# Patent

## Title
Pneumococcal serotype 6D

## Abstract

Disclosed is a new and emerging serotype of Streptococcus pneumoniae designated serotype 6D, and assays and monoclonal antibodies useful in identifying same. Also disclosed is a novel pneumococcal polysaccharide with the repeating unit →2) glucose 1 (1→3) glucose 2 (1→3) rhamnose (1→4) ribitol (5→phosphate. This new serotype may be included in pneumococcal vaccines.

## Background
RELATED APPLICATIONS
This application is related to and claims the benefit of PCT Application No. US08/064,951, filed 28 May 2008, and U.S. Provisional Patent Application No. 60/924,703 and No. 60/924,704, both filed May 29, 2007.


This invention was made with U.S. governmental support under Contracts No. AI 30021 and No. AI-031473 awarded by the National Institutes of Health. The U.S. government has certain rights in this invention.


FIELD OF THE INVENTION
This invention relates to bacteriology, immunology, and epidemiology. More specifically, this invention relates to new and emerging serotypes of Streptococcus pneumoniae and assays and monoclonal antibodies useful in identifying these serotypes.
BACKGROUND
Streptococcus pneumoniae is a well known human pathogen and a major etiologic agent for pneumonia, meningitis, otitis media as well as sepsis, among primarily young children and older adults. S. pneumoniae has been divided into ninety serotypes based on its expression of serologically distinct carbohydrate capsules. Antibodies to a capsular polysaccharide (PS) may provide protection against pneumococci expressing the same capsular serotype. Currently available pneumococcal vaccines contain a mixture of capsular PS of multiple serotypes. For example, one pneumococcal vaccine (called PS vaccine) contains capsular PS from twenty-three commonly found serotypes. The most recently developed type of vaccine (called conjugate vaccine) contains capsular PS from seven to thirteen serotypes that are conjugated to a protein molecule. A seven-valent conjugate vaccine was introduced in 2000 for clinical use in the USA and has reduced the incidence of invasive pneumococcal diseases in children and in adults.
The distribution of pneumococcal serotypes is useful in estimating vaccine efficacy. Ideally, an effective pneumococcal vaccine would reduce the prevalence of pneumococci expressing the serotypes included in the vaccine and leave the prevalence of the pneumococci expressing non-vaccine serotypes the same. In reality, the prevalence of the pneumococci expressing non-vaccine types increases to replace those expressing the vaccine serotypes. Further, the prevalence of specific serotypes may change over time for unknown reasons. Consequently, accurate and efficient serotyping of pneumococcal isolates is important for monitoring the efficacy of pneumococcal vaccines. Indeed, identifying emerging pneumococcal serotypes and producing more efficacious pneumococcal vaccines remain crucial goals in public health.
SUMMARY OF THE INVENTION
An embodiment of the present invention provides for the identification of a new and emerging pneumococcal serotype and means for identifying same. More specifically, the present invention provides for a novel pneumococcal serotype closely related to serotypes 6A, 6B, and 6C, identified herein as serotype 6D.
An additional feature provides for an isolated culture of a bacterium designated Streptococcus pneumoniae 6D.
Another embodiment provides for a novel pneumococcal capsular polysaccharide with the repeating unit {→2) glucose 1 (1→3) glucose 2 (1→3) rhamnose (1→4) ribitol (5→phosphate}, which corresponds to S. pneumonia serotype 6D. A related embodiment provides for an antigenic composition comprising the novel 6D polysaccharide. Another embodiment relates to antigen-binding proteins, such as antibodies, specific for serotype 6D or serotype 6D polysaccharide.
Another feature provides for monoclonal antibodies (mAbs) useful in identifying emerging pneumococcal serotypes, particularly monoclonal antibodies that distinguish serotype 6D, identified here as mAb Hyp6BM6, mAb Hyp6BM7, and mAb Hyp6BM8.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts the results of an inhibition ELISA. Antibody bound to pneumococcal serotype 6A-coated ELISA plates (Y-axis) against dilution of pneumococcal lysates (X-axis). Lysates include three 6C isolates (solid symbols with continuous lines), three 6A isolates (open symbols with dotted lines), and two 6β isolates (dashed connecting lines). Antibodies used for the assay were Hyp6AG1 (Panel A), Hyp6AM3 (Panel B), rabbit Pool serum Q (Panel C) and rabbit “factor 6b” serum (Panel D).
FIG. 2 depicts opsonization assay data with various pneumococci. The number of surviving bacteria measured as a percentage of the bacteria added to the reaction well at the beginning of the opsonization assay reaction (Y-axis) was plotted against the dilution of a human serum (X-axis) used in an opsonophagocytosis killing assay. The assay used various pneumococci including a 6B isolate (open circle), two 6A isolates (open square, open triangle), and seven 6C isolates (data points connected with dashed lines). The seven 6C isolates include those from Brazil, Korea, and the U.S.
FIG. 3 illustrates an opsonization titer comparison. Opsonization titer against a 6A subtype (Y-axis) vs. opsonization titer against 6B serotype (X-axis). Circles and triangles indicate opsonization titers against 6A (labeled 6Aα) or 6C (labeled 6Aβ), respectively. The study used sera from twenty adults who were not vaccinated (left panel) or twenty adults who were vaccinated (right panel) with a conjugate vaccine (solid symbol) or a 23-valent polysaccharide vaccine (open symbol). There were ten subjects in each vaccine group. The detection limit of the assay is 4 and a sample with undetectable opsonization titer was assigned to have a titer of 2. When there were multiple data points at one spot, data points were artificially spread out to show the number of data points.
FIG. 4 presents DNA sequences of a part of the wciP gene from various pneumococcal isolates (serotype 6C is labeled 6Aβ)
FIG. 5 is a photograph of an agarose gel showing PCR products obtained with nine 6A isolates (lanes 1-9) and six 6C (labeled 6Aβ) isolates (lanes 10-15). Two “M” lanes designate DNA size marker. The two lanes show that molecules in the right side of the gel moved faster than those in the left. The two marker bands above and below the pneumococcal PCR products are 2.036 Kb and 1.636 Kb long respectively. The 6A and 6C yielded PCR products that were about 2 Kb and 1.8 Kb long respectively.
FIG. 6 presents a diagram of wchA, wciN, wciO region of the pneumococcal capsule gene locus of isolates AAU9 (middle bar) and ST745 (bottom bar in two pieces). For comparison, the top bar shows a diagram of wchA, wciN, wciO region of pneumococcal capsule gene locus based on CR931638 (a GenBank entry). Genes wchA, wciN, and wciO are labeled above the top bar along with their lengths. Nucleotide sequence positions are indicated below the top bar and the sequence position 1 shown here corresponds to the sequence position 4902 of CR931638. The ST745 strain sequence is 193 base pairs short and the shortage is shown as a gap between position 2398 and 2591.
FIG. 7 depicts the carbohydrate composition (Panel A) of capsular PS from 6A (top two panels) and 6C (6Aβ, bottom two panels) before and after periodate treatment. The monosaccharides are identified in the top chromatogram. In this GLC analysis, a monosaccharide can produce multiple peaks with characteristic retention times and relative proportions. For instance, galactose should have three peaks: first peak (short), second peak (tallest), and third peak (intermediate). Panel B shows normalized peak areas of each monosaccharide for 6A (grey bar) and 6C (6Aβ, black bar). The peak areas of all monosaccharides from each PS are normalized to the peak area of the associated rhamnose. The 6C shows no galactose peaks but shows twice as much glucose as 6A does.
FIG. 8 depicts the mass spectrum of the repeating units of 6A (Panel A) and 6C (Panel B, labeled 6Aβ) and their daughter ions (Panels C and D respectively). Mass to charge ratio (m/z) was rounded off to two decimal points.
FIG. 9 shows the mass spectrum of the repeating unit of 6C PS after oxidation and reduction (Panel A) and their daughter ions (Panels B and C). The sample used for Panel B was reduced with NaBH4 and that for Panel C was reduced with NaBD4. Mass to charge ratio (m/z) was rounded off to two decimal points. R1 and R2 (in Panels B and C) indicate that the peaks correspond to ions derived by reverse fragmentations. Numbers following the delta symbol indicate the m/z unit differences between the peaks and associated with the names of the fragments. All the peaks in Panel C correspond to the peaks in Panel B except for a peak at 136.98, which was not reproduced in Panel B and may be a contaminant.
FIG. 10 presents the proposed chemical structures of 6C capsular polysaccharide and the structure of its cleavage products. Proposed structure of the 6C repeating unit is shown in Panel C. Panels A and B shows possible molecular ions if the phosphate group is attached to ribitol and if the phosphodiester is linked to the second carbon of glucose 1. Panels D, E, and F indicate potential cleavage patterns of the repeating unit if the phosphodiester is linked to the second (Panel D), the fourth (Panel E), or the sixth carbon (Panel F) of glucose 1. Hydrated forms are shown and the residues involved in hydration are shown in parentheses. Periodate sensitive sites are shown in bold and cleavage products are shown in Panels A and F. Potential molecular ions are shown with dotted lines with arrows along with their atomic mass units. Gx and Gy are potential glucose 1 fragments and Rx is the remaining ribose fragment after oxidation and reduction reactions. Their atomic mass units are shown in parenthesis.
FIG. 11. This figure depicts the wciN region exchange experiment diagram: In step A, wchA/wciNα/wciO-P region of TIGR6A4 was replaced with Cassette 1. Cassette 1 has three parts (central core and two flanking regions) and each part is about 1 kb long. The central core has antibiotic susceptibility genes, kanR and rpsL+. The two flanking regions were made with wchA and wciO-P regions from AAU33 strain. In step B, Cassette 1 in TIGR6AX was replaced with Cassette 2. Cassette 2 has wciNβ gene, wchA and wciO-P regions from a 6C strain (CHPA388, regions labeled Aβ). TIGR6C4 shows the final product that is obtained after Cassette 2 is inserted. XbaI and BamHI sites in the PCR primers, which were introduced to simplify genetic manipulations, are shown.
FIG. 12 shows the electrophoresis pattern of the PCR products of wciN region of 6A and 6C isolates. Primers used for the PCR were 5106 and 3101, which are located in wchA and wciO genes respectively. Lanes marked M has DNA ladders. Standard markers with 2000 and 1650 bps were indicated in the left. Lanes 1-13 contain PCR products of 6C isolates, which are CHPA37 (lane 1), CHPA388 (lane 2), BG2197 (lane 3), BZ17 (lane 4), BZ39 (lane 5), BZ86 (lane 6), BZ650 (lane 7), KK177 (lane 8), CH66 (lane 9), CH158 (lane 10), CH199 (lane 11), MX-67 (lane 12), and ACA-C21 (lane 13). Lanes 14-18 contain PCR products of 6A isolates, which are CHPA67 (lane 14), CHPA78 (lane 15), BZ652 (lane 16), KK58 (lane 17) and AAU33 (lane 18).
FIG. 13 presents the nucleotide sequence of wciNβ (sometimes referred to as wciN6C) ORF along with the nucleotide sequences of the 3′ end of wchA and the 5′ end of wciO genes. The potential amino acid sequence of wciNβ ORF (SEQ ID NO:45) is shown below the nucleotide sequence (SEQ ID NO:44). Also shown are putative termination sites of wchA and wciNβ as well as putative initiation sites of wciNβ and wciO genes. The wciO gene has two potential initiation sites.
FIG. 14 shows the DNA sequences of wciNα and wciNβ regions of a 6A strain (GenBank CR931638) (bases 931-1430: SEQ ID NO:47; bases 2523-3223: SEQ ID NO:49) and a 6C strain (CHPA388) (bases 931-1430: SEQ ID NO:46; bases 2130-3330: SEQ ID NO:48). The sequence of the non-homologous mid-region of wciN (about 900-1110 bases) is not shown. Sites of PCR primers (5106, 3101, 5114, and 3113) are shown. Also shown are potential termination sites of wchA and wciN.beta.; and potential initiation sites of wciNβ and wciO.
FIG. 15 presents the genetic map of the capsule gene loci surrounding the wciN gene of 6A and 6C isolates. The map shows wchA (hatched), wciN (horizontal bars or black), wciO (checkered), and wciP (wavy) genes. The 6A locus has two unexpressed DNA fragments (indicated with arrows) in the upstream of (95 bases long) or downstream (312 bases long) to the wciNα (sometime referred to as wciN6A) gene. An alternative initiation site for wciO gene is 32 bases upstream to the initiation site shown (position 2721 for 6A). For 6C isolates, old DNA (1222 bases, region with horizontal bars) in wciNα region is replaced with a new DNA (1029 bases, black region). The replacement creates a new ORF (named wciNβ) that has 1125 bases.
FIG. 16 (panels A to D) presents the DNA sequence of the 6C serotype (isolate CHPA388) capsule gene locus (SEQ ID NO:50).
FIG. 17 indicates the chemical structure of the polysaccharide repeating units of pneumococcal serotypes 6A, 6B, 6C, and 6D.
FIG. 18 depicts the capsule gene loci of 6A (GenBank CR931638) and 6C serotypes (strain CHPA388). All open reading frames (ORFs) involved in the capsule synthesis are shown as horizontal arrows, and their direction indicates the transcriptional orientation. For both the 6A and 6C loci, the putative transcription initiation sites (bent arrow) and putative termination sites (vertical line with a solid circle) are identified using fgenesB, BPROM and FindTerm (Softberry Inc.) available at the molquest website. Transposase sequences (black boxes, labeled “tnp”) are found at both ends of the capsule gene locus. The two capsule gene loci have strikingly different wciN genes (indicated with circles). The wciN gene of 6A (and 6B) serotype is labeled as wciNα and the wciN of 6C is labeled as wciNβ.
FIG. 19 is a schematic showing the creation of a 6D strain using wciNβ gene exchange. The target DNA of Cassette 3 contains the kanamycin-resistance (kanAR) (sometimes referred to as kanR) and streptomycin-sensitivity (rpsL+) genes of the Janus cassette. The two flanking regions of Cassette 3 have the wchA and wciO-P genes from a 6B strain (strain DS2212-94), which was obtained by PCR using the primer pairs described in the figure. The three DNA fragments in Cassette 3 were then linked together by digestion with the restriction enzymes, followed by ligation with T4 DNA ligase (New England BioLabs, Beverly, Mass.). The ligation product was then amplified by PCR using primers 5113 and 3102. Cassette 2 was prepared by PCR of CHPA388 (a 6C strain, GenBank Access No. EF538714: 6522.7646) DNA using primers 5113 and 3102.
FIG. 20 shows the binding of monoclonal antibodies to 6B PS-coated ELISA plates (Y axis) in the presence of varying dilutions of bacterial supernatants containing different capsular PS (X-axis). The names of the monoclonal antibodies used for each experiment are indicated at the top of each panel. TIGR6A, TIGR6B (or TIGR6B4), TGR6C, and TIGR6D respectively produce 6A, 6B, 6C, and 6D capsular PS. The strains were prepared by replacing the capsule locus of TIGR4 with the capsule locus of serotype 6A, 6B, 6C, and 6D, respectively. TIGR6BX (or “TIGR6B-JS”) indicates a variant of TIGR6B without the wciN gene.
FIG. 21. Panel A shows the structure of the hydrated form of the repeating unit of 6D capsular PS. The calculated molecular weight is 701 AMU. Panel B shows Mass spectrum of the repeating units. The peaks at 683.3 m/z and 701.3 m/z respectively correspond to the anhydrous and hydrated forms of the repeating units. Panel C shows the daughter ions of the ion with 683.3 AMU shown in Panel B. Daughter ions are identified at the bottom of Panel C. The peaks with 270.825, 574.758, and 632.756 AMUs and their satellite peaks (separated by 2 AMUs due to chloride isotopes) represent sodium chloride salt clusters. The peaks at 270.825 represents (NaCl)4Cl−. Peaks at 574.758 AMU probably represent another salt cluster, (NaCl)9Cl−, with a water molecule, like salt clusters with organic solvent molecules. The peaks at 632.7 have one more NaCl (i.e., 58 AMUs) than the peaks at 574.758 AMU.
FIG. 22 depicts the ability of various capsular PS (2 mg/ml) to inhibit binding of mAb to ELISA plates (Y-axis) after the PS was hydrolyzed for various time periods (X-axis). “Titers” indicate the dilution of a sample necessary to inhibit the binding by 50%. For 6A and 6C PSs, ELISA plates are coated with 6A PS and mAb Hyp6AG1 is used. For 6B and 6D PSs, ELISA plates are coated with 6B PS and mAb Hyp6BM8 is used.



