Comp. lmmun. Microbiol. infect. Dis. Vol. 14, No. 2, pp. 97-105, 1991 Printed in Great Britain. All rights reserved
0147-9571/91 $3.00+0.00 Copyright © 1991 Pergamon Press plc
H U M O R A L I M M U N E RESPONSE TO H U M A N C Y T O M E G A L O V I R U S PROTEINS: A BRIEF REVIEW M . P. LANDINI* a n d M . LA PLACA Institute of Microbiology, Faculty of Medicine, University of Bologna, Bologna, Italy Abstract--Although human cytomegalovirus (HCMV) has a genome of 150 x 106Da, and a protein-coding content of over 200 open reading frames, few viral proteins seem able to elicit a strong antibody response in the natural host during viral infection. The immunodominant polypeptides include a component of 72 kDa among immediate early proteins, a polypeptide of 52 kDa among delayed early proteins and a glycoprotein complex of 58 and 93 130 kDa and two phosphoproteins of mol. wt 150 and 65 kDa among the structural proteins. Following a general overview of the humoral immune response, this brief survey mainly deals with the antibody response to these proteins. As significant epitopes of the major HCMV immunogenic polypeptides have been expressed in procaryotic cells over the last few years, an overview of the state of the art in this particular field will also be given. Key words: cytomegalovirus, proteins, antigens, antibody response, diagnosis, recombinant pro-
teins, acute infection, latent infection, primary infection, secondary infection.
LA RI~PONSE HUMORALE AUX CYTOMI~GALOVIRUS DE L'HOMME:
PROTI~INES DU UNE BRI~VE REVUE
Rfsumf~-Le cytomfgalovirus humain, avec un gfnome de 150 x 1 0 6 Da, peut thforiquement coder pour environ 200 protfines. Cependant les protfines fortement immunogfniques dans l'infection naturelle sont relativement peu nombreuses. En particulier, elles sont reprfsentfes par une protfine de 72 kDa, parmi les protfines prfcoces immfdiates, par une protfine de 52 kDa, parmi les protfines prfcoces, et par un complexe de glycoprotfines de 58 et 93-130 kDa et deux phosphoprotfines de 150 et 65 kDa, parmi les protfines structurales. Darts cette brfve revue, aprfs une description gfnfrale de la rfponse immunitaire dans l'infection naturelle, il sera fait mention de la rfponse humorale vers les difffrents polypeptides immunogfniques et de la possibilit6 d' utilisation pratique des polypeptides immunog6niques exprimfs dans des cellules procaryotes. Mots-clefs: cytomfgalovirus, protfines, antigfnes, rfponse anticorps, diagnose, protfines
recombinantes, infection aigu~, infection latente, infection primaire, infection secondaire.
INTRODUCTION C y t o m e g a l o v i r u s e s ( C M V ) are u b i q u i t o u s a g e n t s t h a t c o m m o n l y infect m a n y a n i m a l s , s u c h as g u i n e a pigs, m i c e , rats, h a m s t e r s , m o n k e y s , m o l e s , voles, c h i m p a n z e e s , d o g s a n d horses. T h e s e v i r u s e s a r e h i g h l y species-specific a n d by a n d large, t h e r e is v e r y little i n t e r s p e c i e s c r o s s i n g b e t w e e n C M V s (for r e v i e w see Ref. [1]). *To whom all correspondence should be addressed at Institute of Microbiology, Faculty of Medicine, S. Orsola General Hospital, Via Massarenti 9, 40138 Bologna, Italy. 97
98
M.P. LANDINI and M. LA PLACA
Human CMV (HCMV) is an important pathogen for humans. Infection can occur throughout life, and between 28 and 100% of a given population over 35 years old will be seropositive for CMV. Similar to other herpesviruses, CMV remains in the host in a latent state, after primary infection. Usually infection of the immunocompetent host with this agent does not cause clinical symptoms, whereas infection of the immunocompromised host often give rise to severe and even fatal disease. Morbidity and mortality associated with primary CMV infection or with reactivation from latency is common in immunosuppressed transplant recipients and in patients with immunodeficiency caused by HIV infection. Furthermore intrauterine HCMV infections are second only to Down's syndrome as a known cause of mental retardation. In modern medicine, therefore, HCMV is a major pathogen whose control by means of immunization, drug therapy and early diagnosis has become an important objective. Viral diagnosis is best accomplished either by direct detection of the infectious virus by its isolation in human fibroblast cell cultures or by demonstration of the viral genome or viral antigens with molecular probes or immunological reagents respectively. Other procedures for indirect demonstration of the virus are based on the determination of antiviral antibodies by means of serological techniques, but none of these procedures has ever proved completely straightforward. It is generally accepted that one of the problems hampering HCMV serodiagnosis is the use of poorly defined viral antigens. HCMV phospho- and glyco-proteins represent dominant antigens for the humoral immune response. Therefore a detailed characterization of the most immunogenic proteins is crucial not only to understand the immune response to the virus but also to develop better diagnostic reagents. In this brief review we describe results, mainly obtained in our laboratory, regarding the humoral immune response to the major HCMV antigenic proteins. Furthermore as significant epitopes of these polypeptides have been expressed in procaryotic cells over the last 5 years, overview of the results obtained in this particular field will also be given. HCMV PROTEINS The virus has a genome of 150 x 106 Da, which has been almost completely sequenced [2]. A preliminary analysis of the HCMV genome has recently shown that it has a potential protein-coding content of over 200 open reading frames. Furthermore each protein may be subject to post-translational modification by cleavage phosphorylation, glycosylation or sulphation. The spectrum of HCMV proteins is therefore complex, but our understanding of this field has improved greatly in the last 5 years, mainly as a result of the application of monoclonal antibodies and recombinant DNA technology. After infection of a susceptible cell, the temporal expression of the viral genome is closely controlled. Viral genes are expressed at immediate early (1-2 h), delayed early (2-24 h) and late (after 24 h) times after infection and the presence of the earlier gene products is a prerequisite for the expression of the later products [3]. Most early proteins are non-structural proteins and have defined or potential regulatory functions, while the majority of the structural proteins are transcribed after viral DNA synthesis (late or gamma proteins). Molecular studies have shown that the first transcription products from the three immediate early genes are translated in vitro into seven polypeptides (alpha proteins), a
99
Immune response to CMV proteins
72 kDa being the most prominent. In vivo three additional polypeptides are found in the first hour after infection (for review see Ref. [4]). Twenty-five delayed early proteins (also called beta proteins) have been described of which there is particular interest in the viral DNA polymerase (mol. wt 145 kDa) and a major DNA binding protein (mol. wt 52 kDa) whose function has yet to be defined [4]. The genomic location of the genes coding for the major immediate and delayed early proteins is shown in Fig. I(A).
