INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

During HSV-1 neuronal latency, only one viral gene is consistently observed to be expressed at high levels (6, 39). This LAT (latency-associated transcript) gene has a powerful promoter that is active in most cell types (23, 27, 37). LAT viruses appear to be unimpaired during acute infection (35). Thus, insertion of a foreign gene under the LAT promoter in place of the structural portion of LAT can produce a useful recombinant vector. In this study, we inserted the gene for murine interleukin-4 (IL-4) into both copies of the LAT gene (one in each viral long repeat), under control of the LAT promoter, in place of the 5' end of the LAT gene. The recombinant virus carrying this gene, HSV-IL-4, expressed high levels of IL-4 and allowed us to examine the effect of high exogenous IL-4 levels on HSV-1 pathogenicity in mice.

IL-4 is a pleiotropic lymphokine synthesized primarily by activated T helper lymphocytes (26, 34). IL-4 enhances the development of TH2 responses and inhibits TH1 development (1, 21, 43). TH2 cells are involved in humoral (antibody-mediated) immunity and produce IL-4, IL-5, and IL-10 (31, 34). IL-4 is also an important regulator of isotype switching and the stimulation of immunoglobulin E production in B lymphocytes (8-10).

Different reports have provided contradictory evidence suggesting that IL-4 may have either protective or detrimental effects during viral infection (4, 11, 12, 24, 29, 42). Recently, a recombinant mousepox virus expressing IL-4 was reported to have greatly increased pathogenicity in infected mice (22). Even vaccinated mice were not protected against the recombinant virus.

In contrast to the results with the IL-4-expressing mousepox virus, we report here that a recombinant HSV-1 expressing IL-4 had decreased pathogenicity. We found that (i) despite wild-type (wt) replication in tissue culture, HSV-IL-4 replication in mouse eyes and brains was decreased compared to that of wt virus; (ii) HSV-IL-4 infection did not kill any mice; and (iii) in mice depleted of CD4⁺ T cells, HSV-IL-4 had wt pathogenicity, suggesting that a CD4⁺-T-cell response was involved in protecting mice against HSV-IL-4 infection.

View larger version (14K): [in this window] [in a new window]   FIG. 1.   Construction of the pHSV-IL-4 plasmid containing the murine IL-4 gene under control of the LAT promoter. Details of the preparation of the recombinant transfer vector are given in Materials and Methods. The plasmid contains LAT nucleotides 1041 to +4656 (thick line) with a deletion from nucleotides 76 to 1667. The complete gene for mouse IL-4, including the stop codon and its poly(A), site is inserted into the deleted region. The IL-4 gene is under the control of the LAT promoter. Insertion of enhanced green fluorescent protein into HSV-1 in the same location resulted in high-level long-term expression throughout both acute and latent infection (36).

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Viruses and cells. Triple-plaque-purified wt McKrae and recombinant dLAT2903 strains of HSV-1 have been described previously (35). Rabbit skin (RS) cells, used for preparation of virus stocks, culturing mouse tear films, and determining growth kinetics, were grown in Eagle's minimal essential medium supplemented with 5% fetal calf serum. L929 cells, used for enzyme-linked immunosorbent assay titers, were grown in RPMI 1640 supplemented with 10% fetal calf serum.

Mice. Inbred BALB/c, homozygous BALB/c-IL-4^/, and C57BL/6 mice (Jackson Laboratory, Bar Harbor, Maine) were used. All mice used were between 5 and 8 weeks old.

