International Immunology

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International Immunology, Vol. 11, No. 11, 1763-1773, November 1999
© 1999 Japanese Society for Immunology

Protective immune correlates can segregate by vaccine type in a murine herpes model system

Jeong-Im Sin, Velpandi Ayyavoo, Jean Boyer, Jong Kim, Richard B. Ciccarelli and David B. Weiner

Department of Pathology and Laboratory Medicine, 505 Stellar-Chance Lab, University of Pennsylvania, 422 Curie Drive, Philadelphia, PA 19104, USA
¹ WLV, Malvern, PA 19355, USA

Correspondence to: D. B. Weiner

	Abstract

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A central tenet of vaccine development is to identify immune correlates of protection. Both plasmid-encoded gD as well as recombinant protein gD can protect mice from lethal herpes simplex virus (HSV) challenge. It is known that different vaccine modalities should induce different immune phenotypes. Yet, paradoxically, it is also thought that the basis for protection should rely on exploitation of vulnerabilities of the pathogen and therefore that the overlapping properties of these different vaccines would reveal insight into common immune mechanisms responsible for protection. We sought to investigate this question by comparing two different vaccine modalities in the HSV-2 mouse model. We observed that gD protein was a strong inducer of T_h2-type immune responses, and overall antibody titers of IgG, IgE and IgA were significantly higher than those induced by plasmid gD vaccines. In contrast, the plasmid gD vaccine induced a strong T_h1 bias. Following high-dose challenge the gD protein was most effective at providing protection. However, at lower lethal dose challenge, while both vaccines were protective with regards to survival, only the plasmid-vaccinated animals were protected from HSV-2 infection-induced morbidity. These studies suggest that these different vaccine modalities induce protection through unique non-overlapping mechanisms, supporting that vaccine correlates are associated with the types of immunogen rather than solely the pathogen.

Keywords: DNA vaccine, herpes simplex virus infection, protective correlates, subunit vaccine, T_h1, T_h2

	Introduction

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Herpes simplex viruses (HSV) type 1 and 2 are large DNA viruses which can cause cold sores, ocular infections, encephalitis and genital inflammation in humans (1). The surface antigens have been the focus for development of a prophylactic immunogen for this infectious agent. Among HSV surface glycoproteins, gB, gC, gD, gI, gE and gH-I as subunit (purified protein) vaccines have been reported to induce partial or complete protection from lethal herpes challenge in animals (2–7). For example, immunizing animals with recombinant gD protein emulsified in Freund's adjuvant provides complete protection against lethal challenge with either HSV-1 or HSV-2 in mice (7). However, clinical trials with gD subunit vaccines appear problematic for inducing protection from HSV infection in human (8). Such studies underscore the need for a greater understanding of the basis for protective immunity which could aid in improving current experimental vaccine strategies for this pathogen.

DNA vaccination has been a recent addition to immunization techniques. Direct delivery of plasmid vectors in vivo has induced immune responses in multiple pathogenic systems (9–21). Similarly, this technique has induced immune responses in primates including humans (22–24). To date, DNA vaccines expressing gB, gD and ICP27 of HSV-2 have been reported to provide protective immunity against lethal HSV challenge in animals (16,17,25–27). Furthermore, we recently reported that co-injection with gD DNA vaccine plus T_h1-type cytokine cDNAs reduces both mortality and morbidity due to HSV-2 infection, whereas co-injection with T_h2-type cytokine cDNAs results in increased susceptibility to viral infection (28). Thus, direction and magnitude of specific immune responses appear related to protection in this model.

