Departments of Medical Microbiology and Immunology1 and Clinical Virology2, Göteborg University, Guldhedsgatan 10A, 413 46 Göteborg, Sweden
Author for correspondence: Kristina Eriksson. Fax +46 31 820160. e-mail kristina.eriksson{at}microbio.gu.se
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Abstract |
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Introduction |
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Innate immune mechanisms such as natural killer (NK) cells, neutrophils, macrophages and complement proteins participate in the natural defence against HSV infections (Adler et al., 1999 ; Benencia & Courreges, 1999
; Da Costa et al., 1999
; Milligan, 1999
; Milligan et al., 1998
). In addition, protection against genital HSV-2 infection and disease requires an adequate adaptive immune response. Acquired protective immunity can be mediated by both humoral and cell-mediated immune mechanisms. In experimental animals, protection against genital disease can be achieved by passive administration of either monoclonal or polyclonal HSV-specific antibodies (Eis-Hübinger et al., 1993
; Parr & Parr, 1997
; Whaley et al., 1994
). Furthermore, three lines of evidence support the contention that T cells are important in both control of a primary HSV infection and protection against re-infection: (i) in humans, clearance of HSV-2 from recurrent genital lesions correlates with infiltration of HSV-2-specific cytotoxic T cells (Koelle et al., 1998
), (ii) in vivo depletion of T cells from HSV-2-immunized mice abrogates their ability to resolve an HSV-2 vaginal re-infection (Milligan et al., 1998
; Parr & Parr, 1998
) and (iii) resistance to intravaginal challenge with HSV-2 can be transferred to naive recipient mice with genital lymph node cells from immune mice (McDermott et al., 1989
). Both CD4+ and CD8+ T cell subsets have been implicated in immune protection (Martin & Rouse, 1987
; Milligan et al., 1998
).
The present study was undertaken to elucidate further the requirement for B, CD4+ and CD8+ T cells in control of primary infection and protection against genital re-infection, for which purpose we used mice deficient in IgM heavy chain (µMT), CD4+ T cells (CD4-/-) or CD8+ T cells (CD8-/-). We used a mouse genital infection model system in which a single vaginal inoculation of a thymidine kinase-deficient attenuated strain of HSV-2 TK- confers protective immunity to a subsequent vaginal challenge with a lethal dose of a wild-type (WT) HSV-2 strain (McDermott et al., 1984 ; Parr et al., 1994
). The results indicate that B cells secreting natural antibodies are involved in the innate control of local virus replication after primary genital HSV-2 TK- infection. Furthermore, CD4+ but not CD8+ T cells were shown to play a crucial role in adaptive immune protection against genital HSV-2 re-infection. Moreover, we demonstrated that exogenous IFN-
could be used to compensate for the lack of CD4+ T cells during HSV-2 re-infection.
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Methods |
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Viruses.
The attenuated mutant of HSV-2, strain Lyon, which contains a partial deletion of the thymidine kinase gene (TK-), was kindly provided by E. De-Clercq (Rega Institute for Medical Research, K. U. Leuven, Belgium) (Andrei et al., 1997 ). HSV-2 TK-, strain Lyon and WT HSV-2, strain 333 (Seth et al., 1974
) were grown and titrated in monolayers of African green monkey kidney cells (GMK-AH1) and prepared by one cycle of freezethaw and subsequent removal of cellular debris by centrifugation.
HSV-2 antigen preparation.
HSV-2 strain 333 was grown in GMK-AH1 cell monolayers. One cycle of freezethaw and centrifugation for the removal of cellular debris was used to recover virus from the culture. Control cell extract (mock antigen) was prepared in a similar fashion from uninfected GMK-AH1 cell monolayers. Virus preparations containing 1·5 to 2x107 p.f.u./ml of HSV-2 and control cell extracts were inactivated by UV light for 30 min (Koelle et al., 1994 ). Inactivated HSV-2 and mock antigen were used at 1:100 final dilution for in vitro experiments.
Primary and secondary infections.
