Original Articles. Donald Henson, Ralph Helmsen, Karl E. Becker, Alfonso J. Strano, Margery Sullivan, and Duron Harris

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1 Original Articles Ultrastructural localization of herpes simplex virus antigens on rabbit corneal cells using sheep antihuman IgG antihorse ferritin hybrid antibodies Donald Henson, Ralph Helmsen, Karl E. Becker, Alfonso J. Strano, Margery Sullivan, and Duron Harris Sheep antihuman antiferritin hybrid antibodies were used to localize at the ultrastructural level herpes simplex virus (HSV) antigens on rabbit corneal (SIRC) cells. HSV antigens were distributed over the entire surface of infected cells, although they were often concentrated over focal areas of plasma membrane thickening. Morphologically, this focal plasma membrane thickening resembled the envelope of HSV. In disrupted cells infected with HSV, ferritin label was found along many of the internal membranes and at focal sites on the nuclear membrane. Intranuclear labeling was not observed. It is suggested that the development of HSV-induced antigens on the surface of infected cells causes these cells to be recognized as foreign by the host, much like an incompatible tissue graft. Results of this study indicate that sheep hybrid antibodies are useful for localizing antigenic changes on virus-infected cells. H erpes virus (HSV) keratitis represents a leading cause of corneal disease in the United States. Although the pathogenesis of infection is still controversial, it presently revolves around two unrelated problems: (1) persistence and periodic activation of latent HSV infection in the presence of circulating antibody and (2) mechanisms of the acute corneal lesion and its From the Laboratory of Pathology, National Cancer Institute, Laboratory of Vision Research, National Eye Institute, Laboratory of Immunology NIAMDD, National Institutes of Health, and Armed Forces Institute of Pathology, Washington, D. C. Submitted for publication May 7, Reprint request: Dr. D. Henson, Building 10, Room 2A-29, National Cancer Institute, Bethesda, Md complications stromal ulceration and perforation. There is experimental and clinical evidence to suggest that stromal keratitis is the result of a delayed-type hypersensitivity reaction in the cornea. In HSV-sensitized experimental animals, the development of corneal opacification after inoculation of HSV antigens into the cornea coincides with the development of delayed hypersensitivity to HSV antigens but not with the appearance of circulating antibody. 3 ' - Indirect evidence comes from clinical observations which have shown that corticosteroids which impair cell-mediated immune reactions effectively suppress disciform keratitis in man: 1 It is generally suspected, that in delayed hypersensitivity reactions occurring in virus infections, the

2 820 Henson et al. Investigative Ophthalmology November 1974 HOURS AFTER INFECTION Fig. 1. Replication of HSV-1 in SIRC cells. Cultures in 60 mm. Petri dishes were infected with HSV, washed, fed, and incubated at 37 C. At times indicated, two cultures were frozen and the virus subsequently titered by the plaque method using methylcellulose as the overlay. Solid line represents intra- and extracellular virus. immune response is associated with virusinduced antigens on the surface of infected cells rather than with free extracellular virus. In HSV infection, new antigens are known to develop on the surface of productively infected cells, 1 " 7 and presumably similar antigens appear on the surface of corneal cells infected with HSV. In this report, we describe the localization of antigens on membranes of HSV-infected rabbit corneal cells by electron microscopic immunolabeling using sheep antihuman gamma globulin (IgG) antihorse ferritin hybrid antibodies. Materials and methods Antiserum to HSV. Serum was obtained from a laboratory assistant who had recurrent herpes labialis. The HSV complement-fixation titer was 512. Immunoglobulin G (IgG) was separated by DEAE chromatography and concentrated to approximately 25 mg. per milliliter by pressure 20 filtration. This IgG will be referred to as anti-hsv IgG. Cell line. A cell line (SIRC) derived from normal rabbit cornea was used. 8 Cells were purchased from North American Biologicals Inc. (Rockville, Md.) and grown in 32 ounce bottles on RPMI Medium with 10 per cent fetal calf serum. Preparation of antihorse ferritin antihuman IgG hybrid antibodies. Sheep hybrid antibodies were prepared using procedures developed for rabbit" and human hybrid antibodies. 