Intranasal Immunization of Mice with PspA - Semantic Scholar

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Intranasal Immunization of Mice with PspA (Pneumococcal Surface Protein A) Can Prevent Intranasal Carriage, Pulmonary Infection, and Sepsis with Streptococcus pneumoniae Hong-Yin Wu, Moon H. Nahm, Y. Guo, Michael W. Russell, and David E. Briles

Departments of Microbiology and Pediatrics, University of Alabama at Birmingham; Department of Pathology, Washington University School of Medicine, St. Louis, Missouri; Massachusetts Public Health Biologic Laboratories, Boston

Many pathogens, including Streptococcus pneumoniae, are carried asymptomatically on the nasopharyngeal mucosa and spread among individuals by close contact. Clinical disease results when pneumococci escape from the mucosa and invade sterile sites. Although systemic immunity can prevent invasive disease, control of person-to-person spread is probably dependent on immunity acting at the mucosal surface. Intranasal immunization of mice with PspA (pneumococcal surface protein A) or a capsular 6B polysaccharide – tetanus toxoid conjugate induced mucosal and systemic antibody responses and provided long-lasting protection against carriage of S. pneumoniae. Resistance to carriage was dependent on mucosal rather than systemic immunity and was effective against heterologous strains of heterologous PspA types. Intranasal immunization with PspA also protected against systemic infection following intravenous, intratracheal, and intraperitoneal challenge.

The majority of infectious diseases directly affect or are acquired through the mucosal surfaces. For many organisms, colonization of the respiratory, gastrointestinal, and urogenital tracts is the first step in pathogenesis, and prevention of colonization should effectively block both transmission and infection. Specific immune protection of mucosal surfaces is mediated by the mucosal immune system and by the small amounts of serum immunoglobulin that reach these sites. The most conspicuous product of the mucosal immune system is secretory IgA. Generation of specific secretory IgA antibodies usually requires stimulation of specialized inductive sites of the mucosal immune system. Only in recent years have effective strategies for immunization at these sites been developed [1], and the development of mucosal vaccines has lagged behind conventional parenteral vaccination [2]. Streptococcus pneumoniae cause pneumonia, meningitis, otitis media, ocular infections, bacteremia, and sinusitis in humans and are a major cause of fatal infections worldwide [3 – 5]. Capsular polysaccharide (CPS) [6, 7] and pneumococcal surface protein A (PspA) [8] are both virulence factors in pneu-

Received 3 August 1996; revised 24 October 1996. Animal studies followed appropriate guidelines of the University of Alabama at Birmingham (UAB) Animal Care and Use Committee under the supervision of veterinary staff, Dept. of Comparative Medicine, Animal Resources Program Division. UAB animal facilities are accredited by the American Association for the Accreditation of Laboratory Animal Care. Grant support: NIH (AI-21548, HL-51646, and DE-06746); WHO. Reprints or correspondence: Dr. David Briles, Dept. of Microbiology, University of Alabama at Birmingham, 658 BBRB, Mail Box 10, Birmingham, AL 35294-2170. The Journal of Infectious Diseases 1997;175:839–46 q 1997 by The University of Chicago. All rights reserved. 0022–1899/97/7504–0015$01.00