Mice were injected subcutaneously with 2·0 mg of Depo-Provera (DP) (Pharmacia) in 100 µl PBS, anaesthetized 6 days later using methofane (Schering-Plough Animal Health) and vaccinated by intravaginal inoculation of 3·6x106 p.f.u. of attenuated HSV-2 TK- in 20 µl Hanks balanced salt solution (HBSS). Four weeks after HSV-2 TK- vaccination, vaccinated and unvaccinated mice were pre-treated with DP as above, followed 6 days later by an intravaginal inoculation of 4x104 p.f.u. (100 LD50) of virulent HSV-2 strain 333 in 20 µl HBSS.
Monitoring of infection
(a) Virus replication.
Following intravaginal HSV-2 infection, vaginal fluids were collected by pipetting 40 µl of sterile HBSS in and out of the vagina until a discrete clump of mucus was retrieved. A second wash was then performed. The two washes were pooled and immediately centrifuged to clarify the vaginal washes and then stored at -70 °C. HSV-2 titres were determined by plaque assay on GMK-AH1 cell monolayers.
(b) Inflammation and disease.
Mice were examined daily for vaginal inflammation, neurological illness and death after HSV-2 infection. The severity of disease was graded as healthy (0), genital erythema (1), moderate genital inflammation (2), severe and purulent genital lesion (3), hind-limb paralysis (4) and death or sacrifice due to paralysis (5), as described previously (Morrison et al., 1998 ).
Reconstitution of µMT mice.
Groups of DP-treated µMT mice were injected, 0·5 ml intraperitoneally (i.p.) and 0·2 ml intravenously, with pooled serum from naive control (C57BL/6) mice. These mice, together with non-reconstituted µMT mice, were vaginally inoculated 2 h later with 3·6x106 p.f.u. of attenuated HSV-2 TK-.
In vivo IFN-
treatment.
Chinese hamster ovary (CHO) cells or CHO cells transfected with the gene encoding murine IFN- (kindly provided by M. Taube, Göteborg University, Sweden) were used as sources of control supernatent and recombinant IFN-
(rIFN-
), respectively. The supernatants were dialysed against PBS and further concentrated using polyethylene glycol (PEG 3000) as described (Zhao et al., 1998
). HSV-2 TK--vaccinated, DP-treated CD4-/- mice and naive, DP-treated WT mice were given either 6 µg of rIFN-
or the same volume of control supernatant i.p. The mice were injected intravaginally 6 h later with either 0·4 µg of rIFN-
in 10 µl or the same volume of control supernatant and then challenged intravaginally with 4x104 p.f.u. of HSV-2 strain 333. The challenged mice were treated i.p. with rIFN-
or control supernatant as above on days 2, 4, 6 and 8 post-challenge.
Proliferation assays.
Mononuclear cell suspensions of spleen cells isolated 4 weeks after vaccination with attenuated HSV-2 TK- were seeded in triplicate wells at a concentration of 1x105 cells per well in Iscoves medium supplemented with L-glutamine, 50 µM 2-mercaptoethanol, gentamycin and 10% foetal calf serum (complete Iscoves medium) and incubated at 37 °C for 5 days in 96-well round-bottom plates in the presence of UV-inactivated HSV-2 or mock antigen. Cells were pulsed with 1 µCi [3H]thymidine (Amersham Pharmacia) for the last 6 h of culture and the cellular DNA was harvested on glass-fibre filters and then assayed by liquid scintillation counting. Data are expressed as stimulation indices (SI), corresponding to the mean c.p.m. of UV-inactivated HSV-2-treated cultures divided by the mean c.p.m. of mock-treated cultures.
Cytokine measurements.
