INFECTION AND IMMUNITY, Jan. 1991, p. 222-2280019-9567/91/010222-07$02.00/0Copyright © 1991, American Society for Microbiology

PspA, a Surface Protein of Streptococcus pneumoniae, Is Capableof Eliciting Protection against Pneumococci of More

Than One Capsular TypeLARRY S. McDANIEL,l* JEANNE S. SHEFFIELD,' PAMELA DELUCCHI,2 AND DAVID E. BRILES13,4Departments of Microbiology,' Pediatrics,3 and Comparative Medicine,4 University ofAlabama at Birmingham,

Birmingham, Alabama 35294, and Clontech Laboratories, Inc., Palo Alto, California 943032

Received 9 July 1990/Accepted 16 October 1990

Monoclonal antibodies against pneumococcal surface protein A (PspA) have been shown to protect mice fromfatal pneumococcal infection. PspA is highly variable serologically, raising the possibility that PspA from one

strain might not be able to elicit protective responses against strains which possess serologically different PspA.We have prepared a Agtll library of pneumococcal genomic DNA and identified a clone expressing PspA. Therecombinant PspA in this phage lysate elicited protection against pnehmococcal infections with three strains oftwo different capsular serotypes. This finding demonstrated that PspA could elicit a protective response in theabsence of other pneumococcal antigens. The observed protection was probably antibody mediated because itcould be passively transferred with immune sera. Lambda lysates producing pneumococcal proteins other thanPspA failed to elicit protection against fatal pneumococcal infection.

It is well established that immunity to Streptococcuspneumoniae can be mediated by specific antibodies againstthe polysaccharide capsule of the pneumococcus (16). Im-munization of persons over 65 years of age with capsularpolysaccharide results in protection, with an efficacy ofabout 60% (3). Unfortunately, neonates and young childrenfail to develop an immune response against polysaccharideantigens (1, 7, 9, 14), are not effectively immunized with thepolysaccharide pneumococcal vaccine, and can have re-peated infections involving the same capsular serotype (10).Young children have been immunized against Haemophi-

lus influenzae, another encapsulated bacterium, by conjugat-ing the capsular polysaccharide to proteins to make itimmunogenic (13). However, there are over 80 knowncapsular serotypes of S. pneumoniae, of which 23 accountfor most of the disease. For a pneumococcal polysaccharide-protein conjugate vaccine to be successful, the capsulartypes responsible for most pneumococcal infections wouldhave to be made adequately immunogenic.An alternative approach to protecting children from pneu-

mococcal infections would be to identify protein antigensthat could elicit protective immune responses. Such proteinsmight serve as a vaccine by themselves but more likely couldbe used in conjunction with successful polysaccharide-pro-tein conjugates or as carriers for polysaccharides. SuchT-cell-dependent vaccines might also be beneficial for theelderly.

In previous studies, we have shown that monoclonalantibodies to PspA (pneumococcal surface protein A) areable to protect mice from fatal pneumococcal infection (18,20). We have also shown that immunization of X-linkedimmunodeficient (XID) mice with nonencapsulated pneumo-cocci expressing PspA will protect mice from subsequentfatal infection with pneumococci but that immunization withisogenic pneumococci lacking PspA will not (23).No other pneumococcal protein has been identified that

can protect mice from fatal infection with pneumococci,

* Corresponding author.

although immunizations with both pneumolysin and neur-aminidase result in a statistically significant delay to time ofdeath when mice are infected with a normally lethal dose ofpneumococci (15, 25). In our attempts to make antibodies topneumococcal surface proteins by immunizing with wholepneumococci or pneumococcal cell wall extracts (CWEs),we have observed that over half of the antiprotein monoclb-nal antibodies produced are to PspA (8, 20). Of a panel of 9anti-PspA monoclonal antibodies, we found 6 to be protec-tive (3a, 20). Ten monoclonal antibodies to at least four otherpneumococcal proteins have been found not to be protective(19; unpublished results). Thus, on the basis of the presentdata, PspA is the most effective protection-eliciting proteinof the pneumococcus.

In this study we have prepared a Agtll pneumococcalgenomic DNA library to search for cloned genes producingprotection-eliciting antigens. Clones producing PspA andother pneumococcal antigens were tested to see whetherthey could elicit protection against fatal pneumococcal in-fection with strains of pneumococci virulent in mice.

MATERIALS AND METHODS

Mice. CBA/N (xid) mice were obtained from the JacksonLaboratory, Bar Harbor, Maine. These mice carry the XIDtrait, which renders them virtually unable to respond topolysaccharide antigens, but they do respond with normallevels of antibodies against protein antigens (29).

Bacterial strains and growth conditions. S. pneumoniaestrains included type 2 strain D39 (2), type 3 strains WU2 (6)and A66 (2), type 6A strain EF-6796 (obtained courtesy ofCatharina Svanborg-Eden, University of Goteborg, Gote-borg, Sweden), and the nonencapsulated strains Rxl (27)and WG44.1 (23). WG44.1 was a mutant of Rxl that failed toexpress PspA, while all other strains were PspA+. The 50%lethal doses (LD50s) of D39, WU2, A66, and EF-6796 in XIDmice have all been determined to be between 1 and 10 CFU(4).

