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WO2000077254A1 - Detection de streptococcus pneumoniae et immunisation contre l'infection par streptococcus pneumoniae - Google Patents

Detection de streptococcus pneumoniae et immunisation contre l'infection par streptococcus pneumoniae Download PDF

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Publication number
WO2000077254A1
WO2000077254A1 PCT/US1999/013421 US9913421W WO0077254A1 WO 2000077254 A1 WO2000077254 A1 WO 2000077254A1 US 9913421 W US9913421 W US 9913421W WO 0077254 A1 WO0077254 A1 WO 0077254A1
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Prior art keywords
streptococcus pneumoniae
nucleic acid
seq
oligonucleotide
amplicon
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Inventor
Jacqueline S. Sampson
Katherine E. Morrison
Jennifer Crook
Edwin E. Ades
George M. Carlone
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US Department of Health and Human Services
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US Department of Health and Human Services
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Priority to US10/168,602 priority Critical patent/US6869767B1/en
Priority to AU46832/99A priority patent/AU4683299A/en
Publication of WO2000077254A1 publication Critical patent/WO2000077254A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C07K14/3156—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci from Streptococcus pneumoniae (Pneumococcus)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria

Definitions

  • This invention relates to the detection and treatment of Streptococcus pneumoniae infection.
  • the present invention relates to diagnostic assays and kits for detecting the presence of Streptococcus pneumoniae in a sample and to vaccines for use against Streptococcus pneumoniae .
  • the present invention relates to PCR assays for the presence of Streptococcus pneumoniae surface adhesin A protein (PsaA) and to vaccines raised against a portion of PsaA encoded by a PCR product that is provided using specific primers.
  • PsaA Streptococcus pneumoniae surface adhesin A protein
  • Streptococcus pneumoniae is frequently isolated from the young, the elderly, and the immunocompromised as the etiologic agent of a broad range of diseases (Centers for Disease Control and Prevention, "Prevention of pneumococcal disease: Recommendations of the Advisory Committee on
  • Streptococcus pneumoniae normally colonizes the nasopharynx and can be found in 5-10 % of healthy adults and 20-40 % of healthy children.
  • S. pneumoniae is responsible for a variety of infections including common "strep throat," otitis media, sinusitis, meningitis, tracheitis, bronchitis, rheumatic fever, and community- acquired pneumonia. It can also spread from the nasopharyngeal cavity into the central nervous system, heart valves, bones, joints, and peritoneal cavity. The organism typically binds to the upper respiratory tract. It is transmitted person-to- person via droplets in the air: transmission through direct contact is also thought to occur.
  • streptococcal infections affect the lungs in less than 5% of cases. Such lung infections, however, progress with great rapidity and severity, despite treatment with antibiotics. This form of pneumonia is difficult to control and may last for weeks. Although severe pneumonia can be caused by a wide variety of bacteria, as well as by viruses, streptococcal infection accounts for about 55% to 76% of cases. About 2 million Americans get pneumonia each year, with 40.000 to 70,000 deaths reported. Pneumonia ranks sixth among all categories of disease as a cause of death. Despite the many testing technologies available to physicians, it is sometimes very difficult to determine which species of bacteria is responsible for the illness; in up to half the cases no etiological agent can be identified.
  • the gene encoding PsaA from S. pneumoniae strain R36A (an unencapsulated strain) has been cloned in E. coli and sequenced.
  • This serotype does not. however, contain a nucleic acid encoding a 37 kDa protein that is highly conserved among the various serotypes (Sampson et al. 1994).
  • This particular nucleic acid and polypeptide therefore, are of limited value for use as diagnostic reagents, in prevention or treatment of infection, or in vaccine development.
  • U.S. Patent 5,854.416 provides a nucleic acid encoding the 37-kDa protein from serotype 6B of Streptococcus pneumoniae. Isolated nucleic acids comprising a unique fragment of at least 10 nucleotides of the 37-kDa protein are also provided. The patent further discloses purified polypeptides encoded by the nucleic acid encoding the 37-kDa protein, as well as nucleic acids comprising a unique fragment of at least 10 nucleotides coding for the 37-kDa protein.
  • antibodies are provided which selectively bind the polypeptides encoded by the nucleic acid encoding the 37-kDa protein and the nucleic acids comprising a unique fragment of at least 10 nucleotides of the 37-kDa protein.