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Fig. 1. Genomic locations of the major non-structural and structural protein genes of HCMV. The restriction map for the enzymes HindllI and EcoRl is given for strain ADI69. (A) Locations of the genes coding for the major non structural proteins, gp = glycoprotein; IEP = immediate early protein; DB = DNA-binding protein; pol = DNA-polymerase; EP = delayed early protein. (B) Locations of the genes coding for the major structural glycoproteins are shown. EP = early protein; gC = glycoprotein complex; IMP = integrate membrane protein. (C) Locations of the genes coding for the major structural phosphoproteins. PK = protein kinase; AP = assembly protein.
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M . P . LANDINI and M. LA PLACA
After viral DNA replication approx. 55 proteins (late or gamma proteins) have been described, with 32 descriptions of glycopeptides and 23 of phosphopolypeptides. Of particular interest are the proteins composing the viral structure whose number has yet to be ascertained. However, there is general agreement about the presence of several major polypeptides of the following mol. wt: 200, 150 (2 proteins), 130, 116, 86, 74, 68, 64, 58, 55, 52, 49, 45, 38 and 28 kDa [4]. The genomic locations of the regions coding for the major structural glycoproteins and phosphoproteins are indicated in Fig. I(B) and (C) respectively. It is also noteworthy that HCMV acquires its final envelope either from the plasma membrane or from the intracytoplasmatic vacuoles and saccules of the Golgi apparatus [5, 6] and that several proteins of cellular origin have been repeatedly found associated with extensively purified viral particles. In this respect it is particularly interesting that Michelson et al. [7, 8] found that a mouse monoclonal antibody which neutralizes HCMV-infectivity reacts with a normal cell membrane component also present in the viral envelope [7]. HUMORAL IMMUNE RESPONSE TO NONSTRUCTURAL PROTEINS At least four alpha proteins (mol. wt 78, 72, 68 and 30 kDa) are immunoprecipitated from cell extracts mainly by antibody from acutely infected individuals. Among them the 72kDa seems the most important immunogen for both humoral and cell mediated immunity [8-10]. Within the first 6 h after infection and before viral DNA synthesis, 17 other polypeptides are immunoprecipitated by HCMV immune sera. Among them, a protein of mol. wt 52 kDa strongly reacts with antibodies present in sera from patients with ongoing CMV infection [i 1]. In general the reactivity to nonstructural proteins is higher with sera from acutely infected individuals than with sera from long-term seropositive subjects. This is consistent with data previously obtained by immunofluorescence showing that antibodies to immediate early and early antigens are preferentially associated with the acute phase of the infection [12]. HUMORAL IMMUNE RESPONSE TO STRUCTURAL GLYCOPROTEINS The humoral immune response to viral structural glycoproteins is particularly interesting and clinically important as glycoproteins are mainly localized in the viral envelope. They are the target for virus neutralizing antibody (for review see Ref. [13] and are therefore the best candidates in vaccine development for prevention of HCMV infection [14]. As summarized in Table 1 there are three glycoprotein complexes present in the viral envelope, recently named gCI, gCII and gCIII [15-18]. At least two of these complexes (gCI and gCIII) trigger the synthesis of HCMV-neutralizing antibodies when inoculated into experimental animals. While antibodies reacting with gCI do need the complement Table 1. Glycoprotein complexes of the HCMV envelope Mol. wt ( × 103 Da) Complexes gCl gCn gCln
Non-reduced
Reduced
References
250-300, 190, 160 93-300 240
93-130, 55 58 39-48, 47-63 145, 86
[13, 16, 19, 33] [13, 16, 17, 18] [13, 16, 43]
I m m u n e response to C M V proteins
101
to neutralize the viral infectivity, antibodies to gCIII are complement independent as are monoclonal antibodies to gCII. Very recent results presented at the 15th International Herpesvirus Workshop (Washington, D.C., August 1990) support the possibility that gCI is involved in the binding to the cellular receptor (30 kDa), while gCIIl is responsible for fusion between viral envelope and cell membrane through the binding to a cellular protein of mol. wt 92 kDa. During natural infection as well as after experimental vaccination, gCI seems to be the immunodominant antigen among the glycoproteins [14, 19]. In fact antibodies to gCI can be detected at high frequencies in patients having medium or high anti-HCMV ELISA titres: much and often most [between 50 and 70%] of human serum neutralizing activity has been shown to be directed against a single gCI complex [19]. Another glycoprotein present in an integrated form in the envelope of HCMV (45 kDa) and which is conserved among herpesviruses has been shown to stimulate the production of antibodies in vivo [20] but their appearance and evolution has not yet been studied. HUMORAL IMMUNE RESPONSE TO STRUCTURAL PHOSPHOPROTEINS Viral structural phosphoproteins are mainly localized in the viral tegument and/or in the capsid. As recently reviewed by Jahn and Mach [21] and summarized in Table 2, the HCMV virion contains four major immunogenic phosphopolypeptides of the following ml. wts 150, 65, 38 and 28 kDa. There is evidence in the literature [22-25] indicating that a polypeptide of mol. wt 150kDa is the most immunogenic. In fact high antibody titres to this protein have repeatedly been found irrespective of the stage of infection in nearly 100% of HCMV seropositive subjects. All four IgG subclasses of antibodies are induced by this polypeptide [24] and the overall immune response persists for years after convalescence when the antibody to the other HCMV polypeptides has disappeared. There are two proteins with an apparent molecular weight of 150 kDa (the phosphorylated structural component of the viral tegument and the major capsid protein) and differential migration in high bisacrylamide gels has shown that the phosphorylated polypeptide is responsible for the strong antigenic stimulation [25]. Another immunogenic phosphoprotein is the most abundant structural constituent of HCMV, the so-called lower matrix protein which is particularly plentiful in the viral dense bodies [26]. The antibody response to this protein is very high during the acute phase of infection, as well as during the early convalescence, but decreases rapidly thereafter [22]. Both p150 and p65 are highly recognized by anti-HCMV specific IgM during acute infection. Early during the course of a naturally occurring primary CMV infection as well as after injection of seronegative individuals with the Toledo strain vaccine [14] a very low or Table 2. Main phosphoproteins present in HCMV virions Protein mol. wt ( x 103 Da) p150 p65 p38 p28 *DB = dense bodies.
