WO2015017823A1 - Tetravalent pneumococcal serogroup 6z - Google Patents

Abstract

The present embodiments provide for a recombinant pneumococcal serotype designated (6Z) characterized as having the genotype for a tetravalent hybrid capsular polysaccharide having the capsular polysaccharide repeating unit of serotypes (6A, 6B, 6C and 6D). In at least one embodiment, the recombinant tetravalent pneumococcus produces a capsular polysaccharide in which a single polymer contains the repeating units characteristic of serotypes (6A, 6B) and (6D); and may also include the repeating unit characteristic of serotype (6C).

Description

TETRAVALENT PNEUMOCOCCAL SEROGROUP 6Z

FEDERAL FUNDING

[0001] This invention was made with governmental support under grant R01 Al-31473, awarded by the National Institutes of Health. The U.S. Federal Government has certain rights in the invention.

RELATED APPLICATIONS

[0002] This application claims priority benefit of Application No. 61/861,705, filed 2 August 2013, and Application No. 61/896,203, filed 28 October 2013, which are incorporated entirely herein.

FIELD OF THE INVENTION

[0003] The present embodiments provide for recombinant hybrid Streptococcus pneumonia serotypes in which bacteria have a hybrid genotype encoding several capsular polysaccharide repeating units in pneumococcal serogoup 6. In some embodiments, a serotype designated 6Z has the genotype for serotype 6 A, 6B, 6C and 6D capsular polysaccharide repeating units. The hybrid serotype provides for a pneumococcal isolate that produces several serotype 6 capsular polysaccharide repeating units in a single polymer.

BACKGROUND

[0004] Streptococcus pneumonia (pneumococcus) is an important human pathogen that is often responsible for sepsis, meningitis, otitis media, and pneumonia. Pneumococcal disease is the leading cause of death in children under the age of five (according to a 2004 estimation by the World Health Organization). According to the Global Alliance for Vaccines and

Immunization (GAVI), pneumonia is the world's "biggest child killer." The crisis is particularly lethal in children of economically emerging countries, where pneumococcal vaccines are not yet integrated into a routine vaccination schedule. According to Helen Evans, interim CEO of the GAVI Alliance, "Introducing the new pneumococcal conjugate vaccine in developing countries is a critical step that can prevent millions of bouts of illness and countless deaths in children from the terrible disease that pneumonia is." Hence, there is an urgent need for understanding pneumococcal disease and emerging strains, and for developing better and more

affordable vaccines.

[0005] Pneumococcal virulence is pathogenic largely determined by a capsular polysaccharide (PS) that protects it from the host immune system. Because antibodies (Abs) to the capsular PS can opsonize pneumococci for phagocytic cells, pneumococcal conjugate vaccines (PCVs) are designed to elicit anti-capsule Abs and provide capsule-type (or serotype- specific) protection. In the U.S., a PCV with capsular PSs from seven serotypes (PCV7) was licensed in 2000, and a 13-valent PCV (PCV13) was approved for use in children in 2010 and in adults in 2011. Use of PCVs has resulted in a significant reduction in the number of deaths due to pneumococcal infection in children and adults. Thus, PCVs have an enormous impact on health care.

[0006] PCVs provide protection in a serotype-specific manner, so these vaccines must contain PSs of serotypes that are prevalent in the region where the vaccine is going to be administered. Ideally, if PCVs are effective, their use should reduce the prevalence of PCV serotypes, but not raise the population of pathogenic non-PCV serotypes. Thus, design and use of PCVs requires persistent monitoring of the epidemiology of pneumococcal serotypes circulating in the region in which the PCVs are administered. Epidemiologic studies, however, require accurate knowledge of capsular PSs. For example, PCV7, which contains serotype 6B PS, is effective against serotype 6A but not against serotype 6C. Consequently, as use of PCV7 has increased, 6A has become rare but 6C has become prevalent. Yet, prior to the

characterization of 6C serotype, it was mistyped as 6A serotype, and PCVs' cross-protection against group 6 pneumococcus was confusing. Clearly, there is a need for accurate knowledge of capsular PSs in order to design PCVs, and monitor cross-reactivity and emerging strains. In particular, there remains a need for a recombinant pneumococcal strain that carries the genotype for, and may express several if not all of, the serogroup 6 capsular polysaccharide

repeating units.

SUMMARY

[0007] The present embodiments provide for a recombinant a tetravalent pneumococcal serotype 6 that encodes the capsular polysaccharide repeating units characteristic of

serotypes 6A, 6B, 6C, and 6D. This novel recombinant construct, called serogroup or serotype 6Z, provides an efficient strain for the production of several immunogenic group 6 polysaccharides in a single polymer; and a single bacterial strain that can provide for an immunogenic construct for serogroup 6. In particular, the tetravalent recombinant

pneumococcus produces a triplevalent or tetravalent capsular polysaccharide in which the repeating units characteristic of serotypes 6A, 6B and 6D, and to a lesser extent 6C, are produced and included on a single polymer, although not is a specified order. There has been no previous description of a recombinant pneumococcal strain that encodes the four known members of serogroup 6, capable producing a single capsular polysaccharide polymer with at least three different repeating units characteristic of the pneumococcal group 6 serotypes. The present embodiments provide an important advance in monitoring emerging pneumococcal strains, and provides another weapon (a multi-hybrid polysaccharide vaccine component) in the arsenal against pneumococcal disease. The multi-hybrid strain also simplifies design and preparation of pneumococcal vaccines, and permits the production of more precise serotyping reagents. The novel, recombinant bacterium may also be used as a whole, killed immunogenic preparation; or as an attenuated vaccine preparation. The strategy of expressing several repeat units in a single polymer may be applied to streamline or broaden vaccine formulations by eliminating the need for multiple polysaccharide sources to cover multiple serogroup members (e.g., serotypes 6A and 6B).

[0008] At least one embodiment provides for a recombinant pneumococcal serotype designated 6Z characterized as having the genome that encodes a tetravalent hybrid capsular polysaccharide made up of the serotype 6A repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→3) ribitol (5→phosphate}, the 6B repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→4) ribitol (5→phosphate}, the 6C repeating unit {→2) glucose (1→3) glucose(l→3) rhamnose (1→3) ribitol (5→phosphate} and the 6D repeating unit {→2) glucose(l→3) glucose(l→3) rhamnose (1→4) ribitol (5→phosphate}, wherein the order of the repeating units is not limited to a particular order.

[0009] Additionally, at least one embodiment provides for a recombinant pneumococcal serotype designated 6Z characterized as producing a hybrid capsular polysaccharide made up of the serotype 6A repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→3) ribitol (5→phosphate}, the 6B repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→4) ribitol (5→phosphate}, and the 6D repeating unit {→2) glucose(l→3) glucose(l→3) rhamnose (1→4) ribitol (5→phosphate}. In an aspect of the embodiments, the several polysaccharide repeating units are produced, in any order, in a single polymer. In an additional aspect of this embodiment, the hybrid capsular polysaccharide further comprises the serotype 6C repeating unit {→2) glucose (1→3) glucose(l→3) rhamnose (1→3) ribitol (5→phosphate}. In a particular embodiment, the hybrid polysaccharide exhibits the serological profile shown in FIG. 13A. In another particular embodiment,

[0010] Another embodiment provides for an immunogenic composition comprising a pharmaceutically acceptable carrier or an adjuvant and a purified or isolated capsular polysaccharide derived from a recombinant pneumococcal strain, wherein the capsular polysaccharide is characterized as a single polymer including hybrid capsular polysaccharides having, in any order, the 6A repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→3) ribitol (5→phosphate}, the 6B repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→4) ribitol (5→phosphate} , and the 6D repeating unit {→2) glucose(l→3) glucose(l→3) rhamnose (1→4) ribitol (5→phosphate}. In an additional aspect of this embodiment, the hybrid capsular polysaccharide further includes the serotype 6C repeating unit {→2) glucose (1→3)

glucose(l→3) rhamnose (1→3) ribitol (5→phosphate}. The capsular polysaccharide can be conjugated to a protein carrier. The adjuvant can be a protein or an aluminum salt. The immunogenic composition can be included in a vaccine.

[0011] Another embodiment relates to antigen-binding molecules, such as antibodies, specific for the recombinant multiple hybrid serotype polysaccharide. A related embodiment provides for a panel of antigen-binding proteins (e.g., antibodies), that differentiates multiple hybrid serotype from other serogroup 6 pneumococci in an immunoassay.

[0012] A further embodiment provides for an isolated recombinant strain of

Streptococcus pneumonia, designated serotype 6Z, characterized as encoding a multivalent hybrid capsular polysaccharide comprising the 6A repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→3) ribitol (5→phosphate}, the 6B repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→4) ribitol (5→phosphate} , the 6C repeating unit {→2) glucose (1→3) glucose(l→3) rhamnose (1→3) ribitol (5→phosphate} and the 6D repeating unit {→2) glucose(l→3) glucose(l→3) rhamnose (1→4) ribitol (5→phosphate}.

[0013] Another embodiment provides for a gene cassette including at least a portion of a mutated pneumococcal wciP gene in which the mutated WciP expresses C195, A192, R254, or all three of A192-C195-R125 (ACR). Another embodiment provides for a gene cassette including at least a portion of a mutated pneumococcal wciNa gene in which the mutated WciNa expresses SI 50. Another embodiment provides for a gene cassette including at least a portion of a mutated pneumococcal wciP gene, in which the mutated WciP expresses C195, A192, R254, or ACR, and at least a portion of a mutated pneumococcal wciNa gene in which the mutated WciNa expresses SI 50. These gene cassettes are useful in constructing a panel of isogenic target strains by transferring particular cassettes or combinations of cassettes to a recombinant TIGR4 genomic background.

[0014] The recombinant multivalent serotype, such as serotype 6Z, expresses nonspecific glycosyltransferases, produces capsular polysaccharides with multiple types of repeating units as a single polymer, and may produce polysaccharides that elicit broadly cross-reactive antibodies that can simplify pneumococcal conjugate vaccine development. Because

pneumococcal infections are often caused by serogroup 6 pneumococci, accurate knowledge of serogroup 6 is critical to updating or using pneumococcal conjugate vaccines, which are important health care tools in fighting pneumococcal disease. DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1 presents structural models for the capsular polysaccharide repeating units of serotypes 6A, 6B, 6C and 6D. Glc' indicates the second glucose residue in the repeating units of 6C and 6D PSs.

[0016] FIG. 2 shows a scheme for an approach to constructing two recombinant tetravalent S. pneumoniae (MB0189 and MBP190) that express the capsular polysaccharide repeating units of serotypes 6A, 6B, 6C and 6D. Primer binding sites are indicates with black circle and primer name. Allelic exchange is described by dashed lines. Nucleotide exchanges are indicated as #: D38N; $: A150S; *: A150T.

[0017] FIG. 3 shows that 6X11 and 6X12 are serologically distinct from other members in Serogroup 6. Flow cytometry histograms of various pneumococcal strains (indicated at the left of each row) that were stained with different monoclonal antibodies (mAbs) (indicated at the bottom of each column).

[0018] FIG. 4 shows that 6X11 and 6X12 PS have chemical structures different from other known Serogroup 6 PS. ^]H NMR spectra of 6A, 6C, 6X12 PSs, 6B, 6D and 6X11 PSs. NMR spectra were aligned at 5.6 ppm, normalized to have equal-sized Rha peaks, and identified with labels. Glc' indicates the second glucose residue in the repeating units of 6C and 6D PSs. Note that 6X11 and 6X12 PSs have both Gal and Glc' peaks.

[0019] FIG. 5 presents an overlay of ^-"C HMQC spectra for PS representing serotypes 6A (black) and 6X12 (gray) and 6B (black) and 6X11 (gray). For 6X12 and 6X11, new glucose (Glc') signals appear indicating their PSs are a mixture of two different repeating units (RU). Panel A: The chemical shifts (ppm) ofthe labeled Gal and Glc' peaks of 6X12 and 6A overlap at 5.60 ppm indicating 75% of 6X12 RUs are identical to 6A while 25% are 6C- like because they contain Glc' . Panel B: Anomeric signals of Gal and Glc' 6X11 and 6B overlap at 5.12 ppm indicating 40% of 6X11 RUs are identical to 6B while 60% are 6D-like because they contain Glc' . Panel C: Proposed structural models of 6X11 and 6X12 PSs.

[0020] FIG. 6 shows that 6X12 and 6X11 cps loci differ from a 6 A cps by very few residues. Comparison of 6X11 and 6X12 cps loci with two published 6 A and 6C loci (GenBank Accession Numbers EF538714 and CR931638). The scale at the top indicates nucleotide sequence positions. The ends of 6X11 and 6X12 cps sequences were shown as jagged edges at dexB and aliA genes. The wciN and wciP alleles were indicated by a and β. 6X12 and 6X11 cps did not have wciNfi allele but were 99.99% identical to a 6A cps. The dashed or dark lines respectively indicate the difference at codons 38 or 150 of wciNa. [0021] FIG. 7 is a schematic for creation of isogenic mutant strains. Primer binding sites are indicated with black circles and primer names. Allelic exchanges are described by dashed lines. Mutations are indicated by symbols (*, A150T; #, D38N; $, A150S).

[0022] FIG. 8 is a comparison of immunologic (Panel A) and chemical (Panel B) properties of the four WciNa mutants with three reference strains (6A, 6B, and 6X12). For immunological comparison, all the strains were stained with 6A (Hyp6AG4) or 6C (Hyp6DM5) specific mAbs, the amount of mAb bound to bacteria were determined with a flow cytometer and the amount (mean fluorescent intensity) were plotted in both axes. The amount of Hyp6AG4 bound to MB0182 was artificially reduced by 20% to provide better visual separation between MB0182 and MB0177. For chemical comparison, the capsular PSs were purified from the mutants and were analyzed by NMR to obtain chemical shifts in the anomeric region.

[0023] FIG. 9 shows flow-cytometric stains of four pneumococcal strains (identified in each panel) with mAbs specific for 6 A (thin line), 6B (thick line), and serogroup 6 (dashed line). Note that MNZ1130 carrying the S195C mutation is stained with both 6A- and 6B- specific mAbs.

