Measles virus vaccination of measles seropositive individuals suppresses lymphocyte proliferation and chemotactic factor production

CLINICA,

IMML’NOLOGY

AND

1MMUNOPATHOI.OGY

21, 341-350 (1981)

Measles Virus Vaccination of Measles Seropositive individuals Suppresses Lymphocyte Proliferation and Chemotactic Factor Production ROBERT L. HIRSCH, FOROOZAN MOKHTARIAN, DIANE E. GRIFFIN, BENJAMIN RIX BROOKS, JAY HESS, AND RICHARD T. JOHNSON Ho\cwrd Hughe& Medical Microbiology. The Johns

Institute Hopkins

Laboratories. Departments of‘ Nrurolog+v. University Sc,hool of Medkine. Baltimore,

Medicine, Maryland

and 21205

Measles virus vaccination of adults seropositive for measles resulted in suppression of chemotactic factor production in response to concanavalin A (Con A), purified protein derivative (PPD), and purified measles virus antigen, and of lymphoproliferative responses to high doses of phytohemagglutinin (PHA). Persons receiving the vaccine had no clinical signs of infection, no detectable viremia, and no rise in antibody titers to measles virus. The suppression of both chemotactic factor production and lymphoproliferation was of shorter duration than that observed in studies of acute measles virus infection. Suppression could not be related to increases in the number of circulating ceils capable of suppressing the Con A or PHA responses of normal lymphocytes. These studies indicate that measles virus may cause immunosuppression in the absence of detectable virus replication in mononuclear cells.

Natural infections with several viruses have been observed to suppress skin test responses and lymphoproliferative responses (l-4) to a number of antigens and mitogens. Von Pirquet first recognized the immunosuppression caused by measles virus in reporting the activation of tuberculosis in children presenting with acute measles virus infections (5). Subsequent studies have demonstrated that skin test responses are depressed not only in children with natural measles virus infections (6. 7) but also in those receiving the live attenuated measles virus vaccine (6, S- 13). Infection of lymphocytes by measles virus has been suggested as a cause of the immunosuppression (14- 16). However, depressed skin test responses after immunization with live attenuated measles virus vaccine occur independent of the immune status of the recipient (8-12). In addition, the observations that many subpopulations of mononuclear cells can be infected in vitro (17, 18), that the effect of measles virus on lymphocyte function is dependent upon the time of addition of virus (19), and that measles virus may cause proliferation of T suppressor cells (20), raise the possibility that measles virus has suppressive effects in the absence of lymphoid cell infection. To further examine the suppression of in vitro correlates of cell-mediated immunity after measles virus vaccine, we have studied production of mononuclear chemotactic factor, lymphoproliferation, and circulating suppressor cells after vaccination of seropositive adults. 341 009@1229/81/120341-lO$Ol.OtYO Copyright 0 1981 by Academic Press. Inc. All rights of reproduction in any fom nserved.

342

HIRSCH

El’

Al...

