FEMS MicrobiologyImmunology89 (1992)97-104 © 1992Federation of European MicrobiologicalSocieties0920-8534/92/$05.00 Published by Elsevier

97

FEMSIM 00193

Passive immunization protects guinea pigs from lethal toxoplasma infection Charles S. Pavia 1, Susan J. Bittker 1 and K at hi e E. Curnick 2 1 Department of Medicine, Division of Infectious Diseases and Department of Microbiology and Immunology, New York Medical College, Valhalla, New York, and 2 Trudeau Institute, Saranac Lake, New York, U.S.A.

Received2 October 1991 Accepted 9 October 1991 Key words: Transfer of immunity to lethal toxoplasmosis; anti-Toxoplasma immunity; Toxoplasma gondii; Immunotherapy to toxoplasmosis

1. SUMMARY The cellular and humoral interactions that contribute to protective immunity in toxoplasmosis were studied by adoptive transfer of selective cell populations or immune serum and its fractions into normal syngeneic strain 2 guinea pigs. The results of this study with the RH strain of Toxoplasma gondii confirm and extend the findings of previous studies by showing that the passive transfer of parasite-sensitized T cells or of immune serum from previously infected donors protected recipient guinea pigs against lethal toxoplasmosis. An additional key finding was that similar levels of complete protection against lethal infection occurred in guinea pigs receiving partially purified anti-Toxoplasma immunoglobulins or immune ceils that had been enriched for B ceils prior to transfer. Cells residing in the spleen,

Correspondence to: C.S. Pavia, Divisionof InfectiousDiseases,

Room 209SE, Macy Pavilion, Westchester Medical Center, Valhalla, NY 10595 U.S.A.

lymph nodes and peritoneal cavity, but not the thymus, were equally effective in conferring immunity to challenged recipients. In addition, cell titration experiments revealed that guinea pigs could survive T. gondii infection by infusing them with as little as 2 x 10 7 sensitized T cells or B cells. Unlike protection mediated by T cells, protection against lethal disease occurring in the B cell recipients was associated with the formation of Toxoplasma antibodies. These findings illustrate the major role of both humoral and cellmediated immunity in affording protection against toxoplasmosis based on a guinea pig model of the human disease.

2. INTRODUCTION With the use of appropriate animal models and passive transfer systems, past studies [1-4] have shown that acquired resistance to infection with the RH strain of Toxoplasma gondii depends upon activation of both humoral and cellular components of the host's immune system. In this

98 regard, while there is convincing evidence for the T cell-mediated nature of anti-Toxoplasma immunity [2,3,5,6] there is now mounting evidence that antibodies or other humorat factors [1,2,7] are also protective against toxoplasmosis. However, the manner in which T cells interact with each other as well as with B cells and macrophages in providing protection or in limiting effective immune mechanisms throughout various stages of toxoplasmosis is not fully known. Recent experiments from our laboratory [2,3] have demonstrated the feasibility of using immunocompetent and T cell-deficient inbred guinea pigs for delineating the nature of the immune mechanisms that may be operative during a local, intradermally introduced Toxoplasma infection. As an extension of this experimental system, we report here the results of passive transfer experiments designed to determine the role of humoral and cell-mediated immunity in affording protection against the usually lethal (intraperitoneally induced) infection [2] caused by the RH strain of T. gondii. Our results also demonstrate that whole serum or partially purified immunoglobulins, as well as both T cells and B cells, from guinea pigs resistant to Toxoplasma can confer complete protection against lethal T. gondii infection.

3. MATERIALS AND METHODS

3.1. Animals Strain 2 guinea pigs were bred under barriersustained conditions at the Animal Breeding Facility at the Trudeau Institute from animals originally obtained from the Division of Research Services, National Institutes of Health, Bethesda, MD. Adult (3-20-month-old) animals weighing 400-1100 g and having negative reactions in the indirect hemagglutination (IHA) assay (TPMTest; Wampole Laboratories, Division of CarterWallace, Ind., Cranbury, N J) were used in these experiments. Male and female 12-20-week-old B6D2 and AB6 mice were used for the routine maintenance of T. gondii parasites by serial peritoneal passage [2,3].

3.2. Detection of antibodies Whole blood was collected from guinea pigs before infection and at various intervals postinoculation. Serum was assayed for the presence of anti-Toxoplasma antibodies by using the IHA test performed in accordance with the instructions supplied by the manufacturer.