All strains were maintained as frozen stocks in 10%glycerol at -80°C. Pneumococcal cultures were grown ac-

222

Vol. 59, No. 1

on Septem

ber 29, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

RECOMBINANT PspA ELICITS PROTECTIVE IMMUNITY

cording to procedures routinely used in our laboratory (17)and harvested by centrifugation (4,000 x g, 10 min). TheCFU of pneumococci were determined by plating on bloodagar. Escherichia coli cultures were grown in LB medium(26) or LB supplemented with 10 mM MgSO4.Monoclonal antibodies. All hybridoma cell lines secreting

antibody were obtained from fusions with the myelomaP3-X63-Ag8.653 (12, 20). Monoclonal antibodies used in thisstudy included antibodies to PspA, Xi64 (immunoglobulinM), and Xi126 (immunoglobulin G2b), which have previ-ously been shown to protect mice against pneumococcalinfection (20, 21), as well as additional antibodies producedin this study by using methods previously described (20).Monoclonal antibodies were used as unfractionated tissueculture supernatants for detection of PspA or as dilutedascites in protection experiments as previously described(17, 22).Rabbit immunization. A rabbit antiserum reactive with

non-PspA pneumococcal surface components was preparedby immunizing a rabbit with CWE from pneumococcal strainWG44.1. CWE was prepared as previously described (21),diluted to 1.0 mg of total protein per ml with Ringer's lactate,mixed 1:1 with complete Freund adjuvant (CFA), and in-jected subcutaneously. Booster injections were given every2 weeks without adjuvant. The rabbit was bled monthly, andthe serum was examined for reactivity with pneumococcalproteins in an immunoblot of WG44.1 CWE. When reactiv-ity was detected at a dilution of greater than 1/30,000, 40 mlof serum was collected, aliquoted, and stored at -20°C.

Library construction and screening. A genomic DNA li-brary was prepared in the phage vector Xgtll by using DNAfrom pneumococcal type 2 strain D39. High-molecular-weight DNA was isolated from D39 as previously described(23). The library was constructed according to the protocolsof Huynh, Young, and Davis (11). The D39 DNA wasmechanically sheared to produce fragments between 0.5 and7.0 kb. The fragments were treated with EcoRI methylase,the ends were filled in with Klenow fragment, and EcoRIlinkers were added. The sample was cut with EcoRI and sizefractionated to isolate fragments greater than 0.5 kb. Thefragments were ligated into Xgtll arms, packaged, andinfected into E. coli Y1088.

Screening of clones reactive with anti-PspA monoclonalantibodies or the rabbit antiserum was performed by infect-ing E. coli Y1090 cells with the Agtll D39 genomic library(approximately 2 x 103 plaques per plate) at 42°C for 3.5 h(31). The plates were overlaid with nitrocellulose filterspreviously soaked in 10 mM isopropyl-,3-D-thiogalactopy-ranoside (IPTG) and incubated overnight at 370C. The filterswere removed from the plates and incubated in a blockingsolution of 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) at room temperature for 1 h. The filterswere then incubated at room temperature with either Xi64,Xi126 (each diluted 1/10), or the rabbit antiserum (1/15,000dilution). The filters were then sequentially incubated withbiotinylated goat anti-mouse or goat anti-rabbit immunoglob-ulin (0.2 ,ug/ml) (Southern Biotechnology Associates, Inc.,Birmingham, Ala.) and streptavidin peroxidase (SouthernBiotechnology Associates) with washes between each stepwith PBS plus 0.05% Tween 20. After a final wash with PBSplus 0.05% Tween 20, the filters were incubated with theperoxidase substrate 4-chloro-1-naphthol until the color wasfully developed (approximately 30 min). Plaques producingprotein reactive with antibody were picked and used toinfect Y1090. Each positive plaque was carried through three

screening procedures as described above, and stocks weremade of the stable clones.

Preparation of phage lysates. To prepare recombinantproteins for mouse immunization, phage clones were platedat about 5 x 103 plaques per plate and the plates wereallowed to go to confluent lysis by incubation at 37°C for 7 h.Then 2.5 ml of phage dilution buffer (10 mM Tris [pH 8.0], 10mM MgCl2, 0.1 mM EDTA, and 0.01% gelatin) was added toeach plate, and the plates were incubated overnight at 4°C.The supernatant was decanted from the plates, centrifugedfor 10 min at 10,000 x g, aliquoted, and stored at -80°C.Immunoblot analysis. Sodium dodecyl sulfate-polyacryla-

mide gel electrophoresis was carried out on unlabeled pro-teins of phage lysates prepared from plates of PspA+ Agtllclones or on pneumococcal CWE. Samples containing 10 to30 ,ug of total proteins (Bio-Rad protein assay; Bio-RadLaboratories, Richmond, Calif.) were electrophoresedthrough a 9% resolving gel using a Mini-Protean II gelsystem (Bio-Rad). The electrophoresed proteins were trans-ferred to nitrocellulose by using the Mini-Trans-Blot system(Bio-Rad) at 100 V (250 mA) for 1 h in a 25 mM Tris-192 mMglycine-20% methanol buffer (pH 8.3). The membrane wasprocessed as described above for the plaque screening,except that streptavidin alkaline phosphatase was used andthe antibody-reactive bands were visualized by incubatingthe membrane in a 0.5-mg/ml solution of 5-bromo-4-chloro-3-indolyl phosphate (Sigma) in 1 M Tris (pH 8.8) with 0.01%Nitro Blue Tetrazolium (Sigma).