10 Separate adult sheep were immunized sequentially at 3-week intervals. The first two injections consisted of 1 mg. of either human IgG (Cohn Fraction II, Pentex, Kankakee, 111.) or horse ferritin (6X recrystallized, Pentex, Kankakee, 111.) injected intramuscularly. The third injection consisted of 0.5 mg. of the respective antigens in phosphatebuffered saline (ph 7.4) injected intravenously. Serum was collected 7 to 10 days later and stored at -70 C. Sheep antihuman IgG was absorbed onto a Sepharose human-igg column containing approximately 5 mg. of IgG per milliliter of Sepharose. After extensive washing with borate buffer (0.2 M in isotonic saline, ph 8.0), the bound antibody was eluted with. 0.1 N acetic acid and the protein eluate neutralized with crystalline tris (hydroxymethyl) aminomethane and concentrated. Approximately 7 to 8 mg. of antibody were recovered per milliliter of sheep serum. Antiferritin antibodies were purified in the same way except that the Sepharose-ferritin absorbent contained only 1 mg. of ferritin per milliliter of Sepharose. Seven to 8 mg. of antiferritin antibody were recovered from each milliliter of sheep serum. In both cases the purified antibodies gave strong precipitin lines against the respective antigens when tested by Ouchterlony immunodiffusion. Both antibody preparations were digested with pepsin and the F(ab') fragments recovered in the same way in separate experiments. Antibody (250 mg.) (10 mg. per milliliter) was dialyzed into a 0.15 M Na acetate, 0.05 M NaCl, ph 4.5, buffer. Two per cent (w/w) pepsin was added and the solutions incubated at 37 C. for 16 to 18 hours. The digestion was stopped by bringing the ph to 8 with 1.0N NaOH. The digestion mixture was next applied to a 590 ml. (3.0 by 82 cm.) column of G-150 Sephadex equilibrated with borate buffer. The main component eluted at approximately 50 per cent bed volume. It gave a strong precipitin line on gel diffusion against the appropriate antigen before but not after reduction and alkylation. Yields averaged 50 per cent. The F(ab') 2 fragments of the anti-igg and antiferritin (at 10 mg. per milliliter) were mixed and dialyzed against 0.1

3 Volume 13 Number 11 Ultrastructural localization of HSV antigens 821 f Fig. 2. Infected corneal cell containing intranuclear virus (long arrows), virus in the process of budding into perinuclear space (short arrow), and enveloped virus in the perinuclear space. Uranyl and lead citrate stain. (x45,500.) M Na acetate, ph 5.0. The solution was made M in mercaptoethylamine and incubated for one hour at 37 C. under N-. The solution was then applied to a AG50x2 ion exchange column (90. ml.) equilibrated with the same Na acetate buffer. Approximately 73 per cent of the applied protein was recovered. The reduced proteins were brought to ph 8.0 with 1.0N NaOH and gently stirred at room temperature for two hours under CX>. The solution was next applied to a 550 ml. (3 by 80 cm.) column of G-100 Sephadex equilibrated with borate buffer. Approximately 135 mg. of protein eluted at 36 per cent bed volume, F{ab') 2? and. 23 mg. at 50 per cent bed volume, F(ab'). One-half of the F(ab')u material was processed at a time. It was first applied to a Sepharoseferritin column. After washing with borate, about 39 per cent of the applied material was recovered in the 0.1 N acetic acid eluate. This consisted of antiferritin-antiferritin and antiferritin-anti-igg hybrid molecules. The mixture was applied to a Sepharose-human IgG column and the specifically bound protein eluted with 0.1 N acetic acid and neutralized as before. This material (8 mg.) precipitated with a mixture of human IgG and horse ferritin but with neither antigen alone. The overall yield calculated on the basis of the initial amount of F(ab') = antibody used was 7.5 per cent. The hybrid antibody preparation was stored at 4 C. in borate buffer. In addition to the sheep antihorse ferritin antihuman IgG hybrid antibodies, rabbit antihorse ferritin antihuman IgG hybrid antibodies were also