Construction of IL-4 plasmid. The parental virus for this construct was dLAT2903, a mutant of HSV-1 strain McKrae in which the region of LAT from 161 to +1667 relative to the LAT transcription start site (EcoRV-HpaI) was deleted from both copies of LAT (35). This LAT null mutant is thus missing approximately 0.2 kb of the LAT promoter and 1.6 kb of the 5' end of the primary 8.3-kb LAT. To make the IL-4 plasmid, the BamHI B fragment of McKrae was digested with SwaI-BamHI to produce a 5.5-kb DNA fragment including the region from 1041 to +4656 of the HSV-1 LAT (Fig. 1) (35). A PacI linker was added to the 5.5-kb fragment and ligated into the PacI site of modified pNEB193 (New England Biolabs) lacking its internal BamHI site. The resulting plasmid was digested with StyI-HpaI to remove the 1.6-kb LAT fragment corresponding to LAT +76 to +1667 nucleotides. A BamHI linker was added, and the resulting plasmid was designated pLAT. pLAT contained 880 bp upstream of the BamHI site and 2,989 bp downstream of the BamHI site. A plasmid containing the murine IL-4 gene was digested with Tsp509I (American Type Culture Collection clone 37561). This insert contained the complete 130-amino-acid coding region of the IL-4 gene plus 27 and 33 bp of noncoding sequence in its 5' and 3' regions, respectively. After the addition of a BamHI linker, the insert was ligated into the BamHI site of pLAT, and the resulting plasmid was designated pLAT-IL-4. This plasmid contains the 484-bp IL-4 gene bounded by 880- and 2,989-bp LAT fragments.

Construction of HSV-IL-4. HSV-IL-4 was generated by homologous recombination, as we previously described (35). Briefly, pLAT-IL-4 was cotransfected with infectious dLAT2903 DNA by the calcium phosphate method. Viruses from the cotransfection were plated, and isolated plaques were picked and then screened by restriction digestion and Southern blot analysis for insertion of the IL-4 gene. Selected plaques containing the IL-4 gene were plaque purified eight times and reanalyzed by restriction digestion and Southern blot analysis to ensure that the IL-4 DNA was present in the LAT region. A single plaque meeting this criterion was chosen for purification and designated HSV-IL-4. The final recombinant virus contains the murine IL-4 gene under control of the LAT promoter in the normal LAT location in the viral genome. Thus, there are two copies of LAT promoter-IL-4 (one in each viral long repeat).

Virus replication in tissue culture. RS cell monolayers at 70 to 80% confluence were infected with 0.01 PFU/cell. The virus was harvested at various times by two cycles of freeze-thawing of the cell monolayers with medium. Virus titers were determined by standard plaque assays on RS cells as we described previously (17).

Ocular challenge. Mice were challenged ocularly with 2 × 10⁷, 2 × 10⁶, 2 × 10⁵, 2 × 10⁴, 2 × 10³, or 2 × 10² PFU of HSV-1 strain McKrae, dLAT2903, or HSV-IL-4 per eye in 5 µl of tissue culture medium (17). In some experiments, mice were challenged ocularly with a mixture of 2 × 10⁴ PFU of HSV-IL-4 and 2 × 10⁴ PFU of HSV-1 strain McKrae/eye in 5 µl of tissue culture medium (17).

Titration of virus in tears. Tear films were collected from both eyes of five mice per group at various times as described previously (13). Each swab was placed in 0.5 ml of tissue culture medium, and the amount of virus in the medium was determined by a standard plaque assay on RS cells.

Detection of infectious virus in whole eye, brain, and TG. BALB/c mice were challenged ocularly with 2 × 10⁵ PFU of HSV-IL-4, dLAT2903, or McKrae/eye. On day 3, 5, or 7 postinfection, the mice were euthanized and individual trigeminal ganglia (TG), eyes, and brains were isolated. The TG, eyes, and brain from each mouse were homogenized individually using an IKA Works Inc. (Wilmington, N.C.) T25 homogenizer at 10,000 rpm for 30 s on ice. The debris was removed by centrifugation at 3,000 rpm for 10 min in a Beckman TA10 rotor. The viral titer in the supernatant was then measured on RS cells as described previously (18).

Depletion of CD4⁺ or CD8⁺ T cells. Each mouse received an intraperitoneal injection of 100 µg of purified GK1.5 (anti-L3T4 [CD4⁺]), 2.43 (anti-Lyt-2 [CD8⁺]), or both monoclonal antibodies (NCCC, Minneapolis, Minn.) in 100 µl of phosphate-buffered saline 96 and 24 h before ocular challenge. The injections were repeated 24 and 96 h after ocular challenge. The efficiency of CD4⁺- and CD8⁺-T-cell depletion was monitored by fluorescence-activated cell sorter analysis 24 h after the second depletion, as described previously (14, 15).