It has been generally believed that protective correlates are unique to a pathogen and that determining those correlates is critical for developing improved vaccines. However, the protective correlates or immune mechanisms could be at least partially dependent upon vaccine types rather than the pathogen. In this circumstance, individual vaccines would have unique correlates that may not be clearly generalized to other vaccine modalities. In an effort to clarify this important issue, we directly compared the protective correlates elicited by a DNA versus a protein vaccine in the well-established HSV mouse model. Here we observe that protective correlates are directed by both vaccine types through apparently different mechanisms. DNA vaccine induced T_h1-type cellular responses which played a major role in reducing both mortality and also morbidity following challenge. In contrast, subunit vaccine induced T_h2-type cellular and strong humoral responses which were focused on reducing mortality from early viral infection, but played a more limited role in reducing morbidity. These data support that correlates of protection are not uniquely transferable from one vaccine type to another.

	Methods

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Mice
Female BALB/c mice, 4–6 weeks old, were purchased from Harlan Sprague-Dawley (Indianapolis, IN). Their care was under the guidelines of the National Institutes of Health (Bethesda, MD) and the University of Pennsylvania Institutional Animal Care and Use Committee (Philadelphia, PA).

Reagents
HSV-2 strain 186 (a kind gift from P. Schaffer, University of Pennsylvania, Philadelphia, PA) was propagated in the Vero cell line (ATCC, Rockville, MD). The DNA vaccine pAPL-gD2 (pgD) encoding HSV-2 gD protein was as previously described (27). Plasmid DNA was produced in bacteria and purified by double-banded CsCl preparations. Recombinant HSV-2 gD proteins, a generous gift from G. H. Cohen and R. J. Eisenberg (University of Pennsylvania, Philadelphia, PA), were used as recombinant antigens in these studies.

Protein and DNA immunization of mice
For DNA immunization, the quadriceps muscles of BALB/c mice were injected i.m. with 60 µg of pgD DNA constructs formulated in a final volume of 100 µl of PBS and 0.25% bupivacaine–HCl (Sigma, St Louis, MO) using a 28-gauge needle (Becton Dickinson, Franklin Lakes, NJ). For subunit vaccine immunization, mice were first vaccinated i.p. with 1 µg of gD proteins emulsified with 100 µl of complete Freund's adjuvant. In the second vaccination, mice were immunized i.p. with 1 µg of gD proteins emulsified with 100 µl of incomplete Freund's adjuvant.

ELISA
HSV-2 gD protein (0.75 µg/ml in PBS) was adsorbed onto 96-well microtiter plates. After blocking the plates for 1 h with blocking buffer (PBS/0.05% Tween X/2% BSA), sera were serially diluted to a final volume of 50 µl in PBS and the plates were incubated at 37°C for 1 h. After addition of anti-murine IgG conjugated with horseradish peroxidase (HRP), the plates were incubated for 1 h at 37°C. For the determination of IgA levels, anti-murine IgA–HRP (Zymed, San Francisco, CA) was substituted for anti-murine IgG–HRP (Sigma). For the determination of IgE levels, rat IgG (Zymed) raised against murine IgE and anti-rat IgG–HRP (Sigma) were substituted for anti-murine IgG–HRP. For the determination of relative levels of gD-specific IgG subclasses, anti-murine IgG1, IgG2a, IgG2b or IgG3 conjugated with HRP (Zymed) were substituted for anti-murine IgG–HRP. This was followed by addition of the ABTS substrate solution (Chemicon, Temecula, CA). In each step, plates were washed 3 times with the wash buffer (PBS/0.05% Tween X). The plates were read on a Dynatech MR5000 plate reader with the optical density at 405 nm. ELISA titer was determined as the reverse of highest sera dilution showing similar OD values to naive mice.

