AU734488B2 - Mucosal administration of pneumococcal antigens - Google Patents

Description

AUSTRALIA

Patents Act COMPLETE SPECIFICATION

(ORIGINAL)

Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: Name of Applicant: UAB Research Foundation Actual Inventor(s): David E. Briles Hong-Yin Wu O Address for Service: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Invention Title: MUCOSAL ADMINISTRATION OF PNEUMOCOCCAL ANTIGENS :i Our Ref 569473 POF Code: 328353/207806 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): IP Australia Documents received on: o I APR 1999 1 'z ;11 Batch No: la MUCOSAL ADMINISTRATION OF PNEUMOCOCCAL ANTIGENS The present application is a divisional application from Australian patent number 698724 the entire disclosure of which is incorporated herein by reference.

FIELD OF THE INVENTION This invention relates to mucosal immunization of animals with pneumococcal antigens to provide protection against pneumococcal colonisation and systemic infection.

BACKGROUND TO THE INVENTION Streptococcus pneumoniae causes more fatal infections world-wide than almost any other pathogen (refs. 1,2 a list of the reference appears at the end of the disclosure). In the deaths caused by S. pneumoniae rival in numbers those caused by AIDS (ref. In the most fatal pneumococcal infections occur in individuals over 65 years of age, in whom S.

pneumoniae is the most common cause of community-acquired pneumonia. In the developed world, most pneumococcal deaths occur in the elderly, or in immunodeficient patents including those with sickle cell disease. In the less- 20 developed areas of the world, pneumococcal infection is one of the largest causes of death among children less than 5 years of age (refs. The increase in g o

*I

C:\WINWORD\STACVICPJCSPECIMR569473.DOC the frequency of multiple antibiotic resistance among pneumococci and the prohibitive cost of drug treatment in poor countries make the present prospects for control of pneumococcal disease problematical (refs. 7, 8, 9).

The reservoir of pneumococci that infect man is maintained primarily via nasopharyngeal human carriage.

Humans acquire pneumococci through aerosols or by direct contact. Pneumococci first colonize the upper airways and can remain in nasal mucosa for weeks or months. As many as 50% or more of young children and the elderly are colonized. In most cases, this colonization results in no apparent infection (refs. 10, 11, 12). Studies of outbreak strains have suggested that even highly virulent strains can colonize without causing disease (refs. 13, 14, 15, 16). These expectations have been recently confirmed using molecular probes to fingerprint individual clones J. Crain, personal communication to one of the inventors). In some individuals, however, the organism carried in the nasopharynx can give rise to symptomatic sinusitis or middle ear infections. If pneumococci are aspirated into the lung, especially with food particles or mucus, they can cause pneumonia.

Infections at these sites generally shed some pneumococci into the blood where they can lead to sepsis, especially 25 if they continue to be shed in large numbers from the original focus of infection. Pneumococci in the blood can reach the brain where they can cause meningitis.

Although pneumococcal meningitis is less common than other infections caused by these bacteria, it is *30 particularly devastating; some 10% of patients die and greater than 50% of the remainder have life-long neurological sequelae (refs. 17, 18).

In elderly adults, the present 23-valent capsular "polysaccharide vaccine is about 60% effective against invasive pneumococcal disease with strains of the capsular types included in the vaccine (refs. 19, The 23-valent vaccine is not effective in children less than 2 years of age because of their inability to make adequate responses to most polysaccharides (refs. 21, 22). Improved vaccines that can protect children and adults against invasive infections with pneumococci would help reduce some of the most deleterious aspects of this disease. A vaccine that protected against disease but did not reduce pneumococcal carriage rates would not, however, be expected to control the disease in immunocompromised (ref. 20) and in unimmunized individuals.

Such a vaccine would also not be expected to affect the rates of infection in immunized children prior to the development of an adequate anti-vaccine response.

A strategy that could control infections in all of these individuals would be any form of immunization that prevented or greatly reduced carriage, and hence transmission of pneumococci. In the case of immunization of young children with Haemophilus influenzae group b polysaccharide-protein conjugates, it has been observed that carriage is reduced from about 4% to less than 1%, (ref. 23), a possible explanation of concomitant herd immunity (ref. 24). If a vaccine could prevent colonization by pneumococci, such vaccine would be expected to prevent virtually all pneumococcal infections 25 in the immunized patients. Since even unimmunized patients must acquire pneumococci from others, a vaccine that reduced carriage should reduce infections in immunocompromised as well as unimmunized patients. In fact, an aggressive immunization program, coupled with antibiotic 30 treatment of demonstrated carriers, might be able to largely eliminate the human reservoir of -this organism.

It may not be possible, however, to totally eliminate pneumococci since there are a number of reports that they have been found in laboratory rodents (ref. 25). Whether these pneumococci are infectious for man, easily transmittable to man, or even pathogens in wild rodents (V is not known. S. Pneumoniae does not live free in the environment.

