amounts of biologically functional and highly immunogenic glycoproteins (15-21). We vaccinated mice with an equal

Transcription

1 JOURNAL OF VIROLOGY, Apr. 1994, p Vol. 68, No X/94/$4.+ Copyright 1994, American Society for Microbiology Expression of Seven Herpes Simplex Virus Type 1 Glycoproteins (gb, gc, gd, ge, gg, gh, and gi): Comparative Protection against Lethal Challenge in Mice HOMAYON GHIASI, 12* RAVI KAIWAR,' ANTHONY B. NESBURN,1'2 SUSAN SLANINA,' AND STEVEN L. WECHSLER 12 Ophthalmology Research, Cedars-Sinai Research Institute, Cedars-Sinai Medical Center, Los Angeles, California 948,1 and Department of Ophthalmology, UCLA School of Medicine, Los Angeles, California 9242 Received 12 October 1993/Accepted 7 January 1994 We have constructed recombinant baculoviruses individually expressing seven of the herpes simplex virus type 1 (HSV-1) glycoproteins (gb, gc, gd, ge, gg, gh, and gi). Vaccination of mice with gb, gc, gd, ge, or gi resulted in production of high neutralizing antibody titers to HSV-1 and protection against intraperitoneal and ocular challenge with lethal doses of HSV-1. This protection was statistically significant and similar to the protection provided by vaccination with live nonvirulent HSV-1 (9 to 1%o survival). In contrast, vaccination with gh produced low neutralizing antibody titers and no protection against lethal HSV-1 challenge. Vaccination with gg produced no significant neutralizing antibody titer and no protection against ocular challenge. However, gg did provide modest, but statistically significant, protection against lethal intraperitoneal challenge (75% protection). Compared with the other glycoproteins, gg and gh were also inefficient in preventing the establishment of latency. Delayed-type hypersensitivity responses to HSV-1 at day 3 were highest in gg-, gh-, and ge-vaccinated mice, while on day 6 mice vaccinated with gc, ge, and gi had the highest delayed-type hypersensitivity responses. All seven glycoproteins produced lymphocyte proliferation responses, with the highest response being seen with gg. The same five glycoproteins (gb, gc, gd, ge, and gi) that induced the highest neutralization titers and protection against lethal challenge also induced some killer cell activity. The results reported here therefore suggest that in the mouse protection against lethal HSV-1 challenge and the establishment of latency correlate best with high preexisting neutralizing antibody titers, although there may also be a correlation with killer cell activity. There are 11 known antigenically distinct glycoproteins in herpes simplex virus type 1 (HSV-1) virions: gb, gc, gd, ge, gg, gi, gh, gj, gk, gl, and gm (3, 29, 3, 38). These glycoproteins are the primary inducers and targets of humoral immune responses in HSV infection and are also important inducers and targets of cell-mediated immune responses (48). In addition, some of the HSV glycoproteins have been shown to interact with specific components of the immune system (12, 31). Most immunological and vaccine studies of the HSV-1 glycoproteins have focused on gd and/or gb (4, 23). This is at least partially due to the fact that these are the most abundant HSV glycoproteins and therefore were the first two glycoproteins to be expressed and to be shown to provide protection against HSV infection in animal models (36, 5). Until our recent work (15-21), no laboratory had undertaken to express most of the individual glycoproteins in a baculovirus expression system capable of producing large quantities of each glycoprotein so that meaningful comparative studies could be done. Previous comparative analysis of the HSV-1 glycoproteins with vaccinia virus-expressed glycoproteins obtained from different laboratories showed very low or no protective responses for ge, gg, gh, and gi (7). Therefore, to carry out comparative studies, we have individually expressed each of the first seven identified HSV-1 glycoproteins (gb, gc, gd, ge, gg, gh, and gi) in a baculovirus expression system that produces large * Corresponding author. Mailing address: Ophthalmology Research, Halper Bldg. Rm. 124, Cedars-Sinai Medical Center, 87 Beverly Blvd., Los Angeles, CA 948. Phone: (31) Fax: (31) amounts of biologically functional and highly immunogenic glycoproteins (15-21). We vaccinated mice with an equal amount of each of these recombinants. We report here the results of our comparative studies of the ability of each glycoprotein to (i) protect against lethal intraperitoneal (i.p.) challenge with HSV-1, (ii) protect against lethal ocular challenge, (iii) protect