Journal o f General Virology (1992), 73, 2273-2281.

2273

Printed in Great Britain

Potential signifcance of the cellular immune response against the macaque strain of simian immunodeficiency virus (SIVMAC)in immunized and infected rhesus macaques Gerald Voss,* Sigrid Nick, Christiane Stahi-Hennig, Cheick Coulibaly, Harald Petry, Wolfgang Liike and Gerhard Hunsmann Deutsches Primatenzentrum, Abteilung J~r Virologie und Immunologie, Kellnerweg 4, W-3400 Gfttingen, Germany

The cellular immune response of seven rhesus macaques immunized with Tween-ether-treated macaque strain of simian immunodeficiency virus (SIVMAc) and three non-vaccinated control animals was investigated. Immunization elicited antigen-specific proliferating CD4 ÷ cells in five of seven monkeys. Proliferating T cells were found in all animals protected from a first virus challenge. Cytotoxic T lymphocytes (CTLs) were not induced by the immunization. After the second challenge, the four formerly protected animals became infected, despite a strong proliferative CD4 ÷ cell activity in three of them. All animals lost their proliferative activity 2 weeks after infection. After the first challenge four of the six infected animals exhibited a CTL response and after the second challenge, one of

four newly infected macaques acquired a CTL response. The five animals with a CTL activity against SIVMAc proteins were protected from severe thrombocytopenia, which appeared in the five CTL-negative animals after infection. Our data show the induction of proliferative T cells by immunization with soluble SIVMAc antigen. This T cell reactivity was found in all animals protected from the first virus challenge, but did not confer protection from the second challenge. Interestingly, the proliferative T cell reactivity disappeared 2 weeks after virus infection. Furthermore a CTL response against viral proteins seems to protect infected animals from severe thrombocytopenia which is an early sign of AIDS in monkeys.

Introduction

(Nixon et al., 1988, 1990; Littaua et al., 1991 ; Walker et al., 1989; Hosmalin et al., 1990; Koenig et al., 1990; Clerici et al., 1991 ; Earl et al., 1991 ; Hammond et al., 1991 ; Culmann et al., 1991). Immunization with HIV-1

Human (HIV) and simian immunodeficiency viruses (SIV) induce a cellular immune response in the infected host. Antigen-specific T helper cells were found in HIV1-infected humans (Wahren et al., 1987; Ahearne et al., 1988), chimpanzees (Eichberg et al., 1987) and in SIVMAc-infected macaques (McGraw et al., 1990; Ahmed-Ansari et al., 1990). Immunization of chimpanzees (Zarling et al., 1987; Mannhalter et al., 1991), macaques (Mills et al., 1990) and humans (Berzofsky et al., 1988) also induced a specific T helper cell response. T helper cell epitopes were identified on viral structural gag, pol and env proteins (Mills et al., 1990; Berzofsky et al., 1988; Schrier et al., 1988, 1989). Virus-specific cytotoxic T lymphocytes (CTLs) against various viral proteins were detected in HIV-l-infected individuals (Walker et al., 1987, 1988 ; Plata et al., 1987; Shepp et al., 1988; Nixon et al., 1988; Koenig et al., 1988; LangladeDemoyen et al., 1988; Chenciner et al., 1989; Hoffenbach et al., 1989; Riviere et al., 1989; Gotch et al., 1990; Achour et al., 1990; Littaua et al., 1991) and several CTL epitopes have been identified on different viral proteins 0001-1004 © 1992 SGM

gpl60 induced CD4+-specific CTLs in humans (Orentas et al., 1990) and gpl60-containing immune-stimulating complexes generated specific CD8 ÷ CTLs in mice (Takahashi et al., 1990). SIVMAc gag-specifc CTLs were found in infected rhesus macaques (Macaca mulatta) (Miller et al., 1990) and their respective epitope was identified on p26 (Yamamoto et al., 1990a). Likewise, in cynomolgus macaques (Macaca fascicularis) immunized with gag-expressing recombinant vaccinia virus (VV) (Gotch et al., 1991) gag-specific CTLs were induced. Furthermore it was shown that SIVMAc env proteins are recognized by CTLs from infected animals (Yamamoto et al., 1990b). Although the role of virus-specific CTLs in vivo is still unclear, it was shown that CD8 ÷ lymphocytes from seropositive donors can prevent HIV-1 infection in vitro (Tsubota et al., 1989; Kannagi et al., 1990). SIVMAcspecific CD8 ÷ cells exhibit similar in vitro inhibition (Tsubota et al., 1989; Kannagi et al., 1988). From this knowledge it can be concluded that, if

