WO2019157188A2 - Compositions and methods for cross-protection against pneumococcal disease - Google Patents

Abstract

Disclosed are compositions comprising one or more nonpneumococcal commensal organisms that express a pneumococcal-specific serotype for administration a subject in need thereof, according to embodiments of the invention. Also disclosed are kits comprising the inventive compositions according to embodiments of the invention. Also disclosed are methods for immunizing a host against infection comprising administering an effective amount of the inventive compositions, according to embodiments of the invention.

Description

COMPOSITIONS AND METHODS FOR CROSS-PROTECTION AGAINST

PNEUMOCOCCAL DISEASE

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62/627602, filed February 7, 2018, and U.S. Provisional Patent Application No. 62/769884, filed November 20, 2018, the disclosures of which are incorporated by reference in their entirety.

STATEMENT REGARDING

FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with Government support under Grant Number CDC- RFA-CK17-1701, awarded by the U.S. Centers for Disease Control and Prevention. The Government has certain rights in this invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED

ELECTRONICALLY

[0003] Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 13,604,250 bytes Byte ASCII (Text) file named "74l8l4_ST25.txt," created on February 7, 2019.

BACKGROUND OF THE INVENTION

[0004] Streptococcus pneumoniae, or pneumococcus, the type of invasive bacterium that causes pneumococcal disease, is a major cause of morbidity and mortality globally. In some cases, S. pneumoniae can harmlessly colonize the respiratory tract, sinuses, and nasal cavity of healthy carriers. However, in susceptible individuals, such as young children, the elderly, or immunocompromised individuals, the bacterium can become pathogenic. Pneumococcal infections can range from ear and sinus infections to life threatening illnesses such as pneumonia and meningitis. Similar to other major invasive bacterial pathogens, S.

pneumoniae are encapsulated with a polysaccharide layer that lies outside the cell envelope. Such polysaccharide capsules protect against phagocytosis by the host, provide antigenic variation, and facilitate virulence. Encapsulated species typically express one of a number of immunochemically distinct capsular polysaccharides that define serotypes. Presently, there are over 90 known serotypes of S. pneumoniae, of which only a minority produce the majority of pneumococcal infections.

[0005] Currently available pneumococcal vaccines contain a mixture of capsular polysaccharides of multiple pneumococcal serotypes. When successful, these pneumococcal vaccines induce the production of antibodies to specific capsular polysaccharides in the host, which can opsonize the bacteria in a serotype-specific manner, leading to complement- dependent phagocytosis. Exposure to a specific capsular polysaccharide(s) and the production of antibodies specific for the capsular serotype can provide protection against future infection from pneumococci expressing the same capsular serotype(s). One currently marketed pneumococcal vaccine contains capsular polysaccharides from twenty-three commonly found serotypes (PPSV23 or PNEUMOVAX 23^®). Another currently marketed pneumococcal vaccine contains capsular polysaccharides of thirteen serotypes that are conjugated to a non-immunogenic and non-toxic protein (PCV13 or PREVNAR 13^®).

[0006] However, currently available pneumococcal vaccines suffer from drawbacks. For example, host immune responses can be inconsistent among all serotypes provided in a vaccine. Additionally, certain populations do not respond to vaccine antigens. Furthermore, although certain serotypes are rare in the United States, such as serotype 1, in other countries, serotype 1 has been the leading cause of invasive pneumococcal disease. Additionally, serotype 1 has the potential to cause large outbreaks and epidemics even in countries that have already introduced PCV13. Accordingly, the prevention and treatment of

pneumococcal disease both inside the United States and internationally continues to be a challenge, such that there is a need for improved compositions and methods for the prevention and treatment of pneumococcal disease.

BRIEF SUMMARY OF THE INVENTION

[0007] An embodiment of the invention provides a composition comprising (a) a pharmaceutically acceptable carrier and (b) one or more nonpneumococcal commensal organisms, wherein the commensal organisms are (i) Streptococcus mitis (ii) Streptococcus oralis, or (iii) Streptococcus infantis, and wherein the commensal organisms express a pneumococcal-specific serotype for administration to a subject in need thereof. [0008] An embodiment of the invention provides a kit for administering an immunogenic composition, the kit comprising: a composition comprising (a) a pharmaceutically acceptable carrier and (b) one or more nonpneumococcal commensal organisms, wherein the commensal organisms are (i) Streptococcus mitis (ii) Streptococcus oralis, or (iii) Streptococcus infantis, and wherein the commensal organisms express a pneumococcal-specific serotype, for administration to a subject in need thereof, wherein the immunogenic composition comprises a vaccine or a probiotic, and (c) at least one container for holding the immunogenic composition.

[0009] Further embodiments of the invention provide methods for immunizing a host against infection from pneumococcal disease comprising administering an effective amount of the inventive composition.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0010] Figure 1 is a schematic showing a comparison of 2 cpsl-L polysaccharide synthetic cluster regions from A mitis strains L6/L164 (16,613 bp) (SEQ ID NO: 1)

,Ll 15/Ll 16 (l6,6l7bp) (SEQ ID NO: 2) and Ll 21 (16,626 bp) (SEQ ID NO: 3) with the corresponding pneumococcal cpsl sequence. White rectangles represent open reading frames not shared between the species. The top diagram depicts ranges of sequence identity between pneumococcal cpsl genes from GenBank accession CR931648 (SEQ ID NO: 9) and the S. mitis counterparts depicted on the below diagram. Ranges of sequence identity between the S. mitis alleles are given below the S. mitis cpsl diagram. L6 and L164 shared identical 16613 sequence. L115 and L116 shared identical l66l7bp sequence. The connecting lines depict the alignment of the polysaccharide synthesis clusters. Black regions in the pneumococcal diagram depict remnants of transposase genes. Two inactive pneumococcal genes, (aliB and rmlD, the latter of which renders the rhamnose biosynthesis cluster inactive) containing frameshift mutations are indicated, as well as the partial 5’ and 3’ ends of the dexB and aliA genes in both species. The region detected through use of PCR assays to first detect the presence of these strains in upper respiratory specimens is depicted by arrows above the wzy gene.

[0011] Figure 2 is a schematic showing a comparison of cps4-L polysaccharide synthetic cluster regions from A infantis strain GA0067 (SEQ ID NO: 4) with the corresponding pneumococcal cps4 reference sequence (GenBank accession CR931635: SEQ ID NO. 10). The 3’ ends of the pneumococcal and S. infantis dexB genes are shown upstream of both regions. The downstream regions are flanked by aliA and ftsA in S. pneumoniae and in S. infantis, respectively. The percent sequence identity of the polysaccharide synthetic genes relative to the pneumococcal homologs are shown below the depiction of the S. infantis cps4- L region. The connecting lines depict the alignment of the polysaccharide synthesis genes. Black regions depict remnants of transposase genes. The 2 PCR primers used to first detect the presence of the strains in upper respiratory specimens are depicted by arrows on the S. infantis counterpart (bottom), with the number of nucleotide matches to the wzy sequence shown for each.

[0012] Figure 3 is a schematic showing a comparison of cps9 polysaccharide synthetic cluster regions from S. infantis (SEQ ID NO: 6) with the corresponding pneumococcal cps9V reference sequence (GenBank accession CR931648, SEQ ID NO: 11). The percent sequence identity of the polysaccharide synthetic genes relative to the pneumococcal homologs are shown below the depiction of the S. infantis cps9 region. The connecting lines depict the alignment of the polysaccharide synthesis genes. The 3’ ends of the pneumococcal and S. infantis dexB genes are shown upstream of both regions. The downstream regions are flanked by aliA and ftsA in S. pneumoniae and in S. infantis, respectively. Black regions depict remnants of transposase genes. The 2 PCR primers used to first detect the presence of the strains in upper respiratory specimens are depicted by arrows, with the number of nucleotide matches to the wzy sequence shown for each.

[0013] Figure 4 is a schematic showing the phylogenetic tree of Streptococcal strains and the closest pneumococcal and nonpneumococcal species.

[0014] Figure 5 is a bar graph showing the results of competitive absorption testing of S. mitis and serotype 1 S. pneumoniae isolates for capsular homology. In the figure,“SM” refers to S. mitis ST1;“Pnc” refers to S. pneumoniae ST1; and“SM ATCC” refers to S. mitis ATCC confirmed to be cpsl -negative. The antibody source for this assay was rabbit ST1 pneumococcal antiserum. Absorption studies clearly demonstrated the homology between the pneumococcal and non-pneumococcal capsule.

[0015] Figure 6 is a bar graph showing S. mitis capsule reaction specificity to rabbit pneumococcal anti-serum. In the figure,“SM” refers to S. mitis ST1;“Pnc” refers to S. pneumoniae ST1; and“SM ATCC” refers to S. mitis ATCC confirmed to be cpsl -negative.

[0016] Figure 7 is a bar graph showing the results of an opsonophagocytosis assay of two distinct S. mitis serotype 1 clones. Serotype 1 S. pneumoniae was the positive control; S. mitis ATCC strain confirmed to be cpsl -negative was the negative control. The antibody source for OPK assay was 007 S. pneumoniae human reference serum and baby rabbit complement (Pelfreez) was used. The Y axis in the figure is the OPK titer. OPK titer is defined as reciprocal of serum dilution with > 50% growth compared to serum free complement control. (S. mitis strain L006 and L121 representing two distinct S. mitis clones tested). The X axis is the serotype.

[0017] Figure 8 is a graph showing the results of an opsonophagocytosis assay of S. mitis clones and pneumococcal isolates of various serotypes. The antibody source for the assay was 007 SP human pneumococcal reference serum and baby rabbit complement (Pelfreez) was used. The OPK titer (y-axis) is defined as the reciprocal of serum dilution with >50% growth compared to serum complement control (N > 5). The x-axis is the serotype and species.

[0018] Figure 9 is a photograph showing the results of a Quellung reaction performed on the S. mitis cpsl isolates to assess capsule expression. The assay verified serotype 1 capsule expression.

[0019] Figure 10 is a photograph showing the results of a double immunodiffusion assay testing cross-reactivity of serotype 1 S. mitis and S. pneumoniae capsule extracts with serotype 1 S. pneumoniae anti-serum. S. Mitis L006 strain used in reaction. In the figure, “Blank” = wells with no bacteria, and“1 AS” = serotype 1 S. pneumoniae anti-serum. The assay verified serotype 1 capsule expression. The image demonstrates the cross reactivity between antibodies against pneumoccal serotype 1 capsule and S. mitis serotype 1 capsule.