Description Basic phosphoprotein Lower matrix protein Assembly protein
Intraviral location
References
Matrix Matrix DB* Capsid Capsid
[21, 25, 30] [21, 25, 27] [26, 29] [27, 30, 31, 34]
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M.P. LAND1NIand M. LAPLACA
completely absent antibody response to p150 was detected, while Ig are found reacting with p65 [14, 28]. This suggests that the lack of antibody to p150 or its presence in lower amounts than Ig to p65 could be a useful marker for primary HCMV infection [28]. While the antibody response to p150 and p65 is due to both IgG and IgM, the response to other structural phosphoproteins is almost exclusively due to only one antibody class. The reactivity to p38 (which corresponds to the assembly protein described by Gibson, Ref. [29]) is mainly due to IgM and in several cases of secondary (reactivated or exogenous reinfection) CMV acute infection IgM exclusively reacting with p150 and p38 are detectable [23]. On the contrary, antibody response to the capsidic phosphoprotein p28, is almost solely due to IgG and evolves in a similar fashion to the response already described for p150 although the antibody titres detected in patients sera are generally lower [30, 31]. HUMORAL IMMUNE RESPONSE TO RECOMBINANT HCMV PROTEINS Within the last 5 years, several fusion proteins containing significant epitopes of the major HCMV immunogenic polypeptides have been expressed in procaryotic cells and studied by some European research groups: Institute of Clinical and Molecular Virology of the University of Erlangen (Germany), Laboratory of Biotechnology in Braunschweig (Germany) and Institute of Microbiology, Medical Faculty, University of Bologna (Italy) ([32-40], W. Lindenmeier, A. Necker, S. Krauss, R. Bonewald and J. Collins, unpublished data). Several studies have been carried out in cooperation in order to evaluate the effectiveness of the expressed epitopes as antigenic material for serological procedures. In a recent study a large number of human sera with different anti-HCMV antibody titres as detected by ELISA were tested for IgG and IgM reactivity to 10 different fusion proteins containing antigenic epitopes of the major structural proteins of 150, 74, 65, 38 and 28 kDa and the nonstructural DNA-binding protein of mol. wt 52 kDa [40]. The results obtained showed that HCMV can be replaced by some well characterized recombinant viral proteins in the serological evaluation of anti-HCMV antibodies. Significant portions of p150, p65, p52 and p38 were shown to represent an antigenic complex specific enough to detect HCMV IgM. Furthermore accurate evaluation of anti-HCMV IgG was best accomplished using a higher number of recombinant proteins. In addition to the clones necessary for IgM detection, another clone containing a different portion of p150 and a clone containing a large part of p28 seemed to be necessary to obtain an antigenic complex sensitive enough to detect even low levels of antibodies due to a past infection. In other experiments the antibody reactivity of two clones (G2 and DI) was studied in more detail [36, 39, 40]. One clone expressed a portion (from aminoacid 1071 to 1301) of the major DNA binding non-structural protein of mol. wt 52 kDa (G2) and the other a small fragment (the last 25 aminoacids at the carboxy terminus) of the major structural phosphoprotein of tool. wt 150kDa (DI). While antibodies to G2 were shown to be preferentially present during acute infection, antibodies to D1 were abounded during convalescence. Comparison of the reactivity obtained with acutely infected patients and long-term seropositive people disclosed a lack of reactivity to D1 in the first group and a lack of reaction to G2 in the second group. Therefore the differential reactivity to these two recombinant antigens was shown to be able to distinguish between a latent and an active HCMV infection. It is well known that acute HCMV infection include primary and secondary infection (ex novo exogenous reinfection or, more frequently, reactivated from a latent state). The
Immune response to CMV proteins
103
study of IgG and IgM reactivity to G2 and D1 in sera from patients with primary or secondary infections showed that IgM to G2 are preferentially present during primary HCMV infection and therefore these antibodies could represent a very important marker of acute primary infection. In view of the results obtained we think that in the near future it will be possible to use bacteria-produced recombinant HCMV proteins in the serodiagnosis of HCMV infection. This should make it easier to standardize antigenic material to be used in serological tests such as ELISA or latex agglutination. Standardization of antigenic complexes to be used in CMV serodiagnosis should greatly reduce the occurrence of conflicting serological results with the same serum sample and two different antibody detection kits [41, 42]. A drawback to the large scale use of bacterially purified HCMV antigens in serological tests could arise from the presence of antibodies against E. coli proteins in human sera that could give rise to a high number of false-positive reactions. In this respect 3% of sera showing the presence of anti-E, coli beta-galactosidase antibody was found [40] suggesting that it would be useful to express important epitopes as nonfusion proteins. Furthermore some reactivity to other E. coli proteins was often found, indicating that it could also be advantageous to purify the fusion proteins from the whole cell lysates. CONCLUSIONS
Many problems still remain especially in the interpretation of the data regarding antibody recognition. In fact all the studies on the humoral immune response to CMV proteins are carried out by immunoblotting or immunoprecipitation and both techniques have negative aspects. It is known that in immunoblotting some important epitopes (especially glycosidic residues) involved in the reaction with antibody can be destroyed [43] and therefore the reaction of some proteins can be underestimated. On the other hand the reactivity of IgG3 cannot be evaluated by immunoprecipitation as IgG3 do not bind Staphylococcus-protein A, leading to an underestimation of the antibody reactivity with proteins that induce an IgG3 response [24]. In addition some proteins of similar molecular weight can easily co-migrate in SDS-PAGE and antibody reactivity may overlap. An unequivocal analysis of the humoral immune response needs technical improvements and an increased use of cloned gene products. However, we think that the studies summarized in this brief review represent an important step towards our understanding of the immune response to HCMV and to the development of more reliable diagnostic tests. Acknowledgements--Personal work presented in this review has been partially supported by C.N.R. and by Italian Ministry of Health (AIDS projects 1990, 1991).
REFERENCES 1. Ho M. Cytomegalovirus. In Biology and Infection. Current Topics in Infectious Disease (Edited by Greenough III and Merigan T.). Plenum Press, New York (1982). 2. Chee M. S., Bankier A. T., Beck S., Bohni C. M., Brown C. M., Cerny R., Hornsell T., Hutchinson III C. A., Kouzarides T., Martignetti J. A., Preddie E., Satchwell S. C., Tomlinson P., Weston K. M. and Barrell B. G. Analysis of the protein content of the sequence of human cytomegalovirus strain ADI69. In Current Topics in Microbiology and Immunology (Edited by McDougall J. K.), Vol. 54, pp. 125-169. Springer, Berlin (1990). 3. Whaten M. W., Thomsen D. R. and Stinski M. F. Temporal patterns of human cytomegalovirus transcription: mapping the viral RNAs synthesized at immediate early, early and late times after infection. J. Virol. 41, 462--477 (1981). 4. Landini M. P. and Michelson S. Human cytomegalovirus proteins. Prog. med. Virol. 35, 152-185 (1988).
