Comparison of Adjuvant Efficacy of Herpes Simplex Virus Type 1 Recombinant Viruses Expressing T_H1 and T_H2 Cytokine Genes

Abstract

The adjuvant effects of cytokines in humoral and cell-mediated immunity to herpes simplex virus type 1 (HSV-1) have been examined in mice using HSV-1 recombinant viruses expressing murine interleukin-2 (IL-2), IL-4, or gamma interferon (IFN-γ) gene. Groups of naive BALB/c mice were immunized intraperitoneally with one or three doses of the HSV-1 recombinant viruses expressing IL-2, IL-4, or IFN-γ or with parental control virus. Despite similar replication kinetics, these three recombinant viruses elicited different immune responses to HSV-1 on immunization. Immunization with the recombinant virus expressing IL-4 elicited a humoral response of greater magnitude than immunization with the recombinant viruses expressing IL-2 or IFN-γ or with parental virus. In contrast, immunization with recombinant virus expressing IL-2 elicited a higher cytotoxic T-cell response than immunization with viruses expressing IL-4 or IFN-γ. Stimulation in vitro of splenocytes obtained from the mice immunized with UV-inactivated HSV-1 McKrae resulted in a T_H1 pattern of cytokine expression irrespective of the recombinant virus used in the immunization. As observed for the parental virus, both CD4⁺ and CD8⁺ T cells contributed equally to the production of IL-2 by the splenocytes of mice immunized with any of the three recombinant viruses. However, the pattern of IFN-γ production by CD4⁺ and CD8⁺ T cells differed according to the recombinant virus used. After lethal ocular challenge, all immunized mice were protected completely against death and manifestations of eye disease caused by HSV-1, which are typical responses in unimmunized mice. Mice immunized with IL-4-expressing virus cleared the virus from their eyes more rapidly than mice immunized with IL-2- or IFN-γ-expressing virus. Taken together, our results suggest that, in contrast to IFN-γ which did not exhibit an adjuvant effect, both IL-4 and IL-2 act as adjuvants in immunization with HSV, with IL-4 showing greater efficacy.

Numerous vaccines that provide protection against herpes simplex virus type 1 (HSV-1) infection-associated encephalitis and eye disease have been developed (21, 28, 40). However, these vaccines exhibit only low efficacy in the prevention of HSV-1 replication in the eye and the subsequent establishment of latency in the trigeminal ganglia. Similarly, immunization of individuals with a cocktail of gB-plus-gD subunit vaccine in a phase III clinical trial was not successful in protection against the acquisition of genital HSV, even though high neutralizing antibody titers, high cytokine titers by enzyme-linked immunosorbent assay (ELISA), and high cell-mediated immunity (CMI) based on lymphocyte proliferation assays, were achieved (9).

The development of vaccines against HSV-1 infection has, in general, focused on the use of live attenuated virus (21, 28, 40) or virus subunits (9, 14) as the antigen. The efficacy of the vaccines has been shown to be affected by both the number of immunizations and the amount of antigen used (12, 13, 16). More recently, it has been shown that inclusion of cytokines in a vaccine cocktail can further alter the response to the vaccine by pushing it towards or away from specific immune responses (18, 44, 45, 50). For example, addition of interleukin-2 (IL-2) as an adjuvant shifts the vaccine-induced response towards IL-2- and IL-2-related responses (17, 50). Similarly, the use of IL-4 as an adjuvant shifts the vaccine-induced immune response towards IL-4- and IL-4-related responses (49). Gamma interferon (IFN-γ) favors elicitation of a T_H1 response (3, 20). This body of literature suggests that cytokines can induce a T_H1 response or a T_H2 response, with the production of IL-2 and IFN-γ being indicative of a T_H1 response and the production of IL-4 being indicative of a T_H2 response (33, 36, 43).

To determine whether inclusion of cytokines in an HSV-1 immunization regimen can improve the efficacy of the vaccine in protection against ocular HSV-1 infection by shifting the immune system towards or away from a T_H1 cytokine response, we constructed HSV-1 recombinant viruses expressing murine IL-2, IL-4, or IFN-γ. We used these recombinant viruses to evaluate and compare the adjuvant effect of each cytokine and its ability to shift the relative balance between the T_H1 versus T_H2 immune responses in immunized mice and its potential for improving vaccine efficacy in protection against subsequent HSV-1 infection. We observed that recombinant HSV-1 expressing IL-4 enhanced the humoral immune response more efficiently than recombinant HSV-1 expressing IL-2 or IFN-γ or the parental virus. Overall, the ability of the recombinant viruses expressing IL-2 or IFN-γ to enhance the humoral responses was no greater than that of the parental virus. All three recombinant viruses, irrespective of whether the cytokine genes they carried favor a T_H1 or T_H2 immune response, induced a T_H1 response in immunized mice. Finally, IL-2-expressing recombinant HSV-1 vaccine induced a higher cytotoxic T-lymphocyte (CTL) response. The results of these studies support the concept that not only can recombinant virus vaccines expressing cytokine genes alter the immune response but that they can also increase the efficacy of protection against infection from disease. Thus, the use of cytokine gene-delivered adjuvants, in particular IL-4, could be important in the development of vaccines that are more efficacious in preventing ocular HSV-1 infection and subsequent disease.