[0020] Figure 11 is a schematic showing a comparison of the pneumococcal cps9V reference sequence (GenBank accession CR931648: SEQ ID NO. 11) with polysaccharide synthetic cluster regions from non-pneumococcal strains S. infantis GA64 (SEQ ID NO: 6) and S. oralis GA3m (SEQ ID NO: 7). The percent sequence identity of the polysaccharide synthetic genes relative to the pneumococcal homologs are shown below each non- pneumococcal cps operon (S. infantis and S. oralis). The connecting lines depict the polysaccharide synthesis genes that share the same order in all 3 strains. The 3’ ends of the dexB genes are shown upstream of all 3 strains. The downstream region is flanked by aliA in S. pneumoniae and strain GA3m, while GA64 lacks aliA and has the ftsA gene at the downstream end. Black regions depict remnants of transposase genes. The qPCR-positive region that revealed the presence of the strains in upper respiratory specimens is depicted by a black line (coordinates 14489-14642 for strain GA3m and 14787-14940 for strain GA64) above the wzx genes. [0021] Figures 12A-12C are gene alignments of cps5 operons from 5 strains of non- pneumococcal species S. mitis strain 67013 (SEQ ID NO: 29) (Figure 12A), S. oralis strains US0049 (SEQ ID NO: 31) and F0392 (SEQ ID NO: 30) (Figure 12B), and & infantis strains US969j (SEQ ID NO: 32) and US0024 (SEQ ID NO: 33) (Figure 12C). The percent sequence identity of the entire biosynthetic cluster (wzg through fnlC) with the pneumococcal serotype 5 reference sequence (GenBank accession CR931637, SEQ ID NO: 34) is underlined at left. The percent identity of the allele over its entire overlap with the pneumococcal reference is indicated within each rectangle representing the indicated gene. For the S. oralis and S. infantis alignments that each depict a pair of distinct strains, each gene has the indicated conserved translational start and/or end. S. mitis 67013 is the only strain showing close linkage of the cps5 locus with upstream pbp2x and downstream pbpla, while the two S. oralis cps5 loci he 2 - 17 kb upstream of pbpla. The position of conventional (c) and real time (rt) serotype 5 detection assays are indicated (c assay positive for all 4 strains tested, rt assay positive for all but S. mitis US67013). The 5 cps5 genes that appear entirely serotype 5-specific (< 54% identical to all other known pneumococcal cps genes) are indicated by checkered pattern. Phylogenetic analysis shows the relative relatedness of the 5 serotype 5 specific genes between the 4 species. Gene functions listed at bottom left are taken from accession CR931637. Black rectangles indicate transposase gene remnants. The orientations of pbp2x and pbpla relative to cps5 are indicated at the right and left end, respectively, of each cps5 operon.

[0022] Figure 13 is a photograph of an immunodiffusion experiment showing reactivity of typing antisera raised against serotype 5 pneumococcal strain Ambrose (middle well) against wzy5-positive strains of S. infantis, S. oralis, S. mitis, and serotype 5 S. pneumoniae Ambrose. A serotype 4 pneumococcal strain is included as a negative control.

[0023] Figures 14A-14C are photographs of immunodiffusion experiments showing reactivity of pneumococcal typing antisera against various non-pneumococcal strains.

[0024] Figure 14A shows the reactivity of pneumococcal typing antisera against S. mitis 67013 (top, center well contains type 5 antisera; wells 1 and 2 contain type 5 pneumococcal extract. Wells 3 and 4 contain strain KE67013 extract.

[0025] Figure 14B shows the reactivity of indicated dilutions of pneumococcal typing antisera (peripheral wells) against S. mitis KE67013 in the center well. [0026] Figure 14C shows the reactivity of indicated dilutions of pneumococcal typing antisera (peripheral wells) against and serotype 5 S. pneumoniae strain Ambrose extracts in the center well.

[0027] Figure 15A is a photograph showing a positive Quellung reaction of serotype 5 pneumococcal strain Ambrose when reacted with antisera raised against wzy5-positive S. mitis strain KE67013.

[0028] Figure 15B is a photograph showing a positive Quellung reaction of serotype 5 pneumococcal strain Ambrose when reacted with standard pneumococcal serotype 5 typing antiserum.

[0029] Figure 15C is a photograph showing a negative Quellung reaction of serotype 5 pneumococcal strain Ambrose reacted against antisera prepared against an S. mitis strain containing a cps operon unrelated to cps5.

[0030] Figure 16 is a graph showing the opsonophagocytic killing activity of rabbit antisera raised against serotype 5 S. mitis KE67013 in 3 separate rabbits (labeled 1 - 3) and of pooled, clarified antisera from the three rabbits (pooled). GMT values of titers across 5-6 assay runs are shown with >50% killing compared with the growth in the complement control wells. Initial dilution was 1:400 with subsequent 2-fold dilutions down to 1:51200. Testing of the rabbit antiserum generated against serotype 5 S. pneumoniae gave optimal titer at 1 :960 (not shown).

[0031] Figures 17-20 are gene alignments showing general features of additional homologs of cps loci from non-pneumococcal species (SO = S. oralis ; SM = S. mitis) compared to pneumococci of known capsular serotypes. The genes aligned by the slanted lines indicate the polysaccharide synthetic gene cluster. Within each gene of the cluster, the percent identity between the two homologs is shown. In each cps operon shown the gene cluster lies between dexB and aliA. White rectangles indicate open reading frames (ORFs) that lack homology with the respective pneumococcal reference sequence. The checkered open reading frames share > 60% sequence identity only with pneumococcal strains within small serogroups 12F/12A/12B/44/46, 15A/15F, 18C/18A/18B/18F, and 33F/33A/37. Black rectangles do not represent open reading frames, but have spurious homology to transposase structural genes.

[0032] Figure 17 is a gene sequence comparison of S. oralis KE67213 cps 12

polysaccharide biosynthetic locus (bottom) with the 17 gene pneumococcal cpsl2F (top). KE67213 has a single ORF (bases 12726-14978) that corresponds to the distinct pneumococcal ORFs wcxD and wcxE as indicated. Otherwise, the two polysaccharides share exactly the same gene order.

[0033] Figure 18 is a gene sequence comparison of S. oralis KE66813 polysaccharide biosynthetic gene cluster (bottom) with the 16 gene pneumococcal cpsl5A (top). The wciZ contains frameshift and is lacking altogether in strain KE66813. Although KE66813 contains a gene with homology to wchX that encodes a putative glycerol phosphotransferase, strain KE66813 lacks the three glycerol-2 -phosphate synthesis genes (gtpl, gtp2, gtp3).

[0034] Figure 19 is a gene sequence comparison of S. mitis KE667-13 (“cpsl8”) polysaccharide biosynthetic locus (bottom) with pneumococcal cpsl8C (GenBank accession CR931673) (top). The asterisk for the pneumococcal glf gene indicates that it is a pseudogene that contains several stop codons, even though it is of the same exact length as its highly homologous S. mitis counterpart that contains no stop codons. The open reading frame ORF1 is highly homologous to an open reading frame in S. mitis SK667 (GenBank accession JPFV01000015) while ORF2 has limited homology to wcyF (unknown function) within the pneumococcal cps25A, cps25F, and cps38 operons.

[0035] Figure 20 is a gene sequence comparison of S. oralis KE66913 (“cps33”) polysaccharide biosynthetic gene cluster (ORFs wzg through glf) (bottom) with

pneumococcal cps33F (GenBank accession CR931702) (top). The open reading frame wycO (putative acetyltransferase) has indicated homology to the cps2l homolog. Two full-length aliB (oligopeptide-binding protein) genes are indicated.

[0036] Figure 21 presents photographs showing the results of a double immunodiffusion assay testing cross-reactivity of serotype 1 S. mitis and S. pneumoniae capsule extracts with serotype 1 S. pneumoniae anti-serum. Reaction 1 demonstrates the cross-reactivity of serotype 1 S. mitis and S. pneumoniae capsule extracts with serotype 1 S. pneumoniae typing anti-serum. Reaction 2 is a control reaction using serotype 1 S. mitis and serotype 1 and non serotype 1 S. pneumoniae extracts with serotype 5. S. mitis L006 strain was used in the assay. In the figure,“Blank” refers to wells with no bacteria;“1AS” refers to serotype 1 S. pneumoniae anti-serum;“Spn stl” refers to S. pneumoniae serotype 1;“5 AS” refers to serotype 5 S. pneumoniae anti-serum;“Spn st5” refers to S. pneumoniae serotype 5.

DETAILED DESCRIPTION OF THE INVENTION

[0037] An embodiment of the invention provides a composition comprising (a) a pharmaceutically acceptable carrier and (b) one or more nonpneumococcal commensal organisms, wherein the commensal organisms are (i) Streptococcus mitis (ii) Streptococcus oralis, or (iii) Streptococcus infantis, and wherein the commensal organisms express a pneumococcal-specific serotype for administration to a subject in need thereof.

Embodiments of the invention can provide any one or more of a variety of advantages. For example, the inventive composition can be useful for treating or preventing a number of conditions caused by pneumococcal organisms in a host, for example, ear infections, pneumonia, meningitis, and bacteremia. Alternatively, or additionally, the inventive methods may provide inventive diagnostic assays for laboratory and commercial use.

[0038] The one or more nonpneumococcal commensal organisms can be combined with a pharmaceutically acceptable carrier and formulated into compositions. In an embodiment, the compositions are pharmaceutical compositions, such as immunogenic compositions (e.g., vaccine formulations or probiotics). The pharmaceutical compositions provided herein can be in any form that allows for the composition to be administered to a subject in need thereof. In a specific embodiment, the pharmaceutical compositions are suitable for human administration.

[0039] The pharmaceutically acceptable carrier for use in the inventive pharmaceutical composition can be any of those conventionally used and is limited only by chemico-physical considerations, such as solubility, and by the route of administration. The pharmaceutically acceptable carriers for use in the present invention - for example, vehicles, excipients, and diluents - are well-known to those skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert to the active agent(s) (i.e., the one or more nonpneumococcal commensal organisms) and one which has no detrimental side effects or toxicity under the conditions of use. The choice of carrier will be determined in part by the particular compounds used in the pharmaceutical composition, as well as by the particular method used to administer the inventive

composition.

