Identification of paramyosin as a potential protective antigen against Brugia malayi infection in jirds

315

Molecular and Biochemical Parasitology, 49 (1991) 315-324 (Q 1991 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/91/$03.50 ADONIS 016668519100451D MOLB10 01640

Identification of paramyosin as a potential protective antigen against Brugia malayi infection in jirds B e n - W e n Li, R a m a s w a m y Chandrashekar, Raquel M. Alvarez,

Fanya Liftis and Gary J. Weil Department of Medicine, Infectious Disease Division, Washington University School of Medicine and The Jewish Hospital of St. Louis, St. Louis, MO, U.S.A. (Received 10 June 1991; accepted 5 August 1991)

Vaccination of jirds with irradiated infective larvae of Brugia malayi has been reported to provide partial immunity to larval challenge. In the present study, we found that sera from vaccinated animals recognized larval antigens with apparent molecular weights of 97, 55-60, and 10 kDa that were not recognized by sera from infected animals. A B. malayi cDNA expression library in 2gtll was screened to identify clones that were preferentially recognized by sera from immunized animals. One of these clones (BM-5) was chosen for further study. BM-5 contains a 2.1 kb DNA insert and produces a fusion protein with a molecular weight of 185 kDa. Antibody, affinity-purified with the BM-5 fusion protein, binds to a 97 kDa native B. malavi antigen. Immunological studies and partial DNA sequence data confirm that BM-5 encodes paramyosin. Recombinant B. malayi paramyosin is strongly recognized by antibodies in sera from jirds that have been immunized either by injection with irradiated larvae or by chemotherapy-abbreviated infection. Most sera from infected jirds do not contain antibody to paramyosin. Additional studies are needed to determine whether paramyosin is actually protective in this filariasis model. Key words: Filariasis; Immunity; Nematode; Brugia malayi; Paramyosin; Jird

Introduction An estimated 90 million people are infected with Wuchereria bancrofti and Brugia malayi, nematode parasites that cause lymphatic filariasis in the tropics [1]. Very little is known regarding factors responsible for protective immunity in human filariasis. In fact, there is Correspondence address." G.J. Weil, Infectious Disease Division, The Jewish Hospital at Washington University Medical Center, 216 S. Kingshighway, St. Louis, MO 63110, U.S.A. Note: Nucleotide sequence data reported in this paper have been submitted to the GenBank T M data base with the accession numbers M63097 and M63098.

Abbreviations: L3, third stage larvae; PBS/T/FCS, phosphatebuffered saline with 0.05% Tween 20 and 5% fetal calf serum; BCIP/NBT, 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium; IPTG, isopropyl/~-D-thiogalactoside.

no direct evidence that such immunity exists in humans. Partial protective immunity to filarial larval challenge has been induced in animals by immunization with irradiated infective third stage larvae (L3), which are alive but developmentally arrested, and by drug treatment of early infections, so-called chemotherapy-abbreviated infections (reviewed in refs. 2 and 3). These methods of immunization presumably work by stimulating exaggerated immune responses to larval antigens that differ from immune responses seen in animals that have been infected with normal larvae. The mechanism(s) responsible for this immunity are not known, but experience with the more extensively studied Schistosoma mansoni [4] suggests that both humoral and cell mediated immunity may be involved. Little is known regarding the antigenic targets of immunity in animals vaccinated

316

with irradiated filarial larvae. The purpose of the present study was to clone and characterize potentially protective antigens that are preferentially recognized by sera from jirds vaccinated with irradiated B. malayi infective larvae.

studies. Rabbit antibodies to B. malayi adult worm extract were produced as previously described [7]. Purified recombinant Dirofilaria immitis paramyosin [8] and mouse antibodies to this protein were kindly provided by C. Maina and L. McReynolds (New England Biolabs, Beverly, MA).