against the establishment of HSV-1 latency, (iv) induce neutralizing antibody, (v) induce a delayed-type hypersensitivity (DTH) response, (vi) induce a lymphocyte proliferative response, and (vii) induce killer cell activity. MATERUILS AND METHODS Virus and cells. Plaque-purified HSV-1 strains were grown in CV-1 cell monolayers in minimal essential media (MEM) containing 1% fetal calf serum as we described previously (51). KOS, a nonneurovirulent strain of HSV-1, was used as a positive control vaccine. McKrae, a neurovirulent HSV-1 strain, was used as the challenge virus. Baculovirus and baculovirus recombinants were grown in Sf9 cells with TNM-FH media containing 1% fetal bovine serum as previously described (15). Mice. Six- to eight-week-old female BALB/c mice were used. Expressed glycoproteins. Recombinant baculoviruses expressing gb, gc, gd, ge, gg, gh, or gi (all derived from KOS) were developed in our laboratory (15-22). The level of expression for each recombinant glycoprotein was sufficient to enable us to identify the expressed glycoproteins by Coomassie blue staining of sodium dodecyl sulfate-polyacrylamide gels of total cell extracts. The relative proportion of each expressed glyco-

2 VOL. 68, 1994 VACCINATION WITH SEVEN HSV-1 GLYCOPROTEINS 2119 protein was determined by scanning Coomassie blue-stained gels on a laser densitometer. The individual glycoproteins were all expressed at similar high levels representing between 1 and 2% of the total cell protein. Preparation of cell lysates for immunization. Sf9 cell monolayers were infected with individual baculovirus recombinants (gb, gc, gd, ge, gg, gh, and gi) at a multiplicity of infection of 1 PFU per cell. The infected cells were collected after 72 h, washed, suspended in phosphate-buffered saline, and freezethawed for later use. For mock vaccination, Sf9 cells infected with wild-type baculovirus under the same conditions and identically processed were used. Immunization of mice. Mice were either vaccinated once with each individual baculovirus recombinant glycoprotein or vaccinated three times at 3-week intervals with each individual baculovirus recombinant glycoprotein. Because of the similar high expression levels of the baculovirus recombinants, immunization of mice with equal aliquots of the recombinant baculovirus-infected cell extracts resulted in inoculation with similar amounts of each of the expressed glycoproteins (5 to 1,ug per vaccination). For all mice, each vaccination consisted of a subcutaneous and an i.p. inoculation given at the same time. Subcutaneous injections were done with Freund's complete adjuvant on day and with an identical preparation but with Freund's incomplete adjuvant on days 21 and 42. Intraperitoneal injections were done on the same days with the same dose of infected Sf9 cells in PBS. Mock-vaccinated mice were similarly inoculated with wild-type baculovirus-infected Sf9 cells (mock 1 [Ml]) or with PBS (mock 2 [M2]). Positivecontrol mice were immunized i.p. with 2 x 15 PFU of KOS in tissue culture media. Sera from immunized mice were collected 3 weeks after the final vaccination. Ocular challenge. Ocular challenge was done 3 weeks after the final vaccination. HSV-1 McKrae (2 x 15 PFU) in 5 [l of tissue culture media was placed in each eye without corneal scarification, and the lid was held closed and rubbed gently for 3 s. Determination of latency. Mice surviving 28 days after challenge were euthanized. Both trigeminal ganglia were removed and individually explanted onto Vero or RS (rabbit skin) cell monolayers as previously described (25). The monolayers were monitored for 3 days for the presence of infectious virus. Serum neutralizing antibody titers. Serum neutralizing antibody titers were determined by 5% plaque reduction assays as we described previously (15). 5tCr release assay. Spleens from immunized mice were removed aseptically, and single-cell suspensions were prepared and stimulated in vitro with 1.5 PFU of UV-inactivated HSV-1 (KOS) per cell for 3 da/s (35). The CL7 target cells (a cell line compatible at the H-2' locus with BALB/c mice) (a gift from Rafi Ahmed) were infected with HSV-1 (KOS) at a multiplicity of infection of 1 PFU per cell for 3 h and labeled for 45 min with 51Cr (3,uCi per 2 x 16 cells). After labeling, the target cells were washed and 2 x 14 target cells were incubated with different ratios of effector to target cells (1:1, 5:1, and 25:1) for 4 h at 37 C. The specific " Cr release was calculated as previously described (34, 37). Lymphocyte proliferation response. Spleens