[0040] As used herein, the term“commensal organisms” means normal, indigenous microorganisms or microflora which are present on body surfaces covered by epithelial cells such as the gastrointestinal and respiratory tracts. Commensal bacteria co-evolved with their hosts and are thus usually harmless. However, in some cases they may overcome protective host responses and exert pathologic effects. Commensal organisms which are suitable for inclusion in the inventive composition include any nonpneumococcal commensal organisms that express a pneumococcal-specific serotype. In a preferred embodiment, commensal organisms suitable for use in the inventive composition include Streptococcus mitis, Streptococcus oralis and Streptococcus infantis. S. mitis, S. oralis, and S. infantis are commensal organisms of the mucous membranes, which have been isolated from the human oral cavity (Facklam,“What happened to the Streptococci: Overview of Taxonomic and Nomenclature Changes, Clinical Microbiology Reviews, Vol. 15, No. 4: 613-630 (2002) (incorporated herein in its entirety by reference)). S. pneumoniae is closely related to Streptococcus mitis and Streptococcus oralis, sharing >99% homology by 16S rRNA gene analysis (Kawamura et al.“Determination of 16S fRNA Sequences of Streptococcus mitis and Streptococcus gordonii and Phylogenetic Relationships among Members of the Genus Streptococcus. International Journal of Systemic Bacteriology, Vol. 45, No. 2: 406-408 (1995) (incorporated herein in its entirety by reference)).

[0041] In embodiments, the one or more commensal organisms can be“purified” or “isolated.” As used herein, the term“purified” means that the organism, subunits of the organism, or protein antigens of the organism that have been distilled from large amounts of the organism wherein contaminants and undesirable biological elements have been removed in the laboratory prior to incorporation into the inventive composition. As used herein, the term“isolated” means that the organism, subunits of the organism, or protein antigens of the organism have been removed from a natural source, e.g., cells. The terms“purified” and “isolated” can additionally refer to an organism, subunits of the organism, or protein antigens of the organism which are substantially free of contaminating materials from the natural source, e.g., soil particles, minerals, chemicals from the environment, and/or cellular materials from the natural source, such as but not limited to cell debris, cell wall materials, membranes, organelles, the bulk of the nucleic acids, carbohydrates, proteins, and/or lipids present in cells.

[0042] In an embodiment, the inventive composition can comprise a vaccine. As used herein,“vaccine” means an immunogenic composition prepared from small amounts of weakened or inactivated organisms or toxins that can cause disease. In general, there are several types of vaccines which can be employed in connection with the invention for inducing a specific neutralizing antibody against a specific bacterial antigen, (i.e., the polysaccharide capsule) including live, inactivated, and subunit vaccines.

[0043] In an embodiment, the inventive composition can comprise a live commensal organism or an inactivated commensal organism. Vaccines which incorporate live or inactivated organisms include live vaccines and inactivated vaccines described below. Live vaccines

[0044] Live vaccines incorporate live, disease-causing microorganisms which have been isolated and/or purified under laboratory conditions. Such vaccines replicate in a vaccinated host to produce an immune response.

Inactivated vaccines

[0045] “Inactivated vaccines” refers to a vaccine that includes one or more

microorganisms that have been killed through physical (e.g., heat or radiation) or chemical processes. Inactivated vaccines have low residual infectivity following inactivation.

Subunit vaccines

[0046] In an embodiment, the inventive composition can comprise a purified capsular polysaccharide of a commensal organism or a segment of the purified capsular

polysaccharide of the commensal organism. Vaccines which incorporate segments of organisms include subunit vaccines.

[0047] “Subunit vaccine” refers to a vaccine that includes one or more antigen components necessary to elicit a protective immune response, but not a complete organism, such as immunogenic epitopes, proteins, polysaccharides, antigen fusion proteins, or protein fragments. Subunit vaccines, as used herein, can be monovalent (comprise a single antigen) or multivalent (comprise more than one antigen component). Subunit vaccines include conjugate vaccines. “Conjugate vaccine” refers to a vaccine comprising an antigenic component covalently attached to a carrier protein.

[0048] In embodiments, the inventive composition can comprise any or all of the vaccine formulation types described herein.

[0049] In an embodiment, the composition can be a probiotic formulation comprising the commensal organisms described herein. As used herein, the term“probiotic” means a formulation containing microorganisms in sufficient numbers, which alter the microflora in the host and thereby exert beneficial health effects in the host. The probiotic formulations can take the form of any pharmaceutical-type composition (e.g., capsule, tablets, liquid, aerosol, etc.) or in the form of a food supplement. In embodiments, the invention provides a probiotic formulation in oil-containing soft-gels or capsules (which also can be considered “excipients” or“carriers” as used herein with reference to the inventive composition). In other embodiments, the probiotic can be provided in a kit which includes a dose of the probiotic in a container and at least one separate sterile container of diluent.

[0050] The commensal organisms (described herein with respect to other aspects of the invention) can be purified or isolated prior to incorporation into the inventive probiotic formulation. In embodiments, the inventive probiotic can be suitable for human

consumption. In an embodiment, the commensal organisms can comprise between about 0.01% to about 50% by weight of the probiotic formulation; between about 0.01% and about 10% by weight of the probiotic formulation; or between about 0.01% and about 5% by weight of the probiotic formulation, e.g., about 1%, about 2%, about 3%, about 4%, or about 5% by weight of probiotic.

[0051] In embodiments, the microbial make-up of the inventive probiotic formulation comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or any ranges therein (e.g., 1-4, 5-10, 8-20, etc.) of strains and/or species of microbes. In some

embodiments, fewer than 50 microbial strains (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, or any ranges therein (e.g., 1-4, 5-10, 8-20, etc.) are at least 50% (e.g., 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%. 99.9%,

99.99%) of the microbial population (e.g., by mass, by colony forming units (CFU), etc.) of the probiotic formulation. In some embodiments, the bacterial formulation comprises at least 5x10⁶ CFU (e.g., 5xl0⁶ CFU, lxlO.x⁷ CFU, 2xl0⁷ CFU, 5xl0⁷ CFU, lxlOx⁸ CFU, 2xl0⁸ CFU, 5xl0⁸CFU, lxlO⁹ CFU, 2xl0⁹ CFU, 5xl0⁹ CFU, lxl0¹⁰ CFU, 2xl0¹⁰ CFU, 5xl0¹⁰ CFU, lxlO¹¹ CFU, 2xlOⁿ CFU, 5xl0ⁿ CFU, lxlO¹² CFU, 2xl0¹² CFU, 5xl0¹² CFU, or more or ranges there between) of nonpneumococcal commensal organisms. In an embodiment, the probiotic formulation can be administered prophylactically. In another embodiment, the probiotic formulation can be administered therapeutically. These examples are not limiting.

[0052] In additional embodiments, the invention provides a diagnostic assay. In embodiments, the diagnostic assay may comprise a PCR-based diagnostic assay. In an embodiment, the invention provides a PCR-based diagnostic assay for detecting non pneumococcal commensal organisms of a particular serotype in a biological sample.

[0053] In embodiments, live organisms suitable for inclusion in the inventive composition include any nonpneumococcal commensal organism which expresses a pneumococcal specific serotype, for example, one or all of S. mitis, S. oralis, or S. infantis.

In embodiments, purified capsular polysaccharides suitable for inclusion in the inventive method include, for example, purified capsular polysaccharides of one or all of S. mitis, S. oralis, or S. infantis. Extracting and purifying suitable capsular polysaccharides can be performed by any methods known in the art. Traditionally, the purification process for bacterial capsular polysaccharides include precipitation with solvents (e.g., ethanol and phenol) and detergents, and separation of solids by centrifugation.

[0054] Suitable capsular polysaccharides can be identified by, for example, the Quellung reaction, among other methods. The Quellung reaction involves testing a pneumococcal cell suspension with pooled and specific antisera directed against the capsular polysaccharide.

The antigen-antibody reactions are observed microscopically. The protocol has three main steps: 1) preparation of a bacterial cell suspension, 2) mixing of cells and antisera on a glass slide, and 3) reading the Quellung reaction using a microscope (Habib et al,” Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction,” J. Vis. Exp., vol. 84: 51208 (2014) (Incorporated by reference in its entirety)). Additionally, methods for detecting serotype genes include presumptive serotype deduction using conventional multiplex PCR (cmPCR) or genetic sequencing.

[0055] As noted, the S. mitis, S. oralis, and S. infantis organisms for inclusion in the compositions and methods of the present invention express a“pneumococcal serotype.” In the context of the present invention“pneumococcal serotype” refers to the distinct polysaccharide capsule type which surrounds the pneumococcal bacteria and protects it against ingestion by phagocytosis. Over 90 distinct pneumococcal capsular types (serotypes) have been described (Geno, KA et al,“Pneumococcal capsules and their types: past, present, and Future,” Clinical Microbiology Review , 28:871-899 (2015)).

[0056] In one embodiment, the nonpneumococcal S. mitis or S. oralis organism or organisms for use in the present invention express pneumococcal-specific serotype 1. In alternative embodiments, the nonpneumococcal S. mitis, S. oralis, and S. infantis organism or organisms for use in the present invention express one of pneumococcal-specific serotypes 1, 4, 9V or 9A, or 18C. In other alternative embodiments, the nonpneumococcal S. mitis, S. oralis, and S. infantis organism or organisms for use in the present invention express one of pneumococcal-specific serotypes 12F, 15A, or 33F. In another embodiment, the

nonpneumococcal S. mitis, S. oralis, and S. infantis organism or organisms for use in the present invention express pneumococcal-specific serotype 5.

[0057] Within the context of the present invention, the pneumococcal-specific serotype expression by such S. mitis, S. oralis, and S. infantis organisms can be assessed by subjecting the S. mitis and/or S. oralis and/or S. infantis strains to methods previously only used for determining pneumococcal serotypes. As disclosed herein with respect to other aspects of the invention, a combination of the following methods, traditionally only used on pneumococcal organisms, have advantageously been used to determine capsular serotype expression, presence of cell wall polysaccharides, and pneumococcal genes in

nonpneumococcal organisms. Suitable methods are listed below:

(a) serotyping by Quellung reaction; antigen-antibody reactions are observed microscopically; (Austrian, R.,“The quellung reaction, a neglected microbiologic technique,” Mt. Sinai J. Med., 43(6):699-709 (1976) (Incorporated by reference in its entirety));

(b) immunographic testing for cell wall polysaccharides by the BinaxNow

pneumococcal antigen test; (c) polymerase chain reaction (qPCR) to detect the lytA gene (pneumococcal autolysin gene) (Carvalho et al,“Revisiting Pneumococcal Carriage by Use of Broth Enrichment and PCR Techniques for Enhanced Detection of Carriage and Serotypes,” J. Clin. Microb., Vol. 48, No. 5: 1611-1618 (2010)), which is herein incorporated by reference in its entirety); and

(c) multiplexed conventional PCR (cmPCR) to determine capsular serotype.

Thereafter, basic phylogenetic analysis for concatenated housekeeping genes was used to distinguish species. The results are summarized below in tables 2 and 3.