Materials and Methods

Antibody assays. Immunoblot detection of antibodies to parasite antigens was performed essentially as described by Towbin et al. [9]. Briefly, B. malayi L3 antigens were separated by SDS-PAGE on 5 20% gradient slab gels, and proteins were electrophoretically transferred to nitrocellulose. After blocking in 0.01 M phosphate buffered saline, pH 7.4, with 0.05% Tween 20 (Sigma) and 5% non-fat dry milk, nitrocellulose strips were incubated in jird sera diluted in PBS with 0.05% Tween 20 and 5% fetal calf serum (PBS/T/FCS) overnight at 4'C. After extensive washing, strips were incubated in alkaline phosphatase conjugated antibodies to mouse IgG (Promega, Madison, WI) for 3 h at 37°C and developed with 5-bromo-4-chloro3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT, Sigma). Antibodies to fusion proteins were also detected by immunoblot. Escherichia coli Y I090 was infected at high density with recombinant phage on a thin layer of agarose over LB agar to achieve confluent lysis, and synthesis of fusion proteins encoded by cDNA inserts was induced with isopropyl/~-D-thiogalactoside (IPTG) impregnated filters. Top agarose containing bacterial lysate and fusion protein was then gently scraped off and dissolved in SDS-PAGE sample buffer. SDSPAGE, electrophoretic transfer of proteins and antibody detection was performed as described above, except that sera were absorbed with E. coli lysate (Biorad Laboratories, Richmond, CA) prior to testing to decrease nonspecific background antibody reactivity. Antibodies to B. malayi L3 surface antigens were detected by indirect immunofluorescence (IFA). Previously frozen larvae were incubated with jird sera diluted 1:10 in PBS overnight at 4 C in 1.5 ml tubes. After washing 3 times in PBS, larvae were incubated in fluorescein-

Jird inJections and immunizations. Male Mongolian jirds (Meriones unguiculatus) 8 10 weeks old were purchased from Tumblebrook Farms, West Brookfield, MA. All immunizations and infections were performed by TRS Labs, Athens, GA with B. malayi L3 produced by standard methods [5]. Jird infection sera were collected 5 weeks after s.c. injection of 100 B. malayi L3 in the left inguinal region. Other jirds were vaccinated by s.c. injection with 75 B. malayi L3 that had been exposed to 15 kRad from a 6°Co source. Jirds were vaccinated on days 1 and 28, and sera from these animals were collected on day 35. Chemotherapyabbreviated infections were produced in jirds as previously described [6] by administering two daily doses of 25 mg kg J CGP 20376 by stomach tube beginning 5 days after s.c. injection of 200 B. malayi L3. Sera from these animals were collected 1 month after infection. Parasite antigens and anti-parasite antibodies. Soluble B. malayi L3 antigen was prepared by grinding previously frozen larvae (provided by N I A I D supply contract AI 02642, U.S.-Japan Cooperative Medical Sciences Program) with a ceramic mortar and pestle for 5 min. The fragmented larvae were extracted in 0.01 M Tris buffer (pH 8.3) with 1% sodium deoxycholate, 5% fl-mercaptoethanol, and protease inhibitors (1 mM PMSF, 1 mM EDTA, 1 m M EGTA, 25 /,g ml ~ N-tosyl-L-phenylalanine chloromethyl ketone and 25 #g ml ~ N-~-p-tosyl-L-lysine chloromethyl ketone, all from Sigma Chemical Co., St. Louis, MO). The worm homogenate (1000 L3 ml -~) was rocked at 4°C overnight and centrifuged at 15 000 x g for 10 min, and the supernatant was used for immunoblot

317

conjugated goat anti-mouse immunoglobulins (Organon Teknika-Cappel, West Chester, PA) for 3 h at 37°C. Larvae were washed 3 times in PBS and examined with a Nikon Fluphot epifluorescence microscope with a 40 x objective. Surface fluorescence was rated on a scale of 0 4 + .

Production of a B. malayi cDNA expression library in Xgtll. B. malayi adult worms and microfilariae were collected from i.p. infected animals as previously described [1]. Complementary D N A was produced from RNA and ligated into the EcoRI site of 2gtll as previously described in detail by Donelson et al. [11], except that oligo(dt) cellulose enrichment of poly(A) + R N A was omitted. The library contained 2 x 106 independent recombinants, and it had a recombinant frequency of 93% (% white plaques) after amplification. Approximately 1 in 900 plaques in this library produce fusion proteins that are recognized by a pool of high-titered human sera from patients with brugian filariasis. The mean insert size of 10 clones selected with antifilarial antibodies was 1.4 kb with a range of 0.32.8 kb. We have also screened the library with a monoclonal antibody to #-tubulin (provided by J.L. Lessard of the University of Cincinnati); the frequency of positive plaques was 1 in 12 500. Immunoscreening the cDNA expression library. The library was immunoscreened to identify B. malayi clones that were preferentially reactive with sera from immunized jirds essentially as described by Young and Davis [12]. Briefly, phage were plated onto a lawn of E. coli Y1090 at a density of 50 000 phage per petri dish (150 cm 2) and grown at 42°C for 3 h. When plaques were visible, IPTG-impregnated nitrocellulose filters were placed on the plates for 3 h at 37°C. Filters were removed and fresh filters were placed on plates for 3 h to obtain a second plaque lift. After blocking in PBS/T, filters were incubated with gentle rocking overnight at 4°C in jird serum pools from infected or immunized animals diluted 1:50 in PBS/T and developed with alkaline phospha-

tase conjugated goat anti-mouse IgG antibody and NBT/BCIP as described above for immunoblots. These serum pools were prepared with sera collected 5 weeks after immunization or infection as described above. Clones that were preferentially recognized by the serum pool from immunized jirds were selected and purified by repeated cycles of immunoselection.