DETAILED DESCRIPTION OF THE INVENTION
It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such may vary. 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.
As used herein and in the claims, the singular forms “a,” “an,” and “the” include the plural reference unless the context clearly indicates otherwise. Thus, for example, the reference to an antibody is a reference to one or more such antibodies, including equivalents thereof known to those skilled in the art. 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.” 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 is 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.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those commonly understood to one of ordinary skill in the art to which this invention pertains. Although any known methods, devices, and materials may be used in the practice or testing of the invention, the methods, devices, and materials in this regard are described here.
The present invention provides for Streptococcus pneumoniae with a novel polysaccharide capsule layers. Briefly, serogroup 6 of S. pneumoniae includes two serotypes named 6A and 6B with highly homologous capsule gene loci. Recently, and as described herein, serotype 6C was identified. The 6A and 6B capsule gene loci consistently differ from each other by one nucleotide in the wciP gene. Additionally, the 6A capsule gene locus has a galactosyl transferase, but the 6C capsule gene locus has a glucosyl transferase. The present invention provides for a new serotype named “6D”, the in which the galactosyl transferase of the 6B capsule gene locus has been replaced with the glucosyl transferase of 6C. The gene transfer yields a viable pneumococcal strain, and the capsular polysaccharide from this strain has the predicted chemical structure and serologic similarity to the capsular polysaccharide of the 6B serotype. The new strain (i.e., serotype 6D) is typed as 6B by quellung reaction, but it can be distinguished from 6B strains with monoclonal antibodies to 6B polysaccharides. Reexamination of 264 pneumococcal isolates that were previously typed as 6B with classical typing methods revealed no isolates expressing serotype 6D.
The novel 6D isolate provided for herein has a chemically distinct capsular polysaccharide (PS) structure. More specifically, the repeating units of the 6D PS apparently contain one ribitol, one rhamnose, and two glucose moieties. This is the same content as serotype 6C, however the chemical linkages of the carbohydrate moities is different in these serotypes. Further, the distinct chemical structure of the PSs are antigenically distinct. Serotype 6D thus represents the 92nd pneumococcal serotype, with 90 pneumococcal serotypes having been previously recognized, (Henrichsen, 33 J. Clin. Microbiol. 2759-62 (1995)), and the 6C serotype representing the 91st serotype.
Importantly, the novel 6D serotype provided for herein offers an important alternative PS for vaccine development. More specifically, the current pneumococcal vaccines contain 6B PS rather than 6A PS because, in part, 6A PS was not stable enough in vaccine preparations. This chemical instability was due to the 1→3 linkage between rhamnose and ribitol in the 6A PS. Because the 1→4 linkage of 6B PS is more stable than the 1→3 linkage of 6A PS, 6B PS is more stable than 6A PS. The 6C linkage, provided for herein, has the more unstable 1→3 linkage whereas the 6D PS has the more stable 1→4 linkage. Thus, 6D PS may prove even more useful in a vaccine than 6C PS.
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, 271-99 (Plotkin & Mortimer eds., W.B. Saunders Co., Philadelphia, Pa., 1988). The most prominent virulence factor of pneumococcus is the capsular polysaccharide (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. 73-89 (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.
S. pneumoniae has been divided into 91 serotypes based on its expression of serologically distinct chemistry of the PS capsules. Henrichsen, 1995; Park et al., 45 J. Clin. Microbiol. 1225-33 (2007). Antibodies against the PS provide serotype-specific protection from infection, and current vaccines against pneumococcus incorporate capsular PS of the most prevalent strains. Cole, 61 JAMA, 663-66 (1913). Serogroup 6 strains are very common in invasive pneumococcal disease and the current vaccines are formulated to protect against serogroup 6 infections. Hausdorff et al., 30 Clin. Infect. Dis. 100-21 (2000).
Accurate efficient serotyping pneumococcal isolates is important for measuring the efficacy of pneumococcal vaccines. Following the introduction of a new pneumococcal vaccine, pneumococci expressing the serotypes included in the vaccine become less common while the prevalence of the pneumococci expressing non-vaccine types may stay the same. In some cases, pneumococci expressing the non-vaccine types replace those expressing the vaccine serotypes and the prevalence of non-vaccine types may become higher. Pelton, 19(1) Vaccine S96-S99 (2000). Further, the prevalence of serotypes can change over time for unknown reasons. Finland & Barnes, 5 J. Clin. Microbiol. 154-66 (1977). Because these changes influence the clinical effectiveness of a vaccine, serotyping of a large number of pneumococcal isolates is an important part of monitoring pneumococcal vaccines.
Moreover, regarding S. pneumoniae serotype 6A, current vaccine formulations do not carry a 6A PS, but carry the 6B PS, and the antibodies raised against 6B are thought to cross react against 6A. This phenomenon, is not 100%, however: Some vaccines that include the 6B PS do not raise antibodies against 6A. Yu et al., 180(5) J. Infect. Dis. 1569-76 (1999). Indeed, it appears that non-vaccine serotypes such as 6A are still causing disease in vaccinated children. Clover & Klein, Strategies for Prevention & Treatment of Pneumococcal Disease, 44th Ann. ICAAC Meeting (Washington, D.C., 2004). Hence, the emergence of additional group 6 serotypes may become even more important.
Further, the 6A and 6B serotypes account for 4.7% and 7%, respectively, of invasive pneumococcal diseases. Robbins et al., 148 J. Infect. Dis. 1136-59 (1983). Biochemical studies found that serotypes 6A and 6B PS include linear polymers of a repeating unit containing four monosaccharides: rhamnose, ribitol, galactose, and glucose. Kamerling, in S. pneumoniae: Molecular Bio. & Mechanisms of Dis. 81-114 (Tomasz, ed., Mary Ann Liebert, Inc., Larchmont, N.Y., 2000). As noted above, the 6A PS has 1→3 rhamnose to ribitol linkage and the 6B PS has 1→4 rhamnose to ribitol linkage. Id.
Although there are various serotyping methods well known in the art, they are largely manual, slow, and tedious to perform. An improved serotyping “multibead assay”, based on a multiplexed immunoassay that can be performed semi-automatically with a flow cytometer has been described. Park et al., 7 Clin. Diagn. Lab. Immunol. 486-89 (2000). The multibead assay's specificity has been fully established using pneumococcal strains representing all ninety known serotypes. Yu et al., 43(1) J. Clin. Microbiol. 156-62 (2005). This assay provides superior specificity, using many mAbs specific for pneumococcal capsular PS, and is largely automated and can provide a high throughput. Consequently, the assay may be useful in many epidemiologic studies.
The multibead assay is particularly advantageous because monoclonal antibodies are more specific than polyclonal reagents. Regarding 6A serotypes, although most “6A” isolates (defined by quellung reaction and polyclonal reagents) reacted with 6A-specific monoclonal antibodies (Hyp6AG1, Hyp6AM6, and Hyp6AM3), some “6A” isolates reacted with one mAb (Hyp6AG1) but not others (Hyp6AM6 or Hyp6AM3). Other tests described herein confirmed that the 6A isolates that did not react with Hyp6AM6 or Hyp6AM3 were a previously unidentified 6A subtype. In other words, the monoclonal antibodies recognized subtypes within the 6A serotype. See Lin et al., 44(2) J. Clin. Microbiol. 383-88 (2006). Initially, the isolates reacting with both mAbs were identified as 6Aα and those reacting with only Hyp6AG1 were labeled as 6Aβ, but subsequently, but subsequently it was proposed that the 6Aα remain 6A, and the new serotype be identified as 6C rather than 6Aβ. Id.; Park et al., 45 J. Clin. Microbiol. 1225-33 (2007). Thus both 6Aβ and 6C may be used herein and are equivalent. Monoclonal antibodies useful for identifying 6D include those designated Ab Hyp6BM6, mAb Hyp6BM7, and mAb Hyp6BM8.
Further regarding 6C, a consideration in defining a new serotype is its binding characteristics with human antibodies. The various 6A, 6C, and 6B isolates were compared using an opsonization assay and a human serum with a high level of anti-6B antibodies. Although the human serum opsonized 6B and 6A (FIG. 2), it did not opsonize seven different 6C isolates from Brazil, Korea, and the U.S. (FIG. 2), indicating that 6C isolates display distinct but uniform serological characteristics.
Genetic studies report that pneumococci expressing either 6A or 6B serotype have almost identical capsule gene locus (CGL) of about 17.5 Kb in size. Sequence information is available on-line at, for example, the Sanger Institute's Sequencing Genomics Projects site. A consistent difference exists in the wciP gene that encodes for rhamonosyl transferase. Mavroidi et al., 186 J. Bacteriol. 8181-92 (2004). The serotype 6A wciP gene encodes serine at residue 195 but the serotype 6B gene encodes asparagine at that residue. Id.
Genetic studies also confirmed that the 6C isolates were, indeed, members of the 6A serotype (rather than the closely related 6B serotype or some other unrelated serotype). In a study of ten isolates collected from Brazil, Korea, and the U.S., all ten isolates identified as 6C had the serine at residue 195, consistent with the wciP gene in serotype 6A. DNA sequences of the wciP gene of several pneumococcal isolates are presented in FIG. 4. The genetic sequences of transferase genes wciN and wciO were also compared. When wciN region was examined by PCR using primers 5016 and 3101, all nine 6A isolates examined yielded about 200 base pair (bp) longer product than did all six 6C isolates (from Korea, U.S., and Brazil) examined (FIG. 5).
The nucleotide sequences of the PCR products from one 6A isolate (AAU9) and one 6C isolate (ST745) were compared (FIG. 14). All the bases between positions 1203 to 2959 (1757 bases) in AAU9 PCR product were sequenced and found to be homologous to CR931638, which is the capsule locus sequence of a 6A isolate reported in the GenBank database. In contrast, the ST745 (6C) sequence was found to be almost identical to that of 6A up to position 1368, and then again starting from position 2591. The intervening 1029 bp sequence (from 1369 to 2397) is quite different from that of 6A. The intervening sequence contains about 98 bp that is similar to a transferase (EpsG) used for polysaccharide synthesis by S. thermophilus. 
Note that the capsule gene locus of 6C is very similar to the 6A locus except for the wciN gene: 6A strains have the wciNα gene, but 6C strains have the wciNβ gene. The two genes differ in sizes, thus 6A and 6C serotypes can be readily distinguished by PCR. The wciNα gene encodes WCINα with 314 amino acids, while the wciNβ gene produces a 1125 base-long ORF and its product, WCINβ has 374 amino acids. Additionally, these two proteins have little homology at the amino acid level.
Sequences of the putative wciN gene products suggest their glycosyl transferase functions. WCINβ has similarity to the staphylococcal capH gene product and has a 160-amino acid-long transferase domain that belongs to glycosyl transferase group 1 family. In contrast, WCINα belongs to glycosyl transferase family 8 (ex), which includes many galactosyl transferases. Campbell et al., 326 Biochem. J. 929-39 (1997). The present studies show that wciN is responsible for the difference between the 6A and 6C bacteria, as substitution of wciN6C (wciN of 6C) for wciN6A (wciN of 6A) through homologous recombination appears to have caused the serotype switch from 6A to 6C. Park et al., 75 Infect. Immun. 4482-89 (2007). Indeed, a 6A strain can be converted to a 6C strain by replacing the wciNα gene with the wciNβ gene.
Interestingly, the galactose/glucose exchange observed for 6A and 6C is found for other pneumococcal serotypes. The 9L serotype PS of pneumococcus has a galactose molecule, but 9N PS has a glucose molecule. The capsule gene loci of the 9L and 9N serotypes resemble each other but differ in one gene, wcjA, which encodes a galactosyl transferase for 9L and a glucosyl transferase for 9N. The wcjA genes of the 9L and 9N serotypes are very similar; it is likely that one arose from the other by mutation.
In contrast, the wciNα and wciNβ genes are very different, and the wciNβ gene is not homologous to any other pneumococcal genes available in databases. Perhaps, the wciNβ gene may have originated from an organism other than pneumococci, a notion supported by the wciNβ gene's two flanking regions which may have participated in homologous recombination and which are known to be critical for homologous recombination in pneumococci. Prudhomme et al., 99 P.N.A.S. USA 2100-05 (2002). Additionally, studies of antibiotic-resistance genes have shown horizontal gene transfers between S. pneumoniae and another bacterial species. See, e.g., Feil et al., 151(6) Res. Microbiol. 465-69 (2000); Muller-Graf et al., 145(11) Microbiol. 3283-93 (1999); Coffey et al., 5(9) Mol. 2255-60 (1991).
Additionally, a part of the wciNβ gene is similar (81% homology) to the EpsG gene, a gene involved in the synthesis of exopolysaccharide by S. thermophilus. The homology is found for only a very short piece of DNA, however, thus, S. thermophilus may not be the source for wciNβ. The protein sequence of WCINβ resembles the waaG (rfaG) gene product of E. coli K-12 strain and some pneumococcal genes may have come from Gram-negative organisms. Thus, it is possible that the wciNβ gene could have come from a Gram-negative species as well. Nevertheless, S. salivarius, S. mitis, and S. oralis are the leading candidates because they co-exist in the oral cavity with pneumococci and many antibiotic-resistance genes have been linked to S. oralis. 
When the wciNβ region was examined for multiple 6C isolates, their cross-over points and flanking region sequences were found to be identical. Also, their capsule gene locus profiles are highly limited to 9-10-1 in contrast to 6A isolates, which have many different capsule gene locus profiles. Mavroidi et al., 2004. In addition, the 9-10-1 capsule gene profile is unusual among, and largely segregated from, the capsule gene profiles of the 6A and 6β isolates, findings indicating that the capture of the wciNβ gene must have taken place, and that all the 6C isolates found through out the world, and causing many types of diseases have the capsule gene locus from the single bacterium that originally became 6C. Because 6C may provide a unique and clear example of foreign gene capture, it would be a good model for studying bacterial genetic evolution. This may also constitute a stable change, unlike antibiotic resistance genes.
The 6C serotype has only one or two capsule gene locus profile(s) whereas the 6A and 6B serotypes have diverse capsule gene locus profiles. Mavroidi et al., 2004. Thus, the 6C capsule gene locus may have appeared much more recently compared with the 6A or 6B capsule gene loci. Although 6C may have appeared more than twenty-seven years ago, these findings suggest the 6C serotype capsule gene locus appeared “recently” in one place and spread quickly through out the world. When a gene provides strong survival advantage, the gene can spread quickly throughout the world. For example, an antibiotic-resistance gene may spread worldwide within only years. Perhaps natural human antibodies are less effective against 6C than against 6A or 6B. Whether the 6C capsule gene locus provides more survival advantage than 6A or 6B may be investigated.
MLST studies show that 6C expresses multiple independent STs, thus the 6C capsule gene locus must have been exchanged among different pneumococcal isolates. Whether the 6C capsule gene locus may combine with a ST that provides additional survival advantages may be investigated. The spread of 6C and the emergence of the 6C capsule locus among international strains that have multiple resistance genes should be monitored.
As noted above, the 6C pneumococcal isolate has a chemically distinct PS structure. More specifically, monosaccharide analysis indicated that the galactose found in the 6A capsular PS is absent in the 6C PS, which contains glucose instead. The repeating units of the 6C PS apparently contain one ribitol, one rhamnose, and two glucose moieties. Serotype 6C is the third member of serogroup 6 in view of its serological and structural relation to serotype 6A, and represents the 91st pneumococcal serotype.
Galactose and glucose molecules differ only in the orientation of the hydroxyl group attached to their fourth carbon, and the repeating units of 6A and 6C PS differ only in the orientation of one hydroxyl group. This small structural difference explains why 6C was not identified previously with polyclonal antisera. With the elucidation of the chemical structure, 6C can be distinguished biochemically from 6A by carbohydrate composition analysis or by simple proton NMR of anomeric protons. Abeygunawardana et al., 279 Anal. Biochem. 226-40 (2000). Although 6A NMR and 6C NMR patterns do differ, the NMR pattern of the anomeric protons of 6C is very similar to that of 6A. Although chemical and genetic tests can be used, serological methods may be the most useful way to identify 6C using either monoclonal antibodies or polyclonal antisera made specific by absorption.
Serogroup 6 has been known to contain three epitopes: 6a, 6b, and 6c. Henrichsen, 1995. Epitope 6a is known to be present in both serotypes 6A and 6B whereas epitopes 6b and 6c are found only in either serotype 6A or 6B, respectively. Discovery of the 6C serotype indicates the presence of additional epitopes within serogroup 6. The mAb Hyp6AM3, which recognizes 6A and 6B but not 6C, should recognize epitope 6b. Because mAb Hyp6AG1 recognizes 6A and 6C, it may be defined as recognizing a new epitope “6d”. Another mAb binding to all three serotypes (6A, 6B, and 6C) and the shared epitope may be defined as “6e”. A confirmation-dependent epitope for serotypes 6A and 6B has also been described. Sun et al., 69 Infect. Immun. 336-44 (2001). The observation of so many epitopes for serogroup 6 is consistent with a previous observation that even a simple linear homopolymer of sialic acid can have at least three epitopes. Rubenstein & Stein, 141 J. Immunol. 4357-62 (1988). Indeed, pneumococcal PS have many more epitopes than previously defined (Henrichsen, 1995), and that the presence of many epitopes increases chances of altering epitopes during the manufacture of pneumococcal conjugate vaccines.
The discovery of serotype 6C was quite unexpected because serogroup 6 has been extensively studied following its discovery in 1929. Heidelberger & Rebers, 1960. One should therefore consider the possibility that additional subtypes (or serotypes) are present among even well-established and extensively characterized serogroups. For instance, one may need to consider the possible presence of subtypes among serotype 19A because two chemical structures for the 19A capsular PS have been reported. Kamerling, Pneumococcal polysaccharides: a chemical view, in MOL. BIOL. & MECHANISMS OF DISEASE 81-114 (Mary Ann Liebert, Larchmont, 2000). If 19A subtypes are found, their presence may explain the rapid increase in the prevalence of serotype “19A” seen after the introduction of the pneumococcal conjugate vaccine. Pai et al., 192 J. Infect. Dis. 1988-95 (2005). In addition, one should consider the possibility that 6C may have arisen recently. Consistent with this possibility, the genetic studies suggest that the 6C serotype capsule gene locus is not as diverse (Lin et al., 44 J. Clin. Micro. 383-(1988)), as is the 6A locus (Mavroidi et al., 2004). It would be interesting to investigate the origin and spread of 6C strains by studying pneumococcal isolates obtained a long time ago (perhaps 50-100 years ago).
The discovery of 6C increases the evolutionary potential of serogroup 6 capsule gene locus by the logical possibility for a new member of serogroup 6. The new member provided for herein, labeled “6D”, comprises the wciP of 6B and wciN6C. Chemically, the 6D PS has a glucose instead of galactose, and has a 1→4 rhamnose-ribitol linkage. It was unclear, before the present invention, if this novel serotype existed or might emerge in nature. Therefore, the serotype 6D strain was used to examine a laboratory collection of pneumococcal isolates for a 6D strain.
The 6C serotype is also useful to monitor vaccine efficacy: The 6A and 6C serotypes must be distinguished in epidemiological studies involving pneumococcal vaccines and in studies of pneumococcal vaccine efficacy. For example, if a pneumococcal vaccine is effective against 6A but not 6C, the vaccine may not be effective in areas where the 6C serotype is prevalent. This would be the case because pneumococcal vaccines elicit antibodies opsonizing 6C only occasionally. Also, usage of conventional pneumococcal vaccines may well alter the prevalence of 6C: the prevalence of 6C may increase although the prevalence of 6A decreases. Preliminary data shown below suggests that 6C prevalence is unchanged whereas 6A prevalence has decreased with the use of conjugate vaccines since 2000. Without distinguishing between the serotypes, it may be difficult to deploy a vaccine or assess its efficacy. At present, the new serotypes can be identified by the antibodies as disclosed herein, but additional genetic and biochemical tests may be devised and are envisioned by the present invention.
Moreover, the prevalence of the 6C serotype or the emergence of the 6D serotype should be monitored globally, providing valuable information on the emergence of new pneumococci in areas with and without pneumococcal vaccine distribution. The 6C serotype has also been identified in Brazil, Canada, China, Korea, Mexico, Europe (Hermans et al., 26 Vaccine 449-50 (2008)), and the U.S.
To that end, the monoclonal antibodies of the present invention are useful in identifying the 6C serotype. To with, both 6A and 6C are identified by the mAb Hyp6AG1, but the 6C serotype does not react with the mAb Hyp6AM6 or mAb Hyp6AM3. Hence, Hyp6AM6 or Hyp6AM3 may be used as a negative control from which 6A and 6C can be identified. Using these monoclonal antibodies, the prevalence of 6A and 6C among the U.S. pneumococcal isolates submitted to the CDC were analyzed. Approximately the same number of pneumococcal isolates were submitted to the CDC from 1999 to 2006. Specimens typed as “6A” by the old method were reanalyzed using the monoclonal antibodies described herein. Almost all the “6A” specimens received in 1999, 2003, and 2004 were reanalyzed. Only a fraction of the samples the CDC received in 2005 and 2006 were reanalyzed. As seen in the table, the prevalence of 6A decreased but the prevalence of 6C remained the same. This suggests that the currently available pneumococcal vaccine may not be effective against 6C.

