Although intramuscular immunization with capsular polysaccharide vaccines has been effective at reducing the incidence of pneumococcal sepsis in the elderly (ref. 20), it has not been reported to affect pneumococcal carriage rates in children up to 54 months of age (refs 26, 27) Whether the conjugate vaccine will reduce carriage in children is not known. Thus, the search for a vaccine with can reduce rates of nasopharyngeal carriage must include an examination of non-capsular antigens. Since immunity to carriage would be expected to operate at the mucosal surface, any attempt to identify antigens for vaccines against carriage should include immunizations designed to elicit mucosal immune responses. For these reasons, the present disclosure focuses on intranasal immunization with pneumococcal proteins in addition to the evaluation of polysaccharide-protein conjugates.

Throughout the description and claims of the specification the word "comprise" and variations of the word, such as "comprising" and "comprises" is not intended to exclude other additives, components, integers or steps.

SUMMARY OF THE INVENTION In accordance with the present invention, it has now been found that 20 mucosal administration, particularly intranasally, of pneumococcal surface protein A (PspA) or an immunogenic fragment thereof provides protection to a host against pneumococcal colonization and systemic infection.

This invention provides a substance or composition for use in a method of immunization of a host against pneumococcal infection wherein the method 25 includes mucosally administering to the host an immunizing amount of the substance or composition, wherein said substance or composition includes pneumococcal surface protein A (PspA) or at least one fragment of such PspA containing at least one protection-eliciting epitope and wherein said PspA is in the form of killed whole pneumococci or of a lysate of pneumococci.

Accordingly, in one aspect, the present invention relates to a method of protecting a host, particularly a human host, against colonization by A pneumococci and systemic infection by mucosal administration, particularly by intranasal administration, to the host of an effective amount of at least one \\MINNIE1PJC\Elsabeth\PJC\SPECI\14226-9a.DOC 4a pneumococcal. surface protein A and/or an immunogenic fragment thereof containing at least one protection-eliciting epitope.

The PspA may be in the form of killed whole S. Dneumoniae or a lysate, of whole S. pneumoniae.

C:%WNWORD\JOANNE\PJCXSPECI%14225-9g.D()C Alternatively, the PspA may be in the form of purified isolated protein or a fragment thereof. Such purified and isolated protein or fragment thereof may be obtained from bacterial isolates or may be formed recombinantly.

In a preferred aspect of the invention, the PspA is administered with cholera toxin B as an adjuvant.

The mucosal administration preferably is effected intranasally to provide protection to the host against both pneumococcal colonization and systemic infection.

The intranasal administration also may provide protection to the host against pulmonary infection as well as protection to the host against an infection starting as a pulmonary infection.

In a particular aspect of the invention, there is provided a method of colonization of a host against colonization with Streptococcus pneumoniae in the nasopharynx, which comprises intranasally administering to the host an immunizing amount of pneumococcal surface protein A (PspA) in the form of a killed whole pneumococci, a lysate of pneumococci or an isolated PspA or an immunogenic fragment thereof.

The present invention further provides a vaccine composition for intranasal administration to a host to *2 confer protection against colonization with S. pneumoniae 25 in the nasopharynx, which comprises: a vaccine composition for intranasal administration to a host to confer protection against colonization with S. pneumoniae in the nasopharynx, which comprises: an effective amount of a pneumococcal surface 30 protein A (PspA) in the form of a killed whole pneumococci, a pneumococcal lysate, an- isolated and purified PspA or an immunogenic fragment thereof containing at least one protection-eliciting epitope, an adjuvanting amount of the B subunit of cholera .toxin, and a pharmaceutical carrier therefor.

In published International patent application WO 92/14488, there are described the DNA sequences for the psPA gene from S. pneumoniae Rxl, the production of a truncated form of PspA by genetic engineering and the demonstration that such truncated form of PspA confers protection in mice to challenge with live pneumococci.

From sequences of the PsDA gene, it has been shown that PspA proteins are variable in size (roughly The C-terminal 37% of the molecule is largely composed of the 20-amino acid repeats which form a binding site that permits PspA to attach to the phosphocholine residues of the pneumococcal lipoteichoic acids. The central region of PspA is rich in proteins and is suspected to be the portion of the molecule that passes through the cell wall. The sequence of the N-terminal 80% of the molecule is largely a-helical and contains the region of PspA that can elicit antibodies that are protective against sepsis.

Although PspA's are almost always at least slightly different from one another, there is enough crossreactivity between them that antibodies to one PspA detect PspAs on all pneumococci. Moreover, immunization with one PspA can either protect against death or delay death with virtually all different challenge strains.

Accordingly, a mixture of a small number of PspAs could a 25 elicit effective immunity against most pneumococci.

The immunoprotective truncated PspAs described in WO 92/14488 may be used in the present invention as the PspA fragments described above for mucosal administration.

The ability of a vaccine to protect against 30 pneumococcal colonization, as provided herein, means that the active component may protect against disease not only in the immunized host but, by eliminating carriage among immunized individuals, the pathogen and hence any disease it causes may be eliminated from the population as a whole.

In the data presented herein, it is shown that intranasal administration can also prevent sepsis resulting from intratracheal administration of pneumococci, so that the vaccine can protect against both pneumococcal colonization and sepsis.