from immunized mice were removed, and single-cell suspensions were prepared and stimulated in vitro with 1.5 PFU of UV-inactivated HSV-1 (KOS) per cell for 3 days (35). On day 3, 2 x 15 lymphocytes were labeled with 1,uCi of [3H]thymidine and incorporation was determined 18 h later. Controls included unstimulated lymphocytes from vaccinated groups and lymphocytes from mock-vaccinated mice. DTH. Mice were vaccinated three times as described above. Three weeks after the final vaccination, mice were injected in the dorsal side of the right ear with 2 x 1(6 PFU of UV-inactivated HSV-1 (KOS) in 5 p.l of MEM. The DTH reaction was measured, with a micrometer (Mitutoyo, Tokyo, Japan), as net swelling (postchallenge minus prechallenge ear thickness) (42) at, 1, 2, 3, and 6 days after injection. Controls included HSV-1 (KOS)-vaccinated mice (positive control) and wild-type baculovirus (Ml)- and PBS (M2)-vaccinated mice (negative controls). Statistical analyses. Statistical analyses were done by using the personal computer program Instat (GraphPad software, version 2.2). Comparisons with the two mock-vaccinated groups were made separately or they were made with the MI group only, as indicated in the text. For analyses comparing proportions (e.g., 6 of 1 versus 1 of 1 survivors), the Fisher exact test was employed. For analyses comparing the means of two sets of numbers (e.g., a neutralization titer of 61 ± 39 versus a neutralization titer of 179 ± 11), the Student t test was employed. When the standard deviations for the groups being compared were significantly different, the Mann-Whitney rank sum test was used in place of the Student t test, as suggested by the software program. Results were considered statistically significant only if the comparison to the MI (baculovirus mock-vaccinated) group gave a P value of <.5. RESULTS Protection of vaccinated mice from lethal ocular HSV-1 challenge. The construction of baculovirus recombinants, each expressing one of the seven HSV-1 glycoproteins (gb, gc, gd, ge, gg, gh, and gi), was described previously (15-21). BALB/c mice were vaccinated once subcutaneously and i.p. (concomitantly) with individual recombinant baculovirus-infected freeze-thawed whole-insect-cell lysates containing expressed gb, gc, gd, ge, gg, gh, or gi as described in Materials and Methods. Control groups of mice were similarly inoculated with either wild-type baculovirus (MI) or PBS (M2). A positive-control group of mice were immunized i.p. with 2 x 15 PFU of live KOS. Three weeks after vaccination, the mice were challenged ocularly with 2 x 15 PFU of HSV-1 McKrae (1 times the 5% lethal dose [LD5]) as described in Materials and Methods (Fig. IA). Of 1 mice, 1 (1%) survived the lethal challenge in the baculovirus-expressed gb, gd, and gi groups and the KOS positive-control group. In contrast, only 2 of 1 (2%) PBS (M2)-vaccinated mice and 6 of 1 (6%) wild-type baculovirus (M1)-vaccinated mice survived the lethal challenge. The differences between the gb-, gd-, or gi-vaccinated mice and the mock-vaccinated mice were statistically significant (P <.5 by a single-sided Fisher's exact test for comparison with either mock-vaccinated group), indicating that a single vaccination with any one of these three glycoproteins provided significant protection against ocular challenge. Survival following ge vaccination was only slightly lower (9 of 1; 9%), but because of the small number of mice in each group, this was not statistically significant compared with the value for the MI control (P >.5). The survival rate for mice vaccinated with gg (8 of 1; 8%) was also not significant. The survival rate for mice vaccinated with gc or gh was identical to the survival rate for the wild-type baculovirus-vaccinated mice (6 of 1; 6%). Thus, a single vaccination with gb, gd, or gi provided statistically significant protection against lethal ocular challenge compared with results for both control groups, while a single vaccination with gc, ge, gg, or gh did not. To determine whether multiple vaccinations could provide increased protection, identical groups of mice were vaccinated