Southern blot analysis. DNA hybridization was carried outon DNA fragments transferred to nitrocellulose by themethod of Southern (28) as previously described (23), exceptthat hybridized fragments were detected by using theBluGENE nonradioactive system (Bethesda Research Lab-oratories, Gaithersburg, Md.). The probe used in thesestudies was pKSD300 (23), which contains a 550-bp insert ofpneumococcal DNA from pspA. The pKSD300 probe wasbiotin labeled by following the instructions of the BethesdaResearch Laboratories nick translation kit and by usingbiotin-11-dUTP. After hybridization, the blots were proc-essed by following the instructions of the Bethesda ResearchLaboratories BluGENE kit.Mouse immunization studies. In initial studies, XID mice

were immunized either with lysates producing PspA or with1 of 19 pools of lysates that reacted with the rabbit antise-rum. Mice were immunized by subcutaneous injection in thesubinguinal area with antigen emulsified in CFA. After 14days, the mice were injected intraperitoneally with antigenpreps diluted in Ringer's lactate without CFA. Seven dayslater, the mice were challenged intravenously with about 600times the LD50 of pneumococcal strain WU2. The survival ofthe mice was monitored for 10 days. In experiments toassess cross-protection elicited by recombinant PspA, micewere challenged with at least 300 times the LD50 of infectingpneumococci.The relative quantity of PspA in phage lysates used for

immunization was determined by using a previously de-scribed enzyme-linked immunosorbent assay (ELISA) inhi-bition procedure (20). In this assay, phage lysates containingrecombinant PspA were tested for their ability to block thebinding of Xi126 to heat-killed Rxl. Experiments had shownthat two injections of pneumococcal CWE containing 20 ,ugof total protein had sufficient PspA to elicit protection inmice against pneumococcal challenge. Therefore, a dilutionof phage lysate that resulted in inhibition of Xi126 bindingequivalent to that obtained by 20,ug of CWE was designatedas 1 unit of PspA.

VOL. 59, 1991 223

on Septem

ber 29, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

224 McDANIEL ET AL.

As a control for the mouse immunization studies, weselected a recombinant Xgtll clone from the library thatcontained an apparent insert of pneumococcal DNA butfailed to produce any protein detectable by anti-PspA anti-bodies. One unit of the control lysate was defined as thedilution, determined on the basis of PFU per milliliter, thatcorresponded to 1 U of the lysate producing full-lengthPspA.

In the experiments in which pooled lysates were used toimmunize mice, the pools contained 5 to 12 individuallyisolated phage lysates. Each pool was prepared by adding 50,ul of the individual lysates to Ringer's lactate to give a finalvolume of 1.0 ml. This was mixed 1:1 with CFA prior toinjection. As a positive control for these pools of non-PspAproducing clones, a pool was prepared which was composedof equal volumes of lysate from 11 non-PspA-producingclones and from the clone producing full-length PspA.

Anti-PspA response of immunized mice. A direct bindingELISA was used to estimate the amount of anti-PspAantibody in the sera of mice immunized with recombinantPspA. After coating microtitration plates with heat-killedRxl and blocking with 1% BSA, either mouse sera orquantitated purified Xi126, which served as a standard, wasdiluted in duplicate through seven wells. The assay was thendeveloped in a typical fashion, and the bound antibody wasdetected with goat anti-mouse immunoglobulin alkalinephosphatase-conjugated antiserum (Southern BiotechnologyAssociates). The dilution of each serum sample giving 33%maximum binding with respect to Xi126 was determined,and on the basis of the dilution of Xi126 that gave 33%binding, the results for the individual mouse sera wereexpressed (in micrograms per milliliter) (5). As a specificitycontrol for binding to non-PspA antigens, other plates werecoated with heat-killed WG44.1, which has a deletion in theupstream portion of pspA (29a) and fails to produce detect-able PspA (23).