GENERAL DESCRIPTION OF INVENTION The principal determinant of specific immunity at mucosal surfaces is secretory IgA (S-IgA) which is physiologically and functionally separate from the components of the circulatory immune system. S-IgA antibody responses may be induced locally by the application of suitable immunogens to a particular mucosal site. The bulk of mucosal S-IgA responses, however, are the results of immunity generated via the common mucosal immune system (CMIS) (ref. 28), in which immunogens are taken up by specialized lympho-epithelial structures, collectively referred to as mucosa-associated lymphoid tissue (MALT). The best studied immunologic lympho-epithelial structures are the gut-associated lymphoid tissues (GALT), such as intestinal Peyer's patches. It is now clear, however, that other structurally and functionally similar lymphoid follicles occur at other mucosal surfaces, including those of the respiratory tract (ref. 29).

S* 25 Bronchus-associated lymphoid tissue (BALT) was described by Bienenstock (refs. 30, 31) in experimental animals, but is apparently not present in the noninfected human bronchial tree (ref. 32). The upper respiratory tract in humans, however, is furnished with Waldeyer's 30 ring of tonsils and adenoids. In rodents, the functional equivalent of these consists of nasal-associated lymphoid tissue (NALT), a bilateral strip of lymphoid tissue with overlying M-like epithelial cells at the base of the nasal passages (ref. 33).

In the experimental results set forth in the Examples below, it is shown that mice can be effectively immunized by intranasal instillation of bacterial protein immunogens, particularly when conjugated to or mixed with cholera toxin (CT) or its B subunit (CTB) (ref. 34). When CTB is used as an adjuvant for i.n.

immunizations, specific IgA antibodies are induced in secretions of the intestinal, respiratory, and genital tracts, as well as predominantly IgA antibody-secreting cells in the intestinal lamina propria and salivary glands. Strong circulatory immune responses are also induced, with IgG and IgA antibodies in the serum, and IgG and IgA antibody-secreting cells in the spleen. The circulatory (or systemic) immune responses elicited by i.n. administration of antigens along with CTB are comparable with, or even stronger than, those induced by the administration of similar immunogens by the intragastric peroral) route (refs". 34, Accordingly, it appears that i.n. immunization is an effective route for stimulating common mucosal responses as well as circulatory antibody responses and requires less antigen than i.g. immunization.

Most soluble or non-replicating antigens are poor mucosal immunogens, especially by the peroral route, probably because they are degraded by digestive enzymes i and have little or no tropism for the GALT. A notable 25 exception is CT, which is a potent mucosal immunogen (ref. 36), probably because of the GMI gangliosidebinding property of its binding subunit, CTB, that enables it to be taken up by the M cells of Peyer's patches and passed to the underlying immunocompetent 30 cells.- In addition to being a good mucosal immunogen, CT is a powerful adjuvant (refs. 37, 38, 39). When administered in Ag doses, CT greatly enhances the mucosal immunogenicity of other soluble antigens co-administered with it.

In the experimental results contained in the Examples below, it is shown that CTB is a strong adjuvant when given i.n. in mice along with the pneumococcal protein, PspA. Although the inventors cannot completely rule out a role for small amounts of CT in these studies, CT was of the 5 Ag dose of CTB that was administered. Thus, it would appear that when administered CTB may be a stronger adjuvant and act more independently of CT, than when it is given i.g. For example, when given orally or CTB has no direct adjuvant effect, but can act synergistically with CT (ref. The mechanisms by which CT and CTB act as adjuvants are not fully understood, but are certainly complex, and appear to depend on several factors, including: 1) the toxic activity associated with the ADP-ribosylating property of the Al subunit (ref. 41); 2) increased permeability of mucosae (refs. 42, 43), 3) enhanced antigen-presenting cell function (with increased levels of IL-1) (refs. 44, 45), as well as 4) direct stimulation of T and B cell activities (refs. 46, 47, 48, 49). This last point is controversial, however, as the in vitro effects of CT or CTB on T and B cells are generally inhibitory rather than stimulatory (refs. 50, 51, 52).

Nevertheless, numerous reports attest to the in vivo mucosal immunoenhancing effects of CT and of CTB coupled 25 to antigens (refs. 38, 39, 53, 54, 55, 56).

Although carriage of pneumococci can be maintained for long periods in the very young and the elderly, it is generally not a permanent condition. Carriage is much less common in older children and young adults (refs. 30 11, 12, 57, 58). One explanation for these findings is that carriage may be interfered with by immunity (possibly mucosal immunity) to pneumococci (refs. 11, 59). The inventors have shown that most human saliva have IgA antibodies to type 23 capsular polysaccharide and phosphocholine (an immunodominant determinant of pneumococcal cell wall teichoic acids (ref. It seems likely, therefore, that human sera would also contain antibodies to other pneumococcal antigens. In the case of group A streptococci, M proteins have been shown to be required for colonization in rats, and antibodies to M proteins can protect against colonization of the throat (refs. 61, 62). In mice, the inventors have shown herein that antibody to PspA can prevent carriage of S. pneumoniae.

