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Role of Mucosal Immunity in Herpes Simplex Virus Infection¹

Department of Microbiology, University of Tennessee, Knoxville, TN 37996

	Abstract

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This study evaluates whether the vaginal mucosal surface of immunized mice can prevent invasion by herpes simplex virus (HSV) and aims to identify immune components that affect immunity after challenge at the vaginal mucosa. Despite the induction of both IgA and IgG vaginal Ab following immunization with recombinant vaccinia virus vectors expressing either glycoproteins B or D, viral infection occurred in most animals even after minimal viral dose challenge. Challenged immune animals, including those genetically unable to generate anti-HSV Ab, survived and showed few if any clinical signs of infection. Experiments with T cell subtype knockout animals and depletion with T cell subset-specific MAb indicated that immunity following vaginal challenge was principally dependent on the function of CD4⁺ T cells. Our results indicate that anti-HSV vaccines may not provide barrier immunity at the vaginal mucosal site but may be adequate to minimize clinical expression of disease.

	Introduction

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Mucosal surfaces are vulnerable sites for pathogen invasion but if effectively protected by immunization could achieve immune exclusion. This represents an attractive prospect with agents such as herpes simplex virus (HSV)³ and HIV where successful infection sets the stage for later disease. For example, genital infection with HSV is not only a major cause of recurrent venereal disease but is also responsible for significant mortality in infants born to infected mothers (1). Currently, there is no effective HSV vaccine, although many have been advocated for use over the last two decades (2). Past vaccines against HSV were all administered systemically, and it is possible that they failed to prevent infection because of inadequate mucosal immune induction. In this report, we have addressed the issue of mucosal immunity against HSV in a mouse model system, paying particular attention to the effectiveness of barrier immunity. Our results demonstrate that in spite of high levels of vaginal Ab induced by mucosal vaccines, this is rarely sufficient to prevent viral infection. Our report adds further evidence to the concept that immunity to HSV is seldom if ever adequate to prevent viral invasion. In consequence, anti-HSV vaccines should be designed to minimize or prevent the expression of disease. As is also indicated in this report, immunity to disease following vaginal infection appears to be mainly a function of CD4⁺ T cell-mediated immunity.

	Materials and Methods

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Mice

Female BALB/c mice were purchased from Harlan Sprague-Dawley (Indianapolis, IN). B cell-deficient mice (µMT^-/-) made by targeted disruption of the membrane exon of the Ig µ-chain gene were provided By Dr. Werner Muller (Institute for Genetics, University of Cologne, Cologne, Germany) (3). Lack of IgM⁺ B cells in µMT^-/^- mice was confirmed by FACS analysis. ß₂-Microglobulin (ß₂m) knockout mice made by Dr. Oliver Smithies (University of North Carolina) were purchased from The Jackson Laboratory (Bar Harbor, ME) (4). CD4 knockout mice were made by Dr. Nigel Killen (5) (University of California, San Francisco, CA). ß₂m and CD4 knockouts were on B6/129 stock background and were backcrossed to 129 mice for four to five generations. The lack of CD4⁺ or CD8⁺ T cells in these mice was confirmed by FACS analysis.

During experimental procedures, the investigators adhered to guidelines proposed by the Committee of the Care of Laboratory Animal Resources, Commission of Life Sciences, National Research Council.

Virus

HSV strain KOS or McKrae cells were grown on monolayers of Vero cells (ATCC CCL81) and were titrated as described previously (6). Vaccinia virus expressing glycoprotein D (VgD) (7) or glycoprotein B (VgB) (8) and parent vaccinia virus vtk^- (VV) were grown on monolayers of CV1 cells as described previously (7, 8). All viruses were stored in aliquots at -80°C until used.

Immunization of mice

Four- or five-week-old female mice were immunized intranasally (i/nas) with 10⁷ pfu of VV expressing glycoprotein B or D of HSV (VgB, VgD) or VV in 20 µl volume. Some animals were given 10⁶ pfu of HSV-1 KOS live or UV inactivated (titrated before inactivation). In some experiments, the immunization was repeated twice.

Virus challenge

To synchronize the estrous cycle at the progesterone-dominated stage as described by Parr et al. (9), the mice were injected s.c. with DepoProvera (DP) (Upjohn, Kalamazoo, MI) at a concentration of 2 mg/mouse in 50 µl of distilled H₂O. Five days following DP administration, the animals were challenged with 10³, 10⁴, or 10⁷ pfu of HSV-1 McKrae in 20 µl. The mice were examined daily for vaginal inflammation, neurologic illness, and death. The severity of disease was scored 1 to 5 (0, no change; 1, mild inflammation; 2, moderate swelling; 3, severe inflammation; 4, paralysis, death) (10).