3 212 GHIASI ET AL. J. VIROL. A * C1-, B 1] gb gc gd ge gg gh gi KOS MI M2 * * * * * * gb gc gd ge gg gh gi KOS MI M2 (26) (3) (26) (3) (26) (22) (3) (3) (24) (8) FIG. 2. Protection against the establishment of latency in mice vaccinated with baculovirus-expressed gb, gc, gd, ge, gg, gh, or gi. Surviving mice from the experiment shown in Fig. 1 were euthanized on day 28 post-ocular challenge. Both trigeminal ganglia (TG) were removed from each mouse. Each individual TG was analyzed for the presence of latent HSV-1 by cocultivation on monolayers of Vero or RS cells as described in Materials and Methods. The monolayers were examined daily for up to 3 days for the appearance of viral cytopathic effects. For each group, the results for one and three vaccinations were pooled and are presented together (see text). Each bar represents the percentage of TG in all the surviving mice from each vaccination group (one vaccination plus three vaccinations) that harbored latent infections. Ml and M2 represent wild-type baculovirus-vaccinated and PBS-vaccinated groups, respectively. *, statistically significant compared with values for mock controls (P <.5 by Fisher's exact test). The numbers in parentheses indicate the number of TG analyzed for HSV-1 latency in each group. Differences are due to the differences in survival rates among the groups. gb gc gd ge gg gh gi KOS Ml M2 FIG. 1. Lethal ocular challenge of mice immunized with baculovirus recombinants expressing HSV-1 gb, gc, gd, ge, gg, gh, and gi. Groups of mice were vaccinated with each individual glycoprotein (5 to 1 p.g of glycoprotein per vaccine dose) one time (A) or three times (B), as described in Materials and Methods. Three weeks after the final vaccination, the mice were challenged ocularly as described in Materials and Methods and survival was monitored for 28 days. Panel A shows results obtained with 1 mice per group, and panel B shows results obtained with 15 mice per group. Bars gb, gc, gd, ge, gg, gh, and gi represent the survival rates of mice vaccinated with each baculovirus-expressed glycoprotein. KOS, positive control [survival rate of mice vaccinated with live nonvirulent HSV-1 (KOS)]; Ml, survival rate of wild-type baculovirus-vaccinated mice; M2, survival rate of PBS-vaccinated mice; *, value significantly different from that for either mock control (for panel A, P <.5 by a single-sided Fisher exact test; for panel B, P <.3 by a two-sided Fisher exact test). three times at 3-week intervals as described above. The number of mice per group was also increased to 15 for increased statistical power. Because of space constraints, this necessitated pooling the results from two identical experiments, one employing 1 mice per group and one employing 5 mice per group. This was statistically valid because the two sets of results were similar. Three weeks after the third vaccination set, the mice were challenged ocularly as described above (Fig. 1B). The survival rate of 1% for the positive KOS control was unchanged (15 of 15). Survival rates for the two mockvaccination controls (7 of 15 for the Ml wild-type baculovirus group and 3 of 15 for the M2 PBS group) were also similar. Survival in the gb-, gd-, and gi-vaccinated groups was also unchanged (15 of 15; 1%), again indicating significant protection compared with the Ml control (P =.2 by the Fisher exact test). Protection by both gc and ge increased to a statistically significant level (15 of 15; 1% [P =.2 by the Fisher exact test versus control Ml]). Protection with gg (9 of 15) and gh (8 of 15) remained not significant compared with values for the Ml control (P =.72 and 1., respectively). Thus, following three vaccinations, five of the seven expressed glycoproteins (gb, gc, gd, ge, and gi) provided statistically significant protection against ocular challenge, while gg and gh remained unprotective. Effect of vaccination on establishment of latent infection. Mice that survived the ocular challenges described in the legend to Fig. 1A and B (the subset containing 1 mice per group only) were kept for 28 days in order to allow for the establishment of latency. The mice were euthanized, and their trigeminal ganglia were removed and analyzed for the presence of latent HSV-1 by explant cocultivation. This gave a measure of the rate of the establishment of latency in mice vaccinated with the different glycoproteins. The patterns of latency were similar, regardless of whether the mice were vaccinated one or three times, allowing statistical analysis of the combined data (Fig. 2). This was necessary because of the small number of surviving mice in the mock-vaccinated control groups. Vaccinations with gb, gc, gd, ge, and gi all resulted in the detection of latency in fewer trigeminal ganglia (P <.5 by Fisher's exact test versus either control), indicating that these five glycoproteins provided protection against the establishment of latency. In contrast, vaccination with gg or gh did not significantly reduce the rate of establishment of latency (P >.1).