3.3. Parasites and infections The virulent RH strain of T. gondii was obtained from Dr. Jack Remington (Palo Alto Medical Research Foundation, Palo Alto, CA). In passive transfer experiments (described below), guinea pigs were injected i.p. with 10 4 tachyzoites. The latter parasites were derived from peritoneal exudates of infected passage animals and were prepared as detailed elsewhere [2,3].

3.4. Preparation of lymphocytes Spleens and draining lymph nodes (popliteal, inguinal, and iliac-lumbar) from normal and resistant donors were removed aseptically, and single cell suspensions were prepared in RPMI 1640 medium by teasing apart the excised organs with forceps and gently pressing the tissue through a 60-mesh stainless steel screen. After washing once, harvested cells were resuspended in RPMI 1640. Viable nucleated ceils were enumerated after staining with 0.4% trypan blue. Peritoneal exudate cells (PEC) were obtained by injecting 25 ml of sterile light mineral or paraffin oil (Fisher Scientific, Springfield, N J) into the peritoneal cavity of normal or immune donor guinea pigs. Three to four days later, the guinea pigs were injected with 100 ml of heparinized (20 units/ml) RPMI 1640. After washing twice, the harvested cells were resuspended in 2-3 ml RPMI 1640 and were counted after staining with 0.4% trypan blue. Differential staining of thin smears with Giesma stain and phagocytosis of latex particles revealed that 75-80% of the PEC were macrophages or monocytes, 10-15% were lymphocytes and the remainder were granulocytes (neutrophils, eosinophils and basophils).

3.5. Enrichment for T cells and B cells In some experiments, spleen and lymph node cell suspensions were enriched for T cells by a

99 modification [2,8] of the technique of Mage et al. [9] which depletes B ceils by adhering them to anti-IgG-coated plates. For T cell depletion, cell suspensions were treated with anti-T cell 8BE6 monoclonal antibody [10] plus complement [2] and were prepared for inoculation as described previously [2]. Spleen cell suspensions were depleted of macrophages by allowing them to adhere to plastic dishes (No. 4030; Lab-Tek Division, Miles Laboratories, Elkhart, IN) for 1-2 h at 37°C. The high degree of enrichment of these preparations for either T cells or B cells was verified in cytotoxic, immunofluorescent and functional assays as described recently by us [2,3,8] and as shown in Table 1. In all cell transfer experiments (described below), cell preparations used for adoptive immunization were parasitefree based on subinoculation of aliquots of donor cells into test mice as detailed elsewhere [2].

3.6. Preparation of immune and normal serum Normal strain 2 guinea pigs were infected i.d. with a total of 2 × 106 T. gondii followed by an i.d. challenge infection with 106 tachyzoites 5 weeks later [2]. After another week, guinea pigs were bled out by intracardiac puncture to obtain sera. Titers of anti-T, gondii antibody in pooled immune sera, measured by the I H A test, ranged from 1024-2048. A group of uninfected syngeneic guinea pigs with negative I H A titers were similarly bled to obtain normal serum. The pooled immune serum samples and the pooled normal serum were filter-sterilized (pore size, 0.45 Izm)

and stored at - 7 0 ° C until used. In some experiments, high titer ( > 1024) anti-Toxoplasma immune guinea pig serum and normal guinea pig serum were enriched for immunoglobulin by ammonium sulfate precipitation [11]. After extensive dialysis in phosphate buffered saline, pH 7.2, each crude Ig fraction was sterilized by filtration, analysed for reactivity with rabbit anti-guinea pig IgG (Miles Laboratories) based on immunodiffusion in gels [12], and was tested for antibody activity in the I H A assay. Antibody titers in these crude Ig fractions for the Toxoplasma samples usually increased two- to three-fold following ammonium sulfate treatment, whereas supernatant fractions resulting from ammonium sulfatetreated immune serum were almost totally depleted of antibody (see RESULTS).