[0024] FIG. 10 presents the DNA sequence of the genes encoding the capsular polysaccharide of a recombinant 6Z construct, MBO190 (SEQ ID NO:9). WchA: underlined; WciNa: italics, Asp38Asn, Alal50Thr and a samesense mutation: bold and underlined;

HG272: underlined; WciO: italics; WciP: Garamond font, A->G samesense mutation, T->C Metl28Thr, Alal92, Cysl95 and Arg254: bold and underlined; wzy follows immediately after WciP in plain font.

[0025] FIG. 11 presents the DNA sequence of the genes encoding the capsular polysaccharide of a recombinant 6Z construct, MB0189 (SEQ ID NO: 10). WchA: underlined; WciNa: italics, A-T, Asn9Tyr, Asp38Asn, Alal50Ser and a samesense mutation bold and underlined; HG272: underlined; WciO: italicized; WciP: Garamond font, A->G samesense mutation, T^C Metl28Thr, Alal92, Cysl95, Arg254 bold underlined; wzy follows

immediately after WciP in plain font.

[0026] FIG. 12 is a summary of the genotypic and phenotypic features of S. pneumoniae serogroup 6 isolates, including 6Z isolates MBO190 and MB0189. $: the reference WciP sequence is based on a GenBank sequence, CR931638; M128T represents an allelic difference. #: classical 6C and 6D have WciN instead of WciNa. $$: the reference WciNa is based on a GenBank sequence, CR931638. N9Y was introduced during genetic manipulation. *: 6Xd expresses serologic properties of 6A and 6B. 6Z expresses serologic properties of 6A/6B/6C/6D.

[0027] FIG. 13A shows serological profiles of strains used in the some of the present embodiments. Indicated strains (at left) were incubated with supernatants from indicated hybridomas (at bottom) producing antibodies specific for each unique serogroup 6 repeat unit prior to staining with appropriate secondary antibodies. Y-axes represent number of events at a given fluorescent intensity. Gray shaded area represents secondary-only control.

[0028] FIG. 13B illustrates a sandwich ELISA demonstrating expression of 6A and 6B repeat units in the same polymer. Capsular PS was captured by a 6A-specific monoclonal antibody (Hyp6AG3) and detected using a 6B-specific monoclonal antibody (Hyp6BMl) as described herein. A 6D-specific isotype-matched antibody (Hyp6DM3, "irrelevant IgM") was used as a negative control.

[0029] FIG. 13C shows a one-dimensional ^]Η NMR spectragraph of wciP mutants expressing hybrid repeat units. Gal, galactose; Glc, glucose; Rha, rhamnose; Glc', the second glc in serotype 6C/6D repeat units.

DETAILED DESCRIPTION

[0030] All patents and other publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[0031] As used herein and in the claims, the singular forms include the plural reference and vice versa unless the context clearly indicates otherwise. The term "or" is inclusive unless modified, for example, by "either." Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term "about."

[0032] Unless otherwise defined, scientific and technical terms used in connection with the formulations described herein shall have the meanings that are commonly understood by those of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0033] This specification includes a Sequence Listing, which is incorporated fully herein for all purposes. [0034] Streptococcus pneumonia (pneumococcus) is a persistent, opportunistic commensal of the human nasopharynx and is the leading cause of community-acquired pneumonia. It expresses and anti-phagocytic capsular polysaccharide (PS). Musher 14 Clin. Infect. Dis. 801 (1992). Although there are many pneumococcal serotypes, only a few serotypes are primarily associated with invasive disease and current pneumococcal vaccine target those serotypes. Robbins et al., 148 J. Infect. Dis. 1126 (1983). For instance, serogroup 6 includes commonly pathogenic serotypes and is targeted to some extent in commercial vaccines.

Hausdorff et al., 30 Clin. Infect. Dis. 100 (2000). Widespread pneumococcal vaccination exerts selective pressure against the serotypes in the vaccine and promotes the emergence of novel, non- vaccine serotypes. Steenhoff et al., 42 Clin. Infect. Dis. 907 (2006); Singleton et al., 297 JAMA 1784 (2007). Thus, serotype surveys of pneumococcal isolates are performed in many counties. In Germany, for example, about 20,000 invasive pneumococcal disease isolates were collected from 1992 to 2012 and about 7% belong to serogroup 6. van der Linden et al., 8 PLoS One e60848 (2013).

[0035] Genetic variation of the capsular polysaccharide synthesis (cps) locus is the molecular basis for structural and antigenic heterogeneity of the capsule types (serotypes). Serogroup 6 has four known members (6A, 6B, 6C and 6D) with distinct serological properties, homologous cps loci and structurally similar PSs. The characteristic capsular polysaccharide repeating units (RUs) of 6A, 6B, 6C and 6D can be depicted as shown here (see also FIG. 1):

[0036] Thus, the PSs of serogroup 6 have similar yet distinct chemical structures: Gal is present in 6A and 6B PSs, but a second glucose (Glc2) is present in 6C and 6D PSs. The linkage between Rha and Rib is 1→3 for 6A and 6C PSs, but is 1→4 for 6B and 6D PSs. See U.S. Patent No. 8,440,815.

[0037] The four serotypes are also serologically similar, but can be distinguished with either rabbit factor sera or monoclonal antibodies (mAbs) (see FIG. 12). Bratcher & Nahm, J. clin. Microbiol. 3378 (2010); Oftadeh et al., 48 J. Clin. Microbiol. 3378 (2010); Park et al., 2007a; Bratcher et al., 2010. Serological properties can be distinguished according to serum factors as shown below, in which "Fs" denoted "factor serum" and Fs 6b is specific for 6A:

Fs 6a Fs 6b Fs 6c Fs 6d

6A Pos Pos Neg Neg

[0038] The group 6 cps locus, which is -17 kb long and bound by the genes dexB and aliA, has genes ordered in sequence dexB, wchA, wciN, wciO, wciP, wzy, wzx, aliA. The genes wchA, wciN, wciO, and wciP encode transferases; and wzy and wzx, respectively, encode polymerase and flippase.

[0039] Genetically, all the serotype 6 strains have similar cps loci containing fourteen genes (in -17 kb), but differences exist: serotypes 6A and 6B have wciNa belonging to

Pfam01501, whereas serotypes 6C and 6D have wciNfi belonging to Pfam00534. aMore specifically, the cps of serotypes 6A and 6B have wciN a, encoding a- 1,3 -galactosyl-transferase whereas serotypes 6C and 6D have wciNfi encoding a-l,3-glucosyl-transferase. Because wciN a and wciNfi can be distinguished easily, they are commonly used as the genetic marker to distinguish 6C/6D from 6A/6B. Park et al., 2007b.

[0040] Although wciP can be useful in distinguishing 6A/6C from 6B/6D, it is used infrequently for that purpose because wciP sequences are so similar. Jin et al., 47 Clin.

Microbiol. 2470 (2009). In contrast, WciP is the basis for the differentiating 6A/6C from 6B/6D: WciPa links rhamnose to ribitol through an a(l-3) linkage, while WciP mediates an a(l-4) linkage. The two WciP alleles have distinct amino acid triads at positions 192, 195, and 254. Among the three residues however, residue 195 most strictly correlates with serotype; serine in WciPa correlates with 6A and 6C but asparagine in WciP correlates with 6B and 6D. Mavroidi et al., 186 J. Bacterid. 8181 (2004); Park et al., 45 J. Clin. Microbiol. 1225 (2007a); Jin et al., 200 J. Infect. Dis. 1375 (2009); Bratcher et al., 156 Microbiol. 555 (2010); Park et al., 75 Infec. Immun. 4482 (2007b); McEllistrem & Nahm, Clin. Infect. Dis. (2013); Oliver et al., 288 J. Biol. Chem. 25976 (2013); U.S. Patents No. 8,440,815 and No. 8,481,054. Nevertheless, the canonical sequence of the wciP of 6A (named wciPa) codes for A192, S195 and R254, whereas that of the wciP of 6B (named wciPfi) has S192, N195 and G254. Sheppard et al., 17 Clin.

Vaccine Immunol. 1820 (2010); Mavroidi et al., 2004. [0041] The following table shows the wciN and wciP geotypes of serotypes 6A-6D (see also FIG. 12):

[0042] In addition to these serotypes, an additional serotype 6E has been reported, which has a cps locus that differs strikingly from those of known serogroup 6 members, and its serologic and biochemical properties have not yet been reported. Ko et al., 51 J. Clin.

Microbiol. 3395 (2013).

[0043] Recently, two additional atypical serogroup 6 isolates, named 6X11 and 6X12, were discovered in Germany. Using serogroup 6-specific mouse monoclonal antibodies, 6X11 was found to have serological properties of both 6B and 6D, whereas 6X12 has serological properties of both 6A and 6C. Studies of their capsular PSs with NMR methods revealed that 6X12 PS had two different repeating units; about 75% was that of 6A PS and about 25% was that of 6C PS. The 6X11 PS was also found to contain two different repeating units; about 40% of 6B and 60% of 6D. Genetic studies of the two strains revealed mutations in the wciNa gene. The 6X12 isolate had one mutation (A150T) and 6X11 had two mutations (D38N and A150T). Using site-directed mutagenesis, the A150T mutation, but not D38N mutation, was found to make a 6A strain acquire hybrid serologic and chemical profiles like 6X12. An additional recombinant 6X12 was made by introducing an A150S into the WciNa gene product. The hybrid serotypes represented by 6X12 and 6X11 strains were named serotypes 6F and 6G, respectively. Single amino acid changes in cps genes encoding glycosyl-transferases can alter substrate specificities, permit biosynthesis of heterogeneous capsule repeating units, and result in new hybrid capsule type may differ in their interaction with the host immune system. Oliver et al., 2013.

[0044] Additionally, atypical serogroup 6 isolates were discovered in London, that had serological properties of both 6A and 6B. Genetic studies of the wciP glycosyltransferase gene of these isolates revealed that a mutation at position 195, particularly N195S, resulted in a disruption of glycosyltransferase specificity, with additional substitutions at position 192 and 254 affecting complete switching between the serogroups. In particular, strains expressing wciPa A192, C195 and R254 (ACR) (which differs from the canonical wciPa ASR) and wciPfi S192, S195 and G254 (SSG) (which differ from the canonical wciPfi SNG), expressed both 6A and 6B serotype polysaccharides. No further biochemical or site-directed mutation studies were undertaken with these isolates. Sheppard et al., 17 Clin. Vaccine. Immunol. 1820 (2010).

[0045] The present embodiments provide for the site-directed mutagenesis for the construction of isogenic strains in a serotype 6A strain. The constructs differed only in the mutations in their wciPa genes and determined whether either triad was sufficient to impart WciP with the ability to form both a(l-3) and a(l-4) linkages. In particular, it was found that the mutation S195C resulted in a serotype that consistently exhibited the serological properties of both serotypes 6 A and 6B.

[0046] The present embodiments also provide for the engineering of a strain encoding all four genetic variations with the possibility that the strain would be capable of producing three or four unique Serogroup 6 repeating units. For example, the bi-specific variant wciNa' was introduced into a strain encoding bi-specific WciP.

[0047] The discovery of atypical group 6 isolates demonstrates gaps in the knowledge of group 6 pneumococci. One gap concerns the genetic, biochemical, and serological nature of the atypical group 6 isolates. Importantly, the other gap concerns assessing their direct impacts on PCVs, particularly both the epidemiology and ability of current PCVs to cross-protect against these strains. The present embodiments provide for a recombinant atypical, multivalent PS strain that is useful in generating immunogenic compositions, including vaccines that may reduce the number of separate PS strains used in vaccine and immunology studies. A reduction in PCVs complexity provides a solution to a critical problem in current PCVs.

[0048] More specifically, PCVs are very effective and successful therapeutic tools against pneumococcus, a major human pathogen, and our proposed studies will have direct impact on PCVs in many ways. For example, the recombinant pneumococcus will improve epidemiologic studies of group 6 pneumococci by accurately recognizing group 6 variants, which are found in Germany, England, Korea and other countries. Additionally, key nucleotides associated with serotype changes in group 6 can be confirmed and manipulated. This knowledge will greatly improve pneumococcal serotyping because genetic approaches of serotyping are used increasingly. Furthermore, the present embodiments reveal a novel approach to reduce PCVs' complexity because a multivalent repeating unit PS expressing 6 A, 6B and 6D antigens, or a tetravalent repeating unit PS expressing 6A, 6B, 6C and 6D antigens, could replace two (6A and 6B) PSs in PCV13 and one (6B) PS in PCV7.

[0049] The present recombinant bacterium also provides for studies of PCVs that can facilitate the development of new conjugate vaccines that target capsular PS or LPS of other bacteria. For instance, reducing the number of PS in conjugate vaccine is relevant to conjugate vaccines against Shigella, because they would also need to contain many LPS serotypes. [0050] The present invention also highlights the importance of single codon changes in epidemiologic studies of other bacteria. Indeed, changes of one critical codon are able to alter the PS structure of the Neisseria meningitides capsule or the Campylobacter jejuni

lipooligosaccharide. Additionally, the various genetic construct cassettes prove a useful means of manipulating genotype to further explore factors affecting phenotype; and serve as a model for the manipulation of other bacterial species.

[0051] Moreover, the construction of the multivalent or tetravalent 6Z bacterium facilitates the identification of this serotype in nature, whether it exists now or comes to exist in the future. The skilled artisan may then consider the possible existence of multi-RU PSs when studying pneumococcal capsules as well as other bacterial capsules or LPSs. Also, multi-RU PSs may raise new questions in glycobiology, such as regulatory controls for synthesizing and polymerizing different RUs. For instance, nutrient factors may control the relative use of one RU over the other. This invention provides models useful to the investigation of such questions.

[0052] Additionally, the genetic constructs of the present embodiments can be used to manipulate bacterial genotypes for a variety of phenotypes. More specifically, distinct recombinant gene cassettes with the mutations in the wciN or wciP genes, or both, as described herein can be "mixed and matched" to express desired features of the serogroup 6 pneumococci. By examining the expression of the mutated genes serially and in combination, the impact of media and other growth conditions on PS production can be determined. These genetic cassettes may also be inserted into other expression systems to explore their function in alternative hosts.