Statistical analysis. Protective parameters were analyzed by Student's t test and Fisher's exact test using Instat (GraphPad, San Diego, Calif.). Results were considered to be statistically significant when the P value was <0.05.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Structure of HSV-IL-4. We constructed a mutant of HSV-1 expressing IL-4 to examine the effect of exogenous IL-4 on HSV-1 infection. McKrae was used as the original parental virus. The genomic structure of wt HSV-1 McKrae is shown schematically in Fig. 2A. The HSV-1 genome contains a unique long region and a unique short region, both of which are flanked by inverted repeats (long terminal and internal repeats and short terminal and internal repeats). The location of the LAT promoter TATA box is indicated. The transcription start site of the primary 8.3-kb LAT RNA is 28 nucleotides downstream of the TATA box (47). The previously described LAT null mutant, dLAT2903 (Fig. 2B), was derived from McKrae (35). dLAT2903 contains a 1.8-kb deletion in both copies of the LAT gene (one in each long repeat). This deletion consists of 0.2 kb of the LAT promoter and the portion of the LAT gene including the first 1.6 kb of the 8.3-kb primary LAT and extends to LAT nucleotide +1667.

View larger version (13K): [in this window] [in a new window]   FIG. 2.   Construction and structure of the HSV-IL-4 mutant virus. (A) The top schematic shows the HSV-1 McKrae genome in the prototypic orientation. TRL and IRL represent the long terminal and internal (or inverted) repeats, TRS and IRS represent the short terminal and internal (or inverted) repeats. UL and US represent the long and short unique regions, respectively. The solid rectangle represents the very stable 2-kb LAT. The start site for LAT transcription is indicated by the arrow at +1, the relative location of the LAT promoter TATA box 28 nucleotides upstream of the start of transcription. (B) dLAT2903 has a deletion from LAT nucleotides 161 to +1667 (XXXXXX) in both copies of LAT and makes no LAT RNA. (C) HSV-IL-4 was constructed from dLAT2903 by homologous recombination between dLAT2903 DNA and a plasmid containing the complete LAT promoter and the entire structural IL-4 gene [including its 3' poly(A) signal] as described in Materials and Methods.

Expression of IL-4 by HSV-IL-4 in tissue culture. Confluent monolayers of RS cells and L929 cells were infected at a multiplicity of 1 PFU of HSV-IL-4, dLAT2903, or McKrae/cell. The media were collected from 0 to 96 h postinfection and assayed by enzyme-linked immunosorbent assay for the presence of IL-4 protein by using antisera against IL-4 protein, as described in Materials and Methods. In RS cells, the level of IL-4 in the medium peaked 24 h after infection (102 ± 18 pg/ml) and stayed the same throughout the rest of the study period (not shown). The amount of IL-4 detected in the medium from murine L929 cells peaked 48 h after infection (30.5 ± 5 pg/ml) and was higher than in RS cells from 12 to 96 h postinfection (not shown). The media from RS or L929 cells infected with dLAT2903 or McKrae did not contain detectable IL-4 (not shown). Thus (i) HSV-IL-4 expressed significant amounts of recombinant IL-4, (ii) the expressed IL-4 was secreted into the medium, and (iii) the level of expression was higher in the mouse cell line than in the rabbit cell line.

Replication of HSV-IL-4 in tissue culture. RS cells were infected with 0.01 PFU of HSV-IL-4, dLAT2903, or wt McKrae/cell. The monolayers were freeze-thawed at the indicated times, and the virus yield was determined as described in Materials and Methods. Replication of all three viruses appeared similar (Fig. 3A). Thus, expression of IL-4 by HSV-1 did not appear to have a profound effect on virus replication in tissue culture.