4 VOL. 68, 1994 VACCINATION WITH SEVEN HSV-1 GLYCOPROTEINS 2121 TABLE 1. Lethal i.p. challenge of mice immunized with each of the seven baculovirus recombinants expressing HSV-1 gb, gc, gd, ge, gg, gh, and gi Immunization No. of survivors/total Survival (%) gb 18/2" 9 gc 19/19" 1 gd 2/2 1 ge 19/2a 95 gg 29/38" 76 gh 8/19 42 gi 18/2" 9 KOS 5/5" 1 M l 21/58 36 M2 7/38 2 "Survival rate (protection) was significantly different from that for both mock-vaccinated controls (P <.1 by Fisher's exact test). " Survival rate (protection) was significantly different from those for the mock-vaccinated controls (P =.2) and the KOS-infected group (P =.3 by Fisher's exact test). Protection of vaccinated mice from lethal i.p. HSV-1 challenge. The comparative lethal ocular challenge results described above were similar to the results of lethal i.p. challenges that we have previously published separately for each of these glycoproteins (15-21). These results are compiled and presented together for the first time in Table 1. Groups contained 19 to 58 mice (Table 1). These mice were vaccinated three times as described above and challenged i.p. with 2 x 16 PFU of HSV-1 McKrae (4 LD5s) 3 weeks after the third vaccination. High levels of protection, similar to that induced by vaccination with live KOS (P >.4 by Fisher's exact test) and significantly greater than that seen for either mockvaccinated group of mice (P <.1), were seen with the same five glycoproteins (gb, gc, gd, ge, and gi) that provided high levels of protection against ocular challenge following three vaccinations. gh, which provided no protection against ocular challenge, also did not protect against i.p. challenge (8 of 19; 42% survival [P >.1 versus the Ml control]). In contrast to the similarity between the results for the other six glycoproteins, gg provided no statistically significant protection against ocular challenge, but it did provide partial protection against i.p. challenge. This protection was greater than that for either of the mock-vaccinated groups (P =.2) but lower than that for the KOS-vaccinated group (P =.3). Induction of HSV-1 neutralizing antibodies. Sera were obtained from the mice used in the above-described ocular challenge study 3 weeks after the third vaccination, just prior to challenge. Individual serum samples were heat inactivated for 3 min at 56 C, and neutralization titers were determined by a 5% plaque reduction assay on each of 1 serum samples per group, as described in Materials and Methods. The average neutralization titers are shown in Fig. 3. gb, gc, gd, ge, and gi all induced neutralization titers significantly higher than that of either mock-vaccination control (P <.5 by the Student t test). The gd titer was similar to that induced by live KOS (P >.5), while the titers induced by gb, gc, ge, and gi were significantly lower than that induced by KOS (P <.5). The neutralization titer induced by gg (7 ± 25) was not different from the wild-type baculovirus mock-vaccine (Ml) titer ( ). As we previously reported (19), the average gh-induced titer (179 ± 11) was slightly higher than that of the wild-type baculovirus mock vaccine. However, in this particular experiment this difference was not statistically significant (P >.5). Thus, the five glycoproteins that induced the best protection 12 2~~~~~~~~ 1 o * gb gc gd ge gg gh gl KOS MlI M2 FIG. 3. Neutralizing antibody titers in mice vaccinated with individual baculovirus-expressed glycoproteins. Ten mice per group from the mice vaccinated three times (Fig. IB) were bled just prior to challenge. HSV-1 neutralizing antibody titers were determined for each serum sample as described in Materials and Methods. For each bar, the neutralizing antibody titer represents the average of the titers from 1 serum samples. The error bars indicate the standard errors. *, significantly different from values for mock-vaccinated controls (P <.5 by Fisher's exact test); #, similar to KOS values (P >.5). against ocular and i.p. challenge also appeared to induce the highest neutralization titers. Measurement of DTH response in vaccinated mice. The DTH response induced by each of the seven glycoproteins was measured by ear swelling as described in Materials and Methods, with five mice per group (Fig. 4). At day 1 after injection of antigen, no DTH response above that of the mock-vaccinated groups (MI and M2) was detected (Fig. 4A). By day 2, significant positive DTH responses were seen only in the ghand KOS-vaccinated groups (P <.5 by the Student t test) (Fig. 4B). On day 3, DTH responses greater than those of either mock-vaccinated group (P <.5), and comparable to that of the KOS group, were detected in the ge, gg, and gh groups (Fig. 4C). By day 6, the gg and gh DTH responses had decreased back to