[0053] The present embodiments also provide for the possibility that one tetra-RU PS expressing four antigens (6A, 6B, 6C and 6D) or one triple-RU PS expressing three antigens (6A, 6B and 6D) can replace two (6A and 6B) PSs in PCV13, an one (6B) PS in PCV7. This is an important technological innovation that can make PCVs affordable worldwide. Current PCVs are too expensive to be used worldwide - the annual cost of PCVs exceeds $3 billion - because it is difficult to manufacture a PCV with many different PSs. In addition to serotypes 6A and 6B PSs, PCV13 also contains serotypes 19A and 19F PSs, which differ in only one glycosidic linkage, just as with 6A and 6B PSs. Using the present embodiments as a model, the skilled artisan will be able to find or create a pneumococcal strain expressing capsular PS with the RUs of both 19A and 19F. Thus, simplification of PCVs by using multiple-RU PSs may not only apply to serogroup 6, but to serogroup 19 or other pneumococcal serogroups as well. Further, this technological innovation would apply to the development of other vaccines (e.g., shigella LOS vaccine) and may make other conjugate vaccines more affordable.

[0054] The recombinant tetra-valent genotype can be made, for example, by introducing a 150T or 150S mutation into the WciNa gene product and introducing a C195 mutation into the WciP glycosyltransferase gene product. The mutation in the WciNa gene product disrupts the specificity UDP-Gal-only transferase to allow UDP-Gal and UDP-Glc substrates, thus allowing either glucose or galactose to be inserted in the first position of the capsular polysaccharide RU. The mutation in the WciP glycosyltransferase gene product allows the rhamnose-ribitol linkage to be either a 1→3 or 1→4 linkage. This combination is based on the following genetic and phenotypic observations:

[0055] Structural analysis of the tetravalent RU elucidates the novel and unusual structure and potential alternative explanations (e.g., unexpected glycosidic bond elsewhere) through systematic determination of the nature of monosaccharide, monosaccharide

conformations and isomers, glycosidic bonds, and potential modifications (e.g., acetyl group) throughout the entire PS. The verification requires complete NMR signal assignments (e.g., ring region with numerous NMR peaks) and not just the anomeric region. Additional NMR spectra such as NOESY, COSY and TOCSY verify the PS conformation and glycosidic bonds.

Furthermore, ^]H-³¹P HMBC is useful for studying the Rib-(5→P→2)-Gal/Glc2 region of the RU.

[0056] Multiple analytical approaches are employed to confirm the PS structures. The predicted monosaccharide/alditol compositions are analyzed using methanolysis, chemical modification, and GC. Furthermore, analytical approaches will include periodate treatment (to distinguish amounts of Glcl and Glc2), alkali hydrolysis (to distinguish between Rha(l→3)Rib and Rha(l→4)Rib linkages), and Smith degradation (to confirm glycosidic linkages). These approaches have been used extensively in previous studies of 6C and 6D PS structures.

[0057] If the NMR data of the tetra-valent polysaccharides suggest unexpected structures (e.g., novel linkages for Rib-Gal or Rib- Glc2), additional NMR experiments are done

(e.g, ^]Η-³¹Ρ HMBC) to verify the PS structures. Also, the genetic basis for such novel PS structures is identified by genetic recombination studies with various parts of cps locus.

[0058] As noted above, studies suggest that 6X11 and 6X12 arose from 6B and 6A strains respectively, following the mutation of wciNa at codon 150. There are more than 100 additional cps mutations, however, that may alter other parts of capsular PS of 6X11 and 6X12. In addition, genes outside of cps loci can participate in capsular PS production. For instance, extra cps genes are involved in capsule production for pneumococcal serotypes 1 and 37.

Alternatively, a strain may not produce sufficient amounts of UDP-Gal, and the shortage of UDP-Gal may increase the use of UDP-Glc. Thus, it may be interesting to directly determine the role of D38N and A150T mutations of WciNa in atypical capsule production. The same is true for S195C mutations of WciP. Thus, it may be possible to manipulate the expression levels of the wciN and wciP genes by further genetic modification, or by using culture conditions such as substrate availability. For example, the altered wciN and wciP genes could be expressed simultaneously or sequentially; or simultaneously to different extent depending on the quantity of each RU desired for a particular purpose.

[0059] Further, the creation of the serotype 6Z provides for the production and isolation of anti-6Z antigen-binding molecules, such as anti-6Z antibodies. These can be prepared by conventional means well known in the art in light of the current specification. In this regard, the term antigen-binding molecules includes intact immunoglobulin molecules, monoclonal antibodies, purified polyclonal antibodies, as well as portions, fragments, peptides and derivatives thereof, such as, for example, Fab, Fab', F(ab')₂, Fv, fragments, CDR regions, or any portion or peptide sequence of an immunoglobulin molecule that is capable of binding a 6Z antigen, epitope, or mimotope: all of which may also be referred to as an "antigen-binding molecule." An antibody or antigen-binding molecule is said to be "capable of binding" an antigen if it is capable of specifically reacting with the antigen to thereby bind the antigen to the antibody or antigen binding molecule. See, e.g. , WO US2006/014720; WO US2006/015373. The antigens of the 6Z serotype may also be used to raise antibodies that might be used for passive protection. Such methods are also well-known in the art. A panel of antigen-binding molecules (e.g., monoclonal antibodies) can be assembled to differentiate 6Z from other serotypes, and particularly among serogroup 6.

[0060] The tetravalent PS can be used in studies that assess PCVs for their ability to elicit cross-protection against atypical strains by examining the opsonophagocytic capacity (OPA) of immune sera. The mutant cassettes can be used to construct a panel of isogenic target strains expressing all the different typical and atypical group serotypes by transferring the mutant cps loci to a TIGR4 genomic background. These isogenic strains are used to eliminate non-capsule factors affecting OPA results. The panel includes wild type strains, strains with cps of the hybrid A/B, A/C and B/D variants, and the novel recombinant strain 6Z described herein with cps containing mutated wciN and wciP. Any newly discovered novel atypical strains can be included as target strains. Anonymized sera from children who were immunized with PCV13 are collected, and then OPA performed using the target strains, immune sera, and baby rabbit sera as a complement source.

[0061] Such antibody responses can be characterized by ELISA, whole cell ELISA, serum bactericidal assay and a flow cytometric opsonophagocytosis assay using established protocols, the Streptococcus pneumoniae opsonophagocytosis using differentiated HL-60 cells (Promyelocytic Leukemia Cell Line) Laboratory Protocol prepared by Steiner, et al., the Centers for Disease Control and Prevention, and Emory University Atlanta, GA, including the 'draft 2.0 version' which can be found at http://www.vaccine.uab.edu/refer/cdcops3.htm.

[0062] Previous work has indicated that the OPA correctly predicted cross-protection. Lee et al., 16 Clin. Vaccine Immunol. 376 (2009); Yu et al., 180 J. Infect. Dis. 1569 (1999). Also, the ability to provide cross-opsonization by individual sera (id.) or individual mAbs (Sun et al., 69 Infect. Immun. 336 (2001)) is either none or complete; and that the percent of samples providing cross-opsonization reflects cross-protection. Therefore, the percentage of samples providing cross-opsonization is determined. In the past, very few vaccinees produced cross- opsonic activity against 19A; indeed, PCV7 was not cross-protective against 19A. In contrast, PCV7 (which contains conjugate 6B PS) showed cross-opsonization against serotype 6A in about 50% of vaccines; and this was sufficient to provide cross-protection to the U.S.

population, perhaps due to herd immunity. Thus, it is anticipated that cross-opsonic activity by 50% of samples should be sufficient to provide cross-protection to a population.

[0063] Although rabbit and human complement yielded similar pneumococcal OPA results (Romero-Steiner et al., 4 Clin. Diagn. Lab. Immunol. 415 (1997)), OPA results with the new target strains may depend on the source of complement. Thus, the results with OPA can be confirmed using human complement. Anti-capsule antibodies can provide protection in vivo by mechanisms other than opsonophagocytosis. Burns et al., 73 Infect. Immun. 4530 (2005). If cross-opsonophagocytosis is not observed for some atypical group 6 isolates, evaluating cross- protection against the atypical isolates using passive protection in mice can be done. Briles et al., 153 J. Exp. Med. 694 (1981); Briles et al., 182 J. infect. Dis. 1694 (2000); Glover et al., 76 Infect. Immun 2767 (2008). Animal models of pneumococci are well known in the art, as is conducting studies involving passive protection. Immune sera from children is injected into the mice 4 hours before intravenous infection with typical (e.g., 6B) and the atypical strain being tested. This approach requires more serum volume than the OPA and serum pools can be used.

[0064] An important prospect is that the tetravalent serotype may help practitioners overcome the major limitation of PCVs: their complexity. PCV13 has 13 different PS-protein conjugates in a single vaccine, which is considered to be the most complex vaccine in history and is very expensive to produce. In addition to 6B and 19F PS, PCV13 contains 6 A and 19A PS, because the use of PCV7 with 6B and 19F PSs alone led to increased prevalence in 6C and 19A infections. Park et al., 2008; Nahm et al., 2009; Hicks et al., 196 J. Infect. Dis. 1346 (2007). The need for 19A PS was surprising because 19A and 19F PSs differ by only one glycosidic linkage just as do 6A and 6B PSs. Kamerling, in STREPTOCOCCUS PNEUMONIA MOLEC. BIOL. & MECH. DIS. 81 (Tomasz, ed., Mary Ann Liebert, Inc., Larchmont, NY, 2000). If a PS containing RUs of serotypes 6A, 6B, 6C and 6D can elicit cross-protection against serogroup 6, PCVs can be significantly simplified and improved. This hypothesis can be examined by immunizing mice with a protein conjugate vaccine with the tetravalent PS.

[0065] Regarding the importance of vaccine, S. pneumoniae is a well-known human pathogen and a major etiologic agent for pneumonia, meningitis, otitis media as well as sepsis, primarily among young children and older adults. Fedson, VACCINES, 271 (Plotkin & Mortimer, eds., W.B. Saunders Co., Philadelphia, Pa., 1988). The most prominent virulence factor of pneumococcus is the PS, which coats the surface of the bacterium to block antibodies and complement from binding to surface moieties and being recognized by phagocytic cells. Avery & Dubos, 54 J. Exp. Med. 73 (1931). More specifically, the capsule interferes with phagocytosis by preventing C3b opsonization of the bacterial cells. Anti-pneumococcal vaccines are based on formulations of various capsular (polysaccharide) antigens derived from the highly- prevalent strains.

[0066] The novel serotype 6Z bacterium, particularly the triplevalent or tetravalent PS obtained therefrom, as provided herein, may be useful in an immunogenic composition, including a vaccine, or in pneumococcal vaccine development. An immunogenic composition comprises a molecule capable of inducing an immune response directed to the novel, recombinant bacterium 6Z or the capsular polysaccharide purified from the bacterium; or the immunogenic composition may induce cross-reactivity against several of the serotypes 6A-6D. An immunogenic composition elicits an immunological response of a cellular or antibody- mediated immune response to the composition in the subject. The immunogenic composition may reduce the incidence or severity of a clinical symptom associated with pneumococcal infection, stymie pneumococcal colonization, prevent manifestation of symptoms of

pneumococcal disease, or affect pneumococci directly, for example, by directly or indirectly killing the bacteria. The immunogenic composition may confer protective immunity against one or more of the clinical signs of pneumococcal infection.

[0067] One aspect of the present invention is directed to a method of eliciting or inducing, in a mammal, an immune response directed to pneumococci, the method comprising administering to the mammal an effective amount of an immunogenic composition comprising the triplevalent or tetravalent polysaccharide repeating unit, capable of inducing an immune response directed to the 6Z bacterium, the 6Z polysaccharide, or two or more members of the serogroup 6 pneumococci, e.g., 6A, 6B, 6C or 6D. The term "mammal" includes humans, non- human primates, livestock animals, laboratory test animals, companion animals, and captive wild animals.

[0068] Still yet another aspect of the present invention relates to the use of a composition comprising the triplevalent or tetravalent polysaccharide capable of inducing an immune response directed to pneumococci in the manufacture of a medicament for the therapeutic or prophylactic treatment of a mammalian disease condition characterized by infection

with pneumococci.

[0069] For example the 6Z PS, or a mimetic of the 6Z PS may be incorporated into a pneumococcal vaccine. Conjugate vaccines comprising streptococcal and pneumococcal PS are well-known in the art. See e.g. , U.S. Patents No. 7,189,404, No. 6,248,570; No. 5,866,135; No. 5,773,007. Commercially available PCV13 (PREVNAR 13®, Pfizer) is conjugated to diphtheria toxoid. Tetanus toxoid is another common protein for commercially available polysaccharide conjugate vaccines (for example, Pentacel®, which includes Haemophilus b PSconjugated to tetanus toxoid). Additionally, PS mimotopes, such as protein or peptide mimetics of polysaccharide molecules, are also possible as alternative antigens or immunogens. See, e.g. , Pincus et al., 160. J. Immunol. 293 (1998); Shin et al., 168 J. Immunol. 6273 (2002). Additionally, the proteins or nucleic acids of 6Z may serve as antigens or immunogens in vaccine or vaccine development using any number of techniques known in the art.

Pneumococcus -derived protein carriers are also known in the art. See, e.g. , U.S. Patents No. 6,936,252, No. 6,027,734, No. 6,042,838, No. 6,592,876, No. 7,189,404, No. 7,078,042; U.S. Patent Publications No. 2011/0008419, No. 2009/0170162; WO 2009/020391.

[0070] Additionally, one or more adjuvant agents may be included in such vaccines. The adjuvant may be selected from the range of adjuvants known to induce high levels of antibody, including, for example, water in oil emulsions, oil in water emulsions, water in oil in water double emulsions, saponin or derivatives of saponin, DEAE-dextran, dextran sulphate, aluminum salts (e.g., alum, aluminum phosphate, aluminum hydroxide), squalene, and nonionic block co-polymers. The vaccine may include other immunomodulators, such as, for example, muramyl-dipeptide and derivatives, cytokines, and cell wall components from species of mycobacteria or corynebacteria. The vaccine may include a combination of two or more adjuvants or immunomodulators. The level of active component and adjuvants or

immunomodulators are chosen to achieve the desired level and duration of immune response. [0071] The delivery of pneumococcal vaccines, either by parenteral, mucosal, or other administration, and the design, monitoring, and dosing regimens of such vaccines (e.g., more than one vaccination), are also well-known in the art.