Antibodies may be effective against carriage in two ways, namely: 1) they might act at the mucosal surface by opsonizing pneumococci, preventing attachment or surface invasion; 2) they might act via opsonophagocytosis and killing. This latter mechanism could be especially important if nasopharyngeal carriage is dependent on minimal invasion of the nasal mucosal surface. The complement fixing antibodies could prevent the invasion and facilitate the killing of any pneumococci that invaded locally. Alternatively, complement fixing antibodies might be able to act and the mucosal surface if inflammation causes a sufficient release of complement, phagocytes, and possibly serum antibody.

One of these mechanisms might play a role in the observation that carriage of H. influenzae can be prevented by an intramuscular vaccine (ref. 23). It has 25 recently been reported that significant levels of H.

influenzae polysaccharide-specific IgG and IgA are detected in secretions of children following immunization with the group b polysaccharide conjugate vaccine (refs.

24, 63).

30 Although definitive comparisons have not been made in most cases, existing mouse protection data (refs. 64, 66, 67) suggests that antibodies that can opsonize pneumococci those to the capsule) are generally more protective against sepsis than those that block the activities of toxins pneumolysin) or enzymes (e.g.

autolysin or neuraminidase). However, at the mucosal surface, the role played by antibodies that inactivate toxins and enzymes may be greater than that played by opsonic antibodies. The reason to suspect this is that for opsonic antibodies to exert their anti-bacterial effect, complement and phagocytes are required.

Phagocytes are rare on the surface of normal nasopharyngeal tissue, and even if present, the phagocytes do not have the filtering action of the spleen and reticuloendothelial system to increase their chance of interactions with opsonized bacteria. Antibodies that block the virulence enhancing effects of pneumolysin and pneumococcal enzymes should able to bind their antigens just as effectively whether phagocytes were present or not.

The results provided herein show that i.n.

immunization with heat-killed pneumococci, and pneumococcal lysates, and purified PspA can protect mice against nasopharyngeal carriage. As noted earlier, the ability of a vaccine to protect against colonization means that it may protect against disease not only in the immunized host, but, by eliminating carriage among immunized individuals, the pathogen and hence any disease it causes may be eliminated from the population as a *g whole.

25 The vaccine composition which is administered intranasally as provided herein may be formulated in any convenient manner and in a dosage formulation consistent with the mode of administration and the elicitation of a protective response. The quantity of antigen to be 30 administered depends on the subject to be immunized and the form of the antigen. Precise amounts and form of the antigen to be administered depend on the judgement of the practitioner. However, suitable dosage ranges are readily determinable by those skilled in the art and may be of the order of micrograms to milligrams. Suitable regimes for initial administration and booster doses also are variable, but may include an initial administration followed by subsequent administrations.

EXAMPLES

Example 1 This Example illustrates the provision of a model for pneumococcal carriage in mice.

Seven different strains of S. pneumoniae were inoculated in 10 Al volumes with log-phase cultures in the nares of CBA/N XID mice over a period of several minutes using slow delivery from a 20 .l Pipetman. After 6 to 7 days, the mice were sacrificed and their trachea was cut just below the larynx. 50 l of sterile saline was instilled and washed out through the nares. The area washed represents the pharynx and nasal tissues. Six of these strains was able to establish carriage without concomitant sepsis or bacteremia. One of the seven strains, (A66, a virulent capsular type 3 strains) killed all four of the i.n. infected CBA/N mice within 3 days.

The results are shown in the following Table I: TABLE I 25 *0 0..

3

S

.3 @0 0@ 5500 Carriage of S. pneumoniae in CBA/N (XID) mice: 10 7

CFU

i.n.

Strain Capsular Log Alive: Median Max.

type LDso Dead With CFU in CFU/ i.v. Carriage nose 50 pl blood A66 3 <2 0:4 BG9739 4 <2 3:1' 67' 1,000 <3 L82106 6B >7 6:0 100 2,000 <3 BG9163 6B 3.5 2:0 100 2,000 <3 TJ0893 14 .4 4:0 100 4,000 <3 L82013 19 2 4:0 100 200 <3 BG8826 23F 2 2:0 100 20,000 <3 Note: Carriage and blood CFU determined after 7 days. Greater than 10 CFU of pneumococci were recovered from the nose of all mice judged as carriers.

One mouse in this group that grew out a contaminant in large numbers and no pneumococci. It seems likely that the contaminant excluded the pneumococci.

Example 2: The procedure of Example 1 was repeated by inoculating the nares of BARB/By mice with two of three pneumococcal isolates used. Carriage was observed with all three strains, without bacteremia or sepsis. The results are shown in the following Table II: TABLE II Carriage of Streptococcus pneumoniae isolates in BALB/ByJ mice Strain Capsular Log Alive: Median Max.

type LDo Dead With CFU in CFU i.v. Carriage nose in of blood D39 2 7 4:0 100 400 <3 A66 3 4:0 100 4,000 <3 BG7322 6B -7 4:0 75 1,700 <3 L82013 19 >8 4:0 100 6,000 <3 BG8826 23F 8 4:0 75 1,900 <3 Note: Carriage and blood CFU determined after 7 days.