4 822 Henson et al. Investigative Ophthalmology November 1974 HSV Infected Cell Human Anti-HSV IgG HSV- Induced Neo Antigens On Cell Surface Sheep Anti- Human IgG Anti-ferritin F(ab 1 )2 Hybrid Antibody Visual Marker Ferritin Fig. 3. Schematic diagram showing the mechanism of labeling HSV-induced antigens on the surface of an infected cell with sheep antihuman IgG antihorse ferritin hybrid antibodies. (Modified after Neauport-Sautes. 1 ) prepared and used in some experiments. The rabbit hybrid antibodies were prepared in the same manner as the sheep hybrid antibodies. Serum was used from 12 rabbits, six injected with ferritin and six injected with human IgG. Adsorption of HSV anti-igg. Anti-HSV IgG (1.0 ml.) was absorbed for 20 minutes at 4 C. with 2 x 10 uninfected washed corneal cells before use. Adsorbed IgG was used in all experiments. Antibody reaction. Corneal cells in 60 mm. plastic Petri dishes were infected with a high multiplicity of HSV type I, Bramson strain, obtained from Dr. Gordon Plummer. After 12 or 18 hours of incubation, cells were scraped and washed three times in Tris-buffered saline, ph 7.2. The cells were then treated with pre-adsorbed anti-hsv IgG for 40 minutes at 4 C. with gentle agitation. They were then washed twice. Hybrid antibody, 50 Mg per milliliter, was added to the cells and they were further incubated at 4 C. for 30 minutes with gentle agitation. The cells were centrifuged and resuspended in 1.0 ml. of Tris-buffered saline containing recrystallized ferritin, 0.35 mg. per milliliter, for 20 minutes. Cells were washed three times with Tris-buffered saline and the pellet fixed in 3 per cent glutaraldehyde for 24 hours. Electron microscopy. Glutaraldehyde-fixed cells were postfixed in osmium, embedded in Epon, and sectioned on a Sorvall MT II microtome. Unstained and uranyl and lead citrate-stained sections were examined in a Hitachi electron microscope. Results Replication of HSV in rabbit corneal cells. Fig. 1 shows the replication of HSV in rabbit corneal cells after infection with 5.2 x 10 G plaque-forming units per 0.2 ml. Progeny virus appeared six to eight hours after the time of infection and increased in titer for the following 12 to 18 hours. Microscopically, a cytopathic effect was evident by 10 to 12 hours; by 24 hours the cells were rounded and many had detached from the surface of the dish. Electron microscopy. By 12 to 18 hours, infected cells showed degenerative changes in the nucleus and cytoplasm. The chromatin became beaded and marginated, mitochondria were swollen and the cristae fragmented, the endoplasmic reticulum was dilated and formed vesicles which occasionally contained electron-dense material. HSV developed in the nuclei as small dense cores surrounded by capsids. As virus passed into the cytoplasm, it often became invested (enveloped) by a thickened localized area of the inner nuclear membrane (Fig. 2). Less frequently, unenveloped virus was seen in the perinuclear space or in the cytoplasm. In the cytoplasm, enveloped virus was chiefly confined within smooth-surface tubules or vesicles which seemed to be formed by the endoplasmic reticulum. Unenveloped virus in the cytoplasm became enveloped as it budded into these vesicles or tubules. Excessive reduplication of nuclear membranes which occur in HEp-2 cells infected with HSV was not seen in the rabbit corneal cells. Extracellular virus was visible along the external cell surface as single particles or in clumps often associated with cytoplasmic fragments or amorphous material, i.e., the products of disrupted cells. Budding of virus from the cell surface was not observed. Immunolabeling of HSV-infected rabbit corneal cells. The reaction of anti-hsv

5 Volume 13 Number 11 Ultrastructural localization of HSV antigens 823 Fig. 4. Adjacent infected cells with ferritin labeling of surface virus (arrows) and cytoplasmic membranes. Unstained section. (x60,000.) IgG, hybrid antibodies, and ferritin with the cell surface is shown diagrammatically in Fig. 3. As seen in the figure, the hybrid antibody which has two combining sites forms a bridge between the HSV IgG bound to the cell and the visual marker ferritin. Ferritin labeling was evident in both stained and unstained sections, although the label was better demonstrated in the unstained sections. On intact cells, label was visible as small electron-dense grains aligned along the surface of the cell and around extracellular virus (Fig. 4). A clear zone, often visible between the ferritin and cell surface or virus particle, apparently represented the hybrid antibody bridge (Fig. 3). In all experiments, virtually the entire surface of infected cells was labeled with ferritin. However, the label was irregularly distributed over the surface, often being concentrated over areas of focal plasma membrane thickening (Fig. 5). In sections stained with uranyl and lead citrate, these thickened areas morphologically re- Fig. 5. Ferritin labeling along surface of infected cell. Observe ferritin concentrated over area of thickened plasma membrane (arrow). Unstained section. (xl50,000.)

