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. 2001 Oct;75(19):9029–9036. doi: 10.1128/JVI.75.19.9029-9036.2001

Recombinant Herpes Simplex Virus Type 1 Expressing Murine Interleukin-4 Is Less Virulent than Wild-Type Virus in Mice

Homayon Ghiasi 1,2,*, Yanira Osorio 1, Guey-Chuen Perng 1, Anthony B Nesburn 1,2, Steven L Wechsler 1,2
PMCID: PMC114471  PMID: 11533166

Abstract

The effect of interleukin-4 (IL-4) on herpes simplex virus type 1 (HSV-1) infection in mice was evaluated by construction of a recombinant HSV-1 expressing the gene for murine IL-4 in place of the latency-associated transcript (LAT). The mutant virus (HSV-IL-4) expressed high levels of IL-4 in cultured cells. The replication of HSV-IL-4 in tissue culture and in trigeminal ganglia was similar to that of wild-type virus. In contrast, HSV-IL-4 appeared to replicate less well in mouse eyes and brains. Although BALB/c mice are highly susceptible to HSV-1 infection, ocular infection with HSV-IL-4 resulted in 100% survival. Furthermore, 57% of the mice survived coinfection with a mixture of HSV-IL-4 and a lethal dose of wild-type McKrae, compared with only 10% survival following infection with McKrae alone. Similar to wild-type BALB/c mice, 100% of IL-4−/− mice also survived HSV-IL-4 infection. T-cell depletion studies suggested that protection against HSV-IL-4 infection was mediated by a CD4+-T-cell response.

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.

MATERIALS AND METHODS

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.

FIG. 1.

FIG. 1

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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).

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

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.

FIG. 2.

FIG. 2

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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.

HSV-IL-4 was derived from dLAT2903 by insertion of the IL-4 gene and restoration of the LAT promoter so that IL-4 is under the control of the powerful LAT promoter, as described in Materials and Methods (Fig. 2C). The genomic structure of HSV-IL-4 was confirmed by restriction enzyme analysis and partial sequencing. HSV-IL-4 contains the entire sequence of the IL-4 gene, including its polyadenylation signal, under the control of the LAT promoter. There is a noncoding region of 27 nucleotides in front of the first ATG. This is followed by the complete coding region of 390 nucleotides and 33 noncoding nucleotides after the IL-4 termination codon at the 3′ end. There are two complete copies, one in each viral long repeat.

HSV-IL-4 is identical to its LAT null mutant parent, dLAT2903, except that the LAT promoter is restored and is driving expression of IL-4. dLAT2903 is identical to its wt parent, McKrae, for all parameters examined, except those relating to latency and reactivation (35).

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.

FIG. 3.

FIG. 3

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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.

To confirm that the reduced ocular HSV-IL-4 titers were not mouse strain specific, C57BL/6 mice were infected with HSV-IL-4 or dLAT2903. A 10-fold higher challenge dose (2 × 106 PFU/eye) was used because C57BL/6 mice are highly resistant to HSV-1 compared to BALB/c mice. Again, the HSV-IL-4 titers were lower (Fig. 3C). Thus, the presence of exogenous IL-4 (expressed by the recombinant HSV-IL-4) in mouse eyes appeared to significantly decrease the amount of HSV-1 in mouse 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.

FIG. 4.

FIG. 4

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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.

On day 3 postinfection, similar amounts of all three viruses were detected in extracts of whole eyes (Fig. 4A). However, on days 5 and 7, high levels of both dLAT2903 and McKrae were detected while no HSV-IL-4 virus was detected. Similar to the tear film results, this suggests that the IL-4 produced by HSV-IL-4 may have resulted in faster clearance of virus from the eyes.

In contrast to the above-mentioned results, the average amounts of virus detected per TG were not different for HSV-IL-4, dLAT2903, and McKrae (Fig. 4B). In mouse brains no virus was detected on day 3 postinfection (Fig. 4C). On day 5 postinfection, the average amounts of virus detected in the brain were similar for all three viruses. However, by day 7 postinfection, the amount of HSV-IL-4 in brains was significantly reduced compared to the amounts of dLAT2903 and wt McKrae. (Fig. 4C). Thus, HSV-IL-4 appeared to be cleared from mouse eyes and brains more rapidly than dLAT2903 or wt McKrae. This may be related to the reduced pathogenicity of this virus. Interestingly, IL-4 expression did not appear to impact virus replication or clearance in the TG. This may suggest that, whatever the protective immune response stimulated by the recombinantly expressed IL-4, it has little or no effect in the TG during the times examined here.