1999
2003
2004
2005
2006






















All ages
6A
169
132
51
16
16



6C
41
40
57
21
23









Serotype 6C has been deposited with the American Type Culture Collection in accord with the Budapest Treaty.
The actual synthesis of pneumococcal capsular polysaccharides requires cooperation among many different gene products. For instance, a new repeating unit made by a new glycosyl transferase must be compatible with the existing flippase as well as polymerase before it can be expressed as a new capsule. Thus, the new strain expressing serotype 6D (named TIGR6D) was produced by inserting wciN6C into 6B capsule gene locus. This novel strain produces capsular polysaccharide with predicted structure, it displays serological similarity to 6B, and it can grow as well as other members of serogroup 6 in various growth conditions. Thus serotype 6D is possible not only logically but also biologically, and it could exist in nature.
Just as 6C was previously typed as “6A” by the classical typing method (Lin et al., 2006; Park et al., 2007), the quellung reaction method typed the new 6D strain as serotype 6B. Thus, to identify natural isolates expressing serotype 6D, isolates that were classically defined as 6B were re-examined using mAbs. Despite testing more than 250 isolates that were previously typed as 6B, 6D isolates were not found to exist in nature. Additionally, wciN6C was not detected among the CDC isolates that were serotyped as “6B” with the classical typing method. Thus, pneumococcal isolates expressing serotype 6D have not been found to exist in nature. Alternatively, if serotype 6D exists in nature, its prevalence is extremely low (<1% of the 6B prevalence).
Despite its apparent absence in nature, the 6D serotype could emerge in nature by one of two possible mechanisms. One mechanism involves a mutation of the wciP gene of 6C, because the only difference between 6A and 6B serotype appears to be one nucleotide in the wciP gene. The mutation rate for pneumococci is  ̃1×10−8 (del Campo et al., 43 J. Clin. Microbiol. 2207-14 (200); Fedson, 1988; Morosini et al., 47 Antimicrob. Agents. Chemother. 1464-67 (2003)), and a COPD patient with stable pneumonia may have 2.6×108 CFU of pneumococci per mL of sputum. Sethi et al., 176 Am. J. Respir. Crit. Care Med. 356-61 (2007). Thus, the correct mutation should arise in almost all cases of 6C pneumonia and often in other 6C infections with less bacterial load. The alternative mechanism involves the lateral gene transfer of wciN from a 6C strain into a 6B strain as provided for herein. The situation could actually occur in nature because carriage of multiple pneumococcal serotypes can be relatively high among children (Gratten et al., 50 Biol. Neonate 114-20 (1986); Hill et al., 46 Clin. Infect. Dis. 807-14 (2008)), and serotypes 6B and 6C are fairly common in some parts of the world (e.g., Brazil) (Lin et al., 2006; Park et al., 2007). Further, homologous recombination would occur easily because the 17 kb of the capsule gene locus of 6B and 6C are almost identical except for the wciN gene. These considerations strongly suggest that the circumstances for creating 6D serotype exist in nature.
Given that the circumstances for creating 6D do exist in nature, it is interesting to consider reasons for its absence. It is possible that there could be natural immune barriers against 6D, but apparently pre-immune human sera do not kill or opsonize TIGR6D. Alternatively, the 6C could have appeared so recently that there may not have been enough time for 6D to appear. Although it is difficult to estimate the physical time that would permit the appearance of 6D, 6C has existed for decades and is found in several continents. The most likely explanation is that there has not been enough biological pressure for selecting 6D over 6A, 6B, or 6C. In the absence of survival advantage, 6D serotype may have appeared in nature (as it should in almost every case of a 6C infection, as mentioned above) but was not propagated due to competition with more abundant 6A, 6B, or 6C serotypes. In an analogous manner, antibiotic resistant strains survive and propagate when antibiotics are clinically used and they disappear when antibiotics are discontinued. See Katsunuma et al., 102 J. Appl. Microbiol. 1159-66 (2007)
The 6A PS was included in the original 14-valent PS vaccine, but it was replaced with 6B PS when the 23-valent vaccine became available in 1983 because, inter alia, 6B PS is greatly more resistant to hydrolytic breakdowns than 6A PS. Zon et al., 37 Infect. & Immun. 89-103 (1982). The work provided herein shows that 6D PS is as chemically stable as 6B PS, and much more resistant to hydrolysis than 6C PS. Because 6D PS would likely elicit antibodies cross-reactive with 6C PS, 6D may be more useful as a vaccine than 6C PS.
It is interesting to understand the evolution of the capsule gene locus, which encodes pneumococci's most important virulence factor. Even when only two serotypes were known, the evolution of serogroup 6 has been extensively studied (Mavroidi et al., 2004; Robinson et al., 2002). Serogroup 6 has become more interesting following the discovery of serotype 6C: now the serogroup is even more interesting for evolution studies with serotype 6D.
Moreover, although the currently available 23-valent pneumococcal vaccine contains 6B PS, the old 14-valent pneumococcal vaccine contained 6A PS. The PS was replaced in the 23-valent pneumococcal vaccine because 6A PS was not stable in vaccine preparations, and because 6B PS often induced antibodies cross-reacting to 6A PS. Robbins et al., 1983. Investigations found that 6A's PS chemical instability was due to the 1→3 linkage between rhamnose and ribitol. Zon et al., 1982. The 1→4 linkage found in 6B PS is more stable than the 1→3 linkage of 6A PS. The putative structure of 6D PS is identical to that of 6C PS except that 6C PS has the unstable 1→3 linkage whereas 6D PS has the more stable 1→4 linkage. Thus, because of this stability, 6D PS may prove even more useful in a vaccine than 6C PS.
Importantly, the novel serotypes 6C and 6D provided herein may be useful in a vaccine or in pneumococcal vaccine development. For example the 6D PS, a portion of that PS, or a mimetic of the 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. Pat. No. 6,248,570; No. 5,866,135; No. 5,773,007. 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. 293-98 (1998); Shin et al., 168 J. Immunol. 6273-78 (2002). Additionally, the proteins or nucleic acids of 6C and/or 6D may serve as antigens or immunogens in vaccine or vaccine development using any number of techniques known in the art. See, e.g., U.S. Pat. No. 6,936,252. One or more adjuvant agents may be included in such vaccines. The delivery of pneumococcal vaccines, either by parenteral, mucosal, or other administration, and the design, monitoring, and dosing regimens of such vaccines, are well-known in the art.
Additionally, the 6C and 6D serotypes may be useful in vaccine development because the bacteria would be used as the target in an opsonization or ELISA assays using sera or antibodies raised by test vaccines. The antigens of the 6C and 6D serotypes may also be used to raise antibodies that might be used for passive protection. Such methods are also well-known in the art.
Further, the identification of 6C and 6D provides for the production and isolation of anti-6C antibodies and anti-6D antibodies. These can be prepared by conventional means well known in the art in light of the current specification. In this regard, the term antibodies includes both intact immunoglobulin molecules as well as portions, fragments, peptides and derivatives thereof, such as, for example, Fab, Fab′, F(ab′)2, Fv, CDR regions, or any portion or peptide sequence of an immunoglobulin molecule that is capable of binding a 6C 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 embodiments of the invention will now be described further by non-limiting examples.
EXAMPLES
Example 1
Identification of Pneumococcal Serotypes
Collection of pneumococcal lysates: The pneumococci serotype 6Aβ (See Lin et al., 44 J. Clin. Micro. 383-88 (2006)) was isolated in a blinded study using 495 clinical isolates: Fifty isolates were from Mexico, 100 from Denmark, and 345 from Brazil. Twenty-two isolates were from asymptomatic carriers of pneumococci in the nasopharynx and 475 isolates were from patients with invasive pneumococcal infections such as meningitis and sepsis. In addition, control pneumococcal strains expressing serotypes 11A, 11B, 11C, 11D, and 11F were purchased from Statens Serum Institut (Copenhagen, Denmark).
Lysates of the clinical isolates were prepared in the country of origin. Three hundred microliters of Todd-Hewitt medium with 0.5% yeast extract (THY medium) was inoculated with a single colony of pneumococci. After an overnight incubation at 37° C., cells were lysed with 50 μl of lysing solution (0.2% sodium deoxycholate, 0.02% SDS, 0.1% sodium azide, 0.3M sodium citrate, pH 7.8). In Brazil, 400 μl of THY medium was used for bacterial growth and 100 μl was removed to store the bacteria frozen before mixing the remaining 300 μl with 50 μl of lysing solution. In Denmark, 325 μl of THY medium and 25 μl of lysing solution were used. Bacteria were lysed by incubating the mixture at 37° C. The lysates were coded and shipped to the University of Alabama at Birmingham (UAB) laboratory for serotype testing by regular mail at ambient temperature.
To simplify the shipping of bacterial lysates from distant sites to UAB for the multibead assay, the stability of bacterial lysates was compared after storage at room temperature (RT) or 37° C. The work revealed that bacterial lysates can be stored at RT for up to one month or at 37° C. for several days without affecting the results of the multibead assay. Thus, the regular postal mail system was used to ship all the lysates in this study at ambient temperature without any thermal protection.
Serological Reagents: All the polyclonal serotyping sera were made in rabbits and were obtained from Statens Serum Institut. They include twelve serum pools for serogrouping and various type- or factor-specific antisera. Sorensen, 31 J. Clin. Microbiol. 2097-2100 (1993). All the mAbs were produced as described, and hybridoma culture supernatants were used. Yu et al., 2005.
Multibead assay: This assay was performed as described using two different sets of latex beads. Yu et al., 2005. One set of beads (Set 1) was a mixture of fourteen different latex beads, each coated with one pneumococcal PS antigen. The fourteen pneumococcal PS antigens were serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9N, 9V, 14, 18C, 19A, 19F, and 23F. Bead Set 2 was created by coating each of ten bead types with one of ten different pneumococcal PS (serotypes 2, 8, 10A, 11A, 12F, 15B, 17F, 20, 22F, and 33F).
Set 1 beads were mixed with either 5× or 20× diluted bacterial lysate and a mixture of mAbs specific for the pneumococcal capsular PS contained on the beads. After incubation and washing, the bead mixture was reacted with fluorescein-conjugated anti-mouse immunoglobulin antibody. Set 2 beads were used the same as Set 1 beads except that a mixture of polyclonal rabbit antisera (Statens Serum Institut) and fluorescein-conjugated anti-rabbit immunoglobulin antibody were used. After incubation, the amount of fluorescence of each bead type was determined with a flow cytometer (FACSCalibur, Beckton Dickinson, San Jose Calif.). The fluorescence of each bead type was then used to determine its serotype. Fluorescence inhibitions greater than 67% were used as positives.
Neufeld's test: This assay was performed as described (Henrichsen, 33 J. Clin. Microbiol. 2759-62 (1995); Konradsen, 23 Vaccine 1368-73 (2005); Lund, 23 Bull. Wld Hlth Org. 5-13. (1960)) by the reference laboratories in Denmark, Brazil, and Mexico using standard serogrouping (Sorensen, 1993) and serotyping rabbit antisera from Statens Serum Institut.
Dot blot assay: To investigate discrepant results, this assay was performed as described (Fenoll et al., 35 J. Clin. Micro. 764-76 (1997)), using pneumococcal antisera from Statens Serum Institut to the following serogroups/serotypes: 1, 4, 5, 6, 7, 8, 9, 11, 12, 14, 18, and 23. Monoclonal antibodies specific for 6A (Hyp6AM3) and 18C (Hyp18CM1) were also used in some cases. Briefly, heat-killed pneumococci grown in THY medium were spotted on strips of nitrocellulose membranes. After drying, the strips were blocked and washed. Strips were then incubated in a diluted antiserum or mAb solution for 1 hour, washed and exposed to a diluted goat anti-rabbit or mouse immunoglobulin-peroxidase conjugate. After one hour incubation at room temperature, the strips were washed and exposed to 3-amino-9 ethylcarbazole solution. When the spots appeared, the strips were washed and evaluated.
PCR reactions: Pneumococci were grown in THY medium to an OD of 0.8 at 650 nm wavelength. Chromosomal DNA was prepared using the Invitrogen EASY-DNA kit and following the given instructions, beginning with a 4 ml sample of the THY-grown pneumococci concentrated to 1 ml (Invitrogen, Carlsbad, Calif.). For serogroup 6 determination, PCR was performed using chromosomal DNA as template and primers wciP-up, 5′-ATG GTG AGA GAT ATT TGT CAC-3′(SEQ ID NO:1) and wciP-down, 5′-AGC ATG ATG GTA TAT AAG CC-3′ (SEQ ID NO:2). PCR thermocycling conditions were as described in Mavroidi et al., 2004. A Qiagen PCR cleanup column (Qiagen, Valencia, Calif.) was used to remove excess primer from the PCR reactions and the PCR was submitted as DNA template for automated DNA sequencing using the wciP-up primer. Results were analyzed with the aid of the Sequencher (GeneCodes, Inc., Ann Arbor, Mich.) and the MacVector Sequence Analysis (Accelyrs, San Diego, Calif.).
For serotype 11A determination, PCR for a part of the capsule gene locus was performed as described (Mavroidi et al., 2004), using chromosomal DNA as the template, 1 μl of forward primer (50 pmol), and 1 μl of reverse primer (50 pmol). Primers were 11A forward, 5′-GGA CAT GTT CAG GTG ATT TCC CAA TAT AGT G-3′ (SEQ ID NO:3) and 11A reverse 5′-GAT TAT GAG TGT AAT TTA TTC CAA CTT CTC CC-3′ (SEQ ID NO:4). PCR cycling began with 94° C. for 5 min, followed by thirty cycles of 94° C. for 1 min, 50° C. for 1 min and 72° C. for 2 min, followed by a final extension of 72° C. for 10 min. The PCR reaction products were analyzed by agarose gel electrophoresis (Tris-acetate buffer 0.8% agarose) to determine the amplicon size.
Study of fifty isolates from Mexico: The fifty isolates from Mexico were grown in THY medium, lysed, and sent to UAB for typing. When the multibead assay results were compared with the Neufeld's test results, results from ten samples were discrepant. When new lysates of eight of the discrepant samples were obtained and re-examined in a blind fashion, all results matched, suggesting that the discrepancies were largely due to mislabeling. Two isolates (MX24 and MX37) that were typed to be serotype 3 and 10A by the Neufeld's test were originally typed as non-typeable (NT) by the multibead assay. Because the two serotypes should have been identified by the multibead assay, the two bacterial isolates were sent to the UAB laboratory for further study. There they were found to grow well in THY medium, with the new lysates producing results matching the Neufeld's test results. Thus, the two isolates were initially falsely identified as negatives by multibead assay, most likely due to insufficient growth of pneumococci.
Study of 100 isolates from Denmark: When the multibead assay results of one hundred Denmark isolates were compared with the Neufeld's test results, four errors were found in transcribing the Neufeld's test results and one strain (DK94) was typed as serotype 20 by the Neufeld's test and as NT by the multibead assay (Table 1).