Identification of native parasite proteins that correspond to recombinant fusion proteins. Antibody to fusion protein was affinity purified as follows: agarose with bacterial lysate and fusion protein was subjected to SDS-PAGE and electrophoretic transfer as described above. The fusion protein was identified by staining the nitrocellulose with amido black, and this band was cut out with a razor blade. Nitrocellulose strips containing fusion protein (or #-galactosidase as a control protein) were incubated with rabbit anti-B. malayi adult worm antibody diluted 1:2000 in PBS/T overnight at 4°C. After extensive washing with PBS, bound antibody was eluted by immersing the nitrocellulose in 0.15 M glycine-HCl, pH 2.8, for 2 min. The eluates were promptly neutralized with 0.1 M NaOH, and bovine serum albumin was added to a final concentration of 0.5%. The affinity-purified antibodies were used to probe immunoblots of B, malayi L3 antigens as described above. DNA sequencing. 2gtll D N A purified from selected clones was digested with EcoRI and ligated into the EcoRI site of pBluescript II S K - (Strategene Cloning Systems, La Jolla, CA) by standard methods [13], and plasmid D N A was prepared for sequencing. The dideoxynucleotide chain termination method [14] was used for double stranded D N A sequencing using the TaqTrack Sequencing System (Promega) with T3 and T7 pBluescript primers and with synthetic oligonucleotide primers produced by the Washington University Protein Chemistry Laboratory. The PC/ G E N E D N A Sequence Analysis System (IntelliGenectics, Inc., Mountain View, CA) was used to analyze nucleotide and deduced

318

amino acid sequences and to determine sequence homologies with previously reported sequences in the GenBank data base.

Results

Antibodies to B. malayi L3 in sera J?orn vaccinated and infected jirds. Representative results of immunoblots of sera from vaccinated and infected jirds are shown in Fig. 1. Several antigen bands were recognized equally well by sera from both groups. However, most vaccinated animals had antibodies to several antigens that were not recognized by sera from infected animals. These antigens had molecular sizes of approximately 97, 55-60, and 10 kDa (Table I). Sera from vaccinated animals were also much more reactive with L3 surface antigens by IFA than sera from infected animals.

nized animals were used to differentially screen a B. malayi adult worm c D N A expression library in )~gtl 1. Approximately 600 000 clones were screened. Six clones were isolated that were preferentially recognized by sera from immunized animals. Preliminary characterization of these clones included analysis of fusion

12545 kOa 200

-

116 Immune selection of recombinant clones preferentially recognized by sera .from vaccinated jirds. Serum pools from infected and immu-

92.5

-

66TABLE 1

Immune reactivity of jird sera with Brugia rnalayi infective larval antigens

Immunized jirds A B C D E Infected jirds A

IFA

l0 kDa

55 60 kDa

97 kDa

3 u 4u 3u 2u 3u

+ + + + +

+ + + + +

+ + + + +

-

m

-

5121-

B

2p ±p

C

+p

D E

lp _p

-

0 0

-

Uninfected jirds A B

42.5

--

Immune reactivity was assessed by indirect i m m u n o f l u o r escence (IFA) with intact larvae and by i m m u n o b l o t . I F A results are e x p r e s s e d with a score which refers to fluorescence intensity distributed uniformly (u) or in patches (p) over the larval surface. I m m u n o b l o t results for individual antigen bands are expressed as positive ( + ) or negative ( - ) .

14.4 -

Fig. I. Immunoblot of B. malayi L3 antigens developed with lane 1, normal jird serum; 2 3, sera collected from jirds 5 weeks after immunization with irradiated B. rnalayi E3; 4 5, sera collected from jirds 5 weeks after infection with B. malayi L3.

319

proteins by immunoblot and sizing of cDNA inserts (data not shown). Additional studies focused on the most immunoreactive of these clones, BM-5.