Measurement of cytokine production in spleen cells was performed using a modified version of a celELISA method (Beech et al., 1997 ). Briefly, wells of a 96-well flat-bottom plate were coated with 50 µl anti-mouse IFN-
(1·5 µg/ml, R&D Systems), IL-2 (2 µg/ml, Pharmingen) or IL-4 (1 µg/ml, Pharmingen) in PBS. After overnight incubation at 4 °C, wells were blocked with a sterile solution of 1% BSA in PBS. Mononuclear spleen cell suspensions obtained 4 weeks post-vaccination with attenuated HSV-2 TK- were seeded in duplicate at different cell densities in complete Iscoves medium containing UV-inactivated HSV-2 or mock antigen and incubated at 37 °C for 24 h (for IFN-
and IL-2 determinations) or 48 h (for IL-4 determination). The cells were removed by extensive washing and biotinylated anti-mouse IFN-
(1 µg/ml), IL-2 (2 µg/ml) or IL-4 (0·25 µg/ml) was added overnight at 4 °C followed by a 45 min incubation with 2 µg/ml peroxidase-labelled avidin (Sigma) at room temperature. Colour development was achieved with 100 µl peroxidase substrate containing 3,3',5,5'-tetramethylbenzidine (0·1 mg/ml, Sigma) and 0·06% H2O2 in 0·05 M phosphatecitrate buffer at pH 5·0. The reaction was stopped using 25 µl 1 M H2SO4 and absorbance was read at 450 nm. The concentration of each cytokine was determined by extrapolation from a standard curve obtained using recombinant cytokine (IFN-
and IL-4, R&D systems; IL-2, Pharmingen). Results are expressed as the concentration of each cytokine secreted per 1x106 spleen cells.
Measurement of delayed type hypersensitivity (DTH) responses.
DTH reaction was elicited 4 weeks after vaccination with attenuated HSV-2 TK- by injection of UV-inactivated HSV-2 (corresponding to 7x106 p.f.u. in 30 µl medium) in the left (experimental) footpad and 30 µl of mock antigen in the right (control) footpad. Swelling of the footpads was measured 48 h later using a calliper (Oditest) and the specific footpad swelling was determined for each animal as the mean difference between the thickness of experimental and control footpads.
Antibody measurements.
Maxisorp 96-well plates (Nunc) were coated with 100 µl of a deoxycholate-solubilized membrane fraction of HSV-1-infected cells (Jeansson et al., 1983 ) for 4 h at room temperature in 0·05 M carbonate buffer at pH 9·6. The plates were blocked with 2% BSA in PBS for 30 min at 37 °C. Serial dilutions of sera obtained 4 weeks post-vaccination with attenuated HSV-2 TK- were incubated for 1 h at 37 °C. After washing with 0·05% Tween 20, the plates were incubated for 1 h at 37 °C with goat anti-mouse IgG coupled to horseradish peroxidase (1:1000, Southern Biotechnology) in 1% BSA in PBS. The plates were washed with 0·05% Tween 20 and developed using 100 µl of 1 mg/ml O-phenylenediamine dihydrochloride (Sigma) in 0·1 M citrate buffer (pH 4·5) containing 0·04% H2O2. After 20 min incubation at room temperature, the absorbance was read at 450 nm. The sample IgG titre was defined as the reciprocal of the sample dilution giving an absorbance value of 0·4 above the background value.
Statistical analysis.
Statistical analyses were done by Students t-test or Pearsons correlation test.
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Results |
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CD4+ T cells provide protective immunity against genital HSV-2 re-infection
To examine the importance of different lymphocyte subsets for the induction of a protective immune response against a lethal vaginal HSV-2 challenge, µMT, CD4-/-, CD8-/- and WT mice were vaccinated vaginally with HSV-2 TK- 4 weeks prior to vaginal challenge with 100-fold LD50 dose (4x104 p.f.u. per mouse) of HSV-2. All unvaccinated WT, µMT, CD4-/-, CD8-/- and control mice died within 12 days after HSV-2 challenge (median, day 8; range, days 7 to 12; data not shown). Vaccinated CD8-/- mice were similar to vaccinated WT mice in that they had no detectable HSV-2 in vaginal washings obtained day 3 post-challenge (Table 1) and no genital or neurological signs of inflammation during the 30 day observation period (Fig. 2
). In vaccinated µMT mice, a low titre of HSV-2 was seen at day 3 post-challenge (Table 1
); however, these mice displayed resistance to subsequent lethal challenge with HSV-2 (Fig. 2
). The vaccinated CD4-/- mice exhibited significantly increased titres of HSV-2 at day 3 post-challenge (Table 1
). Furthermore, the vaccinated CD4-/- mice all succumbed to a lethal HSV-2 challenge, with vaginal symptoms starting at day 4 (Fig. 2a
), and died as a result of neurological illness within 2 weeks, i.e. at approximately the same time as the non-vaccinated WT control mice (Fig. 2b
). These findings indicate that CD4+ but not CD8+ T cells or B cells are critical for an effective acquired immune response against a lethal HSV-2 challenge in mice, even though B cells/antibodies appear to suppress the local virus replication in the genital tract.