Th cell proliferation assay
Spleens were aseptically removed from each group, weighed and pooled together. Lymphocytes were harvested from spleens, and prepared as the effector cells by removing the erythrocytes and washing several times with fresh media (RPMI 1640 supplemented with 10% FCS). The isolated cell suspensions were resuspended to a concentration of 1x10⁶ cells/ml. A 100 µl aliquot containing 1x10⁵ cells was immediately added to each well of a 96-well microtiter flat-bottom plate. HSV-2 gD protein at the final concentration of 1 and 5 µg/ml was added to wells in triplicate. The cells were incubated at 37°C in 5% CO₂ for 3 days. [³H]Thymidine (1 µCi) was added to each well and the cells were incubated for 12–18 h at 37°C. The plate was harvested and the amount of incorporated [³H]thymidine was measured in a Beta Plate reader (Wallac, Turku, Finland). Stimulation index was determined from the formula: stimulation index = (experimental count – spontaneous count)/spontaneous count. Spontaneous count wells include 10% FCS which serves as irrelevant protein control. To assure that cells were healthy, 5 µg/ml phytohemagglutinin (PHA; Sigma) was used as a polyclonal stimulator positive control.

T_h1- and T_h2-type cytokines, and chemokines
A 1 ml aliquot containing 6x10⁶ splenocytes was added to wells of 24-well plates. Then, 1 and 5 µg of HSV-2 gD protein/ml was added to each well. After 2 days incubation at 37°C in 5% CO₂, cell supernatants were secured and then used for detecting levels of IL-2, IL-10, IFN-, RANTES (regulated on activation, normal T cell expressed and secreted), monocyte chemotactic protein (MCP)-1 and macrophage inflammatory protein (MIP)-1 using commercial cytokine kits (Biosource, Camarillo, CA and R & D Systems, Minneapolis, MN) by adding the extracellular fluids to the cytokine- or chemokine-specific ELISA plates.

Intravaginal (i.vag.) HSV-2 challenge
After DNA injection, mice were challenged i.vag. with HSV-2 strain 186 at lethal doses previously determined (28). Before inoculating the virus, the i.vag. area was swabbed with a cotton-tipped applicator (Hardwood, Guiford, ME) soaked with 0.1 M NaOH solution and then cleaned with dried cotton applicators. Mice were then examined daily to evaluate pathological conditions and survival rates.

	Results

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Systemic humoral immune responses
We compared serology induced by gD recombinant or plasmid vaccine. gD protein (1 µg) emulsified in Freund's adjuvant was used as this dose was previously reported to induce gD-specific antibody in 100% of vaccinated mice and complete protection from lethal HSV challenge (7). In contrast, 60 µg of gD DNA vaccine dissolved in bupivacaine solution was used because the concentration tested generated seroconversion in 100% mice (Fig. 1) and complete protection from lethal HSV challenge (28). As shown in Fig. 2, gD subunit vaccine induced systemic IgG, IgA, and IgE responses significantly higher than gD DNA vaccine alone. ELISA titers of gD DNA vaccine and gD subunit vaccine were determined to be 6400 and 640,000, respectively or 2 log difference. When isotypes of gD-specific IgG were compared, a dramatic phenotypic difference was noted. gD subunit vaccine resulted in significantly increased production of IgG1 isotypes, whereas gD DNA vaccine showed increased induction of antigen-specific IgG2a isotypes (Fig. 3). Similarly, the relative ratio of IgG1 to IgG2a isotypes was determined to be 4.4 (gD subunit vaccine) and 1.4 (gD DNA vaccine) (Fig. 3C). This data is supportive of the notion that DNA vaccine is T_h1-biased and that subunit vaccine is T_h2-biased in this model.

View larger version (23K): [in this window] [in a new window]   Fig. 1. gD-specific IgG production levels over a different dose of gD DNA vaccines. Each group of mice (n = 4) was immunized i.m. with 0.6, 6 and 60 µg of gD DNA vaccines per mouse at 0 week. Mice were bled bi-weekly and sera were diluted to 1:50 for reaction with gD. Absorbance (OD) was measured at 405 nm. The assay was performed in triplicate. Values and bars represent mean and SD. *Statistically significant at P < 0.05 using the paired Student's t-test compared to naive control. , naive; x, pgD, 0.6 µg; pgD, 6 µg; , pgD, 60 µg.