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5. Landini M. P., Severi B., Badiali L., Gonczot E. and Mirolo G. Morphological components of human cytomegalovirus: in situ localization of the major glycoprotein. Intervirology 27, 154-160 (1987). 6. Severi B., Landini M. P. and Govoni E. Human cytomegalovirus morphogenesis: an ultrastructural study of the late cytoplasmic phases. Archs Virol. 98, 51 64 (1988). 7. Michelson S., Tardy-Panit M., Colimon R. and Landini M. P. A human cytomegalovirus neutralizing monoclonal antibody recognizes a normal cell protein. J. gen. Virol. 70, 673 684 (1989) 8. Michelson S., Horodniceanu F., Kress M. and Tardy-Panit M. Human cytomegalovirus immediate early antigen. Analysis in SDS PAGE after immunoprecipitation. J. Virol. 32, 259-267 (1979). 9. Rodgers B., Borysiewicz L., Mundin J., Graham S. and Sissons P. Immunoattinity purification of a 72K early antigen of human cytomegalovirus: analysis of humoral and cell-mediated immunity to the purified polypeptide. J. gen. Virol. 68, 2371-2378 (1985). 10. Borysiewicz L. K., Hickling J. K., Graham S., Sinclair J., Cranage M. P., Smith G. L. and Sissons J. G. P. Human cytomegalovirus-specific cytotoxic T cells. Relative frequence of stage specific CTL recognizing the 72 Kd immediate early protein and glycoprotein B-expressed by recombinant vaccinia viruses. J. exp. Med. 168, 919-931 (1988). 11. Ripalti A., Landini M. P., Mocarski E. and La Placa M. Identification and preliminary use of recombinant lambda gt 11 fusion proteins in Cytomegalovirus diagnosis. J. gen Virol. 70, 1247-I 251 (1989). 12. Landini M. P. Antibody response against early antigens in Herpesviridae infections. Eur. J. EpidemioL 1, 62-66 (1985). 13. Rasmussen L. Immune response to human cytomegalovirus infection. In Current Topics in Microbiology and Immunology (Edited by McDougall J. K.), Vol. 54, pp. 221~54. Springer, Berlin (1990). 14. Gonczol E. and Plotkin S. A. Progress in vaccine development for prevention of human cytomegalovirus infection. In Current Topics in Microbiology and Immunology (Edited by McDougall J. K.), Vol. 54, pp. 254-274. Springer, Berlin (1990). 15. Gretch D. R., Gehrz R. C. V. and Stinski M. F. Characterization of a human cytomegalovirus glycoprotein complex (gCI). J. gen. Virol. 69, 1205 1215 (1988). 16. Gretch D. R., Kari B., Rasmussen L., Gehrz R. C. and Stinski M. F. Identification and characterization of three distinct families of glycoprotein complexes in the envelope of human cytomegalovirus. J. Virol. 62, 875-881 (1988). 17. Gretch D. R., Kari B., Gehrz R. C. and Stinski M. F. A multigene family encodes the human cytomegalovirus glycoprotein complex gCII (gp47-52 complex). J. Virol. 62, 1956 1962 (1988). 18. Kari B., Goertz R. and Gehrz R. C. Characterization of cytomegalovirus glycoproteins in a family of complexes designated gCII with murine monoclonal antibodies. Archs Virol. 112, 55 65 (1990). 19. Britt W. J., Vugler L., Butfiloski E. J. and Stephens E. B. Cell surface expression of human cytomegalovirus (HCMV) gp55 116 (gB): use of HCMV-recombinant vaccinia virus-infected cells in analysis of the human neutralizing antibody response. J. Virol. 64, 1079-1085 (1990). 20. Lehner R., Meyer H. and Mach M. Identification and characterization of a human cytomegalovirus gene coding for a membrane protein that is conserved among human herpesviruses. J. Virol. 63, 3792-3800 (1989). 21. Jahn G. and Mach M. Human cytomegalovirus phosphoproteins and glycoproteins and their coding regions. In Current Topics in Microbiology and Immunology (Edited by McDougall J. K.), Vol. 54, pp. 171-185, Springer, Berlin (1990). 22. Landini M. P., Mirolo G., Baldassarri B. and La Placa M. human immune response to cytomegalovirus structural polypeptides studied by immunoblotting. J. med. Virol. 17, 303-311 (1985). 23. Landini M. P., Mirolo G., Coppoleechia P., Re M. C. and La Placa M. Serum antibodies to individual cytomegalovirus structural polypeptides in renal transplant recipients during viral infection. Microbiol. Immunol. 30, 683-695 (1986). 24. Landini M. P., Baldassarri B., Mirolo G., Ripalti A. and La Placa M. Reactivity of cytomegalovirus structural polypeptides with different subclasses of IgG present in human sera. J. Infect. 16, 163-167 (1988). 25. Jahn G., Scholl B.-C. and Fleckenstein B. The two major structural phosphoproteins (pp65 and ppl50) of human cytomegalovirus and their antigenic properties. J. gen. Virol. 68, 1327-1337 (1987). 26. Gibson W. Protein counterparts of human and simian cytomegaloviruses. Virology 128, 391-406 (1983). 27. Landini M. P., Severi B., Furlini G. and Badiali L. Human cytomegalovirus structural components: intracellular and intraviral localization of p28 and p65-69 by immuno-electron microscopy. Virus Res. 8, 15-23 (1987). 28. Landini M. P., Rossier E. and Schmitz E. Antibodies to human cytomegalovirus structural polypeptides during primary infection. J. Virol. Meth. 22, 309-317 (1988). 29. Irmiere A. and Gibson W. Isolation of human cytomegalovirus intranuclear capsids, characterization of their protein constituents and demonstration that the B-capsid assembly protein is also abundant in noninfectious enveloped particles. J. Virol. 56, 277-283 (1985). 30. Re M. C., Landini M. P., Coppoleechia P., Furlini G. and La Placa M. A 28,000 d human cytomegalovirus structural polypeptide studied by means of a specific monoclonal antibody. J. gen. Virol. 66, 2507 2511 (1985).