[0072] The recombinant tretravalent pneumococcus of the present invention is also useful in monitoring the epidemiology of atypical strains among isolates from around the world. Group 6 commonly causes IPDs and therefore is clinically important; consequently, our understanding of its epidemiology must be accurate. Yet, the recent discovery of atypical group 6 serotypes shows that existing surveys of the serotypes making up group 6 are flawed. Further, the prevalence of atypical group 6 isolates is not only unknown, but also may vary throughout the world. For instance, the prevalence of serotype 6D is extremely low in the U.S. (Massire et al., 50 J. Clin. Microbiol. 2018 (2012)); but is high in Korea (2.7 % (26 6D isolates/948 isolates)) where about half of the Korean 6D isolates may be serologically atypical (factor serum 6b+) (Choi et al., 16 Emerg. Infect. Dis. 1751 (2010); Baek et al., 49 J. clin. Microiol. 765 (2011)). Also, the atypical group 6 isolates are inconsistently mistyped in epidemiologic surveys. For instance, the two German variants were typed as 6C and 6D with serologic typing, but they would have been typed as 6A and 6B, respectively, if genetic typing was used. Thus, there is a major gap in world-wide epidemiologic surveys of this important serogroup.

[0073] Thus, group 6 isolates from four different regions of the world: Korea, Germany, the U.S., and Brazil are obtained. Group 6 isolates will be identified at the collection sites with the quellung method using factor serum 6a, which positively identifies all the known typical and atypical isolates. Henrichsen et al., 33 J. Clin. Microbiol. 2759 (1995). Although each collection differs in composition, the collections will include IPD and nasopharyngeal isolates from adults and children. These isolates are analyzed using a multiplexed assay for all four classical group 6 serotypes. The multiplex assay can distinguish all the known atypical strains from typical group 6 isolates, because the variants display a secondary serotype in addition to the primary serotype, and because the multiplex assay can recognize the presence of even a small amount (-1%) of a secondary serotype in addition to the primary serotype. Yu et al., 18 Clin. Vaccine

Immunol. 1900 (2011). A study of about 1000 serogroup 6 isolates from each region will provide statistical confidence of atypical hybrid isolates with a prevalence of -0.3% among serogroup 6 isolates. Incidentally, the prevalence of the isolates expressing both 6A and 6B serotypes is -0.3% in England. Sheppard et al., 17 Clin. Vaccine Immunol. 1820 (2010). In addition to the observed atypical patterns (i.e., 6A/6B, 6A/6C, 6B/6D), the present invention provides for identifying an as-yet undiscovered atypical group 6 isolate displaying a new pattern of 6Z, namely 6A/6B/6C/6D. If such an isolate is identified, it can be characterized as described herein by studying the isolate' s serologic, genetic, and chemical properties. Such novel atypical strains will also be examined as OPA target strains.

[0074] The prevalence of atypical isolates may be too low to be detected among 1000 isolates. But because the typing assay has high throughput, more isolates from one region, such as the U.S. where the CDC conducts a very large survey, can be screened. Some atypical isolates may express a secondary serotype in minute amounts (e.g., less than 1% of the primary serotype). Thus, to complement current assays that are based on an inhibition-type assay, a capture-type ELISA can be developed for all the group 6 PSs using ELISA wells coated with immobilized mAbs and polyclonal rabbit antisera as the secondary Ab, as described previously. Sheppard et al., 2010. This assay is sensitive and should identify atypical isolates expressing even minimal amounts of secondary serotypes. Antibodies recognizing epitopes of the 6Z tetravalent PS RUs will be particularly useful in these assays for characterizing and monitoring emerging atypical strains.

EXAMPLES

Example 1. Alterations to wciN results in 6A/6C and 6B/6D hybrid RUs

[0075] Bacterial strains and other reagents. Reference strains expressing serotypes 6A, 6B, 6C, 6D or no capsule were respectively TIGR6A, TIGR6B, TIGR6C, TIGR6D and TIGRJS, which were produced in the genomic background of TIGR4 by previously described genetic manipulations. Park et al., 2007a; van der Linden et al., 2013; Trzcinski et al., 69 Appl. Environ. Microbiol. 7364 (2003). MNZ21 is a clinical isolate expressing serotype 6D . Bratcher et al., 2010. The 6X11 and 6X12 strains were clinical isolates from Germany named PS6657 and PS 16864, respectively, and the strains were re-derived from a single colony on blood agar plates to avoid a potential mixture of two different serotypes. All pneumococci were grown at 37°C in 5% C0₂ in Todd Hewitt broth (BD Biosciences, San Jose, CA) containing 0.5% yeast extract (THY), harvested at A₆oo of 0.4-0.6, and aliquoted in THY with 15% glycerol. The aliquots were kept at -80°C until needed.

[0076] Serotype 6B PS was purchased from ATCC (Manassas, VA, U.S.). Serotypes 6A and 6C PSs were purchased from Staten Serum Institute (SSI, Copenhagen, Denmark).

Hyp6BM8, Hyp6AM3, Hyp6AGl, Hyp6BMl or Hyp6DM5 are mouse hybridomas that produce serogroup 6 specific IgM antibodies. Park et al., 2007a; Sun et al., 69 Infect. Immun. 576 (2009); Yu et al., 18 Clin. Vaccine Immunol. 1900 (2011).

[0077] Site-directed mutagenesis: Mutant pneumococcal strains were created by transforming TIGR6A with appropriate genetic constructs. Park et al., 2007b. An

unencapsulated variant, named MB 0163, was created by allelic exchange of the wchA-wciN- wciO gene region with a Janus cassette (Cassette #1) as described. Trzcinski et al, 2003; Sung et al., 67 Appl. Environ. Microbiol. 5190 (2001); Xayarath & Yofer, 189 J. Bacteriol. 3369 (2007). Additional genetic constructs with desired mutations at WciNa were created by overlap extension PCR. Cassette #2 codes for WciNa with A150T mutation, cassette# 3 codes for D38N and A150T mutations, cassette #4 D38N, and cassette #5 codes for A150S mutation.

Replacement of the Janus cassette in MB0163 with cassettes #2, #3, #4 and #5 resulted in four recombinant strains, which were named MB0172, MB0182, MB0184 and MB0177, respectively. All strains had the expected antibiotic sensitivities, were established from well- isolated single colonies, and were confirmed to have only the desired genetic changes by sequencing their cps loci using the UAB Heflin Sequencing Core. The GenBank accession numbers of the deposited DNA sequences that may be relevant to the embodiments herein are 6X12: KC832410 (SEQ ID NO: 11); 6X11 : KC832411 (SEQ ID NO: 12); TIGR6A:

KC832412 (SEQ ID NO: 13); MB0163: KC832413 (SEQ ID NO: 14); MB0172: KC832414 (SEQ ID NO: 15); MB0182: KC832415 (SEQ ID NO: 16); MB0184: KC832416 (SEQ ID NO: 17); and MB0177: KC832417 (SEQ ID NO: 18); each one of which is included in the Sequence Listing incorporated herein.

[0078] Capsular Polysaccharide purification: Capsular PSs were purified from pneumococcal strains MNZ21, 6X11 and 6X12, in addition to the four mutants (MB0172, MB0182, MB0184, and MB0177) that were created as described herein. The strains were grown in two liters of chemically defined medium (JRH Biosciences, Lenexa, KS)

supplemented with choline chloride (1 g/L), sodium bicarbonate (2.5 g/L), and cysteine-HCl (0.73 g/L) and lysed with 0.1% deoxycholic acid. After removing cell debris by centrifugation, PS was precipitated step-wise in 30%, 50% and 70% ethanol and was recovered by dissolution in 200 mM NaCl. After desalting by dialysis against MilliQ water, the PS was loaded onto a 60 ml column of DEAE-Sepharose (Amersham Biosciences, Uppsala, Sweden) and eluted with a linear gradient of NaCl from 0 M to 1 M. The resulting fractions were tested for PS using a multi bead inhibition assay. Calix et al., 287 J. Biol. Chem. 18996 (2012). The PS-containing fractions were pooled, desalted by dialysis, lyophilized, dissolved in 1-2 ml of water and loaded onto a gel filtration column containing 120 ml of Sephacryl S-300 HR (Amersham Biosciences). Using a buffer containing 10 mM Tris-HCl and 100 mM NaCl, the PS was eluted from the column and all fractions were tested for PS with the multiplexed inhibition type immunoassay. Yu et al., 2001. The fractions containing the first PS peak were pooled, lyophilized and used for NMR studies. See also U.S. Patents No. 8,440,815 and 8,481,054.

[0079] NMR Spectroscopy: Briefly, 1-2 mg of lyophilized PS was dissolved in 1 mL of D₂0 for ID or 2D NMR analyses. The ID ^]H NMR data of purified PSs were collected on a Bruker DRX (600 MHz) spectrometer equipped with a cryoprobe at 25 °C. Data were analyzed with ACDINMR Processor Academic Edition (Advanced Chemistry Development, Inc., Toronto, Canada). Chemical shifts were recorded relative to internal phosphocholine eH, 3.22 ppm). The decoupled 2D ^]H-¹³C heteronuclear multiple quantum coherence (HMQC) data were collected at 45 °C on a Bruker Avance IT (700 MHz ^]H) spectrometer equipped with a cryoprobe, processed with NMRPIPE (Delaglio et al., 6 J. Biomol. NMR 277 (1995)), and analyzed with NMRVIEW (Johnson & Blevins, 4 J. Biomol. NMR 603 (1994)). Assignments of ^]H and ¹³C signals for 6X11, 6X12, 6A, and 6B PS were achieved by 2D nuclear Overhauser spectroscopy (¾-¾ NOESY), correlation spectroscopy (¾-¾ COSY), total correlation spectroscopy (¾-¾ TOCSY), and HMQC NMR methods. ^]H and "Cchemical shifts for 6A, 6B and 6C capsular PSs have been reported. Talaga et al., 19 Vaccine 2987 (2001); Cai et al., 351 Carbohydr. Res. 98 (2012).

[0080] Flow cytometry: The flow cytometric serotyping assay was performed as previously described. Bratcher et al., 2010; Calix et al., 2012. Frozen bacteria aliquots were thawed, washed and normalized to -30 of 0.02 in F ACS buffer (PBS) containing 4% fetal bovine serum, (Thermo Scientific Hyclone, Logan, Utah), added to a V -bottom ELISA plate (Nunc, Roskilde, Denmark) and incubated with a culture supernatant of a hybridoma at 1:40 final dilution. After incubation for 30 min at 4°C without shaking, the plates were washed with FACS buffer and incubated again for 30 min at 4°C with phycoerythrin-conjugated goat anti- mouse IgM antibody diluted 1: 1,000. After washing, bacteria were re-suspended in FACS buffer and examined with a flow cytometer (FACSCalibur, Becton Dickinson, Mountain View, California). The data were analyzed with FCS Express V 3.0.

[0081] The serologic properties of the two German isolates with ambiguous serotypes were defined by re-deriving strains from single colonies by flow cytometry to determine their binding to five serogroup 6-specific mAbs Hyp6BM8, Hyp6AM3, Hyp6AGl, Hyp6BMl and Hyp6DM5, which respectively react with serotypes 6A/6B/6C/6D, 6A, 6A/6C, 6B and 6C/6D. As serogroup 6 reference strains, cps variants ofTIGR4 (TIGR6A, TIGR6B, TIGR6C and TIGR6D) were used. See Park et al., 2007a; Bratcher at al., 2010; Sung et al., 67 Appl. Environ. Microbiol. 5190 (2001). Although the reference strains showed expected binding patterns (FIG. 3), 6X11 and 6X12 showed unexpected patterns. 6X11 reacted with Hyp6BM8 and Hyp6DM5 as does serotype 6D, but it also weakly but reproducibly reacted with Hyp6BMl, a 6B-specific marker: 6X11 simultaneously expressed serologic properties of both 6B and 6D. Similarly, 6X12 displayed serologic properties of serotypes 6A and 6C by reacting with Hyp6BM8, Hyp6AM3, Hyp6AGl and Hyp6DM5. These unique serologic findings of 6X11 and 6X12 were confirmed with inhibition ELISA using pneumococcal lysates and Hyp6AM3 and Hyp6BMl. Thus, 6X11 and 6X12 were serologically distinct from serotype 6A, 6B, 6C and 6D strains.

[0082] The structural characterization of 6X11 and 6X12 PS capsular PS was characterized using NMR spectroscopy. The ID and 2D NMR data revealed that the 6X12 PS contains two distinct forms of capsule RUs and that the ^]H and ¹³C chemical shifts of these two forms are essentially identical to those of 6A and 6C PSs. Because the anomeric signals in the ^]H NMR spectra correspond to residues in PS repeating units (RUs), the anomeric signals of 6X12 PS were compared with those of 6A and 6C PSs. Three signals have been observed in the anomeric region of 6A (5.60, 5.10 and 5.02 ppm), corresponding to the anomeric protons of aGal, aGlc and Rha. Cai et al., 351 Carbohydr. Res. 98 (2012).

[0083] Similarly, the ^]H spectrum of 6C has three signals (5.57, 5.10, and 5.02 ppm), which respectively correspond to the anomeric protons of aGlc', aGlc, and Rha. In contrast, 6X12 PS had the three anomeric signals of 6 A PS as well as a fourth signal at 5.57 ppm, which corresponds to aGlc' of 6C PS (FIG. 4). Thus, 6X12 PS appears to contain RUs of both 6A and 6C PSs, even though it is purified from a single bacterial colony.

[0084] To unambiguously determine the structure of 6X12 PS, sets of 2D NMR data (NOESY, COSY, TOCSY, and ^-"C HMQC) were collected for 6X12 and 6A PSs. The assignment of the peaks was greatly facilitated because 6A and 6C PS were analyzed recently in detail. Id. As shown in FIG 4 panel A, ^-"C HMQC NMR spectra of 6X12 and 6A are essentially identical except for additional six signals assig ned to aGlc' in 6C (^]H ¹³C (ppm): 5.57,99.37; 4.01,77.32; 3.84,73.70; 3.52,70.91; 4.05,73.34; and 3.80,62.05). The chemical shifts are summarized in Table 1 :

Table 1. ^]H and ¹³C chemical shifts (ppm) of 6A, 6A12, 6C, 6B and 6X11 PSs at 45°C.