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mococci. Polysaccharide (PS) is antiphagocytic and is the most conspicuous protection-eliciting antigen of pneumococci. The ability of serum antibodies to CPS to provide serotype-specific protection is well established [9]. Since children õ2 years of age respond poorly to immunization with PS vaccines [10], protein conjugates of PS are being investigated as a potential human vaccine for children [11]. There are §90 different CPSs constituting 48 non – cross-reactive PS groups [12]. Thus, to be effective, PS vaccines must contain multiple PS [13]. Pneumococci are acquired through aerosols or by direct contact and first colonize the upper airways, where they can be carried for weeks or months. Some 10% – 20% of adults carry pneumococci in the upper nasopharynx at any time; colonization rates in young children and in the elderly are much higher. In most cases, colonization does not result in apparent disease [14, 15] and even highly virulent strains can colonize without causing disease [16, 17]. PspA is necessary for full virulence of pneumococci and retards their clearance from the blood of mice [8, 18]. Antibody to PspA facilitates clearance and protects against death [8, 19, 20]. Like CPS, PspA is variable in structure. PspA from different isolates usually differ in molecular weight, the combination of epitopes expressed [21 – 23], and pspA-associated restriction fragment length polymorphism patterns [24]. However, PspAs share many cross-reactive epitopes, and immunization with a single PspA is cross-protective in mice against fatal infection with strains of many different capsular types [8, 21, 25, 26]. Whether parenteral immunization with pneumococcal PS or PS-protein conjugates can reduce pneumococcal carriage in humans is still under investigation [27, 28]. Much like Haemophilus influenzae, immunization with serotype b PS protein conjugates reduced carriage rates from Ç4% to õ1% [29, 30]. Although immunity at the nasal mucosa can be elicited through

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JID 1997;175 (April)

Table 1. Characteristics of encapsulated S. pneumoniae studied. Serotype Strain A66 L82016 BG7322 BG8826

LD50 in BALB/c and (CBA/N) mice

Capsule

PspA

3 6B 6B 23F

13 33 24 6

Intravenous 104 ú107 107 ú107

(õ102) (ú107) (õ102) (107)

Intratracheal

Intranasal

Reference

(5 1 103) — — —

ú3 1 107 (õ105) ú108 (ú108) ú107 (ú107) ú107 (107)

[34] [34] [34] This study*

* Collected by B. M. Gray, University of Alabama at Birmingham.

the common mucosal pathway by immunization at other mucosal sites, mucosal immunity is often strongest at or near the site of immunization [31]. Since pneumococci colonize the nasopharynx and infect the lungs and upper airway, we investigated the ability of mucosal immunizations to block infection and colonization at these sites. When pneumococci spread from the nasopharynx to the lung, they must pass through bronchial tissue. Thus, mucosal immunity at both the nasopharynx and bronchial sites might play a significant role in preventing pulmonary infection. We immunized mice intranasally (inl) with isolated PspA [32] to evaluate its ability to block inl or intratracheally (int) acquired infections. To test the ability of immunity to PspA to act at the mucosal surface, we also examined the ability of inl administered PspA to elicit protection against nasal carriage of S. pneumoniae. Immunizations inl used the B subunit of cholera toxin (CTB) as adjuvant. CTBs greatly augment immune responses to protein antigens given inl [33].

Methods Mice. Mice used in these studies were 6–12 weeks old. BALB/ c mice were raised at the University of Alabama at Birmingham from BALB/cJ parents obtained from Jackson Laboratories (Bar Harbor, ME). CBA/CAHN-XID/J (CBA/N) mice were purchased from Jackson Laboratories. Bacterial strains. Table 1 lists S. pneumoniae strains that we used for mouse challenge [34]. The only other strain used was R36A, an avirulent rough variant of capsular type 2 strain D39 [8, 35], which has been used for isolation of PspA. Immunizations, antigens, and ELISAs. Mice received three immunizations inl 10 days apart. On each occasion, 150 ng of PspA or 1500 ng of a conjugate of 6B-PS with tetanus toxoid (6B-TT) in 10–12 mL of Ringer’s lactate was slowly delivered into the nares. CTB (4–5 mg; List Biological Laboratories, Campbell, CA) was included in the first two immunizations. Other mice received two systemic injections of 1 mg of PspA: The first was given subcutaneously with complete Freund’s adjuvant (CFA); the second (20 days later) was given intraperitoneally (ip) without adjuvant. PspA for immunization was isolated from R36A pneumococci on choline-Sepharose columns as described previously [32]. For some control immunizations, we followed the same isolation procedure using strain WG44.1, which contains a mutation that