Note: CFU in nose expressed as CFU in 50 1 of 1ml nasal wash Example 3: This Example shows the effects of i.n. inoculation on lung infection.

With three of the strains examined for carriage in Example 1, the numbers of pneumococci in the lung were examined at the time of sacrifice. The results obtained are shown in the following Table III: TABLE m Carriage of Three Strains of S. pneumoniae in the Nasopharynx of CBA/N Mice Strain L82106 BG9163 BG8826 Capsule 6B 6B 23F Type 10 3 x 10 107 Mouse #1 #2 #1 #2 #1 #2 Nasal 6,600 1,400 1,500 3,300 32,000 10,000 Blood <3 <3 <3 <3 <3 <3 Lungs 600 <60 <60 <60 <60 180 The data shown in this Table III illustrates the fact that, although the carried pneumococci do not generally cause detectable bacteremia or sepsis, they can spread in at least small numbers to the lung. The ability of S. pneumoniae to spread from the nasopharynx to the lung emphasizes the importance of a vaccine blocking carriage in the upper airways.

Example. 4 1: 0 This Example shows the effect of i.n. inoculation on colonization stability.

Colonization appeared to be present for at least 19 days and stable for at least 6 days, as shown by the results in the following Table IV: 0** *o a a.

TABLE IV .CFU recovered from the nasovharnvx of mnice infected iLn. with 101 strain L82106 Experiment N11 Experiment 0f2 ay_ and mouse N) (day and mouse 4) Day I Day 3 -Day 6 Dayl14 Dayl19 Source #11 #2 #1 l f2lI 0 #3 #1 N2 qasal Wash 56,862 5,346 29,160 6,336 41,365 9,504 202 <3 261 67 760 124 Blood <20 1,752 <20 <20 <20 20 <3 3 <3 <3 <3 <3 Lungs <20 .1,920 <20 I<20 <20 .<20 n.d. n.d. n.d. n.d. n.d.

Note: Data expressed as C1'U in the SOIl nasal wash, in the i of Itung flomogenate, or in 50 til Since mice Were killed to obtain the nasal wash, different mice were assayed at each time point.

Of 01000.

To identify the pneumococci from nasal washes in the experiments reported in Examples 1 to 4, they were plated on gentamicin plates since this antibiotic does not kill pneumococci but kills most other bacterial from the nose (ref. 68). Individual colonies from each nasal wash were then picked and replated with an optochin disk to confirm that they were pneumococci. In some cases, the bacteria were capsule-typed to be sure that they were, in fact, the same bacteria with which inoculated the mice were inoculated. Control mice that received no bacteria yielded no bacteria that grew on 0.02% gentamicin and were sensitive to optochin. Subsequent studies have shown that challenge with as few as 2x10 7 colony forming units (CFU) also yields carriage in all mice. Lower doses down to 103 CFU yield comparable carriage in most mice but as many as 1/4 to 1/3 of the mice fail to carry any pneumococci after 1 week. These studies indicate that the ideal dose is probably between 107 and 106 CFU of L81905.

Example This Example illustrates elicitation of protection against carriage by immunization with heat killed pneumococci and with a lysate of pneumococci.

CBA/N mice were immunized i.n. with 2 x 107 heat- 25 killed (60*C) L82016 or a lysate of an equal number of L82016. Mice were given three i.n. immunizations spaced days apart. The first two injections were given with ig of CTB. Two weeks after the last injection the mice were challenged with 108 CFU of live L82016. The results 30 obtained are set forth in the following Table V: 0 .000 TABLE V Elicitation of protection against carriage by immunization with heat killed and autolysed pneumococci Immunogen CFU from individual Geometric P vs P vs mice mean CTB none

CFU

Heat killed 3, 9, 26 4.8 <.01 <.02 L82016

CTB

Autolysed 8, 30 4.5 <.01 <.02 L82016

CTB

CTB 9, 160, 197, 248, 741 139 n.s. n.s.

None 6, 1340, 1400, 1400 354 n.s. n.s.

P. values calculated by Student's t-test. n.s. not significantly different.

Example 6 This Example illustrates protection against carriage by immunization with isolated and purified PspA.

CBA/N mice at 10 weeks of age were immunized i.n.

with PspA purified by passage over a choline-Sepharose column and eluted in 2% choline chloride. The material run over the column is clarified medium from a pneumococcal culture grown to late log phase in a defined medium (Rijn et al, Infect. Immun. 1990, 27, pp 444-448), containing 0.03% ethanolamine and no more than 0.000,001% choline chloride. When pneumococci is grown in this medium, about half of the PspA is released from the cell surface and can be recovered in the medium. Most pneumococci can not grow in this medium and must be adapted by growing them is CDM with successive lower concentrations of choline. The PspA eluted from the column comprised at least 99% of the eluted protein.

The CBA/N mice were immunized i.n. with 150 ng or s.c. with 1 lg of 99.9% pure PspA from strain L82016.