TABLE 1







Serotyping results with both serotyping assays


and the final results after the investigations.












Serotype#
Multibead
Neufeld
Final results
















 1
30
30
30



 2
 1
 1
1



 3
22
22
22



 4
20
20
20



 5
11
11
11



 6A
 16*
21
21



 6B
24
24
24



 7F/A
14
14
14



 8
13
13
13



 9V
18
18
18



 9N/L
12
12
12



10A/B/39/33C
12
12
12



11A/D/F
 8*
10
9



12F/A/B
16
16
16



14
52
52
52



15B/C
10
10
10



17F
 6
 6
6



18C
28
 27*
28



19A
18
18
18



19F
26
26
26



20
  3**
 4
4



22F/A
 6
 6
6



23F
19
19
19



33F/A
 6
 6
6



NT
104 
97
97



Total
495 
495 
495







#NT indicates non-typeable serotypes by the multibead assay. 7F/A means that the isolate may express either 7F or 7A serotypes. 10A/B/39/33C indicates that the isolate may express serotype 10A, 10B, 39 or 33C.



*After additional studies of Brazilian isolates, it was concluded that the multibead assay failed to identify five 6A strains (with Hyp6AM3) and one 11A strain, and that Neufeld's test failed to identify one 18C strain and falsely identified one strain as 11A.



**One Danish strain had high background signal and was not detected during the initial multibead assay.






When the DK94 isolate was re-grown in THY and re-examined, it produced almost no inhibition (9%) at a 1:5 dilution, but it produced more inhibition at higher dilutions (35% at a 1:20 dilution and 50% at a 1:320 dilution). This unexpected behavior suggested the presence of non-specific binding material in the lysate of this specific isolate. When the PS in the lysate was precipitated with 70% ethanol and the ethanol precipitate was examined with the multibead assay, the precipitate produced a clear inhibition for serotype 20 (86% at a 1:5 dilution and 81% at a 1:20 dilution). Thus, the initial discrepancy was due to non-specific binding, which was occasionally observed with the assays performed with polyclonal sera, and there is no intrinsic problem in assay sensitivity and specificity with clinical isolates.
Study of 345 samples from Brazil: When the results of 345 Brazilian isolates obtained with the two assay methods were compared, there were thirty-eight mismatches. When these thirty-eight samples were re-examined by investigating test records and retesting by Neufeld's test in Brazil, seventeen of the mismatches could be explained as typing mistakes or sample misidentification. One of the seventeen mismatches was strain BZ652. This was initially typed as 18B, but was determined to be 6A because it was typed as weakly 6A by Neufeld's test and was typed as serogroup 6 by the dot blot assay using the polyclonal antisera and mAb Hyp6AM3. When the twenty-one remaining mismatched samples were regrown in THY medium and retested by multibead assay, the new results of thirteen isolates matched the Neufeld's test results. When the original multibead assay results of the thirteen isolates were re-examined, three isolates produced weak and incomplete inhibitions (inhibitions were less than 67%) for the appropriate serotype in the original multibead. Although twelve isolates were initially typed as NT, one isolate (BZ52) was initially typed as type 3. It was retyped as NT with the second sample and the result became consistent with the Neufeld's test result (Table 1) (above).
After these re-examinations, eight discrepancies were reproducible and still unexplained (Table 2 and Table 3): five isolates were typed as 6A by the Neufeld's test but as NT by the multibead assay, two isolates (BZ435 and BZ705) were typed as 11A by the Neufeld's test but as NT by the multibead assay, and one isolate (BZ438) was typed as NT by the Neufeld's test but as 18C by the multibead assay. By the Neufeld's test, BZ438 did not react with several lots of serogrouping Poolsera A and Q (Sorensen, 1993), which should react with serogroup 18 pneumococci. It also did not react with several different lots of antisera specific for serogroup 18 or specific for factors 18c, 18d, 18e, and 18f. BZ438 produced positive dot blot results, however, with a serogroup 18-specific polyclonal serum or with mAb Hyp18CM1 (Yu et al., 2005). Thus, the BZ438 isolate was considered to be 18C.
Strains BZ435 and BZ705 were considered to be 11A by the Neufeld's test but not 11A, 11D, or 11F by the multibead assay. Because the standard multibead assay uses a polyclonal antiserum against serogroup 11 (Yu et al., 2005), the two strains were examined with two mAbs (Hyp11AM1 and Hyp11AM2) that are specific for serotypes 11A, 11D, and 11F and that were recently produced in the UAB laboratory (Table 2). Interestingly, Hyp11AM1 recognizes BZ435 but not BZ705. Interestingly, Hyp11AM2 recognized neither strain, suggesting heterogeneity among the strains expressing the 11A serotype. A PCR test produces 463 base pair amplimers with strains for 11A, 11D, and 11F but not for 11B and 11C (Table 2). When both strains were tested by this PCR, BZ435 was positive, but BZ705 was not. Although the Neufeld's test showed that both strains reacted with antisera specific for factor 11c, the Neufeld's test also revealed differences between them: BZ435 but not BZ705 reacted with Poolserum T (Sorensen, 1993), with serogroup 11 antisera, or with 11f factor serum. BZ705 yielded ambiguous results for factor 11b expression and this suggested that it could be serotype 11D. In a dot blot test for serogroup 11 using rabbit typing serum, however, BZ435 was positive but that BZ705 was negative. Considering all of these results, it appeared that BZ435 is an 11A strain and that BZ705 is not 11A, 11D, or 11F. BZ705 may belong to the 11C serotype since BZ705 expresses the 11c epitope (and reacts with 11c antisera) that is not expressed on 11B strains.







TABLE 2







Studies of two strains for the 11A serotype with Neufeld, multibead, PCR, and dot blot assays










Multibead assay















Neufeld's test with
With
With
With

Dot blot assay


Strains
rabbit sera#
rabbit sera#
Hyp11AM1
Hyp11AM2
PCR
with rabbit sera#





BZ435
11A
−
+
−
+
+


BZ705
 11A*
−
−
−
−
−


Control
Not tested
+
+
+
+
Not tested


Strain 11A


Control
Not tested
−
−
−
−
Not tested


Strain 11B


Control
Not tested
−
−
−
−
Not tested


Strain 11C


Control
Not tested
+
+
+
+
Not tested


Strain 11D


Control
Not tested
+
+
+
+
Not tested


Strain 11F





#All the rabbit sera were from Statens Serum Institut (Denmark).


*In the Neufeld's test, BZ705 did not react with Poolserum T and factor serum 11f, but it did react strongly with factor serum 11c and ambiguously with factor serum 11b.






To investigate the remaining discrepant strains that were 6A, the DNA sequence of the wciP gene was examined. A recent study reported that the capsular PS of 6A and 6B has repeating units with rhamnose linked to ribitol. The linkage is 1-3 for 6A and 1-4 for 6B. The study found that the rhamnosyl transferase is likely encoded by the wciP gene in the capsule locus, that wciP for 6A encodes serine at residue 195, and that wciP for 6B encodes asparagine at residue 195 (Mavroidi et al., 2004). Also, wciP alleles 1, 2, 7, 9, and 11 are exclusively associated with serotype 6A, and alleles 3, 4, 5, 6, 8, and 12 are associated with serotype 6B. (Mavroidi et al., 2004).
Bacterial DNA was obtained from the five isolates labeled 6A as well as BZ652, which was considered to be only weakly 6A by the Neufeld's test. This DNA was amplified a part of the wciP gene by PCR, sequenced the amplicon, and examined the sequence (645 base pairs). Five samples were amplified successfully, and their sequences were consistent with a 6A serotype because they expressed alleles associated with the 6A serotype (Table 3) and expressed serine at amino acid residue 195. Compared to the prototypic sequence of allele 2 wciP, the wciP sequence of BZ652 has five base pair changes with three potential amino acid replacements. Three isolates (BZ17, BZ39, and BZ86) express the identical wciP gene sequence with one identical nucleotide variation from the prototypic sequence for allele #9 and may, therefore, be clonally related (Table 3).







TABLE 3







Studies of six strains for 6A serotype with


Neufeld, multibead, and PCR assays.











Neufeld's





test with
PCR for
Multibead assay













polyclonal
wciP

Polyclonal



Names
antisera
allele*
Hyp6AM3
sera
Hyp6AG1





BZ17
6A
#9 (1)
NT
6A
6A


BZ39
6A
#9 (1)
NT
6A
6A


BZ86
6A
#9 (1)
NT
6A
6A


BZ650
6A
#1
NT
6A
6A


BZ652#
NT (6A)$
#2 (5)
6A
6A
6A


BZ1048
6A
Not
NT
6A
6A




done





*The number in parentheses indicates the number of base pairs different from the proband sequence (Mavroidi et al., 2004). BZ652 has five base pair differences that produce three amino acid differences. All these alleles express serine at amino acid residue 195.


$BZ652 was initially typed as NT (non-typeable) but was typed as weakly 6A on re-examination.