 

View larger version (21K): [in this window] [in a new window]   Fig. 2. Levels of systemic gD-specific antibody (IgG, IgA and IgE) in mice immunized with DNA and subunit vaccines. Each group of mice (n = 10) was immunized with 60 µg of gD DNA vaccine or 1 µg of gD subunit vaccine per mouse at 0 and 2 weeks. Mice were bled 2 weeks after the second immunization, and then equally pooled sera were diluted to 1:1000 and 1:10,000 for reaction with gD. Absorbance (OD) was measured at 405 nm. The assay was performed in triplicate. Values and bars represent mean and SD. *Statistically significant at P < 0.05 using the paired Student's t-test compared to naive control. **Statistically significant at P < 0.05 using the paired Student's t-test compared to gD DNA vaccine alone. Open columns, sera dilution, 1:1,000; filled columns, sera dilution, 1:10,000.

 

View larger version (20K): [in this window] [in a new window]   Fig. 3. IgG isotype class switch in mice immunized with DNA and subunit vaccines. Each group of mice (n = 10) was immunized with 60 µg of gD DNA vaccines or 1 µg of gD subunit vaccines per mouse at 0 and 2 weeks. Mice were bled 2 weeks after the second immunization, and then sera were diluted to 1:100 for DNA vaccines and to 1:1,000 for subunit vaccines, respectively. Absorbance (OD) was measured at 405 nm. Relative OD value was calculated as each IgG subclass OD/total OD value. (A) Relative OD levels of mice immunized with gD subunit vaccine alone. (B) Relative OD levels of mice co-immunized with gD DNA vaccine. The relative ratio of IgG1 to IgG2a is shown in (C). Line bars represent the mean (n = 10) of relative OD values of each mouse IgG subclass. *Statistically significant at P < 0.05 using the paired Student's t-test compared to gD protein.

 
Cellular immune responses
T_h cells play an important role in eliciting both humoral and cellular immune responses via expansion of antigen-stimulated B cells and expansion of CD8⁺ T cells respectively. Furthermore, the production patterns of T_h1 cytokines (IL-2 and IFN-) and T_h2 cytokines (IL-4, IL-5 and IL-10) have been a mainstay in our understanding of the polarization of immune responses. We next compared the T_h phenotype induced. As shown in Fig. 4(A), gD subunit vaccine induced T_h cell proliferative responses to gD of greater magnitude than the gD DNA vaccine. We previously reported that gD plasmid vaccination does not result in cytotoxic T lymphocyte (CTL) responses due to a lack of CTL epitope in the BALB/c background (49). However, to evaluate cellular effects in more detail we next investigated whether two immunogens induce T_h1- and/or T_h2-type cytokines. As shown in Fig. 4(B and C), gD DNA vaccine induced production of IL-2 and IFN- after stimulation of splenocytes in vitro, with gD significantly higher than gD subunit vaccine. The gD subunit vaccine induced higher production of IL-10 from splenocytes, as compared to DNA vaccine (Fig. 4D). This data further supports that DNA vaccine is T_h1-biased and that subunit vaccine is T_h2-biased.

View larger version (28K): [in this window] [in a new window]   Fig. 4. Th cell proliferation and cytokine production levels of splenocytes after in vitro stimulation with gD proteins. (A) Each group of mice (n = 2) was immunized with 60 µg of gD DNA vaccines or 1 µg of gD subunit vaccines per mouse at 0 and 2 weeks. Two weeks after the second injection, two mice were sacrificed and spleen cells were pooled. Splenocytes were then stimulated with 1 and 5 µg/ml gD-2 proteins, and as a positive control with 5 µg/ml PHA. After 3 days stimulation, cells were harvested and then c.p.m. was counted. Samples were assayed in triplicate. The PHA control sample showed a stimulation index of 50–60. (B–D) Spleen cells were stimulated with 1 and 5 µg of gD-2 proteins/ml for 2 days, and then cell supernatants were measured for IL-2, IL-10 and IFN-. Samples were assayed in triplicate. This was repeated at least 3 times with similar results. Values and bars represent mean ± SD. *Statistically significant at P < 0.05 using the paired Student's t-test compared to gD DNA vaccine. Open columns, gD protein, 1 µg/ml; filled columns, gD protein, 5 µg/ml.