View larger version (12K): [in this window] [in a new window]   FIG. 3.   Replication of HSV-IL-4 in tissue culture and mouse tears. (A) HSV-IL-4 replication in tissue culture. Subconfluent RS cell monolayers in triplicate from two separate experiments were infected with 0.01 PFU of HSV-IL-4, dLAT2903, or McKrae per cell as described in Materials and Methods. Total virus was harvested at the indicated times postinfection by two cycles of freeze-thawing. The amount of virus at each time for each virus was determined by standard plaque assays on RS cells. Each point represent the mean ± the standard error of the mean (n = 6). (B) Virus titers in BALB/c mouse tears. BALB/c mice were ocularly infected with 2 × 105 PFU of HSV-IL-4 or dLAT2903 per eye. Tear films were collected from days 1 to 7, and virus titers were determined by standard plaque assays. Each point represents the mean ± the standard error of the mean of titers from 10 eyes. (C) Virus titers in C57BL/6 mouse tears. C57BL/6 mice were ocularly infected with 2 × 106 PFU per eye of HSV-IL-4 or dLAT2903. Virus titers were determined as for panel B. Each point represents the mean + the standard error of the mean of the titers from 10 eyes.

Virus titers in mouse tears. BALB/c mice were infected ocularly with 2 × 10⁵ PFU of HSV-IL-4 or dLAT2903/eye (as described in Materials and Methods). Replication of dLAT2903 in mouse eyes was similar to that of wt McKrae (35). Tear films were collected from 10 eyes per group per time point, and the amount of virus was assayed by plaque assays on RS cells (Fig. 3B). dLAT2903 virus had a peak titer of approximately 10³ PFU per eye. In contrast, HSV-IL-4 had a peak titer of less than 100. This difference was highly significant (P < 0.001; Student t test), suggesting that HSV-IL-4 either replicated poorly in mouse eyes or that the IL-4 expressed by HSV-IL-4 induced an immune response that resulted in reduced virus in the tears.

Virus replication in whole eyes, TG, and brains. Fifteen BALB/c mice from two separate experiments were ocularly infected with HSV-IL-4, dLAT2903, or wt McKrae, as described in Materials and Methods. On days 3, 5, and 7 postinfection, five mice per group were sacrificed and eyes, TG, and brains were harvested for analysis of infectious virus as described in Materials and Methods. The data from both experiments were combined and are shown in Fig. 4.

View larger version (16K): [in this window] [in a new window]   FIG. 4.   Virus titers in whole eyes, TG, and brain. BALB/c mice were ocularly infected with 2 × 105 PFU of HSV-IL-4, dLAT2903, or McKrae per eye as described in Materials and Methods. The mice were euthanized on the indicated days. Eyes (A), TG (B), and brain (C) were removed and homogenized, and virus titers were determined as described in Materials and Methods. Each bar represents the mean ± the standard error of the mean of 10 eyes, 10 TG, or 5 brains.

Virulence of HSV-IL-4 in BALB/c mice. Groups of 40 BALB/c mice from two different experiments were challenged ocularly with 2 × 10⁵ PFU of HSV-IL-4, dLAT2903, or McKrae/eye as described in Materials and Methods. All mice (100%) infected with HSV-IL-4 survived ocular infection (Table 1). In contrast, only 5 (2 of 40) and 10% (4 of 40) of mice infected with dLAT2903 and McKrae survived, respectively (P < 0.0001 versus HSV-IL-4-infected mice; Fisher's exact test).

Coinfection of BALB/c mice with both HSV-IL-4 and wt McKrae viruses. To further confirm that the recombinantly expressed IL-4 in HSV-IL-4 was protecting against mortality, groups of 10 to 30 BALB/c mice from two different experiments were challenged ocularly with 2 × 10⁴ PFU of HSV-IL-4 alone, McKrae alone, or both HSV-IL-4 and McKrae/eye. As expected, all of the mice (10 of 10) infected with HSV-IL-4 survived ocular infection, while only 3 of 25 mice (12%) infected with McKrae survived the challenge (Fig. 5A). In contrast, 17 of 30 mice (57%) coinfected with both viruses survived the lethal challenge (P = 0.0007 compared to McKrae alone; P = 0.01 compared to HSV-IL-4 alone). Consistent with the marker-rescued-virus results, these results suggest that the decreased mortality with HSV-IL-4 infection was due to recombinant expression of IL-4.