control levels while the ge response had increased further (Fig. 4D). In addition, significant DTH responses were now seen in the gc- and gl-vaccinated mice (P <.5 versus either control). To summarize, (i) gh and gg induced DTH responses that could be detected only early after challenge (2 to 3 days), (ii) gc and gl induced responses that could be detected only after day 3, (iii) ge induced an early and long-lasting DTH response, and (iv) gd and gb did not induce significant DTH responses. Lymphocyte proliferation. Mice were vaccinated three times as described above, and spleen cells were harvested I week later. Induction of a lymphocyte proliferation response by the individual HSV-1I glycoproteins was determined by the method of measuring incorporation of [3H]thymidine into spleen cells as described in Materials and Methods. In the absence of in vitro priming with UV-inactivated HSV-1, none of the groups showed any incorporation above the mock-vaccination control (MI and M2) levels (Fig. 5). Following stimulation of the spleen cells with UV-inactivated virus, significant lymphocyte proliferation was detected in all seven vaccinated groups compared with results for both mock-vaccinated control groups (P <.5 by the Studentt test or Mann-Whitney nonparametric test). 51Cr release assay. To measure total killer cell activity (combined natural killer activity and CD4 and CD8 cytotoxic T lymphocyte activity), 51Cr release assays were done with lym-

5 e24i c) C 18 2~ 12 7 gb 2122 GHIASI ET AL. C) C)._ 18 A ggk g D g G g l KS MI M C * ca 24 C 18._ B ; 6- I B g D E g g I KO l M =8 18 L LgB gc gd ge gg gh gl KOS M.. I 24'D* * M2 J. VlIROL. G) Ca a) c I..g g g g KS I gb gc gd ge gg gh gl KOS M I M2 gb gc gd ge gg gh gl KOS MI M2 FIG. 4. Induction of DTH responses in mice vaccinated with baculovirus-expressed glycoproteins. Groups of five mice were vaccinated three times with individual expressed glycoproteins. DTH responses were measured by the amount of ear swelling 24 h (A), 48 h (B), 72 h (C), and 6 days (D) following injection of antigen (UJV-inactivated HSV-1) into the dorsal side of the right ear as described in Materials and Methods. Each bar represents the mean ± standard error (error bars) for five mice per group. *, significantly different from the values for MI and M2 controls (P <.5 by the Student t test). phocytes from mice vaccinated three times with each glycoprotein. Assays were done with lymphocytes pooled from three mice per group 21 days after the last vaccination and stimulated in vitro with UV-inactivated HSV-1 as described in Materials and Methods. BALB/c CL7 target cells were infected with 1 PFU of HSV-1 (KOS) per cell. gb, gc, gd, and gi all had significant killer cell activity (compared with values for the Ml and M2 controls) at all effector-to-target ratios tested (Fig. 6) (P <.1 for gb, gc, and gd and P <.5 for gi by the Student t test). The highest levels were seen with gb and gd. ge induced significant killer cell activity at a ratio of 1:1 (P <.5) but not at a ratio of 5:1 or 25:1 (P >.5). gg and gh did not induce any significant killer cell activity (P >.1). DISCUSSION HSV-1 glycoproteins play vital roles in the infection process and in viral pathogenesis (48), including virus attachment (13), penetration (14, 28, 45), egress from infected cells, and mem ~~~~~~~ * 4 36 e * T CX 16 T T X 2I I I 1 I ITiITiI* IiT 12 4 i I_I Mt gb gc gd ge gg gh gi M2 FIG. 5. Lymphocyte proliferation in mice vaccinated with baculovirus-expressed glycoproteins. Mice were vaccinated three times, spleens were harvested, single-cell suspensions of splenocytes were primed by infecting them with 1.5 PFU of UV-inactivated HSV-1 per cell and culturing them for 72 h, and the cells were labeled with [33HJthymidine for 18 h as described in Materials and Methods. Each bar represents the mean ± standard error for seven replicates. Solid bars, primed cells; hatched bars, unprimed cells. *, significant compared with values for mock-vaccinated controls (P <.5 by the Student t test). gb gc gd ge gg gh gi Mt M2 FIG. 6. Killer cell activity induced by individual baculovirus-expressed glycoproteins as measured by a 5'Cr release assay. Mice were vaccinated three times, and 5'Cr release assays were done with in vitro-primed splenocytes as effector cells and HSV-1-infected CL7 cells as target cells as described in Materials and Methods. Spontaneous release was <3% of total release. Effector-to-target-cell ratios were 1:1 (solid bars), 5:1 (open bars), and (25:1) (hatched bars). Each bar represents the mean ± standard error (error bars) for at least eight replicates. *, significantly different from the values for the corresponding MI and M2 controls (P <.1 for gb, gc, and gd and P <.5 for gi and ge by the Student t test).