2274

G. V o s s a n d o t h e r s

functional significance is demonstrated, a potential vaccine against AIDS should induce a cellular immune response. The best established animal model for vaccine s t u d i e s a r e m a c a q u e s i n f e c t a b l e w i t h S I V ( L e t v i n et al., 1985) o r H I V - 2 ( P u t k o n e n et al., 1989; D o r m o n t et al., 1989; S t a h l - H e n n i g et al., 1990). P r o t e c t i o n a g a i n s t S I V or HIV-2 infection by immunization with formalini n a c t i v a t e d ( D e s r o s i e r s et al., 1989; M u r p h e y - C o r b et al., 1989; C a r l s o n et aL, 1990) o r d e t e r g e n t - t r e a t e d v i r u s ( D e s r o s i e r s et al., 1989; P u t k o n e n et aL, 1991; S t a h l H e n n i g et al., 1992) c a n be a c h i e v e d r e p r o d u c i b l y , b u t so f a r t h e r e is n o p a r a m e t e r a v a i l a b l e w h i c h p r e d i c t s p r o t e c t i o n in t h e a n i m a l s . We have therefore examined the cellular immune response of rhesus monkeys immunized with Tweene t h e r ( T E ) - t r e a t e d SIVMA C. T h e r e s u l t w a s t h a t o n l y a n i m a l s w i t h a p r o l i f e r a t i v e T cell r e a c t i v i t y w e r e protected from virus infection and that no other p a r a m e t e r c o r r e l a t e d w i t h p r o t e c t i o n ( S t a h l - H e n n i g et al., 1992). N o w w e r e p o r t t h a t a f t e r a s e c o n d c h a l l e n g e t h e f o r m e r l y p r o t e c t e d a n i m a l s t h o u g h still s h o w i n g a p r o l i f e r a t i v e T cell r e s p o n s e also b e c a m e i n f e c t e d . F u r t h e r m o r e t h e i n f e c t e d a n i m a l s lost t h e i r T cell proliferative activity. Comparison of the immunological d a t a to t h e c l i n i c a l status o f t h e i n f e c t e d m a c a q u e s revealed that monkeys lacking a CTL response acquired a severe thrombocytopenia (TP), while CTL-positive a n i m a l s b e c a m e o n l y m i l d l y o r n o t at all t h r o m b o cytopenic.

Methods Cells, viruses and antigen preparation. The SIVMAc251/32Hisolate (Cranage et al., 1990) obtained after in vivo passage of the original SIVMAc2~1 (Daniel et al., 1985) was used for immunization of the rhesus macaques. For antigen preparation, the virus was grown on C8166 cells, concentrated by differential centrifugation and further purified by column chromatography. The purified antigen was supplied by Dr M. P. Cranage and the programme EVA. The vaccine was prepared by TE extraction. The preparation contained mainly SIV antigens as demonstrated by PAGE and Western blot analysis (for details see Stahl-Hennig et al., 1992). Antigen for proliferation assays was obtained by dissolving purified virus in CG medium (Camon) supplemented with 1~ (v/v) human AB serum, filtration through a 0.45 ~m filter and subsequent heat inactivation (30 min at 56 °C). The recombinant SIVMAc VV used for CTL assays encoded the gag, pol or env gene of the SIVMAcBK28 clone (Kornfeld et al., 1987). VV gag expressed the 57K gag precursor, VV-pol encoded the reverse transcriptase and integrase domain of the pol gene and VV-env contained the entire env gene. The VV constructs were kindly provided by Dr F. Bex, Dr M. Delchambre and Dr A. Burny, Universit6 Libre de Bruxelles, Belgium. Animals, immunization scheme and determination of virus infection. Ten rhesus macaques of Indian origin from the breeding colony of the DPZ were used for the vaccine trial (Stahl-Hennig et al., 1992). Briefly, seven rhesus macaques were immunized four times at 18, 14, 10 and 2 weeks before challenge with 140 p.g purified TE-treated SIVMAc25u32H