MATERIALS AND METHODS

Viruses and cells.

Rabbit skin (RS) cells, used for preparation of virus stocks, culturing of mouse tear films, and determination of growth kinetics, were grown in Eagle's minimal essential medium (MEM) supplemented with 5% fetal calf serum (FCS). Plaque-purified HSV-1 strains and the recombinant viruses derived from these strains were grown in RS cells. McKrae, a neurovirulent strain of HSV-1 causing stromal disease, was used as the ocular challenge virus. KOS and a parental LAT-γ34.5-null mutant of HSV-1 strain McKrae (DM-33), which when given peripherally do not result in fatality and do not produce stromal disease, were used as live-virus vaccines. L929, CL7, and spleen cells were grown in RPMI 1640 medium supplemented with 10% FCS.

Mice.

Inbred female BALB/c mice (Jackson Laboratory, Bar Harbor, Maine) were used. All mice used were between 4 and 6 weeks of age.

Preparation of transfer plasmid.

To prepare the transfer plasmid, the BamHI B fragment of HSV-1 strain McKrae was digested with SwaI-BamHI to produce a 5.5-kb DNA fragment including positions −1041 to +4656 of the HSV-1 latency-associated transcript (LAT) (15). 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 nucleotides +76 to +1667. 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.

Construction of IL-2 plasmid.

A plasmid containing the murine IL-2 gene was digested with PstI-BglI. After addition of a BamHI linker, the insert was ligated into the BamHI site of pLAT, and the resulting plasmid was designated pLAT-IL-2. This insert contained the complete 169-amino-acid coding region of the IL-2 gene plus 7 and 232 bp of noncoding sequence in its 5′ and 3′ regions, respectively.

Construction of IL-4 plasmid.

A plasmid containing the murine IL-4 gene was digested with Tsp509I. 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′ region, 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.

Construction of IFN-γ plasmid.

A plasmid containing the murine IFN-γ gene was digested with BseRI. This released a 656-bp insert, which contained the complete 155-amino-acid coding region of the IFN-γ gene plus 36 and 155 bp noncoding sequences in its 5′ and 3′ regions, respectively. After addition of the BamHI linker, the insert was ligated into the BamHI site of pLAT, and the resulting plasmid was designated pLAT-IFNγ.

Generation of recombinant viruses.

Recombinant viruses expressing IL-2, IL-4, and IFN-γ were generated by homologous recombination as described previously (15, 37, 38). Briefly, pLAT-IL-2, pLAT-IL-4, or pLAT-IFNγ was cotransfected with infectious HSV-1 double mutant (DM-33) DNA by the calcium phosphate method. The DM-33 (LAT-γ34.5-null mutant) virus is a mutant of HSV-1 strain McKrae in which 1.8 kb of LAT and 0.9 kb of γ34.5 have been deleted (42). Viruses from the cotransfection were plated, and the isolated plaques were picked and then screened for the presence of the IL-2, IL-4, or IFN-γ gene using restriction digestion and Southern blot analysis. Selected plaques containing the IL-2, IL-4, or IFN-γ gene were plaque purified eight times and reanalyzed by restriction digestion and Southern blot analysis to ensure that the IL-2, IL-4, or IFN-γ DNA was present in the LAT region. A single plaque meeting this criterion for each recombinant was chosen and designated dbl-IL-2, dbl-IL-4, or dbl-IFNγ. The final recombinant viruses contain either the murine IL-2, IL-4, or IFN-γ gene under the control of the LAT promoter in the normal LAT location in the viral genome. Thus, there are two copies of the LAT promoter-IL-2, -IL-4, or -IFN-γ (one in each viral long repeat) in each virus.

Immunization.

Mice were immunized either once or three times intraperitoneally with 10⁶ PFU of live dbl-IL-2, dbl-IL-4, dbl-IFNγ, parental virus, or HSV-1 strain KOS. Mock-immunized mice were similarly inoculated either once or three times with MEM collected from mock-infected RS cells.

Serum-neutralizing antibody titers.

Serum-neutralizing antibody titers were determined using 50% plaque reduction assays as described previously (14) using sera collected 3 weeks after the final immunization.

Ocular infection.

Mice were challenged ocularly with 2 × 10⁵ or 2 × 10⁶ PFU of HSV-1 strain McKrae per eye, in 5 μl of tissue culture medium without corneal scarification (14).

Titration of virus in tears.

Tear films were collected from both eyes of each mouse (five mice per group) at various times as described previously (11). 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.

Monitoring of eye disease.

The severity of corneal scarring in surviving mice was scored in a blind fashion by examination with a slit lamp biomicroscope following addition of 1% fluorescein as eye drops. Disease was scored on a scale from 0 to 4 as previously described (11), with a score of 0 for no disease and scores of 2, 3, and 4 for 50, 75, and 100% involvement, respectively.

Lymphokine ELISA.

