Department of Medical Microbiology1 and Department of Clinical Immunology4, University of Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands
Rotterdam Eye Hospital2 and Institute of Virology3, Erasmus University Rotterdam, Rotterdam, The Netherlands
Author for correspondence: Sytske Welling-Wester. Fax +31 503633528. e-mail S.Welling-Wester{at}med.rug.nl
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() |
---|
![]() |
Main text |
---|
![]() ![]() ![]() ![]() |
---|
Of the HSV-1 glycoproteins, gD has been studied most extensively with respect to immune responses and immunological properties. The role of HSV gH:gL as a possible target of the immune system has been studied less extensively. It is known that monoclonal antibodies (MAbs) to the gH:gL complex can inhibit HSV-1 infections in vitro (Buckmaster et al., 1984 ; Showalter et al., 1981
). Passive administration of neutralizing antibodies raised against the gH:gL complex protects mice from zosteriform spread of HSV-1 infections (Forrester et al., 1991
; Simmons & Nash, 1985
). However, immunization studies with recombinant forms of gH and gL (Ghiasi et al., 1992
, 1994a
, b
) or recombinant vaccinia viruses expressing gH, gL and gH:gL (Browne et al., 1993
; Forrester et al., 1991
) induced only limited protection. Subsequently, mice immunized with recombinant complexes consisting of truncated gH and full-length gL produced by a mammalian cell line were shown to be protected from a lethal HSV-1 challenge (Peng et al., 1998
).
This latter finding encouraged detailed investigations of immune responses to the HSV-1 gH:gL complex in naturally infected humans. In the present study, a soluble purified recombinant form of the HSV-1 gH:gL complex, produced and secreted by insect cells, was used to analyse the humoral and peripheral blood (PB) T cell responses in seven HSV-1-seropositive and three HSV-1-seronegative healthy volunteers. The seven HSV-seropositive donors had been naturally infected by HSV-1 and most likely not by HSV-2, since none of the sera reacted with recombinant gG2 fragments and gG2 peptides (Oda-Ikoma et al.,1998 ).
The recombinant complex, designated gHt(His):gL, consisted of full-length gL and truncated gH. The gH molecule was truncated before the transmembrane region at amino acid 791 and was tagged with the peptide RSHHHHHH at the C terminus. High Five insect cells (Invitrogen) were infected with recombinant baculoviruses containing the open reading frames of both gHt(His) and gL under control of polyhedrin promoters. After 72 h of infection, the culture medium (Insect Xpress, BioWhittaker) was harvested. The gHt(His):gL complex was isolated in a one-step purification from the culture medium by immobilized metal affinity chromatography (D. F. Westra, unpublished). The purified gHt(His):gL complex was obtained with a high degree of purity, as analysed on SDSpolyacrylamide gels with subsequent silver staining (Fig. 1A). The predicted molecular masses of gHt(His), gL and gDt are 100, 30 and 47 kDa. Western blots with the appropriate antibodies confirmed the identity of the bands seen on the silver-stained gel (Fig. 1B
D
). Size exclusion HPLC (data not shown) showed that the gHt(His):gL complex has a molecular mass of 125 kDa, and confirmed that the complex is a hetero-dimer. The purified gHt(His):gL was recognized by MAb LP11, as assayed by ELISA. Reactivity to LP11 is seen as indicative of correctly folded gH:gL since this antibody recognizes gH only when coexpressed with gL (Hutchinson et al., 1992
).
|
The humoral responses to a crude HSV-1 lysate, and to recombinants gHt(His):gL and gDt were analysed by ELISA. For the preparation of HSV antigen, Vero cells were infected with HSV strain McIntyre (ATCC VR-539) at an m.o.i. of 10. The infected cells were lysed by freezethawing followed by heat-inactivation at 65 °C for 1 h. As a control, lysed mock-infected Vero cells were used. The antigens were stored in aliquots at -80 °C. The presence of specific IgG in human sera was analysed by ELISA. A lysate of HSV-1-infected Vero cells and recombinant proteins gHt(His):gL and gDt were coated overnight in 50 mM sodium bicarbonate buffer pH 9·6. Serial dilutions of the sera were incubated for 1 h. After washing, horseradish peroxidase-conjugated rabbit anti-human IgG antibodies (Dako) were added. The substrate O-phenylenediamine.HCl was used and colour development was measured. The antibody levels were defined as the reciprocal of the serum dilution which gave an A495 of 0·5.