adsorbed onto AI(OH)3 as an adjuvant. The remaining three animals received only adjuvant and are later referred to as controls. Two weeks after the last booster the 10 animals were challenged with 50 monkey infectious doses (MIDs0) of SIVMAc2S1/32H. The challenge virus was kindly provided by Dr M. P. Cranage and Dr P. J. Greenaway. Subsequent virus infection was determined by polymerase chain reaction, virus reisolation, urinary neopterin increase, seroconversion and clinical findings as described elsewhere (Stahl-Hennig et al., 1992). Sixteen weeks after the first challenge the four protected macaques were rechallenged with the same virus dose and became infected. Isolation of peripheral blood mononuclear cells (PBMCs) and preparation of Tcell subsets. PBMCs were separated from whole citrated blood by Ficoll-diatrizoate (Histopaque, Sigma) density gradient centrifugation. CD4 + or CD8 + T cell subsets were separated with immunomagnetic beads. CD4- or CD8-specific beads (Dynal) were incubated at a ratio of 4 : 1 with PBMC for 30 min at 4 °C. The cells bound to the beads were then collected in a magnetic particle separator (MPC-6, Dynal) and the purified cells as well as the remaining cell suspension were used for further assays. The purity of the cell preparations was controlled by flow cytometry as described (Martin et al., 1983). The separated cells were always over 96% pure and the remaining cells never contained more than 2% of the depleted cell population. Antigen-specific proliferative T cell response. An antigen-specific T cell proliferation assay was performed repeatedly during the immunization period and after the virus challenge. PBMC (1 x l0 s) (whole population, purified CD4 + cells or remaining cells) in a total volume of 100 ~tl CG medium supplemented with 1% (v/v) human AB serum, 50 units/ml penicillin and 50 I~g/ml streptomycin per well were seeded in U-bottomed 96-well plates. For antigenic restimulation, 1 ~tg purified heat-inactivated (30 min at 56 °C) virus per well was added. After a 3 day culture period at 37 °C in a humidified 5 ~ CO2 atmosphere, 50 p.1 CG medium was added to each well. Seven days after the initiation of the cultures, 0-5 gCi [3H]thymidine (50 Ci per mmol, Amersham) was added for 6 h. Thereafter the cells were harvested onto glass fibre filters and the incorporated radioactivity was determined in a fl-counter. The mean counts of triplicate cultures were used to calculate the antigenspecific stimulation indices (SI) (values of restimulated cultures v. control cultures). SI over 2.5 were considered positive. For major histocompatibility complex (MHC) blocking experiments 1 ~tl per well of monoclonal antibody (MAb)-containing culture supernatant of the hybridoma cell lines W6/32 (anti-class I) (Parham et al., 1979) or 12G6 (anti-class II DR) (Samstag et al., 1991) were initially added to the cultures. Cytotoxicity assay. To obtain MHC-compatible target cells for cytoxicity assays, herpesvirus papio-transformed B lymphoblastoid cell lines (BLCLs) (Rabin et al., 1977) were generated from each monkey prior to immunization. These target cell lines were infected with 1 p.f.u. per cell of either recombinant or wild-type (wt) VV (strain Copenhagen) 16 h before the assay. Effector cells were generated by cultivation of PBMC in RPMI-1640 supplemented with 10~ (v/v) foetal calf serum (FCS), 50 units/ml penicillin, 50 Ixg/ml streptomycin and 10 ktg concanavalin A per ml at 1 × 106 cells per ml for 3 days (Miller et al., 1990). Thereafter the medium was replaced by RPMI-1640 containing 10~ FCS and 20 units per ml recombinant human interleukin-2. After an additional 4 day culture period the cells were used for the cytotoxicity assays. For the determination of the effector T cell subset, the precultured cells were separated with CD8-specific beads prior to the assay. The MHC restriction of the effector cells was examined by incubation with MHC class I-matched or mismatched target cells. The MHC typing was performed using one-dimensional isoelectric focusing (Gotch et al., 1985).