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blocks PspA production. The resulting material contained essentially no protein, since it lacked PspA, but was administered at the same dilution (based on the starting volume of bacterial culture) as the material from strain R36A [32]. This mock immunization always gave results identical to those obtained with CTB alone (data not shown). 6B-TT was prepared by the method of Schneerson et al. [36] and contained 3 mg of TT per 2 mg of 6B-PS. Mice were bled 10 days after their last immunization. Saliva (Ç100 mL) was collected from mice 10 days after the last immunization by injection ip of 3–5 mg of carbachol to stimulate salivation using a pipette fitted with a plastic tip [37]. Antibody levels were determined by ELISA using plates coated with isolated PspA or 6BPS. PS-specific antibody assays were done in the presence of pneumococcal CPS to prevent the detection of any antibody to cell wall CPS [38]. Isotype specific antibody levels were determined by using alkaline phosphatase– or peroxidase-conjugated antibodies specific for mouse immunoglobulin isotypes [37, 38]. Mouse challenge. Mice were challenged inl 2 weeks or 144 days after the last immunization. Other mice were challenged int, intravenously (iv), or ip 4 weeks after the last injection. Challenges ip and iv were done as previously described using log-phase pneumococci diluted in Ringer’s lactate to the indicated number of colony-forming units (cfu) [32]. Inoculation inl delivered 10 mL of Ringer’s solution containing cfu into the nares of nonanesthetized mice. We did inoculation int by a nonsurgical procedure that used a 69-mm finely drawn gel-loading plastic pipette tip (05541-9; Fisher Scientific, Pittsburgh). Mice were anesthetized with ketamine and xylazine and placed on their backs with heads bent slightly back. Slight tension was applied to the tongue to expose the glottis. The gel-loading tip was inserted into the glottis, and 20 mL of bacteria in Ringer’s injection solution was discharged into the trachea. The course of infection and colonization was followed by monitoring the numbers of cfu in the blood, homogenized lung, and nasal washes at various times after challenge. Bacteria from blood and lungs were plated in serial dilutions on blood agar plates so that cfu could be counted [20]. Nasal wash samples were collected from mice after sacrifice. The trachea was cut at the top of the larynx, and 50 mL of Ringer’s solution was injected and collected from the tip of the nose. The bacteria were plated on blood-agar plates containing 4 mg/mL gentamicin to inhibit the growth of most nonpneumococcal bacteria that might be present [39]. To further ensure that the bacteria observed were pneumococci, samples were also plated on a second set of gentamicin-containing plates that also contained 5 mg/mL optochin (ethyl hydrocupreine hydrochloride; Sigma, St. Louis).

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Table 2. Salivary IgA responses of BALB/c mice to R36A PspA and CTB. Geometric mean mg/mL IgA (SE factor) Immunization*

Route*

PspA / CTB PspA0 / CTB CTB PspA / CFA CFA

inl inl inl sc/ip sc/ip

PspA

CTB

0.9 (1.3) £0.01‡ £0.01‡ £0.01 £0.01

Geometric mean, % specific IgA (SE factor)† Total

3.3 (1.1) 2.8 (1.6) 3.2 (1.2) £0.01 £0.01

6.2 7.4 4.0 1.4 1.1

(1.1) (1.3) (1.2)x (1.2) (1.4)

PspA

CT

14 (1.2) £1§ £1§ £1 £1

53 (1.1) 37 (1.7) 78 (1.2)x £1 £1

* Each group contained 5 mice. PspA0 refers to immunization preparation for mice containing PspA0 strain. Immunizations used cholera toxin B subunit (CTB) or complete Freund’s adjuvant (CFA) as adjuvants. inl, intranasally; ip, intraperitoneally; sc, subcutaneously. † Antibody (10 days after last immunization) to CTB was assayed using microtitration plates coated with cholera toxin (CT). Statistical significance from PspA / corresponding adjuvant by Wilcoxon 2-sample rank test: P õ ‡.0001, §.01, x .05.