Seven HSV-1-seropositive individuals (donors 17) and three HSV-1-seronegative individuals (donors 810) were tested for IgG levels specific for HSV antigen and for gHt(His):gL complex and gDt (Table 1). Six out of seven HSV-1-seropositive individuals had identical antibody levels to the gHt(His):gL complex. None of the HSV-1-seronegative donors had significant IgG levels to the antigens tested. One seropositive individual (donor 1) had a relatively low antibody level to recombinant gHt(His):gL. This response was, however, still higher than those of the seronegative donors. The antibody levels to gDt varied to a larger extent. Two donors (donors 5 and 6) had both very high gDt-specific antibody and very high HSV-specific antibody levels. In general, higher gDt-specific antibody levels correlated with higher HSV-specific antibody levels. This correlation was not seen for the antibody levels specific for the gHt(His):gL complex. In three out of seven HSV-seropositive individuals, the gHt(His):gL antibody levels were higher than the antibody levels for gDt (Table 1
). The data suggest that the HSV gH:gL complex is an important antigen to which humoral immune responses are elicited upon natural HSV infection.
|
Proliferative responses of freshly isolated peripheral blood mononuclear cells (PBMC) to lysates of HSV-1-infected Vero cells and to recombinants gHt(His):gL and gDt were examined in a lymphocyte proliferation assay. PBMC were isolated by density centrifugation of heparinized PB on a Ficoll gradient (Lymphoprep; Nycomed) and were cultured in RPMI 1640 supplemented with 10% heat-inactivated human serum. 105 PBMC per well were incubated with recombinant gDt (1, 5, 10 and 20 µg/ml), recombinant gHt(His):gL (1, 5, 10 and 20 µg/ml) and lysates of mock-infected and HSV-1-infected Vero cells. T cell proliferation was measured as 3[H]thymidine incorporation over the last 16 h of a 6 day culture period. The stimulation index (SI) for HSV was calculated as the quotient of c.p.m. from cultures stimulated with a lysate of HSV-1-infected Vero cells to c.p.m. from cultures stimulated with a lysate of uninfected Vero cells. The SI for gHt(His):gL and gDt was calculated as the quotient of c.p.m. from cultures stimulated with the purified glycoproteins to c.p.m. of unstimulated cultures.
PBMC of seven seropositive and three seronegative healthy adult donors were tested on two or more occasions with the antigens. Data from a representative experiment are shown in Table 1. The PB T cells of all HSV-seropositive donors (donors 17) responded to lysates of HSV-1-infected Vero cells, recombinant HSV-1 gHt(His):gL and gDt. Among the three seronegative donors, two donors did not respond to the HSV antigens and donor 9 demonstrated profound PB T cell responses to the two recombinant HSV-1 glycoproteins with only a marginal response to Vero/HSV-1 lysate. The data indicate that following a natural HSV-1 infection, HSV-1 gD but also gH:gL-specific T cell immunity is elicited. Given the nature of the antigen, e.g. exogenous antigen, the antigen-specific T cell responses measured in vitro were most likely orchestrated by CD4+ T cells.
T helper cells can be divided into subsets, Th1 and Th2, based on their production ofdistinct patterns of cytokines. Typical Th1 and Th2 cytokines are interferon (IFN)- and interleukin (IL)-4, respectively (Biron, 1994
). To determine the Th subset of the antigen-specific PB T cells, the secretion of IFN-
and IL-4 byantigen-stimulated PB T cells of the HSV-seronegative and -seropositive donors was measured (Table 2
). After antigenic stimulation for 6 days, cell-free culture supernatants were assayed for IFN-
and IL-4 with commercial ELISA kits as described by Verjans et al. (1998b)
. PB T cell cultures from donors 17 stimulated with HSV-1 lysate, gDt, and gHt(His):gL secreted large amounts of IFN-
and no detectable IL-4 (Table 2
). Interestingly, PB T cells from donor 9 also secreted significant amounts of IFN-
upon stimulation with HSV-1 antigen and recombinant gHt(His):gL, and, to a lesser extent, gDt (Table 2
). These results indicated, as described previously, that the HSV-1-specific PB T cell response is Th1-like (Ghiasi et al., 1992
; Biron, 1994
; Carmack et al., 1996
; Cher & Mosmann, 1987
; Hendricks et al., 1992
).
|
The role of T cell immunity in controlling systemic and local HSV-1 infections has been studied extensively. HSV-specific CD4+ and CD8+ cells with cytotoxic and/or proliferating activities are present in high frequencies in PB cells and herpetic lesions (Verjans et al., 1998a , b
; Schmid, 1988
; Posavad et al., 1996
; Carmack et al., 1996
; Koelle et al., 1994
). The role of CD8+ T cells may be limited because HSV-infected cells have a reduced surface expression of MHC class I molecules (Hill et al., 1995
; York et al., 1994
). Nonetheless, CD8+ lymphocytes may have an important function in later stages of herpetic lesions. This is because IFN-
secreted by stimulated CD4+ Th1 cells upregulates MHC class I expression on HSV-infected cells (Posavad et al., 1998
). Alternatively, some cells may be less susceptible to the HSV-induced downregulation of MHC class I (Posavad et al., 1996
).