Three i.n. immunizations were given at 10 day intervals.

pg of commercially-obtained 299.9% pure (OTB) (List Biochemical Labs, Inc., Campbell, CA) were given with the first two injections as an adjuvant. In the case of s.c.

immunization, the first injection was given with PspA in Freund's complete adjuvant and a second injection of PspA in saline was given two weeks later. Two weeks after the last injections, the mice were inoculated i.n. with 107 CFU of L82016. Seven days later, the mice were sacrificed, bled and assayed for carriage in the nasopharynx. The results obtained are illustrated in the following Table VI: TABLE VI Intranasal immunization with isolated PspA elicits protection against carriage in CBA/N mice challenged i.n. with 10' CFU of L82016 9* 9 *9 9* 9.

9*9* 9 9* 9* *999 20 Immunization Carriage Immunogen Route Yes: No Log mean P vs.

3 CFU: _3 CFU PspA CFU) factor)

CTB

PspA i.n. 4:0 440 0.014 PspA CTB i.n. 0:4 <3 CTB i.n. 8:0 440 0.002 PspA CFA s.c. 4:0 240 0.014 CFA s.c. 4:0 190 0.014 None None 4:0 1260 0.014 P values calculated by Fisher exact test. A one way ANOVA gave a P value of 0.02.

None of the mice exhibited detectable pneumococci in their blood at the time of assay. As may be seen from Table VI, carriage was seen in all mice except the group of four immunized i.n. with PspA and CTB. Control mice immunized with CTB or PspA alone still exhibited carriage.

In another experiment mice were infected i.n. with ten times the dose (10 S CFU), and immunization with PspA CTB still protected against pneumococcal carriage (Table VII below). In that experiment the control mice were injected with CTB plus a comparable preparation from a PspA- strain WG44.1, made by the identical purification procedures used for PspA. Mice were immunized with a dilution of the WG44.1 material comparable to that of the isolated PspA. The failure of these control mice to be protected against carriage makes it clear that the protection elicited by isolated PspA is due to PspA and not an undetected contaminant co-isolated with PspA.

When mice were immunized although they produced humoral antibody to PspA, they still-exhibited carriage.

TABLE VII Intranasal immunization with isolated PspA elicits protection against carriage in CBA/N mice challenged i.n. with 10' CFU of L82016 Immunogen CFU from individual Geometric P vs P vs mice mean PapA- pooled CFU controls FL-L82016 <3 <3 0.028 <0.0001 PspA

CTB

PspA- 128, 18, 277, 49, 527 4,875 (WG44.1)

CTB

CTB 1,059, 26,720, dead' 11,226 None 426, 11,484, dead' 6154 P. values calculated by Welch's t-test. n.s. not significantly different.

for the purpose of the statistical calculations, these two mice were assigned carriage values of 50,000 since 49,000 was the highest carriage level observed in a live animal.

Example 7 This Example illustrates the elicitation of secretory and systemic immune responses by i.n.

immunization with PspA.

Using the immunization protocol of Example 6, further immunization studies were carried out and mice were bled 10 days after immunization. Secretory and systemic antibody responses were determined and the results appear in the following Table VIII: TABLE VII Antibody to PspA elicited by s.c. and i.t. immunization with PspA Serum Salivary Antigen Route IgM IgG IgG IgA tG 1/g specific specific PspA CTB i.n. <1.5 33 14 8.7+3 13.5+ 2.6 PspA i.n. <1.5 <1.5 n.d. n.d.

WG44.1 CTB i.n. <1.5 <1.5 <2 <2 CTB i.n. <1.5 <1.5 <2 <2 PspA CFA s.c. <1.5 470 217 n.d. <2 CFA s.c. <1.5 1.5 n.d. <2 0 0

U

*9 000.

25 Note: Salivary responses are expressed as percent of total immunoglobulin that is specific to PspA.

As may be seen from this Table, the immunization elicited detectable salivary IgG and IgA antibody responses. Control immunizations with CTB, CFA or a PspA' fraction isolated from PspA' strain WG44.1 did not elicit antibody to PspA. Antibody was not detected when CTB was not used as an adjuvant for i.n. immunization. Assays for antibody to PspA were conducted with ELISA plates coated with isolated PspA.

Example 8 This Example illustrates elicitation of crossprotection against carriage with strains whose PspAs differ from those of immunizing PspA R36A.

Mice were immunized with three i.n. immunizations with 0.15 gg of R36A PspA. In the first two injections immunization was accompanied with 4gg of purified CTB.

Injections were 10 days apart and mice were challenged about 2.weeks after the last immunization. The control mice received the two CTB immunizations and were "immunized" with saline only for the third injection.

These mice were largely protected from challenge carriage with two different challenge strains BG7322 and BG8826 of different capsular types and different PspA types. Both have PspA of a different type than the serotype 25 PspA used for immunization. The R36A PspA immunization came from strain R36A which is a non-encapsulated mutant of capsular type 2 strain D39. Thus, neither the capsular type nor the PspA type of the strains providing the immunizing PspA were the same as the challenge strains.