 
Chemokine production
CC-type chemokines including RANTES, MIP-1 and MCP-1 chemoattract particularly monocytic phagocytes, and activate T cells, basophils, eosinophils and mononuclear phagocytes (29,30). As compared to MCP-1, RANTES and MIP-1 were also reported to be a major HIV suppressive factor (31). We next compared the levels of chemokines (RANTES, MCP-1 and MIP-1) induced by these vaccine types. As shown in Fig. 5, gD DNA vaccine induced production of RANTES and MIP-1 after stimulation of splenocytes in vitro with gD significantly higher than gD subunit vaccine. However, the gD subunit vaccine induced higher production of MCP-1 from splenocytes, as compared to gD DNA vaccine. Background levels of MIP-1, MCP-1 and in particular RANTES, which has a higher background activity, were similar to prior published work (49).

View larger version (15K): [in this window] [in a new window]   Fig. 5. Production levels of RANTES, MIP-1 and MCP-1 from splenocytes in mice immunized with DNA and subunit vaccines. Each group of mice (n = 2) was immunized with 60 µg of gD DNA vaccines or 1 µg of gD subunit vaccines per mouse at 0 and 2 weeks. Two weeks after the second injection, two mice were sacrificed and spleen cells were pooled. Splenocytes were stimulated with 1 µg of gD-2 proteins/ml for 2 days. Samples were assayed in triplicate. This was repeated at least 3 times with similar results. Values and bars represent mean of released chemokine concentrations + SD. *Statistically significant at P < 0.05 using the paired Student's t-test compared to gD DNA vaccine.

 
Mortality rate of gD protein versus gD plasmid
To determine how gD DNA and subunit vaccines influence survival of animals from lethal challenge with HSV-2, animals were immunized with these immunogens and challenged i.vag. with 4, 200 and 400 LD₅₀ of HSV-2 (186). The high lethal dose of HSV-2 was chosen to investigate differences in survival rates among all vaccinated animals. Furthermore, the i.vag. infection route was chosen because HSV-2 infects mucocutaneously, and results in genital infection and quantifiable lesions. Figure 6 shows that gD subunit vaccine resulted in complete protection from challenge with 200 and 400 LD₅₀ of HSV-2, while gD DNA vaccine showed 63 and 34% survival of mice respectively. However, both vaccine types generated 90–100% survival of mice at the challenge dose of 4 LD₅₀ of HSV-2 (Fig. 7A).

View larger version (24K): [in this window] [in a new window]   Fig. 6. Survival rates of mice immunized with DNA and subunit vaccines. Each group of mice (n = 8–10) was immunized with 60 µg of gD DNA vaccines or 1 µg of gD subunit vaccines per mouse at 0 and 2 weeks. Three weeks after the second injection, each group of mice was challenged i.vag. with 200 LD50 (7x105 p.f.u.) (A) and 400 LD50 (1.4x106 p.f.u.) (B) of HSV-2. Surviving mice were scored for 60 days post viral challenge. *Statistically significant at P < 0.05 using ANOVA compared to gD DNA vaccine.

 

View larger version (16K): [in this window] [in a new window]   Fig. 7. Mortality and morbidity of mice immunized with DNA and subunit vaccines. Each group of mice (n = 8–10) was immunized with gD DNA vaccines (60 µg per mouse) or protein (gD, 1 µg per mouse) at 0 and 2 weeks. Three weeks after the second injection, mice were challenged i.vag. with 4 LD50 (1.4x104 p.f.u.) of HSV-2 (186). (A) Surviving mice were counted for 30 days post viral challenge. (B) Surviving mice after viral challenge were examined daily to observe the pathological symptoms. Values represent the number of mice showing lesions/the total number of mice surviving HSV infection. , naive, n = 8; , gD DNA, n = 9; , gD protein, n = 10.