View larger version (8K): [in this window] [in a new window]   FIG. 5.   Survival following infection with HSV-IL-4. (A) Survival of BALB/c mice coinfected with HSV-IL-4 and McKrae. Mice were coinfected ocularly with 2 × 104 PFU of HSV-IL-4/eye and 2 × 104 PFU of wt McKrae/eye. Survival was determined 28 days after infection. (B) Survival of BALB/c IL-4/ mice infected with HSV-IL-4. IL-4/ mice were inoculated ocularly with 2 × 105 PFU of HSV-IL-4 or dLAT2903 as described in Materials and Methods. Survival was measured 28 days after infection. (C) Survival of CD4+-depleted or CD8+-depleted BALB/c mice after ocular infection with HSV-IL-4. CD4+ or CD8+ T-cell depletions were done as described in Materials and Methods. Mice were inoculated ocularly with 2 × 105 PFU of HSV-IL-4/eye, and survival was measured 28 days after infection.

Virulence of HSV-IL-4 in IL-4^/ mice. Groups of 20 BALB/c IL-4^/ mice were challenged ocularly with 2 × 10⁵ PFU of HSV-IL-4 or dLAT2903/eye as described in Materials and Methods. One hundred percent (Fig. 5B; 20 of 20) of IL-4^/ mice survived following infection with HSV-IL-4. In contrast, only 10 of 20 mice (50%) infected with dLAT2903 survived (P = 0.0004; Fisher's exact test). Thus, host IL-4 expression was not required for activity of the recombinant IL-4 expressed by HSV-IL-4.

Effect of CD4⁺- and CD8⁺-T-cell depletion on survival following HSV-IL-4 infection. Twenty mice per group from two separate experiments were depleted of CD4⁺ or CD8⁺ T cells as described in Materials and Methods. All 20 control mice and 19 of 20 CD8⁺-T-cell-depleted mice (95%) survived ocular challenge with 2 × 10⁵ PFU of HSV-IL-4/eye (Fig. 5C; P = 1.0 versus control). In contrast, only 6 of 20 CD4⁺-T-cell-depleted mice (30%) survived the lethal challenge (P < 0.0001 versus control mice or CD8⁺-T-cell-depleted mice; Fisher's exact test). These results suggest that CD4⁺ T cells were involved in the ability of mice to survive lethal challenge with HSV-IL-4.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In general, control of infection by viruses and intracellular microbes is linked to the induction of a TH1 response, while protection against extracellular pathogens correlates with a TH2 response (3, 30). IL-4 has a broad range of biological and immunological activities (33, 34) and is considered an indicator of a TH2 response (31, 34, 43). The functional properties of IL-4 have been evaluated by depletion studies (19, 40), with knockout mice (16, 25), and by exogenous addition of IL-4 in vitro (20) or in vivo (7). Some of these studies have produced conflicting results. Thus, to help clarify the effect of IL-4 on HSV-1 infection, we constructed a recombinant HSV-1 expressing two copies of the IL-4 gene, each under control of the strong LAT promoter. Since the HSV-IL-4 genome differs from that of its parental strain by only a single gene, this virus is a useful tool to study how exogenous IL-4 affects HSV-1 infection.

In both RS and mouse (L929) cells infected with HSV-IL-4, IL-4 was secreted into the media. Larger amounts of IL-4 were detected in the L929 cells. Replication of HSV-IL-4 did not differ from that of the parental strains in tissue culture. However, the amount of HSV-IL-4 seen in the tears of mice was reduced compared to its parental viruses. This is in contrast with previous studies in which delayed virus clearance was seen in mice challenged with influenza virus in the presence of exogenously applied IL-4 (29), following respiratory syncytial virus infection of transgenic mice expressing IL-4 (12), and following infection of mice with a vaccinia virus recombinant expressing IL-4 (46).

At early times postinfection in mouse eyes and brains, the titers of HSV-IL-4 were similar to those of the parental viruses. At later times, the amount of HSV-IL-4 detected in eyes and brains was reduced compared to those of the parental viruses. This suggests that the recombinantly expressed IL-4 resulted in faster clearance of the virus from these tissues. In contrast, the parental viruses and HSV-IL-4 all had similar virus titers in mouse TG at all times examined.