In general, there were few if any colonies on the blood agar plates containing gentamicin plus optochin, confirming that the bacteria observed on the gentamicin-containing plates were pneumococci. In the rare cases in which there were ú15% cfu on the gentamicin plus optochin plates, data from that mouse were discarded. In some initial experiments, colonization of the inoculated pneumococci was also confirmed by capsular typing of the pneumococci recovered from the nasal tissues. Statistical analysis. We expressed antibody levels as geometric means with the SE factor (the number by which a number must be multiplied or divided to obtain the upper or lower SE limits). Statistical differences were calculated with a one-tailed Wilcoxon two-sample rank test. P § .05 was considered nonsignificant.

Results Immune response to immunization inl with PspA. When given inl with CTB, 150-ng doses of PspA elicited significant IgA antibodies in saliva of BALB/cJ mice (table 2). No IgG antibody to PspA was detectable (õ0.01 mg/mL) after immunization inl. No antibody response to PspA was observed in the

absence of CTB (table 2) or if the dose of PspA was reduced to 15 ng (data not shown). Immunization inl with PspA and CTB elicited serum IgG and IgA antibody to PspA (table 3). Although parenteral immunization with 1000 ng of PspA in CFA induced a strong serum IgG antibody response, it failed to induce any serum IgA antibody to PspA and did not elicit detectable anti-PspA of any isotype in saliva (tables 1, 2). Immunization of CBA/N mice using similar intranasal and parenteral protocols elicited essentially the same responses (data not shown) as obtained with BALB/c mice. Protection against invasive disease by immunization inl with PspA. To test the ability of immunization inl with PspA to protect against pulmonary infection, Ç100 LD50 of A66 pneumococci were inoculated intratracheally (int) into BALB/c mice in 20 mL of Ringer’s solution. Protection against acquisition inl of pneumococcal infection was tested by inoculating immunized CBA/N mice inl with 3 1 106 cfu of virulent A66 pneumococci. CBA/N, rather than BALB/c mice were used for challenge inl, because inoculation inl of A66 readily kills CBA/ N mice but does not kill 100% of BALB/c mice, even when

Table 3. Serum antibody responses of BALB/c mice to R36A PspA and CTB. Geometric mean, mg/mL anti-PspA (SE factor)

Geometric mean, mg/mL anti-CTB (SE factor)

Immunization

Route

IgM

IgG

IgA

IgM

IgG

IgA

PspA / CTB PspA0 / CTB CTB PspA / CFA CFA

inl inl inl sc/ip sc/ip

£1 £1 £1 £1 £1

15 (1.2)* õ0.5 õ0.5 291 (1.7)* õ0.5

1.7 (1.3)* õ0.5 õ0.5 õ0.5 õ0.5

£1 £1 £1 £1 £1

4073 (1.1) 6457 (1.3) 6450 (1.2) õ0.5 õ0.5

55 (1.13) 78 (1.13)† 11 (1.07) õ0.5 õ0.5

NOTE. PspA0 refers to immunization preparation for mice containing PspA0 strain. Immunizations used cholera toxin B subunit (CTB) or complete Freund’s adjuvant (CFA) as adjuvants. inl, intranasally; ip, intraperitoneally; sc, subcutaneously. Statistical significance from PspA / corresponding adjuvant by Wilcoxon 2-sample rank test: P õ *.01, †.05.

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Figure 1. Protection against fatal challenge by intranasal (i.n.) immunization with R36A PspA adjuvanted with cholera toxin subunit B (CTB). Statistical comparisons of PspA / CTB vs. respective CTB-only controls are by Wilcoxon 2-sample rank test. A, Intratracheal (i.t.) challenge of BALB/c mice with 5 1 105 A66 (Ç1001 LD50) S. pneumoniae 14 or 144 days after i.n. immunization. §20 mice/group 2 weeks after immunization; 5 PspA immune and 10 CTB only mice 5 months after immunization. B, i.n. challenge of CBA/N mice with 3 1 106 cfu of A66 S. pneumoniae 30 days after i.n. immunization with PspA adjuvanted with CTB. PspA / CTB vs. CTB, P Å .011. C, Intravenous (i.v.) challenge of BALB/c mice with 8 1 104 cfu A66 (101 LD50) S. pneumoniae 2 weeks after i.n. immunization with PspA adjuvanted with CTB. PspA / CTB vs. CTB, P Å .045. D, Intraperitoneal (i.p.) challenge of BALB/c mice with 1750 cfu A66 (Ç901 LD50) S. pneumoniae 2 weeks after i.n. immunization. PspA / CTB vs. CTB only, P Å .004.

