHC-I-) mice by all three vaccines. The neutralizing antibody titres were similar for all three vaccines, but were only approximately 1/4 to 1/3 of that developed in C57BL/6 (parental) mice vaccinated with the same antigens. All three vaccines protected 100% of the wild-type C57BL/6 mice against lethal challenge with 2 x 10(7) plaque-forming units (PFU) of HSV-1. The live virus vaccine and the 7gPs vaccine also protected 80% of the beta 2m% mice against the same lethal HSV-1 challenge dose. In contrast, in Abo/o mice, none of the vaccines provided significant protection against the same lethal challenge dose of HSV-1. However, at a lower challenge dose of 2 x 10(6) PFU, all three vaccines protected 70-80% of the vaccinated Ab% mice (compared to only 10% survival in mock vaccinated controls). Thus, vaccination provided some protection against lethal HSV-1 challenge in both beta 2m% and Ab% mice; however, the protection was less than that seen in the parental C57BL/6 mice. In addition, Ab% mice were less well protected by vaccination than were beta 2m% mice. Our results suggest that (1) both MHC-I and MHC-II are involved in vaccine efficacy against HSV-1 challenge; (2) both types of responses must be present for maximum vaccine efficacy: and (3) the MHC-II-dependent immune response appeared to be more important than the MHC-I-dependent immune response for vaccine efficacy against HSV-I challenge.

Animals↗

PubMed1997

MHC-II but not MHC-I responses are required for vaccine-induced protection against ocular challenge with HSV-1.

PURPOSE: To determine the importance of major histocompatibility complex (MHC) class-I versus MHC class-II immune responses in protecting naive versus vaccinated mice against an ocular HSV-1 challenge. METHODS: Class-II deficient A beta o/o (CD4-CD8+ T cells) knockout mice, which are effectively CD4+ T cells-negative, and class-I deficient beta(2)mo/o (CD4+CD8- T cells) knockout mice, which are effectively CD8+ T cells negative, were either vaccinated or mock-vaccinated and examined for their ability to withstand HSV-1 ocular challenge. RESULTS: Unvaccinated A beta o/o and beta(2)mo/o mice were both more susceptible to lethal ocular HSV-1 infection than the parental wild type C57BL/6J mice, indicating that both MHC-I and MHC-II were required for optimal protection of naive mice against ocular HSV-1 challenge. Vaccinated beta(2)mo/o mice produced significant neutralizing antibody titers, and following ocular challenge, these mice were completely protected against death and corneal scarring. In contrast, vaccinated A beta o/o mice developed no neutralizing antibody titers and vaccination did not provide these mice with any protection against death or corneal scarring. Passive transfer of anti-HSV-1 antibody into A beta o/o mice up to 6 days post ocular challenge resulted in complete protection against death and corneal scarring. CONCLUSIONS: Passive antibody transfer, but not vaccination, protected A beta o/o mice against ocular challenge. In contrast, vaccination completely protected beta(2)mo/o mice. These results suggest for a vaccine to provide optimal protection against ocular HSV-1 challenge in this system, it is not only sufficient, but it is also required, that the vaccine induce an effective neutralizing antibody response.

Animals↗

PubMed1997

Nonneutralizing antibody against the glycoprotein K of herpes simplex virus type-1 exacerbates herpes simplex virus type-1-induced corneal scarring in various virus-mouse strain combinations.