The secretion of IL-2, IL-4, and IFN-γ by spleen cells obtained from mice immunized with each recombinant virus was measured in vitro 3 weeks after the first or third immunization. Three mice per group from three separate experiments were euthanized, and single-cell suspensions of spleen cells were prepared. Spleen cells were cultured in 24-well plates in a humidified 5% CO₂ atmosphere for 72 h at a concentration of 10⁶ cells/well in a total volume of 1 ml. Lymphocytes were cultured in medium alone or medium containing UV-inactivated HSV-1 strain McKrae (10 PFU/cell). The supernatants were collected after 72 h of culture and stored at −80°C until their use in an ELISA. Cell-free culture supernatants were assayed for IL-2, IL-4, and IFN-γ using an ELISA specific for each cytokine (BD PharMingen, San Diego, Calif.). The concentration of each cytokine in the supernatants was estimated by comparing the optical densities of the unknowns to those of the standards and is presented as mean concentration (in picograms per milliliter) ± standard error.

In vitro depletion of CD4⁺ or CD8⁺ T cells.

Mice immunized three times as described above were sacrificed 3 weeks after the third immunization, the spleens were removed, and single-cell suspensions were prepared as described above. Before in vitro lymphocyte culture, the cells (10⁶) were incubated with 100 μg of anti-CD4⁺ (GK1.5) monoclonal antibody (MAb), anti-CD8⁺ (2.43) MAb, or an irrelevant MAb of a similar isotype for 30 min. The treatment was repeated once, and the cells were washed three times with RPMI 1640 medium before the live cells were counted. The CD4⁺-depleted, CD8⁺-depleted, or nondepleted T cells were grown, and the production of cytokines was evaluated as described above. The percentages of IL-2 and IFN-γ produced by each of the T-cell subtypes were calculated on the basis of the total IL-2 and IFN-γ produced by nondepleted spleen T cells.

CTL assay.

Spleens were removed aseptically from mice 3 weeks after the third immunization. Single-cell suspensions were prepared without in vitro stimulation. Prior to the CTL assay, the CD8⁺ effector cells were depleted of CD4⁺ T cells using GK1.5 MAb plus low-toxicity M rabbit complement (Cedarlane) for 30 min as described previously (2, 24). The CL7 target cells were infected with HSV-1 strain McKrae (10 PFU/cell) for 4 h and washed three times with assay medium. The CTL activity was measured by the lactate dehydrogenase (LDH) release assay in 96-well round-bottom plates. Target cells (2 × 10⁴ cells/well) in a 100-μl volume were incubated with 100 μl of effector cells at various effector/target ratios for 4 h in phenol red-free RPMI 1640 medium containing 3% FCS. Supernatant (50 μl/well) was then transferred to the wells on 96-well plates, and lysis was determined by measuring LDH release using the Cytotox 96 assay kit (Promega Corp., Madison, Wis.). The percentages of CTLs were calculated by the following formula: specific lysis = (optical density [OD] of experimental LDH release − OD of spontaneous LDH release from effector cells − OD of spontaneous LDH release from target cells)/(maximum LDH release from target cells − OD of spontaneous LDH release from target cells) × 100%. All determinations were performed in triplicate.

Statistical analysis.

Student's t test and Fisher's exact test were performed using Instat (GraphPad, San Diego, Calif.) to analyze protective parameters. Results were considered to be statistically significant when the P value was less than 0.05.

RESULTS

Structures of the dbl-IL-2, dbl-IL-4, and dbl-IFNγ recombinant viruses.

We constructed three recombinant HSV-1 viruses expressing IL-2, IL-4, or IFN-γ in order to examine the adjuvant effects of virally expressed cytokines on altering and possibly improving live-virus vaccine efficacy. The HSV-1 McKrae strain was used as the original parental virus. The genomic structure of wild-type HSV-1 McKrae is shown schematically in Fig. 1A. The HSV-1 genome contains a unique long region (U_L) and a unique short region (U_S), both of which are flanked by inverted repeats (terminal and internal repeats long [TR_L and IR_L] and terminal and internal repeats short [TR_S and IR_S]). The transcription start site of the primary 8.3-kb LAT RNA is 28 nucleotides downstream of the TATA box (51). The previously described LAT-γ34.5-null mutant (Fig. 1B) was derived from HSV-1 strain McKrae (37, 38). LAT-γ34.5-null mutant (DM-33) virus is a mutant of HSV-1 strain McKrae in which 1.8 kb of LAT and 0.9 kb of γ34.5 have been deleted (42).

FIG. 1.