[0058] Additionally, latex agglutination followed by the Quellung reaction using pneumococcal typing antiserum of specific pneumococcal serotypes, i.e., serotypes 1, 4, 9V, and 18C can be used to assess nonpneumococcal capsule expression. Double

immunodiffusion assays using the same antiserum can be used to assess cross-reactivity of non-pneumococcal streptococci with serotype 1, 4, 9V, and 18C pneumococci (Sorensen et al,“Capsular Polysaccharide Expression in Commensal Streptococcus Species: Genetic and Antigenic Similarities to Streptococcus pneumoniae,” mBio, vol. 7, No. 6: eOl 844-16 (2016) (incorporated herein in its entirety by reference)). As a result of the assays, the positive identification of the S. mitis, S. oralis, or S. infantis organism as expressing pneumococcal- specific serotype can be verified. Finally, the opsonophagocytic killing assay OPK (Romero- Steiner et al,“Standardization of an opsonophagocytic assay for the measurement of functional antibody activity against Streptococcus pneumoniae using differentiated HL-60 cells,” Clin Diagn. Lab Immunol. 4(4):4l5-22 (1997) (Incorporated by reference in its entirety)) can be used to assess functional serotype activity against the non-pneumococcal streptococci strains expressing pneumococcal capsular polysaccharides. [0059] Further, whole genome sequencing can be performed on isolated nonpneumococcal strains using the method according to Metcalf et al.,“Using whole genome sequencing to identify resistance determinants and predict antimicrobial resistance phenotypes for year 2015 invasive pneumococcal disease isolates recovered in the United States,” Clin. Microb. Infect., 22: l002.elel002.e8 (2016) (Incorporated by reference in its entirety). Whole genome sequencing can be used to detect nonpneumococcal genes or gene clusters containing highly similar genes or gene clusters to the pneumococcal reference.

[0060] In an embodiment, the nonpneumococcal commensal organism of the inventive composition provides cross-protection against infection from Streptococcus pneumoniae. As used herein, the term“cross-protection” means that the protection against bacterial infection from a pneumococcal serotype is provided to the host by its prior inoculation with a nonpneumococcal organism expressing the same serotype. Without wishing to be bound, it is thought that the host would be protected against infection due to the host’s previous production of neutralizing antibodies against the specific capsular polysaccharide antigen.

[0061] Without being bound to a particular theory or mechanism it is thought that inclusion of commensal streptococci which express homologs of pneumococcal capsular biosynthetic genes in an immunogenic composition can induce cross-protective immunity from pneumococcal disease in the host. In particular, the S. mitis strains expressing the serotype 1 pneumococcal capsule colonizing the upper respiratory tracts of healthy adults in the United States might induce immunity against serotype 1 pneumococcal disease.

Similarly, the S. mitis strains expressing the serotype 5 pneumococcal capsule colonizing the upper respiratory tracts of healthy adults in the United States might induce immunity against serotype 5 pneumococcal disease. Similarly, without wishing to be bound by theory, it is thought that S. mitis, S. oralis, and S. infantis strains expressing pneumococcal-specific capsule serotypes in addition to serotype 1, might induce immunity against pneumococcal colonization or disease caused by those specific serotypes. For example, pneumococcal serotypes 4, 9V, 12F, 15A, 18C, and 33F are among the serotypes that lead to invasive pneumococcal infections, such as bacteremia and meningitis, as well as pneumonia and common upper respiratory tract infections including otitis media and sinusitis. In

embodiments, the inventive composition can comprise any one or more of the pneumococcal- specific capsule serotypes described herein. [0062] An embodiment of the invention provides a method for immunizing a host against infection from pneumococcal disease comprising administering an effective amount of the inventive composition.

[0063] As used herein, the term“effective amount” in the context of administering a therapy to a subject refers to the amount of a therapeutic composition which has a prophylactic and/or therapeutic effect(s). In embodiments, an“effective amount” in the context of administration of a therapy to a subject in need thereof refers to the amount of a therapy which is sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of an pneumococcal infection, disease or symptom associated therewith; (ii) reduce the duration of an pneumococcal infection, disease or symptom associated therewith; (iii) prevent the progression of a pneumococcal infection, disease or symptom associated therewith; (iv) cause regression of a pneumococcal infection, disease or symptom associated therewith; (v) prevent the development or onset of a pneumococcal infection, disease or symptom associated therewith; (vi) prevent the recurrence of a pneumococcal infection, disease or symptom associated therewith; (vii) reduce or prevent the spread of a pneumococcal infection from one cell to another cell, one tissue to another tissue, or one organ to another organ; (ix) prevent or reduce the spread of S.

pneumoniae from one subject to another subject; (x) reduce sequelae associated with a pneumococcal infection; (xi) reduce hospitalization of a subject; (xii) reduce hospitalization length; (xiii) increase the survival of a subject with a pneumococcal infection or disease associated therewith; (xiv) eliminate a pneumococcal infection or disease associated therewith; (xv) inhibit or reduce pneumococcal infection replication; (xvi) inhibit or reduce the entry of S. pneumoniae into a host cell(s); (xviii) inhibit or reduce replication of the S. pneumoniae genome; (xix) inhibit or reduce synthesis of S. pneumoniae proteins; (xx) inhibit or reduce assembly S. pneumoniae, (xxi) inhibit or reduce release of S. pneumoniae from a host cell(s); (xxii) reduce bacterial titer; (xxiii) enhance or improve the prophylactic or therapeutic effect of a treatment being concurrently administered; and/or (xxiv) prevent acquisition or reduce duration of S. pneumoniae colonization in the pharynx. It is within the level of skill in the art to determine if one or more of these effects has been achieved.

[0064] The immunogenic compositions (i.e., vaccines or probiotics) described herein can be used for prophylactic and/or therapeutic treatment of S. pneumoniae. Accordingly, the compositions provide a method for treating a subject suffering from or susceptible to S. pneumoniae infection, comprising administering an effective amount of any of the formulations described herein. The subject receiving the vaccination can be a male or a female, and can be a child or adult. In some embodiments, the subject being treated is a human. In other embodiments, the subject is a non-human animal.

[0065] In prophylactic embodiments, the composition is administered to a subject to induce an immune response that can help protect against the establishment of S. pneumoniae, for example by protecting against colonization, the first and necessary step in disease. Thus, in some aspects, the method inhibits infection by S. pneumoniae in a non-colonized or uninfected subject. In another aspect, the method can reduce the duration of colonization in an individual who is already colonized.

[0066] In vaccine embodiments, the compositions of the invention confer protective immunity, allowing a vaccinated individual to exhibit delayed onset of symptoms or sequelae, or reduced severity of symptoms or sequelae, as the result of his or her exposure to the vaccine. In particular embodiments, individuals who have been vaccinated can display no symptoms or sequelae upon contact with S. pneumoniae, do not become colonized by S. pneumoniae, or both. Protective immunity is typically achieved by one or more of the following mechanisms: mucosal, humoral, or cellular immunity. Mucosal immunity is primarily the result of secretory IgA (sIGA) antibodies on mucosal surfaces of the respiratory, gastrointestinal, and genitourinary tracts. The sIGA antibodies are generated after a series of events mediated by antigen-processing cells, B and T lymphocytes that result in sIGA production by B lymphocytes on mucosa-lined tissues of the body. Humoral immunity is typically the result of IgG antibodies and IgM antibodies in serum. Cellular immunity can be achieved through cytotoxic T lymphocytes or through delay ed-type hypersensitivity that involves macrophages and T lymphocytes, as well as other mechanisms involving T cells without a requirement for antibodies. In particular, cellular immunity can be mediated by Tm or THI7 cells.

[0067] In therapeutic embodiments, the vaccine can be administered to a patient suffering from S. pneumoniae infection, in an amount sufficient to treat the patient. Treating the patient, refers to reducing S. pneumoniae symptoms and/or bacterial load or bacterial density in an infected individual. Reduced S. pneumoniae symptoms can be assessed by comparing symptoms of a host before and after vaccination. In an embodiment, the vaccine can be administered to repopulate a patient’s microbiome after extended antibiotic use. Bacterial load can be assessed by, for example, counting colonies. In some embodiments, treating the patient refers to reducing the duration of symptoms or sequelae, or reducing the intensity of symptoms or sequelae. In some embodiments, the vaccine reduces transmissibility of S. pneumoniae from the vaccinated patient. In certain embodiments, the reductions described above are at least 25%, 30%, 40%, 50%, 60%, 70%, 80% or even 90%. In therapeutic embodiments, the vaccine can be administered to an individual post-infection, e.g. before symptoms or sequelae manifest, or can be administered during or after manifestation of symptoms or sequelae. Without being bound to a particular theory or mechanism it is believed that therapeutic embodiments of the inventive composition can reduce the intensity and/or duration of the various symptoms or sequelae of S. pneumoniae infection, for example, pneumonia, acute sinusitis, otitis media (ear infection), meningitis, bacteremia, sepsis, osteomyelitis, septic arthritis, endocarditis, peritonitis, pericarditis, cellulitis, or brain abscess.

Vaccine Doses and Routes of Administration

A. Dosage Forms, Amounts, and Timing

[0068] The amount of antigen in each vaccine formulation dose is selected as an effective amount, which induces a prophylactic or therapeutic response, as described above, in either a single dose or over multiple doses. Preferably, the dose is selected to minimize adverse side effects. Such amount will vary depending upon which specific antigen is employed. In embodiments, a dose can comprise one or more live organisms. In embodiments, a dose can comprise 1-1000 pg of each capsular polysaccharide, for example 2-100 pg, for example 4- 40 pg. The appropriate amount of antigen to be delivered can depend on the age, weight, and health (e.g. immunocompromised status) of a subject. When present, typically an adjuvant will be present in amounts from 1-250 pg per dose, for example 50-150 pg, 75-125 pg or 100 fig-

[0069] In embodiments, one dose of the vaccine is administered to achieve the results described above. In other embodiments, following an initial vaccination, subjects receive one or more booster vaccinations, for a total of two, three, four or five vaccinations. A booster vaccination can be administered, for example, about 1 month, 2 months, 4 months, 6 months, or 12 months after the initial vaccination, such that one vaccination regimen involves administration at 0, 0.5-2 and 4-8 months.

[0070] The vaccines described herein can be prepared in a variety of dosage forms. In embodiments, the immunogenic composition is provided in solid or powdered (e.g., lyophilized) form; it also can be provided in solution form. In other embodiments, a dosage form is provided as a dose of immunogenic composition and at least one separate sterile container of diluent. In other embodiments, the dosage form is provided in a kit which includes a dose of immunogenic composition in a container and at least one separate sterile container of diluent.