PURPOSE: To determine whether the exacerbation of herpes simplex virus type-1 (HSV-1) induced corneal scarring that the authors reported previously in HSV-1 glycoprotein K (gK) vaccinated BALB/c mice challenged with HSV-1 strain McKrae was a general phenomenon independent of virus and mouse strains. To determine the gK-induced immune response leading to exacerbation of HSV-1-induced corneal scarring. METHODS: BALB/c or C57BL/6 mice were vaccinated with gK, ocularly challenged with HSV-1 strain KOS or McKrae, and the relative amount of corneal scarring determined 28 days after challenge. The T cells, total serum, or purified immunoglobulin G (IgG) isolated from gK-vaccinated mice was transferred individually to naive mice, and the affects on corneal scarring after HSV-1 challenge were determined. RESULTS: The KOS challenge of gK-vaccinated BALB/c mice resulted in significant corneal scarring (P = 0.0003), despite the fact that KOS normally produces no corneal scarring. McKrae challenge of gK-vaccinated C57BL/6 mice resulted in significant corneal scarring (P < 0.0001), despite the fact that C57BL/6 mice are normally refractory to HSV-1-induced corneal scarring. Passive transfer of total anti-gK mouse sera or purified anti-gK mouse IgG, but not adoptive transfer of total anti-gK T-cells to naive mice, resulted in exacerbation of corneal scarring after HSV-1 challenge (P < 0.0001). Mice defective for T-cell-dependent antibody production were not susceptible to exacerbation of HSV-1-induced corneal scarring by gK vaccination (P < 0.0001). CONCLUSIONS: The ability of gK vaccination to exacerbate HSV-1-induced corneal scarring was not mouse strain or HSV-1 strain specific. The gK-induced exacerbation of corneal scarring was related to anti-gK IgG. How anti-gK IgG exacerbated HSV-1 induced corneal scarring remains to be determined.

Animals↗

PubMed1996

Vaccination with herpes simplex virus type 1 glycoprotein K impairs clearance of virus from the trigeminal ganglia resulting in chronic infection.

Following primary ocular infection, HSV-1 establishes a latent infection in the trigeminal ganglia with the complete absence of detectable infectious virus. Recently, we showed that vaccination of BALB/cJ with HSV-1 glycoprotein K (gK), but not other HSV-1 glycoproteins, significantly exacerbated the severity of HSV-1-induced ocular disease and herpetic dermatitis (H. Ghiasi et al. J. Virol. 68, 2347-2354, 1994). We now report that prior vaccination with gK blocked viral clearance from trigeminal ganglia until at least Day 56 postinfection, resulting in a chronic infection. The significance of this novel finding with respect to potential harmful complications associated with the immune response to gK is discussed.

Animals↗

PubMed1996

The UL3 open reading frame of herpes simplex virus type 1 codes for a phosphoprotein.

Based on sequence analysis, the protein encoded by the UL3 open reading frame (ORF) of herpes simplex virus type 1 (HSV-1) was predicted to contain an N glycosylation site and to be a glycoprotein. To determine if this prediction was correct, we cloned and expressed the DNA encoding the complete sequence of the UL3 ORF in a baculovirus expression system. Western blotting was done using polyclonal antibody raised against synthetic UL3 peptides. Two major baculovirus-UL3 expressed protein bands with apparent molecular weights of 30 kDa and 31 kDa, and two minor protein bands with apparent molecular weights of 29 kDa and 33 kDa were detected. None of the expressed UL3 protein species were susceptible to tunicamycin treatment, suggesting that they were not N-linked glycosylated. Cell fractionation studies indicated that the UL3 protein was localized in the cytoplasmic and nuclear portion of the cells, rather than the cell membrane, again suggesting a lack of glycosylation. In contrast, the baculovirus expressed UL3 protein was phosphorylated as judged by 32Pi-labeling. Immunoprecipitation followed by SDS-PAGE demonstrated a single 32Pi-labeled UL3 related band with an apparent molecular weight of 33 kDa, indicating that the UL3 protein was a phosphoprotein. Antibodies produced in mice vaccinated with baculovirus-UL3 protein reacted with two UL3 related HSV-1 bands on Western blots. These protein bands had apparent molecular weights of 27 and 33 kDa and presumably represent the unphosphorylated and phosphorylated forms of UL3.

Animals↗

PubMed1996

Vaccination with a cocktail of seven recombinantly expressed HSV-1 glycoproteins protects against ocular HSV-1 challenge more efficiently than vaccination with any individual glycoprotein.