Sugar/ribotol 6A PS 6X12 PS *6C PS 6B PS 6X11 PS position: W³C W³C W³C W³c W³C

-2)-a-D-Gal/>

C-l 5.60/99.42 5.60/99.42 None 5.59/99.38 5.59/99.38

C-2 4.31/74.74 4.31/74.74 None 4.28/74.95 4.28/74.95

C-3 3.99/70.26 3.99/70.26 None 3.99/70.19 3.99/70.19

C-4 4.05/71/04 4.05/71.04 None 4.06/7.05 4.06/7.05

C-5 4.30/72.15 4.30/72.15 None 4.31/72.14 4.31/72.14

C-6 3.73/62.51 3.73/62.51 None 3.72/62.45 3.73/62.50

-3)-a-D-Glcp

C-l 5.10/97.07 5.10/97.07 5.12/97.1 5.12/97.05 5.11/97.07

C-2 3.67/71.68 3.67/71.68 3.69/71.7 3.67/71.69 3.67/71.69

C-3 3.96/81/30 3.96/81/30 3.97/81.7 3.96/81.20 3.94/81.46 Sugar/ribotol 6A PS 6X12 PS *6C PS 6B PS 6X11 PS position: W³C W³C W³C W³c W³C

C-4 3.70/71.68 3.70/71.68 3.73/71.7 3.70/71.69 3.70/71.69

C-5 3.96/73.33 3.96/73.33 3.98/73.3 3.97/73.12 3.97/73.12

C-6 3.78/62.17 3.78/62.17 3.81/62.3 3.78/62.16 3.78/62.16

3.83/62.3

-2)-a-O-GlC'p

C-l None 5.57/99.37 5.58/99.3 None 5.56/99.35

C-2 None 4.01/77.32 4.04/771. None 3.98/77.34

C-3 None 3.84/73.70 3.88/73.6 None 3.85/73.69

C-4 None 3.52/70.91 3.53/71.1 None 3.53/70.96

C-5 None 4.05/73.34 4.07/73.2 None 4.05/73.26

C-6 None 3.80/62.05 3.81/62.3 None 3.82.62.13

3.83/62.3

C-l 5.02/101.91 5.02/101.91 5.05/101.7 5.14/101.26 5.14/101.26

C-2 4.21/68.74 4.21/68.74 4.23/68.7 4.26/68.63 4.26/68.63

C-3 3.83/77.04 3.83/77.04 3.85/77.2 3.87/76.85 3.87/76.85

C-4 3.57/71.98 3.57/71.98 3.61/71.96 3.58/72.03 3.58/72.03

C-5 3.85/71.24 3.85/71.24 3.86/71.08 3.78/71.05 3.79/71.16

C-6 ND ND 1.31/18/5 1.30/18.58 ND

-3/4)-D-Ribitol

C-l 3.63/64.48 3.63/64.48 3.65/64.46 3.63/64.46 3.63/64.46

C-2 3.80/64.48 3.80/64.48 3.83/64.46 3.79/64.46 3.79/64.46

C-3 3.97/73.33 3.97/73.33 3.99/73.30 3.83/73.93 3.83/73.93

C-4 3.80/81.15 3.80/81.15 3.83/81.20 3.78/73.93 3.78/73.93

C-5 4.10/71.33 4.10/71.33 4.13/71.50 4.10/78.50 4.10/78.66

C-6 4.04/68.78 4.04/68.78 4.05/68.60 4.10/66.30 4.09/66.30

4.11/68.79 4.11/68.79 4.14/68.60 4.23/66.30 4.23/66.30 aC\ carbonyl; ^bND, not determined; *6C PS chemical shift data as reported.

[0085] Based on the signal assignment of the second monosaccharide in 6A and 6C RUs, aGal and aGlc' respectively, 6X12 PS is a mixture of two RUs; approximately 75% 6A and 25% 6C. Thus, 6X12 strain produces a novel "hybrid" capsular PS (FIG. 5C).

[0086] A similar strategy has been utilized to characterize the molecular structure and sugar composition of 6X11 PS. To aid in the data analysis, ID NMR data for 6B and 6D PS, as well as 2D NMR data for 6B PS, were obtained. As expected, serotype 6B PS had three ^]H resonances at 5.59, 5.16 and 5.14 ppm, assigned to aGal, Rha and aGlc, respectively. Talaga et al., 19 Vaccine 2987 (2001). Serotype 6D PS also had three anomeric ^]H signals at 5.57, 5.16 and 5.14 ppm, which respectively correspond to aGlc' , Rha and aGlc (FIG. 4). Interestingly, 6X11 PS had four anomeric signals: three were identical to those of 6D PS and a small fourth signal at 5.59 ppm, assigned to aGal of 6B PS (FIG. 4). Thus, 6X11 PS purified from a single bacterial colony appears to contain RUs composed mostly of 6D PS with a small amount of 6B PS (FIG. 4). The ^-"C HMQC NMR spectra (FIG. 5, panel B) of 6B and 6X11 PSs are identical except for the additional six signals in 6X11 (W^ (ppm): 5.56,99.35; 3.98,77.34; 3.85,73.69; 3.82,62.13; 3.53,70.96; and 1.05,8.22) (Table 1). These signals were assigned to aGlc' in 6D. Collectively, the NMR data demonstrate that 6X11 PS contains two different RUs (40% 6B and 60% 6D), confirming that 6X11 is a new "hybrid" capsule type. The structural model of 6X 11 is summarized in FIG. 5, panel C.

[0087] The cps loci of 6X11 and 6X12 are nearly identical to those of 6A. To determine the genetic basis for the two anomalous German strains, their capsule gene loci (cps) sequences from dexB to aliA (FIG. 6; GenBank Accession Number KC832410 and KC832411) were determined. Compared with a serotype 6A cps sequence (GenBank Accession Number

CR931638), 6X11 and 6X12 sequences were 99.9% and 98.9% identical, respectively. The sequence differences were limited to -10-100 individual nucleotides that were randomly distributed (FIG. 6). wciP allelism can distinguish serotypes 6A/6C from 6B/6D; the former group has wciPa and the latter has wciPfi. Mavroidi et al., 2004. When the wciP alleles of the two German strains were examined, the 6X12 sequence was identical to a typical 6A wciPa (GenBank Accession Number CR931638) but the 6X11 sequence was identical to the wciPfi of a typical serotype 6B isolate (GenBank Accession Number JF911503). Lin et al., 2006. Thus, this finding explains the association of 6X12 with serotypes 6A/6C, and 6X11 with 6B/6D.

[0088] Next, the sequences of wciN, which genetically distinguishes serotypes 6A/6B from serotypes 6C/6D, were examined. Serotypes 6A/6B have WciNa which adds UDP-Gal whereas serotypes 6C/6D have WciNfi that is completely different from wciNa and adds UDP- Glc. Jin et al., 2009; Bratcher et al., 2010; Park et al., 2007(b). When the two German strains were examined, they had wciNa, not wciNfi. Careful comparison of their wciNa sequences with a canonical 6A wciNa sequence (CR931638) revealed a single nucleotide substitution (G to A) at position 488 of the wciNa coding strand, changing an alanine residue A150 to threonine T150 (FIG. 6). The 6X11 wciNa had two mutations consisting of a substitution (G to A) at position 113, changing aspartic acid D38 to asparagine N38 as well as the aforementioned G448A point mutation resulting in A150T of its corresponding gene product. The two mutations were highly unusual because they were absent among all wciNa sequences of strains expressing serotype 6A or 6B in the literature. One amino acid change can convert a mono-specific glycosyltransferase to a bispecific transferase. Ramakrishnan & Qasba, 6 PLoS One e25018 (2011). Thus, the two mutations, A150T or D38N, may broaden WciNa' s specificity from UDP- Gal only to UDP-Gal and UDP-Glc, and be responsible for the observed serologic and biochemical changes.

[0089] WciNa residue 150 mediates substrate specificity. A site-directed mutagenesis strategy was employed to create four isogenic TIGR6A variants (MB0172, MB0177, MB0182, and MB0184) with mutations at residues 38 or 150 of WciNa (FIG. 7). MB0172 has WciNa (A150T), MB0182 has WciNa (D38N and A150T), MB0184 has WciNa (D38N), and

MB0177 has WciNa (A150S). A150S variant was created since it is found in WciNa of serotype 33B and human glycogenin-1. Chaikuad et al., 108 PNAS 21028 (2011). Flow cytometric studies suggested that all strains expressed equivalent amount of capsule as the parent strain and that the mutations do not alter the amount of capsule synthesized. The mutants were then studied for antigenic changes by flow cytometry using mAbs Hyp6AG4 and

Hyp6DM5, which are specific for serotypes 6 A and 6C respectively. As expected, TIGR6A and TIGR6C strains reacted with only one of the two mAbs whereas 6X12 reacted with both mAbs. Although MB0184 reacted only with the 6A-specific mAb like the 6A strain, MB0172, MB0182, MB0177 reacted with both mAbs like 6X12 (FIG 7 panel A). These findings suggest that the D38N mutation alone does not significantly change WciNa ligand specificity but the A150T or A150S mutation can alter WciNa specificity and capsular PS structure.

[0090] To directly show the change in the molecular structure, the ^-NMR spectra of all four isogenic mutants were obtained. The ^-NMR spectra of 6X12 and MB0172 were identical in all regions, providing evidence that A150T mutation alone was responsible for the altered capsule type seen with 6X12. In contrast, the spectrum of MB 0184, which has D38N mutation, was similar to that of 6 A PS, whereas the spectrum ofMB0182 with both D38N and A150T mutations showed a more prominent Glc' peak compared with Gal peak. Thus, D38N mutation alone does not alter WciNa specificity but enhances its preference for UDP-Glc introduced by A150T mutation. When the ^-NMR spectra of MB0177 was examined, it had a bigger Glc' peak than Gal peak, suggesting it produces more 6C RUs units than 6 A RUs (FIG. 8B). Thus, the A150S mutation is more effective than A150T mutation in altering WciNa substrate specificity. Taken together, the A150T is the key mutation that has altered WciNa specificity observed in the two German strains.

[0091] Although the two German strains clearly belong to serogroup 6, the present work demonstrates that they have serologic properties, biochemical features, and genetic markers that are stable, unique, and distinct from the other four members in serogroup 6. It is well known that some established serotypes differ by one to three nucleotides in their cps. For instance, serotypes 9V and 9 A differ by one single nucleotide (Mavroidi et al., 2007); serotypes 15B/15C and 18B/18C differ by two nucleotides (id.; van Selm et al., 71 Infect. Immun. 6192 (2003)); and serotypes 6A/6B differ by three nucleotides (Mavroidi et al., 2007; Sheppard et al., 17 Clin. Vaccine Immunol. 1820 (2010)). Thus, although the German strains genetically differ from serotypes 6A and 6B by only one or two nucleotides, the genetic changes of the German strains alter the enzyme function and therefore the two German strains represent two new serotypes: 6F and 6G. Serotype 6F is represented by 6X12 and has serologic properties of both serotypes 6 A and 6C. Serotype 6G has properties of 6B and 6D and is represented by the strain 6X11.

[0092] The German strains provide important insights into the molecular basis for the ligand specificity of WciNa. WciNa belongs to Pfam01501, which includes many glycosyl transferases used by viruses, bacteria and eukaryotes, and has the DXD motif well known for binding divalent cations. Wiggins & Munro, 95 PNAS 7945 (1998); Breton et al., 123 J.

Biochem. 1000 (1998). The mutation at residue 38 of WciNa is present only in one of the two German strains and its neighboring residues are not conserved among Pfam01501 members. Indeed, the present site-directed mutagenesis data revealed it not to alter ligand specificity, although it may augment the impact of mutation in residue 150. In contrast, the mutation at residue 150 is present in both German strains and is located within a highly conserved region. For instance, residues 148-152 of WciNa are conserved in human glycogenin-1 except for residue 150 (FNAGV vs FNSGV). Chaikuad et al., 2011. Because the residue 150 is variable, the residues 149-151 are herein named as NXG motif to simplify its description.

Crystallographic studies found that the NXG motif forms part of a ligand binding pocket: the NXG of N. meningitidis LgtC surrounds the "CI" of the donor ligand. Persson et al., 8 Nat. Str. Biol. 166 (2001). The NXG motif of human glycogenin-1 is located in the binding pocket and interacts "C2" and "C3" of Glc. Chaikuad et al., 2011. Molecular modeling of WciNa using PHYRE2 also predicted the NXG motifto form a ligand binding pocket. Kelley & Sternberg, 4 Nat. Protoc. 363 (2009). Pfam01501 members with NAG are often galactosyl transferases like WciNa whereas members with NSG, like glycogenin-1, are often glucosyl transferases. The present studies clearly show that substitution of alanine in NAG to threonine or serine (NTG or NSG, respectively), alters the ligand specificity of WciNa, making it capable of transferring both galactose as well as glucose. Taken together, NXG is critical to WciNa ligand specificity and probably to the specificity of all Pfam01501 members.

[0093] More interesting is that both A150T and A150S mutations turn WciNa into bi- specific transferases and the mutants produce novel PSs with two different RUs. Such a novel PS raises interesting questions such as one RU may favor termination of PS chains or require a higher substrate level than the other. Because of the interesting biochemical properties, bi- specific transferases have been sought after. So far, among eukaryotic transferases, two examples have been reported; one natural (Ramakrishnan & Qasba, 365 J. Mol. Biol. 570 (2007)) and the other that was artificially created (Ramakrishnan & Qasba, 277 J. Biol.