Cellular immune response to SIVMAc

275 g for 5 m i n and 100 ~tl supernatant was harvested. The supernatants were counted in a gamma-counter and the m e a n counts of triplicate samples were used to calculate the specific s lCr release (experimental release - spontaneous release v. m a x i m u m release - spontaneous release). The m a x i m u m release was determined with 5 % (v/v) Triton X-100. The spontaneous release never exceeded 25% of the m a x i m u m release.

SIVMAc-Specific cytotoxicity was determined in a standard slCrrelease assay (Grabstein & Chen, 1980). One-million VV-infected target cells were labelled with 50 ~tCi Na2[ 5~Cr]O, (specific activity 400 m C i / m g Cr; A m e r s h a m ) for 1 h. T h e n I x 104 target cells in 100 ~tl per well were seeded in 96-well U-bottomed plates and the effector cells were added at different effector to target ratios from 100 to 12.5:1 in 100 ~tl. After a 5 h incubation at 37 °C the plates were centrifuged at

T a b l e 1.

CTL reactivities of SIVMAc TE-vaccinated rhesus monkeys Time after first challenge (weeks)

Animal no.

VV*

0

4

8

12

20

ND

NO

ND

ND

22

26

30

3 37 18 18

7 26 26 0

16 22 13 18

Non-vaccinated controls 1639

1673

wt gag pol env wt gag pol env

0~" 0 ND 0

ND~

1 19 ND 21 0

ND

ND

0

2

NO 0 0 0 ND 1

0 5 ND 2

0 0 ND 4

2 9 10 13

wt gag pol env

0 0 ND 5

3 2 ND 6

1 7 ND 16

0 27 16 22

wt gag pol env

27 15 ND 13

lI 26 ND 47

wt gag po1 env

3 4

0 0

0 0

ND

ND

ND

7

1

0

1707

1713

1714

ND

1696

1703

1722

wt gag pol env wt gag pol env wt gag pol env wt gag pol env

ND

4 6 ND 5

8 9 8 13

3 2 2 4

1 2 2 6

7 21 30 41

4 17 20 18

4 5 20 13

0 14 18 18

0 1 0 8

32 36 28 48

18 16 18 18

ND

ND

ND

NO

ND

ND

55 44 ND 54 31 28 ND

31

Vaccinated protected 1663

0

1 10

wt gag 19ol env Vaccinated infected 1709

Infected§ 0 0

0 0

1 3

2 0

ND

ND

ND

ND

1

0

10

0

13 15 13 14

3 0 ND 0

18 36 28 49

ND

ND

l 0 ND 0 0 0 ND 4

ND

3 0 ND 0

2 ND

0

ND 4

ND

0 ND

0

ND 0

2275

18 18 13 16 7 4 10 13

ND

ND

ND

9 15 14 31

ND

ND

ND

ND

3 6 ND 5 6 13 12 21 1 0 ND 3 ND

* Autologous target cells were infected with respective VV and incubated at an E :T ratio of 50:1. t Percent specific lysis. :~ ND, Not determined. § Animals were rechallenged 16 weeks post-first challenge.