Infection with HSV-IL-4 was not lethal to mice, even at an infectious dose of 2 × 10⁷ PFU/eye. This is 100-fold higher than the dose of wt virus that resulted in the death of approximately 90% of the mice. Coinfection of mice with a mixture of HSV-IL-4 and a lethal dose of McKrae resulted in survival of 57% of the mice compared with survival of only 10% of mice infected with the same dose of McKrae alone. This suggested that the recombinantly expressed IL-4 from HSV-IL-4 was able to at least partially protect against lethal infection with wt virus. In addition, marker-rescued virus, in which the IL-4 gene was removed and the original LAT deletion was restored, regained the high lethality of the parental LAT virus (dLAT2903), confirming that the reduced virulence of HSV-IL-4 was due to the recombinantly expressed IL-4 and not to an unexpected secondary mutation in the virus. The additional finding that 100% of IL-4^/ mice survived infection with HSV-IL-4 suggests that host-produced IL-4 was not important in this system.

We previously reported that in IL-4^/ mice, which are deficient in IL-4 production, lack a TH2 response, and have an elevated IL-2 response, HSV-1 replicated to lower titers and ocular HSV-1 replication could be increased by exogenously added recombinant IL-4 (16). This suggested that IL-4 enhanced replication. However, the IL-4^/ mice have increased IL-2 levels, presumably because IL-4 normally down regulates IL-2 (32). Thus, it is likely that IL-2 suppresses HSV-1 replication in the eye, and IL-4 appears to enhance replication in this system because it decreases IL-2 levels. In contrast, in this study HSV-IL-4-infected mice had reduced HSV-1 replication in the eye, suggesting that IL-4 decreased virus replication. However, we found that in contrast to expectations, HSV-IL-4 increased IL-2 and gamma interferon (IFN-) production (not shown). The reason for this is not clear, but it suggests that, consistent with our previous results with IL-4^/ mice, the reduced replication of HSV-IL-4 may be due to increased levels of IL-2. Our result with HSV-IL-4-infected mice is in contrast to mousepox virus expressing IL-4, which has increased virulence (22). This may be because the mousepox virus expressing IL-4 resulted in reduced IFN- gene expression (22) while HSV-IL-4 infection resulted in increased IFN- expression (not shown).

The results presented above strongly suggest that exogenous IL-4 provided protection against HSV-1 infection in BALB/c mice. This appears to be in contrast to a recent study showing that a mutant mousepox virus expressing IL-4 was highly lethal to infected mice (22). It is possible that IL-4 has a different impact on the outcome of HSV-1 infection than it has on mousepox or that the different strains of mice used produced different results. In another study, IL-4 expression by a recombinant vaccinia virus exacerbated infection, and the IL-4-induced exacerbation was T cell independent (4). Detrimental effects on host animals were also observed in experiments in which IL-4-expressing transgenic mice were infected with respiratory syncytial virus (12) and when influenza virus infection was treated in vivo with recombinant IL-4 (29). In contrast, studies with IL-4^/ mice have not revealed any significant role for IL-4 in viral pathology (2, 16, 28). The reasons for these apparent differences remain unclear.

IL-4 is an immunomodulatory cytokine secreted by activated CD4⁺ TH2 cells (5), CD8⁺ T cytotoxic 2 (TC2) cells (41), mast cells (38), and basophils (44, 45). In this study, depletion of CD4⁺ T cells, but not CD8⁺ T cells, reduced survival in infected mice, suggesting that the protection induced by recombinantly expressed IL-4 in HSV-IL-4-infected mice was due to CD4⁺ T cells. We therefore propose that the IL-4 recombinantly expressed by HSV-IL-4 may promote the development of CD4⁺ T cells. This would be consistent with previously published results showing that both CD4-deficient and CD4-depleted mice are more susceptible to HSV-1 infection than CD8-deficient or CD8-depleted mice (14, 15).

In summary, HSV-IL-4 was less virulent than its parental viruses, and virulence was returned to parental levels by marker rescue. Depletion of CD4⁺ T cells also restored the virulence of HSV-IL-4 to parental levels. These findings suggest a role for IL-4 in protection against HSV-1 that is mediated by CD4⁺ T cells. Finally, this study emphasizes the useful role genetically modified HSV-1 can play in helping determine factors involved in the immune response to HSV-1 infection.