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(a) T cell proliferative responses in vitro.
Spleen cells from vaccinated WT mice developed strong HSV-2-specific proliferative responses with SI values of 30 or more. Vaccinated µMT and CD8-/- mice showed marked reductions in their specific proliferative responses compared with WT animals (Fig. 3a). Thus, all vaccinated mice that were protected against subsequent challenge had measurable proliferative response in vitro. In contrast, vaccinated CD4-/- mice, which failed to develop protective immune responses to HSV-2, showed severely impaired HSV-2-specific proliferative responses in vitro (P<0·05 compared with that in vaccinated WT mice) (Fig. 3a
).
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(d) Antibody responses.
To investigate whether the absence of CD4+ or CD8+ T cells might affect the systemic specific IgG response, mice deficient in lymphocyte subsets were examined for specific serum IgG antibodies. Sera obtained from vaccinated WT and CD8-/- mice contained similar titres of HSV-specific IgG, while vaccinated µMT mice, as expected, lacked specific serum IgG antibodies (Fig. 5). Surprisingly, sera from vaccinated CD4-/- mice contained HSV-specific IgG, although at a tenfold lower level than that in WT animals (Fig. 5
). Thus, a specific serum IgG response can be elicited in the absence of CD4+ T cells following HSV-2 TK- inoculation.
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Discussion |
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In WT mice, vaginal inoculation with HSV-2 TK- caused a subclinical infection. In comparison, µMT mice, which lack functional B cells and antibodies, had significantly higher virus loads and 40% of the animals also displayed an apparent genital inflammation. The most likely explanation for the greater virus replication following primary HSV-2 infection is the lack of immunoglobulins including natural antibodies. We found that transfer of normal mouse serum into µMT mice significantly reduced the vaginal load of HSV-2, indicating an early role of natural antibody in control of virus replication. To our knowledge this is the first report to suggest that natural antibodies play an early and beneficial role in primary genital HSV-2 infection. These results are in line with a recent study showing a role for natural antibodies in both innate control of a cutaneous HSV-1 infection (Deshpande et al., 2000 ) and limiting of the spread of lymphocytic choriomeningitis virus during the first days of a primary infection (Ochsenbein et al., 1999
). The beneficial effect of natural antibodies on HSV-2 replication that we observed could depend on the initial virus load, as no differences in virus replication were observed between µMT and WT mice when a low-dose HSV-2 inoculum was used (Dudley et al., 2000
).
Despite the dramatic drop in virus shedding, the serum-reconstituted µMT mice still developed genital inflammation at the same frequency as those not receiving serum transfer. Therefore, we postulate that natural antibodies have little role to play in early control of HSV-2-induced inflammation. We cannot conclusively exclude such a role, however, since a single serum transfer would not mimic the continuous immunoglobulin production of WT mice. It is also possible that B cells themselves could have an anti-inflammatory role. Thus, B cells have been shown to be the first lymphocytes to be recruited to the vagina of mice following HSV-2 TK- inoculation (Parr et al., 1994 ) and provide several functions in addition to antibody production, including: (i) B cells may regulate the appropriate trafficking of other cells to the site of infection via secretion of, e.g. macrophage inflammatory protein-1
(MIP-1
) and MIP-1
(Krzysiek et al., 1999
), (ii) B cells are one of the major sources of IFN-
(Liles & van Voorhis, 1995
), which blocks virus replication and spread, and (iii) B cells may also activate NK cells (Yuan et al., 1994
), which can suppress HSV infection (Minato et al., 1980
).
Previous studies have shown that transfer of specific serum IgG from HSV-2 TK--vaccinated mice to the vaginal lumen of naive mice reduced the virus load and pathological signs of disease in recipient mice following vaginal HSV-2 challenge (Parr & Parr, 1997 ) and also that transfusion of monoclonal antibodies to glycoprotein gB of HSV conferred protection against a subsequent challenge (Eis-Hübinger et al., 1991
, 1993
; Kino et al., 1985
). In contrast to these findings, our study corroborates the findings of Dudley et al. (2000)
in showing clearly that resistance to a lethal vaginal HSV-2 challenge in mice vaccinated with HSV-2 TK- is not critically dependent on B cells and/or antibodies.