Immunological characterization of BM5. BM-5 produced a 185-kDa fusion protein after induction by IPTG (Fig. 2). In addition to this fusion protein, E. coli lysate produced by BM-5 contains a distinctive set of peptide fragments that are reactive with antibody to B. malayi but not reactive with antibody to /?galactosidase. The BM-5 fusion protein and the betagalactosidase product of wild-type 2gtl 1 were used to affinity purify antibodies from rabbit

anti-B, malayi serum to identify immunologically crossreactive native B. malayi antigens by immunoblot (Fig. 3). Antibodies purified with BM-5 bound to parasite antigens with apparent molecular weights of 97 and 200 kDa. Antibodies purified with beta-galactosidase did not bind to any B. malayi antigens by immunoblot. The size of the native antigen that was reactive with antibodies to BM-5

125 kDa 200-

A

B

12

12

kDa 180

92.5 -

66-

116 -

4584-

31-

5848.5

-

36.5

-

Fig. 2. Immunoblot analysis of BM-5//-galactosidase fusion protein. Bacterial lysates of E. coli Y1090 infected with wild-type 2gtll (lane 1 in both panels) or BM-5 (lanes 2) were subjected to SDS-PAGE on 8% gels and electroblotted to nitrocellulose. Nitrocellulose papers were developed with rabbit anti-B, malayi adult worm antibody (panel A) and mouse monoclonal antibody to beta-galactosidase (panel B).

2114.4 -

Fig. 3. Identification of native B. malavi antigens that are immunologically crossreactive with BM-5 fusion protein. immunoblot of B. malayi L3 antigens was developed with: lane 1, antibody to BM-5 affinity purified from rabbit antiB. malayi antibody; lane 2, unfractionated rabbit anti-B. malayi antibody; lane 3, control for lane 1 with antibody affinity purified with beta-galactosidase produced by wildtype 2gtl 1.

320

fusion protein suggested that it might be paramyosin. Additional immunoblot studies performed with Dirofilaria immitis paramyosin reagents confirmed this hypothesis: Antibody to D. immitis paramyosin reacted with the BM5 fusion protein (Fig. 4), and affinity-purified

I 25

456

kDo 200

m

92.5 -

6645-

51-

I

antibody to BM-5 reacted with recombinant D. immitis paramyosin (not shown). Anti-paramyosin antibody was also reactive with all of the other BM clones identified in our original screen as preferentially reactive with sera from immunized animals. This antibody was not reactive with surface antigens of B. malayi L3 by indirect immunofluorescence. Immunoblot studies were performed to determine the reactivity of the BM-5 fusion protein with several types of jird sera (Fig. 4, Table II). Most sera from jirds immunized by injection with irradiated B. malayi L3 or by chemotherapy-abbreviated infection were reactive with BM-5. In contrast, most sera from infected jirds were not reactive with the antigen. Molecular biological characterization of BM5. The BM-5 c D N A insert was determined to be approximately 2.1 kb by agarose gel electrophoresis of BM-5 DNA after digestion with EcoRI. To determine whether BM-5 encoded paramyosin, the insert was subcloned into pBluescript, and partial sequence was obtained from both the 5' and 3' ends (251 bp and 659 bp, respectively; Fig. 5). The close relationship of BM-5 to D. immitis paramyosin [8] is shown in Fig. 6. The level of homology observed for 827 bp of overlapping coding region sequenced was 92% identity at the T A B L E II I m m u n e reactivity o f j i r d sera with BM-5 fusion protein by' immunoblot Jird sera tested ~'

No tested

No. positive

% positive h

Irradiated L3 recipients

1I

10

91

Chemotherapyabbreviated infections

12

8

67

Normal B. malayi infections

16

3

18

Uninfected jirds

Pool

21-

14.4

-

Fig. 4. l m m u n o b l o t analysis o f jird antibodies to BM-5 fusion protein. BM-5 bacterial lysate was separated by S D S - P A G E a n d electroblotted to nitrocellulose. Nitrocellulose strips were developed with: 1, n o r m a l jird serum; 2, s e r u m f r o m a jird i m m u n i z e d with irradiated B. malayi L3; 3 -4, sera from jirds infected with B. malayi; 5, s e r u m from a jird after C G P 20376 t r e a t m e n t o f early B. malayi infection ( c h e m o t h e r a p y - a b b r e v i a t e d infection); 6, m o u s e a n t i b o d y to p a r a m y o s i n .