108 cfu are inoculated. Immunization inl with PspA provided good protection against challenge both inl and int (figure 1A, B). Challenge of mice int 144 days after immunization inl elicited long-term immunity (figure 1A). Mice immunized inl with PspA and CTB were also protected against sepsis and bacteremia; 3 days after challenge int, immune mice had õ30 cfu/mL in blood, whereas CTB control mice had 104 – 108 cfu/mL (data not shown). Challenge iv directly tested for protection against sepsis. Immunization inl with PspA from strain R36A protected BALB/c mice against

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otherwise fatal challenge iv with S. pneumoniae A66 capsular type 3 (figure 1C). Although inoculation ip is not a natural route of pneumococcal infection, it has been widely used as a model for passive immunity. Fewer pneumococci are needed to kill mice ip than by other challenge routes [40]. Previous studies have shown that subcutaneous immunization of mice with PspA can protect against death following challenge ip with S. pneumoniae [32]. In the present studies, we observed that even though immunization inl used õ1/6 the amount of PspA used in the subcutaneous

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Table 4. Protection of CBA/N mice against carriage of capsular type 6B L82016 S. pneumoniae by immunization with L82016 PspA. Carriage Immunization* Immunogen

Route

PspA / CTB CTB PspA PspA / CFA CFA None

inl inl inl sc sc None

Yes : No (§3 cfu : õ3 cfu) 0 8 4 4 4 4

: : : : : :

4 0 0 0 0 0

Log mean cfu (SE factor)*

P for PspA / CTB

õ3 — 440 (2.2) 440 (2.6) 240 (1.8) 190 (1.6) 1260 (4.4)

— .002 .014 .014 .014 .014

NOTE. Adjuvants used in immunizations were cholera toxin B subunit (CTB) or complete Freund’s adjuvant. Mice were challenged intranasally (inl) with 107 L82016 S. pneumoniae in 12 mL 2 weeks after last immunization. Long mean cfu/entire 50 mL of fluid washed from nasal tissue. Pneumococci were detected on blood-agar plates containing 4 mg/mL gentamicin sulfate. sc, subcutaneously. P values calculated by Fisher’s exact test; 1-way analysis of variance, P Å .02. In all cases, there were õ3 cfu of pneumococci/50 mL of blood at time of sacrifice. * SE factor is no. by which no. must be multiplied or divided to obtain upper or lower SE limits.

immunizations [32] and induced significantly less serum antibody to PspA than did subcutaneous immunization (table 3), it still induced enough immunity to significantly delay the time of death after challenge ip (figure 1D). Protection against nasopharyngeal carriage by immunization inl with PspA. With the fatal challenge (inl, iv, int, or ip) models described above, sepsis invariably precedes death. Thus, although mucosal immunity may contribute to protection against death following inoculation inl and int, protection against death may have been due primarily, if not exclusively, to protection against sepsis. To test whether immunity to PspA can act at the nasopharyngeal mucosa, we used a mouse nasopharyngeal carriage model in which colonization occurs in the absence of bacteremia and sepsis. Immunization inl with PspA from the capsular type 6B strain L82016 (plus CTB adjuvant) prevented nasopharyngeal colonization with strain L82016; neither PspA nor CTB alone elicited protection (table 4). Immunity to carriage was still detectable 144 days after boosting (table 5). This 5-month rest eliminated any concern that protection against carriage required concomitant CTB-dependent inflammation. Tables 1 and 2 show clearly that the only mice making anti-PspA responses after immunization inl were those immunized with both PspA and CTB. The requirement for mucosal immunity in the protection against carriage was demonstrated by inl challenge of the mice parenterally immunized with PspA and colonization factor antigen. Although high levels of serum antibody to PspA were elicited, parenteral immunization did not elicit mucosal antibody and did not protect against carriage (table 4). Cross-protection against carriage by immunization inl with PspA. In the experiment shown in table 4, the immunizing PspA was isolated from the same strain of pneumococcus used