6 VOL. 68, 1994 VACCINATION WITH SEVEN HSV-1 GLYCOPROTEINS 2123 brane fusion and envelopment (24, 4). HSV-1 glycoproteins are also important inducers and targets of humoral and cell-mediated immune responses following infection (7, 44, 48). There is a vast literature on the immune response to HSV, on vaccination against HSV, and on the correlation of immune responses and protection. The subject is a confusing one since different workers have concluded that different mechanisms, complement-independent neutralization (5, 11, 26), complement-dependent neutralization (26), class I-mediated (41) or class I1-mediated (8) cytotoxicity, DTH (42, 43), antibodydependent cellular cytotoxicity (32), or cytokine-mediated responses (9, 49), are the most important immune responses for protection or recovery. It is clear that different results have been obtained depending on the vaccine used, the challenge virus and route used, the major histocompatibility complex mouse background, and the method used to measure protection. Thus, interpretation of our results should be qualified by the understanding that although they are valid for the particular experimental system used here, confirmation of these results in other systems awaits additional studies. To examine in a single system the ability of individual HSV-1 glycoproteins used as vaccines to protect against HSV-1 challenge, and to evaluate the humoral and cell-mediated immune responses associated with each glycoprotein, we have expressed gb, gc, gd, ge, gg, gh, and gi in a baculovirus expression system. By expressing these seven glycoproteins in the same system, we not only made them all available for these studies but also ensured that any differences detected would not be due to the mode by which the glycoproteins were expressed. We have previously shown that each of these seven expressed glycoproteins is glycosylated, biologically functional, and highly immunogenic (15-21). We have also previously shown that vaccination with gb, gc, gd, ge, or gi provided good protection against lethal i.p. challenge, while gg provided partial protection against i.p. challenge and gh provided no protection against i.p. challenge. Using these seven baculovirus-expressed glycoproteins, we show here for the first time that mice vaccinated with gb, gc, gd, ge, or gi were also well protected against lethal ocular challenge. Also in agreement with our previous i.p. challenge results, mice vaccinated with gh were not protected against ocular challenge. In contrast to our i.p. results, gg did not show significant protection against ocular challenge. This may indicate that the protective effect of vaccination with gg differs in accordance with the route of challenge. Alternatively, it is possible that this lack of statistical significance was due to the smaller number of mice used here than in our i.p. studies. We also found that mice vaccinated with gb, gc, gd, ge, or gi were significantly protected against the establishment of latency and that they produced high neutralizing antibody titers to HSV-1. In contrast, gg- and gh-vaccinated mice were not protected against the establishment of latency, and only low or no neutralizing antibody titers were induced. To determine whether any immunological responses in addition to high neutralization titers could be correlated with protection against lethal ocular challenge and the establishment of latency, we examined several additional immunological parameters, including DTH, lymphocyte proliferation, and killer cell activity. DTH responses have been suggested to play a major role in recovery from HSV cutaneous infections by releasing lymphokines which in turn stimulate nonspecific effector cells (42). In this study, we did not detect any obvious correlation between DTH responses and protection against ocular challenge. No DTH response could be detected in gd- or gb-vaccinated mice, even though such mice had excellent protection against lethal challenge. In addition, gh and gg both induced highlevel DTH responses within 2 to 3 days, despite providing no protection against ocular challenge. Lymphocyte proliferation induction was observed following vaccination with each of the seven glycoproteins, including those that provided no protection against lethal challenge. Thus, no correlation was seen between protection and lymphocyte proliferation. In fact, the greatest lymphocyte proliferation response was in gg-vaccinated mice, which were not protected against lethal ocular challenge. Killer cell activity was seen in mice vaccinated with gb, gc, gd, or gi at all effector-to-target-cell ratios and in those vaccinated with ge at a ratio of 1:1. No activity was induced by gg or gh. As with neutralization titers, these results may correlate with protection against lethal ocular challenge, since the better-protected mice appeared to have the higher killer cell activities. However, at present, we do not know whether the killer cell activity we measured is due to (i) CD4+ cytotoxic T lymphocytes (2), (ii) CD8+ cytotoxic T lymphocytes (33), (iii) natural killer cells (6), or (iv) a combination of these factors. An interesting observation, unrelated to the relative protection afforded by the individual HSV-1 glycoproteins, is that there appeared to be a difference