Chem. 20833 (2002)). In contrast, studies of bacterial transferases suggest several examples of bispecific transferases. Pneumococci with mixed capsule types have been described. Sheppard et al., 2010) and meningococci (Claus et al., 71 Mol. Micro. 960 (2009). Although they may produce PSs with mixed RUs, their genetic and chemical bases have been incompletely characterized. Better described is LOS produced by C. jejuni with a Cst-II variant (T51N). Yuki, 103(S. 1) J. Neurochem. 150 (2007). The mutation was shown to be responsible for producing two RUs with different glycosidic linkages. Using WciNa variants, the present work provided a clear example that can transfer two different ligands. mAbs that are specific for the different RUs are available, genetic manipulations are easily performed with pneumococci and a simple in vitro substrate for WciNa was found recently (Han et al., 51 Biochem. 5804 (1921)), WciNa is useful for studying molecular bases of bi-specific transferases.

[0094] The significance of PS with multiple RUs in host-pathogen interactions is intriguing. As the most exposed structure for bacteria, capsular PS is critical to host-pathogen interaction. Also a minor structural change can dramatically alter its interaction with host's adaptive or innate immunity. For instance, pneumococcal serotypes 19A and 19F, which differ by one linkage in their RUs, are starkly different in their cross-reactivity with vaccine-induced antibodies (Lee et al., 16 Clin. Vaccine Immunol. 376 (2009)) and also binding of factor H (Hyams et al., 81 Infect. Immun. 354 (2013)). Also, C. jejuni strains with the Cst-II variant elicit a unique autoimmune disease. Yuki, 2007. Thus, survival advantages for serotypes 6F and 6G can be characterized by comparing their complement binding and reaction with Abs with other serogroup 6 members.

[0095] Because capsular PS is important in host-pathogen interaction, capsule evolution has been studied extensively. Most studies found pneumococci regularly switch capsule types by acquiring new DNA from other bacteria through genetic recombination. Coffey et al., 27 Mol. Microbiol. 73 (1998); Wyred et al., 207 J. Infect. Dis. 439 (2013). One of the present embodiments provides for two single base mutations that are synergistic in capsule type alterations. Perhaps there may be the third mutation that may complete serotype change from 6 A to 6C. Thus, if individual mutations give survival benefits to pneumococci, presence of such mutational stepping stones would open an evolutionary pathway for pneumococci to alter its capsule structure without a source of foreign DNA. This serotype shift could be useful in invading deeper tissues from the nasopharynx or in rapid responses to vaccination. Interestingly, evidence for such a capsule type shift by mutation has been recently described for S. iniae infecting vaccinated fish in fish farms. Millard et al., 78 Appl. Environ. Microbiol. 8219 (2012).

[0096] Because protective immunity is associated with the surface PS, pathogens are commonly divided into discrete serotypes based on their surface PS structure. With increased knowledge of the genetic basis for PS synthesis, genetic markers are often used alone to determine serotypes. Pai et al., 44 J. Clin. Microbiol. 124 (2006); Kong et al., 54 J. med.

Microbiol. 351 (2005); Batt et al., 43 J. Clin. Microbiol. 2656 (2005). Genetic differences between two serotypes may be only one or two nucleotides, however. Furthermore, presence of PSs with mixed RUs blurs serologic boundaries and the definitions of serotypes. Thus, analytical approaches are required for accurate identification of serotypes useful for correct prediction of protective immunity.

Example 2. Alterations to wciP results in hybrid 6A and 6B repeating units

[0097] Bacterial strains and other reagents: All strains were made in the TIGR4 background {Tettelin, 2001 #8789 } . Pneumococci were grown on blood agar plates (Remel) at 37°C in 5% C02, in Todd Hewitt broth (BD Biosciences, San Jose, CA) containing 0.5% yeast extract (THY), or in chemically-defined medium (CDM; JRH Biosciences, Lenexa, KS) supplemented with choline chloride (1 g/L), sodium bicarbonate (2.5 g/L), and cysteine-HCl (0.73 g/L) at 37°C in a water bath. Pneumococci were cultured in the liquid media until their OD600 was -0.6 (THY) or 1.2 (CDM). Working samples were generated by mixing cultures with an equal volume of medium, supplementing with 80% glycerol to a final concentration of 16%, and freezing aliquots at -80°C until use.

[0098] Antibiotics were purchased from Sigma-Aldrich (St. Louis, MO) and used at the following concentrations when appropriate: kanamycin, 100 μg/mL; streptomycin, 300 μg/mL; and spectinomycin, 100 μg/mL. Serotype 6A and 6B capsular PS was pur-chased from the Staten Serum Institute (Copenhagen, Denmark). Mouse hybridomas Hyp6AM3 (IgM isotype), Hyp6AG3 (IgG), Hyp6BG3 (IgG), Hyp6BMl (IgM), Hyp6CG6 (IgG), and Hyp6DM3 (IgM) produce monoclonal antibodies specific for 6 A, 6A/6C, 6B, 6C and 6D respectively. Park et al., 2007a; Sun et al., 2001; Yu et al., 2011 ; Oliver et al., 2013.

[0099] Genetic manipulations: Primers used in this example are listed in Table 2:

rom re erence sequence.

[0100] Mutated wciPa genes were introduced into TIGR6A and mutated wciPfi into TIGR6B. TIGR6A and TIGR6B have respective serotypes 6 A and 6B cps loci in the genetic background of TIGR4. More specifically, TIGR6AX (TIGR6A wciN Janus cassette, (Park et al., 75 Infect. Immun. 4482 (2007)) was transformed with a modified Janus cassette (Sung et al., 2001) in which the kanamycin resistance gene was replaced with aad9, a spectinomycin resistance gene (obtained from the plasmid pCLT1242, see Dong et al. 190 J. Bacterid. 2350 (2008)) to generate TIGR6AX2 (TIGR6AX wciP::aad9-rpsL⁺). Constructs 1 and 2, each encoding different triads of residues at positions 192, 195, and 254, were generated using overlap extension PCR from primers described in Table 1 as follows: 5' fragments (wze-wciP') were amplified from TIGR6A chromosomal DNA using primer 5114 and the reverse primer containing the desired mutation(s); 3' fragments ('wciP-wzx) were amplified from TIGR6A (construct 2) or TIGR6B (construct 1) chromosomal DNA using the forward primer containing the desired mutation(s) and primer 3144. The resulting products were mixed and amplified using internal primers 5113 and 3143, yielding constructs 1 and 2. The PCR products were digested with BspHI, and ligated with ligase; and then inserted to pneumococcal strain TIG6AX2 by selecting streptomycin resistant colonies. TIGR6AX2 is a pneumococcal strain that lacks the wcipP gene and is sensitive to streptomycin and resistant to spectinomycin, prepared from TIGRA by inserting a Janus cassette (Park et al., 2007) that lacks the wciP gene but has a spectinomycin resistance gene. Recombinant bacterium MNZ1130 expresses WciPa with S195C; MNZ1126 expresses WciP with N195S. All mutations were confirmed with DNA sequencing by the Heflin Center for Human Genetics, University of Alabama at Birmingham, Birmingham, AL, or Macrogen Company, Seoul, Korea. [0101] Flow cytometry: Flow cytometric serotyping assays (FCSAs) were performed as previously described. See Bratcher et al., 2010; Calix et al., 2012. Frozen bacterial aliquots were washed and resuspended in FACS buffer (phosphate buffered saline [PBS; 140 mM NaCl, 3 mM KC1, 5 mM Na₂HP0₄, 2 mM KH₂P0₄, pH 7.4] with 3% fetal bovine serum and 0.01% sodium azide). Fifty microliters containing ~5 x 105 CFU were incu-bated with 50 μL· hybridoma culture supernatants at 4°C for 30 minutes. After incubation, bacteria were washed twice and incubated with either (1) goat-anti-mouse IgG antibody conjugated with phycoerythrine (Southern Biotech, Birmingham, Ala.), or (2) goat-anti-mouse IgM antibody conjugated with phycoerythrine-Cy7 (Southern Biotech, Birmingham, Ala.). After washing, the bacteria were examined in a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, Calif.).

The data were analyzed with FCS Express versions 3.0 and 4.0 (De Novo Software, Los Angeles, Calif.).

[0102] Sandwich ELISA: To investigate whether multiple epitopes were present in a single carbohydrate polymer, a sandwich ELISA using monoclonal antibodies of differing isotypes was developed. ELISA plates were coated with rabbit-anti-mouse IgGl antibody (Zymed) by an overnight incubation at 4°C. Plates were washed three times with PBST (PBS with 0.05% tween-20) and incubated with Hyp6AG3 (1: 100 in PBST) for 1 hr at 37°C. After washing, the plates were blocked with 3% powdered skim milk in PBST. Bacterial lysates, which was prepared with deoxycholate (see Yu et al., 2011), were serially diluted 10-fold in PBST and were added to the plates for 1 hour incubation at 37°C. After washing with PBST, Hyp6BMl (1 : 100 dilution) was added into the wells and incubated for 1 hr at 37°C. Detection antibody (anti-mouse IgM-alkaline phosphatase, Sigma) was diluted 1 : 10,000 in PBST and added to each well prior to incubation at 37°C for 1 hr. After washing, plates were incubated with p-nitrophenyl phosphate (Sigma) for 2 hours before their optical density at 405 nm was read with a microplate reader (BioTek Instruments Inc., Winooski, VT). (All added volumes were 100 μL· per well except washes.)

[0103] Purification of Capsular PS: Capsular PS was purified from strains MNZ1130 by anion exchange chromatography of mutanolysin-treated cultures as previously described (see Bratcher et al., 2009), with the modification that elution from DEAE-sepharose columns was performed with a step gradient of 0-1 M NaCl in 0.1 M increments. The capsular PS containing fractions were identified by capsule specific ELISA and pooled. Capsular PS in the pool was precipitated with 70% ethanol, dialyzed against H₂0, and lyophilized. The lyophilized capsular PSs were dissolved and fractionated by size with Sephacryl S-300 HR. The fractions containing the first capsular PS peak were isolated, pooled, lyophilized for nuclear magnetic resonance (NMR) analysis. [0104] Nuclear Magnetic Resonance spectroscopy: For ^]H-NMR analysis,

approximately 10 mg of lyophilized capsular PS was dissolved in D20, and spectra were collected with a 500-MHz NMR spectrometer (INOVA-500) at 25 °C. Data were analyzed with ACD/NMR Processor (Advanced Chemistry Development, Inc., Toronto, Canada). Chemical shifts were assigned based on published structures reported elsewhere. See Cai et al., 2012; Oliver et al., 2013.

[0105] Preliminary data showed that MNZ1126 behaved entirely like serotype 6A without trace of serotype 6B. Flow-cytometry data indicated that MNZ1130 has serologic properties of both serotypes 6A and 6B. See FIG. 9. Thus, the S195C mutation in WciPa leads to the atypical serologic properties of the hybrid 6A and 6B RUs: The structural basis for the atypical serologic properties may be due to a novel linkage between Rha and Rib (e.g., 1→2), but is likely due to a di-RU Ps containing 6A and 6B RUs.

[0106] Based on serologic studies (FIG. 9), it appears that MNZ1130 has significant amounts of both 6A and 6B RUs. S. pneumoniae MNZ1130 was received 31 July 2013 at the American Type Culture Collection (ATCC® patent depository, 10801 University Blvd., Manassas, VA 20110, U.S.) and processed in accord with the Budapest Treaty; and has the Accession number PTA-120514.

[0107] Additionally, the structure of MNZ1126 PS is explored using approaches described above. Based on serologic studies (FIG. 9), MNZ1126 may primarily (or only) produce the RU of 6 A PS. To investigate a possible presence of a minimal amount of 6B RU, this PS is oxidized with Periodate then hydrolyzed, and the resulting molecular fragments are examined with mass spectrometry as reported previously. Because 1→3 and 1→4 linkages produce molecular fragments with different sizes, this approach detects the presence of even minimal amounts of 6B RU. If 6B RU is found, this indicates that WciPP N195S is a bi-specific enzyme with a strong preference to forming 1→3 linkages. If no 6B RU is found, this indicates that the N195S mutation has altered the specificity of WciPP and that WciPP N195S behaves like WciPa.

[0108] WciP (ACR) is sufficient for the production of serotype 6A and 6B repeat units in a serotype 6A strain. To determine whether the previously described mutations in wciP (Sheppard et al., 2010) can induce serotype 6A S. pneumoniae to express capsular PS comprised of both serotype 6A and 6B epitopes, TIGR6A-derived isogenic variants encoding WciP (ACR) (MNZ1130) or WciP (SSG) (MNZ1126) were constructed. WciP (ACR) and WciP (SSG) correspond to the mutant WciP enzymes expressed by the previously reported PN6AB4 and PN6AB 1 strains, respectively (Table 2; Sheppard et al., 2010). Through standard Quellung serotyping using factor serum 6b (which recognizes serotype 6A) and factor serum 6c (which recognizes serotypes 6B and 6D), MNZ1130 serotyped as serotype 6B, and MNZ1126 serotyped as 6A, consistent with the reported Quellung serotypes of isolates PN6AB4 and PN6AB 1 (see Table 2, below; Sheppard et al., 2010).

[0109] MNZ1126 and MNZ1130 were further serotyped with monoclonal antibodies. The specificity of the murine hybridomas used to serotype isolates is shown in FIG. 13A ("TIGR6A"-"TIGR6D"). MNZ1130 was bound by 6A- and 6B-specific monoclonal antibodies, indicative of expression of both repeat units; however, MNZ1126 was bound by only 6A- specific antibodies (FIG. 13A). An additional mutation (Y91C) in WciP (SSG) of MNZ1126 prevents conclusively stating that the SSG triad is insufficient for dual RU expression;

nonetheless, the ACR triad is sufficient for alteration of WciP specificity.

[0110] Moreover, MNZ1130 expresses serotype 6A and 6B repeat units within the same polymer chain. To determine whether capsular PS of MNZ1130 is expressed with 6A and 6B RUs within a single polymer, as with hybrid capsules composed of 6A/6C RUs (6F) or 6B/6D RUs (6G) (see Oliver et al., 2013), a sandwich ELISA was developed using mono-clonal antibodies of differing isotypes. Serotype 6A RUs in 6X13 PS were captured on a microtiter plate by a 6A/6C-specific antibody (Hyp6AG3). Hyp6DM3, an isotype-matched negative control antibody that is 6D-specific (FIG. 13A) did not recognize 6X13 PS (FIG. 13B), but captured PS was bound by a 6B-specific antibody (Hyp6BMl) in a concentration-dependent manner, indicating that 6X13 contains both serotype 6A and 6B RUs in a single PS

chain (FIG. 13B).