6 8 4 Benson et al. Investigative Ophthalmology November 1974 Fig. 6. Disrupted cell showing ferritin labeling of intracellular virus (short arrow) and external nuclear membrane (long arrow). Unstained section. (x52,500.) sembled the envelope of HSV virions. Cytoplasmic projections and loose extracellular cytoplasmic fragments from disrupted cells were usually labeled. In cells with disrupted plasma membranes, ferritin was localized along many of the intracytoplasmic membranes and at the nuclear membrane (Figs. 6 and 7). Failure of all cytoplasmic membranes to label is believed to be the result of inadequate diffusion of ferritin and hybrid antibodies through the cytoplasm. Cytoplasmic membranes which contained ferritin appeared to be diffusely labeled while on the nuclear membrane the ferritin was focal in distribution. Fused cytoplasmic membranes were labeled along both sides. Mitochondrial membranes were not labeled. Label was present around extracellular virus and around fully enveloped intracellular virus in disrupted cells (Fig. 6). Label was not seen on unenveloped virus whether intracytoplasmic or intranuclear. Although scattered ferritin grains were often seen in the nucleus of disrupted infected cells, no specific structures were labeled. Ferritin labeling was not present on uninfected corneal cells. To determine if the surface distribution of ferritin on infected corneal cells was similar to the distribution on other cell types, Vero cells, an epithelial cell line derived from the kidney of a Vervet monkey, were infected with HSV and processed for electron microscopy after immunolabeling. The distribution of label on HSV-infected Vero cells was similar to the distribution on HSV-infected corneal cells.

7 Volume 13 Number 11 Ultrastructural localization of HSV antigens 825 Fig. 7. Ferritin labeling of cytoplasmic membrane (arrow) and intracellular enveloped virus in a disrupted corneal cell. Unstained section. (x90,000.) Discussion Three procedures can be used for immunolabeling of cells with ferritin the direct, indirect, and hybrid antibody method. In the direct method, the visual marker, ferritin, is coupled directly to the antibody under study which can then be used to label the cells. The indirect method involves the coupling of ferritin to anti-igg antibody which can then be added to the cells after previous incubation with antibody. In the latter method, the ferritintagged IgG binds to the IgG which has bound to the antigens on the cell. In both the direct and indirect methods, ferritin is coupled directly to IgG by chemical reagents, which might be a disadvantage because these reagents may partially inactivate or reduce antibody activity by reacting with active sites. In contrast, in the hybrid antibody method, ferritin is bound immunologically to IgG. In this method, F(ab') fragments of anti-igg are joined by disulfide bonds to F(ab') fragments of antiferritin antibody to produce an antibody that contains two reactive sites, one directed to IgG and the other to ferritin. Basically an indirect method, the hybrid antibody is added to the cells after addition of the IgG of interest. One of the combining sites of the hybrid binds to the IgG which is bound to the antigens on the surface of the cell while the other site is free to bind ferritin. In our study, we prepared

8 826 Henson et al. Investigative Ophthalmology November 1974 hybrid antibodies from sheep serum and from rabbit serum against human IgG and horse ferritin. Using sheep hybrid antibodies, we have demonstrated HSV antigens on the surface of infected rabbit corneal cells and on extracellular virus. In disrupted infected cells, label was also found along many of the intracytoplasmic membranes and on the nuclear membranes. Assuming that the localization of ferritin label corresponds to the distribution of viral antigens, these results indicate that HSV-induced antigens are associated with and widely