gB glycoprotein complex of human cytomegalovirus envelope induces humoral and cellular immune-responses in human volunteers

Isolated gA/gB glycoprotein complex of human cytomegalovirus envelope induces humoral and cellular immune-responses in human volunteers Eva Gonczol*, John Ianacone*, Wenzhe Ho*, Stuart Starr ~, Bernard Meignier ~ and Stanley Plotkin* Three human cytomegalovirus ( H C M V ) seronegative individuals were immunized with a single dose of H C M V envelope; two individuals developed neutralizing antibodies. Two naturally H C M V seropositive and three H C M V seronegative human volunteers were immunized with a major glycoprotein complex, gA/gB, of H C M V that had been purified by immunoadsorbent column chromatography. After a single injection of the gA]gB preparation, the naturally seropositive individuals developed higher titres of neutralizing antibodies and temporarily higher H C M V-specific lymphocyte proliferation ( H C M V - L P ) responses in vitro. The seronegative individuals developed neutralizing antibodies after the third injection of gA/gB, which were present only transiently, but showed a rapid reappearance and increase in titre after the fourth injection. At 1 year after the

first injection, the neutralizing antibody titres were still comparable with those of the naturally seropositive individuals. HCMV-LP responses to H C M V in the initially seronegative individuals developed after the second or third injection with the gA/gB preparation and remained positive during the 1-year observation period. These results show that the gA/gB protein induces both humoral and cellular immune responses in humans, and might serve as the basis of a subunit vaccine. Keywords: HCMV; Cytomegalovirus envelope; gA/gB glycoprotein; immune response: human immunization

Introduction About 1% of infants are born congenitally infected with human cytomegalovirus (HCMV) and about 10-15% of such infants suffer neurologic sequelae of the infection t. In prospective studies, primary maternal infection is associated with such sequelae, while maternal reactivation infections are usually associated with asymptomatic infections. In later life, H C M V causes disease in organ transplant recipients. The symptoms are especially severe if a seronegative recipient receives an organ or blood products from seropositive donors-'. Thus, protection of seronegative individuals in high-risk ~roups is clearly desirable. The live attenuated Towne strain of HCMV has been shown to protect seronegative renal transplant recipients from severe clinical symptoms of HCMV infection 3''* and to protect initially seronegative healthy individuals from infection and clinical symptoms after subcutaneous challenge with a wild-type strain of HCMV s. However concerns remain about the use of a live HCMV vaccine because of the latency-reactivation phenomenon characteristic of herpesvirus infections in humans and because of the capacity of certain strains of HCMV to transform cells malignantly in t:itro~'~. Although the Towne strain was shown not to reactivate *The Wistar Institute of Anatomy and Biology, 36th Street at Spruce, Philadelphia, Pennsylvania, USA. ~Children's Hospital of Philadelphia, 34th and Civic Center Boulevard, Philadelphia, Pennsylvania, USA. "+lnstitut Merieux, 69280 Marcy I'Etoite, France. (Received 29 June 1989; accepted 3 August t989) 0264-410X/90/020130-07 {~ 1990 Butterworth & Co. (Publishers) Ltd

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in immunosuppressed renal transplant recipients s, a subunit CMV vaccine may be more acceptable for human immunization. Herpesvirus glycoproteins are thought to be important in inducing virus-specific immunity because of their expression on the membranes of infected cells and because monoclonal antibodies to the surface glycoproteins often have neutralizing activity. At least three glycoprotein complexes in the HCMV viral envelope may be involved in the protective immune response. The major envelope glycoprotein complex of HCMV, variously termed gA C~-1-', gp 55-116 t-~ ts, p130 55~-is, gB 19, gcI -'°, and referred to as gAgB in this publication. Virus-neutralizing monoclonal antibodies directed to this complex have been developed by several taboratoriesg.~ ~.~_~.~<2,-2-,. The gA'gB proteins purified using these monoclonal antibodies, or monospecific polyclonal sera, induce humoral t2'~r and cellular ~2 immune responses in animals. Sera with neutralizing activity from naturally seropositive individuals or Towne strain vaccinees recognized the g A g B complex in Western blot assays -'-~, indicating the importance of this complex in the immune response to HCMV in humans. The coding region for the gA/gB protein complex has been mapped to the HindllI F and Hind D DNA fragment of Ad-t69 and Towne strain, respectively, sequenced ~'2~'2:, and cloned into a vaccinia vector ~5~' Experimental animals immunized with the recombinant vaccinia virus produced antibodies that neutralized HCMV infectivity in ~.!itro 15't') A second glycoprotein of 86kDa is probably less immunogenic in mice than the gA/gB complex: to our