Our results in CD8-/- mice show that, in similarity with experiments with B cells, CD8+ T cells are not crucial for control of vaginal HSV-2 re-infections in mice. These results are in agreement with recent findings in humans that show that HSV-2 recurs even in the presence of high levels of specific CD8+ cytotoxic T cells in the genital tract (Posavad et al., 2000 ). Interestingly, the lack of B cells or CD8+ T cells was associated with increased IL-4 responses concomitant with reduced levels of IL-2 and IFN-
, indicating that the presence of both B cells and CD8+ T cells favours Th-1 phenotype responses. However, the shift towards Th-2 phenotype responses, characterized by decreased levels of IFN-
and increased levels of IL-4, did not affect protection.
Our data show that CD4+ T cells are critical for the development of an efficient protective immune response to HSV-2. The failure of CD4-/- mice vaccinated with HSV-2 TK- to combat infection was associated with an enhanced HSV-2-specific T cell-mediated immune response after the initial infection/vaccination, as evidenced by impaired HSV-2-specific cytokine production and proliferative response in vitro and a compromised virus-specific DTH response in vivo. These results support previous findings that vaccination with recombinant antigens could induce protection from genital HSV-2 infection in WT but not CD4-/- mice (Kuklin et al., 1998 ) and extend the observations made by others with HSV-2 infection in T cell-depleted animals (Milligan et al., 1998
; Parr & Parr, 1998
).
Interestingly, our results show that protection against genital HSV-2 re-infection in CD4-/- mice vaccinated with HSV-2 TK- can be restored by administration of exogenous IFN- at the time of HSV-2 re-infection. It was obvious that IFN-
was not anti-viral per se, as exogenous IFN-
treatment could not rescue non-vaccinated WT mice from a lethal genital HSV-2 challenge. Thus, in the absence of CD4+ T cells, IFN-
acts in conjunction with other components of the adaptive immune system in combating HSV-2 infection. This latter finding suggests that CD4+ T cells may be the major source of IFN-
at the time of HSV-2 re-infection, which is supported by the virtual absense of IFN-
in CD4-/- mice and by the decreased levels of genital IFN-
responses in CD4+ T cell-depleted animals (Milligan et al., 1998
). Previous studies have shown an important role for IFN-
in control of genital HSV-2 infection (Milligan & Bernstein, 1997
; Milligan et al., 1998
; Parr & Parr, 1999
) but it has not been reported previously that IFN-
could substitute for the CD4+ lymphocyte population.
Several mechanisms might account for the contribution of IFN- in protection against genital HSV-2 infection including: (i) IFN-
enhanced expression of both MHC class I and II on the cell surfaces, which facilitates the recognition and subsequent destruction of infected cells by CD8+ and CD4+ T cells, (ii) IFN-
inhibition of HSV replication in vitro by blocking transactivation-induced transcription of immediately early genes (De Stasio & Taylor, 1990
) (however, since administration of exogenous IFN-
to naive WT mice did not protect against disease and death, this direct anti-viral effect, if it occurs in vivo, is of relatively minor importance), (iii) IFN-
-mediated chemoattraction and activation of macrophages and neutrophils (Fong & Mosmann, 1989
), and (iv) IFN-
up-regulation of intracellular adhesion molecules such as ICAM-1 on endothelial cells (Dustin et al., 1986
) to direct lymphocytes into the site of HSV infection.
In conclusion, the data presented here indicate that B cells secreting natural antibodies play an important role in innate control of local HSV-2 replication following primary genital infection with HSV-2. The protective immunity following vaginal vaccination with HSV-2 TK- depends on CD4+ T cells. Furthermore, IFN- could compensate for the lack of CD4+ T cells during genital HSV-2 re-infection. These findings have implications for our understanding of immune mechanisms involved in protection against genital HSV-2 infection as well as for immunomodulating therapies against genital herpes infection.
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Acknowledgments |
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References |
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Received 28 September 2000;
accepted 8 December 2000.