Construction and structures of the IL-2, IL-4, and IFN-γ recombinant viruses. (A) The wild-type (wt) HSV-1 strain McKrae genome in the prototypic orientation is shown at the top of the schematic. The rectangles labeled long terminal repeat (TR_L) and long inverted repeat (IR_L) represent the terminal and internal (or inverted) long repeats, while the rectangles labeled short terminal repeat (TR_S) and short inverted repeat (IR_S) represent the terminal and internal (or inverted) short repeats. The unique long (U_L) and unique short (U_S) regions are shown. The expanded presentation of part of the internal long and short repeats indicates the location and direction of LAT and γ34.5 transcription. The ICP0 and γ34.5 mRNA is shown for reference. The black rectangle represents the very stable 2-kb LAT. The LAT TATA box at nucleotide (nt) −28 is shown. pos., positive. (B) Parental virus (DM-33) has a deletion from LAT nucleotides −161 to +1667 and from LAT nucleotides +6309 to +7103 in both copies of LAT and makes no LAT or γ34.5 RNA. The deleted regions are indicated (XXXXX and XXX). neg, negative. (C) dbl-IL-2 was constructed from parental virus by homologous recombination between parental virus DNA and a plasmid containing the complete LAT promoter and the entire structural IL-2 gene [including its 3′ poly(A) signal] as described in Materials and Methods. (D) dbl-IL-4 was constructed in a manner similar to dbl-IL-2 except parental virus DNA and a plasmid containing IL-4 gene were recombined. (E) dbl-IFNγ was constructed in a manner similar to dbl-IL-2 and dbl-IL-4 except parental virus DNA and a plasmid containing the entire structural IFN-γ gene were recombined.

dbl-IL-2 (Fig. 1C), dbl-IL-4 (Fig. 1D), and dbl-IFNγ (Fig. 1E) viruses were derived from the LAT-γ34.5-null mutant by insertion of the IL-2, IL-4, or IFN-γ gene and restoration of the LAT promoter such that the IL-2, IL-4, and IFN-γ genes are under the control of the powerful LAT promoter as described in Materials and Methods. Restriction enzyme analysis and partial sequencing confirmed the genomic structures of the dbl-IL-2, -IL-4, and -IFNγ viruses. dbl-IL-2, -IL-4, and -IFNγ contain the entire sequence of the IL-2, IL-4, and IFN-γ genes, respectively, including the polyadenylation signal, under control of the LAT promoter (Fig. 1C to E). There are two complete copies of each cytokine gene, one in each viral long repeat.

dbl-IL-2, dbl-IL-4, and dbl-IFNγ viruses are identical to their LAT-γ34.5-null mutant parent, except that the LAT promoter is restored and is driving the expression of the IL-2, IL-4, and IFN-γ genes.

To determine whether the three recombinant viruses replicate in tissue culture as efficiently as the parental virus, RS cells were infected in triplicate with 0.01 PFU of dbl-IL-2, dbl-IL-4, dbl-IFNγ, or parental virus per cell. The cell monolayers were freeze-thawed at 12, 24, 48, 72, and 96 h postinfection, and the yield of infectious virus was quantitated by a standard plaque assay. Replication kinetics of the three recombinant viruses appeared to be similar to that of the parental virus (not shown), suggesting that expression of IL-2, IL-4, or IFN-γ by an HSV-1 vector lacking both LAT and γ34.5 regions did not appear to have a profound effect on virus replication in tissue culture.

Expression of IL-2, IL-4, and IFN-γ by recombinant viruses in tissue culture.

Confluent monolayers of murine L929 cells were infected at a multiplicity of 10 PFU of dbl-IL-2, dbl-IL-4, dbl-IFNγ, or parental (LAT-γ34.5-null mutant) virus per cell. Media were collected from 0 to 96 h postinfection and assayed using an ELISA kit for the presence of IL-2, IL-4, and IFN-γ protein using antiserum specific for each cytokine. The recombinant viruses secreted significant amounts of cytokines in the media from L929 cells (Fig. 2). The media from L929 cells infected with parental virus did not contain detectable levels of either cytokine (Fig. 2).

FIG. 2.

Kinetics of expression of IL-2, IL-4, and IFN-γ by recombinant viruses. Subconfluent monolayers of L929 or RS cells were infected with 10 PFU of dbl-IL-2, dbl-IL-4, dbl-IFN-γ, or parental viruses per cell as described in Materials and Methods. Supernatant was harvested at the indicated times postinfection, and the amount of expressed IL-2, IL-4, or IFN-γ was determined by ELISA as described in Materials and Methods. (A) IL-2 expressed by dbl-IL-2 virus, (B) IL-4 expressed by dbl-IL-4 virus, and (C) IFN-γ expressed by dbl-IFNγ virus.

To determine whether these recombinant viruses can also express their IL-2, IL-4, or IFN-γ gene in cell lines derived from species other than the mouse, RS cells were infected with each recombinant virus as described above. All three recombinant viruses were able to secrete their encoded proteins in the RS cells (Fig. 2), whereas RS cells infected with parental virus did not contain detectable levels of either cytokine (Fig. 2). Thus, our results established that these recombinant viruses are capable of expressing and secreting a considerable quantity of each cytokine gene in these cell lines.

Induction of HSV-1 neutralizing antibody titers on immunization of mice.