RESULTS

Isolation and characterization of PspA+ and PspA- clones.The Agtll genomic DNA library from pneumococcal strainD39 was screened with the PspA-binding monoclonal anti-body Xi126 and with a rabbit serum against WG44.1 CWE.The rabbit antiserum was used in an effort to detect clonesproducing non-PspA pneumococcal proteins. Since WG44.1is a mutant of Rxl (a nonencapsulated variant derived fromD39) that fails to produce PspA, the proteins detected by therabbit antiserum would not be expected to include PspA.Two clones reactive with Xi126 were detected from a total

of 1.5 x 104 phage tested. One clone was found to contain aninsert of 3.8 kb of pneumococcal DNA and produced full-length PspA (84 kDa) (Fig. 1). The other clone had an insertof 2.9 kb of pneumococcal DNA and encoded a moleculewith an apparent molecular mass of 47 kDa. There were twoadditional lower-molecular-mass bands, 42 and 38 kDa,produced by this clone which may be breakdown products ofthe 47-kDa band.An immunoblot of the Xi126-reactive phage lysates re-

vealed several features of the antibody-reactive molecules(Fig. 1). The full-length clone gave a banding pattern almostidentical to that of PspA from pneumococcal strain D39.Both the full-length and partial clones produced PspA inde-pendent of induction by IPTG, indicating that expression isoccurring from the pspA promoter. Finally, epitopes de-tected by protective monoclonal antibodies Xi64 and Xi126,which react with different PspA epitopes (20), are present on

2 3 4 5 6

200 kDa -- .69a--..

92 kDa -- S

69 kDa --

ir

46 kDa _-

30 kDa --

FIG. 1. Immunoblot of recombinant PspA reacted with anti-PspA monoclonal antibody Xi126. Lanes: 1 and 2, phage lysate fromthe full-length clone with and without induction by IPITG, respec-tively; 3, phage lysate from a PspA- clone; 4 and 5, phage lysatefrom the partial clone with and without induction by IPTG, respec-tively; 6, CWE from pneumococcal strain D39.

PspA produced in E. coli. The banding pattern with Xi64 wasidentical to that seen with Xi126 in Fig. 1 (data not shown).The observation that PspA was expressed as a non-,B-galactosidase fusion protein has been seen with other pro-teins expressed in Xgtll (24, 30).PspA+ pneumococcal DNA inserts were characterized by

restriction mapping (Fig. 2) and Southern blot analysis. TheSouthern blots of the Xgtll inserts were carried out by usinga previously cloned 550-bp fragment of pspA (23).

In an effort to clone protection-eliciting non-PspA pro-teins, we screened approximately 1.3 x 104 plaques andfound 246 plaques that reacted with the rabbit antiserum toWG44.1. After plaque purification, 139 stable clones werefound to produce proteins reactive with the rabbit antiserum.DNA restriction enzyme analysis of 40 random samples ofthe 139 stable clones revealed DNA inserts of at least 8different sizes, ranging from 1 to 7 kb.Immunization of XID mice with recombinant pneumococcal

antigens. To determine whether the PspA+ recombinantclones could elicit a protective immune response, XID micewere immunized with decreasing amounts of both recombi-nant PspA+ clones. Table 1 shows that immunization with 2and 0.2 U of PspA of the full-length clone protected all themice from 300 times the LD50 of type 3 strain WU2. Injectionof lysate from the PspA partial clone failed to give statisti-

x _P..-4 I p1kb L I 2.9 kb-I

< <

3.8 kb

FIG. 2. Restriction endonuclease map of the Agtll recombinantclones that express PspA. The solid line represents the pneumococ-cal DNA insert. Symbols: 0, the Xgtll vector DNA; ED, thepreviously cloned 550-bp pspA fragment described in the text. The2.9- and 3.8-kb clones produce partial and full-length PspA, respec-tively.

INFECT. IMMUN.

on Septem

ber 29, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

RECOMBINANT PspA ELICITS PROTECTIVE IMMUNITY

TABLE 1. Immunization of XID mice with recombinant PspA

Antigen Dose No. of mice alive/no. dead P(U of PspA)a 10 days postchallengeb

Full-length PspA 2.0 6/0 <2 x 10-40.2 6/00.02 1/2

Partial PspA 2.0 3/3 NS0.2 1/30.02 0/4

PspA- 20.0 0/42.0 1/5

a One unit of PspA equals the amount of PspA in 20 p.g ofCWE from strainRxl. For the PspA- clone, 1 U of PspA equals the same dilution of lysate,determined on the basis of PFU that contained 1 U of the full-length clone.

b Mice were infected with 300 times the LD50 of WU2.I Pooled data for 2.0 and 0.2 U of full-length or partial clones were

compared by the Cochran corrected chi square test with the pooled data forPspA- at 20.0- and 2.0-U doses. NS, Not significant.

cally significant protection, but three of six mice did survivewhen immunized with the higher dose of the partial clone.Mice immunized with an equivalent or even a 10-fold excessof lysate from a PspA- clone were not protected frominfection with pneumococci, providing evidence that theprotection seen with the PspA+ clones was not due toinjection of E. coli antigens. The data are a composite of twosimilar experiments. The protection-eliciting effects of PspAagainst 300 times the LD50 ofWU2 were also apparent whenit was present as only 1 of a pool of 12 lysates from Agtllclones (Table 2).To determine the relative protection-eliciting ability of