3. 7. Passive transfer experiments in guinea pigs Recipient guinea pigs were injected i.v. with 8 - 1 0 ml of immune or normal serum per kilogram of body weight or with 3 - 4 ml of the relevant ammonium sulfate-treated fraction. For cell transfer experiments, age- and sex-matched syngeneic recipients were infused i.v. with 8 × 10 8 unfractionated spleen and lymph node cells, or 2 x 10 8 PEC, or with 4 × 10 8 fractionated (T-enriched or T-depleted) cells suspended in 2 - 3 ml of R P M I 1640; serum, serum fractions, or cells were transferred within 2 h after i.p. challenge with 1 x 10 4 tachyzoites (obtained from freshly harvested, infected peritoneal exudates of syngeneic guinea pigs or AB6 mice).

Table 1 Evidence for enrichment of T cells by the panning technique and of B cells by treatment with 8BE6 plus complement (C) Experiment

Cell group a

Proliferative response to: No mitogen Phytohemagglutinin

1 2 3 4

Unfractionated cells Nonadherent (panned) cells Cells treated with C alone Cells treated with 8BE6+ C

1028 c 757 1231 949

72177 95 545 83 708 4451

Lipopolysaccharide 57221 8937 51684 77660

% Fluorescent positivecells t~ 23-31 4- 9 21-30 92-97

a Cells were derived from the draining lymph nodes of normal or immune guinea pigs. b % of viable cells (range) reacting with a fluorescent anti-guinea pig IgG reagent. c Data are reported as mean counts per minute of tritiated thymidine incorporation of triplicate cultures from six separate experiments. The standard deviations were + 15% or less and are not shown. Culture conditions were identical to those described in references 18 and 19.

100 --A--A--Za O O

6

4. R E S U L T S

•- - O - - O - -

4.1. Passive transfer of anti-T, gondii immunity An earlier study conducted from this laboratory [2] revealed that i.v. infusion of T. gondiisensitized T cells or immune anti-Toxoplasma serum conferred partial protection against local (i.d.) and disseminated T. gondii infection in recipient guinea pigs. It also showed that, in guinea pigs, the R H strain of T. gondii causes a dose-dependent fatal infection when parasites are inoculated i.p. whereas a self-healing infection occurs and long-lasting immunity is generated and sustained when parasites are introduced by the i.d. route. It was, therefore, of interest to determine whether immunity to lethal infection ever becomes expressed in animals surviving an i.d. infection. Three groups of guinea pigs (previously infected) were challenged i.p. with 10 4 T. gondii tachyzoites at 5, 10, or 15 weeks after a primary cutaneous infection with 2 million parasites. Five weeks was the earliest time-point to be tested since prior experiments [2,3] had indicated that by 5 weeks of a primary T. gondii infection, parasites had been cleared from all organ sites and maximum resistance to cutaneous (secondary) challenge occurs in such previously infected guinea pigs. Table 2 shows that all previously infected guinea pigs survived an i.p. challenge with the R H strain of T. gondii, irrespective of the duration of the primary immunizing infection. In sharp contrast, all normal, previously uninfected guinea pigs died within nine days after i.p. parasite challenge. In the next series of experiments, groups of 5 - 6 guinea pigs were infused i.v. with either T.

~--A----.-~

O

O~---O--

\ 0

L

--~ I m m u n e cell recipients --~ I m m u n e T-cell r e c i p i e n t s

O--O

",%_°

L I I 4 8 Days of t o x o p l o s m o

t

-4,- N o r m a l cell recipients

112

//

218

in~ection

Fig. 1. Evidence that guinea pigs receiving unfractionated, immune spleen and lymph node cells (zx), or immune T cells (e) enriched by the panning technique, are fully protected against an i.p. challenge with 1 × 10 4 T. gondii relative to challenged recipients of normal spleen/lymph node cells or normal T cells (©).

gondii-immune lymphocytes, normal lymphocytes, immune serum or normal serum 2 h before i.p. infection with 104 T. gondii tachyzoites. It was found that all recipients of immune material survived T. gondii infection whereas 83-100% of the control guinea pigs had died by day 10 of infection (Figs. 1 and 2). These studies were subsequently repeated using fractionated immune cells. All guinea pigs receiving immune T cells or immune B cells survived lethal parasite challenge but recipients of non-immune cells died shortly after challenge infection (Figs. 1 and 2). Additional experiments revealed that this transfer of protection against lethal i.p. infection can be accomplished using ceils taken from either the spleen, lymph nodes or peritoneal exudate, but not from the thymus, of T. gondii-immune donor animals (Table 3). The minimum cell requirement