Groups of 10 mice from two separate experiments (5 mice per experiment) were immunized either once or three times with the recombinant viruses as described in Materials and Methods. Three weeks after the final immunization, sera were collected from 10 immunized mice per group, and the neutralization titers were determined using a 50% plaque reduction assay (Table 1). The average neutralizing antibody titer of the dbl-IL-4 group after one immunization was significantly higher than that of the dbl-IL-2, dbl-IFNγ, parental, or KOS virus (the most effective attenuated live-virus vaccine) group (P < 0.03 by Student's t test). Similarly, after three immunizations, the average neutralizing antibody titer for the dbl-IL-4 group was significantly higher than that of the dbl-IL-2, dbl-IFNγ, or parental virus group (P < 0.05 by Student's t test) but was similar to that of the KOS group (P = 0.35 by Student's t test). The mice immunized with dbl-IL-2 and dbl-IFNγ exhibited neutralizing antibody titers that were similar to those of mice immunized with the parental virus (P > 0.05 by Student's t test) (Table 1). Finally, mice immunized with dbl-IFNγ, dbl-IL-4, or KOS virus had higher neutralizing antibody titers after three immunizations than mice immunized once with the same virus (Table 1). In contrast, mice immunized with dbl-IL-2 or parental virus showed similar neutralizing antibody titers after one or three immunizations (Table 1). The neutralizing antibody titers for all immunized groups were significantly higher than those for the mock-immunized mice (P < 0.01 by Student's t test), except for the dbl-IFNγ group after one immunization.

TABLE 1.

Neutralizing antibody titers in mice immunized with recombinant viruses expressing different IL-2, IL-4, or IFN-γ genes^a

Virus	Neutralizing antibody titerb		P valuec
	One immunization	Three immunizations	One immunization	Three immunizations
dbl-IL2	237 ± 47	259 ± 11
dbl-IFNγ	102 ± 49	313 ± 79
dbl-IL4	422 ± 55	772 ± 94
Parental	189 ± 46	211 ± 30
KOS	174 ± 46	914 ± 108
None (mock)	27 ± 11	23 ± 14
Virus groups
dbl-IL2 vs dbl-IFNγ			0.06	0.51
dbl-IL2 vs dbl-IL4			0.02	<0.0001
dbl-IL2 vs parental			0.35	0.15
dbl-IL2 vs KOS			0.47	<0.0001
dbl-IFNγ vs dbl-IL4			0.0004	0.001
dbl-IFNγ vs parental			0.21	0.24
dbl-IFNγ vs KOS			0.30	0.0003
dbl-IL4 vs parental			0.004	<0.0001
dbl-IL4 vs KOS			0.003	0.33

^aMice were immunized once or three times intraperitoneally with 10⁶ PFU of the specified recombinant virus. Ten mice per group were bled 3 weeks after the first or third immunization.

^bHSV-1 neutralizing antibody titers were determined for each serum sample as described in Materials and Methods. The neutralizing antibody titer represents the average of the titers from 10 serum samples ± standard error.

^cP values for the neutralizing antibody titers for the groups of mice immunized with the different viruses by Student's t test.

Protection of immunized mice from lethal ocular challenge.

To determine whether immunization with recombinant viruses expressing IL-2, IL-4, or IFN-γ gene had any effect on survival, 10 mice per group were immunized three times as described above. Immunized mice were challenged ocularly with 2 × 10⁵ PFU of HSV-1 McKrae per eye as described in Materials and Methods. All 10 mice (100%) in each group of mice immunized with the recombinant viruses survived ocular infection. In contrast, only 4 of 10 (40%) of the mock-immunized mice survived the lethal challenge. The protection provided by the three recombinant viruses was statistically significant compared to the mock-immunized mice (P = 0.01 by Fisher's exact test). However, the protection afforded by immunization with the recombinant viruses in this test was similar to that afforded by the parental virus and KOS strain of HSV-1 (P = 1 by Fisher's exact test).

The dose of HSV-1 used to challenge mice in the above experiment failed to reveal possible differences in the efficacy of protection among the recombinant virus vaccines. Therefore, an additional 10 mice per group were immunized three times and challenged with a 10-fold-higher dose of infectious virus. Our results suggest that, even after ocular infection with a higher dose of infectious virus, all immunized mice were protected against HSV-1 infection. However, none of the 10 (0%) mock-immunized mice survived the lethal challenge.

Finally, to determine whether one immunization, rather than three immunizations, would further alter survival, 10 mice per group were immunized once, and three weeks later, they were challenged by ocular infection with 2 × 10⁵ PFU of HSV-1 McKrae per eye. As observed for mice immunized three times, all of the immunized mice were protected completely against lethal infection. This protection was statistically significant compared to that of the mock-immunized mice (P = 0.0007 by Fisher's exact test).

Titration of HSV-1 replication in the eye.

To determine the effect of immunization with recombinant viruses expressing IL-2, IL-4, or IFN-γ on prevention of virus replication in the eye after ocular infection with a virulent HSV-1 strain (McKrae), tear films from mice vaccinated once or three times and infected ocularly with 2 × 10⁵ PFU of McKrae per eye were collected on days 1, 2, 3, 4, 5, 6, and 7 postchallenge, and the amount of infectious HSV-1 was determined. In all groups, the virus titers were highest on days 3 and 4 (Fig. 3A and B). The mock-immunized mice had the highest average peak virus titer. A significant difference between the mice immunized with dbl-IL-4 and KOS compared to the other immunization groups was observed on days 3 and 4 (P < 0.05 by Student's t test). Thus, immunization with the dbl-IL-4 recombinant virus reduced virus replication in the eye more efficiently than immunization with either the dbl-IL-2 or dbl-IFNγ recombinant virus.