PspA compared with other pneumococcal proteins, micewere immunized with the 139 lambda lysates that producednon-PspA pneumococcal antigens. Lysates prepared fromthe 139 stable clones were used to form 19 pools, and eachpool was used to immunize groups of three XID mice whichwere subsequently challenged with 300 times the LD50 oftype 3 strain WU2. Because of this small sample size, we didnot expect to see statistically significant protection even if allthree injected mice survived. Since any pool that protectedall three mice would be studied further, we expected thisprocedure to identify any pool that contained a clone pro-ducing a protection-eliciting antigen.To make sure that it was possible to detect a highly

immunogenic clone capable of eliciting protection, even aspart of a pool, XID mice were immunized with a mixturecontaining both 11 non-PspA-producing lysates that hadpreviously been shown not to elicit protection and phagelysate from the recombinant PspA clone. This pool of 12lysates, 1 of which produced full-length recombinant PspA,protected five of five mice. None of five mice injected withthe same pool lacking recombinant PspA lysate survived.Of the 57 mice vaccinated with the other pools of non-

PspA lysates, only 14 mice survived to the end of theexperiment at 10 days postchallenge. However, in 1 of the 19pools (group 3), all three mice survived to 10 days postch-allenge (Table 2). To determine if group 3 might contain aneffective protection-eliciting clone, each of the 5 lysates thatmade up group 3 were injected separately into groups of 8 to17 XID mice. As a control, 21 XID mice were immunizedwith the PspA- clone that previously did not elicit protec-tion. The immunized mice were challenged with 600 timesthe LD50 and monitored for 10 days. None of the clones

TABLE 2. Initial immunization of XID mice with pooled Agtlllysates reactive with anti-WG44.1 rabbit antiseruma

No. of mice alive/no. deadc

pool' 3 days 10 dayspostchallenge postchallenge

3 3/0 3/05 2/1 2/18A 2/1 1/27 2/1 1/22A 2/1 1/21 1/2 1/22 1/2 1/24 1/2 1/26 1/2 1/24A 1/2 1/27A 1/2 1/29A 1/2 0/36A 1/2 0/31A 0/3 0/33A 0/3 0/35A 0/3 0/31OA 0/3 0/311A 0/3 0/312A 0/3 0/313d 0/5 0/513+rPspAd 5/0 5/0

a Mice were given a subcutaneous injection of a pool of Agtll lysates withCFA. Fourteen days later, the mice were boosted with the same preparationwithout adjuvant and then challenged 7 days later with 300 times the LD50 ofWU2 pneumococci.

I Each pool contained a group of 5 to 12 different lysates. No lysate wasincluded in more than one pool. Altogether, the 19 pools contained 139lysates.

I After infection with WU2.d This control pool is composed of equally mixed lysates pooled from 11

non-PspA-producing Agtll clones that do not elicit protection by themselves.The 13+rPspA is composed of the 11 lysates with 2 U of recombinant PspAadded. The results for the pool with PspA versus the pool without PspA weresignificant at P < 0.005.

protected more than 25% of the challenged mice wheninjected individually. In no case was the observed survivalstatistically significant when compared with that of thecontrol group, in which 2 of 21 mice lived. Thus, none of thefive clones in this group appears to produce a pneumococcalprotein that by itself can elicit protection as effectively asrecombinant PspA.

Capacity of recombinant PspA to elicit protection. Todetermine the extent of protection elicited by recombinantPspA, groups of at least four XID mice were immunized with2 U of full-length recombinant PspA lysate and challengedwith 3 x 103, 3 x 105, or 3 x 106 CFU of type 3 WU2pneumococci. We observed that all of the immunized micewere protected from fatal infection when challenged with 3X 103 or 3 x 105 pneumococci and that three of fiveimmunized mice survived challenge with 3 x 106 WU2.Nonimmunized, control mice all died when infected with 50CFU of WU2.To assess the ability of PspA to elicit a cross-protective

immune response, additional mice were immunized with 2 Uof PspA of the full-length clone and subsequently challengedwith 4 different pneumococcal isolates of three differentcapsular types. Immunization with recombinant PspA elic-ited cross-protection against three of the four isolates testedrepresenting two different capsular types (Table 3). Thisresult was particularly significant since PspA has beenshown to be highly variable among different isolates (of 57

VOL. 59, 1991 225

on Septem

ber 29, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

226 McDANIEL ET AL.

TABLE 3. Cross-protection of XID mice immunized with recombinant PspA

PspA-a rPspAbChallenging Capsular

strain serotype No. of mice alive/ No. of days No. of mice alive/ No. of days P valueno. dead to death no. dead to death

WU2 3 1/9 1.32 8/2 >16 <0.005A66 3 0/7 1.70 11/0 >16 <0.001EF-6796 6A 1/11 5.48 9/5 >16 <0.001D39 2 0/9 1.18 0/13 2.4 >0.2

a PspA- is a Agtll lysate that was shown not to produce a protein detectable by anti-PspA monoclonal antibodies.b rPspA is 2 U of full-length recombinant PspA phage lysate described in the text.c The P value was calculated by the two-sample rank test.

pneumococcal isolates tested, 31 different serologic proteintypes were observed) (8, 23). In fact, PspA of strain EF-6796was unreactive with the anti-PspA antibodies Xi64 and Xi126(data not shown). The observation that recombinant D39PspA elicited protection against EF-6796 indicated that D39PspA contained protection-eliciting epitopes other thanthose recognized by the monoclonal antibodies Xi64 andXi126.