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31. Meyer H., Bankier A., Landini M. P., Ruger B. and Mach M. Identification and prokaryotic expression of the gene coding for the highly immunogenic 28 Kd structural phosphoprotein (pp28) of human Cytomegalovirus. J. ViroL 62, 2243-2250 (1988). 32. Jahn G., Kouzarides T., Mach M., Scholl B. C., Plachter B., Predy R., Satchwell S. C., Traupe B., Fleckenstein B. and Barrell B. G. Map position and nucleotide sequence of the gene for the large structural phosphoprotein of human cytomegalovirus. J. Virol. 61, 1358-1367 (1987). 33. Mach M., Utz U. and Fleckenstein B. Mapping of the major glycoprotein gene of human cytomegalovirus. J. gen. Virol. 67, 1461-1467 (1986). 34. Meyer H. A., Bankier A. T., Landini M. P., Brown C. M., Barrell B. G., Ruger B. and Mach M. Identification and prokaryotic expression of the gene coding for the highly immunogenic 28-kilodalton structural phosphoprotein (pp28) of human cytomegalovirus. J. Virol. 62, 2243-2250 (1988). 35. Mocarski E. S., Pereira L. and Michael N. Precise localization of genes on large animal virus genomes: use of lambda gtl 1 and monoclonal antibodies to map the gene for a cytomegalovirus protein family. Proc. natn. Acad. Sci. U.S.A. 82, 1266-1270 (1985). 36. Ripalti A., Landini M. P., Mocarski E. S. and La Placa M. Identification and preliminary use of recombinant lambda gtll fusion proteins in human cytomegalovirus diagnosis. J. gen. Virol. 70, 1247-1251 (1989). 37. Scholl B. C., Von Hintzenstern J., Borisch B., Traupe M., Broker M. and Jahn G. Prokaryotic expression of immunogenic polypeptides of the large phosphoprotein (ppl50) of human cytomegalovirus. J. gen. Virol. 69, 1195-1204 (1988). 38. Plachter B., Klages S., Hagelmann S., Britt W. Landini M. P. and Jahn G. Procaryotic expression of phosphorylated tegument protein pp65 of human cytomegalovirus and application of recombinant peptides for immunoblot analyses. J. clin. Microbiol. 28, 1229-1235 (1990). 39. Landini M. P., Lazzarotto T., Ripalti A., Guan M. X. and La Placa M. Antibody response to recombinant lambda gtll fusion proteins in Cytomegalovirus infection. J. clin. Microbiol. 27, 2324-2327 (1989). 40. Landini M. P., Guan M. X., Jahn G., Lindenmaier W., Mach M., Ripalti A., Necker A., Lazzarotto T. and Plachter B. Cytomegalovirus recombinant antigens: large scale screening with human sera. J. clin. Microbiol. 28, 1375-1379 (1990). 41. Re M. C. and Landini M. P. IgM to human cytomegalovirus: comparison of two enzyme immunoassays and lgM reactivity to viral polypeptides detected by immunoblotting. J. clin. Lab. Analysis 3, 169-173 (1989). 42. Miller H., McCulloch B., Landini M. P. and Rossier E. Comparison ofimmunoblotting with other serological methods and virus isolation for the early detection of primary cytomegalovirus infection in allograft recipients. J. clin. Microbiol. 27, 2672-2677 (1989). 43. Rasmussen L., Nelson M., Neff M. and Merigan T. Characterization of two different human cytomegalovirus glycoproteins which are targets for virus neutralizing antibody. Virology 163, 308 318 (1988).
0147-9571/91 $3.00+0.00 Copyright © 1991 Pergamon Press plc
H U M O R A L I M M U N E RESPONSE TO H U M A N C Y T O M E G A L O V I R U S PROTEINS: A BRIEF REVIEW M . P. LANDINI* a n d M . LA PLACA Institute of Microbiology, Faculty of Medicine, University of Bologna, Bologna, Italy Abstract--Although human cytomegalovirus (HCMV) has a genome of 150 x 106Da, and a protein-coding content of over 200 open reading frames, few viral proteins seem able to elicit a strong antibody response in the natural host during viral infection. The immunodominant polypeptides include a component of 72 kDa among immediate early proteins, a polypeptide of 52 kDa among delayed early proteins and a glycoprotein complex of 58 and 93 130 kDa and two phosphoproteins of mol. wt 150 and 65 kDa among the structural proteins. Following a general overview of the humoral immune response, this brief survey mainly deals with the antibody response to these proteins. As significant epitopes of the major HCMV immunogenic polypeptides have been expressed in procaryotic cells over the last few years, an overview of the state of the art in this particular field will also be given. Key words: cytomegalovirus, proteins, antigens, antibody response, diagnosis, recombinant pro-
teins, acute infection, latent infection, primary infection, secondary infection.
LA RI~PONSE HUMORALE AUX CYTOMI~GALOVIRUS DE L'HOMME:
PROTI~INES DU UNE BRI~VE REVUE
Rfsumf~-Le cytomfgalovirus humain, avec un gfnome de 150 x 1 0 6 Da, peut thforiquement coder pour environ 200 protfines. Cependant les protfines fortement immunogfniques dans l'infection naturelle sont relativement peu nombreuses. En particulier, elles sont reprfsentfes par une protfine de 72 kDa, parmi les protfines prfcoces immfdiates, par une protfine de 52 kDa, parmi les protfines prfcoces, et par un complexe de glycoprotfines de 58 et 93-130 kDa et deux phosphoprotfines de 150 et 65 kDa, parmi les protfines structurales. Darts cette brfve revue, aprfs une description gfnfrale de la rfponse immunitaire dans l'infection naturelle, il sera fait mention de la rfponse humorale vers les difffrents polypeptides immunogfniques et de la possibilit6 d' utilisation pratique des polypeptides immunog6niques exprimfs dans des cellules procaryotes. Mots-clefs: cytomfgalovirus, protfines, antigfnes, rfponse anticorps, diagnose, protfines
recombinantes, infection aigu~, infection latente, infection primaire, infection secondaire.
INTRODUCTION C y t o m e g a l o v i r u s e s ( C M V ) are u b i q u i t o u s a g e n t s t h a t c o m m o n l y infect m a n y a n i m a l s , s u c h as g u i n e a pigs, m i c e , rats, h a m s t e r s , m o n k e y s , m o l e s , voles, c h i m p a n z e e s , d o g s a n d horses. T h e s e v i r u s e s a r e h i g h l y species-specific a n d by a n d large, t h e r e is v e r y little i n t e r s p e c i e s c r o s s i n g b e t w e e n C M V s (for r e v i e w see Ref. [1]). *To whom all correspondence should be addressed at Institute of Microbiology, Faculty of Medicine, S. Orsola General Hospital, Via Massarenti 9, 40138 Bologna, Italy. 97
98
M.P. LANDINI and M. LA PLACA
Human CMV (HCMV) is an important pathogen for humans. Infection can occur throughout life, and between 28 and 100% of a given population over 35 years old will be seropositive for CMV. Similar to other herpesviruses, CMV remains in the host in a latent state, after primary infection. Usually infection of the immunocompetent host with this agent does not cause clinical symptoms, whereas infection of the immunocompromised host often give rise to severe and even fatal disease. Morbidity and mortality associated with primary CMV infection or with reactivation from latency is common in immunosuppressed transplant recipients and in patients with immunodeficiency caused by HIV infection. Furthermore intrauterine HCMV infections are second only to Down's syndrome as a known cause of mental retardation. In modern medicine, therefore, HCMV is a major pathogen whose control by means of immunization, drug therapy and early diagnosis has become an important objective. Viral diagnosis is best accomplished either by direct detection of the infectious virus by its isolation in human fibroblast cell cultures or by demonstration of the viral genome or viral antigens with molecular probes or immunological reagents respectively. Other procedures for indirect demonstration of the virus are based on the determination of antiviral antibodies by means of serological techniques, but none of these procedures has ever proved completely straightforward. It is generally accepted that one of the problems hampering HCMV serodiagnosis is the use of poorly defined viral antigens. HCMV phospho- and glyco-proteins represent dominant antigens for the humoral immune response. Therefore a detailed characterization of the most immunogenic proteins is crucial not only to understand the immune response to the virus but also to develop better diagnostic reagents. In this brief review we describe results, mainly obtained in our laboratory, regarding the humoral immune response to the major HCMV antigenic proteins. Furthermore as significant epitopes of these polypeptides have been expressed in procaryotic cells over the last 5 years, overview of the results obtained in this particular field will also be given. HCMV PROTEINS The virus has a genome of 150 x 106 Da, which has been almost completely sequenced [2]. A preliminary analysis of the HCMV genome has recently shown that it has a potential protein-coding content of over 200 open reading frames. Furthermore each protein may be subject to post-translational modification by cleavage phosphorylation, glycosylation or sulphation. The spectrum of HCMV proteins is therefore complex, but our understanding of this field has improved greatly in the last 5 years, mainly as a result of the application of monoclonal antibodies and recombinant DNA technology. After infection of a susceptible cell, the temporal expression of the viral genome is closely controlled. Viral genes are expressed at immediate early (1-2 h), delayed early (2-24 h) and late (after 24 h) times after infection and the presence of the earlier gene products is a prerequisite for the expression of the later products [3]. Most early proteins are non-structural proteins and have defined or potential regulatory functions, while the majority of the structural proteins are transcribed after viral DNA synthesis (late or gamma proteins). Molecular studies have shown that the first transcription products from the three immediate early genes are translated in vitro into seven polypeptides (alpha proteins), a
99
Immune response to CMV proteins
72 kDa being the most prominent. In vivo three additional polypeptides are found in the first hour after infection (for review see Ref. [4]). Twenty-five delayed early proteins (also called beta proteins) have been described of which there is particular interest in the viral DNA polymerase (mol. wt 145 kDa) and a major DNA binding protein (mol. wt 52 kDa) whose function has yet to be defined [4]. The genomic location of the genes coding for the major immediate and delayed early proteins is shown in Fig. I(A).