Monoclonal antibodies

For T cell depletion, anti-CD4 (G.K. 1.5 ascites fluid; ATCC TIB 20F) or anti-CD8 (2.43 ascites fluid; ATCC TIB 210) mAbs were used. The concentration of the Abs was determined by ELISA. PharMingen (San Diego, CA) reagents, namely, rat anti-mouse CD4 FITC (catalog no. 01064D), rat anti-mouse CD8-phycoerythrin (catalog no. 01045B) and as isotype controls rat IgG2a-FITC (catalog no. 11024C) and rat IgG2a-phycoerythrin (catalog no. 11025A) were used for flow cytometry analysis. Additionally, for detection of IgM⁺ cells, biotinylated affinity pure goat anti-mouse IgM (catalog no. 115-065-075) (Jackson ImmunoResearch, West Grove, PA) and streptavidin-FITC (catalog no. 13024D) (PharMingen) were used.

In vivo T cell depletion

µMT^-/- or BALB/c mice were given 10⁵ pfu of HSV-1 KOS i/nas. One group of mice received PBS and was used as negative control. Thirty days after the immunization, the mice were injected s.c. with 2 mg/mouse DP. At day 36, all animals were challenged intravaginally with 5 x 10⁶ pfu of HSV-1 McKrae (100 LD₅₀). Depleting anti-CD4 or anti-CD8 mAbs (0.5 mg/mouse) were administered i.v. on days 34, 38, and 40 (2 days before challenge, 2 and 4 days after challenge). Ten days after the third depletion, the mice were killed, spleens and vaginal draining lymph nodes were removed, and cells were pooled together and analyzed by two-color flow cytometry for CD4, CD8, and B220 markers (Consort-30 FACScan flow cytometer; Becton Dickinson, Mountain View, CA).

Antibody assays

Serum was collected by retroorbital bleeding. Vaginal washings were collected with a micropipette by introducing 100 µl of PBS into the vaginal cavity, and 50 to 70 µl were recovered per animal. The vaginal lavage sediments were subsequently removed by centrifugation. Fecal samples were weighed and suspended in PBS, 0.1% sodium azide at a concentration of 100 mg/ml. All samples were stored at -70°C until used. Ab detection was performed as previously described (6). Briefly, ELISA plates were coated overnight at 4°C with gB or gD protein, 2 µl/ml (kindly provided by Dr. Rae Lyn Burke, Chiron, Emeryville, CA) or anti-mouse IgG or IgA (Southern Biotechnology Associates (SBA). Birmingham, AL) in bicarbonate buffer. After blocking and washing the ELISA plates, standard mouse IgG or mouse IgA (SBA) was added at a concentration of 100 ng/ml to the anti-IgG- or anti-IgA-coated wells. Serum fecal or vaginal samples were added to the gB- or gD-coated wells and were subsequently diluted twofold into the ELISA plates. After 2 h of incubation at 37°C and subsequent washing, goat anti-mouse IgG- or IgA-conjugated horseradish peroxidase (SBA) was added, and plates were incubated for 1 h at 37°C. Substrate 2,2'-azino-bis(ethylenethiazoline-6-sulfonic acid) was used for color development. The quantity of Ab bound was computed from a standard curve run simultaneously (6).

Statistics

Wherever specified, data obtained were analyzed for statistical significance by Student’s t test.

	Results

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Generation of vaginal Ab after intranasal immunization with recombinant vaccine virus expressing gB and gD

Previous reports showed that i/nas exposure to wild-type HSV or to recombinant viral vectors expressing HSV proteins leads to the generation of Ab responses at both systemic and at distal mucosal sites such as the vaginal tract (11, 12). In the present report, we have used two recombinant vaccinia virus vectors expressing, respectively, gB and gD. As is evident in Table I, immunization with both vectors led to the production of high titers of glycoprotein-specific IgA and IgG Ab in vaginal washings. The Ab levels observed were of the same magnitude as occurs in vaginal washings of mice immunized i/nas with infectious virus.

Immunized mice were given progesterone to standardize their susceptibility to HSV challenge as described by Parr et al. (9). This procedure actually changes the ratio of HSV-specific IgG to IgA in animals (13). In fact, in two experiments, we found ratios to change from an average of 0.6 to 4.5. Synchronized immune and control mice were challenged with different doses of infectious HSV. Animals were followed for signs of disease, and vaginal washings were collected for both virus titration and Ab measurement. It was reasoned that if levels of vaginal Ab were sufficient to neutralize virus and prevent viral invasion, then virus should be rapidly removed from vaginal tissues and animals should show no evidence of a systemic secondary immune responses to HSV. However, as indicated in Table II, which compares the results of such infection in VgB and control vaccinia (vtk^-) virus-immune animals, the vaginal immunity was insufficient to prevent viral invasion in the majority of animals. Thus, all animals challenged with 10⁷ pfu (200 LD₅₀) of virus, and even one-third of those exposed to 10³ pfu, developed Ab responses to gD. In a separate experiment, VgB-immunized mice were challenged with 10⁵ pfu (before inactivation) of UV-inactivated virus. None of the animals developed gD-specific Ab (see Table III, Footnote b). Consequently, we assume the presence of gB Ab in the vaginal tract does not prevent viral infection of mucosal cells and the induction of an immune response. However, immunity following vaccination was sufficient to control the expression of clinical disease. Thus, even challenge of VgB vaccinees with 10⁷ pfu of HSV failed to cause symptoms. In contrast, VV-immune animals all succumbed to 10⁷ as well as 10⁵ pfu challenge, and 20% of animals succumbed to as little as 10⁴ pfu challenge.