 
Morbidity rate of gD protein versus gD plasmid
In addition to direct mortality, HSV-2 challenge can result in significant and easily detected lesions, paralysis and scarification following i.vag. challenge. To investigate effectiveness of subunit and DNA vaccines on reducing morbidity resulting from HSV-2 challenge, the HSV-2-challenged animals were checked daily and scored for herpetic lesions. Herpetic lesions can be observed on the epithelial layers of the skin around the vaginal area. To determine morbidity of animals after viral challenge, mice were challenged i.vag. with a lethal dose (4 LD₅₀) of HSV-2. As shown in Fig. 7(B), herpetic lesions and pathological symptoms were observed in 56–70% of mice immunized with subunit vaccine over the time period of observation. In contrast, no or 11% mice immunized with DNA vaccine showed only insignificant signs of morbidity resulting from HSV-2 infection. When herpetic lesions were scored, the magnitude of subunit vaccine-immunized animals showed severe lesion formation, whereas DNA-vaccinated animals showed little if any lesion formation (data not shown). Similarly, when animals were first challenged i.vag. with a sub-lethal dose of HSV-2 and secondly challenged with a lethal dose (40 LD₅₀) of HSV-2 after 13 days following the first viral challenge, gD DNA vaccine showed no lesions (Fig. 8). However, 60–70% mice immunized with gD subunit vaccines alone showed severe lesions over the same time period of observation.

View larger version (18K): [in this window] [in a new window]   Fig. 8. Mortality and morbidity of mice immunized with DNA and subunit vaccines. (A) Schematic diagram of DNA and subunit immunization and HSV-2 challenge. Ten mice per group were immunized with gD DNA vaccines (60 µg per mouse) or protein (gD, 1 µg per mouse) at 0 and 2 weeks. Three weeks after the second injection, mice (n = 10) were challenged i.vag. with sublethal dose of HSV-2 186 strain and then rechallenged at 13 days following the first viral challenge with 40 LD50 (1.4x105 p.f.u.) of HSV-2 (186). (B) Surviving mice after viral rechallenge were examined daily to observe the pathological symptoms. Values represent the number of mice showing lesions/the total number of mice surviving HSV infection. (C) Degrees of severity of herpetic lesions were recorded as tiny lesions (1+), mild to less severe lesions (2+), severe lesions (3+) and paralysis (4+). Values and bars represent the mean of degrees of severity ± SD respectively. *Statistically significant at P < 0.05 using ANOVA compared to gD DNA vaccine. , naive, n = 3; x, gD DNA, n = 10; , gD protein, n = 10.

 

	Discussion

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In designing HSV vaccines, mortality has been considered to be a less important factor since normal individuals are not susceptible to death by HSV infection. Efforts have been focused on the control of HSV-derived morbidity and recurrent infections. HSV is the causative agent of a spectrum of human diseases, such as cold sores, ocular infections, encephalitis and genital infections (1). HSV can establish viral latency with frequent reoccurences in the host (32). During the viral infection, neutralizing antibody inactivates viral particles, but is unable to control intracellular HSV infection (33). Rather, antibody-dependent complement-mediated and antibody-dependent cell-mediated cytotoxicity are both involved in cytolysing HSV-infected cells (33–35). However, cellularmediated immunity is a major effector function which kills HSV-infected cells (25,33,36–38). From animal studies, it has been suggested that humoral and cellular immune responses are both involved in controlling HSV infection (36,39–41). For example, adoptive transfer of antibodies raised against HSV antigens provided animals complete protection from following lethal HSV challenge (40). However, some patients with a high titer of HSV-specific antibodies still develop recurrent HSV infection and particularly the incidence is more common when cellular immune responses are suppressed (42,43). Similarly, the ability of B cell-suppressed mice to control primary HSV infection (44) or the ability of adoptively transferred T cells to prevent subsequent viral infection (36) further suggests that cell-mediated immunity might be directly related to inhibition of viral infection and its spread. It has also been well documented that both CD4⁺ and CD8⁺ T cells are involved in prevention of HSV infection (45,46). Furthermore, there have been several reports (including ours) that T_h1-type CD4⁺ T cells play a more crucial role for protection from HSV-2 challenge (25,45,47–49).