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Fig. 1. Genomic locations of the major non-structural and structural protein genes of HCMV. The restriction map for the enzymes HindllI and EcoRl is given for strain ADI69. (A) Locations of the genes coding for the major non structural proteins, gp = glycoprotein; IEP = immediate early protein; DB = DNA-binding protein; pol = DNA-polymerase; EP = delayed early protein. (B) Locations of the genes coding for the major structural glycoproteins are shown. EP = early protein; gC = glycoprotein complex; IMP = integrate membrane protein. (C) Locations of the genes coding for the major structural phosphoproteins. PK = protein kinase; AP = assembly protein.
100
M . P . LANDINI and M. LA PLACA
After viral DNA replication approx. 55 proteins (late or gamma proteins) have been described, with 32 descriptions of glycopeptides and 23 of phosphopolypeptides. Of particular interest are the proteins composing the viral structure whose number has yet to be ascertained. However, there is general agreement about the presence of several major polypeptides of the following mol. wt: 200, 150 (2 proteins), 130, 116, 86, 74, 68, 64, 58, 55, 52, 49, 45, 38 and 28 kDa [4]. The genomic locations of the regions coding for the major structural glycoproteins and phosphoproteins are indicated in Fig. I(B) and (C) respectively. It is also noteworthy that HCMV acquires its final envelope either from the plasma membrane or from the intracytoplasmatic vacuoles and saccules of the Golgi apparatus [5, 6] and that several proteins of cellular origin have been repeatedly found associated with extensively purified viral particles. In this respect it is particularly interesting that Michelson et al. [7, 8] found that a mouse monoclonal antibody which neutralizes HCMV-infectivity reacts with a normal cell membrane component also present in the viral envelope [7]. HUMORAL IMMUNE RESPONSE TO NONSTRUCTURAL PROTEINS At least four alpha proteins (mol. wt 78, 72, 68 and 30 kDa) are immunoprecipitated from cell extracts mainly by antibody from acutely infected individuals. Among them the 72kDa seems the most important immunogen for both humoral and cell mediated immunity [8-10]. Within the first 6 h after infection and before viral DNA synthesis, 17 other polypeptides are immunoprecipitated by HCMV immune sera. Among them, a protein of mol. wt 52 kDa strongly reacts with antibodies present in sera from patients with ongoing CMV infection [i 1]. In general the reactivity to nonstructural proteins is higher with sera from acutely infected individuals than with sera from long-term seropositive subjects. This is consistent with data previously obtained by immunofluorescence showing that antibodies to immediate early and early antigens are preferentially associated with the acute phase of the infection [12]. HUMORAL IMMUNE RESPONSE TO STRUCTURAL GLYCOPROTEINS The humoral immune response to viral structural glycoproteins is particularly interesting and clinically important as glycoproteins are mainly localized in the viral envelope. They are the target for virus neutralizing antibody (for review see Ref. [13] and are therefore the best candidates in vaccine development for prevention of HCMV infection [14]. As summarized in Table 1 there are three glycoprotein complexes present in the viral envelope, recently named gCI, gCII and gCIII [15-18]. At least two of these complexes (gCI and gCIII) trigger the synthesis of HCMV-neutralizing antibodies when inoculated into experimental animals. While antibodies reacting with gCI do need the complement Table 1. Glycoprotein complexes of the HCMV envelope Mol. wt ( × 103 Da) Complexes gCl gCn gCln
Non-reduced
Reduced
References
250-300, 190, 160 93-300 240
93-130, 55 58 39-48, 47-63 145, 86
[13, 16, 19, 33] [13, 16, 17, 18] [13, 16, 43]
I m m u n e response to C M V proteins
101
to neutralize the viral infectivity, antibodies to gCIII are complement independent as are monoclonal antibodies to gCII. Very recent results presented at the 15th International Herpesvirus Workshop (Washington, D.C., August 1990) support the possibility that gCI is involved in the binding to the cellular receptor (30 kDa), while gCIIl is responsible for fusion between viral envelope and cell membrane through the binding to a cellular protein of mol. wt 92 kDa. During natural infection as well as after experimental vaccination, gCI seems to be the immunodominant antigen among the glycoproteins [14, 19]. In fact antibodies to gCI can be detected at high frequencies in patients having medium or high anti-HCMV ELISA titres: much and often most [between 50 and 70%] of human serum neutralizing activity has been shown to be directed against a single gCI complex [19]. Another glycoprotein present in an integrated form in the envelope of HCMV (45 kDa) and which is conserved among herpesviruses has been shown to stimulate the production of antibodies in vivo [20] but their appearance and evolution has not yet been studied. HUMORAL IMMUNE RESPONSE TO STRUCTURAL PHOSPHOPROTEINS Viral structural phosphoproteins are mainly localized in the viral tegument and/or in the capsid. As recently reviewed by Jahn and Mach [21] and summarized in Table 2, the HCMV virion contains four major immunogenic phosphopolypeptides of the following ml. wts 150, 65, 38 and 28 kDa. There is evidence in the literature [22-25] indicating that a polypeptide of mol. wt 150kDa is the most immunogenic. In fact high antibody titres to this protein have repeatedly been found irrespective of the stage of infection in nearly 100% of HCMV seropositive subjects. All four IgG subclasses of antibodies are induced by this polypeptide [24] and the overall immune response persists for years after convalescence when the antibody to the other HCMV polypeptides has disappeared. There are two proteins with an apparent molecular weight of 150 kDa (the phosphorylated structural component of the viral tegument and the major capsid protein) and differential migration in high bisacrylamide gels has shown that the phosphorylated polypeptide is responsible for the strong antigenic stimulation [25]. Another immunogenic phosphoprotein is the most abundant structural constituent of HCMV, the so-called lower matrix protein which is particularly plentiful in the viral dense bodies [26]. The antibody response to this protein is very high during the acute phase of infection, as well as during the early convalescence, but decreases rapidly thereafter [22]. Both p150 and p65 are highly recognized by anti-HCMV specific IgM during acute infection. Early during the course of a naturally occurring primary CMV infection as well as after injection of seronegative individuals with the Toledo strain vaccine [14] a very low or Table 2. Main phosphoproteins present in HCMV virions Protein mol. wt ( x 103 Da) p150 p65 p38 p28 *DB = dense bodies.