[0111] Bispecific WciP (ACR) preferentially forms a(l-4) linkages. To investigate the chemical structure of 6X13 capsular PS, ^-NMR was utilized to examine the ratio of 6A and 6B RUs in MNZ1130. Spectra were consistent with previously describe shifts for the anomeric carbons of serogroup 6 RUs. See Cai et al., 2012; Oliver et al., 2013. Based on the area under the anomeric peak corresponding to rhamnose for 6A (5.05 ppm) compared to that of 6B (5.17 ppm), 6X13 capsular PS is disproportionately composed of 6B RUs relative to 6A (-9: 1) (FIG. 13C). The strong bias towards 6B could account for the relatively weak Quellung reaction for MNZ1130 (corresponding to PN6AB4 described by Sheppard, et al., see Table 2) observed with factor serum 6c (sero-type 6B/6D-specific) relative to factor serum 6b (serotype 6A- specific), as described previously.

[0112] The work described in this example demonstrated that the ACR triad is sufficient for expression of a 6A+6B hybrid, but the SSG triad did not appear to be capable of expressing this hybrid. The residue at position 195 of WciP appears to be a key determinant of WciP specificity, modulating the ability to link rhamnose to ribitol with either an a(l-3) or a(l-4) linkage. Because this new serotype appears to relevant in identifying clinical isolates, this hybrid serotype expressing epitopes for both 6A and 6B (6X13) may be designated serotype 6H. The same bispecificity may be conferred to WciP in serotype 6C in vitro, permitting a strain to express 6C and 6D units in the same polymer chain (termed 6X14), but such an isolate has yet to be identified in vivo. When and if it is discovered, it may be designated serotype 61.

[0113] The change in triad from ASR to ACR, is in essence, a S195C substitution. Serine and cysteine are identical except for a single atom: serine contains oxygen in a hydroxyl group (R-OH) while cysteine contains sulfur in a sulfhydryl group (R-SH). It is extraordinary and unexpected that the exchange of a single atom of similar character is sufficient to alter the linkage specificity of WciP. The D194 residue of WciP is expected to be an active site residue, forming a hydrogen bond to orient the ribitol substrate. See Charnock & Davies 38

Biochem. 6380 (1999). The side group of the residue at position 195 would extend in the opposite direction, making a direct role in catalysis unlikely. The cysteine sulfhydryl group would be expected to form weaker hydrogen bonds than the more polar hydroxyl of serine, and perhaps this reduction relaxes the tertiary structure of the enzyme to accommodate altered substrate orientation and linkage formation.

[0114] Genes for at least three bispecific enzymes are currently known to occur within S. pneumoniae cps loci. Two such enzymes, WciN (serogroup 6) and WcrL (serotype 11D) mediate the addition of multiple sugars (Oliver et al., 2013; Oliver et al., 288 J. Biol.

Chem. 21945 (2013b); the third, described here and previously (Sheppard et al, 2010), mediates two different linkages of its substrates. These are part of a growing repertoire of described and impli-cated bispecific transferases in eukaryotes and prokaryotes alike. Ramakrishnan et al., 2002; Ramakrishnan et al., 2007; Oliver, 2013; Oliver, 2013(b); Claus et al., 2009;

Yuki et al., 2007.

[0115] The present work shows that bispecific transferases can arise from single nucleotide changes within their respective genes. The ability of single nucleotide differences to alter capsular PS serotype presentation has important ramifications for serotyping by qualitative genetic methods (e.g., PCR) alone. Indeed, as shown herein, small changes in nucleotide sequences have proven troublesome for typing by genetic means; single base or short changes have caused entire serotype changes in serogroup 6, as has been explored in Serogroup 11 (Calix et al., 2010; Oliver et al., 2013(b)), and a variant wzy allele in serogroup 19 resulted in mistyping of serotypes 19F and 19 A, which vary by the Wzy-mediated linkage, in multiplex PCR serotyping schemes. Pimenta et al., 47 J. Clin. Microbiol. 2353 (2009); Siira et al., 50 J. Clin. Microiol. 2727 (2012); Menezes et al., 51 J. Clin. Microbiol. 2470 (2013). Example 3. Construction of tetravalent pneumococcus by combining the wciNa and wciP genetic variants

[0116] Strains used for final experiments are described in Table 3, and strains used during the genetic manipulations are also described herein.

Table 3. Strains used in examples

[0117] To generate IPZ2002, wciNa was replaced with wciNfi in MNZ1130 through allelic exchange with Janus cassette. See Park et al., 2007. Replacement of wciNa with wciNfi in MNZ1130 results in expression of capsule polysaccharide containing serotype 6C and 6D epitopes. A serotype 6A strain can be induced to express serotype 6C capsular PS (or a serotype 6B strain to express serotype 6D capsular PS) when the wciNa gene is replaced with wciNfi {Park, 2007 #5579;Bratcher, 2010 #5984}. The wciNa of MNZ1130 was replaced with the wciNfi gene from TIGR6C to generate IPZ2002, which should express capsular PS with epitopes for both serotypes 6C and 6D (termed 6X14). FCSA showed that IPZ2002 reacts with 6C- and 6D-specific monoclonal antibodies (FIG. 13A, "IPZ2002"). NMR spectra confirmed that galactose (5.63 ppm) was replaced with glucose (5.60 ppm) in IPZ2002 and that the serotype 6C and 6D RUs were present at a ratio similar to that be-tween serotype 6A and 6B RUs in MNZ1130 (FIG. 13C).

[0118] To generate MBO 190, a wciN allele encoding the bispecific WciNa ' (D38N, A150T) was introduced into MNZ1130 through allelic exchange with Janus cassette as described. See Oliver et al., 2013. A partial cps locus sequence for MBO190, including wciNa' and wciP, was submitted to GenBank (accession no. KF597302). [0119] A serotype 6A strain encoding bispecific WciNa' and WciP (ACR) expressed a hybrid capsule comprising at least serotype 6A, 6B, and 6D repeat units. Combining bispecific wciNa ' and wciP (ACR) alleles may permit a serogroup 6 strain to produce capsular PS containing all four unique serogroup 6 RUs. To investigate this possibility, MBO190, a MNZ1130 derivative encoding the bispecific glucosyl/galactosyl transferase WciNa' and WciP (ACR), was created. FCSA clearly demonstrated the presence of 6A, 6B, and 6D repeat units (FIG. 13A, "MBO190"); but the 6C repeat unit was not reproducibly detected using this serological approach.

[0120] A recombinant pneumococcal isolate having both variants as constructed in herein encodes four different RUs representing all four serotypes in group 6. Such a complex PS has not been reported, and its biologic and serologic properties are unknown. One interesting possibility is that such a complex PS that expresses all the serologic properties of group 6 may elicit Abs that cross-react with all group 6 PSs, and thus may be valuable as a PCV component.

[0121] A recombinant strain is constructed in the laboratory by inserting the wciN Janus cassette from MB0177 (see Example 1) into MNZ1130, which already expresses WciPa with the S195C mutation (see Example 2). After confirming the genetic alteration by sequencing the involved region of the cps locus, the recombinant bacterium is tested for having broad serologic properties; and then examined for the chemical structure of its PS. An alternative approach in generating the tetravalent strain involves mutating the wciP of MB0177 using primers as taught herein (see Example 2).

[0122] Two particular 6Z constructs, MBO190 and MB0189, were constructed as illustrated in FIG. 2. A summary of the DNA sequence and mutations resulting in particular amino acid substitutions for MBO190 is shown in FIG. 10. A summary of the DNA sequence and mutations and particular amino acid substitutions for MB0189 is shown in FIG. 11. The sequences of these constructs were also deposited with GenBank, and have the following accession information: Bankltl657682 MB0189: KF597301; Bankltl657682

MBO190: KF597302. Recombinant pneumococci containing the 6Z genotype were deposited with the ATCC® patent depository (10801 University Blvd., Manassas, VA 20110, U.S.), received 17 September 2013 and processed in accord with the Budapest Treaty, and have the Accession numbers: S. pneumoniae MBO190, PTA-120596 and S. pneumoniae

MB0189, PTA-120597.

[0123] Pneumococci unable to export the RUs cannot recycle undecaprenol and are not viable; unless their RU synthesis is completely blocked through a secondary mutation in cpsE. Thus, the genetic recombination described herein can be done in a pneumococcal strain possessing defective cpsE. In such a case, the cpsE mutation would completely block the production of any RU and would thus permit free manipulation of cps genes without having an impact on viability. If genetic recombination can be readily created with cpsE mutants, that finding excludes technical difficulties of genetic manipulation as the reason for the failure to create a recombinant strain by inserting wciNa from MB0177 into MNZl 130 or mutating wciP in MB0177. It also provides another avenue for mixing and matching various altered cps cassettes for example for assay development, or before introducing such cassettes into production strains.

[0124] Capsular PSs from mutant 6Z strains are purified and conjugated the PSs to keyhole limpet hemocyanin (KLH) using a commercially available kit for protein conjugation (Imject® mcKLH, Pierce Biotech., Inc., Rockford, 111.). Three different conjugates for each PS are designed by varying the PS to protein conjugation ratios. 6C and 6D PS-KLH conjugates have been prepared using this conjugation kit, which has been found it to be reliable, easy to use, and to yield conjugates that were immunogenic. Conjugation of pneumococcal PS by other techniques are well-known in the art, see e.g. , U.S. Patent No. 8,440,815. To monitor the serologic properties of the new conjugates, the conjugate can be mixed with alum adjuvant, and used to immunize Balb/c mice, e.g., subcutaneously three times with the alumized conjugate; and the mouse sera obtained before and after each immunization. In this test, ~1 μg of PS per dose is used, with five mice per experimental group. One additional group of five mice is immunized with the commercially available PCV13, as a positive control. These regimens have been previously successful using the commercially available PCV7 and PS-protein conjugates.

[0125] Following immunization, ELISA is used to test the serum samples for the presence of IgG and IgM Abs cross-reacting to the four serogroup 6 serotypes. If increased Ab responses are observed, the antigen-specificity of the Abs is examined by testing the sera for binding other randomly chosen, unrelated serotypes such as serotypes 14 or 23F. Because cross- reactive Abs by ELISA may not be cross-protective, one can also determine if the immune sera can opsonize all four serotypes using OPA as well-known in the art. Burton & Nahm, 13 Clin. Vaccine Immunol. 1004 (2006); Burton et al., 19 Clin. Vaccine Immunol. 835 (2012). Following the cross-opsonization studies with serotypes 6A-6D, cross-opsonization against the

tetravalent 6 serotype using the mutant strains as OPA targets is investigated.

[0126] If the immunization does not yield a robust Ab response, antigen doses can be changed or a different adjuvant, such as Quil A, can be tested, which have been found to be useful in the past. Alternatively, the poor immunization may be caused by the instability of the new tetravalent PS. In the past, when the 14-valent PS vaccine was replaced with the 23-valent PS vaccine, 6A PS was replaced with 6B PS. Robbins et al., 148 J. Infect. Dis. 1136 (1983). The replacement was made because the Rha(l→3)Rib linkage in 6A PS is less stable than the Rha(l→4)Rib linkage in 6B PS. Some of the RUs in the tetravalent PS have the 1→3 linkage, but other RUs of the PS has only the 1→4 linkage. The tetra-valent strain, which has the RUs of all four serotypes, may elicit a more broadly cross-reactive Ab response.

[0127] Additionally, a mouse challenge model can be used to evaluate protective immunity from disease or colonization following immunization. See, e.g., U.S. Patent Pubs. No. 2011/0002962; No. 2009/0170162.

[0128] The major limitation of the conjugate vaccine strategy is increasing the number of serotypes covered. Bispecific enzymes present a novel means of simplifying conjugate vaccine production and provide a means for the inclusion of more serotypes in vaccine formulation without increasing the number of capsular PS sources required. Additional examination of residue 192 in WciP may reveal a point for optimizing WciP towards more equitable linkage formation. For example, asp-38 of WciN was shown to alter the ratio of 6A to 6C RUs in serotype 6F, although asp-38 did not determine specificity of the enzyme. With carefully applied mutagenesis and optimization, the present work shows that it may be feasible to engineer strains within this and other serogroups that produce multiple serotypes useful for vaccines, lowering the cost of vaccine production and addressing concerns over cross-protection.

Example 4. Monoclonal antibodies directed against 6Z polysaccharides

[0129] Purified 6Z PS is prepared as described herein or by methods known in the art. Conjugates of the recombinant 6Z polysaccharide to ovalbumin are prepared as follows.

Cyanogen bromide- activated PS is coupled to ovalbumin during an overnight incubation. The PS -protein conjugate is purified from the reaction mixture with a molecular weight sizing column. Alternatively 6Z PS is conjugated to Imject Blue Carrier Protein (Pierce Cat No 77130) using a CDAP procedure described. Lees et al., 14 Vaccine 190 (1996). Additional methods of PS conjugation are known in the art, and indeed pneumococcal conjugate vaccines are in commercial use.

[0130] Mouse hybridomas are produced as described previously as known in the art. See, e.g. , Yu et al., 180 J. Infect. Dis. 1569 (1999) (citing Sun et al., 69 Infect. Immun. 336 (2001)). Briefly, BALB/c mice are immunized twice subcutaneously with PS-protein conjugate (days 0 and 21) and once intraperitoneally on day 59. Each dose contains 1 μg - 10 μg of PS. The primary and secondary immunogens contain 10 μg of Quil A (Sigma Chemical, St. Louis, Mo.). Alternatively, mice (Balb/c strain) are immunized subcutaneously with the PS-protein conjugate three times at 2- week intervals (2-4 μg of PS/mouse/dose).

[0131] Three days after the last immunization, the mice are sacrificed, the spleens harvested, and the splenocytes fused with SP2/0 Ag-14 as described previously or as known in the art. See, e.g. , Nahm et al., 129 J. Immunol. 1513 (1982). Primary culture wells are screened for the production of desirable antibodies, using a pneumococcal antibody ELISA as described or known in the art. See, e.g. , Wernette et al., 10 Clin. Diagn. Lab. Immunol. 514 (2003). Wells producing such antibodies are cloned twice by limiting dilution.