2276

G. Voss and others

Results

Table 3. MHC restriction of SIVMAc-Specific separated CD8 + cells

SIVMAc-SpecifiC CTLs in the rhesus macaques occurred only after infection Seven immunized rhesus macaques and three nonvaccinated controls were examined for SIVMAc-Specific C T L activity during the immunization period and after a first virus challenge (Table 1). Prior to the challenge no C T L activity was detectable in any monkey. After the first virus challenge six of the 10 monkeys became infected, the three controls ( M m 1639, 1673 and 1709) and three vaccinated animals (Mm 1707, 1713 and 1714). Four of them (Mm 1639, 1707, 1709 and 1713) developed virus-specific CTLs starting 8 weeks post-challenge (p.c.). The C T L s were directed against the SIVMAc gag, pol and env proteins. Two monkeys ( M m 1707 and 1639) exhibited virus-specific C T L s during the whole investigation period up to 36 weeks p.c. In contrast, M m 1709 showed only a transient C T L reactivity 12 weeks p.c. Animal 1713 reacted against gag and env initially, but later developed a very high non-specific cytolytic activity. This activity was apparently related to VV infection of the target cells though uninfected target BLCLs were not lysed (data not shown). The other two infected ( M m 1673 and 1714) and the four protected macaques ( M m 1663, 1696, 1703 and 1722) did not show any specific C T L response. After the rechallenge with 50 MIDs0 16 weeks after the first challenge the four formerly protected animals also became infected. In response to infection one additional animal ( M m 1696) mounted a virus-specific CTL-reactivity.

The CTLs were CD8 + and their activity was M H C class 1-restricted To define the cells that mediated the cytolytic activity, the precultured P B M C s were separated with CD8specific beads. As shown in Table 2 for the animal 1707

Table 2. SIVMAc-specific lysis mediated by separated CD8 + cells 51Cr release (%) Effector cells*

VV (wt)l"

VV-gagt

VV-polt

VV-envl

Whole -CD8 ÷ CD8+

5 7 4

17 8 18

13 5 20

23 20 17

* Whole precultured PBMC of Mm 1707 or CD8+ cells with immunomagnetic beads (CD8÷) or the cells remaining after depletion ( - CD8÷) were incubated at an E :T ratio of 50 : 1. t Autologous target cells were infected with VV (wt), VV-gag, VVpol or VV~nv.

51Cr release (%) Target cells* Autologous Matched Mismatched

VV (wt)t 2 4 3

VV-gagi" 22 24 4

VV-pol'~

VV-envt

13 11 3

16 18 5

* Precultured isolated CD8+ cells of Mm 1639 were incubated with autologous, MHC class I-matched (Mm 1709) or mismatched (Mm 1696) target cells at an E :T ratio of 50 : 1. i" Target cells were infected with VV (wt), VV-gag, VV-pol or VVenv.

the isolated CD8 ÷ cells exhibited cytotoxicity against gag, pol and env. The remaining cells ( - C D 8 ÷) did not lyse target cells infected with V V - g a g or VV-pol, but still reacted with VV-env-infected targets. This shows that CD8 ÷ cells were responsible for the lysis of target cells expressing the different viral proteins. Furthermore, the env-specific lysis was also mediated by an additional cell population. To characterize the virus-specific lytic activity further, isolated CD8 ÷ cells were incubated with autologous, M H C class I-matched or mismatched target cells (Table 3). Purified CD8 ÷ cells of animal 1639 lysed autologous and M H C - m a t c h e d target cells expressing the SIVMAc gag, pol or env proteins. In contrast, the same effector cells were unable to lyse M H C - m i s m a t c h e d target cells. Thus, the cytotoxic activity of the CD8 + cells was obviously M H C class I-restricted. Similar results were also obtained for other animals.

Antigen-specific proliferative T cell response did not correlate with protection against the second virus challenge As a second parameter of the cellular immune response, antigen-specific proliferating T cells were measured. As reported previously, all animals protected from a first virus challenge possessed a proliferative T cell response whereas two out of three infected vaccinees did not react in the assay (Fig. 1) (Stahl-Hennig et al., 1992). At 2 weeks p.c. only the four protected animals displayed a proliferative activity whereas the infected M m 1707 had lost its proliferative activity (Fig. 1). To confirm the importance of a proliferative T cell reactivity the four protected animals were rechallenged 16 weeks after the first challenge, when three of them (Mm 1663, 1696 and 1703) still showed a considerable proliferative response. However the animals were not protected any longer and all four monkeys became infected despite their good