What protects mucosally immunized mice from clinical disease?

Since vaginal Ab was insufficient to prevent viral invasion, yet immunized animals were protected from clinical disease and in fact cleared virus more rapidly than control mice, the question arose as to possible immunologic mechanisms involved in the control of vaginal infection. Two types of experiments indicated a major function for CD4⁺ T lymphocytes. In the first series of experiments, mice were immunized i/nas with HSV and all were shown to respond systemically and vaginally with specific Ab production. Animals were then hormonally synchronized, depleted of either CD4⁺ or CD8⁺ T cells by mAb treatment, and then challenged intravaginally with 5 x 10⁶ pfu HSV (100 LD₅₀). Vaginal wash samples were subsequently collected for the measurement of viral titers. The results in Figure 1 show that the CD4⁺-depleted animals had viral titers 2.5 logs higher than the CD8⁺-depleted ones. In HSV-immune nondepleted mice, only one of five vaginal samples tested at day 2 postchallenge had detectable virus. These results indicate that CD4⁺ T cells are more important for vaginal immunity than are CD8⁺ T cells.

View larger version (58K): [in this window] [in a new window]   FIGURE 1. Influence of CD4+ and CD8+ T cell depletion on viral clearance in HSV-immune BALB/c mice. a, Groups of six 4- to 5-week-old female BALB/c mice were immunized i/nas with 1 x 106 pfu of HSV-1 KOS. Thirty days later, the mice were injected s.c. with DP (2 mg/mouse). At day 36, all animals (naive or HSV immune) were challenged with 1 x 107 pfu of HSV-1 McKrae. Depletion of CD4+ and CD8+ T cells was performed by i.v. administration of 0.5 mg/mouse anti-CD4 (1.5) or anti-CD8 (2.43) mAbs on days 34, 38, and 40 (2 days before and 2 and 4 days after virus challenge). Vaginal wash samples were collected at days 2 and 5 postchallenge, and virus titer was determined by plaque assay. The data represent one of two experiments performed with similar results. Groups a and b differ significantly (p < 0.01). FACS data confirming the efficacy of in vivo depletion, using splenocytes (the depletion was also confirmed using cells from vaginal draining lymph nodes): a, 2.6% CD4 and 16.1% CD8+ T cells; b, 28.8% CD4 and 0.6% CD8+ T cells; c, 14.8% CD4+ and 8.8% T cells.

Taken together, the above experiments indicate that protection against vaginal viral challenge depends on the activity of T cell immunity, particularly CD4⁺ T cells. To evaluate whether B cell-mediated resistance plays an additional role in defense, the outcome of vaginal viral challenge was compared in BALB/c and immunocompromised µMT^-/- mice of the same genetic background. The data presented in Table V indicate that the failure to generate anti-HSV Ab responses did not impair the ability to resist infection. After intravaginal challenge with a low dose of HSV (10⁵ pfu), VgB-immunized µMT^-/- had marginally higher vaginal virus titers, but when animals were challenged with a high dose of HSV (10⁷ pfu), titers were approximately the same. Additionally, the time of viral clearance was similar in immune µMT^-/- and BALB/c mice. Furthermore, both immune B cell-immunocompromised and control mice survived low and high dose intravaginal challenge, whereas all naive animals succumbed to infection (Table V).

	Discussion

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The conclusion that Ab at the site of mucosal infection was usually inadequate to prevent invasion came from experiments in which mice immunized i/nas with recombinant vaccinia expressing HSV glycoproteins were challenged vaginally with HSV. Despite high titers of both IgA and IgG, vaginal Ab against the immunizing glycoprotein, infection following viral challenge was assumed to occur in many animals. Thus, Ab responses against other glycoproteins were induced, and in addition challenged animals developed secondary Ab responses to the immunizing glycoprotein. This pattern of events was evident even in some immune animals challenged with a minimal dose (10³ pfu) of virus. Our results indicate either that Ab, at least against gB or gD, the current favored candidates for subunit vaccines, fails to neutralize HSV in the vagina or that virus-Ab complexes remain infectious to FcR-bearing cells in the vaginal mucosa. Our observations do stand in contrast with some previous reports (18, 19, 20). For example Eis-Hubinger et al. (18) observed vaginal protection in mice following the i.v. administration of monoclonal, although not polyclonal, Ab. However, these workers failed to exclude whether or not viral invasion occurred. Thus we also routinely observed protection from disease in animals that did succumb to infection. In addition, disease following ocular infection may not become evident in mice previously given high concentrations of mAb (21). Nevertheless, such animals still establish latent infection.