HCMV gA/gB glycoprotein vaccine in human volunteers: E. Gonczol et al.

knowledge, only two neutralizing monoclonal antibodies have been produced to this protein 2s'29. The purified 86 kDa glycoprotein and the deglycosylated form of it also induced neutralizing antibodies in guinea pigs iv'is The coding region has been identified to the HindIII fragment of Ad-169 strain and recombinant vaccinia viruses that expressed this gene were constructed 29. A third 47-52 kDa glycoprotein complex 22"~'*, which seems to be antigenically unrelated to the gA/gB glycoprotein complex, is the least studied. Monoclonal antibodies directed to the 47-52kDa complex have very high neutralizing activityz2'24, but the immunogenicity of the isolated glycoprotein complex has not been tested. The gene encoding this complex is identified in the HindlII X fragment of the Towne strain 3°. In the present report, the ability of the purified virus envelope and gA/gB glycoprotein complex to induce neutralizing antibodies and a cellular immune response in humans is described. M a t e r i a l s and m e t h o d s

Cells and viruses MRC-5 human embryo fibroblasts (from the American Type Culture Collection) were used throughout the experiments. Strain Ad-169 of HCMV 31 was used for the purification of gA/gB protein. Strains Ad-169 and Towne 32 and a low-passage Toledo strain 33 of HCMV were used for neutralizing assays. The Towne strain was used for immunoprecipitation, Western blot, immunofluorescence and lymphocyte proliferation assays.

Preparation of gA/gB protein All steps of the protein preparation were carried out under aseptic conditions. Monoclonal antibody CH-380 was generated by fusing the spleen cells of Balb/c mice immunized with purified gA/gB protein with a myeloma cell line ~~. Antibody from the hybridoma cell line immune precipitated the family of gA/gB proteins and reacted with denatured 138-142 and 58.5kDa forms of the glycoprotein immobilized on nitrocellulose 1~. Monoclonal antibody CH-380 produced in ascites was purified and coupled to CNBr-activated Sepharose 4B according to the manufacturer's instructions (Pharmacia, Piscataway, NJ, USA). Extracellular HCMV, grown in MRC-5 cells in roller bottles, was cleared of cellular debris by low-speed centrifugation of culture medium of infected cells, collected by ultracentrifugation at 30 0009 for 2 h (Beckman type 19 motor Fullerton, CA, USA) and resuspended in phosphate buffered saline (PBS) containing 0. l mM phenylmethylsulphonyl fluoride (PMSF; Sigma St Louis, MO. USA) as a proteinase inhibitor. After a l h treatment with 2% of the dialysable non-ionic detergent, n-octyl-glycoside (Sigma), the viral envelope was prepared as previously described 3"~. About 8-10rag envelope protein were first incubated for 2 h with 4ml CNBr-activated Sepharose 4B beads (precolumn), then with 4ml of the immuno-adsorbent beads for 2h by end-over-end rotation at 4°C, which were then loaded on to a column. The column was washed with at least 2.51 of PBS containing n-octyl-glycoside. Bound viral proteins were eluted with 3.0M KSCN (Sigma) in PBS (pH 7.0) containing 0.1% n-octyl-glycoside. Samples were dialysed at 4°C against several changes of PBS containing Bio-beads SM-2, 20-50 mesh (Bio-Rad Richmond, CA, USA), to remove n-octyl-glycoside and

KSCN, and concentrated in an Amicon (Danvers, MA, USA) concentrator, After protein determination3~, samples were aliquoted and stored at - 80'~C. From about 35 mg of envelope protein preparation, 1.8 mg of gA/gB protein were isolated. Sterility tests established the absence of any bacterial or yeast contamination in the protein preparation, and use of the limulus amebocyte lysate kit (E-Toxate Kit No 210-A, Sigma) according to the manufacturer's instructions, indicated the absence of endotoxin contamination. The absence of contamination with mouse proteins was determined by a direct dot blot assay on the isolated gA/gB preparation (2/~g protein per dot) with peroxidaseconjugated goat anti-mouse IgG (Cappel, West Chester, PA, USA), developed with 4ochloro-l-naphthol (Sigma) and 0.02% hydrogen peroxide (Sigma). The tests showed that the preparation was free of the mouse IgG used to purify it. Further, sera from guinea-pigs immunized with the gA/gB preparation (see Results) were used in dot blot assay against 2/xg protein per dot containing mouse IgG, fetal calf serum (used to grow the infected cells) and uninfected MRC-5 cell.lysates. The reactions were detected by treatment of the blots with peroxidaseconjugated goat anti-guinea-pig IgC, and developed with 4-chloro-l-naphthol and 0.02% hydrogen peroxidase. The results with the guinea-pig sera were negative for mouse, bovine, and human protein contaminants (data not shown).