Because the DNA study suggested that these isolates may belong to the 6A serotype, these isolates were examined with the multibead assays using polyclonal antisera. All six isolates were typed as 6A (Table 3). When they were typed with nineteen different 6A-specific mAbs in addition to Hyp6AM3, one mAb (Hyp6AG1) identified the six isolates as 6A (Table 3). When Hyp6AG1 was used to retest forty-six 6A isolates (twenty-one from this study and twenty-four in the Univ. Alabama Birmingham laboratory collection), it was found that this mAb identified all of them as 6A and that it did not recognize any of the eighty-nine non-6A serotypes, including the forty-three isolates expressing the 6B serotype. Thus, it was clear that all these six isolates are 6A and that Hyp6AG1 recognizes all 6A isolates. Also, mAb Hyp6AM3 recognizes a subset of 6A isolates, although that subset is very large.
Example 2
Pneumococcal Serotype 6C Isolates from Different Countries have the Molecular Characteristics Associated with 6A
As described above, Brazilian isolates that did not react with both mAbs previously associated with serotype 6A were shown to belong to the 6A serotype by examining the wciP allele. Thus, the inventors examined wciP gene of ten 6C isolates from geographically diverse locations. Brazilian isolates collected in 2003 and in 2004, USA isolates and one isolate from Korea were examined. The sequences clearly showed that all ten isolates have the genetic characteristics associated with 6A serotype.
Example 3
6C Isolates from Different Areas have Uniform Serological Characteristics
To investigate serological characteristics of the 6C isolates in a quantitative manner, isolates were examined using an inhibition assay. The assay measured inhibition by bacterial lysates of various anti-6A antibodies binding to 6A PS-coated ELISA plates. Briefly, the wells of ELISA plates (Corning Costar Corp., Acton, Mass.) were coated at 37° C. with 5 μg/mL of 6A capsular PS (a gift of G. Schiffman, Brooklyn, N.Y.) overnight in PBS. After washing the plates with PBS containing 0.05% of Tween 20, previously diluted bacterial culture supernatant (or lysates) was added to the wells along with an anti-6A antibody. Pneumococcal lystates were prepared by growing pneumococci in 10 mL of Todd-Hewlett broth supplemented with 0.5% yeast extract (THY) without shaking until the tubes became turbid and then incubating the tubes for 15 minutes at 37° C. with a lysis buffer (0.1% sodium deoxycholate, 0.01% SDS, and 0.15M sodium citrate in deionized water). Hyp6AG1 mAb was used at a 1:250 dilution, and Hyp6AM3 mAb was used at 1:100 dilution. Pool Q and factor “6b” rabbit antisera from Staten Serum Institute (Copenhagen, DK) were used at a 1:500 dilution. After thirty minutes of incubation in a humid incubator at 37° C., the plates were washed and incubated for two hours with alkaline phosphatase-conjugated goat anti-mouse Ig (Sigma, St. Louis, Mo.) or alkaline phosphatase-conjugated-goat anti-rabbit Ig (Biosource, Camarillo, Calif.). The amount of the enzyme immobilized to the wells was determined with paranitrophenyl phosphate substrate (Sigma) in diethanolamine buffer. The optical density at 405 nm was read with a microplate reader (BioTek Instruments Inc. Winooski, Vt.).
Because the qualitative nature of the quellung reaction may have prevented detection of 6A subtypes, it was determined whether the subtypes might be distinguishable with a quantitative assay using the rabbit sera used for quellung reactions. This was determined by adapting the rabbit sera to an inhibition assay, in which pneumococcal lysates were allowed to inhibit the binding of rabbit antisera to 6A PS immobilized on ELISA plates (FIG. 1). As a control, pneumococcal lysates were tested for inhibition of the two mAbs: Hyp6AG1 and Hyp6AM3 (FIGS. 1A and 1B). Lysates of three 6Aα isolates (CHPA378 from the U.S.A., KK58 from Korea, and ST558 from Brazil) inhibited both mAbs, and lysates of two 6B isolates (strains ST400 and ST518 from Brazil) inhibited neither mAb (FIGS. 1A and 1B). Three lysates of 6C isolates (including strains BZ17 and BZ650 from Brazil) clearly inhibited the binding of Hyp6AG1, even at a 1:1000 dilution (FIG. 1A). They showed almost no inhibition, however, of Hyp6AM3, even at a 1:10 dilution (FIG. 1B).
When the pneumococcal lysates were examined for inhibiting Pool Q (a rabbit antiserum often used for serotyping (Sorensen, 31 J. Clin. Microbiol. 2097-2100 (1993)), both lysates of 6A and 6C could inhibit equally well, but the 6B lysates could not inhibit (FIG. 1C). When a “6b”-factor-specific rabbit serum was tested, all 6A, 6C, and 6B isolates could inhibit the factor serum equally well (FIG. 1D). Because the 6b-factor serum is designed to be 6A-specific, this was unexpected. The factor serum is designed to be specific in quellung reactions, however, not in this inhibition assay. Nevertheless, this experiment showed that rabbit antisera commonly used for pneumococcal typing do not distinguish between the 6A and 6C subtypes.
Various 6A, 6C, and 6β isolates were compared using an opsonization assay and a human serum with a high level of anti-6B antibodies. Although the human serum opsonized 6B as well as 6A (FIG. 2), it did not opsonize seven different 6C isolates from Brazil, Korea, and the United States (FIG. 2).
Example 4
Human Antisera are not Equally Protective Against the 6A and 6C
Because a human antiserum can opsonize 6A and 6B but not 6C, serum samples from twenty adults for were examined for opsonizing 6A, 6B, and 6C serotypes (FIG. 3A). None of the serum donors were vaccinated with a pneumococcal vaccine at least for five years. Although most individuals have low opsonization titers, four individuals had opsonization titers greater than 100 for serotype 6B. Sera from the four individuals had significant opsonization titers against 6A, but only one had a significant titer against 6C. The observation suggests that the adult population has less natural immunity against 6C than against 6A.
To examine whether immunization with 6B induces antibodies cross-reacting with 6C, the sera from twenty adults who were immunized with a pneumococcal vaccine were studied (FIG. 3B). Ten adults were immunized with a 9-valent pneumococcal conjugate vaccine (PCV) and ten with a 23-valent PS vaccine (PPV). Eight of the ten persons immunized with PCV had a high (>100) opsonization titer for 6B. Of these eight, seven persons had an opsonization titer against 6A commensurate with 6B titer but only one person had 6C titer commensurate with 6B titer. Because the person's serum opsonized 6A almost as well as 6B, it is likely that the elicited anti-6B antibodies that were cross-reacting with 6C. When PPV vaccines were examined, five persons had a high opsonization titer (>100) against 6B, two persons had a high titer against 6A, but none had a high titer against 6C (FIG. 3B). Taken together, these findings suggest that currently available pneumococcal vaccines may provide protection against 6A better than against 6C infections.
Example 5
Development of Monoclonal Antibodies Useful to Identify Pneumococci
Mouse hybridomas were produced as described previously. Yu et al., 2005 (citing Sun et al., 69 Infect. Immun. 336-44 (2001)). Briefly, BALB/c mice were immunized twice subcutaneously with PS-protein conjugate (days 0 and 21) and once intraperitoneally on day 59. The immunogen for seven serotypes (4, 6B, 9V, 14, 18C, 19F and 23F) was Prevnar (Wyeth Lederle Vaccines, Pearl River, N.Y.). Conjugates used for serotypes 5 and 7F were prepared at the U.S. Food and Drug Administration (Bethesda, Md.), the 6A conjugate was a gift of Porter Anderson (Rochester, N.Y.), and conjugates of serotypes 1, 3, and 9N to ovalbumin were prepared as follows. Cyanogen bromide-activated PS was coupled to ovalbumin during an overnight incubation. The PS-protein conjugate was purified from the reaction mixture with a molecular weight sizing column. Each dose contained 1 μg of PS for serotypes 4, 9V, 18C, 19F, and 23F; 2 μg for serotypes 3 and 6B; and 10 μg for serotypes 1, 5, 6A, 7F, and 9N. The primary and secondary immunogens contained 10 μg of Quil A (Sigma Chemical, St. Louis, Mo.).
Three days after the last immunization, the mice were sacrificed, the spleens harvested, and the splenocytes fused with SP2/0 Ag-14 as described previously. Nahm et al., 129 J. Immunol. 1513-18 (1982). Primary culture wells were screened for the production of desirable antibodies, and the wells producing such antibodies were cloned twice by limiting dilution. A human-mouse hybridoma, Dob9, was produced by hybridizing peripheral blood lymphocytes from a person immunized with a 23-valent PB vaccine, as described previously. Sun et al., 67 Infect. Immun. 1172-79 (1999).
The human-mouse hybridoma is specific for pneumococcal serotypes 19A and 19F. All hybridomas produced either IgM or IgG antibodies, excepting one IgA producer. Hyp6AG1 is IgG and Hyp6AM6 is IgM.
A total of twenty-one hybridomas specific for 6A serotypes were isolated. Many have similar serological behavior and some may be sister clones (i.e., some may have the identical variable region structure). Names of 6A-specific hybridomas produced are Hyp6A1, Hyp6AM1, Hyp6AM2, Hyp6AM3, Hyp6AM4, Hyp6AM5, Hyp6AM6, Hyp6AM7, Hyp6AM8, Hyp6AM9, Hyp6AM10, Hyp6AM11, Hyp6AM12, Hyp6AM13, Hyp6AG1, Hyp6AG2, Hyp6AG3, Hyp6AG4, Hyp6AG5, Hyp6AG6, Hyp6AG7.
Example 6
Genetic Study of 6C
A non-capsulated pneumococcal strain might be transformed with genes from a 6C isolate. This finding suggests that 6C capsule synthesis requires one (not multiple) gene fragment, most likely the capsule gene locus. To identify the gene(s) responsible for 6Aβ expression, three transferases (wciN, wciO, and wciP) were examined. The wciP gene may be identical between 6A and 6C isolates (as discussed above). When wciN region was examined by PCR using primers 5016 and 3101 (5106: 5′-TAC CAT GCA GGG TGG AAT GT (SEQ ID NO:5) and 3101: 5′-CCA TCC TTC GAG TAT TGC (SEQ ID NO:6)), all nine 6Aa isolates examined yielded about 200 base pair (bp) longer product than did all six 6C isolates examined (FIG. 5). The six isolates included 6C isolates from Korea, USA, and Brazil. Thus, this PCR can be used as a genetic test for 6A subtypes.
The nucleotide sequences of the PCR products from one 6A isolate (AAU9) and one 6C isolate (ST745) were then determined (FIG. 6). All the bases between positions 1203 to 2959 (1757 bases) in AAU9 PCR product were sequenced and the sequence was found to be homologous to CR931638, which is the capsule locus sequence of a 6A isolate reported in the GenBank database. By contrast, the ST745 sequence was found to be almost identical to that of 6Aα up to position 1368, and then again starting from position 2591. The intervening 1029 bp sequence (from 1369 to 2397) is quite different from that of 6A. The intervening sequence contains about 98 bp that is similar to a transferase used for polysaccharide synthesis by Streptococcus thermophilus. 
Example 7
The 6C Isolates have Chemically Distinct Capsules
Two 6C isolates (BZ17 and BZ650), four 6A strains (SP85, ST558, and CHPA378), and two 6B strains (ST400 and ST518) were compared. All pneumococcal isolates had colony morphologies typical of pneumococci, and were both optochin-sensitive and bile-soluble. Subtyping assays were conducted as described in Example 3, above.
Polysaccharide isolation and purification: A pneumococcal strain (SP85 or BZ17) was grown in two liters of a chemically defined medium (van de Rijn et al., 27 Infect. Immun. 444-49 (1980)) from JRH Biosciences (Lenexa, Kans.), which was supplemented with choline chloride, sodium bicarbonate and cysteine-HCl, and lysed with 0.05% deoxycholate. After removing cell debris by centrifugation, PS was precipitated in 70% ethanol and was recovered by dissolving it in 120 mL of 0.2 M NaCl. After dialyzing the PS in 10 mM Tris-HCl (pH 7.4), the PS was loaded onto a DEAE-Sepharose (Amersham Biosciences, Uppsala, Sweden) column (50 ml) and eluted with a NaCl concentration gradient. The resulting fractions were tested for 6Aα or 6Aβ PS with the inhibition assay described above. The PS-containing fractions were pooled, concentrated by ethanol precipitation (70%), dialyzed, and lyophilized. The lyophilized PS was dissolved in 3 ml of water and loaded onto a gel filtration column containing 120 ml of Sephacryl S-300 HR (Amersham Biosciences). The PS was eluted from the column with water and all the fractions were tested for 6Aβ PS with the inhibition assay. The fractions containing the first 6Aα or 6Aβ PS peak were pooled and lyophilized.
Monosaccharide analysis: The lyophilized capsular PS was subjected to methanolysis in 1.5 M HCl at 80° C. for 16 hr. After evaporating the methanolic HCl, the residue was treated with Tri-Sil reagent (Pierce Biotech. Inc. Rockford, Ill.) for 20 min at room temperature. The reaction products were analyzed on a GLC/MS (Varian 4000, Varian Inc. Palo Alto, Calif.) fitted with a 30 m (0.25 mm in diameter) VF-5 capillary column. Column temperate was maintained at 100° C. for 5 min, and then increased to 275° C. at 20° C./min, and finally held at 275° C. for 5 min. The effluent was analyzed by mass spectrometry using the electron impact ionization mode.
Oxidation, reduction, and hydrolysis: Capsular PS (1 mg/mL) was treated with 40 mM sodium periodate in 80 mM sodium acetate buffer (pH=4) for four days at 4° C. in the dark. After neutralizing the excess periodate with ethylene glycol, the sample was dialyzed and lyophilized. Stroop et al., 337 Carbohydr. Res. 335-44 (2002). The PS (1 mg/mL) was reduced with 200 mg/mL of sodium borohydride (NaBH4) or its deuterium form (NaBD4) for three hours at RT, dialyzed, and lyophilized. The oxidized/reduced 6C PS was hydrolyzed in 0.01M NaOH at 85° C. for thirty minutes, neutralized by adding 0.01M HCl, and then directly used for mass spectrometry without desalting.
Tandem mass spectrometry: The tandem mass spectral analysis of native and oxidized/reduced 6C were performed in the Mass Spectrometry Shared Facility at the University of Alabama at Birmingham with Micromass Q-TOF2 mass spectrometer (Micromass Ltd. Manchester, UK) equipped with an electrospray ion source. The samples, dissolved in distilled water, were injected into the mass spectrometer along with running buffer (50/50 acetonitrile/water containing 0.1% formic acid) at the rate of 1 μL/min using a Harvard syringe pump. The injected sample was negatively ionized with electrospray (needle voltage=2.8 kV) and detected with a TOF mass spectrometer. The injected sample was negatively ionized with electrospray (needle voltage=2.8 kV) and detected with a TOF mass spectrometer. For MS/MS, the parent ion was fragmented into daughter ions by energizing it to 40 eV before collision with argon gas. The daughter ions were analyzed with a TOF mass spectrometer. The MS/MS spectra were processed using the Max-Ent3 module of MassLynx 3.5.
Smith degradation and glycerol detection: Periodate treated 6A and 6C PSs were reduced with 10 mg/ml Sodium borodeuteride in 1M ammonium hydroxide for 16 hr. Excess sodium borodeuteride was removed by addition of glacial acetic acid and 0.5 ml of methanol:acetic acid (9:1) was added. Samples were dried under a stream of nitrogen and washed twice with 0.25 ml of methanol. Dried samples were suspended in 0.5 ml of 1.5M methanolic HCl and incubated at 80° C. for 16 hr. Samples were dried under a stream of nitrogen and washed twice with 0.25 ml of methanol. Dried samples were suspended in 0.1 ml of Tri-Sil (Pierce) and incubated at 80° C. for 20 min. The 1 μl of samples were injected into a Varian 4000 gas chromatograph mass-spectrometer (Varian Inc., Palo Alto, Calif.) equipped with a 60 m VF-1 column. Helium was used as the carrier gas at a constant flow rate of 1.2 ml/min. The oven conditions were an initial temperature of 50° C. held for 2 min, temperature increase at 30° C./min to 150° C., then another increase at 3° C./min to 220° C., which was held for two minutes. The injector temperature was kept at 250° C. and the MS transfer-line at 280° C. MS data acquisition parameters included scanning from m/z 40 to 1000 in the electron impact (EI) mode or in the chemical ionization (CI) mode using acetonitrile.
The chromatography of 6A PS showed all the peaks that are characteristic of ribitol, rhamnose, glucose, and galactose (FIG. 7A), consistent with a previous publication. Kim et al., 347 Anal. Biochem. 262-74 (2005). For instance, galactose yields three major peaks appearing between 11.2 and 11.6 min retention times with the second peak being the tallest. Kim et al. (2005). When 6C PS chromatogram was examined, characteristic peaks of ribitol, rhamnose, and glucose were found but galactose peaks was absent. When the areas of each carbohydrate peaks were normalized to rhamnose peak area and compared between 6A and 6C (FIG. 7B), 6A and 6C PS have the equivalent areas of ribitol peaks. The glucose peak area of 6C, however, was twice of that of 6A (FIG. 7B). This finding suggested that the repeating unit of 6C has one ribitol and one rhamnose as 6A but it has two glucose molecules instead of one each of glucose and galactose molecules. Thus, 6C produces a capsular PS that is chemically different from the PS produced by 6A by using glucose instead of galactose.
To further investigate the two glucose molecules presumed to be present in 6C PS, 6A and 6C PS were treated with periodate, which selectively destroys vicinal glycols. As expected from the published structure of 6A PS, the galactose and ribitol peaks of 6A PS became undetectable while the glucose and rhamnose peaks were undisturbed. Kamerling, Pneumococcal polysaccharides: a chemical view, in Mol. Biol. & Mechanisms of Disease 81-114 (Mary Ann Liebert, Larchmont, 2000); Kim et al., 347 Anal. Biochem. 262-74 (2005); Rebers & Heidelberger, 83 J. Am. Chem. 3056-59 (1961). When 6C PS was periodate-treated, its ribitol became undetectable and its glucose peak was reduced by about half while its rhamnose peaks remained undisturbed (FIG. 7B). This finding strongly suggests that the 6A PS structure is identical to the 6A PS structure published in the literature. Also, it indicates that 6C PS is chemically different from 6A PS and that 6C PS has two glucose molecules, one of which is sensitive to periodate and the other of which is not.
Example 8
Determination of Monosaccharide/Ribitol Sequence within the Repeating Units
A mild alkali hydrolysis of 6A PS breaks the phosphodiester bond in each repeating unit and produces a repeating unit with a negative charge, which can then be examined with tandem mass spectrometry. The hydrolysis product of 6A PS (from strain SP85) showed three well-defined peaks with a negative charge: peaks with 683.21, 701.21, and 759.19 mass to charge ratio (m/z) units (FIG. 8A). The peak at 683.21 m/z units represents anhydrous form of the peak at 701.21 m/z units and the peak at 759.19 represents the molecule with 701.21 m/z unit with NaCl salt. This indicates that the mass of the repeating unit is 683.21 mass units as described. Kamerling, 2000; Kim, 2005. The daughter ions (product ions) of the 701.21 peak were examined and yielded daughter ions with masses of 539.13, 377.08, and 212.99 m/z units, which respectively correspond to the masses of glucose-rhamnose-ribitol-P, rhamnose-ribitol-P, and ribitol-P fragments (FIG. 8C). Also their anhydrous counterparts at 701.21, 539.14, 377.08 and 212.99 m/z units. Additional peaks observed at 96.94 m/z and 78.93 m/z units represent H2PO4− and PO3− ions (FIG. 8C).
Analysis of 6C PS, using the same procedure used for 6A PS, showed three major peaks at 683.24, 701.25, and 759.22 m/z units, corresponding to the three major peaks found for 6A PS (FIG. 8B). Also, the 6C cleavage products had a mass spectrum identical to those of 6A (FIG. 8D). This finding indicated that the mass of the repeating unit of 6C PS is 683.2 m/z units and that the carbohydrate sequence of the 6C repeating unit is glucose 1-glucose 2-rhamnose-ribitol-P. (To distinguish between the two glucoses, they are labeled as glucose 1 and glucose 2. Glucose 1 corresponds to the galactose of 6A. Thus, the monosaccharide sequence of 6C is identical to that of 6A except for the replacement of galactose with glucose 1.
Example 9
Determination of the Linkages Between Carbohydrate and Ribitol of the 6C Repeating Unit
To identify the 6C glucose that is periodate-sensitive, 6C PS was oxidized and reduced to repeating units by mild alkali hydrolysis, and the repeating units studied with tandem mass spectrometry. Their mass spectrum showed several major (and dominant) peaks between 650 and 700 m/z units (FIG. 9A). The dominant peaks were at 655.23, 659.73, 661.24, 664.25, 673.25, and 675.24 m/z units. Due to natural isotopes, each dominant peak has satellite peaks with one or two additional mass units and these satellite peaks can be used to determine the charge states and the true mass of the dominant peaks. Cole, ELECTROSPRAY IONIZATION MASS SPECTROMETRY: FUNDAMENTALS, INSTRUMENTATION, & APPLICATIONS (Wiley, New York, 2000). For instance, the dominant peak at 661.24 m/z units has a satellite peak with 661.57 m/z units. Because these two peaks are separated by 0.33 m/z units, the 661.24 peak represents a molecular ion with three negative charges and 1983.72 mass units (i.e., three repeating units with one water molecule; 655.23*2+673.76=1983.72). Similarly, the 664.25 and 675.24 peaks represented two repeating units with two negative charges, but the 675.24 peak has a sodium ion replacing a proton. The 673.25 and 655.23 peaks represent one repeating unit with one negative charge with or without a water molecule. Because the mass of the anhydrous repeating unit prior to oxidation/reduction was 683.26, the repeating unit lost 28 mass units due to oxidation and reduction. To identify the periodate reaction products of ribitol and glucose, the ribitol fragment was named the Rx fragment and the two glucose fragments were named the Gx and Gy fragments (FIG. 10A).
Daughter ions were obtained by fragmenting the parent ion with 673.25 m/z units (FIG. 9B). During the fragmentation, one fragment may exchange one atomic mass unit (AMU) with the other fragment. Grossert et al., 20 Rapid Commun. Mass. Spectrom. 1511-16 (2006); McLafferty 31 Anal. Chem. 82-87 (1959). Also, molecular ions become variably hydrated within argon collision cells. Sun et al., 69 Infect. Immun. 336-44 (2001). Indeed, the daughter ions could be grouped into hydrated and anhydrous peaks based on differences of 18 m/z units (FIG. 9B). The peaks found at 673.25, 581.16, 509.13, 347.07, and 200.99 m/z units are hydrated peaks, each of which has a corresponding anhydrous peak that is 18 AMU less. Also, the peaks at 200.99, 347.07 and 509.13 m/z units correspond to the fragments with 200, 346, and 508 AMUs with one hydrogen atom added to the fragmentation site (FIG. 10B) during the fragmentation. The peak at 200.99 m/z unit confirms that ribitol lost CH2OH during the periodate treatment. The peaks at 347.07 and 509.13 indicate that rhamnose and glucose 2 are periodate resistant. Presence of a peak at 581.16 indicates that glucose 1 is cleaved.
Periodate cleavage divided glucose 1 into two parts (which were named Gx and Gy in FIG. 10A). The combined mass of the two parts is 164 instead of 162 (mass of intact glucose) because glucose 1 lost no carbon but acquired two hydrogen atoms at the breakage site during the oxidation and reduction reactions. The mass spectrum shown in FIG. 9 is consistent with Gx and Gy having 91 and 74 AMUs respectively. The peak at 581.16 m/z units indicates that a repeating unit lost Gx and one extra proton (FIG. 10). Neutral loss of both Gx and Gy (74 AMUs) results in additional loss of 72 m/z units because Gy already lost one hydrogen to Gx and leaves one hydrogen with glucose 2. The same patterns were found for the anhydrous peaks: i.e., 655.22, 563.16, and 491.12 m/z units. Furthermore, when the 6Aβ PS was reduced with NaBD4, the two additional mass units were associated with glucose 1: the neutral loss of Gx fragment was 93 instead of 92, and that of Gy was 73 instead of 72 (FIG. 4C). These findings clearly indicated that glucose 1 cleaves into Gx and Gy with sizes shown in FIG. 10A.
The mass spectrum of daughter ions also provided information about the glycosidic linkages of 6Aβ PS. Glucose and rhamnose must be linked to the preceding carbohydrate at their first carbon. Rebers & Heidelberger, 1961. Also, they must be linked to the succeeding carbohydrate at the third carbon in order to be resistant to periodate. Rebers & Heidelberger, 1961 Thus, 6C PS must have glucose 1 (1→3) glucose 2 (1→3) rhamnose (1→). Further examination of the daughter ions shows that their glucose 1 has the phosphodiester bond at its second carbon. To be periodate sensitive, glucose 1 must have its phosphodiester link only at positions 2, 4, or 6. The phosphodiester bond linkage is not at position 6 because the linkage at 6 results in a loss of a carbon atom in glucose 1 (FIG. 10F). If the phosphodiester linkage is at position 4, the breakage occurs between the second and the third carbon. Gx and Gy should then have 120 and 42 AMUs, and a peak with 552 m/z units should be detected instead of the peak at 581 m/z unit (FIG. 10E).
Although hydrolysis cleaves the phosphodiester bond with glucose 1, it occasionally breaks the phosphodiester bond with ribitol instead. Examination of this reverse cleavage products further confirms that the phosphodiester linkage must be at the second carbon of glucose 1. The peaks with 150.95 and 243.00 m/z units are reverse cleavage products of glucose 1 (FIG. 9B) since products with these m/z units can be produced from glucose 1 with the phosphodiester bond at the second carbon (FIG. 10D) and these peaks have one (150.95151.97) or two (243.00245.02) more m/z units if reduction was performed with NaBD4 instead of NaBH4 (FIG. 9C). An ion at 120.95 m/z units can be also obtained if the ion at 150 m/z units loses the terminal methanol group. These peaks cannot be explained if the phosphodiester bond is at the fourth or the sixth carbon (FIGS. 10E and 10F). Thus, the data with the reverse cleavage products also indicate that the phosphodiester bond is linked to the second carbon of glucose 1.
Additional examination of the mass spectra showed that the rhamnose-ribitol linkage must be (1→3). Because pneumococci use CDP-5-ribitol that is produced for teichoic acid synthesis for their capsule synthesis as well (Pereira & Brown 43 Biochem. 11802-12 (2004)), the linkage between ribitol and glucose 1 must be ribitol (5→P→2) glucose 1. The peaks at 78.94 and 96.94 correspond to PO3− and H2PO4−, while the peaks at 182.98 and 200.99 (FIG. 9B) correspond to the Rx fragment attached to PO3− and H2PO4− (FIG. 10A). Thus, ribitol must lose a hydroxymethyl group during the oxidation and reduction reaction and the linkage between rhamnose and ribitol must be rhamnose (1→3) ribitol. Considering all of the above, the 6Aβ repeating unit should be {P→2) glucose 1 (1→3) glucose 2 (1→3) rhamnose (1→3) ribitol (5→} (FIG. 10C).
When 6A PS was analyzed, peaks identical to the 6C PS peaks were found, which indicate that galactose and ribitol were destroyed by periodate but that glucose 2 and rhamnose remained intact. Thus, the structure of 6A PS must be {→2) galactose (1→3) glucose 2 (1→3) rhamnose (1→3) ribitol (5→P), which is identical to the 6A PS structure published in the literature. Kamerling, 2000; Rebers & Heidelberger, 1961. In summary, the structural difference between 6A and 6C PS is the orientation of the hydroxyl group at the fourth carbon of glucose 1 (or galactose).
Classically, the phosphodiester bond of 6A PS was determined to be at the second carbon of galactose by demonstrating that glycerol is released after a Smith degradation of the 6A PS that was oxidized and reduced. Rebers & Heidelberger, 1961. To confirm the position of the 6C phosphodiester bond using this classical approach, the Smith degradation of 6A and 6C PSs after oxidation and reduction was performed as described above. The reaction products of 6A and 6C PSs indicated glycerol from the two PSs. Thus, glucose 1 has a phosphodiester bond at the second carbon of glucose 1.
Example 10
Genetic Origin of Serotype 6C
Bacterial Strains and Culture:
The pneumococcal strains used in the 6C study are listed in Table 4:







TABLE 4







List of pneumococcus strains











Strain


Country of origin



names
Serotype
Tissue location
(year of isolation)
Source or reference





CHPA37
6C
Nasopharynx
USA
McEllistrem et al., 40 Clin.





(1999-2002)
Infect. Dis. 1738-44 (2005)


CHPA388
6C
Nasopharynx
USA
Id.





(1999-2002)


BGO-2197
6C
Nasopharynx
USA (1979)
Hollingshead, unpublished


MX-67
6C
Bronchus
Mexico (1996)
Robinson et al., 184 J.


CMN



Bacteriol. 6367-75 (2002)


ACA-C21
6C
Nasopharynx
Canada (1995)
Id.


BZ17
6C
CSFa
Brazil (2003)
Lin et al., 176 J. Bacteriol.






7005-16 (1994)


BZ39
6C
CSF
Brazil (2003)
Id.


BZ86
6C
CSF
Brazil (2003)
Id.


BZ650
6C
CSF
Brazil (2003)
Id.


ST260
6C
CSF
Brazil (2003)
presented herein


KK177
6C
Oropharynx
Korea (2005)
presented herein


CH66
6C
Nasopharynx
China (1997)
Robinson et al., 2002


CH158
6C
Nasopharynx
China (1997)
Id.


CH199
6C
Nasopharynx
China (1998)
Id.


CHPA67
6A
Nasopharynx
USA
McEllistrem et al., 2005





(1999-2002)


CHPA78
6A
Nasopharynx
USA
Id.





(1999-2002)


BZ652
6A
CSF
Brazil (2003)
Lin et al., 1994


KK58
6A
Oropharynx
Korea (2005)
presented herein


AAU-33
6A
Blood
USA (1998)
Mavroidi et al., 186 J.






Bacteriol. 8181-92 (2004)


TIGR4JS4
Non-
derived from TIGR4b
Not applicable
Trzcinski et al., 2003. 69 Appl.



capsulated


Environ. Microbiol. 7364-70






(2003)


TIGR6A4
6A
derived from TIGR4JS4
Not applicable
presented herein


TIGR6AX
Non-
derived from TIGR6A4
Not applicable
presented herein



capsulated


TIGR6C4
6C
derived from TIGR6A4
Not applicable
presented herein





aCSF, cerebrospinal fuid.


bTIGR4 was originally isolated from blood. Tettelin al., 293 Science, 498-506 (2001).






In addition to the 6C isolates from Brazil that were reported earlier (Lin et al., 2006), additional 6C strains were identified by retyping the preexisting pneumococcal isolates archived in the laboratory as the “6A” serotype. The collection includes 6A isolates used for studies by Robinson et al., 184 J. Bacteriol. 6367-75 (2002) and Mavroidi, 2004. One strain (BGO-2197) was isolated in 1979 in Birmingham, Ala., U.S. The TIGR4JS4 strain is a non-capsulated variant of the TIGR4 strain (Tettelin et al., 293 Science 498-506 (2001)), and was produced by replacing type 4 capsule gene locus with Janus cassette (kanR-rpsL+) and backcrossing 3 times to wildtype TIGR4 (Trzcinski et al., 69 Microbiol. 7364-70 (2003); Hollingshead (unpublished). TIGR6AX, TIGR6A4, and TIGR6C4 are TIGR4JS4 variants expressing, respectively, no, 6A, or 6C capsule types. These variants were produced as described below.
PCR and DNA Sequencing:
All the PCR primers used in this study are listed in Table 5. The primers used for multi-locus sequence typing (MLST) were as described by Enright & Spratt, 144(11) Microbiol. 3049-60 (1998), and the primers used to amplify the wciN, wciO, and wciP genes were described by Mavroidi et al., 2004. Additional primers were designed using the DNA sequences of the 6A and 6B capsule gene loci in GenBank (accession numbers CR931638 and CR931639, respectively).







TABLE 5







List of PCR primers












Primer site


Source


Primer
of No.


or


name
CR931638
Description*
Sequence
reference










Forward primers











5101
6949-6966
in wciN, for
5′-ATTTGGTGTACTTCCTCC
Mavroidi




INDEL detection
(SEQ ID NO: 7)
et al.,






2004


 


5103
8146-8168
in wciO, for
5′-AAACATGACATCAATTACA
presented




sequencing 6C
(SEQ ID NO: 8)
herein




capsule gene




 


5106
5897-5916
in wchA, for wciN
5′-TACCATGCAGGGTGGAATGT
presented




detection
(SEQ ID NO: 5)
herein


 


5108
8350-8370
in wciP, for wciP
5′-ATGGTGAGAGATATTTGTCAC
presented




allele detection
(SEQ ID NO: 1)
herein


 


5112
Not applicable
in KanR-rpsL+ with
5′-CTAGTCTAGAGTTTGATTTTTAATGG
presented




XbaI site
(SEQ ID NO: 9)
herein


 


5113
4870-4894
in wze, for
5′-GGGAAAAATAAAAAATAGGTCGGG
presented




Fragment C
(SEQ ID NO: 10)
herein


 


5118
7613-7636
in wciO with
5′-CGCGGATCCAGAAAAACTATGTCGCCTGCTAAA
presented




BamHI site
(SEQ ID NO: 11)
herein


 


5120
 1-30
in dexB, for
5′-TGTCCAATGAAGAGCAAGACTTGACAGTAG
Trzcinski




Fragment A
(SEQ ID NO: 12)
et al.,






2003


 


5122
2187-2206
in wzg, for
5′-TTCGTCCATTCACACCTTAG
presented




Fragment B
(SEQ ID NO: 13)
herein


 


5123
8775-8794
in wciP, for
5′-TGCCTATATCTGGGGGTGTA
presented




Fragment D
(SEQ ID NO: 14)
herein


 


5124
11274-11293
in wzx, for
5′-AATGATTTGGGCGGATGTTT
presented




Fragment B
(SEQ ID NO: 15)
herein


 


5125
13864-13883
in rmlC, for
5′-AGTGATTGATGCGAGTAAGG
presented




Fragment F
(SEQ ID NO: 16)
herein


 


5140
9531-9551
in wzy, for wzy
5′-CCTAAAGTGGAGGGAATTTCG
Mavroidi




allele detection
(SEQ ID NO: 17)
et al.,






2004


 


5141
11459-11478
in wzx, for wzx
5′-TTCGAATGGGAATTCAATGG
Id.




allele detection
(SEQ ID NO: 18)



 







Reverse primers











3101
7888-7905
in wciO, for
5′-CCATCCTTCGAGTATTGC
Mavroidi




INDEL and wciN
(SEQ ID NO: 6)
et al.,




detections

2004


 


3103
9468-9487
in wzy, for Janus
5′-AACCCCTAACAATATCAAAT
presented




cassette and
(SEQ ID NO: 19)
herein




Fragment C




 


3107
9226-9245
in wciP, for wciP
5′-AGCATGATGGTATATAAGCC
presented




allele detection
(SEQ ID NO: 2)
herein


 


3112
Not applicable
in KanR-rpsL+ with
5′-CGCGGATCCGGGCCCCTTTCCTTATGCTTTTGG
presented




BamHI site
(SEQ ID NO: 20)
herein


 


3113
6203-6224
in wchA with XbaI
5′-CTAGTCTAGAAATAAAATTTCAATATCTTTCCAG
presented




site
(SEQ ID NO: 21)
herein


 


3121
3676-3660
in wzd, for
5′-GATTGCGATTCACTACG
presented




Fragment A
(SEQ ID NO: 22)
herein


 


3122
5380-5361
in wchA, for
5′-AACTCCCCAACAACCTCATT
presented




Fragment B
(SEQ ID NO: 23)
herein


 


3123
12978-12959
in rmlA, for
5′-AAAATCAAGGCAACGCTATC
presented




Fragment D
(SEQ ID NO: 24)
herein


 


3124
14618-14600
in rmlB, for
5′-ACGGAGAGCTTGGGTTGTA
presented




Fragment B
(SEQ ID NO: 25)
herein


 


3126
17611-17584
in aliA, for
5′-CAATAATGTCACGCCCGCAAGGGCAAGT
Trzcinski




Fragment F
(SEQ ID NO: 26)
et al.,






2003


 


3143
10135-10115
in wzy, for wzy
5′-CCTCCCATATAACGAGTGATG
Mavroidi




allele detection
(SEQ ID NO: 27)
et al.,






2004


 


3144
12068-12049
in wzx, for wzx
5′-GCGAGCCAAATCGGTAAGTA
Id.




allele detection
(SEQ ID NO: 28)





*Fragments A through F refers to the fragments of serotype 6C capsule gene locus used for capsule gene locus sequencing.






For capsule gene locus PCR, the reaction mixture had 10 ng to 30 ng of chromosomal DNA, 1 μl of each primer from a 100-pmol stock, 2 μl of 10 mM dNTP, 5 μl of 10× buffer solution, 0.5 μl (2.5 U) of Taq polymerase (Takara Biomedical, Shiga, Japan), and 39.5 μl of sterile water (Sigma, St Louis, Mich.). The reaction mixture for multi-locus sequence typing had 10 ng to 30 ng of chromosomal DNA, 1 μl of each primer from a 50-pmol stock, 2 μl of MgCl2, 5 μl of Q-solution (Qiagen, Chatsworth, Calif.), 12.5 μl of Master Mix (Qiagen), and 4 μl sterile water (Sigma). Chromosomal DNA was isolated with a Wizard Genomic DNA Purification Kit (Promega, Madison, Wis.) according to the manufacturer's instruction. Thermal cycling conditions were: initial denaturation at 95° C. for 3 min, 30 cycles of denaturation at 95° C. for 1 min, annealing at 52° C.-58° C. for 1 min, extension at 72° C. for 2 min, and a final extension at 72° C. for 10 min. Multi-locus sequence typing used 30 cycles, and capsule locus gene PCR used 35 cycles. The size of the PCR products was determined by electrophoresis in a 1%-1.5% agarose gel.
The DNA sequence of the PCR products was determined by the genomics core facility at the University of Alabama using an automated DNA sequencer, and the PCR products were purified with a Wizard PCR Cleanup Kit (Promega). DNA sequences were analyzed with Lasergene v. 5.1 software (DNASTAR, Madison, Wis.) and the Basic Local Alignment Search Tool (BLAST) located on-line at the NCBI NLM NIH website.
The sequences from the capsule gene locus were compared with the sequences previously reported. Mavroidi et al., 2004. Alleles of each sequence type were assigned using the on-line pneumococcal Multi Locus Sequence Typing (MLST) website. When the sequences were different, new allele numbers were assigned. All the wciNβ sequences are then deposited in the pneumococcal MLST. The entire capsule gene locus of the pneumococcal isolate CHPA388 is deposited in GenBank.
Genetic profiles of 6C strains collected from global sources are presented in Table 6:







TABLE 6







Global sources of serotype 6C isolates









Multilocus sequence typing (MLST)

















Capsule gene







Seq.



locus profile







Type





















Strains
Origin
wciP
wzy
wzx
aroE
gdh
gki
recP
spi
xpt
ddl
(ST)
