Negative

~'Jird sera were collected 5 weeks after infection or i m m u n i z a t i o n , as described in Materials a n d M e t h o d s . bDifferences between the top 3 g r o u p s are statistically significant; Z2 - 7.12 with 2 d.f., P - 0.028.

321

nucleotide level and 98% for deduced amino acids. Sixty-one of 68 differences (90%) in nucleotide sequence identified occurred in the third codon position, and only 3 of these changes resulted in a change in the translated

A

T GT AT A I ~ G G ~ C G T T T T G ~ G ~ T C G ~ T ~ G C T T T G ~ A A E R F E A Q T I E L S N K V E A L N 2 0

A

C A T G 121T~GAGA~A~G~GTA~GAT~T~CA~T~TATCA L K E I B D Q K V Q L D N ~ Q H V K Y Q 6 0

TCAACTTCAAG Q L Q

83

CG G T A GC AT AACT GCAGCAGATGAGCGTG~TCGTGCA~AG~GATGCACGTCGCGCAG~AGCAG~ A A D E R A N R A L A D A R R A V E Q L 2 0 A

61CATGAGGAGCAGG~CA~CCATGAAAA~GATG~CAGG~TCA~GG~GAG~G H E E Q E H S M K I D A L R K S L E E Q G A A A CGT 121GTG~GC~G~C~CA~G~CGGCAGCTTTATTGGG~T~ V K Q L Q V Q I Q E A E T A A L A 181CGTGT~~G~CCCGTATACG~A~GG~CA~AGA~G~ R V I A K L E T R I R D L E 241

4

0

6

0

$

0

A L

G

G

K TG

T

A

L

D

E

E

o

5;o I__J

,o;o

,5~o

B M - 5 cDNA 2089 bp

5'

2o~o

25~o• I

peptide sequence. The 5' end of BM-5 is homologous with base 456 of the published D. immitis paramyosin sequence [8] and continues approximately 2100 bp downstream. Based on sequence homology and the sizes of the insert and its fusion protein, we conclude that BM-5 contains a 2-kb open reading frame. This is equivalent to 79% of the coding region published for D. immitis paramyosin. The 3' portion of BM-5 has 12 bp in its open reading frame and 71 bp of noncoding DNA that are not present in the published D. immitis paramyosin sequence. The noncoding D N A has a stop codon (TAG), a polyadenylation signal (AATAAA), and a 15 bp poly(A) tail.

TA A T G C 1 8 1 A ~ C G ~ G A ~ ~ T T T T G ~ G A C G ~ C G ~ C 8 0 L A Q Q L E E A R R R F E D A Q R E R S

1

D. immitis Paramyosin cDNA 2545 bp

Fig. 6. This figure illustrates the relationship between c D N A insert of clone BM-5 and the published sequence of D. immitis paramyosin [8]. Portions of BM-5 that have been sequenced are shown with brackets. The beginning of BM-5 is homologous with position 456 of the D. immitis paramyosin sequence. The BM-5 sequence contains 12 bp of coding sequence and 71 bp of non-coding D N A at its 3' end (cross-hatched area) with a poly(A) tail (A) that are not present in the D. immitis paramyosin sequence.

T T AT C 6 1 T C G A ~ T ~ A ~ A G C C ~ G ~ C ~ A ~ G ~ T ~ T G A T ~ R H V N D L A Q Q R Q R L Q A E N N D L 4 0

241

5'

A C ACACGTCGACAT~GG~CGC~GGTGCG~CG~G~GATCGA~TATCAAAGAG T R R H K E T Q G A L R K K D R R I K E I 0 0

A

T 301G~C~TGC~G~GATG~GAGCAT~GATG~GTGATGG~C~GATAC~AT V Q M Q V D E E H K M F V M A Q D T A D I 2 0 G C TG T 361AGA~GAAAAA~TA~CAAAAGAGAC~GGAG~G~G~TCA~CA R L L E K L N I Q K R Q L G E A E S L T 1 4 0 421

GA ATGG~T~C~CGAGTACGTCGATATC~CGTG~AGAGGATG~GGACGA M A N L Q R V R R Y Q R E L E D A E G R 1 6 0

G

Discussion

TT

T TA G 481G~GATC~G~G~G~CA~ACAT~CA~CGTGC~GCATCG~CATCAG~G~ A D Q A E S S L H L I R A K H R S S V V 1 8 0 541

601

AT ACGGGCAAAAATG~CAGCGTCAAAAA~ACG~CTCG~GATGAGC~TAGAGAT~ T G K N A S A S K I y V L E D E Q * TTAATTAA TT AC G TTAATTGT CA G CAAT AAAA TTT A CTAATGAG A

J

197

~

Fig. 5. Partial D N A sequence o f c D N A insert of clone BM-. 5 is shown from the N-terminal end (5' to 3', panel A) and from the C-terminal end (3' to 5', panel B) with the predicted amino acid sequence encoded by its open reading frame. Nucleotide and amino acid numbers are shown in the left and right margins, respectively. The sequence begins in a coding region. The stop codon is shown with an asterisk, and the polyadenylation signal is underlined. Nucleotides from the published sequence for D. imrnitis paramyosin [8] that do not agree with the BM-5 sequence are shown above the BM-5 sequence.