  • the patent additionally provides vaccines comprising immunogenic polypeptides encoded by the nucleic acid encoding the 37-kDa protein and by the nucleic acids comprising a unique fragment of at least 10 nucleotides of the 37 kDa protein.
  • a method of detecting the presence of Streptococcus pneumoniae in a sample comprising the steps of contacting a sample suspected of containing Streptococcus pneumoniae with nucleic acid primers capable of hybridizing to a nucleic acid comprising a portion of the nucleic acid encoding the 37-kDa protein, amplifying the nucleic acid and detecting the presence of an amplification product, the presence of the amplification product indicating the presence of Streptococcus pneumoniae in the sample.
  • nucleic acid primers capable of hybridizing to a nucleic acid comprising a portion of the nucleic acid encoding the 37-kDa protein
  • PsaA pneumococcal surface adhesin A
  • the gene encoding PsaA from an encapsulated strain of pneumococcal serotype 6B was cloned and sequenced. The peptide sequence was compared to that of homologs found in S. pneumoniae serotype 2, viridans streptococci, and Enterococcus faecalis. Identity values among the deduced peptides ranged from 57 to 98%.
  • the polymorphism of psaA was examined among the 23 encapsulated vaccine serotypes by using PCR-restriction fragment length polymorphism analysis. This analysis showed that restriction sites within the gene were highly conserved. The lack of variation for the other restriction sites within the gene indicates that PsaA is genetically conserved, an important characteristic necessary for a candidate common protein vaccine.
  • a pneumococcal polysaccharide vaccine was developed with 23 types of pneumococcal bacteria which cause 88 percent of bacteremic pneumococcal disease. Vaccinated, healthy adults are protected from some or all pneumococcal pneumonia bacteria types. However, the carriage rates of the organism do not decline significantly in vaccinated people. Additionally, adverse reactions from this vaccine may occur in older persons, children under the age of two years, and those stricken with long-term illnesses. Furthermore, it is found that the polysaccharide vaccine fails to provide protection in young children, the elderly and the immunocompromised. This led to development of a second-generation protein-conjugate vaccine.
  • This vaccine composed of the seven most frequent invasive disease-causing capsular serotypes, may overcome the problems of poor immunogenicity associated with the 23-valent vaccine.
  • this protein-conjugate vaccine may not prevent replacement carriage of serotypes not contained in the vaccine (Mbelle et al. 1997. "Immunogenicity and impact on carriage of 9-valent pneumococcal conjugate vaccine given to infants in Soweto, South Africa", Abstr. L8-12. p. 13.
  • a culture assay is currently the Gold Standard for detecting Streptococcus pneumoniae .
  • Patient sputum, throat swab, or blood specimens can be cultured for Streptococcus pneumoniae if they are handled correctly.
  • Such diagnostic tests are generally time-consuming, requiring at least overnight incubation.
  • cultures also lack the certainty of positive identification, providing positive diagnoses only 20% to 30% of the time.
  • the current Gold Standard diagnostic assay has important deficiencies associated with it. Because of the speed with which streptococcal pneumoniae can develop and potentially lead to death, there is a need for rapid testing resulting in earlier treatment of infection. This has the potential to significantly abbreviate the course of the disease and reduce mortality.
  • Rapid assays for Streptococcus are available in a number of formats. Older format kits involve the use of a multistep latex agglutination procedure or an EIA format, and employ samples such as serum, whole blood, or sputum swabs. Most take less than eight minutes to perform, and show results through the appearance of a colored visual indicator.
  • assay reagents representing the 90 different components, such as are used in serologic assays including latex agglutination and counterimmunoelectrophoresis, would be needed.
  • Pneumococcal diagnostic assays are based primarily on detection of pneumococci, pneumococcal antigens, DNA or RNA in blood or body fluids. Although blood culture is currently the most accurate method by which to diagnose pneumococcal disease, only approximately 30% of specimens test positive with this method (Kalin et al. 1983.
  • Automated serological testing is used in diagnostic testing for Streptococcus pneumoniae infection. These assays identify and quantify the existence of antibodies to bacterial pathogens. All antibody detecting assays ma ⁇ ' miss active infection if the level of antibodies in a patient's blood is too low. as occurs with certain immunocompromised individuals, or if infection is too recent for antibodies to have been produced. Additionally, as with all antibody-based assays, because levels of antibodies produced against a previous infection fall slowly, testing may indicate an infection that no longer exists.
  • the present invention discloses, in a first aspect, an oligonucleotide including a sequence chosen from among SEQ ID NOT, SEQ ID NO:2, the complement of SEQ ID NOT . and the complement of SEQ ID NO:2.