FIG. 3.

Virus titers in mouse eyes after ocular challenge of immunized mice. BALB/c mice were immunized once or three times with dbl-IL-2, dbl-IL-4, dbl-IFNγ, parental, or KOS virus or mock immunized as described in Materials and Methods. Three weeks after the first or third immunizations, mice were challenged ocularly withHSV-1 strain McKrae, and the presence of HSV-1 in tear films was monitored daily as described in Materials and Methods. (A) Ocular infection with 2 × 10⁵ PFU of McKrae per eye after one immunization; (B) ocular infection with 2 × 10⁵ PFU of McKrae per eye after three immunizations; and (C) ocular infection with 2 × 10⁶ PFU of McKrae per eye after three immunizations. The virus titer (y axis) is the average of the individual titers from 10 eyes, and the error bars indicate the standard errors.

To determine whether mice vaccinated with dbl-IL-2, dbl-IL-4, or dbl-IFNγ recombinant virus would be protected against higher doses of challenge virus, additional mice were vaccinated or mock vaccinated as described above, and vaccinated mice were challenged ocularly with 2 × 10⁶ PFU of HSV-1 strain McKrae per eye. Tear films from infected mice (10 eyes/group) were collected (Fig. 3C). As reported above, mock-vaccinated mice had significantly higher virus titers in their eyes than mice immunized with recombinant virus or control virus (P < 0.05) (Fig. 3C). Thus, regardless of immunization status or challenge dose, immunization with any of the three recombinant viruses appeared to increase clearance of HSV-1 from the eye.

Protection of immunized mice from eye disease.

The eyes of all the mice that survived ocular challenge were examined for corneal scarring on day 28 and scored on a 0 to 4 scale as described in Materials and Methods. All of the immunized mice were completely protected against corneal scarring, whereas the average corneal scarring scores for each of the mock-vaccinated groups ranged from 2.9 ± 0.5 to 3.0 ± 0.7 after three and one immunization, respectively. The differences between the immunized groups compared to the mock-immunized groups were statistically significant (P < 0.0001 by Student's t test).

In vitro cytokine secretion by splenocytes of immunized mice.

Mice were immunized once or three times with the recombinant viruses as described above, and 3 weeks after immunization or mock immunization, the splenocytes from three mice from three separate experiments per group were obtained, and single cell suspensions were stimulated in vitro with 10 PFU of UV-inactivated HSV-1 strain McKrae per cell. Subsequently, the amounts of IL-2, IL-4, and IFN-γ secreted into the media were analyzed using an ELISA kit (as described in Materials and Methods). As shown in Fig. 4A, splenocytes from mice immunized once with dbl-IL-4 or KOS produced the largest amounts of IL-2. Similarly, the splenocytes from these two groups also produced the largest amounts of IL-2 after three immunizations. In all of the groups, except for the dbl-IFNγ-immunized group, the levels of IL-2 expression had declined by the third immunization, compared to the expression elicited by one immunization (Fig. 4A). The lymphocytes from mock-immunized mice secreted only very small amounts of IL-2 after one immunization and no IL-2 after three immunizations (Fig. 4A). In contrast, all of the immunized groups failed to produce significant amounts of IL-4 after either one or three immunizations except for the mock-immunized group (Fig. 4B).

FIG. 4.

Cytokine production by splenocytes of immunized mice. BALB/c mice were immunized once or three times as described in Materials and Methods. Three weeks after the final immunization, mice were euthanized, spleens from three mice per group were harvested, and single-cell suspensions of splenocytes were prepared and stimulated in vitro for 72 h with UV-inactivated HSV-1 strain McKrae, as described in Materials and Methods. The concentrations of IL-2 (A), IL-4 (B), and IFN-γ (C) in the supernatants were measured by ELISA. Each point represents the mean of the titers from three experiments ± standard error (error bar).

The amounts of IFN-γ secreted by cultured spleen cells from mice immunized once with dbl-IL-4, parental, or KOS virus were significantly larger than those for mice immunized once with dbl-IL-2 or dbl-IFNγ (Fig. 4C). After three immunizations, the amounts of IFN-γ secreted by the mice immunized with dbl-IL-4, parental, and KOS virus declined, whereas an increase was observed in the mice immunized with dbl-IL-2 (Fig. 4C). The amounts of IFN-γ produced by the dbl-IFNγ group were similar after one or three immunizations. The lymphocytes from mock-immunized mice secreted only very small amounts of IFN-γ (Fig. 4C).

Sources of IL-2 and IFN-γ in immunized mice.