Cellular immune response to SIVMA C

25~

i

i

r

I

I

I

(a)

I

(a)

I1639 ~7511673

20

8

~ ~

No MAb 12G6 W6/32

(b)

2277

~ Whole ~ CD4 + D -CD4+-

D'7"~1709

15

O

10

~.~'~

5 2 0 .g4 25

i

.... i

I

ii l l

~

i

I

(b)

MHC blocking

CD4 separation

Fig. 2. Proliferative response of separated CD4 + cells and MHC restriction. PBMCs of monkey 1663 were restimulated with purified SIVMac without or in the presence of MAbs 12G6 or W6/32 (a). Cells from the same monkey were separated with CD4-specific immunomagnetic beads and the isolated cells as well as the remaining population were restimulated separately with SIVMac. The SI values are compared to those obtained with unseparated PBMCs in (b).

1707

1~]1713 20

0

i

P'7"/'~1714

.~ 15 ¸ O

10'

The proliferating cells were CD4 ÷ and inhibited by antibodies against M H C class H

2 0 25

i

(c)

i

i

Challenge First Second

20

i 15

-9

0

i

1:X'~]1696 ~'7"~1703 ~1722

2 16 18 20 Time post-challenge (weeks)

22

28

Fig. I. Proliferative response of SIVMAc TE-immunized rhesus macaques. The proliferation of PBMCs from 10 rhesus monkeys in response to purified SIVMAc was determined repeatedly during the immunization period and after the two challenges. The reactivities of the control animals are shown in (a) (ND, not done). (b) Proliferative responses of the immunized macaques that were not protected from the first challenge; the protected macaques are represented in (c). The c.p.m, of the control cultures, which were used to calculate the SI, ranged between 400 and 2400.

proliferative activity. Like the one macaque (Mm 1707) not protected from the first challenge, the four animals being infected after the second challenge exhibited a remarkable decrease of their proliferative activity. Two weeks after the second challenge none of them reacted in the assay. The macaques have now been followed up to 36 weeks p.c. and four vaccinated and subsequently infected animals only transiently developed a proliferative activity (Mm 1663, 1713, 1714 and 1703).

For a more detailed examination of the antigenresponding cells PBMCs were separated with CD4specific immunomagnetic beads. The cells remaining after depletion as well as the purified CD4 ÷ cells adhering to the beads were used for proliferation assays. When purified CD4 ÷ cells of Mm 1663 were tested they gave SI values comparable to those obtained with unfractioned PBMCs (Fig. 2b). In contrast, the CD4depleted remaining cells failed to proliferate in response to the viral antigen preparation. These results indicated that the proliferative response to viral antigen was attributed to the CD4 + cells. This proliferation of CD4 ÷ cells was also observed with PBMCs from the other animals (data not shown). To demonstrate the MHC restriction of the antigen stimulus required for proliferation MHC blocking experiments with MAbs to MHC class I or class II were performed (Fig. 2a). The MHC class I-specific MAb W6/32 induced a slight reduction of the antigen-specific proliferation of PBMCs from Mm 1663, whereas the MHC class II DR-directed MAb 12G6 completely inhibited proliferation. Thus the antigen presentation to the proliferating cells was MHC class II-dependent. Correlation o f the cellular immune response with clinical observations

After the virus challenges the 10 rhesus macaques were monitored for clinical manifestations of SIVMAc-induced disease. Two of the three controls and all three vaccinees, which became infected after the first challenge, de-

2278

G. Voss and others

Time after challenge (weeks) 1st 0

Mm1639

TP C T L [-

4

8

÷

2nd 0 12 16

I

4 20

8 24

12 28

16 32

I _ _ ]

] ÷

Mm1673 TP CTLI

+

?

(+]

Mm1709 TP C T L t-

Mm1707 TP C T L E-

+

+

+

1

]

?