[0071] In some embodiments, the immunogenic composition will be administered in a dose escalation manner, such that successive administrations of the vaccine contain a higher concentration of vaccine than previous administrations. In embodiments, the vaccine will be administered in a manner such that successive administrations of the vaccine contain a lower concentration of vaccine than previous administrations.

B. Vaccine Routes of Administration

[0072] The inventive compositions described herein can be delivered by administration to an individual, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, intradermal, subcutaneous, subdermal, transdermal, intracranial, intranasal, mucosal, anal, vaginal, oral, buccal route or they can be inhaled) or they can be administered by topical application. In an embodiment, the route of administration is intramuscular. In other embodiments, the route of administration is subcutaneous. In yet other embodiments, the route of administration is mucosal. In certain embodiments, the route of administration is transdermal or intradermal.

C. Vaccine Formulations

[0073] The vaccine formulation can be suitable for administration to a subject in need thereof. In an embodiment, the vaccine formulation or immunogenic composition is suitable for administration to a mammal. In an embodiment, the vaccine is substantially free of either endotoxins or exotoxins. Endotoxins can include pyrogens, such as lipopolysaccharide (LPS) molecules. The vaccine or immunogenic composition can also be substantially free of inactive protein fragments which can cause a fever or other side effects. In embodiments, the composition contains less than 1%, less than 0.1%, less than 0.01%, less than 0.001%, or less than 0.0001% of endotoxins, exotoxins, and/or inactive protein fragments. In some embodiments, the vaccine or immunogenic composition has lower levels of pyrogens than industrial water, tap water, or distilled water. Other vaccine or immunogenic composition components can be purified using methods known in the art, such as ion-exchange chromatography, ultrafiltration, or distillation. In other embodiments, the pyrogens can be inactivated or destroyed prior to administration to a patient. Raw materials for vaccines, such as water, buffers, salts and other chemicals can also be screened and depyrogenated. All materials in the vaccine can be sterile, and each lot of the vaccine can be tested for sterility.

[0074] In embodiments, the preparation comprises less than 50%, 20%, 10%, or 5% (by dry weight) contaminating protein. In embodiments, capsular polysaccharide is present in the substantial absence of other biological macromolecules, such as proteins (particularly proteins which can substantially mask, diminish, confuse or alter the characteristics of the component proteins either as purified preparations or in their function in the subject reconstituted mixture).

[0075] In preferred embodiments, the vaccine has low or no toxicity, within a reasonable risk-benefit ratio. In certain embodiments, the vaccine or immunogenic composition comprises ingredients at concentrations that are less than LD50 measurements for the animal being vaccinated. LD50 measurements can be obtained in mice or other experimental model systems, and extrapolated to humans and other animals. Methods for estimating the LD50 of compounds in humans and other animals are well-known in the art. A vaccine formulation or immunogenic composition, and any component within it, might have an LD50 value in rats of greater than 100 g/kg, greater than 50 g/kg, greater than 20 g/kg, greater than 10 g/kg, greater than 5 g/kg, greater than 2 g/kg, greater than 1 g/kg, greater than 500 mg/kg, greater than 200 mg/kg, greater than 100 mg/kg, greater than 50 mg/kg, greater than 20 mg/kg, or greater than 10 mg/kg.

[0076] The formulations suitable for introduction of the vaccine vary according to route of administration. Formulations suitable for parenteral administration, such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, intranasal, oral, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials.

[0077] Solutions and suspensions for injection can be prepared from sterile powders, granules, and tablets. Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the polysaccharide capsule suspended in diluents, such as water, saline or PEG 400; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable emulsions. Tablet forms can include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, com starch, potato starch, tragacanth, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art. The pharmaceutical compositions can be encapsulated, e.g., in liposomes, or in a formulation that provides for slow release of the active ingredient.

[0078] The antigens, alone or in combination with other suitable components, can be made into aerosol formulations (e.g., they can be“nebulized”) to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. Aerosol formulations can be delivered.

D. Preparation and Storage of Vaccine Formulations

[0079] The S. pneumoniae vaccines and immunogenic compositions described herein can be produced using a variety of techniques. For example, a polypeptide can be produced using recombinant DNA technology in a suitable host cell. A suitable host cell can be bacterial, yeast, mammalian, or other type of cell. The host cell can be modified to express an exogenous copy of one of the relevant genes. Typically, the gene is operably linked to appropriate regulatory sequences such as a strong promoter and a polyadenylation sequence. In some embodiments, the promoter is inducible or repressible. Other regulatory sequences can provide for secretion or excretion of the polysaccharide of interest or retention of the polysaccharide of interest in the cytoplasm or in the membrane, depending on how one wishes to purify the polysaccharide. The gene can be present on an extrachromosomal plasmid, or can be integrated into the host genome. One of skill in the art will recognize that it is not necessary to use a nucleic acid 100% identical to the naturally-occurring sequence. Rather, some alterations to these sequences are tolerated and can be desirable. For example, the nucleic acid can be altered to take advantage of the degeneracy of the genetic code such that the encoded polypeptide remains the same. In some embodiments, the gene is codon- optimized to improve expression in a particular host. The nucleic acid can be produced, for example, by PCR or by chemical synthesis. Once a recombinant cell line has been produced, a polypeptide can be isolated from it. The isolation can be accomplished, for example, by affinity purification techniques or by physical separation techniques (e.g., a size column).

[0080] The immunogenic compositions described herein can be packaged in packs, dispenser devices, and kits for administering the compositions to a mammal. For example, packs or dispenser devices that contain one or more unit dosage forms can be provided. Typically, instructions for administration of the compounds will be provided with the packaging, along with a suitable indication on the label that the compound is suitable for treatment of an indicated condition, such as the prophylaxis or treatment of pneumococcal disease, as described herein with respect to other aspects of the invention.

[0081] In an embodiment, the inventive composition can be provided to a subject in need thereof. As used herein,“a subject in need thereof’ refers to a subject in need of therapeutic or prophylactic therapy. In embodiments, the subject can be a mammal. The term “mammal” as used herein refers to any mammal, including, but not limited to, mice, hamsters, rats, rabbits, cats, dogs, cows, pigs, horses, monkeys, apes, and humans.

Preferably, the mammal is a human.

[0082] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

EXAMPLES

[0083] The following examples describe the isolation and characterization of distinct strains of commensal species which express capsular polysaccharides that are structurally related to or identical to those expressed by pneumococcal serotypes. The strains were recovered from specimens from two carriage studies, one in the United States, and one in Kenya, as described below.

EXAMPLE 1

[0084] This example describes the carriage study in the United States, i.e., the study design and population used for collecting and screening nasopharyngeal (NP) and oropharyngeal (OP) specimens to assess cross-reactivity of non-pneumococcal streptococci strains with serotype-specific pneumococcal antisera. [0085] Healthy adults 65 years of age and older were enrolled in this carriage study. Adults were excluded if severe immunocompromising conditions were reported (e.g., hematologic malignancies, transplant recipient, end-stage renal disease) or if they were residents in nursing homes or skilled nursing facilities (i.e., institutionalized individuals).

For each eligible adult who consented to participation, one nasopharyngeal (NP) and one oropharyngeal (OP) swab were collected, and an interview was conducted to obtain demographic, clinical and vaccination data. Data from participants without both an NP and an OP specimen was excluded from the final analysis. Trained staff followed up with healthcare providers to confirm PCV13 and PSV23 receipt and dates. The Center for Disease Control and Prevention’s (CDC’s) and local institutional review boards approved the study. Written informed consent was obtained from all study participants.

Specimen collection and processing

[0086] NP and OP specimens were collected using COPAN sterile nylon tipped flocked swabs (catalog number 503CS01 and 502CS01; Copan Diagnostics INC, Murrieta, CA). If the initial collection was not successful, a second attempt was made. NP and OP specimens were placed separately into cryovials containing 1.0 ml skim milk-tryptone-glucose-glycerol (STGG) medium, vortexed for 10 to 20 seconds to disperse the organisms from the swab, and frozen at -70°C within 4 hours after collection.

[0087] Each specimen was processed separately. For pneumococcal isolation and identification, 200 pl of the STGG-NP inoculated medium was transferred into 5.0 ml Todd Hewitt broth (THY) (Sigma, St. Louis, MO) containing 0.5 % yeast extract and 1 ml of rabbit serum, and then incubated at 37 °C in CO2 for 6 hours. After incubation, 10 mΐ of cultured broth was streaked on tryptone soy agar plates with 5% sheep blood (BAP) (Trypticase Soy Agar with 5 % sheep blood II, Becton Dickinson and Company, Sparks, MD and incubated at 37 °C in CO2 for 18-24 hours as previously described by Carvalho et al, 2010) (incorporated in its entirety herein by reference). Alpha-hemolytic colonies resembling pneumococci were tested for susceptibility to optochin and bile solubility and serotyped by Quellung as previously described (Austrian, R.,“The quellung reaction, a neglected microbiologic technique,” Mt. Sinai J. Med., 43(6):699-709 (1976) (Incorporated by reference in its entirety) using CDC pneumococcal typing antisera. Pneumococcal-culture negative NP and OP specimens underwent real-time PCR targeting lytA for detection of pneumococcal DNA (Carvalho et al,“Evaluation and Improvement of Real-Time PCR Assays Targeting lytA, ply, and psaA Genes for Detection of Pneumococcal DNA. ./ Clin. Microbiology , Vol. 45, No. 8: 2460-2466 (2007) (Incorporated by reference in its entirety). DNA extraction was performed using a MAGNA PURE COMPACT system with an external lysis protocol using buffer #4 (Roche isolation kit III, Roche Diagnostics Corp., Indianapolis, IN) or

NUCLISENS Easy MAG automated system (bioMerieux, Hazelwood, MO) for total nucleic acid extraction, according to manufacture instructions. Twenty-one real-time multiplex PCR assays encompassing 37 serotypes (including PCV13 serotypes) (Pimenta, et al,“Sequential Triplex Real-Time PCR Assay for Detecting 21 Pneumococcal Capsular Serotypes That Account for a High Global Disease Burden,” J. Clin. Microbiology. Vol. 51, No. 2: 647-652 (20l3)(Incorporated by reference in its entirety) were performed on 53 /v -positive and 395 ytA -negative specimens using DNA extracted from 200 ul of each NP and OP swab- inoculated STGG media. Conventional multiplex PCR was also performed for detection of 33 additional serotypes (Carvalho et al, 2010) (incorporated in its entirety herein by reference).