[0132] The specificity of monoclonal antibody secreted by identified hybridoma(s) can be tested by a variety of known methods, such as using a multiplexed immunoassay described previously. Yu et al., 18 Clin. Vac. Immunol. 1900 (2011). This assay tests a monoclonal antibody for binding to 26 different pneumococcal PSs, which includes 6A, 6B, 6B, 6D, and multivalent PSs such as 6Z. These tests show whether the hybridoma produces antibody that binds only to 6Z PS, and whether the antibody binds to any other PSs including 19F, 19A, 6A, 6B, 6C, and 6D PS. Binding specificity can be confirmed using a additional assays (e.g., an inhibition assay). This test evaluates whether only 6Z PS can inhibit antibody binding to 6Z PS, e.g., at 1-10 μg/ml, while 6A, 6B, 6C and 6D PS may not inhibit the antibody binding to what degree at what concentration. Thus, 6Z PS may have a 6Z-specific epitope(s) despite its structural similarity to serotypes 6 A, 6B, 6C and 6D PSs; and a novel monoclonal antibody recognizes one such 6Z-specific epitope. Use of the 6Z antibody in opsonization assays will provide further insight into the activity and potential cross-reactivity of the novel antibody, and the potential of 6Z PS to provide protective immunogenicity in a vaccine.

[0133] A human- mouse hybridoma is produced by hybridizing peripheral blood lymphocytes from a person immunized with a 23-valent vaccine by methods known in the art, such as that described previously by Sun et al., 67 Infect. Immun. 1172 (1999).

[0134] Those skilled in the art will appreciate that the embodiments described herein are susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more said steps or features.

Claims

1. A recombinant pneumococcal serotype designated 6Z characterized as encoding a tetravalent hybrid capsular polysaccharide having the serotype 6A repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→3) ribitol (5→phosphate}, the 6B repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→4) ribitol (5→phosphate}, the 6C repeating unit {→2) glucose (1→3) glucose(l→3) rhamnose (1→3) ribitol (5→phosphate}, and the 6D repeating unit {→2) glucose(l→3) glucose(l→3) rhamnose (1→4) ribitol (5→phosphate}.

2. The recombinant pneumococcal serotype of claim 1, wherein the pneumococcal serotype produces a hybrid capsular polysaccharide having, in any order:

(a) the 6A repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→3) ribitol (5→phosphate}, the 6B repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→4) ribitol (5→phosphate}, and the 6D repeating unit {→2) glucose(l→3) glucose(l→3) rhamnose (1→4) ribitol (5→phosphate}; or

(b) the 6A repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→3) ribitol (5→phosphate}, the 6B repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→4) ribitol (5→phosphate}, the 6C repeating unit {→2) glucose (1→3) glucose(l→3) rhamnose (1→3) ribitol (5→phosphate}, and the 6D repeating unit {→2) glucose(l→3) glucose(l→3) rhamnose (1→4) ribitol (5→phosphate}.

3. A purified or isolated tetravalent capsular polysaccharide obtained from a recombinant pneumococcal strain, wherein the capsular polysaccharide is characterized as a hybrid capsular polysaccharide having, in any order, the 6A repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→3) ribitol (5→phosphate}, the 6B repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→4) ribitol (5→phosphate}, and the 6D repeating unit {→2) glucose(l→3) glucose(l→3) rhamnose (1→4) ribitol (5→phosphate}.

4. The purified or isolated tetravalent capsular polysaccharide of claim 2, further characterized including the 6C repeating unit {→2) glucose (1→3) glucose(l→3) rhamnose (1→3) ribitol (5→phosphate}.

5. A purified or isolated tetravalent capsular polysaccharide obtained from a recombinant pneumococcal strain, wherein the capsular polysaccharide is characterized as a tetravalent hybrid capsular polysaccharide having, in any order, the 6 A repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→3) ribitol (5→phosphate}, the 6B repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→4) ribitol (5→phosphate}, the 6C repeating unit {→2) glucose (1→3) glucose(l→3) rhamnose (1→3) ribitol (5→phosphate}, and the 6D repeating unit {→2) glucose(l→3) glucose(l→3) rhamnose (1→4) ribitol (5→phosphate}.

6. The capsular polysaccharide of claim 3 to 5, conjugated to a protein carrier.

7. The capsular polysaccharide of claim 3 to 5, bound to a solid support.

8. An immunogenic composition comprising a pharmaceutically acceptable carrier or adjuvant and a purified or isolated capsular polysaccharide obtained from a recombinant pneumococcal strain, wherein the capsular polysaccharide is characterized as a hybrid capsular polysaccharide having, in any order:

(a) the 6A repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→3) ribitol (5→phosphate}, the 6B repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→4) ribitol (5→phosphate}, and the 6D repeating unit {→2) glucose(l→3) glucose(l→3) rhamnose (1→4) ribitol (5→phosphate}; or

(b) the 6A repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→3) ribitol (5→phosphate}, the 6B repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→4) ribitol (5→phosphate}, the 6C repeating unit {→2) glucose (1→3) glucose(l→3) rhamnose (1→3) ribitol (5→phosphate}, and the 6D repeating unit {→2) glucose(l→3) glucose(l→3) rhamnose (1→4) ribitol (5→phosphate}.

9. The immunogenic composition of claim 8, wherein the multivalent capsular polysaccharide is conjugated to a protein carrier.

10. A preparation for immunizing a mammal characterized as being prepared from any one of the preceding claims.

11. A method of immunizing a mammal comprising the step of administering an immunogenic preparation derived from any one of the preceding claims.

12. An immunoassay that distinguishes a hybrid serotype characterized as having:

(a) the 6A repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→3) ribitol (5→phosphate}, the 6B repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→4) ribitol (5→phosphate} , and the 6D repeating unit {→2) glucose(l→3) glucose(l→3) rhamnose (1→4) ribitol (5→phosphate}, or

(b) the 6A repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→3) ribitol (5→phosphate}, the 6B repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→4) ribitol (5→phosphate} , the 6C repeating unit {→2) glucose (1→3) glucose(l→3) rhamnose (1→3) ribitol (5→phosphate}, and the 6D repeating unit {→2) glucose(l→3) glucose(l→3) rhamnose (1→4) ribitol (5→phosphate} ,

from other Serogroup 6 pneumococci.

13. A method of producing a mulitvalent pneumococcal polysaccharide having the repeating units of serotypes 6A, 6B, 6D, and optionally 6C, comprising culturing a recombinant bacterium that expresses C195 in WciP and S150 in WciNa of the pneumococcal cps alleles.

14. An artificial genetic construct comprising at least a portion of a mutated

pneumococcal wciP gene in which the mutated WciP is CI 95.

15. An artificial genetic construct comprising at least a portion of a mutated

pneumococcal wciNa gene in which the mutated WciNa is S150.

16. A recombinant vector comprising the artificial gene construct of one or both of claims 14 or 15.

17. A host cell comprising the recombinant vector of claim 16.

18. The host cell of claim 17, wherein the host cell is a Streptococcus cell.

19. An antigen-binding protein that specifically binds to a hybrid capsular

polysaccharide repeating unit characterized as including, in any order:

(a) the 6A repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→3) ribitol (5→phosphate}, the 6B repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→4) ribitol (5→phosphate} , and the 6D repeating unit {→2) glucose(l→3) glucose(l→3) rhamnose (1→4) ribitol (5→phosphate}, or

(b) the 6A repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→3) ribitol (5→phosphate}, the 6B repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→4) ribitol (5→phosphate} , the 6C repeating unit {→2) glucose (1→3) glucose(l→3) rhamnose (1→3) ribitol (5→phosphate), and the 6D repeating unit {→2) glucose(l→3) glucose(l→3) rhamnose (1→4) ribitol (5→phosphate).

20. A panel of antibodies that distinguishes the pneumococcal serotype 6Z of claim 1.

1. A recombinant pneumococcal serotype designated 6Z characterized as encoding a tetravalent hybrid capsular polysaccharide having the serotype 6A repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→3) ribitol (5→phosphate}, the 6B repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→4) ribitol (5→phosphate}, the 6C repeating unit {→2) glucose (1→3) glucose(l→3) rhamnose (1→3) ribitol (5→phosphate}, and the 6D repeating unit {→2) glucose(l→3) glucose(l→3) rhamnose (1→4) ribitol (5→phosphate}.

2. The recombinant pneumococcal serotype of claim 1, wherein the pneumococcal serotype produces a hybrid capsular polysaccharide having, in any order:

(a) the 6A repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→3) ribitol (5→phosphate}, the 6B repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→4) ribitol (5→phosphate}, and the 6D repeating unit {→2) glucose(l→3) glucose(l→3) rhamnose (1→4) ribitol (5→phosphate}; or

(b) the 6A repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→3) ribitol (5→phosphate}, the 6B repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→4) ribitol (5→phosphate}, the 6C repeating unit {→2) glucose (1→3) glucose(l→3) rhamnose (1→3) ribitol (5→phosphate}, and the 6D repeating unit {→2) glucose(l→3) glucose(l→3) rhamnose (1→4) ribitol (5→phosphate}.

3. A purified or isolated tetravalent capsular polysaccharide obtained from a recombinant pneumococcal strain, wherein the capsular polysaccharide is characterized as a hybrid capsular polysaccharide having, in any order, the 6 A repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→3) ribitol (5→phosphate}, the 6B repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→4) ribitol (5→phosphate}, and the 6D repeating unit {→2) glucose(l→3) glucose(l→3) rhamnose (1→4) ribitol (5→phosphate}.

4. The purified or isolated tetravalent capsular polysaccharide of claim 2, further characterized including the 6C repeating unit {→2) glucose (1→3) glucose(l→3) rhamnose (1→3) ribitol (5→phosphate}.

5. A purified or isolated tetravalent capsular polysaccharide obtained from a recombinant pneumococcal strain, wherein the capsular polysaccharide is characterized as a tetravalent hybrid capsular polysaccharide having, in any order, the 6 A repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→3) ribitol (5→phosphate}, the 6B repeating unit {→2)

39 galactose (1→3) glucose (1→3) rhamnose (1→4) ribitol (5→phosphate}, the 6C repeating unit {→2) glucose (1→3) glucose(l→3) rhamnose (1→3) ribitol (5→phosphate}, and the 6D repeating unit {→2) glucose(l→3) glucose(l→3) rhamnose (1→4) ribitol (5→phosphate}.

6. The capsular polysaccharide of claim 3 to 5, conjugated to a protein carrier.

7. The capsular polysaccharide of claim 3 to 5, bound to a solid support.

8. An immunogenic composition comprising a pharmaceutically acceptable carrier or adjuvant and a purified or isolated capsular polysaccharide obtained from a recombinant pneumococcal strain, wherein the capsular polysaccharide is characterized as a hybrid capsular polysaccharide having, in any order:

(a) the 6A repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→3) ribitol (5→phosphate}, the 6B repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→4) ribitol (5→phosphate}, and the 6D repeating unit {→2) glucose(l→3) glucose(l→3) rhamnose (1→4) ribitol (5→phosphate}; or

(b) the 6A repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→3) ribitol (5→phosphate}, the 6B repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→4) ribitol (5→phosphate}, the 6C repeating unit {→2) glucose (1→3) glucose(l→3) rhamnose (1→3) ribitol (5→phosphate}, and the 6D repeating unit {→2) glucose(l→3) glucose(l→3) rhamnose (1→4) ribitol (5→phosphate}.

9. The immunogenic composition of claim 8, wherein the multivalent capsular polysaccharide is conjugated to a protein carrier.

10. A preparation for immunizing a mammal characterized as being prepared from any one of the preceding claims.

11. A method of immunizing a mammal comprising the step of administering an immunogenic preparation derived from any one of the preceding claims.

12. An immunoassay that distinguishes a hybrid serotype characterized as having:

(a) the 6A repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→3) ribitol (5→phosphate}, the 6B repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→4)

40 ribitol (5→phosphate} , and the 6D repeating unit {→2) glucose(l→3) glucose(l→3) rhamnose (1→4) ribitol (5→phosphate}, or

(b) the 6A repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→3) ribitol (5→phosphate}, the 6B repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→4) ribitol (5→phosphate} , the 6C repeating unit {→2) glucose (1→3) glucose(l→3) rhamnose (1→3) ribitol (5→phosphate}, and the 6D repeating unit {→2) glucose(l→3) glucose(l→3) rhamnose (1→4) ribitol (5→phosphate} ,

from other Serogroup 6 pneumococci.

13. A method of producing a mulitvalent pneumococcal polysaccharide having the repeating units of serotypes 6A, 6B, 6D, and optionally 6C, comprising culturing a recombinant bacterium that expresses C195 in WciP and S150 in WciNa of the pneumococcal cps alleles.

14. An artificial genetic construct comprising at least a portion of a mutated

pneumococcal wciP gene in which the mutated WciP is CI 95.

15. An artificial genetic construct comprising at least a portion of a mutated

pneumococcal wciNa gene in which the mutated WciNa is S150.

16. A recombinant vector comprising the artificial gene construct of one or both of claims 14 or 15.

17. A host cell comprising the recombinant vector of claim 16.

18. The host cell of claim 17, wherein the host cell is a Streptococcus cell.

19. An antigen-binding protein that specifically binds to a hybrid capsular

polysaccharide repeating unit characterized as including, in any order:

(a) the 6A repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→3) ribitol (5→phosphate}, the 6B repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→4) ribitol (5→phosphate} , and the 6D repeating unit {→2) glucose(l→3) glucose(l→3) rhamnose (1→4) ribitol (5→phosphate}, or

(b) the 6A repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→3) ribitol (5→phosphate}, the 6B repeating unit {→2) galactose (1→3) glucose (1→3) rhamnose (1→4) ribitol (5→phosphate} , the 6C repeating unit {→2) glucose (1→3) glucose(l→3) rhamnose

41 (1→3) ribitol (5→phosphate), and the 6D repeating unit {→2) glucose(l→3) glucose(l→3) rhamnose (1→4) ribitol (5→phosphate).

20. A panel of antibodies that distinguishes the pneumococcal serotype 6Z of claim 1.

42