The results of the above studies, as shown in Fig. 4, suggested that immunization of the mice with different recombinant viruses resulted in production of IL-2 and IFN-γ. It is known that, depending on the response, these cytokines can be produced primarily by the CD4⁺ T cells, primarily by the CD8⁺ T cells, or by both CD4⁺ and CD8⁺ T cells. Therefore, to identify the source of IL-2 and IFN-γ production, mice were immunized three times as described above, and 21 days after the third immunization, spleens from three mice per group were obtained, and the CD4⁺ or CD8⁺ T cells were depleted by incubation of the spleen cells with anti-CD4⁺ MAb, anti-CD8⁺ MAb, both anti-CD4⁺ and anti-CD8⁺ MAbs, or an irrelevant MAb as described in Materials and Methods. The individual T-cell subtypes thus obtained were stimulated as described above, and their secretion of cytokines was evaluated and compared with that of the total nondepleted T cells that had been treated with the irrelevant MAb. The results for each cytokine are shown in Fig. 5 as the percentage of the amount of cytokine secreted by the nondepleted T-cell population. Our results suggest that after immunization with any of the viruses, the pattern of IL-2 production for all groups was the same, with both CD4⁺ and CD8⁺ T cells contributing equally to its production (Fig. 5A). Similarly, after immunization with dbl-IL-4, parental, or KOS virus, both CD4⁺ and CD8⁺ T cells produced equivalent amounts of IFN-γ (Fig. 5B). However, after immunization with dbl-IL-2, the CD8⁺ T cells produced 75% of the IFN-γ, while only 25% was produced by the CD4⁺ T cells (Fig. 5B). In contrast, after immunization with dbl-IFNγ, only 13% of the IFN-γ was produced by the CD8⁺ T cells, whereas 87% was produced by the CD4⁺ T cells (Fig. 5B). Lymphocytes depleted of both T cells did not secrete detectable amounts of IL-2 or IFN-γ (not shown).

FIG. 5.

Sources of IL-2 (A) and IFN-γ (B) production in immunized mice. Spleens from immunized mice were harvested 3 weeks after the third immunization. Single-cell suspensions of CD4⁺ T cells, CD8⁺ T cells or both T-cell subpopulations were prepared and subjected to in vitro stimulation for 72 h with 10 PFU of UV-inactivated HSV-1 strain McKrae per cell. Cytokines in the supernatants were determined by ELISAs. Each value is the percentage of each cytokine that was produced by CD4⁺ T cells or CD8⁺ T cells in relation to the amount of each cytokine produced by both T cells. Values that were significantly different from each other as well as other vaccine groups (P < 0.05 by Student's t test) (*) and values that were similar to each other and significantly different from other vaccine groups (P > 0.05 by Student's t test) (#) are indicated.

CTL assay.

The above results revealed that on immunization of mice with dbl-IL-2 virus, the CD8⁺ T cells are responsible for the production of IFN-γ by splenocytes in this group, which may indicate higher CTL activity in this group of mice. Therefore, we examined the CTL responses in immunized mice. The mice were immunized three times as described above, and the CTL activity was measured as described in Materials and Methods. Figure 6 shows that the mice immunized with dbl-IL-2 virus exhibited significantly higher HSV-specific cytotoxicity than did those immunized with dbl-IL-4, dbl-IFNγ, or parental virus (P < 0.001). The mock-immunized mice did not exhibit any significant CTL response (Fig. 6). Thus, immunization of mice with dbl-IL-2 elicits a significantly higher CTL response than immunization with dbl-IL-4 or dbl-IFNγ.

FIG. 6.

CTL activity in immunized mice. Splenocytes from immunized and mock-immunized mice were prepared as described in Materials and Methods. CD4⁺ T cells were removed by incubating the splenocytes with L3T4 MAb for 30 min. LDH release assays were performed in triplicate with splenocytes as effector cells and HSV-1-infected CL7 cells as target cells (as described in Materials and Methods). Each point represents mean ± standard error of the mean from three experiments. E:T ratio, effector-to-target cell ratio.

DISCUSSION

The induction of optimal humoral and CMI responses against HSV-1 infection is a critical determinant in the development of an efficacious vaccine. Previously, we showed that the amount of antigen in a vaccine and the number of immunizations can influence the type and nature of the immune response to HSV-1 infection (16). In addition, different groups have shown that the immune responses to immunization can be further altered when cytokines (i.e., protein or DNA) are included with the antigen (30, 31). The present study had two aims: (i) to determine whether immunization with HSV-1 recombinant viruses expressing IL-2, IL-4, or IFN-γ would alter the immune responses compared with those obtained on immunization with their parental virus; and (ii) to determine whether the humoral and CMI responses induced by IL-4 (as an indicator of a T_H2 response) differ from the immune responses induced by IL-2 or IFN-γ (as indicators of a T_H1 response).