Because of the small number of mice in each group the results with the individual strains are not quite significant. However, pooling the data lead to a highly significant demonstration that PspA can elicit protection against challenge pneumococci with a PspA different from the immunizing PspA. The data is summarized in the following Table IX:

P*

9*pp **o *e e o** 9 9 9 9 9 *9 Table IX Intranasal immunization with isolated R36A PspA (1'spA type 25) elicits crossprotection against carriage in CBAIN challenged i.n. with 10 7 CFU of strains BG7322 (PspA type 24) and BG8826 (PspA type Streptococcus pneumoniae CFU recovered from ChallengeStrain Immun- naohrn P versus Max. CFU Name Caps. type PspA type ized individual median CTB only /5Oil of with mice blood BG7322 6B 24 PspA <3 0.057- <3 CTB 12 CTB 1500, 1700 <3 1800,4000 BG8826 23F 20 PspA <3 0.057 <3.

CTB 3 CTB 29, 1900 <3 3700,5800 BG7322 encaps- not 25 PspA+ <3 0.013 <3 BG8826 ulated CTB <3, 3,12 CTB 29, 1700 <3 1500, 1900, 3700,4000, L5800 P value calculated by the Wilcoxon two sample rank test using one degree of freedom.

Note: CFU in nose expressed as CFU in S5jil of 1 ml nasal wash.

Example 9 This Example illustrates elicitation of protection against intratracheal challenge by intranasal immunization with PspA.

Whole-length PspA was recovered from S. pneumoniae R36A strain (which provides the same PspA as the Rxl strain). The strain was grown in 100 Al chemicallydefined medium (Rijn et al, Infect. Immun. 1990, vol.

27, pp 444-448), except that the medium contained 0.03% choline chloride. The bacteria were harvested in late log phase (about 5x10 7 CFU/ml) and washed five times with ml of saline followed by centrifugation at 2000xg for minutes. With each wash, the bacteria was saved and the supernatant discarded. The washed cells then were eluted with 5 ml of 2% choline chloride and the eluted material was shown to contain PspA by dot blot using monoclonal antibody XiR 278.

An identical procedure was carried out on the S.

pneumoniae strain WG44.1 (McDaniel et al which does not produce PspA because of the absence of an upstream portion of the pspA gene. This material provided a control in that the preparation should contain the same g general, impurities that might be in the extract from R36A. The material recovered from the washed WG44.1 25 cells by elution with 2% choline chloride did not contain detectable PspA by dot blot, as expected.

For the purpose of administration, the PspA preparation from R36A was diluted 1:2. 12 .l of the Usolution contained 5 pg of added cholera toxin B subunit 30 (CTB) as an adjuvant and was instilled into the nose of each BALB/cJ mouse. Thirty-two and forty-two days later the immunization was repeated in an identical manner. As a control, some mice were immunized with an identical preparation from the WG44.1 S.pneumoniae strain. A final group was left unimmunized. Seven days after the third dose, the immunized mice were challenged with 2x10 6

CFU

(100xLD 50 of A66 S. pneumoniae intratracheally.

Activity assays also were performed on sera obtained seven days after the third immunization on separate groups of mice immunized by protocols almost identical to those noted above.

The results obtained are set forth in the following Tables X and XI: TABLE X Protection against challenge with 106 A66 after i.n. or s.c.

immunization with 150 tsg doses of PspA Antigen Immunization Challenge Median P value Route Route Alive day of vs.

death matched control -PspA CTB i.n. i.t. 100 >16 <0.0001 PspA only i.n. i.t. 25 4 PspA- CTB i.n. i.t. 11 4 CTB i.n. i.t. 0 4 None i.t. 0 4 PspA 'CFA s.c. i.t. 80 16 0.02 CFA s.c. i.t. 0 4 *9.

9 d.

9 9*9* 9* 0* a...r a 9* P values calculated by the Fisher exact test.

TABLE XI Intranasal Immunization with Ril PspA and Intratracheal Challenge with Capsular Type 3 Strain A66 Immunogen Adjuvant IgG anti- Challenge CFU/ml Day of* PspA CFU of at day 3* Death (jg/ml) A66_ FL PspA CTB 2 x1Hr 102 1 (R36A) of17.8 of<102 12 +o 3.3 to<102 12 102 >12 PspA- CTB 2 x10 6 2 x10' 3 (WG44. 1) 0.4 of4 x10 4 to of2 x10' 4 Saline Saline .2 x 106 N.D. 4 oof.<0.4 toN.D. 4 tto2 x10' N.D. 4 ofN.D. **FL-PspA vs. Saline (or WG44. 1) at P <.005 *Results with individual mice As can be seen in Table X, 24 hours after infection the unimmunized' and the mock immunized mice had high levels of pneumococci in their blood. These mice all died on day 3 post challenge. The mice immunized with PspA exhibited no detectable pneumococci on day 3 and all survived infection. Although the data clearly indicate 4

C.

C

C

C C

C.

C C C C that i.n. immunization with PspA can protect against pulmonary challenge, the strains of S. pneumoniae used, survives well in blood and causes rapid death in mice when injected i.v. (LDs 0 102). Thus, the protection against death may have been due to protection against sepsis as well as pulmonary infection protection had been elicited in the lung.

Example Mucosal immunization with R36A PspA can provide protection against intratracheal and systemic (intravenous) challenge with capsular type 3 A66 Streptococcus pneumoniae.