Table 2 Temporal development of resistance to lethal Toxoplasmainfection Status of guinea pigs Normal, previouslyuninfected Infected i.d. 5 to 15 weeks earlier (immunized)

Number of survivors at week 1 5a 10 a

15 a

Mean survival time in days_+SD

2/4 b Not done

0/4 4/4

7.8 +_0.96 >_28 c

0/4 4/4

0/4 4/4

a Week of primary i.d. infection with 2 million parasites for the immunized group. b Number of survivors/number challenged with 10 4 T. gondii i.p. c All survivors were healthy at 28 days and selected members of each group were observed for well beyond 2 months after challenge.

101 Table 3

Table 4

Ability of lymphocytes from the spleen, lymph nodes, peritoneal cavity or thymus to transfer anti-Toxoplasma immunity

Titration of transferred protective spleen and lymph node cells in challenged recipients

Status of donors Normal, uninfected Previously infected b (immunized) a

Source of guinea pig lymphoid cells Spleen

Lymph Peritoneal nodes cavity

Thymus

Number of transferred immune cells ~

0/4 a

0/4

0/4

0/4

2x108 1 x 108 5 X 107 2 X 107

4/4

4/4

4/4

0/4

Number of survivors/number challenged i.p. with 104 T,

gondii; all non-survivors died between 7 to 9 days after challenge. b Guinea pigs from this group had undergone a primary i.d. infection of at least 5 weeks duration and each of the various cell preparations from these donors were found to be parasite-free based on subinoculation into test mice as previously described [2].

for the t r a n s f e r of p r o t e c t i o n against lethal toxoplasmosis is illustrated by the results of a subseq u e n t e x p e r i m e n t that tested the effect, o n adoptive i m m u n i z a t i o n , of infusing g r a d e d n u m b e r s of sensitized cells. T a b l e 4 shows that the i n f u s i o n of at least 2 x 10 7 sensitized T cells o r B ceils c o n f e r r e d p r o t e c t i o n in T. gondii-challenged recipients. It was also f o u n d that g u i n e a pigs given B ceils a l o n e or B + T ceils, but n o t T cells alone, p r o d u c e d relatively high levels of a n t i b o d y ( T a b l e 4).

1 X 10 7

0b

Cell type B cell

T cell

B + T cell

3/3c (256) 3/3 (256) 3/3 (128) 2/3 (128) 0/3 ( < 64) 0/3 (-)

3/3 3/3 3/3 3/3 0/3 0/3

3/3 3/3 3/3 3/3 0/3 0/3

( < 64) ( < 64) ( < 64) ( < 64) ( < 64) (-)

(512) (256) (256) (256) ( < 64) (-)

Number of B or T cells transferred to recipients challenged i.p. with 104 T. gondii. This group received 2 x 10s normal cells instead of immune cells, prior to parasite challenge. Number of survivors remaining at 28 days after challenge infection/total number infected guinea pigs. Values in parentheses are mean IHA titers of recipients on day 7 of challenge; a minimum titer of 64 is considered the lowest positive value in the IHA test.

4.2. Antibody-mediated transfer of anti-Toxoplasma immunity Since c o m p l e t e p r o t e c t i o n against toxoplasmosis was achieved by a d m i n i s t r a t i o n of i m m u n e g u i n e a pig serum, passive t r a n s f e r e x p e r i m e n t s were d e s i g n e d in o r d e r to d e t e r m i n e w h e t h e r a c o m p a r a b l e degree of p r o t e c t i o n could be conferred to recipient g u i n e a pigs i n f u s e d with fractions of i m m u n e s e r u m e n r i c h e d for i m m u n o g l o b ulins or with i m m u n e s e r u m lacking high levels of antibodies. A f t e r i.p. challenge with 10 4 T. gondii,

Table 5 Immune properties and activities of various fractions of anti-Toxoplasma guinea pig serum Experimental groups

No. of survivors a No. infected

Mean IHA titer of donor fraction

1. Guinea pigs given normal Ig fraction after ammonium sulfate precipitation

0/6

negative

2. Guinea pigs given anti-T, gondii Ig fraction after ammonium sulfate precipitation

6/6 b

4096

3. Guinea pigs given anti-T, gondii non-Ig supernatant after ammonium sulfate precipitation

0/6

_