Our previous studies have shown that of seven HSV-1 glycoproteins (gB, gC, gD, gE, gG, gH and gI) individually expressed in baculovirus, vaccination with gD provides the best protection against HSV-1 challenge. To establish whether vaccination with a mixture of these seven expressed glycoproteins would provide better protection against HSV-1 challenge than vaccination with gD alone, we determined the level of protection afforded by vaccination with a cocktail of the seven expressed glycoproteins. The amount of each of the seven expressed glycoproteins in the mixture was equivalent to one-seventh the amount of gD used in the gD alone vaccination. Thus, the total amount of glycoprotein was the same for the cocktail and gD alone vaccine. For neutralizing antibody titer, delayed-type hypersensitivity (DTH), and survival following lethal challenge, no difference was observed between mice vaccinated with all seven glycoproteins and those vaccinated with gD. However, for other criteria, vaccination with all seven glycoproteins appeared to provide better protection than vaccination with gD. Following ocular challenge, virus was not detected at any time in the tears of mice vaccinated with all seven glycoproteins. In contrast, virus was detected in the tears of gD vaccinated mice for up to 3 days post challenge. Mock vaccinated mice had virus in their tears for as long as 10 days. Mice vaccinated with all seven glycoproteins had no eye disease, while gD vaccinated mice had a significant amount of blepharitis. Finally, compared to gD vaccinated mice, the mice vaccinated with all seven glycoproteins were more efficiently protected against the establishment of HSV-1 latency following ocular infection. Our results therefore suggest that while for some protective criteria there was no significant difference between vaccination with gD or seven glycoproteins, vaccination with seven glycoproteins was more efficient in protecting challenged mice against some forms of eye disease, the duration of infection and the establishment of latency.

Animals↗

PubMed1996

The region of the herpes simplex virus type 1 LAT gene that is colinear with the ICP34.5 gene is not involved in spontaneous reactivation.

The goal of this report was to determine if the region of the LAT gene that is colinear with ICP34.5 (kb 6.2 to 7.1 of LAT) is involved in spontaneous reactivation of herpes simplex virus type 1. We inserted one copy of the ICP34.5 gene into the unique long region of a herpes simplex virus type 1 (strain McKrae) mutant lacking both copies of ICP34.5 (one in each viral long repeat) and the corresponding 917-nucleotide colinear portion of LAT (kb 6.2 to 7.1). Rabbits were ocularly infected with this mutant, and spontaneous reactivation relative to that for the wild-type virus and the original mutant was measured. As we previously reported, the original ICP34.5-deleted virus (d34.5) was significantly impaired for spontaneous reactivation and virulence (G. C. Perng, R. L. Thompson, N. M. Sawtell, W. E. Taylor, S. M. Slanina, H. Ghiasi, R. Kaiwar, A. B. Nesburn, and S. L. Wechsler, J. Virol. 69:3033-3041, 1995). In contrast, we report here that restoration of one copy of ICP34.5 at a distant location completely restored the wild-type level of in vivo spontaneous reactivation, despite retention of the deletion in LAT (spontaneous reactivation rate = 0.3 to 1.4% for the ICP34.5 deletion mutant, 7.7 to 19.6% for the wild type, and 9 to 16.1% for virus with one copy of ICP34.5). Thus, the 917-nucleotide region of LAT from kb 6.2 to 7.1 was not involved in the LAT function required for wild-type spontaneous reactivation. We also found that restoration of a single ICP34.5 gene in a novel location did not restore wild-type virulence (rabbit death rate = 0% [0 of 15] for the original ICP34.5 deletion mutant, 8% [2 of 24] for the single-copy IPC34.5 virus, and 52% [14 of 27] for wild-type virus; P < 0.001 for one versus two copies of ICP34.5). It is likely that either two gene doses of ICP34.5 or its location in the long repeat is essential for full functionality of ICP34.5's virulence function. Furthermore, the ability of the single-copy ICP34.5 virus to reactivate at wild-type levels despite being significantly less virulent than wild-type virus separates the spontaneous reactivation phenotype from the virulence phenotype.