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).

TABLE 1.

Survival of BALB/c mice following ocular challenge with HSV-IL-4a

Virus Survivalb
2 × 102 2 × 103 2 × 104 2 × 105 2 × 106 2 × 107
HSV-IL-4 5/5 (100) 5/5 (100) 5/5 (100) 40/40 (100) 10/10 (100) 10/10 (100)
dLAT2903 5/5 (100) 1/5 (20) 0/5 (0) 2/40 (5) 0/5 (0) 0/5 (0)
McKrae 5/5 (100) 0/5 (0) 0/5 (0) 4/40 (10) 0/5 (0) 0/5 (0)
HSV-IL-4R NDc ND ND 0/5 (0) ND ND
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a

BALB/c mice were ocularly challenged with specified virus titers and survival was determined 28 days after challenge. 

b

Survival is shown as the number surviving/number challenged (percent) after challenge with the indicated PFU per eye. ND, not done. 

Groups of 5 to 10 BALB/c mice were ocularly infected with 2 × 106 or 2 × 107 PFU of HSV-IL-4, dLAT2903, or wt McKrae/eye. One hundred percent of the mice infected with HSV-IL-4 survived the infection at both doses. In contrast, all of the mice infected with dLAT2903 or wt McKrae died at both of these higher challenge doses (Table 1). We have found that even at the low challenge doses of 2 × 104 or 2 × 103 PFU/eye, dLAT2903 and McKrae both kill at least 80% of the BALB/c mice (Table 1). Thus, compared to the parental viruses, HSV-IL-4 appeared to have greatly reduced virulence as measured by survival.

A rescued HSV-IL-4, in which the IL-4 gene was removed and the original deletion of the LAT gene was restored, was constructed. The rescued virus, HSV-IL-4R, was generated by cotransfection and homologous recombination of infectious HSV-IL-4 DNA with the original pLAT plasmid. Following ocular infection of five BALB/c mice with 2 × 105 PFU of HSV-IL-4R/eye, all of the mice died (Table 1). Thus, marker rescue of the HSV-IL-4 virus restored virulence to that of dLAT2903 and McKrae. This confirms that the absence of mortality in HSV-IL-4-infected mice was due to the presence of IL-4 rather than a defect in the recombinant virus.

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.

FIG. 5.

FIG. 5

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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

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-γ) 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-γ gene expression (22) while HSV-IL-4 infection resulted in increased IFN-γ 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.

ACKNOWLEDGMENTS

This work was supported by grants from the Discovery Fund for Eye Research and the Skirball Program in Molecular Ophthalmology to H.G.