Polyacrylamide gel electrophoresis and immunological identification of the gA[gB protein preparation The isolated gA/gB protein was mixed 1: 1 with loading buffer (0.05 M tris-hydrochloride (Sigma), pH 7.0, 2% SDS (Bethesda Research Lab, MD, USA), 5% 5mercaptoethanol (Sigma), 10% glycerol (Sigma), bromophenol blue (Baker Chemical, N J), boiled for 3rain, loaded onto a 7.5% polyacrylamide gel, electrophoretically separated using the method of Laemmli 36 and stained with Coomassie brilliant blue R-250 (Bethesda Research Laboratories). For immunological identification the isolated proteins were electrophoretically separated and transferred to nitrocellulose sheets as described 37 and probed in a Western blot assay with monoclonal antibody CH-380~ 1, 7-1713; (kindly provided by Dr Britt, The University of Alabama at Birmingham, Birmingham, Alabama) and polyclonal monospecific guinea-pig serum 12 to the gA/gB complex. Peroxidase labelled anti-mouse or anti-guinea-pig IgG (Cappel), 4-chloro-1-naplitol and 0.02% hydrogen peroxide were used to develop the reaction. The molecular weight of the proteins was estimated by comparison with molecular weight protein standards (Bio-Rad).

Immunization with the virus envelope and gA [gB preparation The immunogenicity of the isolated gA/gB glycoprotein was first tested in guinea-pigs. Animals were injected with 40 ~g protein in 0.5 ml volume/dose three times at 2-week intervals, mixed with AI(OH)3 (Superfos, Denmark) diluted so that the aluminum content of the final product did not exceed 0.85mg per injection (two animals) or mixed with 0.5ml of complete (first injection) or incomplete (second and third injection) of Freund's adjuvant (one animal). Three healthy seronegative adult human volunteers

Vaccine, Vol. 8, April 1990 131

HCMV gA/gB glycoprotein vaccine in human volunteers: E. Gonczol et al.

were inoculated i.m. in the upper arm with a single dose of 80/~g envelope preparation given together with AI(OH)3 in a total volume of 0.5ml. Three healthy seronegative adults were given serial injections with 80 #g doses of the gA/gB preparation given with AI(OH)~ in the same way as described for the immunization of guinea-pigs. Two of the three individuals received an identical fourth injection 7 months after the third injection. Two seropositive volunteers received a single injection of the gA/gB preparation given together with AI(OH)3 adjuvant. Except a slight discomfort in the injected arms 1-3 days after the injection there was no side effect of the gA/gB immunization.

were dispensed into round-bottomed 96-well microtitre plates. Antigens were added in 10#1 aliquots to give final concentrations of 1:100. Cultures were incubated for 6 days at 37°C in 5% COz. Tritiated thymidine (1 #Ci/0.2 ml; 6.7 Ci/mmol; ICN Biomedicals, Inc., Costa Mes, CA, USA) was added for the final 4h of incubation, after which the cells were collected using a multiple sample harvester and processed for liquid scintillation counting. All determinations were in triplicate. Results are expressed as a lymphocyte stimulation index (LSI) defined as counts min-1 in H C M V antigen-stimulated cultures/counts min -1 in control antigen-stimulated culture.