The observation that a natural HSV-1 infection elicits both a T cell (Th1-like) and humoral response to the HSV-1 gH:gL-complex may be of importance for the development of a subunit vaccine. Taking into account the finding that immunization with soluble recombinant HSV-1 gH:gL protects mice from a lethal HSV-1 challenge (Peng et al., 1998 ), the soluble gH:gL complex may be an important candidate as a component of an effective subunit vaccine for the prevention and/or control of HSV infections.
![]() |
Footnotes |
---|
c Present address: NKI, Amsterdam, The Netherlands.
![]() |
References |
---|
![]() ![]() ![]() ![]() |
---|
Beninga, J., Kalbacher, H. & Mach, M. (1996). Analysis of T helper cell response to glycoprotein H (gpUL75) of human cytomegalovirus: evidence for strain-specific T cell determinants.Journal of Infectious Diseases173, 1051-1061.[Medline]
Biron, C. A. (1994). Cytokines in the generation of immune responses to, and resolution of, virus infection.Current Opinion in Immunology6, 530-538.[Medline]
Browne, H., Baxter, V. & Minson, T. (1993). Analysis of protective immune responses to the glycoprotein Hglycoprotein L complex of herpes simplex virus type 1.Journal of General Virology74, 2813-2817.[Abstract]
Buckmaster, E. A., Gompels, U. & Minson, A. (1984). Characterization and physical mapping of an HSV-1 glycoprotein of approximately 115x103 molecular weight.Virology139, 408-413.[Medline]
Carmack, M. A., Yasukawa, L. L., Chang, S. Y., Tran, C., Saldana, F., Arvin, A. M. & Prober, C. G. (1996). T cell recognition and cytokine production elicited by common and type-specific glycoproteins of herpes simplex virus type 1 and type 2.Journal of Infectious Diseases174, 899-906.[Medline]
Cher, D. J. & Mosmann, T. R. (1987). Two types of murine helper T cell clone. II. Delayed-type hypersensitivity is mediated by TH1 clones.Journal of Immunology138, 3688-3694.
Forrester, A. J., Sullivan, V., Simmons, A., Blacklaws, B. A., Smith, G. L., Nash, A. A. & Minson, A. C. (1991). Induction of protective immunity with antibody to herpes simplex virus type 1 glycoprotein H (gH) and analysis of the immune response to gH expressed in recombinant vaccinia virus.Journal of General Virology72, 369-375.[Abstract]
Forrester, A., Farrell, H., Wilkinson, G., Kaye, J., Davis-Poynter, N. & Minson, T. (1992). Construction and properties of a mutant of herpes simplex virus type 1 with glycoprotein H coding sequences deleted.Journal of Virology66, 341-348.[Abstract]
Fuller, A. O. & Lee, W. C. (1992). Herpes simplex virus type 1 entry through a cascade of viruscell interactions requires different roles of gD and gH in penetration.Journal of Virology66, 5002-5012.[Abstract]
Ghiasi, H., Kaiwar, R., Nesburn, A. B. & Wechsler, S. L. (1992). Baculovirus-expressed glycoprotein H of herpes simplex virus type 1 (HSV-1) induces neutralizing antibody and delayed type hypersensitivity responses, but does not protect immunized mice against lethal HSV-1 challenge.Journal of General Virology73, 719-722.[Abstract]
Ghiasi, H., Kaiwar, R., Nesburn, A. B., Slanina, S. & Wechsler, S. L. (1994a). Expression of seven herpes simplex virus type 1 glycoproteins (gB, gC, gD, gE, gG, gH, and gI): comparative protection against lethal challenge in mice.Journal of Virology68, 2118-2126.[Abstract]
Ghiasi, H., Kaiwar, R., Slanina, S., Nesburn, A. B. & Wechsler, S. L. (1994b). Expression and characterization of baculovirus expressed herpes simplex virus type 1 glycoprotein L.Archives of Virology138, 199-212.[Medline]
Hendricks, R. L., Tumpey, T. M. & Finnegan, A. (1992). IFN-gamma and IL-2 are protective in the skin but pathologic in the corneas of HSV-1-infected mice.Journal of Immunology149, 3023-3028.