REFERENCES

  • 1.Abehsira-Amar O, Gibert M, Joliy M, Theze J, Jankovic D L. IL-4 plays a dominant role in the differential development of Tho into Th1 and Th2 cells. J Immunol. 1992;148:3820–3829. [PubMed] [Google Scholar]
  • 2.Bachmann M F, Schorle H, Kuhn R, Muller W, Hengartner H, Zinkernagel R M, Horak I. Antiviral immune responses in mice deficient for both interleukin-2 and interleukin-4. J Virol. 1995;69:4842–4846. doi: 10.1128/jvi.69.8.4842-4846.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Biron C A. Cytokines in the generation of immune responses to, and resolution of, virus infection. Curr Opin Immunol. 1994;6:530–538. doi: 10.1016/0952-7915(94)90137-6. [DOI] [PubMed] [Google Scholar]
  • 4.Cheers C, Janas M, Ramsay A, Ramshaw I. Use of recombinant viruses to deliver cytokines influencing the course of experimental bacterial infection. Immunol Cell Biol. 1999;77:324–330. doi: 10.1046/j.1440-1711.1999.00829.x. [DOI] [PubMed] [Google Scholar]
  • 5.Cherwinski H M, Schumacher J H, Brown K D, Mosmann T R. Two types of mouse helper T cell clone. III. Further differences in lymphokine synthesis between Th1 and Th2 clones revealed by RNA hybridization, functionally monospecific bioassays, and monoclonal antibodies. J Exp Med. 1987;166:1229–1244. doi: 10.1084/jem.166.5.1229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Dobson A T, Margolis T P, Sedarati F, Stevens J G, Feldman L T. A latent, nonpathogenic HSV-1-derived vector stably expresses beta-galactosidase in mouse neurons. Neuron. 1990;5:353–360. doi: 10.1016/0896-6273(90)90171-b. [DOI] [PubMed] [Google Scholar]
  • 7.Fanslow W C, Clifford K N, Park L S, Rubin A S, Voice R F, Beckmann M P, Widmer M B. Regulation of alloreactivity in vivo by IL-4 and the soluble IL-4 receptor. J Immunol. 1991;147:535–540. [PubMed] [Google Scholar]
  • 8.Finkelman F D, Holmes J, Katona I M, Urban J F, Jr, Beckmann M P, Park L S, Schooley K A, Coffman R L, Mosmann T R, Paul W E. Lymphokine control of in vivo immunoglobulin isotype selection. Annu Rev Immunol. 1990;8:303–333. doi: 10.1146/annurev.iy.08.040190.001511. [DOI] [PubMed] [Google Scholar]
  • 9.Finkelman F D, Katona I M, Urban J F, Jr, Holmes J, Ohara J, Tung A S, Sample J V, Paul W E. IL-4 is required to generate and sustain in vivo IgE responses. J Immunol. 1988;141:2335–2341. [PubMed] [Google Scholar]
  • 10.Finkelman F D, Katona I M, Urban J F, Jr, Paul W E. Control of in vivo IgE production in the mouse by interleukin 4. Ciba Found Symp. 1989;147:3–17. doi: 10.1002/9780470513866.ch2. [DOI] [PubMed] [Google Scholar]
  • 11.Finkelman F D, Shea-Donohue T, Goldhill J, Sullivan C A, Morris S C, Madden K B, Gause W C, Urban J F., Jr Cytokine regulation of host defense against parasitic gastrointestinal nematodes: lessons from studies with rodent models. Annu Rev Immunol. 1997;15:505–533. doi: 10.1146/annurev.immunol.15.1.505. [DOI] [PubMed] [Google Scholar]
  • 12.Fischer J E, Johnson J E, Kuli-Zade R K, Johnson T R, Aung S, Parker R A, Graham B S. Overexpression of interleukin-4 delays virus clearance in mice infected with respiratory syncytial virus. J Virol. 1997;71:8672–8677. doi: 10.1128/jvi.71.11.8672-8677.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Ghiasi H, Bahri S, Nesburn A B, Wechsler S L. Protection against herpes simplex virus-induced eye disease after vaccination with seven individually expressed herpes simplex virus 1 glycoproteins. Investig Ophthalmol Vis Sci. 1995;36:1352–1360. [PubMed] [Google Scholar]
  • 14.Ghiasi H, Cai S, Nesburn A B, Wechsler S L. MHC-II but not MHC-I responses are required for vaccine-induced protection against ocular challenge with HSV-1. Curr Eye Res. 1997;16:1152–1158. doi: 10.1076/ceyr.16.11.1152.5104. [DOI] [PubMed] [Google Scholar]
  • 15.Ghiasi H, Cai S, Perng G C, Nesburn A B, Wechsler S L. Both CD4+ and CD8+ T cells are involved in protection against HSV-1 induced corneal scarring. Br J Ophthalmol. 2000;84:408–412. doi: 10.1136/bjo.84.4.408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Ghiasi H, Cai S, Slanina S M, Perng G C, Nesburn A B, Wechsler S L. The role of interleukin (IL)-2 and IL-4 in herpes simplex virus type 1 ocular replication and eye disease. J Infect Dis. 1999;179:1086–1093. doi: 10.1086/314736. [DOI] [PubMed] [Google Scholar]