Immune response to H C M V after immunization with the virus envelope and gA [gB preparation

Results

Microneutralization assays were carried out as described previously 38. Briefly, serum samples were serially diluted in 96-well flat-bottomed microtest culture plates (Costar, Cambridge, Massachusetts, USA) using a multichannel pipette. Four parallel wells of each serum dilution received 2000 p.f.u, of H C M V and complement. After a 1 h incubation at 37°C in a CO 2 incubator, a cell suspension ( 3 - 5 x 104 cell well -~) was added and the plates were incubated for an additional 2 days. The results were read as reciprocals of the highest dilution of the serum that fully inhibited viral cytopathic effect (CPE) compared with 100% CPE in control wells. All samples were tested three times and no differences between tests were observed. ELISA assays were carried out by using an H C M V - E L I S A kit according to the manufacturer's instructions {Abbott, Chicago, IL, USA) and results were read as negative or positive. Radioimmunoprecipitation assays of protein in HCMV-infected cells was performed as described previously ~z'-~s. For Western blot assays, extracellular virus purified by sucrose gradient ~'~, virus envelope antigens prepared by NP-40 treatment of the virus 3"~ and infected and uninfected cell lysates ~s were used as desc'ribed earlier in Materials and methods except that the nitrocellulose strips were treated with the sera of human vaccinees and immunized guinea-pigs as first antibodies and with peroxidase-conjugated goat antihuman (Cappel) or anti-guinea-pig IgG, respectively as second antibodies. Indirect immunofluorescence assays were done using cells grown on glass coverslips, infected with H C M V at a m.o.i, of 1 2 and either fixed in acetone on the fourth day after infection for intracellular viral antigens or used without fixation for the detection of membrane antigens. In experiments to detect intracellular antigens, incubation of cells with human sera and with the fluorescein isothiocyanate-conjugated goat anti-human IgG (Cappel), as well as washing of the cells, were carried out at 37°C. All steps of the immunofluorescence assays to detect cell membrane antigens were carried out at 4°C. HCMV-specific lymphocyte proliferation (HCMVLP) assays were carried out as described 39. Briefly, H C M V and control antigens were prepared from Towne strain-infected and non-infected MRC-5 cells, the cells were sonicated and the resultant sonicates clarified by centrifugation and then exposed to 56~C for l h. Mononuclear cells were obtained by Ficoil-Hypaque centrifugation of peripheral blood and suspended at a concentration of l × l O 6 cells ml -~ in RPMI-1640 containing 15% autologous plasma. Aliquots (0.2mll

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Composition of the virus envelope and glycoprotein complex used for immunization The molecular masses of the virus envelope proteins (47-52, 58, 82-86, 116-130 kDa) were in agreement with previous results 3"*. The electrophoretic pattern of the isolated gA/gB complex indicated a mixture of 58 and 130kDa proteins (Fi~jure l, lane 1). The molecular weights of the components are in good agreement with those of isolated gA/gB in other studies ~-'t~'zs In Western blot assays, monoclonal antibody CH-380 reacted strongly with the 5 8 k D a and weakly with the 130 kDa components (Figure I, lane 2), both thought to be cleavage products of a higher molecular weight glycosylated precursor of gA/gB complex t 6, ~8--,o. Further, monoclonal antibody 7-17 recognized the 58 kDa protein and a monospecific polyclonal guinea-pig serum to the gA/gB protein ~-" recognized the 58 and 130kDa isolated proteins (data nc~ shown).

1

•

2

I

--

130

--

58

Figure 1 Presence of individual proteins in the gA/gB preparation. Lane 1: The gA/gB preparation (2/~g/lane-') was mixed 1:1 with loading buffer containing 5% 2-mercaptoethanol and boiled for 3 rain. The individual proteins were electrophoretically separated on a 7.5% polyacrylamide gel and stained with Coomassie blue. Lane 2: For the Western blot assay, the gA/gB preparation was separated by SDS-PAGE and electrotransferred to nitrocellulose sheet. Monoclonal antibody CH-380 was added to the nitrocellulose strip at the dilution of 1:20. Numbers on the right are molecular weights ( x 103)

HCMV gA/gB glycoprotein vaccine in human volunteers: E. Gonczol et al. Table 1 Immune response to HCMV immunized with purified gA/gB protein

in seropositive

volunteers

Neutralization titre of volunteers

Lymphocyte stimulation index of volunteers

Time (weeks)