In this study, gD subunit vaccine emulsified in Freund's adjuvant induced humoral immune responses (IgG, IgA and IgE) and T_h cell proliferative responses significantly higher than equivalent gD DNA vaccines. This demonstrates that subunit vaccines are superior to DNA vaccine in inducing humoral responses. Antibody phenotypes were differentially altered by the different immunogens. In particular, gD subunit vaccines showed significantly higher production of gD-specific IgG1 isotype, as compared to IgG2a type. However, gD DNA vaccine displayed increased production of IgG2a isotype, as compared to gD subunit vaccines. The relative ratio of IgG1 to IgG2a of gD subunit vaccine and gD DNA vaccine was determined to be 4.4, and 1.4 respectively, i.e. a >3-fold spread. This demonstrates that DNA vaccines elicit T_h1 phenotypic changes, whereas subunit vaccines induce T_h2 phenotypic responses. Our data is in line with other observations in different experimental systems (50).

We also observed that gD proteins are more effective at inducing T_h cell proliferative responses than gD plasmid. Moreover, cytokine production levels were altered by these vaccine types. The gD DNA vaccine enhanced both IL-2 and IFN- secretion, whereas gD subunit vaccines showed minimal effects on production of these T_h1-type cytokines. However, IL-10 levels were enhanced most significantly by gD subunit vaccines. This confirms the notion that DNA vaccines induce T_h1-type cellular immune responses while subunit vaccines induce T_h2-type humoral immune responses. It appears that DNA vaccine delivery was capable of activating T cells which generate T_h1-type cytokines (IL-2 and IFN-) but that gD protein was capable of activating T cells which generate a T_h2-type cytokine profile. The role of chemokines as factors in the T_h1 versus T_h2 responses has recently been reported (51,52). Here again we observed immune polarization. gD plasmid vaccination enhanced the production of RANTES and MIP-1 in an antigen-specific fashion in contrast to splenocytes taken from animals vaccinated with gD protein. In contrast, the MCP-1 production pattern dramatically correlated with protein immunization. Furthermore, protein vaccine may have affected RANTES production to below the level produced from naive mouse splenocytes, further suggesting an interesting effect of protein vaccine on immune cells. Recently, ocular inflammatory disease mediated by HSV infection was suppressed by topical administration of T_h2-type cytokine protein (IL-10). This application resulted in suppressed chemokine production (53). The disease (inflammation in the eye) was also ameliorated by injection with anti-MIP-1 but not anti-MCP-1, indicating that MIP-1 again segregates as a T_h1-type chemokine. These studies imply that DNA and protein vaccine utilize different mechanisms of activating T cells which result in a unique cytokine, chemokine and isotype profile as well as magnitude shifts in immune responses of both serology as well as the magnitude of the proliferation response.

The mortality profiles induced by both types of vaccines were positive as the both provided 90–100% protection from infection with 4 LD₅₀ of HSV-2 strain 186. However, gD DNA vaccine showed 63% survival rates at the challenge inoculum of 200 LD₅₀ of HSV-2, whereas gD subunit vaccines resulted in complete protection. At the dose of 400 LD_50, 38% survival rates were noted by gD DNA vaccination while complete protection was again observed by gD subunit vaccination. Taken together, it seems that a high amount of gD-specific antibodies induced by subunit vaccine are likely responsible for complete survival from high lethal challenge with HSV-2. Of interest, most mortality was observed within 7–8 days following high lethal challenge, supporting that early protection from HSV is again antibody mediated.