Animals↗

PubMed1996

The spontaneous reactivation function of the herpes simplex virus type 1 LAT gene resides completely within the first 1.5 kilobases of the 8.3-kilobase primary transcript.

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) gene is essential for efficient spontaneous reactivation of HSV-1 from latency. We report here that although the LAT gene is 8.3 kb in length, the first 1.5 kb of the LAT gene alone is sufficient for wild-type levels of spontaneous reactivation. We began with a LAT deletion mutant of HSV-1 strain McKrae in which the LAT promoter and the first 1.6 kb of the 5' end of the LAT gene had been deleted from both copies of LAT (one in each viral long repeat). As we previously reported, this mutant (dLAT2903) was significantly impaired for spontaneous reactivation (G. C. Perng, E. C. Dunkel, P. A. Geary, S. M. Slanina, H. Ghiasi, R. Kaiwar, A. B. Nesburn, and S. L. Wechsler, J. Virol. 68:8045-8055, 1994). We then inserted the LAT promoter and the first 1.5 kb of the LAT gene into a location in the unique long region of dLAT2903 far removed from the normal location of LAT in the long repeats. This resulted in a virus (LAT15a) whose capacity for transcribing LAT RNA was limited to the first 1.5 kb of the 8.3-kb LAT primary transcript. Rabbits were ocularly infected with this mutant, and spontaneous reactivation was measured in comparison to those of the original LAT-negative mutant and its marker-rescued (wild-type) virus, dLAT2903R. LAT15a had an in vivo spontaneous reactivation rate of 12%, compared with a rate of 11% for the marker-rescued virus and 0% for the LAT-negative virus. Southern analysis confirmed that the spontaneously reactivated LAT15a virus retained the original deletions in both copies of LAT and the 1.5-kb LAT insertion in the unique long region. Thus, insertion of the first 1.5 kb of LAT (and its promoter) at a site distant from the normal LAT location appeared to completely restore in vivo spontaneous reactivation to wild-type levels, despite the remaining inability of the original LAT genes to transcribe any LAT RNA. The function of LAT involved in efficient spontaneous reactivation therefore appeared to map completely within the first 1.5 kb of the LAT gene.

Animals↗

PubMed1996

A 371-nucleotide region between the herpes simplex virus type 1 (HSV-1) LAT promoter and the 2-kilobase LAT is not essential for efficient spontaneous reactivation of latent HSV-1.

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) gene is essential for efficient spontaneous reactivation of HSV-1 from latency. However, neither the mechanism by which LAT carries out this function nor the region of LAT responsible for this function in known. LAT is transcribed as an unstable 8.3-kb RNA that gives rise to a very stable 2-kb LAT RNA that is readily detected in latently infected sensory neurons. We show here that 371 of the 662 nucleotides located between the start of LAT transcription and the 5' end of the 2-kb LAT RNA do not appear to be essential for wild-type levels of spontaneous reactivation in the rabbit ocular model of HSV-1 latency. We deleted LAT nucleotides 76 to 447 from both copies of the LAT gene (one in each viral long repeat) to produce the mutant dLAT371. Rabbits were ocularly infected with dLAT371, and spontaneous reactivation was measured in comparison with the marker-rescued virus dLAT371R. Both dLAT371 and dLAT371R had spontaneous reactivation rates of approximately 13 to 14%. This was consistent with the parental McKrae wild-type virus (11.7%; P = 0.49) and significantly higher than the LAT transcription-negative mutant dLAT2903 (2.4%; P < 0.0001). Southern analysis confirmed that the spontaneously reactivated dLAT371 virus retained the deletion in both copies of LAT. Therefore, it appeared that the function of LAT involved in efficient spontaneous reactivation mapped outside the 371-nucleotide region deleted from the LAT gene of dLAT371.

Animals↗