  • 17.Ghiasi H, Kaiwar R, Nesburn A B, Slanina S, Wechsler S L. Expression of seven herpes simplex virus type 1 glycoproteins (gB, gC, gD, gE, gG, gH, and gI): comparative protection against lethal challenge in mice. J Virol. 1994;68:2118–2126. doi: 10.1128/jvi.68.4.2118-2126.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Ghiasi H, Perng G C, Hofman F M, Cai S, Nesburn A B, Wechsler S L. Specific and nonspecific immune stimulation of MHC-II-deficient mice results in chronic HSV-1 infection of the trigeminal ganglia following ocular challenge. Virology. 1999;258:208–216. doi: 10.1006/viro.1999.9710. [DOI] [PubMed] [Google Scholar]
  • 19.Heinzel F P, Sadick M D, Mutha S S, Locksley R M. Production of interferon gamma, interleukin 2, interleukin 4, and interleukin 10 by CD4+ lymphocytes in vivo during healing and progressive murine leishmaniasis. Proc Natl Acad Sci USA. 1991;88:7011–7015. doi: 10.1073/pnas.88.16.7011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Hofman F M, Brock M, Taylor C R, Lyons B. IL-4 regulates differentiation and proliferation of human precursor B cells. J Immunol. 1988;141:1185–1190. [PubMed] [Google Scholar]
  • 21.Hsieh C S, Heimberger A B, Gold J S, O'Garra A, Murphy K M. Differential regulation of T helper phenotype development by interleukins 4 and 10 in an alpha beta T-cell-receptor transgenic system. Proc Natl Acad Sci USA. 1992;89:6065–6069. doi: 10.1073/pnas.89.13.6065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Jackson R J, Ramsay A J, Christensen C D, Beaton S, Hall D F, Ramshaw I A. Expression of mouse interleukin-4 by a recombinant ectromelia virus suppresses cytolytic lymphocyte responses and overcomes genetic resistance to mousepox. J Virol. 2001;75:1205–1210. doi: 10.1128/JVI.75.3.1205-1210.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Javier R T, Stevens J G, Dissette V B, Wagner E K. A herpes simplex virus transcript abundant in latently infected neurons is dispensable for establishment of the latent state. Virology. 1988;166:254–257. doi: 10.1016/0042-6822(88)90169-9. [DOI] [PubMed] [Google Scholar]
  • 24.Keane-Myers A, Maliszewski C R, Finkelman F D, Nickell S P. Recombinant IL-4 treatment augments resistance to Borrelia burgdorferi infections in both normal susceptible and antibody-deficient susceptible mice. J Immunol. 1996;156:2488–2494. [PubMed] [Google Scholar]
  • 25.Kopf M, Le Gros G, Bachmann M, Lamers M C, Bluethmann H, Kohler G. Disruption of the murine IL-4 gene blocks Th2 cytokine responses. Nature. 1993;362:245–248. doi: 10.1038/362245a0. [DOI] [PubMed] [Google Scholar]
  • 26.Le Gros G, Ben-Sasson S Z, Seder R, Finkelman F D, Paul W E. Generation of interleukin 4 (IL-4)-producing cells in vivo and in vitro: IL-2 and IL-4 are required for in vitro generation of IL-4-producing cells. J Exp Med. 1990;172:921–929. doi: 10.1084/jem.172.3.921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Margolis T P, Sedarati F, Dobson A T, Feldman L T, Stevens J G. Pathways of viral gene expression during acute neuronal infection with HSV-1. Virology. 1992;189:150–160. doi: 10.1016/0042-6822(92)90690-q. [DOI] [PubMed] [Google Scholar]
  • 28.Mo X Y, Sangster M Y, Tripp R A, Doherty P C. Modification of the Sendai virus-specific antibody and CD8+ T-cell responses in mice homozygous for disruption of the interleukin-4 gene. J Virol. 1997;71:2518–2521. doi: 10.1128/jvi.71.3.2518-2521.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Moran T M, Isobe H, Fernandez-Sesma A, Schulman J L. Interleukin-4 causes delayed virus clearance in influenza virus-infected mice. J Virol. 1996;70:5230–5235. doi: 10.1128/jvi.70.8.5230-5235.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Mosmann T R. Cytokine patterns during the progression to AIDS. Science. 1994;265:193–194. doi: 10.1126/science.8023139. [DOI] [PubMed] [Google Scholar]
  • 31.Mosmann T R, Coffman R L. TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu Rev Immunol. 1989;7:145–173. doi: 10.1146/annurev.iy.07.040189.001045. [DOI] [PubMed] [Google Scholar]
  • 32.Noben-Trauth N, Kropf P, Muller I. Susceptibility to Leishmania major infection in interleukin-4-deficient mice. Science. 1996;271:987–990. doi: 10.1126/science.271.5251.987. [DOI] [PubMed] [Google Scholar]
  • 33.Paul W E, Ohara J. B-cell stimulatory factor-1/interleukin 4. Annu Rev Immunol. 1987;5:429–459. doi: 10.1146/annurev.iy.05.040187.002241. [DOI] [PubMed] [Google Scholar]