[0088] The results were as follows: Of the 1,229 adults enrolled, 67.9% were female, the median age was 74 years old (range: 65-102), and 73.9%. Pneumococcus was isolated from 16 (1.2%) patients; 12 from NP, 2 from OP and 2 from both NP and OP. Of the 1,283 culture-negative patients, 75 (5.8%) were /v -positive: 1 from NP, 71 from OP, and 3 from both NP and OP.

[0089] PCR-based serotype deduction performed on 53 /y -positive and 395 lylA - negative specimens yielded 8 (15.1%) and 41 (10.4%) c w 7-positive specimens, respectively. Additionally, both /v -positive and lytA- negative specimens were positive for other serotype-specific pneumococcal genes, as shown in Table 1, below. OP specimens were more likely to be PCR-positive for serotype-specific pneumococcal genes compared to NP specimens for both /v -positive (50.9% [27/53] vs. 3.8% [2/53]; P < 001) and L' -negative (32.7% [129/395] vs. 2.3% [9/395]; P < 001) specimens. Adults who had previously received PCV13 were less likely to carry vaccine-type pneumococcal genes compared to those who did not receive PCV13 (26.0% [45/173] vs. 35.8% [90/251]; P=0.03). Table 1: PCR Serotyping Results of lytA- positive and lytA- negative Specimens

PCR Serotyping Results lytA positive specimens /)7 l negative specimens n (%) n (%)

(N=53) (N=395)

001, n (%) 8 (15.1) 41 (10.4)

004, n (%) 9 (16.9) 37 (9.4)

9V/9A^#, n (%) 6 (11.3) 32(8.1)

005, n (%) 1 (1.9) 11 (2.8)

18 A/ 18B/ 18C/18F^#, n(%) 7 (13.2) 25 (6.3)

23 A, n (%) 2 (3.8) 4 (1.0)

19F, n (%) 1 (1.9) 0 (0)

14, n (%) 0 2 (0.5)

12F/12 A/ 12B/44/46^#, n (%) 1 (1.9) 9 (2.3)

Others, n (%) 2 (3.8) 13 (3.3)

Negative for the serotypes tested by 26 (49.1) 264 (66.8)

PCR, n (%)

* Not mutually exclusive

^# PCR reaction groups these serotypes

EXAMPLE 2

[0090] This example demonstrates the methods used for the isolation and characterization of the non-pneumococcal species described in Example 1.

[0091] OP specimens were cultured using broth enrichment followed by plating upon blood agar for isolation of alpha-hemolytic streptococcal species. Non-pneumococcal alpha- hemolytic streptococci colonies that were optochin resistant and bile insoluble were screened for the serotype 1 specific wzyl gene known to he within the cpsl operon by real-time and conventional multiplex PCR. Non-pneumococcal isolates that were wzy 1 -positive, (referred to here as cy?.s7-positive) were subjected to lytA screening by PCR and to BINAXNOW immunochromatographic testing (BinaxNOW® Streptococcus pneumonia Antigen Card, Alere, Ottawa, Ontario, Canada). The OP specimens were also screened for additional serotype genes as described in Example 1. [0092] Whole genome sequencing was performed for all the non-pneumococcal isolates according to the methods disclosed in Metcalf et al, 2016 (incorporated in its entirety herein by reference). Sequences of non-pneumococcal streptococci cpsl operons were compared with the sequence of the cpsl operon of the pneumococcal reference (GenBank accession CR931648: SEQ ID NO. 9), as shown in Figure 1. Sequences of non-pneumococcal streptococci cps4 operons were compared with the sequence of the cps4 operon of the pneumococcal reference (GenBank accession CR931635: SEQ ID NO. 10), as shown in Figure 2. Sequences of non-pneumococcal streptococci cps9A-L operons were compared with the sequence of the cps9V operon of the pneumococcal reference (GenBank accession CR931648: SEQ ID NO. 11), as shown in Figures 3 and 11. Sequences of non- pneumococcal streptococci cpsl8C operons was compared with the sequence of the cpsl8C operon of the pneumococcal reference (GenBank accession CR931648: SEQ ID NO. 12).

[0093] With respect to pneumococcal serotype 1, whole genome sequence analysis of the S. mitis isolates revealed three closely similar, although distinct, cpsl operons and adjacent genes, shown in Figure 1. The lengths of the 3 loci, corresponding to the region between the dexB and ctliA segments of the 22,182 bp pneumococcal reference were closely similar (16,613-16,626) and shared 98.0 - 98.2% sequence identity with one another. Each of the 11 genes within the polysaccharide synthetic cluster ( wzg - ugd) were highly homologous to the S. pneumoniae serotypel counterparts and shared the same organization (Figure 1). The 3 different cpsl operons shared 98.0 - 98.2% sequence identity with, and corresponded to 3 distinct S. mitis clones. While 16 different pneumococcal strains representing 6 different serotypes formed a tight phylogenetic cluster based upon phylogenetic analysis of 3063 bp concatenated housekeeping gene segments (SEQ ID NOS: 13-22) (Bishop et al,“Assigning strains to bacterial species via the internet,” BMC Biology, Vol. 7(3) (2009) (Incorporated by reference in its entirety), the S. mitis strains formed a much more divergent cluster. At the level of direct DNA sequence comparison, the 3 serotype 1 S. mitis clones shared 97.1% - 97.3% sequence identity over the 3063 bp, while the 16 pneumococcal strains shared 99.5% - 99.9% sequence identity. Thus, the genetic distance displayed between S. mitis strains restricted to serotype 1 is much greater than it is between even randomly selected pneumococcal strains that express different serotypes. These S. mitis strains were all macrolide-resistant (all contained emz-methylase or mef genes) and cotrimoxazole-resistant, which corresponded to alterations within folA and/or folP (Metcalf et al, 2016) (incorporated in its entirety herein by reference). As in pneumococci, the cps loci were situated between the penicillin binding protein genes pbp2x and pbpla, which often confer selectable resistance to b-lactam antibiotics. Strains GA115 and GA116 shared the PBP transpeptidase sequences PBP2b-l2l and PBP2x-8, both of which have been associated with pneumococcal resistance to b-lactams (Metcalf et al, 2016) (incorporated in its entirety herein by reference). The cpsl strains lacked the pneumococcal-specific target piaA and contained pneumolysin genes with 61-97% sequence identity to the pneumococcal ply gene. In addition, strain GA121F (identified as isolate ID L-121 below in Table 2) contained a lylA gene highly homologous (82% sequence identity) to pneumococcal counterparts.

[0094] With respect to pneumococcal serotype 4, whole genome sequence analysis of the S. infantis isolates revealed a closely similar, although distinct, cps4-L polysaccharide synthesis gene cluster and adjacent genes, as shown in Figure 2. Additionally, with respect to pneumococcal serotype 9V, whole genome sequence analysis of the S. infantis isolates revealed a closely similar, although distinct, cps9 polysaccharide biosynthetic gene cluster and adjacent genes, as shown in Figure 3. With respect to pneumococcal serotype 18C, whole genome sequence analysis of the S. oralis isolates revealed a closely similar, although distinct, cps 18C biosynthetic gene cluster and adjacent genes.

[0095] With respect to pneumococcal serotype 5, a relatively high number of wzy5- positive upper respiratory specimens from lytA- negative (indicative of pneumococcal negative) specimens were found (11/395, 2.8%), and a single inzo-positive from a less common /v -positive specimen (1/53, 1.9%), as shown in Table 1. Serotype 5 pneumococci was not isolated in the U.S. study, however, two distinct clones of v o-positive S. infantis (two strains) and one S. oralis strain from lytA- negative specimens were isolated.

Characterization of the strains carrying cps 5 loci isolated from both carriage studies is described below in Example 4.

[0096] Species assignment was done by phylogenetic analysis of seven previously described concatenated housekeeping gene sequences (total length of 3063 bases) (Bishop et al,“Assigning strains to bacterial species via the internet,” BMC Biology, Vol. 7(3) (2009) (Incorporated by reference in its entirety)). A selection of the closest matching 3063 bp sequences from designated species within the cited reference to the non-pneumococcal streptococci strains were used to construct the phylogenetic tree (Figure 4). In addition, sequences were extracted from genomic sequences from well-the characterized strains S. pseudopneumoniae (ATCCBAA960; SEQ ID NO. 24), S. mitis (B6 and NCTC 12261; SEQ ID NOs. 25 and 26), S. infantis (ATCC 700779; SEQ ID NO. 27), and S. oralis (Uo5; SEQ ID NO. 28), which are present in the NCBI GenBank. The evolutionary distances were computed using the Maximum Composite Likelihood method (Tamura et al.“Prospects for inferring very large phylogenies by using the neighbor-joining method. PNAS (USA) 101 : 11030-11035 (2004) (Incorporated by reference in its entirety)). The analysis involved 135 nucleotide sequences. Evolutionary analyses were conducted in MEGA7 according to known methods (Kumar et al.,“MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets,” Mol. Biol. Evol., 33 (7): 1870-1874 (2016) (Incorporated by reference in its entirety)). The results are provided below in Table 3.

Capsule expression of non-pneumococcal streptococci isolates

[0097] Latex agglutination followed by the Quellung reaction using polyclonal serotype 1 pneumococcus typing antiserum prepared in rabbits was performed on the S. mitis cpsl isolates to assess capsule expression as shown in Figure 9. In addition, double

immunodiffusion assays using the same antiserum assessed cross-reactivity of non- pneumococcal streptococci with serotype 1 pneumococci using the methods described by Sorensen et al. (Figure 10, see also Figure 21). Briefly, bacterial suspension extracts were tested either untreated (i.e. crude) or treated with sodium metaperiodate. Double

immunodiffusion in agarose was used to examine cross-reactivity between non- pneumococcal and pneumococcal polysaccharide extracts with serotypel typing antiserum. Pierce standard Ouchterlony agarose gel plates (Pierce #31111, Thermofisher Scientific, Waltham, MA) were used as previously described by Oudin and Ouchtherlony. (Oudin, L’analyse,“immunochemique qualitative. Methode par diffusion des antigens au sein de Fimmunoserum precipitant gelose,” Premiere Parte Inst Pasteur, 75:30-52 (1948)

(Incorporated by reference in its entirety); Ouchterlony,“Antigen-antibody reactions in gels and the practical applications of this phenomenon in laboratory diagnosis of diphtheria,” Med Diss Stockholm (1949) (Incorporated by reference in its entirety)).

[0098] In most experiments, 15 pl undiluted serotype 1 pneumococcal antiserum (AS) was applied to a center well (2.8 mm), and l5ul extract samples were applied to the four surrounding wells, placed at a distance, edge to edge, of 3.2 mm. Two wells were left empty without any bacterial extract. Plates were kept for 2 days at 5°C and then observed and photographed.