H

G

H

G

--* 0 1 2 4 8 12 28 36 44 52

32 ND 128 256 128 128 ND ND 128 64

64 ND 128 256 256 256 256 128 128 128

9.6 20.2 19.2 4.7 5.3 6.0 ND ND 8.6 6.9

ND 11.4 9.1 8.2 ND 5.0 ND ND ND 3.9

gA/gB protein and 2 weeks later showed an increase from 1:50 to 1:200 in titre to the 58 kDa species in Western blot assays using purified virions as antigen (Figure 2). Table 2 gives the results of the neutralization and lymphocyte stimulation assays on samples from three initially seronegative individuals immunized with the gA/gB preparation. Neutralizing antibodies were detectable at a titre of 1:16-1:64 after the third dose, but they waned rapidly so that at 16 weeks after the first dose, the sera showed very little neutralizing activity and at 24 weeks, none. Despite three doses of gA/gB protein, no anti-HCMV antibodies were detectable by ELISA assays. Eight months after the first injection, two volunteers (volunteers T and W) received an additional fourth dose

Arrow indicates the time of injection with gA/gB protein. ND, not done

1

Immune response to H C M V in the virus envelope and gA]gB immunized human volunteers No significant differences were observed in the microneutralization assays using Ad-169 and Towne laboratory strains and the Toledo low-passage HCMV strain. Thus, only data obtained with the Towne strain are presented. Of the three volunteers who received the virus envelope preparation, two volunteers developed HCMV neutralizing antibodies with a titre of 1:128 and 1:32 at 8 weeks after inoculation. One volunteer remained negative for neutralizing antibodies. Two initially seropositive individuals were boosted with the gA/gB preparation. Table I summarizes the results of the neutralization and lymphocyte stimulation assays on samples taken at different times after the booster injection. Both LSI and neutralization titre were increased within 2 weeks after a single injection with the gA/gB protein. The LSI decreased to original levels by 4 weeks after injection, but the neutralization titre remained elevated throughout the 1 year examination period. Sera obtained from the initially seropositive individuals at the time of injection with

5

6

7

8

I.

Immune response to H C M V in gA ]gB immunized guinea-pigs At 3 weeks after the last injection with the gA/gB preparation the guinea-pigs developed neutralizing antibodies to the Ad-169 and Towne strains at a titre of 1:256 (in animals injected with gA/gB mixed with AI(OH)3 ) or 1:512 (in animals injected with gA/gB mixed with Freund's adjuvant). This high serum neutralizing activity lasted for at least I year. Because our preliminary experiments revealed no significant differences in the results of lymphocyte stimulation assays of gA/gBimmunized guinea-pigs using either the Ad-169 or the Towne strain as antigen (unpublished data), only the Towne strain was used in the lymphocyte stimulation assays in the present study. The LSI with Towne strain antigen increased to 5.1 and 4.8 from < 2 in the AI(OH)3 adjuvant inoculated animals and to 5.3 from < 2 in the Freund's adjuvant-inoculated animals as detected 8 weeks after the last injection. Human volunteers were immunized with the gA/gB preparation after the immune responses in guinea-pigs to H C M V were evaluated and the guinea-pig sera showed no reactivity in dot blot assays with possible contaminants of the gA/gB preparation, i.e. mouse IgG, fetal calf serum and uninfected MRC-5 cells (data not shown).

234

--

150

--

66

--

58

7~!

.

,

Fig,,re 2 Antibody response to HCMV virion proteins of an initially seropositive individual after a single injection with gA/gB protein. For the Western blot assay, purified virions (Towne-strain) were used as antigen. Viral proteins (15,ug lane -1) were electrophoretically separated and transferred to a nitrocellulose sheets. Strips were incubated with different dilutions of pre-booster and post-booster sera (taken 2 weeks after the gA/gB injection) of volunteer H. Lanes 1-4, pre-booster sera; lanes .Sq~, post-booster sera; lanes 1 and 5: dil. 1:100; lanes 2 and 6: dil. 1:200; lanes 3 and 7: dil. 1:400; lanes 4 and 8: dil. 1:800. Numbers on the right are molecular weights ( x 1~)

Table2 Immune response to HCMV in seronegative volunteers immunized with purified gA/gB protein Neutralization titre of volunteers

Lymphocyte stimulation index of volunteers

Time (weeks)

T

W

S"

T

W

S

--* 0 --, 2 3 ---* 4 8 16 24 ~ 32 33 34 35 40 44 52

<4 <4 <4 16 64 4 <4 <4 64 256 256 256 64 64

<4 <4 <4 <4 16 4 <4 <4 32 64 128 128 64 64

<4 <4 <4 <4 16 <4 <4 ND ND ND ND ND <4 ND

0.9 3.0 1.8 1.8 6.7 3.3 3.4 3.9 1.9 4.7 5.3 4.1 9.6 9.6

1.2 1.4 1.2 8.6 3.6 1.2 3.9 4.3 3.7 3.5 ND 3.7 8.3 5.4

0.8 1.6 2.2 1.4 5.1 1.3 ND ND ND ND ND ND 6.4 ND

Arrows indicate the time of injection with gA/gB protein. "Volunteer S did not receive a fourth injection. ND, not done

V a c c i n e , V o l . 8, A p r i l 1990

133

HCMV gA/gB glycoprotein vaccine in human volunteers: E. Gonczol et al.