Prior studies have shown that a degree of protective immunity to brugian filariasis can be induced in jirds by immunization with irradiated infective larvae [2,15]. In contrast, single or multiple injections with normal Brugia infective larvae confer little or no resistance to reinfection in jirds [16,17]. Little is known regarding the antigenic targets and mechanisms of protective immunity after vaccination, but one study suggested that antibody-dependent cellular cytotoxicity directed to larval surface antigens was involved [2]. In that study, jirds immunized with irradiated B. malayi L3 produced antibodies to larval surface antigens, and dead larvae recovered

I

322

after i.p. challenge of immunized animals were encased in degranulating eosinophils. The purpose of the present study was to identify, clone, and characterize B. malayi antigens that are preferentially recognized by sera from jirds immunized with irradiated infective larvae since these antigens might be targets of protective immune responses. This basic strategy [18] has not been previously used to study protective immunity in filariasis. Our immunological studies identified differences in antiparasite antibody reactivity between sera from immunized and infected jirds. Immunized jirds had stronger antibody responses to larval surface antigens. This finding was consistent with results reported by Yates and Higashi [2]. lmmunoblot studies identified several L3 antigens that were preferentially recognized by sera from immunized animals. These IFA and Western blot studies were performed with sera collected 5 weeks after immunization or infection to focus on immune responses to L3 antigens during this critical, early period. Antibody reactivity to L3 antigens could certainly change over time. However, we have observed in other studies that antibodies to L3 are fairly stable between 1 and 3 months after immunization with irradiated L3 and after infection with normal L3 (authors' unpublished observations). In the next phase of the study, serum pools from immunized and infected animals were used to differentially screen a B. malayi cDNA expression library to select clones that were preferentially recognized by sera from immunized animals. Several related clones were isolated, and one of these, BM-5, was studied in detail. Immunological studies and partial DNA sequence data showed that BM-5 encodes a portion of B. malayi paramyosin. Paramyosin is an integral component of invertebrate muscle (reviewed in ref. 19), and as it is not exposed on the cuticular surface of intact nematodes [20], our finding that antibody to paramyosin failed to bind to intact B. malayi L3 was expected. However, this internal location does not rule out paramyosin as a vaccine candidate for filariasis. Although paramyosin is not exposed on the surface of

schistosomula, immunization with paramyosin has been clearly shown to induce a degree of protective immunity in murine schistosomiasis that is believed to be T cell-dependent [4]. Paramyosin has also been previously proposed as a vaccine candidate for f~lariasis; mice immunized with native paramyosin show enhanced clearance of transfused microfilariae [21], and sera from patients with filarial infections often contain antibodies to paramyosin (ref. 22 and authors' unpublished observations). However, one difference between those studies and the present one is that we have shown that paramyosin is preferentially recognized by sera from animals that have been immunized by two methods that have been shown to induce protective immunity to filarial larval challenge, namely vaccination with irradiated L3 and chemotherapy-abbreviated infection [2,3]. Before concluding, we would like to emphasize two points about our results. First, we did not specifically look for paramyosin in this study. We were surprised to find that all of the clones that we initially identified with our differential immunoscreen of the B. malayi c D N A expression library were reactive with antibody to paramyosin. Second, it should be noted that to date there is no direct evidence that paramyosin can induce protective immunity against larval challenge in filariasis. An alternate explanation for the enhanced antibody reactivity to paramyosin observed in immunized or treated animals in the present study is that this highly antigenic internal protein is preferentially released by larvae killed by drug treatment and by irradiated larvae (which may not live long in the host): the antibody response to paramyosin may not be causally related to the protective immunity induced by irradiated larvae. Despite this reservation, we believe that additional studies should be performed to directly test the protective activity of paramyosin in animal models of filariasis.

323

Acknowledgements This work was supported in part by National Institute of Health grant AI25587.

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