[0099] Quellung and immunodiffusion verified serotype 1 capsule expression (Figures 9 and 10). Five OP specimens, three lytA- negative (ID# GA006, GA115, GA116) and two lytA- positive (ID# GA121, GA164) with cycle threshold values for wzyl between 22.0 and 27.5 yielded S. mitis on culture. S. mitis isolates were positive for serotypel by real-time and conventional PCR (cyw /-positive). and by latex agglutination for serotyping (Table 2). Two S. mitis isolates were Binax positive.

[00100] The nonpneumococcal cpsl strains specifically reacted with typing sera against serotype 1 in a latex agglutination assay (Table 2). Although weak, pneumococcal serotype 1 capsule was visualized by Quellung reaction in S. mitis strains L006, L121, L164 (Table 2). Strains L006 and L121 representing two of the three S. mitis clones were tested using double immunodiffusion assays and showed strong cross-reactivity with S. pneumoniae serotype 1 typing anti-serum. S. mitis serotype 1 antigen and S. pneumoniae serotype 1 antigen generated symmetrical, fused precipitates in reaction with serotype 1 typing antisera, consistent with the two species expressing identical capsular polysaccharide antigens.

Numerous control experiments were performed using typing antisera for other pneumococcal serotypes, and various control strains were also employed in immunodiffusion experiments (data not shown). The results are shown below in Table 2.

Table 2: Results of characterization analyses for non-pneumococcal Streptococus mitis serotype 1 strains

Table 2. continued

|0100j Additional experiments conducted according to the protocol discussed herein revealed a nonpneumococcal serotype 4 strain, serogroup 9 strain, and a serotype 18 strain, shown below in Table 3.

Table 3. Results of characterization analyses for non-pneumococcal strains of serotypes 1, 4, and serogroups 9V/9A and 18.

Homology between pneumococcal and non-pneumococcal streptococcus capsules

[0101} Sero-competition assay was performed to determine the homology between the ST1 capsules expressed by the non-pneumococcal streptococci strains (SMST1) and the serotype 1 pneumococcus (PncSTl). Overnight broth cultures of SMST1, PncSTl or S. mitis ATCC (control) were centrifuged and the pellet was resuspended in 500 mΐ rabbit serotype 1 pneumococcal antisera for overnight absorption at 4 °C in a rotator. Anti-STl antibodies in bacteria absorbed and unabsorbed serum were quantified using standard luminex technique (Lal et al,“Development and validation of a nonaplex assay for the simultaneous quantitation of antibodies to nine Streptococcus pneumoniae serotypes,” J. Immunol.

Methods, Vol. 296, Issues 1-2: 135-147 (2005) (Incorporated by reference in its entirety)). Comparative analysis of reduction in antibody concentration in absorbed sera was used to demonstrate the homology between SMST1 and PncSTl capsules.

|0102 j Absorption studies clearly demonstrated the homology between the pneumococcal and non-pneumococcal capsule (Figure 5). There was no statistical significance (p>0.05) in percent reduction in ST1 specific antibodies between SMST1 and PncSTl absorbed sera.

The absence of S. mitis ATCC control strain (cpsl -negative) reactivity with ST1 serum confirms the specificity of SMST1 capsule to anti- pneumococcal ST1 antibodies.

[0103} Additional experiments to determine S. mitis capsule reaction specificity to rabbit pneumococcal anti-serum were performed according to the methods described above, to determine homology between pneumococcal and nonpneumococcal serotypes 1 and 4 and serogroups 9 (employing antipneumococcal typing sera factors 9d, 9g, 9e), serogroup 18 (employing factors l8cf, and l8ce). The results are shown in Figure 6.

Opsonophagocytosis of non-pneumococcal streptococci isolates

[0104] An opsonophagocytic killing assay (Romero- Steiner et al.,“Standardization of an opsonophagocytic assay for the measurement of functional antibody activity against

Streptococcus pneumoniae using differentiated HL-60 cells,” Clin Diagn. Lab Immunol. 4(4):4l5-22 (1997) (Incorporated by reference in its entirety)) was used to assess functional serotype 1 activity against the non-pneumococcal streptococci strains expressing serotype 1 capsular polysaccharide (SMST1) and the serotype 1 pneumococcus (PncSTl) (Figure 7). Briefly, in a 96-well micro titer plate, 20 mΐ of SMST1 or PncSTlwas pre-opsonized with 10 mΐ anti-pneumococcal serotype 1 specific human polyclonal serum (8-dilutions, diluted 2-fold starting neat) for 15 minutes at 37°C with 5% CC . S. mitis ATCC strain PCR negative for wzyl gene (c/»7 -negative) was used as a control. After pre-opsonization, 5 mΐ baby rabbit complement was added followed by 40 mΐ of human pro-myelocytes (HL60) derived neutrophils (effector cells). Complement, neutrophil, and bacterial controls were maintained. After incubation for 45 minutes at 37°C, 5 mΐ from each well was transferred onto air-dried Todd-Hewitt yeast extract agar. Opsonophagocytic titers were the reciprocal of the serum dilution with >50% killing compared with the growth in the complement control wells (Figure 8).

[0105] Based on the opsonophagocytosis killing (OPK) assays, capsules expressed by serotype S. mitis and S. pneumoniae strains triggered anti-serotype 1 antibody mediated opsonophagocytosis while the S. mitis control strain (c/»7 -negative) was unaffected. (Figure 8).

[0106] An additional opsonophagocytosis killing assay was conducted with respect to serotypes 4, 9, and 18, according to the same protocol described above. As shown in Figure 8, the results of OPK testing show that S. mitis pneumococcal serotypes 1 and 4 undergo opsonophagocytosis in the presence of anti-Pnc type 1 and type 4 antibodies, respectively. S.mitis serotypes 9 and 18 were non-reactive in OPK and comparable to the S. mitis control.

Data Analyses

[0107] All analyses were performed using SAS software, version 9.3. The prevalence of pneumococcal carriage by culture was calculated. Among culture-negative specimens, serotype distribution for lytA- positive and lytA -negative specimens is presented. Comparison of the prevalence of pneumococcal vaccine-type homolog genes (serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F) by PCV13 status was done using chi-square test. Patients were considered vaccinated if PCV13 was received at least 14 days prior to swab collection. Serotypes not distinguished by PCR (e.g. 9A/9V, 7A/7F, 18A/18B/18C/18F) were grouped.

EXAMPLE 3

[0108] This example describes the carriage study used for collecting and screening NP and OP specimens to assess cross-reactivity of non-pneumococcal streptococci strains with serotype-specific pneumococcal antisera in Kenya.

[0109] In Kenya, an area with high pneumococcal carriage among adults (median age 32 years; 43.2% and 26.8% frequency for pneumococcal isolation from combined oropharyngeal and nasopharyngeal specimens from HIV-positive and HIV -negative adults, respectively), a high percentage of upper respiratory specimens from adults were positive for the serotype 5 specific wzy5 gene when employing a conventional PCR assay (32.3%; 51 of 158 specimens), even though serotype 5 pneumococcal isolates from specimens taken from adults were not recovered. The materials and methods for specimen collection, pneumococcal isolation, serotyping, conventional multiplexed PCR-serotyping (cmPCR), real time 1 ytA PCR, cmPCR amplicon sequencing, species approximation of non-pneumococcal strains are described in Carvalho et al,“Non-Pneumococcal Mitis-Group Streptococci Confound Detection of Pneumococcal Capsular Serotype-Specific Loci in Upper Respiratory Tract,” Peer ,l :e97; DOIl0.77l7/peerj.97 (2013)), which is incorporated herein by reference in its entirety.

[0PQ] A Streptococcus mitis strain that was PCR-positive for the serotype 5-specific wzy5 gene was isolated by culturing an aliquot of the original sample that had been stored frozen and looking for S. mitis using conventional microbiological techniques to identify the S. mitis among the colonies that grew on the plate (strain KE67013) (SEQ ID NO: 29) from the collected specimens, the analysis of which is discussed below in Example 4.

EXAMPLE 4

[0111 j This example describes the separate analysis of four commensal strains (three isolated from the United States study described in Example 1, and one isolated from the Kenya study described in Example 3) that were positive for the serotype-5 specific wzy5 gene, which demonstrates the antigenic relatedness of these strains to pneumococcal serotype 5.

Bacterial Strains

[0112| Methods for isolation of the non-pneumococcal strains that were PCR-positive for serotype 5 and other known pneumococcal serotypes from upper respiratory specimens are as described in Carvalho, 2013 (incorporated in its entirety herein by reference). Conventional and real time PCR assays were performed. The cps5-positive strains US0024, US969j l, and US0049 were recovered during this study from the stored US carriage study specimen collection. The genome sequence of strain F0392 was obtained from GenBank project accession AFUOOL Genomic sequencing

[0113] Genomic DNA samples from all isolates were prepared and sequenced as multiplexed libraries on the ILLUMINA MISEQ platform to produce paired end reads (as described in Metcalf et al.,“Using whole genome sequencing to identify resistance determinants and predict antimicrobial resistance phenotypes for year 2015 invasive pneumococcal disease isolates recovered in the United States,” Clin. Microbiol. Infect. 22: l002.el-l002.e8, (2016)) (incorporated herein by reference).

Preparation of streptococcal vaccines.

[0114] Antiserum against formalin-fixed S. mitis strain KE67013 was prepared. Three rabbits were inoculated over a period of six weeks to yield the three antiserum sources used. A pooled, chloroform clarified sample that combined all three sources was also used (Figure 16).

Serology

[0115] Latex agglutination and the Quellung reaction employing rabbit polyclonal typing antiserum were used to assess serotype expression in commensal streptococci. Double immunodiffusion assays employing pneumococcal typing sera and antisera prepared against commensal streptococci were carried out as described above (Figures 14A-14C and 15A- 15C).

Species assignments

|0!!6] Strains were assigned species by virtue of clustering with previously speciated strains employing whole genomic kSNP3.0 analysis to generate core genomic single nucleotide polymorphism and a maximum parsimony phylogenetic tree. Node support was assessed by using 500 bootstrap replicates. Phylogenetic clustering of concatenated housekeeping gene sequences (multilocus sequence analysis; MLSA) was achieved with the MEGA7 program as described in Kumar et al. (2016) (incorporated in its entirety herein by reference).

Opsonophagocytosis (OPK) Assays [0117] The standard OPK assay was performed employing HL-60 cells and complement source (baby rabbit serum; Pel-Freez, Brown Deer, WI). Initial dilution was determined at 1 :400 based on optimization testing with serotype 5 S. pneumoniae- induced antisera against type 5 S. mitis KE67013. Complement control wells included all the test reagents except antibodies to pneumococci. Opsonophagocytic titers taken for GMT reflect the serum dilution with >50% killing compared with the mean growth in the complement control wells (Figure 16).