~xxxx~+

Mm1713 TP CTL ~

+

Mm1714 TP C T L ~-

i

Mm1663 TP CTL

L-

t+l

Mm1696 TP CTL

t

+

Mm1703 TP CTL

L

7

Mm1722 TP CTL

[

~+J

20 36

+

+

¢.xxxxxx~ + r

, ~

~ ]

r

]

1

7-

+

]

+

E:3 ]

j

}

Fig. 3. Clinical findings and their relationship to the cellular i m m u n e response. Seven i m m u n i z e d rhesus m a c a q u e s and three control animals were examined after virus challenge for TP and C T L activity. These two parameters were c o m p a r e d to each other. T P was defined as a d r o p of the thrombocyte counts under the physiological level of 2 × 10 5 to 3.5 x 10 s per ~tl ([]). TP was considered to be severe w h e n the counts were under 1 x 10 5 per p.l ( m ) . T h r o m b o c y t o s i s ( 1 ~ ) was a s s u m e d at platelet counts over 4 x 10 5. The C T L response was considered positive w h e n the animal reacted against at least two viral proteins simultaneously as indicated by + . A response against one protein is m a r k e d with ( + ) . An uncertain reaction because of high non-specific lysis against VV (wt) is indicated by ?

veloped a lymphadenopathy (LA) (data not shown). The animal Mm 1709 only transiently exhibited a mild LA from 8 to 12 weeks p.c. Two of the four formerly protected animals infected following the second challenge showed a progressive LA (Mm 1696 and 1722) and the other two developed only a mild LA (Mm 1663, Mm 1703 only transiently). The second important clinical

finding was a TP, which was induced by the virus infection (Fig. 3). Interestingly only animals that did not show a CTL response to SIVMAc proteins exhibited a thrombocyte count lower than 1 x 105 per p.l. The CTLreactive Mm 1713 developed a pronounced TP 12 weeks p.c., but recovered 4 weeks later, reaching a less severe level. In contrast to the frequently observed loss of thrombocytes in the other infected animals, Mm 1707 developed a thrombocytosis. The occasional proliferative response of four macaques after virus infection did not correlate with clinical parameters.

Discussion The cellular immune response of S I V M A C TE-vaccinated macaques was investigated during the immunization period and after two virus challenges. Immunization with SIVMAc TE did not induce virus-specific CTLs (Stahl-Hennig et al., 1992). Such cells were detectable only after virus infection and were shown to be CD8 ÷ and MHC class I-restricted. CTLs against the major viral proteins gag, pol and env could be detected simultaneously, indicating a broad reactivity against S I V M A C. Gag-specific CTLs were also detected by Miller et al. (1990) in infected macaques and by Gotch et al. (1991) in macaques immunized with recombinant VV coding for gag proteins. In both cases, the cytolytic activity was also attributed to CD8 + cells and was MHC class I-restricted. So far CTLs directed against pol proteins have been reported only in humans infected with HIV-I (Walker et al., 1988). Our data indicate that the pol proteins serve as CTL targets in SIVMAc-infected macaques. The lytic activity against env was mediated by two different cell populations. Besides CD8 + MHCrestricted CTLs a second CDS- cell population was able to lyse VV-env-infected target cells. This is in agreement with Yamamoto et al. (1990b) who found that two different cell populations contribute to env-specific lysis. In that case the MHC-independent lytic activity was mediated by CD16 + cells. Similar MHC-unrestricted cytotoxicity was also demonstrated in HIV-l-infected humans (Koenig et aL, 1988; Riviere et al., 1989; Weinhold et aL, 1988). Furthermore, CD4-positive and MHC class II-restricted env-specific CTLs were observed in immunized humans (Orentas et al., 1990; Hammond et al., 1991). Our observations of the cellular immune response of immunized rhesus monkeys demonstrate that protection can be achieved without a detectable virus-specific CTL response. It seemed rather that virus-specific CTLs are important during the course of SIV infection. Comparison of the CTL responses with the clinical data suggest a link between the lack of virus-specific CTLs and a severe