Figure 3 Surface antigens in infected cells detected by anti-gA/gB human sera. Cells grown on coverslips were infected with Towne strain (m.o.i. of 1-2) and indirect immunofluorescence assays were carried out on unfixed cells on the fourth day after infection using a serum sample (dil. 1:20) from an initially seronegative individual (volunteer T) obtained on the second week after the fourth dose of gA/gB protein

1

2 3 4 5 6

o~,

7 8

--

~t

~

>200

~

130

--

58

Figure 4 Antibody response to HCMV virion proteins of the two im[ially seronegative individuals after the fourth (booster) dose of gA/gB preparation. For the Western blot assay viral proteins (15 Izg lane-1)were electrophoretically separated and transferred to a nitrocellulose sheet. Strips were then incubated with serum samples diluted 1:50 obtained at different times of vaccinee T (lanes 1-3) and of vaccinee W (lanes 4q~). Lanes 1 and 4, serum samples obtained in week 32 before the booster injection; lanes 2 and 5, serum samples obtained in week 33; lanes 3 and 6, serum samples taken in week 35; and as controls, lane 7, monoclonal antibody CH-380 (dil. 1:20), lane 8, monoclonal antibody 7-17 (undiluted hybridoma supernatant)

of the gA/gB protein. There was a rapid increase in neutralization titre after the booster injection. Sera of volunteers T and W became positive after the fourth injection by ELISA and immunofluorescence assays as well, with intracytoplasmic antigens detected in infected and acetone fixed cells (data not shown), and cell surface antigens on the infected, unfixed cells (Figure 3). Preimmunization sera were negative with the infected cells and post-booster sera were negative with uninfected cells in the immunofluorescence assays.

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V o l . 8, A p r i l 1990

The LSI rose transiently after the second or third injection, fell to low levels at 4 months, and then rose again with a second peak at 3 months after the booster injection (Table 2). The immunoreactivity of sera from gA/gB-immunized volunteers who were initially seronegative was further analysed by immunoprecipitation of infected cell lysates and by Western blot assays using purified virions, envelope preparations and H C M V infected cell lysates as antigens. Sera taken within 8 months of the first injection showed no reactivity in immunoprecipitation assays using infected cell lysates, or in Western blot assays using purified virions or infected cell lysates as antigens. However, when an envelope preparation enriched in gA/gB proteins was used as antigen, sera taken at 4 weeks after the third injection detected a 5 8 k D a band in Western blot assays {data not shown}. Fi,qure -I demonstrates that sera taken 3 weeks alter volunteers T and W received the fourth injection showed reactivity with the 5 8 k D a protein in Western plot assays using purified virions as antigen. Monoclonal antibodies CH-380 and 7-17 included as controls also recognized the 58 kDa protein (Figure 4, lanes 7 and 8). Monoclonai antibody CH-380 weakly recognized additional higher molecular weight proteins of the gA/gB glycoprotein complex in the virion preparation (Fi#ure 4, lane 7).

Discussion In the present study, it was shown for the first time that a single dose of H C M V envelope preparation induced neutralizing antibodies and that repeated injections of an isolated glycoprotein complex induced both cellular and humoral immune responses in humans. In initially seronegative individuals, who were immunized with the gA/gB preparation but who lost antibody, a clear priming effect on antibody production after the booster dose was observed, indicating the generation and activation of immune memory cells. This might be important for immunization of high-risk individuals with the gA/gB complex since very rapid production of antibody might occur after challenge with natural H C M V infection. Several studies have demonstrated such a "priming' effect as part of the immune response to repeated injection of VZV 4° or HSV 4t proteins. Note that the neutralization antibody level after the fourth dose reached the values observed in natural infections and exceeded the values observed after Towne vaccination es. In contrast to the undetectable humoral immune response 4 6 months after the first course of immunization, the cellular immune response, as measured in uitro by the lymphocyte stimulation response to H C M V antigen, was only transiently negative at 4 months. A decline in H C M V - L P 3 months after immunization of healthy adults with live attenuated H C M V vaccine followed by a rebound was observed previously "~2. Peak H C M V - L P responses after the fourth-dose of gA/gB were similar to those detected in naturally seropositive individuals, but lower than those detected in recipients of live attenuated Towne strain H C M V vaccine {Ref. 43: unpublished observation). Also, peak responses in recipients of gA/gB occurred later than in recipients of live attenuated vaccine whose peak responses occur 2 4 weeks after immunization "~3. The increased lymphocyte proliferation observed after gA/gB immunization in humans is in agreement with previous results that