Results

[0118] Genome sequencing of the four v o-positive isolates revealed that all lacked the pneumococcal-specific piaA iron transporter determinant. The genome sequence from strain F0392, isolated from a Kenya specimen (SEQ ID NO: 30) is included herein for comparison. Strain KE67013 (SEQ ID NO: 29), isolated from a Kenya specimen, contained recognizable homologs of the major pneumococcal autolysin (lytA) and of the pneumolysin gene (ply), with 79% and 60% sequence identity, respectively. Comparison strain F0392 contained a lytA homolog (72% identity), while strains US0049 (SEQ ID NOs: US0049, US969j l, and US0024h, isolated from U.S. specimens (SEQ ID NOs: 31, 32, and 33, respectively) lacked recognizable homologs of all 3 genes.

[0119] Phylogenetic analysis employing kSNP3.0 (described in Gardner et al,“kSNP3.0: SNP detection and phylogenetic analysis of genomes without genome alignment or reference genom Q,” Bioinformatics , 31 :2877-2878 (2015), herein incorporated by reference) revealed that the five vmo-positive strains (including comparison strain F0392) were genetically highly diverse, clustering with representatives of S. infantis, S. mitis, and S. oralis.

Phylogenetic analysis employing concatenated housekeeping gene fragments was in close agreement with the depicted whole genomic kSNP.O analysis.

[0120] All five non-pneumococcal vmo-positive strains (including comparison strain F0392, for which the genome sequence is available but corresponding strain serology has not yet been not characterized) contained cps loci highly similar to the corresponding cps5 locus from the serotype 5 pneumococcal reference strain Spn Ambrose5, with 87.3% - 90.5% sequence identity over the entire operon (Figures 12A-12C).

[0121] Highly homologous (78 - 99.2% sequence identity) counterparts of each of the previously described 15 cps 5 genes from the pneumococcal serotype 5 reference strain were apparent in the same relative order. The first four genes of the cps 5 locus ( wzg , wzh. wzd. and wze) have widely conserved functions in pneumococcal capsular polysaccharide synthesis, corresponding with high similarity (> 98% sequence identity, regardless of serotype) to a large number of counterparts from pneumococcal cps operons. An additional six cps 5 genes (wcil. wciJ fnlA, [nlB.fnlC. and ugd) with more specialized functions are conserved among a subset of pneumococcal serotypes. The centrally situated five cps 5 genes (wzy. wzx, whaC, whaD, and whaE ) which include the wzy and wzx genes that encode highly substrate-specific flippase and polymerase functions, share little or no sequence similarity with pneumococcal strains of other known capsular serotypes; however these pneumococcal genes shared 76 - 99% sequence identity among the five non-pneumococcal strains. The highly conserved five-gene segment was exactly 5,429 bases in length and all six strains (including S. pneumoniae strain Ambrose) shared the same spacings of translational start and stop codons.

J0122] The presence of the fnlA-C genes in serotype 5 (Figures 13A-13C) is consistent with N-acetyl-a-L-fucosamine found within serotype 4, 5, 12A, and 12F capsule

polysaccharides. Further, N-acetyl-L-pneumosamine and 4-keto-N-acetyle-D- quinovosamine, intermediates within the N-acetyl-a-L-fucosamine pathway, are both uniquely present in the pneumococcal serotype 5 capsule. The near-identical fnlA sequences of the five v o-positive commensal strains of the three different species and serotype 5 pneumococci are consistent with structural similarity or identity between the capsular polysaccharides.

| 123j The location of cps 5 in the genome relative to pbpla and pbp2x varied between the commensal strains (Figures 12A-12C), although the S. mitis cps5 locus showed the same orientation and relative genomic location between the upstream pbp2x and downstream pbpla genes as the serotype 5 pneumococcal reference strain. The two S. oralis cps5 loci were not closely linked to pbp2x, but linkage to the downstream convergent pbpla gene was observed. Genomic proximity of cps5 from the two S. infantis strains to pbp2x was also lacking, however cps5 from one of the two strains (US0024h) was situated 35 kb upstream of the convergent pbpla.

|0124] As with pneumococcal cps loci, the cps 5 locus from S. mitis KE67013 and the two S. oralis strains were situated between dexB and amiA. The cps 5 loci from the two S. infantis strains isolated within the United States differed from the other three species in that it was situated immediately upstream of the cell division gene ftsA. [0125] While a positive Quellung reaction for the four c/rs -positive non-pneumococcal strains with pneumococcal typing serum specific for serotype 5 was not observed, immunodiffusion experiments demonstrated specific reactivity of each strain with anti- pneumococcal type 5 typing serum (Figures 13 and 14). In addition, antiserum produced against strain KE67013, the strain which yielded the weakest immunodiffusion results with anti -pneumococcal type 5 serum (Figures 13 and 14), exhibited strong specific reactivity with pneumococcal serotype 5 strains in immunodiffusion (not shown) and in the Quellung reaction (Figure 15).

[0126] The antiserum raised against strain KE67013 was highly and specifically active in opsonophagocytosis killing (OPK) assays directed against serotype 5 pneumococci (Figure 16), and showed no OPK activity against pneumococci of serotypes 1 and 4. The OPK activity of 3 of the 4 antiserum samples (lst, 3rd, and pooled depicted in Figure 16) against serotype 5 S. pneumoniae was actually higher than our typing antisera prepared against type 5 S. pneumoniae, as shown in Table 4. Additionally, antisera prepared against a control strain of S. mitis carrying a full-length cps operon unrelated to cps5 showed no OPK activity against serotype 5 pneumococci.

Table 4. Opsonophagocytic killing activity of rabbit antisera raised against serotype 5 S. mitis KE67013 in 3 rabbits (sera 1 - 3) and of pooled, clarified antisera from the three rabbits (pooled).

Coefficient of variation (CV) = standard deviation/GMT;

SE (s) = GMT*(exp(standard deviation)-l)/sqrt(N);

(where x. is an individual natural-log transformed value m is the mean/expected value and N is the total number of values);

CV = s!e°² - 1. EXAMPLE 5

[0127] This example describes additional non-pneumococcal strains recovered in samples from the Kenya study that were PCR-positive for pneumococcal serotypes other than serotype 5 and were assigned non-pneumococcal species. The isolation and characterization methods used are described above in Example 4.

[00101] The additional non-pneumococcal strains were PCR positive for serogroups including 18 (assigned as S. mitis KE66713), 15A/15F (S. oralis KE66813), 33 (S. oralis KE66913, and 12 (S. oralis KE67213). When comparing the genomic locations of cps operons from A mitis (cpsl8) and S. oralis (cpsl5A, cps33F, and cpsl2F) with cps loci highly similar to those found in pneumococci of the same specific serotypes , only cps 18 from S. mitis KE66713 showed close linkage to both pbp2x and pbpla. S. mitis KE66713 (cps 18) was the only commensal isolate described here that was from a child and was recovered from a nasopharyngeal specimen.

[0128] As shown in Table 5, immunodiffusion experiments showed specific reactivity of S. mitis strain KE66713 indicative of being weakly positive for serotype 18A (reactivity with serotyping factor 18d). S. oralis KE66913 (cps33) showed pool E reactivity (pool E consists of all factors for resolution of serogroups 12 and 33, along with serotypes 13, 44, and 46) which could only be narrowed to very weak serogroup 33 reactivity. Similarly, KE67213 (cps 12) demonstrated strong reactivity with pool E, but the reactivity could only be narrowed to serogroup 12, due to no reactivity with individual serogroup 12 factors. No detectable S. oralis strain KE66813 (cps 15 A) reactivity with pneumococcal serogroup 15 antiserum was observed.

Table 5. Non-pneumococcal strains of known serotypes

Biological Sequences

[0129} The sequences referred to herein, and submitted herewith in the computer- readable nucleotide sequence listing (txt. file), are listed below:

[0130] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0131] The use of the terms“a” and“an” and“the” and“at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term“at least one” followed by a list of one or more items (for example,“at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms“comprising,”“having,”“including,” and“containing” are to be construed as open-ended terms (i.e., meaning“including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0132] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments can become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIM(S):

1. A composition comprising (a) a pharmaceutically acceptable carrier and (b) one or more nonpneumococcal commensal organisms, wherein the commensal organisms are (i) Streptococcus mitis (ii) Streptococcus oralis, or (iii) Streptococcus infantis, and wherein the commensal organisms express a pneumococcal-specific serotype for administration to a subject in need thereof.

2. The composition of claim 1, wherein the composition is a vaccine.

3. The composition of claim 1, wherein the composition is a probiotic.

4. The composition of any one of claim 1-3, wherein the composition comprises a live commensal organism.

5. The composition of any one of claims 1-4, wherein the composition comprises a purified capsular polysaccharide of a commensal organism or a segment of the purified capsular polysaccharide of the commensal organism.

6. The composition of any one of claims 1-5, wherein the pneumococcal-specific serotype is serotype 1.

7. The composition of any one of claims 1-5, wherein the pneumococcal-specific serotype is serotype 4.

8. The composition of any one of claims 1-5, wherein the pneumococcal-specific serotype is serotype 9A or serotype 9 V.

9. The composition of any one of claims 1-5, wherein the pneumococcal-specific serotype is serotype 18C or 18A.

10. The composition of any one of claims 1-5, wherein the pneumococcal-specific serotype is serotype 5.

11. The composition of any one of claims 1-5, wherein the pneumococcal-specific serotype is serotype 12F.

12. The composition of any one of claims 1-5, wherein the pneumococcal-specific serotype is serotype 15 A.

13. The composition of any one of claims 1-5, wherein the pneumococcal-specific serotype is serotype 33.

14. The composition of any one of claims 1-13, wherein the nonpneumococcal commensal organism provides cross-protection against infection from the pneumococcal organism Streptococcus pneumoniae.

15. A method for immunizing a host against infection from pneumococcal disease comprising administering an effective amount of a composition according to any one of claims 1-14.

16. A kit for administering an immunogenic composition, the kit comprising:

(a) a composition comprising (a) a pharmaceutically acceptable carrier and (b) one or more nonpneumococcal commensal organisms, wherein the commensal organisms (i) Streptococcus mitis (ii) Streptococcus oralis, or (iii) Streptococcus infantis, and wherein the commensal organisms express a pneumococcal-specific serotype, for administration to a subject in need thereof,

wherein the immunogenic composition comprises a vaccine or a probiotic, and

(b) at least one container for holding the immunogenic composition.