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in the challenge. The study depicted in table 5 clearly shows that immunization inl with PspA can elicit cross-protection against carriage. PspA for immunization, isolated from nonencapsulated strain R36A (PspA serotype 25), elicited immunity to carriage of strains BG7322 and BG8826 (PspA serotypes 24 and 6, respectively) [21] (table 5). Results in figure 1 show that R36A PspA can protect against death following int, inl, iv, and ip challenge and illustrate cross-protection, since challenge strain A66 is PspA type 13. PspA types are determined from patterns of reactivity with a panel of seven monoclonal antibodies. PspA serotypes 6, 13, and 24 differed from that of R36A PspA (serotype 25) by 4, 4, and 2 epitope reactivities, respectively [21]. Protection against nasopharyngeal carriage by immunization inl with CPS. Protein conjugates of pneumococcal PS are being investigated as a potential human vaccine [11]. To test the ability of PS-protein conjugates to protect against carriage, 2-month-old BALB/c mice were immunized inl with pneumococcal type 6B PS conjugated to 6B-TT in the presence of CTB. Mice immunized inl three times with 1.5 mg of conjugate produced readily detectable levels of antibody to 6B PS (geometric mean Å 120 ng/mL; SE factor Å 3.4) in sera but barely detectable levels in saliva (geometric mean Å 2.8 ng/ mL; SE factor Å 2.0). Serum antibodies were almost exclusively IgG, but the only salivary antibodies detected were IgA. Mice immunized with three doses of 0.3 mg of conjugate generated unmeasurable levels of serum or saliva immunoglobulins to 6B PS. The highest levels of carriage were in the control mice given CTB only. Mice immunized with the highest dose of 6B-TT had the fewest pneumococci in the nasopharynx (figure 2). The numbers of pneumococci recovered from the nasopharynx of mice immunized with 6B-TT were significantly lower (P Å .03) than those from control mice. Discussion We observed that immunization inl of mice with PspA was efficacious against otherwise fatal infection when challenged with pneumococci int or inl. For challenge int, protection lasted §5 months after immunization. Protection against fatal infection could have resulted from mucosal immunity, systemic immunity, or both. Systemic immunity to PspA can protect against pneumococcal sepsis [20, 32]. From the present studies, immunization inl with PspA resulted in protective immunity in the systemic (protection from challenge iv and ip) and mucosal (protection from carriage) compartments. It is thus likely that both mucosal and systemic immunity may have contributed to protection against fatal challenge inl and int. To study immunity at the mucosal surface, we used a model in which carriage could be observed in the absence of sepsis. It should be noted that the ability of pneumococci to cause sepsis versus carriage only after challenge inl of mice is a function of both the mouse strain and the strain of pneumococci. In these studies fatal infections following inl inoculation

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Table 5. Ability of serotype 25 PspA from strain R36A to cross-protect CBA/N mice against carriage with two unrelated pneumococci. Carriage†

Challenge strain Immunogen

BG-

Capsule type

PspA type

PspA / CTB CTB PspA / CTB CTB PspA / CTB CTB

7322

6B

6

8826

23F

36

8826

23F

36

Days before challenge* 14 14 14 14 144 144

Yes : No 1 3 1 3 4 8

: : : : : :