H C M V g A / g B g l y c o p r o t e i n vaccine in h u m a n volunteers." E. G o n c z o l et al.

naturally seropositive adults show similar proliferative responses to this complex in an in v i t r o assay '~'~. In initially seropositive individuals, an immediate rise in antibody production and HCMV-specific LSI was observed after the booster injection, suggesting the possible usefulness of a gA/gB subunit HCMV vaccine as a booster in certain seropositive individuals, such as weakly HCMV-positive organ transplant recipients. The low yield of purified viral proteins makes it difficult to obtain the gA/gB protein in sufficient quantities for large-scale immunization. Our focus in the present study was on the immunogenicity of the gA/gB complex in humans, rather than refinement of the immunization protocol (i.e., optimal dose, schedule, etc.). The gA/gB gene has already been mapped and sequenced 19'26'27 so that the development of a vaccine for human use theoretically awaits only a suitable expression vector. The protective effect of such a vaccine could become evident in a short time if tested in seronegative and weakly seropositive individuals awaiting transplantation with organs from seropositive donors. No significant differences were detected in our neutralization assays of antibodies generated in initially seronegative gA/gB-immunized individuals to the Ad169, Towne and Toledo strains. This is consistent with previous studies using monoclonal antibodies directed to the gA/gB complex, which showed that neutralizing epitopes on the gA/gB protein are highly conserved among the Ad-169, Towne and Toledo strains x6'22 Further, a 95-94.2% homology at the amino acid level between the gA/gB coding sequences of the Ad-169 and Towne strains has been observed (Ref. 27 and Dr Charles de Taisne, State of New York Department of Health, Albany, New York, personal communication). On the other hand, our previous studies, in which the Towne and Toledo strains were tested against sera from individuals infected with single HCMV stains, indicated qualitative strain-specific differences 2s, perhaps reflecting immune responses to neutralizing epitopes carried on polypeptides other than the gA/gB protein present in the virion. The immunofluorescence assays using the anti-gA/gB human sera showed that the gA/gB proteins are inserted into the plasma membrane of HCMV-infected cells or in the envelope of virions budding through the membrane of infected cells ~s. The presence of the gA/gB proteins, or some epitope(s) of it, in the membrane of infected cells might be important in the induction of neutralizing antibodies and in the recognition of the infected cells by cytotoxic T lymphocytes, but we did not test the latter function. In the Western blot assays, the sera obtained after the fourth injection from initially seronegative individuals showed strong reactivity with the 58 kDa protein in the purified virion preparation. The 130 kDa component of gA/gB complex might have been present in a very small quantity in the purified virions used as an antigen in the Western blot assay, since the human sera did not and the monoclonal antibody CH-380 recognized only weakly the 130 and a > 2 0 0 k D a components. As mentioned earlier, the gA/gB is only one of the envelope glycoprotein complexes that induce neutralizing antibodies in animals or is recognized by neutralizing monoclonal antibodies. The 86 k Da glycoprotein 17.18.28.29 and the 47-52 kDa glycoprotein complex 22'2'*'3° might also be important in protective immunity and might be

considered as subunit vaccines. Moreover, the studies of Borysiewicz e t al. :~6''~7 and Rodgers et al. 48 suggest that some of the non-structural immediate early proteins are also involved in the cellular immunity to HCMV. All of these possibilities must be carefully evaluated before an optimal subunit H C M V vaccine can be designed.

Acknowledgements The authors are grateful to Leopoldo Luistro, Debbie Miller and Marilyn Lewis for excellent technical assistance and to Marina Hoffman for editing the paper. This work: was supported by grant HD-18957 from the National Institute for Child Health and Development, and by the Institut Merieux, Vrance.

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