between the mock-vaccinated controls. In all assays except for those measuring DTH levels, the wild-type baculovirus (Ml)-vaccinated control mice appeared to have a higher level of protection or a higher antibody titer than the PBS (M2)-vaccinated mice. This difference was statistically significant for lethal ocular challenge (P =.2 by Fisher's exact test) and killer cell activity (P =.2 by the Student t test) and was almost statistically significant for lymphocyte proliferation (P =.54 by the Student t test). We do not know the reason for these differences. However, they are likely due either to cross-reactivity between wild-type baculovirus-infected Sf9 cells and HSV-1 or to a nonspecific adjuvant effect of Freund's adjuvant (and/or the baculovirusinfected cells). A summary of our results is shown in Table 2. If for the sake of argument the assumption is made that the effect of each type of immune response was independent of the glycoprotein it was directed against, then it would appear that production of neutralizing antibody was the most important immune response in protecting mice against lethal ocular or i.p. challenge and the establishment of latency. The rationale is as follows. DTH was not important since gb and gd, both of which provided high levels of protection, had no significant DTH. Killer activity was not crucial, since ge, which had a moderately high level of protection, had killer cell activity only at the highest effector-to-target-cell ratio. Lymphocyte proliferation titers were induced by all glycoproteins, including those that did not protect, and therefore were not important. This leaves only neutralization, the importance of which is further supported by the high neutralization titers induced by the five glycoproteins that protected well and the nonsignificant titers induced by the two glycoproteins that did not protect. The above argument is, of course, overly simplistic. It is likely that all the immune responses described above play roles. In particular, if the "important" response is missing, another response may become more consequential. Thus, since gg did not induce any neutralization titer, the partial protection it provided against i.p. challenge may have been the result of either a high-level DTH response or a high-level lymphocyte proliferation response, neither of which appeared important in

7 2124 GHIASI ET AL. J. VIROL. Antigen or control TABLE 2. Summary of protection and immune responses in mice vaccinated with recombinant baculoviruses expressing each of the seven expressed HSV-1 glycoproteinsa Protection against HSV-1 by challenge route Value for immune response Ocular Latency Neutralization DTH Lymphocyte Killer cell i.p. Ocular ~protection NetaiainDHproliferation activity gb gc gd ge gg gh _ gi ++ KOS Ml M a , very high level of protection or a very high immune response; + + +, high protection or a high response; + +, moderate protection or a moderate response; +, small amount of protection or a small response; -, no protection or no immune response. the presence of high neutralizing antibody titers. In addition, gc, ge, and gi, all of which induced lower neutralizing titers than gd and gb, may have required one or more additional immune responses in order to have afforded the high level of protection seen. It is also overly simplistic to assume that all specific immune responses are totally independent of the glycoproteins they are directed against. Thus, although the overall patterns of immune response were similar for gg and gh (minimal neutralization, high-level DTH, significant lymphocyte proliferation, and no killer cell activity), gg was able to afford significant protection against i.p. challenge while gh produced no protection. Another complication in the comparative analysis of the abilities of the HSV-1 glycoproteins to protect against challenge is that three of the glycoproteins (gc, ge, and gi) are known to interact with specific components of the immune system. gc can bind the C3b fragment of the third component of complement (46, 47). ge and gi form a complex that can bind the Fc portion of immunoglobulin G (1, 31). The binding of these glycoproteins to C3b and Fc may block some functions requiring Fc (1, 52) or C3b (27, 39) recognition. This may result in modulation of the immune response to HSV-1 infection, providing the virus with a means to partially or temporarily escape from some aspects of immune attack (27, 39, 52). This possibility is supported by our observation (data not shown) that some mice vaccinated with gc, ge, or gi showed significant signs of viral infection early after lethal HSV-1 challenge but nevertheless recovered fully. On the basis of the analyses presented here, these seven HSV-1 glycoproteins can be classified into three categories: (i) gb and gd, giving excellent protection against lethal challenge and establishment of latency, high neutralizing antibody titers, and no DTH; (ii) gc, ge, and gi, giving very good protection against lethal challenge and establishment of latency, moderately high neutralizing antibody titers, and high-level DTH; and (iii) gg and gh, giving little or no protection, minimal neutralizing antibody titers, high-level DTH, and no killer cell activity. 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