  • 34.Paul W E, Seder R A. Lymphocyte responses and cytokines. Cell. 1994;76:241–251. doi: 10.1016/0092-8674(94)90332-8. [DOI] [PubMed] [Google Scholar]
  • 35.Perng G C, Dunkel E C, Geary P A, Slanina S M, Ghiasi H, Kaiwar R, Nesburn A B, Wechsler S L. The latency-associated transcript gene of herpes simplex virus type 1 (HSV-1) is required for efficient in vivo spontaneous reactivation of HSV-1 from latency. J Virol. 1994;68:8045–8055. doi: 10.1128/jvi.68.12.8045-8055.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Perng G C, Slanina S M, Yukht A, Ghiasi H, Nesburn A B, Wechsler S L. The latency-associated transcript gene enhances establishment of herpes simplex virus type 1 latency in rabbits. J Virol. 2000;74:1885–1891. doi: 10.1128/jvi.74.4.1885-1891.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Perng G C, Zwaagstra J C, Ghiasi H, Kaiwar R, Brown D J, Nesburn A B, Wechsler S L. Similarities in regulation of the HSV-1 LAT promoter in corneal and neuronal cells. Investig Ophthalmol Vis Sci. 1994;35:2981–2989. [PubMed] [Google Scholar]
  • 38.Plaut M, Pierce J H, Watson C J, Hanley-Hyde J, Nordan R P, Paul W E. Mast cell lines produce lymphokines in response to cross-linkage of Fc epsilon RI or to calcium ionophores. Nature. 1989;339:64–67. doi: 10.1038/339064a0. [DOI] [PubMed] [Google Scholar]
  • 39.Rock D L, Nesburn A B, Ghiasi H, Ong J, Lewis T L, Lokensgard J R, Wechsler S L. Detection of latency-related viral RNAs in trigeminal ganglia of rabbits latently infected with herpes simplex virus type 1. J Virol. 1987;61:3820–3826. doi: 10.1128/jvi.61.12.3820-3826.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Ruth J H, Warmington K S, Shang X, Lincoln P, Evanoff H, Kunkel S L, Chensue S W. Interleukin 4 and 13 participation in mycobacterial (type-1) and schistosomal (type-2) antigen-elicited pulmonary granuloma formation: multiparameter analysis of cellular recruitment, chemokine expression and cytokine networks. Cytokine. 2000;12:432–444. doi: 10.1006/cyto.1999.0595. [DOI] [PubMed] [Google Scholar]
  • 41.Sad S, Marcotte R, Mosmann T R. Cytokine-induced differentiation of precursor mouse CD8+ T cells into cytotoxic CD8+ T cells secreting Th1 or Th2 cytokines. Immunity. 1995;2:271–279. doi: 10.1016/1074-7613(95)90051-9. [DOI] [PubMed] [Google Scholar]
  • 42.Sadick M D, Heinzel F P, Holaday B J, Pu R T, Dawkins R S, Locksley R M. Cure of murine leishmaniasis with anti-interleukin 4 monoclonal antibody. Evidence for a T cell-dependent, interferon gamma-independent mechanism. J Exp Med. 1990;171:115–127. doi: 10.1084/jem.171.1.115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Seder R A, Paul W E. Acquisition of lymphokine-producing phenotype by CD4+ T cells. Annu Rev Immunol. 1994;12:635–673. doi: 10.1146/annurev.iy.12.040194.003223. [DOI] [PubMed] [Google Scholar]
  • 44.Seder R A, Paul W E, Ben-Sasson S Z, LeGros G S, Kagey-Sobotka A, Finkelman F D, Pierce J H, Plaut M. Production of interleukin-4 and other cytokines following stimulation of mast cell lines and in vivo mast cells/basophils. Int Arch Allergy Appl Immunol. 1991;94:137–140. doi: 10.1159/000235345. [DOI] [PubMed] [Google Scholar]
  • 45.Seder R A, Paul W E, Dvorak A M, Sharkis S J, Kagey-Sobotka A, Niv Y, Finkelman F D, Barbieri S A, Galli S J, Plaut M. Mouse splenic and bone marrow cell populations that express high-affinity Fc epsilon receptors and produce interleukin 4 are highly enriched in basophils. Proc Natl Acad Sci USA. 1991;88:2835–2839. doi: 10.1073/pnas.88.7.2835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Sharma D P, Ramsay A J, Maguire D J, Rolph M S, Ramshaw I A. Interleukin-4 mediates down regulation of antiviral cytokine expression and cytotoxic T-lymphocyte responses and exacerbates vaccinia virus infection in vivo. J Virol. 1996;70:7103–7107. doi: 10.1128/jvi.70.10.7103-7107.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Zwaagstra J C, Ghiasi H, Nesburn A B, Wechsler S L. Identification of a major regulatory sequence in the latency associated transcript (LAT) promoter of herpes simplex virus type 1 (HSV-1) Virology. 1991;182:287–297. doi: 10.1016/0042-6822(91)90672-x. [DOI] [PubMed] [Google Scholar]

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