MATERIALS AND METHODS S~&et,ts. Ten healthy laboratory personnel (22-40 years of age) were injected with a commercial live attenuated, Enders strain, measles virus vaccine (Attenuvax, Merck, Sharp and Dohme). Another group of six volunteers (26-40 years of age) received the diluent supplied with the lyophilized vaccine. Oral temperatures were recorded daily for 7 days after injection. Preparation oj’1)1ot10tlN(.l~~(lr ~~~~11s. Prior to and I, 2. 3, and 4 or 5 weeks after inoculation 20-30 ml of blood were collected in preservative free heparin (Panheparin, 20 units/ml of blood). The mononuclear cell fraction was obtained by centrifugation of blood on % Ficoll-33.% Hypaque gradients (21). The mononuclear cell fraction was washed three times in calcium and magnesium free Hanks’ balanced salt solution (HBSS) and resuspended at a concentration of I x 10” cells/ml in RPM1 1640 containing 5% mouse serum (Pel-Freeze Biologicals, Rogers, Ark.), 20 kg/ml gentamicin, 10 mM Hepes buffer, and 10 mM glutamine (complete RPMI). Chcmntactk jbctor produc.tion atld assay. Mononuclear cells from six recipients of vaccine and five recipients of diluent were also cultured with Con A (3 lLg/ml), sucrose gradient purified, uv-inactivated measles virus (2 pg/ml) (22). purified protein derivative (PPD) from M. tuhrrculosis (1 pg/ml) (Parke-Davis, Detroit, Mich.), or PBS. Two milliliters of cells were placed in 12 x 75mm tubes with antigens or Con A and cultured at 37°C. After 4 days the cultures were centrifuged at 4OOg and the supernates were removed and diluted 1:5 in RPM1 1640 containing 25 n&f Hepes buffer. Chemotactic assays were performed as previously described (23) with the exception that human mononuclear cells from a single uninoculated donor were used as indicator cells throughout the study. Dried filters were mounted in immersion oil onto glass slides and the mononuclear cells in 10 high-power fields were counted. The chemotactic response was expressed as the ratio of the mean number of mononuclear cells per oil field which migrated in response to supernates from antigen- or mitogen-stimulated cultures divided by the number of mononuclear cells which migrated per oil field in response to supernates from cultures incubated with PBS only. Significance was calculated using the one-tailed Student’s t test. L?,mphoprol~~r~~ti,,cJ assays. Responses of mononuclear cells to several mitogens were tested. One-milliliter aliquots of cells were placed in 12 x 75-mm plastic tubes (Falcon No. 2054) to which 0.2 ml of phytohemagglutinin-P (PHA, Difco Labs, Detroit Mich.), pokeweed mitogen (PWM, GIBCO, Grand Island, N.Y.), concanavalin A (Con A, Pharmacia Fine Chemicals, Piscataway, N.J.), or phosphate-buffered saline (PBS) was added. PHA was used at 100 and 10 pg/ml, PWM was used at 100 and 20 pg/ml, and Con A was used at 3 pg/ml. After the addition of mitogens, the cells in each tube were placed in five replicate wells of flat-bottom 96-well microtiter plates (Falcon No. 3042). Forty-eight hours after initiation of cultures, cells were labeled with 1 PCi of tritiated thymidine ([3H]TdR, Amersham, 20-40 Ci/mmol) per well. Eighteen hours later cells were harvested onto glass fiber filter paper with a multiple automatic sample harvester (Mash II, Microbiological Associates) and prepared for liquid scintillation count-

MEASLES

VIRUS-INDUCED

IMMUNOSUPPRESSION

343

ing in Omniflour (New England Nuclear, Boston, Mass.)-toluene. Stimulation indices (SI) were calculated as previously described (24). Suppressor cell assays. Washed mononuclear cells (106/ml) in HBSS were, without prior culturing, treated with mitomycin C (50 &ml) at 37°C. After 45 min the cells were washed three times with HBSS and readjusted to lo6 cells/ml in complete RPMI. Aliquots (0.5 ml) of these cells were placed in 12 x 75mm tubes with 0.5 ml fresh cells (106/ml) from an individual donor used throughout the course of the study. The responses of the donor’s cells to PHA (10 &ml) and Con A (3 pg/ml) in the presence of cells from recipients of vaccine or diluent were tested. A portion of cells from the donor were also treated with mitomycin C and mixed with fresh donor cells to serve as a control for the above studies. The lymphoproliferative response of the donor cells to the above mitogens was evaluated using [3H]TdR incorporation, as described above. Identijication of mononuclear cells. Total T cells, B cells, macrophages, and cells bearing the receptor for the Fc portion of IgG (Fc y) were identified as previously described (25). Antibody assays. Plasma was assayed for antibody to measles virus in either hemagglutination inhibition assays or in an enzyme-linked immunosorbent assay (ELISA) for measles virus antibody. Virus isolution. Lymphocytes, obtained 3 and 7 days after vaccination were cocultured with Vero cells in microtiter plates for 16 hr. The lymphocytes were then removed and Vero cells were monitored daily for 7- 10 days for the appearance of measles virus cytopathic effect. The vaccine used was shown to cause cytopathic effect on Vero cells. RESULTS

Recipients of the vaccine did not demonstrate an increased temperature or any subjective clinical symptoms. Furthermore, virus could not be isolated from lymphocytes 3 or 7 days after vaccination. Chrmotactic

Assays

Supernates from cells cultured for 4 days in the presence of Con A, PPD, and measles virus were tested for the presence of chemotactic activity for normal human mononuclear cells (Table 1). There was an apparent total disappearance in the production of chemotactic factor(s) in response to Con A, purified measles antigen. and PPD in five of five vaccine recipients tested in this assay. The absence of chemotactic factor production was evident 1, 2. and 3 weeks after vaccination and returned to prevaccine levels for all antigens tested by 4 weeks after vaccination. The production of chemotactic factors in recipients of diluent showed weekly variability but did not change significantly over the course of this study. Lyr?lphoproliferati~lp Rrsponscs

Lymphocytes from 10 of 10 subjects receiving measles virus vaccine demonstrated depressed proliferative responses to the high dose of PHA tested (100 pg/ml). SIs were significantly decreased 1 and 2 weeks after vaccination but

344

tiIRSC

ti

k I‘ AI

4 215 2 54 ” Recipients of vaccine. ’ Recipients of diluent. :> P < 0.005, compared to preinjection

47 * 22:‘: 188 2 50

values.