BALB mice were immunized i.n. three times with 150ng of PspA at 10 day intervals. With each injection they also received 5gg of purified CTB. The last immunization was given in saline. Control mice received the two CTB injections but no PspA. Four weeks after the last injection the mice were challenged with strain A66 type 3 S. pneumoniae by the i.t. or i.v. route at the doses indicated in Table XII. We observed that i.n.

immunization could protect against systemic infections by both the i.t. and i.v. route of challenge. In the case of the i.t. route, the immunization protected against the fatal effects of pulmonary infections and probably also 25 sepsis. In the case of the i.v. infection where the pneumococci are injected directly into the blood, the immunization protected against sepsis.

o 9* a.oa a a .5 4. 5 4 5 *5 Sfl~ a 4. *4 5* *5 4 5 5* *4 6 4 6 4 4 0 5 4 *b0 *44S*e*~ ge TABLE XII Intranasal immunization with R36A PspA can protect against systemic infection with S. pneumoniae.

Challenge Days to Death Survival Route Log Immunogen Individual Median P vs. Alive: P vs.

Dose control 1 Dead control 2 5.1 PspA CTB >21, >21, >21, >21, >21 5 :0- CTB 3,3,4,4,4,5,6, >21, 4.5 0.02 3 :7 0.02 >21, >21 ixv. 4.9 PspA CTB >21, >21, >21, >21, >21 0.045 5 :0 0.042 CTB 4,4,4,4,5, 5, >21, 5 4:6 >21, >21, >21 '6 1 calculated by one tailed Wilcoxon two sample rank test.

2 calculated by one tailed Fisher exact test.

SUMMARY OF THE DISCLOSURE In summary of this disclosure, the present invention provides a method of preventing colonization of pneumococci in a host and for protecting against systemic infection by pneumococci, by mucosal, particularly intranasal (intranasopharyngeal), administration of PspA in various forms. Modifications are possible within the scope of the invention.

*S

0* 29

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Claims (10)

1. A substance or composition for use in a method of immunization of a host against pneumococcal infection wherein the method includes mucosally administering to the host an immunizing amount of the substance or composition, wherein said substance or composition includes pneumococcal surface protein A (PspA) or at least one fragment of such PspA containing at least one protection-eliciting epitope and wherein said PspA is in the form of killed whole pneumococci or of a lysate of pneumococci.

2. A substance or composition for use in a method of immunization according to claim 1 wherein said PspA is in the form of an isolated and purified PspA.

3. A substance or composition for use in a method of immunization according to claim 1 wherein said PspA is administered in conjunction with an adjuvant.

4. A substance or composition for use in a method of immunization according to claim 3 wherein the adjuvant is the B subunit of cholera toxin. A substance or composition for use in a method of immunization according to claim 1 wherein said mucosal administration is effected 20 intranasally to provide protection to the host against both pneumococcal colonization and systemic infection.

6. A substance or composition for use in a method of immunization according to claim 1 wherein said mucosal administration is effected intranasally to provide protection to the host against pulmonary infection. 25 7. A substance or composition for use in a method of immunization 0 according to claim 1 wherein said mucosal administration is effected intranasally to provide protection to the host against infection starting as a pulmonary infection.

8. A substance or composition for use in a method of immunization of a host against colonization with S. pneumoniae in the nasopharynx wherein the method comprises intranasally administering to the host an immunizing amount Sof said substance or composition, wherein said substance or composition r comprises S. pneumoniae pneumococcal surface protein A (PspA) in the form WINNIEPJC\EisabethTPJC\SPECI 14226-99aDOC 18/04 '01 WED 15:16 FAX 613 9622 2294 PHILLIPS ORMONDE IP COMMISSIONER [a004 LA.c-CMJr" 37 of a killed whole pneumococci, a lysate of pneumococci or an isolated and purified PspA or an immunogenic fragment thereof.

9. A substance or composition for use in a method of immunization according to claim 8 wherein said PspA is administered intranasally in conjunction with an adjuvanting amount of the B subunit of cholera toxin (CTB). A substance or composition for use in a method of immunization according to claim 8 for the prevention of carriage of pneumococci and prevention of pneumococcal disease.

11. A substance or composition for use in a method of immunization according to claim 8 for the prevention of pulmonary infection and pneumococcal disease.

12. A substance or composition for use in a method of immunization according to claim 8 for the prevention of carriage of pneumococci and prevention of pneumococcal disease originating from pulmonary infection. S. 15 13. A substance or composition for use in a method of immunization according to claim 1 substantially as herein described. se* S14. A use of pneumococcal surface protein A (PspA) or at least one fragment of such PspA containing at least one protection-eliciting epitope, substantially as herein described.

15. Use of pneumococcal surface protein A (PspA) in the form of a killed whole pneumococci, a lysate of pneumococci or an isolated and purified PspA or immunogenic fragment thereof, substantially as herein described. DATED: 18 April, 2001 PHILLIPS ORMONDE FITZPATRICK Attorneys for: UAB RESEARCH FOUNDATION S:;AL. r .l .OC 18/04 '01 WED 14:12 [TX/RX NO 9179]