distributed along surface and internal membranes of infected SIRC cells. Although scattered ferritin grains were occasionally seen in the nucleus, no specific structures were labeled. This lack of intranuclear label seems to indicate that human IgG (at least from our serum donor) does not contain antibody against capsomere protein or unenveloped virus. However, another explanation for lack of intranuclear labeling is that ferritin and/or antibody may not have diffused through the nuclear pores in sufficient concentration to label intranuclear antigens. Nonetheless, the distribution of label in corneal cells corresponds to the distribution of ferritin label in HSV-infected HeLa and human amnion (FL) cells as demonstrated by the direct method of immunolabeling. 5 Although the entire cell surface contained label, the ferritin tended to be concentrated over focal areas of plasma membrane thickening which morphologically resembled the envelope of extracellular virus. It seems reasonable that these altered segments of the plasma membrane contained the same antigens or similar reactive antigens that are components of the envelope of HSV. Studies have indicated that during replication, HSV as well as other members of the herpes group of viruses code for antigens which become incorporated or inserted into the cellular membranes and that these altered membranes invest the virus forming the envelope or outer coat Although we used a tissue culture line of rabbit corneal cells (SIRC), there is no reason to suspect that new antigens do not form on the surface of infected human corneal cells during HSV keratitis. By a variety of experimental methods, HSV-induced antigens have been demonstrated on the surface of many types of cells productively infected with HSV. 1 " 7 In studies in which the direct method of immunolabeling was used, FL and HeLa cells infected with HSV showed extensive ferritin labeling of both nuclear and cytoplasmic membranes." From these reported data, it is reasonable to conclude that human corneal cells productively infected with HSV acquire new antigens on their surface. Biological implications of HSV-induced antigens. Data have suggested that stromal keratitis results from a delayed hypersensitivity reaction to antigens associated with HSV infection. 1 ' 2 Indeed, since HSV antigens appear on the surface of infected cells, the possibility should be considered that these antigens are recognized as foreign by the host much like the HL-A antigens of an incompatible tissue graft are recognized as foreign. If this is the case, then the pathogenesis of stromal keratitis could be considered analogous to a host-versusgraft type reaction. In HSV infection, individual infected cells would be considered by the host to be an incompatible graft because of the development of foreign surface antigens. The concept that stromal keratitis is analogous to a host-versus-graft reaction is consistent with available information on HSV infection in the eye. Studies indicate that HSV infects not only corneal epithelium, but also endothelium and keratocytes, 15 " 18 and that chronic inflammatory cells infiltrate the corneal stroma during keratitis. Although mechanisms by which lymphocytes or other inflammatory cells terminate viral infections are unknown, in vitro studies indicate that lymphocytes release several cytotoxic substances following specific or nonspecific mitogenic stimulation Furthermore, a cytotoxic factor