3 1 3 1 4 0

median CFU õ3 1650 õ3 1865 4 1035

NOTE. In 50-mL blood samples, all mice had õ3 cfu. In all cases, there were õ3 cfu of pneumococci/50 mL of blood at time of sacrifice. * Days between immunization and intranasal challenge with 107 cfu in 12 mL. † Carriage evaluated as in table 4. Yes, §3 cfu; no, õ3 cfu. For 14-day data, all P Å .01 by 1-tailed Wilcoxon test for PspA / CTB vs. all CTB only. For 144-day data, P Å .0001 for PspA / CTB vs. CTB.

were observed only when A66 pneumococci were used to inoculate the hypersusceptible CBA/N mice. Most strains of pneumococci, however, can cause carriage in mice without accompanying sepsis (unpublished data). The absence of sepsis in the carriage model was critical to our ability to determine that protection could act at the mucosal surface. Since mice carrying the pneumococci in the nasal tissues were not septic, it was clear that the pneumococci found in the nasopharynx were present because they had colonized and not just contaminated (a function of generalized sepsis). The protection against carriage appeared to result from local rather than systemic immunity. For example, both the

Figure 2. No. of bacterial colonies recovered from mice immunized 3 times with 1.5 mg of 6B polysaccharide and tetanus toxoid (6BTT) plus cholera toxin subunit B (CTB), 0.3 mg of 6B-TT plus CTB, or CTB alone. Dose refers to polysaccharide content of conjugate.

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inl and the subcutaneous/ip immunization routes elicited systemic antibody responses, but only immunization inl elicited detectable antibody in secretions. Although it seems likely that the large amount of IgA antibody to PspA observed in the secretions plays a role in protection at mucosal sites, these data do not rule out the possibility that the much smaller (largely undetectable) levels of IgG in the secretions or some type of cell-mediated response may contribute to the resistance observed here. Our demonstration that immunization inl with 6B-TT conjugate can reduce carriage of pneumococci indicates that immunity to antigens other than PspA can also protect against carriage. CPS, PspA, and IgA protease show the highest degree of serologic variation of any pneumococcal antigens [12, 21, 41]. Since the human reservoir of pneumococci is thought to be maintained primarily by nasopharyngeal carriage, it might be expected that the evolutionary selection that has driven serologic variation in CPS, PspA, and IgA protease acts at the level of carriage, rather than at systemic infection. Mouse IgA is not cleaved by pneumococcal IgA1 protease, and we did not attempt to assess the protectioneliciting role of this potential virulence factor in mice. In the case of immunity to PspA and capsule, however, protection against carriage was observed. The ability of PspA and the 6B-TT conjugate to elicit protection against nasopharyngeal carriage in mice raises the possibility that mucosal immunity to these antigens might confer protection against carriage in humans. The observation that immunization with PspA can cross-protect against carriage strains bearing different PspAs enhances the prospects for the use of PspA as a mucosal vaccine. Moreover, in tests of the cross-protection elicited by individual PspA immunogens, the carriage model may have one advantage over the systemic infection model. Strains of many capsular types, including some of the major childhood capsular types 14, 19F, and 23F, are quite avirulent when injected into mice [34] but are readily carried in the nasopharynx (unpublished data).

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Protection against both inl carriage and challenge int was still apparent §5 months after immunization. This long duration of protection provides support for the hypothesis that mucosal immunity may play an important role in protection against pneumococcal colonization and disease. It is important to note, however, that the observations reported here have been made with mouse models of pneumococcal disease. Knowledge of the exact relevance of these data to immunity to human carriage and systemic pneumococcal disease must await detailed human studies. Nonetheless, the observation the PspA can elicit longlasting mucosal immunity to infection and carriage provides encouragement that this easily produced, highly immunogenic protein may be useful as a human vaccine. The observation that immunization inl with PspA and 6B-TT can provide immunity to carriage strengthens arguments for testing mucosal immunization as a potential route for the delivery of pneumococcal vaccines in humans.

12. 13.

14.

15.

16.

17.

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Acknowledgments

19.

We thank Janice King for technical assistance, D. Ashley Robinson and Marilyn J. Crain for PspA typing strain BG8826, and Anni Virolainen for her interest in these studies.

20.

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