In this study, a direct comparison of the protective immune responses elicited by the three different recombinant vaccines (dbl-IL-2, dbl-IL-4, and dbl-IFNγ) was performed. The types of immune responses generated by these three recombinant virus vaccine strategies were distinctly different. While immunization with dbl-IL-2 or dbl-IFNγ virus induced a weak antibody response, immunization with dbl-IL-4 induced a strong humoral response. Many reports indicate that the use of IL-2 (8, 22, 27, 29, 39), IL-4 (6, 7), or IFN-γ (7, 23) as an adjuvant increases vaccine efficacy in a variety of systems. In contrast, other studies have shown that IL-2, IL-4, or IFN-γ does not play a role in improving vaccine efficacy (23, 45). In this study, we have shown that immunization with dbl-IL-4 virus resulted in a significant enhancement of the humoral response compared with immunization with the parental, dbl-IL-2, or dbl-IFNγ virus. This is similar to the previously published results suggesting that coinjection of the IL-4 gene with a hepatitis B virus (HBV) DNA vaccine significantly enhanced the development of the humoral response (7). However, our results obtained on immunization with dbl-IL-2 or dbl-IFNγ differ from the previously published reports that have suggested enhancement of the humoral immune responses on coinjection of the IL-2 gene (8) or IFN-γ gene (7) with the HBV DNA vaccine or IL-2 DNA with HSV-2 gD DNA (45). These discrepancies between our results and the literature are probably due to our use of a different virus or to use of a live-virus vaccine rather than DNA immunization. Also, it has been shown that immunization with DNA induces a different immune response than that elicited by immunization with a live virus (10, 25). Thus, both the type and nature of the immunogen that is used to stimulate the immune response can affect the magnitude of the immune response.

On the basis of the strong IL-2 and IFN-γ responses in immunized mice, we characterized the responses induced by the three recombinant virus vaccines as involving type 1 T_H response. Furthermore, in line with a T_H1 type of cytokine response, the immunoglobulin G2a (IgG2a) responses of mice immunized with all recombinant viruses and the parental virus were greater than that of mock-immunized mice (not shown). T_H1 cells produce both IL-2 and IFN-γ and are involved in the activation of macrophages, delayed-type hypersensitivity, and production of IgG2a antibody (1, 33). The T_H1 nature of immune responses induced by dbl-IL-4 was unexpected, since IL-4 should enhance T_H2 responses and reduce T_H1 responses (33). However, IL-4 is an important molecule driving IL-12, which is an indicator of a T_H1 immune response (19). Thus, it appears that IL-4 may also contribute to a T_H1 response in immunized mice as we reported here. It is possible that there is negative-feedback inhibition between humoral immunity (neutralizing antibody titer) and IL-4 production by cellular immunity. The results of this study are consistent with those that show that T-cell stimulation of HSV-seropositive individuals results in a prominent T_H1 response with little IL-4 production (4). In addition, it has been shown that, in the presence of an antibody titer to HSV-1, IL-4 was not detectable in the corneas of mice on recurrent HSV-1 infection (47).

On stimulation by foreign antigens, CD4⁺ and CD8⁺ T-cell clones of mice and humans produce cytokines in a characteristic pattern (32, 33). In this study, both CD4⁺ and CD8⁺ T cells in the spleens from immunized mice contributed equally to IL-2 secretion whether the vaccine carried the IL-2, IL-4, or IFN-γ gene. However, the sources of IFN-γ production clearly differed depending on the vaccine. In the spleens of mice immunized with dbl-IL-4, parental, or KOS virus, both CD4⁺ and CD8⁺ T cells produced equivalent amounts of IFN-γ. In contrast, in the spleens of mice immunized with dbl-IL-2 virus, the CD8⁺ T cells produced two-thirds of the secreted IFN-γ, while in mice immunized with dbl-IFNγ recombinant virus, more than two-thirds of IFN-γ was produced by CD4⁺ T cells. The higher IFN-γ production by CD8⁺ T cells in the mice immunized with dbl-IL-2 virus is probably the reason that these mice have higher CTL activity than the mice immunized with dbl-IL-4 or dbl-IFNγ virus.

The greatest efficacy in vaccine protection against virus replication in the eye was achieved with the dbl-IL-4 vaccine. The ability of the dbl-IL-2 vaccine to elicit higher major histocompatibility complex class I-restricted CTL response probably contributed to a reduction in the virus load in the eyes of infected mice despite having a lower neutralizing antibody titer than mice immunized with dbl-IL-4 or KOS. This is in agreement with a previously published report showing that IL-2 plays a major role in CTL activity in vitro (26). IL-2 is involved in the growth, differentiation, and function of lymphocytes (35, 46). Similar to this study, it been shown that administration of IL-2 both in vitro (48) and in vivo (41) causes lysis of a broad array of cells. All immunized mice regardless of the vaccine used, the number of immunizations, or the dose of the challenge virus were completely protected against death and corneal scarring. The effect of vaccination on prevention of latent infection in mice that survived ocular challenges was measured by explant cocultivation and reverse transcription-PCR. However, we found no significant differences in the rate of establishment of latency between the three vaccine groups compared with the parental virus control group (not shown).

In conclusion, the data presented here suggest that HSV-1 recombinant viruses expressing IL-2, IL-4, or IFN-γ induce both humoral and CMI responses. However, the HSV-1 recombinant virus expressing IL-4 was superior to the recombinant viruses expressing IL-2 or IFN-γ in providing protection against viral replication in the eye. This higher efficacy of IL-4-expressing virus is probably associated with higher antibody-mediated immunity. This is in agreement with previously published reports showing that IL-4 is involved in the development of humoral immunity, in the differentiation and maturation of B cells, and in the switch from IgE to IgG (5, 34). Finally, this study also demonstrates that cytokine genes can be used to enhance specific immune responses to live-virus vaccines, thus enabling the manipulation of vaccines such that they target the entire spectrum of responses involved in infection and its sequelae.