70 2 27 84 k 33

13.5 lr 37

77 f 2.54:

I

2 3

C”

169 _i 41

V”

238 _f 43

0

(100 @ml)

Weeks after vaccine

PHA

Stimulation

MITOGEN

33 2 9

37 2 25 34 -c 6

57 -t 9

25

PWM V 83 i

indices

II

17

MEASLES

2

-

31 + 10 48 -c 16

80 t

662

C

AFTER

(20 &ml)

STIMULATION

TABLE

52 -t I2

56 2 33 42 i 12

62 t_ IO

107 + 40

V

Con C I8

-

51 _t 20 50 2 16

702

78 + 8

A (3 p*piml)

VACCINATION

1054 f 204

2260 f 1187 1344 t 256

727 -t 117

895 t 340

V

(cpm/? PBS

C

-

656 _f 189 901 t 280

534 r+ 260

803 + 348

x 10” cells)

z

s m

z 2

5 P

f3

z 0

‘f 2

m’ w

346

HIRSCH

E.1’ AL.

returned to prevaccine levels at 3 weeks after vaccination (Table 2). Lymphocytes from persons receiving diluent demonstrated a slight, but not significant. depression in high-dose PHA responsiveness 2 weeks after injection. Responses of lymphocytes from vaccine recipients to PWM and Con A were also slightly, but not significantly depressed. The baseline incorporation of IRH]TdR into cultures to which PBS was added did not change significantly after vaccination (Table 2), indicating that the observed depressions in SIs were not a reflection of an increase in background incorporation of 13H]TdR by lymphocytes obtained after vaccination (26, 271.

Cells from five of the patients receiving vaccine and three receiving diluent were obtained prior to and at various times after immunization. These cells were treated with mitomycin C and then cultured with cells from a normal donor in the presence of PHA and Con A. This test, designed to evaluate the presence of circulating cells capable of suppressing mitogenic responses (411, showed no significant changes after vaccination in levels of circulating suppressor cells (Table 3). Antibody Rrsponses

All subjects had antibody to measles prior to inoculation of vaccine and no significant changes in antibody levels were observed after vaccination using either hemagglutination inhibition or ELISA assays for detection of antibody. Cell Populations

Levels of total T cells, B cells, macrophages, and Fcr cells were evaluated at 0, I, 2, and 3 weeks after vaccination. There were no significant changes over time in the percentages of any of these cell populations (Table 4). DISCUSSION

These studies show that vaccination of normal, measles seropositive adults, with live attenuated measles virus results in a significant suppression of

EFFECTS

OF CELLS

FROM

TABLE 3 RECIPIENTS ON NORMAL

VACCINE

Weeks

after

0

84.8

Con

-+ 12.9

56. I r

16.1

LVMPHOCYTE

A

3

PHA

70.6

t

61.4

100.3

72.2

r

26.6

97.3

Con

_t 10.8 t

9.5

RESPONSES”

inoculation

2

PHA Vaccine recipients Diluent

DONOR

108 99.4

i

A 112.4

_t 7.1

PHA

Con

A

98.8

-c 7.0

99.8

i

12.7

101.3

? 6.6

88.2

-+ 26.3

recipients ” Results express the ratio (x 100) of 13H]Tdr incorporated by lymphocytes from a normal donor in the presence of mitomycin C-inactivated cells from subjects, relative to the amount of ]“H]TdR incorporated by the donor in the presence of an equal number of autologous mitomycin C-inactivated lymphocytes.

38.8

3

” Percentage ” Mononuclear Mononuclear ” Mononuclear ’ Mononuclear ’ V. Vaccine

32.0 _c 3.3 27.6 k 12.2 38.1 f 6.2

0 I 2 6.6 t 2.9

40.5 ? 12.7

V

4

6.3 -+ 3.0

beads (Immunobeads. of cells incorporating

1.8 + 1.3

4.0 r 1.0 2.0 -t 1.2 2.3 k 0.6

8.0 r 1.8 7.8 k 6.3 8.8 2 2.6

Fey’/

AFTER

V

BLOOD

C

IN PERIPHERAL

TABLE

B total’

PRESENT

7.8 _c 2.5 8.6 c 3.2 5.3 2 2.3

POPULATIONS

39.0 _t 13.9 28.8 t 10.9 30.5 ? 13.1

C

CELL

of at least 200 cells counted + SD. cells binding SRBC. cells binding rabbit anti-human IgG (H and L chain)-coated latex cells binding rabbit IgG anti-OX RBC-coated OX RBC (exclusive cells incorporating latex (0.8 pm) beads. recipients: C. diluent recipient controls.