Antibody elicited by recombinant PspA. By using a directbinding ELISA, we examined the antibody response to PspAof the mice immunized with the recombinant PspA clones(Fig. 3). The elicited response appeared to be specific forPspA, since no increase above background levels of bindingwas detected when the sera were reacted with the PspA-pneumococcal strain WG44.1. When sera from the miceimmunized with recombinant PspA were used in an immu-noblot against CWE of the encapsulated strain D39, thesame bands were detected by the mouse sera and by Xi126(data not shown).Immunity elicited to the recombinant PspA could be

passively transferred with serum. Unimmunized XID micewere given 162 ,ug of anti-PspA antibody per mouse, asdetermined by ELISA, by passively transferring sera fromXID mice immunized with recombinant PspA. Five of fiveXID mice that received immune sera were protected from 55

4

.09_

I

C 0)

Qcr

cn J

*_p

3

2

1 2 3 4

WeeksFIG. 3. Concentration of anti-PspA antibodies in the sera of mice

immunized with 2 U of the full-length clone, the partial clone, or thePspA- clone. Antibody levels indicate the amount of antibodybound to a heat-killed pneumococcal PspA+ strain Rxl, comparedwith a known concentration of anti-PspA monoclonal antibodyXi126 bound to the same strain in an ELISA procedure. The controlrepresents background binding when the most reactive anti-PspAsera were reacted with the PspA- pneumococcal strain WG44.1.

CFU of WU2, while five mice that received sera fromnonimmunized mice died.An additional demonstration of anti-PspA specificity of

the elicited antibody came from an examination of thespecificity of hybridoma antibodies produced by fusion ofsplenic lymphocytes from two mice immunized with thefull-length recombinant PspA. Each of the 16 hybridomasthat secreted antibodies reactive with D39 detected PspA, asevidenced by immunoblotting in parallel with lanes devel-oped with Xi126. All 16 antibodies reacted with the partialPspA clone that expresses the amino-terminal portion ofPspA. Eleven of the 16 antibodies were tested for theirability to protect mice from infection with 300 CFU of WU2.Antibodies XiR278, XiR1323, XiR1325, and XiR1526 werefound to protect mice from fatal pneumococcal infection.

DISCUSSION

In previous studies we demonstrated that heat-killed non-encapsulated PspA+ pneumococci in Ringer's lactate solu-tion injected intravenously elicited protective immunity inXID mice but that isogenic PspA- pneumococci did not (23).This was taken as evidence that PspA could elicit protection.However, the immunizing pneumococci may have carriedother antigens capable of eliciting responses that contributedto the protective effect of injection with the PspA+ strain.Our present observation that immunization with recombi-nant PspA can increase the LD50 to pneumococcal infectionby at least 5 logs provides a solid demonstration that PspA inthe absence of other pneumococcal antigens can elicit pro-tection.These studies also demonstrate that recombinant PspA

cloned from strain D39 is able to elicit cross-protectionagainst three pneumococcal isolates of two different sero-types. This indicates that, unlike the capsule polysaccharidevaccine, immunization with a single PspA will be able toprovide protection against strains of more than one capsulartype. It is significant, in this regard, that protection waselicited against strain EF-6796, which lacks the distinctepitopes detected by protective monoclonal antibodies Xi64and Xi126 (20, 23). Therefore, additional protection-elicitingepitopes not detected by these antibodies must be present onPspA from strain D39.We observed that the protective monoclonal antibodies

Xi64 and Xi126, which recognize different epitopes (20),reacted with recombinant PspA from both clones in animmunoblot. These antibodies also reacted with PspA pro-duced by the partial clone, indicating that protection-elicit-ing epitopes reside in this amino-terminal portion of themolecule. The failure of the partial clone to elicit protection,even though it reacts with two protection-conferring anti-

INFECT. IMMUN.

1

on Septem

ber 29, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

RECOMBINANT PspA ELICITS PROTECTIVE IMMUNITY

bodies, is perplexing, and at the present time we have no

demonstrated explanation. However, we have noticed thatthe partial PspA clone, compared with the full-length clone,is very unstable when stored as an unfractionated E. colilysate. Therefore, the failure of the partial PspA clone toimmunize might be explained if it were more susceptiblethan the full-length PspA clone to proteases.Recombinant PspA cloned from strain D39 was unable to

elicit protection against strain D39, even though it did elicitprotection against type 3 and type 6A strains. Mice immu-nized with recombinant PspA and challenged with D39survived longer than control mice, but this increased sur-vival was not statistically significant. In previous studies, wefound that protection against D39 required far more Xi126than protection against other pneumococcal isolates did (20).Therefore, it appears that D39 may differ from the otherpneumococcal isolates in its resistance to anti-PspA-medi-ated protection. The poor protection against D39 is consis-tent with experiments in which PspA- mutants of D39 havebeen produced. The insertional inactivation of pspA in D39results in a rapid initial decrease of pneumococci in the bloodof mice during the first hour postinjection. However, thebacteria are apparently able to compensate for the lack ofPspA and ultimately go on to kill the mice (23). This resultwith D39 stands in contrast to the results of a similar studywith PspA- WU2 in which the loss of PspA resulted in a