1
CHPA37
US
9 (1)
10 (0)
1 (0)
1
13
1
43
5
TD*
20
—


2
CHPA388
US
9 (1)
10 (0)
1 (0)
10 
13
1
43
98
1
20
1390


3
BGO2197
US
9 (1)
10 (0)
1 (0)
2
13
2
1
6
19
14
1092


4
ACA-C21
CA
9 (0)
10 (0)
1 (0)
13 
1
1
5
6
1
18
1715


5
MX67
MX
9 (0)
10 (0)
1 (1)
7
25
4
4
15
20
28
NT


6
BZ17
BR
9 (1)
10 (0)
1 (0)
—
—
—
—
—
—
—
—


7
BZ39
BR
9 (1)
10 (0)
1 (0)
—
—
—
—
—
—
—
—


8
BZ86
BR
9 (1)
10 (0)
1 (0)
 7**
13
8
6
1
1
8
NT


9
BZ650
BR
9 (1)
10 (0)
1 (0)
—
—
—
—
—
—
—
—


10
ST260
BR
9 (1)
10 (0)
1 (0)
1
5
9
43
5
1
6
NT


11
KK177
KR
9 (0)
 1 (0)
1 (0)
7
30
8
6
6
6
14
NT


12
CH66
CN
9 (0)
10 (0)
1 (1)
 7**
42
4
39
25
104
14
NT


13
CH158
CN
9 (0)
10 (0)
1 (1)
—
—
—
—
—
—
—
—


14
CH199
CN
9 (0)
10 (0)
1 (1)
—
—
—
—
—
—
—
—









Production of TIGR4 Variants with 6A and 6C Capsule Gene Loci:
To investigate the role of the wciN gene in 6C capsule expression, desired genes or gene fragments were inserted into the TIGR4JS4 strain, which is derived from TIGR4 but which has lost the capsule gene locus (FIG. 11). Aliquots of frozen, transformation-competent TIGR4JS4 were made by growing it in THY broth at 37° C. until the optical density at 600 nm was about 0.4-0.5; by diluting it 1:100 in Todd-Hewitt broth (pH7.2) supplemented with 0.5% yeast extract, 0.2% bovine serum albumin, 0.01% CaCl2, and 13% glycerol; and by freezing it in 250 μl aliquots at −80° C.
To transform TIGR4JS4, a frozen bacterial aliquot was thawed and mixed with 50 ng of competence-stimulating peptide variant 2. Trzcinski et al., 2003. After 14 min incubation at 37° C., 100 μl of TIGR4JS4 was mixed with 10 μl of bacterial lysate (AAU33 strain) or 100 ng of DNA cassettes. After 2 hr incubation at 37° C., the bacteria were plated on sheep blood agar plates containing 200 μg/ml kanamycin or 300 μg/ml streptomycin and incubated at 37° C. in a candle jar. Colonies of transformants growing in the antibiotic media were harvested and backcrossed three times with DNA-recipient competent bacteria.
To prepare a bacterial lysate of AAU33 for transformation, 10 ml of THY broth was inoculated with the AAU33 strain and cultured for about 5 hr at 37° C. until the optical density at 600 nm was  ̃0.4-0.5. The THY broth was centrifuged to obtain a bacterial pellet, and the pellet was lysed by resuspending it in 0.1 ml of sodium citrate buffer (0.15M, pH 7.5) containing 0.1% sodium deoxycholate and 0.01% sodium dodecylsulfate and then incubating it for 10 min at 37° C. The lysate (0.1 ml) was then mixed with 0.9 ml of normal saline buffered with 0.015M sodium citrate (pH 7.0) and heat-inactivated at 65° C. for 15 min.
To replace the wciNα gene region of TIGR6A4 with the wciNβ gene region from CHPA388, two different DNA cassettes were prepared, labeled Cassette 1 and Cassette 2 in FIG. 11. Each cassette has three parts: the central core containing the target DNA and two flanking DNAs. The two flanking DNAs are for homologous recombination, are about 1 Kb each, and were obtained from either wchA or wciO-P genes. The central core of Cassette 1 has kanamycin-resistance (kanAR) and streptomycin-sensitivity (rpsL+) genes and is obtained by PCR using TIGR4JS4 strain DNA as the template. The flanking DNA fragments were obtained by PCR using chromosomal DNA of AAU33 as the template. All the primer pairs, which are shown in FIG. 11 and Table 5, have restriction enzyme sites to facilitate linking the three DNA fragments. The three DNA fragments were linked together by digestion with an appropriate restriction enzyme and ligation with T4 DNA ligase (New England BioLabs, Beverly, Mass.). The ligation product was amplified by PCR using primers 5113 and 3102. The PCR product was purified by the Wizard PCR Cleanup Kit (Promega) and subjected to nucleotide sequencing. The PCR product was then used as donor DNA in the transformation.
Cassette 2 was used to replace the antibiotic selection genes with the wciNβ gene. The central core has the wciNβ gene from CHPA388. The wchA and wciO-P DNA fragments were obtained by PCR from AAU33 as described for Cassette 1 (FIG. 11).
Identification of Additional 6C Strains Among “6A” Collections:
To obtain a representative collection of 6C serotypes from various locations, the preexisting collection of “6A” strains were re-tested by quellung reaction (Mavroidi et al., 2004; Robinson et al., 2002) and identified nine additional 6C isolates from five countries on three different continents (Table 4). These isolates were obtained from spinal fluid, blood, and the nasopharynx samples, indicating that 6C can be associated with invasive pneumococcal infections as well as asymptomatic carriage. One isolate (BG02197) was obtained in 1979 at Birmingham, Ala. This finding shows that the 6C serotype, identified and isolated for the first time as described herein, may have been in existence for more than twenty-seven years and is now found throughout the world.
Many 6C strains have the identical capsule gene locus profile but different sequence types: To begin investigating the genetic basis for the serotype 6C, the capsule gene locus profiles and the sequence types (STs) of the twelve isolates were examined. Similar to what was observed previously for the Brazilian 6C isolates (Lin et al., 2006), all 6C isolates have allele 9 of the wciP gene with either no or one nucleotide difference. Similarly, all 6C isolates have allele 1 of the wzx gene with either no or one nucleotide difference. All 6C isolates have allele 10 for the wzy gene except for one isolate, which expresses allele 1. In contrast to the 6C isolates' restricted capsule gene locus profile, multi-locus sequence typing shows that 6C isolates express diverse STs. The fact that 6C is associated with multiple STs but with one single capsule gene locus profile (except for one isolate) suggests that the gene(s) responsible for the 6C serotype is probably in the capsule gene locus.
The capsule gene loci of 6A and 6C differ in the region between the wchA and wciO genes: It was hypothesized that the genetic difference between serotypes 6A and 6C is a glycosyl transferase gene, the same gene that is responsible for the difference between serotypes 6A and 6B. When PCR was used to compare the sizes of their glycosyl transferase genes, it was found that the sizes of their wciN genes were different. The wciN PCR products of all 6C isolates were about 1.8 kb long whereas the wciN PCR products of all 6A isolates were about 2 kb long (FIG. 12). To distinguish between the two wciN genes from the 6A and 6C serotypes, they have been named wciNα and wciNβ, respectively.
To further investigate wciNβ gene, the DNA sequences of the wciNβ gene region including the wchA and wciO genes from five 6C strains (BZ17, BZ86, CHPA388, KK177, and ST 260) were compared. Because their sequences were almost identical, the actual DNA sequence is shown for only CHPA388 (FIG. 13) and the sequences of other isolates are deposited in GenBank. The sequence of the wciNβ gene from CHPA388 was then compared with the 6A sequence of the corresponding region available at the GenBank (No. CR931638) (FIG. 13). A summary of the comparison is shown in FIG. 14.
The sequence comparison revealed clear differences in wciNα and wciNβ genes: The 6C serotype has 1029-bp-long DNA in place of 1222-bp-long DNA in 6A (FIG. 14 and FIG. 15). The two wciN genes are completely different, with the sequence homology being only about fifty percent. The DNA difference begins immediately after the termination of wchA gene (position 1368) and ends 130 bases upstream to the beginning of the wciO gene (positions 2398 for 6C and 2631 for 6A) (FIG. 14 and FIG. 15). When the DNA sequences flanking the replaced gene were compared between 6A and 6C, significantly more DNA polymorphisms were found in the flanking regions than in the regions outside of the two flanking regions. For instance, the 300 bases upstream from the replaced gene have 25 different nucleotides, but the 150 bases located immediately upstream from the 300 bases have only one different base (p<0.001 by Fisher's exact test) (FIG. 15). Similarly, in the 3′ direction, 20 bases differ in the proximal 110 bases but only 1 base differs in the next 300 bases (p<0.001 by Fisher's exact test) (FIG. 15). These findings are not unique to this particular 6A sequence (CR931638) because similar results were obtained with the sequence of seven different 6A strains AAU33, D020-1B, HS3050, CHPA78, KK65, ST19, and ST558. These findings suggest that the two flanking regions were parts of the new gene that has been inserted into 6A to create 6C.
The flanking regions may have been involved in the homologous recombination of the wciNβ gene to the 6A capsule locus. Furthermore, all 6C isolates have the identical flanking region sequences. This suggests that the genetic replacement took place only once and that all the 6C isolates must be progeny of this single founder bacterium.
With this gene replacement, wciNβ has a new open reading frame (ORF) that is 1125 bases long and encodes a peptide with 374 amino acids, which is named the WCINβ protein (FIG. 13). The termination codon of the new ORF is between the two potential start codons for the wciO gene, which are located at positions 2497 and 2528 of 6C. When the sequence of the wciNβ gene was compared with the sequences in the database, 110 bases (from 1627 to 1736 in 6C) of 6C demonstrated 81% homology to the 90 bases of the exopolysaccharide synthesis gene of Streptococcus thermophilus strain CNRZ1066 (Bolotin et al., 22 Nat. Biochem. 1554-58 (2004) (FIG. 13). Also, the translated sequence of wciNβ gene has 22% amino acid identity and 44% similarity to the translated sequence of capH gene of Staphylococcus aureus. Lin et al., 176 J. Bacteriol. 7005 16 (1994). The wciNβ gene product is a member of the waaG family. Incidentally, the waaG gene product of E. coli K-12 is an α1,3-glucosyltransferase involved in LPS synthesis. Heinrichs et al., 30 Mol. Microbiol. 221-32 (1998).
The sequences of the capsule gene loci of the 6A and 6C serotypes differ only slightly in regions other than the wciN gene: To determine if the 6A and 6C capsule gene loci differ only in the wciN region, the sequence of the entire capsule locus of a 6C isolate (CHPA388) was analyzed by PCR amplifying the entire capsule gene locus between dexB and aliA loci in six overlapping DNA fragments. FIG. 16 shows the genetic map of the sequence of the capsule gene locus. The entire CHPA388 locus is presented in FIG. 16. The 6C capsule gene locus contained fourteen ORFs involved in the capsular PS synthesis. The ORFs are in the same transcription orientation and correspond exactly to those found for the 6A capsule gene locus. The ORFs of 6C begin with cpsA gene at the 5′ end and end with rmlD gene at the 3′ end. As shown in FIG. 17, a putative promoter binding region and a transcription start site for 6C capsule gene locus are found 5′ to the cpsA gene and a putative transcription terminator site is found 3′ to the rmlD gene. Additionally, there are insertion element (or “tnp” or “transposase”) sequences at both ends of the capsule gene locus, as are commonly found for many pneumococcal capsule gene loci. Bentley et al., PLoS Genet. 2:e31 (2006). The nucleotide sequence of the entire locus are deposited in GenBank.
When the sequence was compared with the capsule gene locus of a 6A strain (GenBank accession No. CR31638), except for the wciN region described above, the capsule gene locus of 6C was very homologous (−98%) to that of 6A. Also, homology was significantly low (about 78%) for about 60 bp in the middle of the cpsA ORF and the “tnp's” found at either end of the capsule gene loci were different between the 6A and 6C capsule gene loci. The 6C capsule gene locus did not have the INDEL that is present upstream to the wciO gene in some 6A or 6B capsule gene loci. Mavroidi et al., 2004. Despite these differences, the most prominent difference between 6A and 6C capsule gene loci is found in the wciN region.
The wciN gene region is responsible for conversion from the 6A to 6C serotype. Although the above comparison of the capsule gene loci showed that the major difference is in the wciN region, minor differences are present in the entire capsule gene region (e.g., cpsA ORF). It is possible that some other small genetic differences outside of the capsule locus could be involved in the 6C expression. To show that only the wciN region is involved, whether the interchange of the wciNα region with the wciNβ region could convert the 6A serotype to the 6C serotype (FIG. 11) was examined. TIGR6A was produced by replacing the capsule locus of TIGR4 with the 6A capsule gene locus from strain AAU33. The wciNα gene was then removed from TIGR6A by transforming it with Cassette 1. The resulting strain, named TIGR6AX, was non-capsulated and was found, via PCR, to have lost the wciNα gene between positions 1325 and 2518. The wciNβ region was then inserted into TIGR6AX using Cassette 2, which contained the wciNβ gene from CHPA388. PCR confirmed that the resulting strain, TIGR6c, had wciNβ at the expected location. TIGR6c was found to express serotype 6C and this confirmed that the wciNβ gene region is sufficient for the serotype conversion.
Example 11
Novel Pneumococcal Serotype 6D
Previous studies of the chemical structure of serotypes 6A and 6B capsular polysaccharides (PS) showed them to be polymers of repeating units containing galactose, glucose, rhamnose, and ribitol phosphate (FIG. 17). The chemical difference between 6A and 6B PS is in the linkage between rhamnose and ribitol: 6A PS has a 1→3 linkage, but 6B PS has a 1→4 linkage. Studies of the genes responsible for the formation of 6A PS are summarized in FIG. 18. All the genes involved in the synthesis of 6A PS are in one genetic locus (termed the “capsule gene locus”), which is about 17 kb long and contains fourteen genes. A consistent genetic difference between the 6A and 6B capsule gene loci appears to be a single nucleotide at position 584 of the wciP gene (584GA; S195N [SerAsn]). Mavroidi et al., 2004. This genetic change is thought to convert the rhamnosyl transferase encoded by wciP from having the ability to make the 1→3 linkage for 6A PS to having the ability to make the 1→4 linkage for 6B PS.
As discussed herein, the 6C PS has a glucose residue in place of the galactose residue of 6A PS (FIG. 17) and the 6C capsule gene locus is almost identical to the 6A capsule gene locus except for a different wciN gene (FIG. 18). The wciN of 6A was renamed to wciNα and the wciN of 6C was renamed to wciNβ. As noted above, the study suggests that the 6A capsule gene locus might have become the 6C capsule gene locus by replacing the wciNα gene with the wciNβ gene.
Because the 6A and 6B capsule gene loci are also almost identical except for one nucleotide difference in the wciP gene, the 6B capsule gene locus has wciNα just as 6A does. The 6B capsule gene locus might capture the wciNβ gene and may form a new capsular PS, named 6D. It was unknown whether the 6D serotype exists in nature because there had been no search for 6D among natural isolates.
Although the currently available 23-valent pneumococcal vaccine contains 6B PS, the old 14-valent pneumococcal vaccine contained 6A PS. The PS was replaced in the 23-valent pneumococcal vaccine because 6A PS was not highly stable in vaccine preparations, and because 6B PS induced antibodies cross-reacting to 6A PS. Robbins et al., 1983. Investigations found the chemical instability to be due to the 1→3 linkage between rhamnose and ribitol. Zon et al., 1982. Because the 1→4 linkage found in 6B PS is more stable than the 1→3 linkage of 6A PS, 6B PS is more stable than 6A PS. The putative structure of 6D PS should be identical to that of 6C PS except that 6C PS has the unstable 1→3 linkage whereas 6D PS should have the more stable 1→4 linkage. Thus, it is likely that 6D PS would be more useful in a vaccine than 6C PS.
PCR and DNA Sequencing:
PCR reaction mixtures contained 38.80 of sterile water, 2 μl of each 5-pmol primer, 2 μl of 10 mM dNTP, 5 μl of 10× buffer solution, and 0 μl of LA Taq polymerase (2.5 U/μl, Takara Biomedical, Shiga, Japan). For template, either chromosomal DNA isolated with Wizard genomic DNA purification kit (Promega, Madison, Wis.) or colonies grown on blood agar plates were used. Thermal cycling conditions varied depending on the primer set used. PCR products were analyzed by electrophoresis in 1% agarose gels. The primers used are listed in Table 7. PCR products were purified using Wizard PCR Clean-up System (Promega), and the DNA sequencing was performed by the genomics core facility at the University of Alabama at Birmingham (UAB). DNA sequences were analyzed with Lasergene v. 5.1 software (DNASTAR, Madison, Wis.) and were compared to the previously reported sequences of the 6B and 6C cps loci in GenBank (access numbers CR931639, EF538714, respectively). Mavroidi et al., 2004.
Creation of 6D by a Genetic Manipulation:
Capsule synthesis involves the cooperation of many genes. For instance, if the repeating unit of 6D PS cannot be exported to the outside of the bacteria to be assembled into a long chain of capsular PS, the repeating unit would accumulate in the bacteria, creating a lethal condition for bacteria with the capsule gene loci for the 6D serotype. If this is so, it is unlikely that the 6D serotype is present in nature. Thus, it is important to demonstrate that the 6D serotype is biologically possible.
The strategy for creating TIGR6D is depicted in FIG. 19. First, TIGR6B expressing serotype 6B was first prepared by inserting the 6B capsule gene locus region into TIGR4 genetic background using Janus-cassette system as previously described in detail. Park et al., 45 J. Clin. Microbiol. 1225-33 (2007). Second, wciN gene was removed from TIGR6B by transforming it with Cassette 1 and selecting for kanamycin resistant isolates. Cassette 1 has the Janus Cassette, which contains a kanamycin resistance gene (kanAR) and a streptomycin sensitivity gene (rpsL+), and two flanking regions designed for homologous recombination to 6B capsule gene locus. A kanamycin resistant strain was obtained and backcrossed into TIGR6B 3 times. The resulting strain, which was labeled TIGR6BX, lost wciN and did not produce capsular PS. To insert wciN6C, TIGR6BX was transformed with Cassette 2. Both Cassette 1 and Cassette 2 were prepared from the genomic DNAs of TIGR6AX and CHPA388 using primer set 5113 and 3102, respectively. Although Cassette 2 contained a part of wciP in addition to wciN6C, it did not contain the wciP codon responsible for distinguishing 6A and 6B serotypes (FIG. 19). After selection for streptomycin resistance and backcrossing against TIGR6B 3 times, a streptomycin resistant strain was labeled TIGR6D. When the capsule gene locus of TIGR6D was sequenced from wchA to wciP, the sequence showed that wciN6B is replaced with the wciN6C gene as intended. For this sequencing, primer sets 5114-3141, 5138-3104, and 5106-3105 were used to produce amplicons, and primers 5103, 5108, and 5129 were used in sequencing:







TABLE 7







List of PCR primers









Primer Names
Sequence (5′ to 3′)
Source





Forward primers




5103
AAACATGACATCAATTACA
Park et al., 2007



(SEQ ID NO: 8)



 


5106
TACCATGCAGGGTGGAATGT
Id.



(SEQ ID NO: 5)



 


5108
ATGGTGAGAGATATTTGTCAC
Id.



(SEQ ID NO: 1)



 


5113
GGGAAAAATAAAAAATAGGTCGGG
Id.



(SEQ ID NO: 10)



 


5114
TTAGTGACGGAGGCAGGTGAA
presented herein



(SEQ ID NO: 29)



 


5129
TCCTACTTACAGCAACTTCTCGTG
presented herein



(SEQ ID NO: 30)



 


5138
AAAGCTATGTCGCCTGCTAAAAAAGCAATGGCTA
presented herein



(SEQ ID NO: 31)



 


Reverse primers




3102
CTGGCATGTCATCTTTAGAAAA
presented herein



(SEQ ID NO: 32)



 


3104
CCTGAAAACAATACTACTT
presented herein



(SEQ ID NO: 33)



 


3105
TTCCCATCTCTAAACATTCTCCT
presented herein



(SEQ ID NO: 34)



 


3141
GGCGACATAGCTTTTCTTTCAATATCTT
presented herein



(SEQ ID NO: 35)









The TIGR6D cps locus sequence is deposited in Genbank (accession number EU714777). TIGR6D was morphologically indistinguishable from TIGR6B when grown on blood agar plates. Also, TIGR6D grew as well as other pneumococcal strains in THY broth. Serotype 6D was deposited in the American Type Culture Collection (10601 University Blvd., Manassas, Va. 20110) on Oct. 5, 2010, and is commercially available as BEI Number NR-20806.
Quellung Reaction:
Bacterial colonies from blood agar plates were suspended in a small volume of phosphate-buffered saline (PBS), and 2 μl of this broth was combined with 2 μl of serum and 2 μl of methylene blue dye solution (3 mg/mL methylene blue and 1.5 mg/mL NaCl in sterile water) on a glass microscope slide. After adding a coverslip, mixtures were examined under bright-field microscopy using a 100× oil immersion lens. The rabbit antisera specific for serotypes 6A and 6B were prepared by the CDC.
Example 12
Inhibition ELISA Used to Distinguish Serotypes 6B and 6D
The two serotypes were distinguished using an inhibition-type-ELISA. Briefly, the wells of ELISA plates (Corning Costar Corp., Acton, Mass.) were coated at 37° C. with 5 μg/mL of 6B capsular PS (ATCC, Manassas, Va.) overnight in PBS. After washing the plates three times with PBS containing 0.05% Tween 20, 50 μl of a previously diluted bacterial culture supernatant (or lysates) was added to the wells along with 50 μl of an anti-6B mAb. Pneumococcal lysates were prepared by growing pneumococci overnight in 1 ml of THY broth without shaking and then incubating the tubes for 15 min at 37° C. with a lysis buffer (0.1% sodium deoxycholate, 0.01% sodium dodecyl sulfate, and 0.15M sodium citrate in deionized water). Culture supernatants of 6B-specific hybridomas Hyp6BM7 and Hyp6BM8 were used at dilutions of 1:50 and 1:100, respectively. After 30 min of incubation in a humid incubator at 37° C., the plates were washed three times and incubated for 30 min with alkaline phosphatase-conjugated goat anti-mouse immunoglobulin (Sigma, St. Louis, Mo.). The plates were washed three times and then 100 μl of paranitrophenyl phosphate substrate (Sigma) in diethanolamine buffer at a concentration of 1 mg/ml was added, and allowed to incubate at room temperature for 1-2 hours. The optical density at 405 nm was read with a microplate reader (BioTek Instruments Inc, Winooski, Vt.).
Example 13
Purification and Characterization of 6D Capsular PS
Capsular PS expressing serotype 6D was purified in two different ways. One method (ethanol precipitation method) was to purify the PS by ethanol precipitation, ion exchange chromatography, and molecular weight sizing chromatography as described previously. Park et al., 45 J. Clin. Microbiol. 1225-33 (2007). The other method, which is faster than the first method and is to purify capsular PS after removing protoplasts, is described below. TIGR6D was grown in 1 liter of THY broth without shaking until the culture reached an OD600 of  ̃0.4. The culture was then centrifuged at 15,000 g for 10 min. The cell pellet was washed twice with 1 L PBS, and resuspended in 30 mL of protoplast buffer {20% sucrose, 5 mM Tris-HCl (pH 7.4), and 2.5 mM Mg2SO4 in deionized water} with mutanolysin (Sigma) at a concentration of 20 U/mL and allowed to incubate overnight at room temperature. The next day, the bacterial cells were examined under a phase contrast microscope to ensure “protoplasting” had occurred, then protoplasts were removed by centrifugation at 27,000 g for 15 min. The supernatant was sterilized through a 0.22 micron filter, diluted 1:1 in deionized water, and was loaded onto a DEAE-Sepharose column (Amersham Biosciences, Uppsala, Sweden) with a 2 ml bed volume. The column was washed with 4 ml of 50 mM ammonium acetate, and the PS was eluted from the column with 4 ml of 500 mM ammonium acetate. After lyophilization, the eluted PS was loaded on a Sephacryl S-300 HR column (Amersham Biosciences) with a bed volume of 130 mL and the PS was eluted with 10 mM Tris-HCl (pH 7.4). The fractions were tested for the presence of PS by the inhibition assay using Hyp6BM8. The first 1 mL of fractions, which contains majority of PS, were pooled and lyophilized.
Monosaccharide Composition Analysis of PS:
1 mg of lyophilized capsular PS prepared by the protoplast method was dissolved in 500 μl of 1M HCl and incubated at 80° C. for 16 hr. After drying the sample under a nitrogen stream, the remaining PS was washed twice with 250 μl of methanol. The sample was then incubated with 200 μl of Tri-Sil Reagent (Pierce Biotech Inc., Rockford, Ill.) to trimethylsilylate all the monosaccharides. The reaction products were analyzed on a gas-liquid chromatograph/mass spectrometer (GLC/MS) (Varian 4000; Varian Inc., Palo Alto, Calif.) fitted with a 30-m (0.25-mm-diameter) VF-5 capillary column. Column temperature was maintained at 100° C. for 5 min and then increased to 275° C. at 20° C./min and finally held at 275° C. for 5 min. The effluent was analyzed by mass spectrometry (MS) using the electron impact ionization mode. The areas of each monosaccharide peaks in GLC/MS were determined using Varian MS Workstation v6.5 software.
Analysis of PS by Tandem Mass Spectrometry:
Intact capsular PSs prepared by the ethanol precipitation method were hydrolyzed to their repeating units before analysis by mass spectrometry. A 2 mg aliquot of PS was hydrolyzed in 1 mL of 10 mM NaOH at 85° C. for 120 hours followed by another hydrolysis at 50 mM NaOH at 85° C. for 120 hr. At the end of hydrolysis, all samples were neutralized with 0.1M HCl.
Tandem mass spectrometry (MS/MS) was performed in the Mass Spectrometry Shared Facility at the UAB with a Micromass Q-TOF2 mass spectrometer (Micromass Ltd., Manchester, United Kingdom) equipped with an electrospray ion source. The samples dissolved in distilled water were injected into the mass spectrometer with running buffer (50/50 acetonitrile-water containing 0.1% formic acid) at a rate of 1 μl/min using a Harvard syring pump. The injected sample was negatively ionized with electrospray and detected with a time-of-flight mass spectrometer. For MS/MS, the parent ion was fragmented into daughter ions by energizing it to either 35 eV or 40 eV before collision with argon gas. The daughter ions were analyzed with a time-of-flight mass spectrometer. The MS/MS spectra were processed using the Max-Ent3 module of MassLynx 2.5. The study showed that ribitol and glucose 1 are cleaved by periodate while glucose 2 and rhamnose are not. The mass of daughter ions showed that the phosphodiester bond is made to the 2nd position of glucose 1 and all other glycosidic bonds are same as 6B PS). The MS/MS studies supported the proposed structure shown in FIG. 21 A.
More specifically, to determine if the monosaccharide sequence of the 6D PS is as proposed in FIG. 21 A, a mild alkali hydrolysis which breaks the phosphodiester bonds and produces repeating units was used. As observed for 6C PS, the hydrolysis yields two types of repeating units with identical mass, one with the phosphate ion linked to ribitol (labeled forward fragmentation) or another linked to glucose (labeled reverse fragmentation). The phosphate ion endows the repeating unit with a negative charge. When the alkali hydrolysis product was analyzed for negative ions by MS/MS, the results showed two prominent peaks with 683 and 701 AMUs (FIG. 21B), which is respectively identical to the anhydrous and hydrated mass of the predicted repeating unit of 6D PS (FIG. 21A). The peak at 260.902 AMU was absent in other MS/MS attempts and may represent a contaminant.
Additionally, the ion with 683 AMU (i.e., intact repeating unit) was subjected to argon collision and identified its daughter ions with MS/MS analysis. Daughter ions were observed at 521, 359, and 213 AMU, which respectively represents daughter ions that lost the first glucose, the second glucose, and rhamnose (FIG. 21C). Peaks were also observed at 549, 403, and 241 AMUs, which also correspond to the daughter ions formed after reverse fragmentation by losing ribitol, ribitol-rhamnose, and ribitol-rhamnose-glucose 2, respectively (FIG. 21C).
Three peaks with 113, 127, and 145 AMUs were absent in other MS/MS analyses and may represent contaminants. Thus, the monosaccharide sequence of the 6D PS repeating unit is glucose 1-glucose 2-rhamnose-ribitol as proposed in FIG. 21A. The two glucose residues were labeled 1 and 2 for clarity.
Oxidation and Reduction of PS:
Capsular PSs were dissolved in 80 mM sodium acetate buffer (pH 4) at a concentration of 1 mg/mL. Sodium periodate was added to the PS solution to a final concentration of 40 mM and the reaction mixture was incubated in the dark at 4° C. for 72 hours. Excess periodate was destroyed by adding ethylene glycol. To determine intact monosaccharides of the oxidized capsular PS, PS was then lyophilized then analyzed using GLC/MS as described above. To investigate the glycosidic bonds, sample was reduced with sodium borohydride or sodium tetradeuteroborate as previously described (Park et al., 45 J. Clin. Microbiol. 1225-33 (2007)), before subjecting to MS/MS as described above. 6D PS was prepared with protoplast method and 6B PS was obtained from ATCC.
Hydrolytic Stability Assay:
0.9 mL of 2 mg/mL PS in water was mixed with 0.1 mL of 0.1 M NaOH and this solution was split into two eppendorf tubes and incubated at 85° C. At the indicated times, 0.1 mL was removed from these samples, neutralized with 0.1M HCl, and then stored at 4° C. until used in the inhibition ELISA. Using the same buffers and incubation conditions described for the inhibition ELISA above, plates were coated with 100 μl of 5 μg/mL 6A, 6B, 6C, or 6D PS. The ELISA was performed with the hydrolyzed samples on plates coated with their respective PSs. For 6A and 6C PSs, Hyp6AG1 was used as the primary antibody (as performed in Park et al., 2007), and for 6B and 6D, Hyp6BM8 was used (as described above). Data shown is the average of samples run in duplicate (FIG. 22).
During the alkali hydrolysis experiments for mass spectrometry, 6D PS was very resistant to alkali hydrolysis. To measure resistance to hydrolysis, the ability of 6A, 6B, 6C and 6D PSs to inhibit binding of Hyp6BM8 (for 6B and 6D PSs) or Hyp6AG1 (for 6A and 6C PSs) to target PS after alkali hydrolysis for various time periods were compared. The 6A and 6C PSs completely lost their ability to inhibit after only 1 hour of hydrolysis. In contrast, 6B PS lost 90% of its inhibitory ability in 8 hours and more than 100 hours of hydrolysis was needed for 6D PS to lose its inhibitory ability by 90% (FIG. 22). Thus, 6D PS is much more resistant than 6A and 6C PS to alkali hydrolysis, and may be more resistant than 6B PS.
Example 14
Screening for the Existence of Serotype 6D Among Isolates
To determine whether 6D exists in nature, 264 pneumococcal isolates that were previously serotyped as “6B” by classical means were re-serotyped for serotypes 6B or 6D using mAbs. The isolates were a part of a University of Alabama, Birmingham, laboratory collection of 6β isolates, which have originated from Africa, Asia, Australia, South America, North America, and Europe. In addition to these, TIGR6A, TIGR6AX, and TIGR6c, which are isogenic strains of TIGR4 expressing the 6A-type capsule, no capsule, and 6C-type capsule (Park et al., 75. Infect. Immun. 4482-89 (2007)), were used as assay controls or a source of DNA. Additional TIGR4 variants, which are TIGR6B, TIGR6BX, and TIGR6D, were prepared as described below. All bacteria were grown in Todd-Hewitt broth (BD Biosciences, San Jose, Calif.) supplemented with 0.5% yeast extract (THY) and kept frozen at −80° C. until used.







TABLE 8







Re-analysis of clinical isolates of S. pneumoniae previously typed as “6B”









strains

















264 clinical isolates


Serotyping Assay
TIGR6A
TIGR6B
TIGR6C
TIGR6D
typed as 6B





Quellung Reaction
negative
positive
negative
positive
positive*


for 6B serotype


Hyp6BM8 ELISA
negative
positive
negative
positive
positive


Hyp6BM7 ELISA
negative
positive
negative
negative
positive





*Many isolates were originally typed as 6B by quellung reaction but some were typed as 6B using an agglutination assay.






When the serological properties of TIGR6D were examined by the quellung reaction using polyclonal rabbit antisera, it was typed as 6B. When TIGR6D PS was examined for binding various mAbs to 6A and 6B PS using an inhibition ELISA, it was reactive with many mAbs to 6B PS. For instance, TIGR6D inhibited Hyp6BM8 binding to 6B PS. These observations clearly demonstrated that 6D PS is serologically very close to 6B PS. In contrast, a mAb specific to 6B PS (Hyp6BM7) was not reactive with 6D PS (FIG. 20). Thus, 6D PS is serologically distinct from 6B PS.
Above serological studies showed that if pneumococcal isolates expressing 6D PS are present in nature, they would have been typed as 6B serotype. To look for the presence of serotype 6D isolates in nature, 264 pneumococcal isolates that were previously typed as serotype 6B were examined using an inhibition ELISA capable of distinguishing between the 6B and 6D serotypes (FIG. 20). These 6β isolates came from six continents (North America, South America, Europe, Asia, Africa, and Australia), and were isolated from patients with bacteremia, meningitis, pneumonia, and otitis media as well as from healthy carriers. None of the 264 6B strains exhibited the antibody binding profile of 6D (Table 8). Thus, the prevalence of serotype 6D, if it exists, is much less than that of the prevalence of 6B.

## Claims

1. A purified polysaccharide comprising the repeating unit {→2) glucose 1 (1→3) glucose 2 (1→3) rhamnose (1→4) ribitol (5→phosphate}.