+ 7.6

V’

Weeks after inoculation

T total”

MONONUCLEAR

1.0

Bio-Rad). latex beads).

4.0 t

V

MonocytesY

34.8

t 6.4

26.8 _f 2.0 28.4 2 10.4 18.7 2 6.5

VACCINATION

4.0 f 1.3 4.3 + 2.9 2.0 + 0.8

C

MEASLES

33 t

10.6

7.5 25.8 r 20.5 ‘- 11.8 24.5 i 7.9

C

%

z m C : zm

it z

c” 2 Q

‘f I?

b c

348

1tlRSC.H

k. I ,\I

lymphokine production and a limited suppression of lymphoproliferative rcsponses. This was demonstrated by inhibition of chemotactic factor production in response to Con A, PPD. and uv-inactivated purified measles virus and by inhibition of lymphoproliferative responses to high doses of PHA t 100 pg/ml). The immunosuppression was evident in the absence of a detectable viremia. clinical signs of infection. or an increased antibody response to measles. Although in t’itro addition of live measles virus to cultures of human peripheral blood mononuclear cells regularly inhibits lymphoproliferation in response to mitogens (14, 28, 29) this has not been a consistent finding using an i/l ~,ir,o virus challenge (16. 30-32). Measles virus has been demonstrated to infect lymphocytes after primary infection i/r r,ir~j (3 2, 33) and both monocytes and lymphocytes after primary or secondary infection in r’if,‘o (15. 17, 18, 34). This infection may account for at least part of the immunosuppression observed in these situations. However, when the host challenged has preexisting high titers of neutralizing antibody mononuclear cell infection seems less likely. Although it is possible that virus may have been present, infection of mononuclear cells was not detected in the vaccinated individuals in this study. In suppression of immune responses after vaccination of previously immune subjects, skin test responses are most consistently affected (8- 13). These delayed type hypersensitivity responses reflect complex interactions between accessory cells, sensitized T cells. lymphoproliferation. lymphokine production, and monocytes. The bulk of the cells in the skin test reaction are nonsensitized monocytes most likely responding in part to lymphokines produced by sensitized T cells in contact with antigen (23. 35). Lymphokine production is a T-cell function which is independent of lymphoproliferation (36, 37) and has been observed to be more severely impaired during primary measles virus infections (3 I ). Our data demonstrate that lymphokine mononuclear chemotactic factor production is also more severely affected after measles virus vaccination. In this study chemotactic factor production was suppressed to stimulation either with specific antigens (measles virus, PPD) or with Con A. The vaccine-induced immunosuppression is less profound and of shorter duration than that observed with primary natural measles virus infection and some different mechanisms are probably involved. One possible mechanism of suppression is the stimulation of a suppressor subset of cells and/or the production of a factor which in turn suppresses cellular responses. Measles virus causes a modest proliferation of T cells bearing receptors for the Fc portion of 1gG (Ty cells) (20). cells which correspond with those suppressing mitogen responses i/l lvitro. However, peripheral blood mononuclear cell populations did not change in percentage in the vaccine-inoculated volunteers. Likewise, a functional assay for circulating cells capable of suppressing mitogen responses did not demonstrate any increased activity. 111 1’itro studies with several viruses, not including measles, have demonstrated a depression in PHA responsiveness related to a suppressive factor produced by the adherent cell population in the presence of live or uv-inactivated virus (38). This factor has not been characterized but it is possible that phagocytes challenged with measles virus, even though they do not replicate the virus, produce

MEASLES

VIRUS-INDUCED

IMMUNOSUPPRESSION

349

mediators similar to interferon which in turn inhibit lymphokine production as well as lymphoproliferation (39,40). Although the mechanism of suppression after measles virus vaccine is not yet understood, the suppression seen in these studies may have a relationship to the lengthy suppression seen in acute measles infections (2-6 weeks after rash) at a time when virus is no longer recoverable. ACKNOWLEDGMENTS Supported in part by USPHS Grant NS 15721. R. L. H. and F. M. are Research Associates of the Howard Hughes Medical Institute. D. E. G. is an Investigator of the Howard Hughes Medical Institute. B. R. B. is supported by TIA No. K07-NS-00385. We would like to thank Norman Karasanyi for technical assistance and Linda Kelly for preparation of the manuscript.

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May

14, 1981; accepted

with

revision

July

IO, 1981

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1972. Exp. Med.

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151