total loss of virulence (29a).Our approach to identifying other protection-eliciting mol-

ecules of D39 was based on the use of a rabbit antiserum tocell wall components prepared from a PspA- mutant of thenonencapsulated D39 variant, Rxl. This strain was chosenfor immunization because preliminary data (unpublishedobservation) demonstrated that CWE from this strain couldelicit protective immunity to pneumococci when injectedwith adjuvant into XID mice. Since we already knew thatPspA was an immunodominant antigen of pneumococci, we

expected that by using a PspA- derivative we would favorthe production of an antiserum to non-PspA proteins. Sincenone of the non-PspA clones elicited protection comparableto that of PspA, our findings emphasize the special ability ofPspA to elicit protection against pneumococcal infection.

ACKNOWLEDGMENTS

We thank Hernan Grenett and Olga McDaniel for assistance inisolating Agtll DNA, David Voellinger and Tim Hughes for experttechnical assistance, Bill Benjamin for the use of his rabbits, andJanet Yother, Marilyn Crain, and Susan Hollingshead for criticalreading of the manuscript.

This work was supported by grants AI27201A, A121548, andP0lHD17812 from the National Institutes of Health.

REFERENCES1. Anderson, P., D. H. Smith, D. L. Ingram, J. Wilkins, P. F.

Wehrle, and V. M. Howie. 1977. Antibody to polyribophosphateof Haemophilus influenzae type b in infants and children. J.Infect. Dis. 136:S53-S62.

2. Avery, 0. T., C. M. MacLeod, and M. McCarty. 1944. Studieson the chemical nature of the substance inducing transformationof pneumococcal types. Induction of transformation by a des-oxyribonucleic acid fraction isolated from pneumococcus typeIII. J. Exp. Med. 79:137-158.

3. Bolan, G., C. V. Broome, R. R. Facklam, B. D. Plikaytis, D. W.Fraser, and W. F. Schlech III. 1986. Pneumococcal vaccineefficacy in selected populations in the United States. Ann.Intern. Med. 104:1-6.

3a.Briles, D. E., M. J. Crain, L. S. McDaniel, and C. Forman.Unpublished data.

4. Briles, D. E., J. Horowitz, L. S. McDaniel, W. H. Benjamin, Jr.,

J. L. Claflin, C. L. Booker, G. Scott, and C. Forman. 1986.Genetic control of the susceptibility to pneumococcal infection,p. 103-120. In D. E. Briles (ed.), Current topics in microbiologyand immunology, vol. 124. Springer-Verlag, New York.

5. Briles, D. E., and J. F. Kearney. 1985. Idiotype antibodies, p.174-189. In S. P. Colwich and N. 0. Kaplan (ed.), Methods inenzymology, vol. 116. Academic Press, Inc., New York.

6. Briles, D. E., M. Nahm, K. Schroer, J. Davie, P. Baker, J. F.Kearney, and R. Barletta. 1981. Antiphosphocholine antibodiesfound in normal mouse serum are protective against intravenousinfection with type 3 Streptococcus pneumoniae. J. Exp. Med.153:694-705.

7. Cowan, M. J., A. J. Ammann, D. W. Wara, V. M. Howie, L.Schultz, N. Doyle, and M. Kaplan. 1978. Pneumococcal polysac-charide immunization in infants and children. Pediatrics 62:721-727.

8. Crain, M. J., W. D. Waltman H, J. S. Turner, J. Yother, D. F.Talkington, L. S. McDaniel, B. M. Gray, and D. E. Briles. 1990.Pneumococcal surface protein A (PspA) is serologically highlyvariable and is expressed by all clinically important capsularserotypes of Streptococcus pneumoniae. Infect. Immun. 58:3293-3299.

9. Gotschlich, E. C., I. Goldschneider, M. L. Lepow, and R. Gold.1977. The immune response to bacterial polysaccharides inman, p. 391. In E. Haber and R. Krause (ed.), Antibodies inhuman diagnosis and therapy. Raven, New York.

10. Gray, B. M., H. C. Dillion, and D. E. Briles. 1983. Epidemio-logical studies of Streptococcus pneumoniae in infants: devel-opment of antibody to phosphocholine. J. Clin. Microbiol.18:1102-1107.

11. Huynh, T. V., R. A. Young, and R. W. Davis. 1985. Construc-tion and screening cDNA libraries in XgtlO and Agtll, p. 49-78.In D. M. Glover (ed.), DNA cloning: a practical approach. IRLPress, Oxford.

12. Kearney, J. F., A. Radbuch, B. Liesegang, and K. Rajewsky.1979. A new mouse myeloma cell line that has lost immunoglob-ulin expression but permits the construction of antibody-secret-ing hybrid cell lines. J. Immunol. 123:1548-1550.