Cellular i m m u n e response to S I V M A c

TP. Since TP is an important parameter for disease progression in human A I D S (Karpatkin, 1990) the S I V macaque model may serve as an experimental system to clarify the role of virus-specific CTLs for pathogenicity. Surprisingly M m 1707 exhibited a thrombocytosis, whereas for most of the other animals their thrombocyte counts dropped. We cannot yet explain this phenomenon, since we observed only one other case among 48 SIVMAc-infected macaques. Although a milder stage of TP was correlated with a C T L response, the C T L reactivity did not delay the development of L A in the animals. Thus it cannot generally be concluded that virus-specific CTLs prevent disease symptoms. In contrast to the C T L response, antigen-specific proliferating T cells could be induced by immunization with TE-treated SIVMAc (Stahl-Hennig et al., 1992). The responding cells were C D 4 ÷ and the proliferation could be inhibited by a M A b directed against M H C class II molecules. The type of responding cells was similar to those detected in SIVMAc-infected macaques ( M c G r a w et al., 1990; Ahmed-Ansari et al., 1990). Unlike those investigators we found only occasionally a transient proliferative response following infection. All five animals with a proliferative T cell response lost their reactivity 2 weeks after the first or the second virus challenge and did not regain this T cell function. Nevertheless the animals exhibited an anamnestic antibody response (data not shown). Since an antiviral B cell response requires T cell help, it remains unclear how the macaques mounted an antibody response. Since there was only a slow decline of the T4 :T8 ratio and the relative T4 cell counts (data not shown), virus infection selectively inhibits the antigen-specific C D 4 + cells. This may be due to a preferential infection of virus-specific helper T ceils or the activity of suppressor T cells. We have observed an apparent discrepancy regarding the connection between proliferative T ceils and protection from the two virus challenges. All animals protected from the first virus challenge possessed proliferative T cells, but the same animals were not protected from the second challenge, although they still exhibited a good proliferative activity. This result indicates that there is no overall correlation between proliferative T cells and protection. We speculate that proliferative reactivities against distinct viral proteins or epitopes m a y indicate protection, but these reactivities cannot be determined with the assay employed. The dissection of the proliferative response with single purified proteins or peptides using cloned C D 4 ÷ cells (Mills et al., 1990) might lead to the recognition of epitopes correlated with protection. Recently, Stott et al. (1991) reported protection from SIVMAc infection in macaques immunized with fixed uninfected C8166 cells and a correlation with anti-cell antibody titres. Le G r a n d et al. (1992) and Osterhaus et

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al. (1992) also observed anti-cell antibodies but did not find a relationship with protection. Hu et al. (1992)

induced protection using a SIV gpl60 subunit vaccine. These results indicate that viral as well as cellular components may contribute to the protection against SIV challenge. The role of cellular components could not be addressed by our investigations. Indeed, although the highly purified SIVMAc antigen was used to immunize the animals and to restimulate the CD4 ÷ cells, it cannot be excluded that the proliferative responses were in part directed against cellular components. Nevertheless, this assumption does not invalidate our two main observations: (i) all animals protected from the first virus challenge exhibited a proliferative response and there was no correlation with either neutralizing antibodies or with anti-cell antibodies (data not shown); (ii) the immunized animals lost their proliferative T cell activity after virus infection. Furthermore additional vaccine experiments showed that pooled SIVMAc peptides or purified recombinant SIV gpl40 used for restimulation induced proliferating T cells specific for SIVMAc (data not shown). However it is possible that anti-cell-directed proliferative T cells contributed to protection from the first virus challenge. We would like to thank Dr F. Bex, Dr M. Delchambre and Dr A. Burny, Department of Molecular Biology, Universit6 Libre de Bruxelles, Belgium for providing us with recombinant SIVMAcVV and Dr T. Dengler and Dr S. Meuer, Deutsches Krebsforschungszentrum, Heidelberg, Germany, for supplying us with hybridoma cell lines W6/32 and 12G6. We would also like to thank Dr M. P. Cranage and Dr P. J. Greenaway, CAMR, Porton Down, U.K. for providing us with the challenge virus stock. The SIV antigen was provided by programme EVA. This paper contains parts of the doctoral thesis of G.V.

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(Received 13 January 1992; Accepted 12 May 1992)