9 Volume 13 Number 11 Ultrastructural localization of HSV antigens 827 which has a nonspecific killing effect can be released from the spleen cells of immunized mice after secondary stimulation with lymphocytic choriomeningitis virus.- 1 According to our concept, lymphocytes or other inflammatory cells responding to the presence of antigens on infected cells infiltrate the cornea and in the process of eliminating infected cells elaborate cytotoxic or lytic substances which cause tissue necrosis or destruction of collagen. In addition to demonstrating HSV-induced antigens on membranes of infected rabbit corneal cells, these studies have demonstrated the utility of using sheep antihuman IgG antihorse ferritin F(ab')- hybrid antibodies to localize antigenic changes on virus-infected cells. REFERENCES 1. Swyers, J. S., Lausch, R. N., and Kaufman, H. E.: Cornea] hypersensitivity to herpes simplex, Br. J. Ophthalmol. 51: 843, Meyers, R. L., and Pettit, T. H.: Corneal immune response to herpes simplex virus antigens, J. Immunol. 110: 1575, Kaufman, H. E.: The Cornea World Congress, Washington, D. C, 1964, London, 1965, Butterworths, p Roane, P. R., Jr., and Roizman, B.: Studies of the determinant antigens of viable cells. II. Demonstration of altered antigenic reactivity of HEp-2 cells infected with herpes simplex virus, Virology 22: 1, Nii, S., Morgan, C, Rose, H. M., et al.: Electron microscopy of herpes simplex virus. IV. Studies with ferritin-conjugated antibodies, J. Virol. 2: 1172, Brier, A. M., Wohlenberg, C, Rosenthal, J., et al.: Inhibition or enhancement of immunological injury of virus-infected cells, Proc. Nat. Acad. Sci. 68: 3073, Rosenthal, J., Hayashi, K., and Notkins, A. L.: Virus antigens on the surface of infected cells: binding and elution of (T-^I)-labeled antivirus antibody, J. Gen. Virol. 18: 195, Leerh0y, J.: Cytopathic effect of rubella virus in a rabbit-cornea cell line, Science 149: 633, Hammerling, U., Aoki, T., deharven, E., et al.: Use of hybrid antibody with anti-gamma C and antiferritin specificities in locating cell surface antigens by electron microscopy, J. Exp. Med. 128: 1461, Neauport-Sautes, C, Silvestre, D., Niccolai, M.-C, et al.: Ultrastructural localization of human HL-A membrane antigens by use of hybrid antibodies, Immunology 22: 833, Nii, S., Morgan, C, and Rose, H. M.: Electron microscopy of herpes simplex virus. II. Sequence of development. J. Virol. 2: 517, Underwood, P. A., and Greenham, L. W.: Herpes simplex infection of HEp-2 and L-929 cells. I. Comparative cytopathic effects and election microscopic studies, Microbios. 5: 121, Mark, G. E., and Kaplan, A. S.: Synthesis of proteins in cells infected with herpesvirus. VIII. Absence of virus-induced alteration of nuclear membrane in arginine-deprived cells, Virology 49: 102, Heine, J. W., Spear, P. G., and Roizman, B.: Proteins specified by herpes simplex virus. VI. Viral proteins in the plasma membrane, J. Virol. 9: 431, Irvine, A. R., and Kimura, S. J.: Experimental stromal herpes simplex keratitis in rabbits, Arch. Ophthalmol. 78: 654, Pavan-Langston, D., and Nesburn, A. B.: The chronology of primary herpes simplex infection of the eye and adnexal glands, Arch. Ophthalmol. 80: 258, Pettit, T. H., and Johnson, H. M.: A study of experimental herpes simplex keratitis, IN- VEST. OPHTHALMOL. 5: 112, (Abstr.) 18. Dawson, C, Togni, B., and Moore, T. E., Jr.: Structural changes in chronic herpetic ikeratitis studied by light and electron microscopy, Arch. Ophthalmol. 79: 740, Granger, G. A., and Kolb, W. P.: Lymphocyte in vitro cytotoxicity: mechanisms of immune and nonimmune small lymphocyte mediated target L-cell destruction, J. Immunol. 101: 111, Williams, T. W., and Granger, G. A.: Lymphocyte in vitro cytotoxicity: mechanism of lymphotoxin-induced target cell destruction, J. Immunol. 102: 911, 1969'. 21. Granger, G. A., Moore, G. E., White, J. G., et al.: Production of lymphotoxin and migration inhibitory factor by established human lymphocytic cell lines, J. Immunol. 104: 1476, Chandler, J. W., Heise, E. R., and Weiser, R. S.: Induction of delayed-type sensitivitylike reactions in the eye by the injection of lymphokines, INVEST. OPHTHALMOL. 12: 400, Oldstone, M. B. A., and Dixon, F. J.: Tissue injury in lymphocytic choriomeningitis viral infection: virus-induced immunologically specific release of a cytotoxic factor from immune lymphoid cells, Virology 42: 805, 1970.