Description Basic phosphoprotein Lower matrix protein Assembly protein
Intraviral location
References
Matrix Matrix DB* Capsid Capsid
[21, 25, 30] [21, 25, 27] [26, 29] [27, 30, 31, 34]
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M.P. LAND1NIand M. LAPLACA
completely absent antibody response to p150 was detected, while Ig are found reacting with p65 [14, 28]. This suggests that the lack of antibody to p150 or its presence in lower amounts than Ig to p65 could be a useful marker for primary HCMV infection [28]. While the antibody response to p150 and p65 is due to both IgG and IgM, the response to other structural phosphoproteins is almost exclusively due to only one antibody class. The reactivity to p38 (which corresponds to the assembly protein described by Gibson, Ref. [29]) is mainly due to IgM and in several cases of secondary (reactivated or exogenous reinfection) CMV acute infection IgM exclusively reacting with p150 and p38 are detectable [23]. On the contrary, antibody response to the capsidic phosphoprotein p28, is almost solely due to IgG and evolves in a similar fashion to the response already described for p150 although the antibody titres detected in patients sera are generally lower [30, 31]. HUMORAL IMMUNE RESPONSE TO RECOMBINANT HCMV PROTEINS Within the last 5 years, several fusion proteins containing significant epitopes of the major HCMV immunogenic polypeptides have been expressed in procaryotic cells and studied by some European research groups: Institute of Clinical and Molecular Virology of the University of Erlangen (Germany), Laboratory of Biotechnology in Braunschweig (Germany) and Institute of Microbiology, Medical Faculty, University of Bologna (Italy) ([32-40], W. Lindenmeier, A. Necker, S. Krauss, R. Bonewald and J. Collins, unpublished data). Several studies have been carried out in cooperation in order to evaluate the effectiveness of the expressed epitopes as antigenic material for serological procedures. In a recent study a large number of human sera with different anti-HCMV antibody titres as detected by ELISA were tested for IgG and IgM reactivity to 10 different fusion proteins containing antigenic epitopes of the major structural proteins of 150, 74, 65, 38 and 28 kDa and the nonstructural DNA-binding protein of mol. wt 52 kDa [40]. The results obtained showed that HCMV can be replaced by some well characterized recombinant viral proteins in the serological evaluation of anti-HCMV antibodies. Significant portions of p150, p65, p52 and p38 were shown to represent an antigenic complex specific enough to detect HCMV IgM. Furthermore accurate evaluation of anti-HCMV IgG was best accomplished using a higher number of recombinant proteins. In addition to the clones necessary for IgM detection, another clone containing a different portion of p150 and a clone containing a large part of p28 seemed to be necessary to obtain an antigenic complex sensitive enough to detect even low levels of antibodies due to a past infection. In other experiments the antibody reactivity of two clones (G2 and DI) was studied in more detail [36, 39, 40]. One clone expressed a portion (from aminoacid 1071 to 1301) of the major DNA binding non-structural protein of mol. wt 52 kDa (G2) and the other a small fragment (the last 25 aminoacids at the carboxy terminus) of the major structural phosphoprotein of tool. wt 150kDa (DI). While antibodies to G2 were shown to be preferentially present during acute infection, antibodies to D1 were abounded during convalescence. Comparison of the reactivity obtained with acutely infected patients and long-term seropositive people disclosed a lack of reactivity to D1 in the first group and a lack of reaction to G2 in the second group. Therefore the differential reactivity to these two recombinant antigens was shown to be able to distinguish between a latent and an active HCMV infection. It is well known that acute HCMV infection include primary and secondary infection (ex novo exogenous reinfection or, more frequently, reactivated from a latent state). The
Immune response to CMV proteins
103
study of IgG and IgM reactivity to G2 and D1 in sera from patients with primary or secondary infections showed that IgM to G2 are preferentially present during primary HCMV infection and therefore these antibodies could represent a very important marker of acute primary infection. In view of the results obtained we think that in the near future it will be possible to use bacteria-produced recombinant HCMV proteins in the serodiagnosis of HCMV infection. This should make it easier to standardize antigenic material to be used in serological tests such as ELISA or latex agglutination. Standardization of antigenic complexes to be used in CMV serodiagnosis should greatly reduce the occurrence of conflicting serological results with the same serum sample and two different antibody detection kits [41, 42]. A drawback to the large scale use of bacterially purified HCMV antigens in serological tests could arise from the presence of antibodies against E. coli proteins in human sera that could give rise to a high number of false-positive reactions. In this respect 3% of sera showing the presence of anti-E, coli beta-galactosidase antibody was found [40] suggesting that it would be useful to express important epitopes as nonfusion proteins. Furthermore some reactivity to other E. coli proteins was often found, indicating that it could also be advantageous to purify the fusion proteins from the whole cell lysates. CONCLUSIONS
Many problems still remain especially in the interpretation of the data regarding antibody recognition. In fact all the studies on the humoral immune response to CMV proteins are carried out by immunoblotting or immunoprecipitation and both techniques have negative aspects. It is known that in immunoblotting some important epitopes (especially glycosidic residues) involved in the reaction with antibody can be destroyed [43] and therefore the reaction of some proteins can be underestimated. On the other hand the reactivity of IgG3 cannot be evaluated by immunoprecipitation as IgG3 do not bind Staphylococcus-protein A, leading to an underestimation of the antibody reactivity with proteins that induce an IgG3 response [24]. In addition some proteins of similar molecular weight can easily co-migrate in SDS-PAGE and antibody reactivity may overlap. An unequivocal analysis of the humoral immune response needs technical improvements and an increased use of cloned gene products. However, we think that the studies summarized in this brief review represent an important step towards our understanding of the immune response to HCMV and to the development of more reliable diagnostic tests. Acknowledgements--Personal work presented in this review has been partially supported by C.N.R. and by Italian Ministry of Health (AIDS projects 1990, 1991).