In humans, some observations do indicate a role for Abs in protection from infection. For example, Brown et al. (22) observed that Ab may protect against invasion of the human infant. They observed that some infants born to infected mothers suffering recurrent disease were protected from infection during birth by their maternal Ab. However, not all infants born to immune mothers were protected from infection (22, 23). Other studies have indicated that the presence of cervical IgA in humans may diminish the duration of viral shedding in women (24, 25).

Whether or not Ab can fully protect against invasion is an important issue in HSV pathogenesis. Thus, if immunity is not sterile, then viral invasion can establish latency and thus set the scene for eventual recurrent disease. It is sobering to realize that no prophylactic vaccine against HSV has yet been deemed satisfactory by independent evaluation (2). Recently, one major company abandoned their prophylactic vaccine trial for thus far unknown reasons, and the results of a second trial using a subunit vaccine approach are still being evaluated.

We also readdressed the issue of which immune defenses are involved in protection against disease following mucosal challenge. This topic was addressed previously by others using animal models usually in a primary infection mode. For instance, McDermott et al. (26) demonstrated transfer of protection against intravaginal challenge by T cells from the genital lymph nodes draining the vaginal mucosa, but not by Ab-producing B cells. More recently, Milligan and Bernstein (27, 28) have assessed T cell role in vaginal protection against HSV-1 in naive mice and concluded that CD4⁺ cells are the principal means of defense and that the cells likely function by IFN- production. IFN- has been advocated as the mediator of systemic immunity conducted by both CD4⁺ and CD8⁺ T cells (29), although animals genetically unable to produce IFN- can still be protected from disease (30). More recently, one group have advocated that CD8⁺ T cells act as the major for mucosal defense entities against HSV infection (31).

The present study using immune animals makes a strong case that T cells, mainly CD4⁺, act as the principal mediators of mucosal defense and that Ab likely plays little or no role. Firstly, we observed that immunized µM knockout mice, lacking B cells and incapable of producing anti-HSV Ab, were equally as well protected against HSV challenge as were immunocompetent animals. Additionally, such µM knockout mice immunized with live HSV and subsequently depleted of CD4⁺ or CD8⁺ T cells were able to survive lethal intravaginal HSV challenge. However, significantly higher virus titers were recovered from the vaginal tract of the CD4⁺ than that of CD8⁺ T cell-depleted µM knockout mice. These results indicate that although CD8⁺ T cells may have some protective role, CD4⁺ T cells act as the principal mediators of vaginal immunity. Moreover, in the recombinant vaccinia experiments, the constructions used that provided protection were not able to induce CD8⁺ CTL responses in BALB/c mice (32, 33). Also arguing for a principal role for CD4⁺ T cells in mucosal immunity was the observation that whereas immunized ß₂M knockout (low CD8⁺) were well protected from HSV vaginal challenge, immune CD4⁺ knockout animals were more susceptible. Thus, our studies add up to the conclusion that mucosal immunity to HSV, at least in the mouse, is principally the domain of CD4⁺ T cells. How such cells control infection in the vaginal tract still must be established, but we favor the idea that it involves multiple cytokines elaborated during the course of an inflammatory response orchestrated principally by CD4⁺ T cells. Whether vaccines can be designed that effectively enhance CD4⁺ T cell immunity so that infection, exogenous or reactivated, remains subclinical must be evaluated.

	Acknowledgments

 
We thank Dr. W. Gerhard (Wistar Institute, Philadelphia, PA) for providing us the µMT^-/- mice and Dr. Rae Lyn Burke (Chiron) for supplying the recombinant gB. Additionally, we thank Paula Rutherford and Kim Cummings for typographic assistance.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grants AI 33511 and AI 14981.

² Address correspondence and reprint requests to Dr. Barry T. Rouse, Department of Microbiology, M409 Walters Life Sciences Building, University of Tennessee, Knoxville, TN 37996-0845. E-mail address:

³ Abbreviations used in this paper: HSV, herpes simplex virus; ß₂m, ß₂-microglobulin; VgD, vaccinia virus expressing glycoprotein D; VgB, vaccinia virus expressing glycoprotein B; VV, vaccinia virus vtk^-; i/nas, intranasally; pfu, plaque-forming units; DP, DepoProvera; gB, glycoprotein B; gD, glycoprotein D.

Received for publication November 21, 1997. Accepted for publication February 6, 1998.

	References

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