13. Kim, K. S., V. K. Wong, R. Adler, and E. A. Steinberg. 1990.Comparative immune responses to Haemophilus influenzaetype b polysaccharide and a polysaccharide-protein conjugatevaccine. Pediatrics 85:S648-S650.

14. Klein, J. O., D. W. Teele, J. L. Sloyer, Jr., J. H. Ploussard, V.Howie, P. H. Makela, and P. Karma. 1982. Use of pneumococ-cal vaccine for prevention of recurrent episodes of otitis media,p. 305-310. In J. B. Robbins, J. C. Hilland, and J. C. Sadoff(ed.), Bacterial vaccine. Thieme-Stratton Inc., New York.

15. Lock, R. A., J. Paton, and D. Hansman. 1988. Comparativeefficacy of pneumococcal neuraminidase and autolysin as im-munogens protective against Streptococcus pneumoniae. Mi-crob. Pathog. 5:461-467.

16. MacLeod, C. M., R. G. Hodges, M. Heidelberger, and W. G.Bernhard. 1945. Prevention of pneumococcal pneumonia byimmunization with specific capsular polysaccharides. J. Exp.Med. 82:445-465.

17. McDaniel, L. S., W. H. Benjamin, Jr., C. Forman, and D. E.Briles. 1984. Blood clearance by anti-phosphocholine antibodiesas a mechanism of protection in experimental pneumococcalbacteremia. J. Immunol. 133:3308-3312.

18. McDaniel, L. S., and D. E. Briles. 1986. Monoclonal antibodiesagainst surface components of Streptococcus pneumoniae, p.143-164. In A. J. L. Macario and E. C. de Macario (ed.),Monclonal antibodies against bacteria. Academic Press, Inc.,Orlando, Fla.

19. McDaniel, L. S., and D. E. Briles. 1988. A pneumococcal surfaceprotein (PspB) that exhibits the same protease sensitivity asstreptococcal R antigen. Infect. Immun. 56:3001-3003.

20. McDaniel, L. S., G. Scott, J. F. Kearney, and D. E. Briles. 1984.Monoclonal antibodies against protease sensitive pneumococcalantigens can protect mice from fatal infection with Streptococ-cus pneumoniae. J. Exp. Med. 160:386-397.

21. McDaniel, L. S., G. Scott, K. Widenhofer, J. M. Caroll, andD. E. Briles. 1986. Analysis of a surface protein of Streptococ-

VOL. 59, 1991 227

on Septem

ber 29, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

228 McDANIEL ET AL. INFECT. IMMUN.

cus pneumoniae recognized by protective monoclonal antibod-ies. Microb. Pathog. 1:519-531.

22. McDaniel, L. S., W. D. Waltman II, B. Gray, and D. E. Briles.1987. A protective monoclonal antibody that reacts with a novelantigen of pneumococcal teichoic acid. Microb. Pathog. 3:249-260.

23. McDaniel, L. S., J. Yother, M. Viayakumar, L. McGarry,W. R. Guild, and D. E. Briles. 1987. Use of insertional inacti-vation to facilitate studies of biological properties of pneumo-coccal surface protein A (PspA). J. Exp. Med. 165:381-394.

24. Morris, S. L., D. A. Rouse, D. Hussong, and S. D. Chaparas.1990. Isolation and characterization of recombinant Xgtll bac-teriophages expressing four different Mycobacterium intracel-lulare antigens. Infect. Immun. 58:17-20.

25. Paton, J. C., R. A. Lock, and D. C. Hansman. 1983. Effect ofimmunization with pneumolysin on survival time of mice chal-lenged with Streptococcus pneumoniae. Infect. Immun. 40:548-552.

26. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecularcloning: a laboratory manual, 2nd ed., p. A.1. Cold Spring

Harbor Laboratory, Cold Spring Harbor, N.Y.27. Shoemaker, N. B., and W. R. Guild. 1974. Destruction of low

efficiency markers is a slow process occurring at a heteroduplexstage of transformation. Mol. Gen. Genet. 128:283-290.

28. Southern, E. M. 1975. Detection of specific sequences amongDNA fragments separated by gel electrophoresis. J. Mol. Biol.98:503-517.

29. Wicker, L. S., and I. Scher. 1986. X-linked immune deficiency(xid) of CBA/N mice, p. 86-101. In D. E. Briles (ed.), Currenttopics in microbiology and immunology. Springer-Verlag, NewYork.

29a.Yother, J., and D. E. Briles. Unpublished data.30. Young, D. B., L. Kent, and R. A. Young. 1987. Screening of a

recombinant mycobacterial DNA library with polyclonal anti-serum and molecular weight analysis of expressed antigens.Infect. Immun. 55:1420-1425.

31. Young, R. A., and R. W. Davis. 1983. Efficient isolation of genesby using antibody probes. Proc. Natl. Acad. Sci. USA 80:1194-1198.

on Septem

ber 29, 2020 by guesthttp://iai.asm

.org/D

ownloaded from