In the case of morbidity, we observed that gD DNA vaccines resulted in no or significantly less mice presenting herpetic lesions, whereas gD subunit vaccines showed a significantly higher number of mice (56–70%) displaying lesions. The herpetic lesions were observed to be far more severe after subunit vaccination while insignificant lesions were noted following DNA immunization. This difference in morbidity rates between DNA and subunit vaccinated groups was also observed when mice were challenged first and rechallenged a second time with HSV-2, which might be of some relevant to recurrent HSV infection in humans. We also reported that T_h1-type cytokine (most significantly IL-12)-mediated cellular immune responses play a critical role in reducing the emergence of herpetic lesions and in enhancing the recovery time from the lesions (28). In particular, IL-12 co-injection inhibited antibody production significantly lower than gD DNA vaccine alone, but enhanced survival from challenge with HSV-2. This suggests that antigen-specific cellular immune responses could be enhanced by co-injection with T_h1-type cytokine. In this study, we observed that gD DNA vaccines induce significantly higher production of IFN-, as compared to gD subunit vaccines. T_h1-type CD4⁺ T cells generate a large amount of IFN- (45). IFN- up-regulates MHC class expression on HSV-infected cells to allow better recognition by cytotoxic CD4⁺ T cells (54) and CD8⁺ CTL (38), and has a direct anti-HSV effect (55). Koelle et al. recently reported that CD4⁺ T cells infiltrating genital herpetic lesions possess cytotoxic activity against HSV-infected cells (56), underscoring the importance of T cell-mediated protective immunity against HSV infection. Furthermore, IFN- is able to enhance an NK cell activity, resulting in suppressing HSV infection in vivo and in vitro (57,58). Treatment of mice receiving HSV-specific T cells with anti-IFN- antibodies also reduces the ability to clear HSV infection in cutaneous tissue (59), indicating that IFN- is responsible in part for clearing cutaneous HSV infection. Thus, our data implies that one problematic feature of subunit vaccines with regard to HSV infection may be due to their polarization towards T_h2-type immunity, which might not be effective for preventing viral morbidity in humans.

In conclusion, protective immune correlates for HSV-2 infection could be directed by the two types of subunit and DNA vaccines: subunit vaccine-induced humoral responses (T_h2) are more related to decreased mortality in a high lethal HSV challenge, while DNA vaccine-induced cellular immune responses (T_h1) play a more major role in reducing HSV-2-derived morbidity in animals. These results suggest that these different vaccine modalities induce protection through unique non-overlapping mechanisms, supporting that protective correlates are associated with the type of immunogen. Due to the increasing diversity and complexity of immunogens it should not be a surprise to find unique correlates that appear to segregate uniquely with a particular immunogen rather than in any clear manner with the pathogen. Such a hypothesis suggests caution regarding the usefulness or failure of a novel vaccine approach based solely on initial immune profiles.

	Acknowledgments

 
We wish to thank Drs G. H. Cohen and R. J. Eisenberg for providing HSV-1, 2 gD (306t). We also thank Drs P. Schaffer and R. Jordan for providing a stock of HSV-2 (186) for this study. J.-I. S. would like to thank Mr M. Merva for his helpful technical assistance for this study. DBW is supported by grants from the NIH.

	Abbreviations

 

CTL cytotoxic T lymphocyte

HRP horseradish peroxidase

HSV herpes simplex virus

MCP monocyte chemotactic protein

MIP macrophage inflammatory protein

PHA phytohemagglutinin

RANTES regulated on activation, normal T cell expressed and secreted

	Notes

 
Transmitting editor: M. Feldmann

Received 27 February 1999, accepted 21 July 1999.

	References

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