REFERENCES 1. Ho M. Cytomegalovirus. In Biology and Infection. Current Topics in Infectious Disease (Edited by Greenough III and Merigan T.). Plenum Press, New York (1982). 2. Chee M. S., Bankier A. T., Beck S., Bohni C. M., Brown C. M., Cerny R., Hornsell T., Hutchinson III C. A., Kouzarides T., Martignetti J. A., Preddie E., Satchwell S. C., Tomlinson P., Weston K. M. and Barrell B. G. Analysis of the protein content of the sequence of human cytomegalovirus strain ADI69. In Current Topics in Microbiology and Immunology (Edited by McDougall J. K.), Vol. 54, pp. 125-169. Springer, Berlin (1990). 3. Whaten M. W., Thomsen D. R. and Stinski M. F. Temporal patterns of human cytomegalovirus transcription: mapping the viral RNAs synthesized at immediate early, early and late times after infection. J. Virol. 41, 462--477 (1981). 4. Landini M. P. and Michelson S. Human cytomegalovirus proteins. Prog. med. Virol. 35, 152-185 (1988).
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5. Landini M. P., Severi B., Badiali L., Gonczot E. and Mirolo G. Morphological components of human cytomegalovirus: in situ localization of the major glycoprotein. Intervirology 27, 154-160 (1987). 6. Severi B., Landini M. P. and Govoni E. Human cytomegalovirus morphogenesis: an ultrastructural study of the late cytoplasmic phases. Archs Virol. 98, 51 64 (1988). 7. Michelson S., Tardy-Panit M., Colimon R. and Landini M. P. A human cytomegalovirus neutralizing monoclonal antibody recognizes a normal cell protein. J. gen. Virol. 70, 673 684 (1989) 8. Michelson S., Horodniceanu F., Kress M. and Tardy-Panit M. Human cytomegalovirus immediate early antigen. Analysis in SDS PAGE after immunoprecipitation. J. Virol. 32, 259-267 (1979). 9. Rodgers B., Borysiewicz L., Mundin J., Graham S. and Sissons P. Immunoattinity purification of a 72K early antigen of human cytomegalovirus: analysis of humoral and cell-mediated immunity to the purified polypeptide. J. gen. Virol. 68, 2371-2378 (1985). 10. Borysiewicz L. K., Hickling J. K., Graham S., Sinclair J., Cranage M. P., Smith G. L. and Sissons J. G. P. Human cytomegalovirus-specific cytotoxic T cells. Relative frequence of stage specific CTL recognizing the 72 Kd immediate early protein and glycoprotein B-expressed by recombinant vaccinia viruses. J. exp. Med. 168, 919-931 (1988). 11. Ripalti A., Landini M. P., Mocarski E. and La Placa M. Identification and preliminary use of recombinant lambda gt 11 fusion proteins in Cytomegalovirus diagnosis. J. gen Virol. 70, 1247-I 251 (1989). 12. Landini M. P. Antibody response against early antigens in Herpesviridae infections. Eur. J. EpidemioL 1, 62-66 (1985). 13. Rasmussen L. Immune response to human cytomegalovirus infection. In Current Topics in Microbiology and Immunology (Edited by McDougall J. K.), Vol. 54, pp. 221~54. Springer, Berlin (1990). 14. Gonczol E. and Plotkin S. A. Progress in vaccine development for prevention of human cytomegalovirus infection. In Current Topics in Microbiology and Immunology (Edited by McDougall J. K.), Vol. 54, pp. 254-274. Springer, Berlin (1990). 15. Gretch D. R., Gehrz R. C. V. and Stinski M. F. Characterization of a human cytomegalovirus glycoprotein complex (gCI). J. gen. Virol. 69, 1205 1215 (1988). 16. Gretch D. R., Kari B., Rasmussen L., Gehrz R. C. and Stinski M. F. Identification and characterization of three distinct families of glycoprotein complexes in the envelope of human cytomegalovirus. J. Virol. 62, 875-881 (1988). 17. Gretch D. R., Kari B., Gehrz R. C. and Stinski M. F. A multigene family encodes the human cytomegalovirus glycoprotein complex gCII (gp47-52 complex). J. Virol. 62, 1956 1962 (1988). 18. Kari B., Goertz R. and Gehrz R. C. Characterization of cytomegalovirus glycoproteins in a family of complexes designated gCII with murine monoclonal antibodies. Archs Virol. 112, 55 65 (1990). 19. Britt W. J., Vugler L., Butfiloski E. J. and Stephens E. B. Cell surface expression of human cytomegalovirus (HCMV) gp55 116 (gB): use of HCMV-recombinant vaccinia virus-infected cells in analysis of the human neutralizing antibody response. J. Virol. 64, 1079-1085 (1990). 20. Lehner R., Meyer H. and Mach M. Identification and characterization of a human cytomegalovirus gene coding for a membrane protein that is conserved among human herpesviruses. J. Virol. 63, 3792-3800 (1989). 21. Jahn G. and Mach M. Human cytomegalovirus phosphoproteins and glycoproteins and their coding regions. In Current Topics in Microbiology and Immunology (Edited by McDougall J. K.), Vol. 54, pp. 171-185, Springer, Berlin (1990). 22. Landini M. P., Mirolo G., Baldassarri B. and La Placa M. human immune response to cytomegalovirus structural polypeptides studied by immunoblotting. J. med. Virol. 17, 303-311 (1985). 23. Landini M. P., Mirolo G., Coppoleechia P., Re M. C. and La Placa M. Serum antibodies to individual cytomegalovirus structural polypeptides in renal transplant recipients during viral infection. Microbiol. Immunol. 30, 683-695 (1986). 24. Landini M. P., Baldassarri B., Mirolo G., Ripalti A. and La Placa M. Reactivity of cytomegalovirus structural polypeptides with different subclasses of IgG present in human sera. J. Infect. 16, 163-167 (1988). 25. Jahn G., Scholl B.-C. and Fleckenstein B. The two major structural phosphoproteins (pp65 and ppl50) of human cytomegalovirus and their antigenic properties. J. gen. Virol. 68, 1327-1337 (1987). 26. Gibson W. Protein counterparts of human and simian cytomegaloviruses. Virology 128, 391-406 (1983). 27. Landini M. P., Severi B., Furlini G. and Badiali L. Human cytomegalovirus structural components: intracellular and intraviral localization of p28 and p65-69 by immuno-electron microscopy. Virus Res. 8, 15-23 (1987). 28. Landini M. P., Rossier E. and Schmitz E. Antibodies to human cytomegalovirus structural polypeptides during primary infection. J. Virol. Meth. 22, 309-317 (1988). 29. Irmiere A. and Gibson W. Isolation of human cytomegalovirus intranuclear capsids, characterization of their protein constituents and demonstration that the B-capsid assembly protein is also abundant in noninfectious enveloped particles. J. Virol. 56, 277-283 (1985). 30. Re M. C., Landini M. P., Coppoleechia P., Furlini G. and La Placa M. A 28,000 d human cytomegalovirus structural polypeptide studied by means of a specific monoclonal antibody. J. gen. Virol. 66, 2507 2511 (1985).
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