Hill, A., Jugovic, P., York, I., Russ, G., Bennink, J., Yewdell, J., Ploegh, H. & Johnson, D. (1995). Herpes simplex virus turns off the TAP to evade host immunity.Nature375, 411-415.[Medline]
Hutchinson, L., Browne, H., Wargent, V., Davis-Poynter, N., Primorac, S., Goldsmith, K., Minson, A. C. & Johnson, D. C. (1992). A novel herpes simplex virus glycoprotein, gL, forms a complex with glycoprotein H (gH) and affects normal folding and surface expression of gH.Journal of Virology66, 2240-2250.[Abstract]
Koelle, D. M., Corey, L., Burke, R. L., Eisenberg, R. J., Cohen, G. H., Pichyangkura, R. & Triezenberg, S. J. (1994). Antigenic specificities of human CD4+ T-cell clones recovered from recurrent genital herpes simplex virus type 2 lesions.Journal of Virology68, 2803-2810.[Abstract]
Mikloska, Z. & Cunningham, A. L. (1998). Herpes simplex virus type 1 glycoproteins gB, gC and gD are major targets for CD4 T-lymphocyte cytotoxicity in HLA-DR expressing human epidermal keratinocytes.Journal of General Virology79, 353-361.[Abstract]
Oda-Ikoma, M., Glazenburg, K. L., The, T. H. & Welling-Wester, S. (1998). A fragment of glycoprotein G of herpes simplex virus type 2 (gG2) expressed in the baculovirus expression system for detection of HSV-2 antibodies. Abstract 189, 23rd International Herpesvirus Workshop, 17 August, 1998, York, UK.
Peng, T., Ponce de Leon, M., Jiang, H., Dubin, G., Lubinski, J. M., Eisenberg, R. J. & Cohen, G. H. (1998). The gHgL complex of herpes simplex virus (HSV) stimulates neutralizing antibody and protects mice against HSV type 1 challenge.Journal of Virology72, 65-72.
Posavad, C. M., Koelle, D. M. & Corey, L. (1996). High frequency of CD8+ cytotoxic T-lymphocyte precursors specific for herpes simplex viruses in persons with genital herpes.Journal of Virology70, 8165-8168.[Abstract]
Posavad, C. M., Koelle, D. M. & Corey, L. (1998). Tipping the scales of herpes simplex virus reactivation: the important responses are local.Nature Medicine4, 381-382.[Medline]
Roop, C., Hutchinson, L. & Johnson, D. C. (1993). A mutant herpes simplex virus type 1 unable to express glycoprotein L cannot enter cells, and its particles lack glycoprotein H.Journal of Virology67, 2285-2297.[Abstract]
Schmid, D. S. (1988). The human MHC-restricted cellular response to herpes simplex virus type 1 is mediated by CD4+, CD8- T cells and is restricted to the DR region of the MHC complex.Journal of Immunology140, 3610-3616.
Showalter, S. D., Zweig, M. & Hampar, B. (1981). Monoclonal antibodies to herpes simplex virus type 1 proteins, including the immediate-early protein ICP4.Infection and Immunity34, 684-692.[Medline]
Simmons, A. & Nash, A. A. (1985). Role of antibody in primary and recurrent herpes simplex virus infection.Journal of Virology53, 944-948.[Medline]
Urban, M., Klein, M., Britt, W. J., Haßfurther, E. & Mach, M. (1996). Glycoprotein H of human cytomegalovirus is a major antigen for the neutralizing humoral immune response.Journal of General Virology77, 1537-1547.[Abstract]
Verjans, G. M., Feron, E. J., Dings, M. E., Cornelissen, J. G., Van der Lelij, A., Baarsma, G. S. & Osterhaus, A. D. (1998a). T cells specific for the triggering virus infiltrate the eye in patients with herpes simplex virus-mediated acute retinal necrosis.Journal of Infectious Diseases178, 27-34.[Medline]
Verjans, G. M., Remeijer, L., van Binnendijk, R. S., Cornelissen, J. G., Volker-Dieben, H. J., Baarsma, S. G. & Osterhaus, A. D. (1998b). Identification and characterization of herpes simplex virus-specific CD4+ T cells in corneas of herpetic stromal keratitis patients.Journal of Infectious Diseases177, 484-488.[Medline]
York, I. A., Roop, C., Andrews, D. W., Riddell, S. R., Graham, F. L. & Johnson, D. C. (1994). A cytosolic herpes simplex virus protein inhibits antigen presentation to CD8+ T lymphocytes.Cell77, 525-535.[Medline]
Received 17 December 1999;
accepted 19 April 2000.