  • the oligonucleotide may include SEQ ID NOT, or SEQ ID NO:2.
  • the invention discloses a pair of oligonucleotide primers that prime the universal and specific amplification of a nucleic acid that includes at least a portion of the psaA gene of Streptococcus pneumoniae.
  • one oligonucleotide of the pair includes the sequence given by SEQ ID NOT .
  • the second oligonucleotide of the pair includes the sequence given by SEQ ID NO:2.
  • the invention in a third aspect, relates to a diagnostic kit for the specific diagnosis of Streptococcus pneumoniae that includes reagents for amplification of a nucleic acid, as well as a pair of oligonucleotide primers that prime the universal and specific amplification of a nucleic acid containing at least a portion of the psaA gene of Streptococcus pneumoniae.
  • one oligonucleotide of the pair includes the sequence given by SEQ ID NOT
  • the second oligonucleotide of the pair includes the sequence given by SEQ ID NO:2.
  • the kit further includes a second primer pair wherein one oligonucleotide of the pair comprises the sequence given by SEQ ID NO:2. and the second oligonucleotide of the pair comprises the sequence given by SEQ ID NO:3.
  • a method of specifically detecting the presence of Streptococcus pneumoniae in a biological sample includes the steps of:
  • step (iv) detecting whether the amplicon is present; whereby, if the amplicon is determined to be present, the sample contains Streptococcus pneumoniae.
  • an enrichment step is conducted on the biological sample obtained in step (i) prior to the contacting of step (ii).
  • one oligonucleotide of the pair includes the sequence given by SEQ ID NOT .
  • the second oligonucleotide of the pair includes the sequence given by SEQ ID NO:2; additionally a second primer pair may further be included in step (ii). in which one oligonucleotide of the pair includes the sequence given by SEQ ID NO:2, and the second oligonucleotide of the pair includes the sequence given by SEQ ID NO:3
  • the present invention discloses an isolated nucleic acid including a nucleic acid amplicon obtained when nucleic acid from a serotype of Streptococcus pneumoniae is contacted with a pair of oligonucleotide primers that prime the universal and specific amplification of at least a portion of the psaA gene of Streptococcus pneumoniae and the Streptococcus pneumoniae nucleic acid so primed is amplified to provide the amplicon which thereby includes the portion of the psaA gene.
  • the portion of the psaA gene is operably linked to control elements that promote the expression of a polypeptide comprising the portion of the PsaA protein of a serotype of Streptococcus pneumoniae encoded by the gene.
  • the invention discloses a polypeptide including an amino acid sequence encoded by a nucleic acid amplicon that includes at least a portion of the psaA gene obtained when nucleic acid from a serotype of Streptococcus pneumoniae is contacted with a pair of oligonucleotide primers that prime the universal and specific amplification of the at least a portion of the psaA gene of Streptococcus pneumoniae and the Streptococcus pneumoniae so primed is amplified to provide the amplicon.
  • the invention discloses a method of stimulating an immune response against a serotype of Streptococcus pneumoniae in a mammal including administering to the mammal an amount of a polypeptide effective to stimulate the immune response, wherein the polypeptide includes an amino acid sequence encoded by a nucleic acid amplicon including at least a portion of the psaA gene obtained when nucleic acid from a serotype of Streptococcus pneumoniae is contacted with a pair of oligonucleotide primers that prime the universal and specific amplification of the portion of the psaA gene of Streptococcus pneumoniae and the Streptococcus pneumoniae so primed is amplified to provide the amplicon.
  • the invention discloses a method of stimulating an immune response against a serotype of Streptococcus pneumoniae in a mammal including administering to the mammal a nucleic acid including a nucleic acid amplicon including at least a portion of the psaA gene obtained when nucleic acid from a serotype of Streptococcus pneumoniae is contacted with a pair of oligonucleotide primers that prime the universal and specific amplification of the portion of the psaA gene of a serotype of Streptococcus pneumoniae and the Streptococcus pneumoniae so primed is amplified to provide the amplicon, wherein the amount of the nucleic acid administered is effective to express an immunostimulatory amount of the portion of the PsaA protein of a serotype of Streptococcus pneumoniae encoded by the amplicon in cells of the mammal.
  • FIG. 1 Amplification of ATCC 16F using different primer pairs. Lane 1, DNA marker; Lane 2. serotype 6B amplified with P3 and P2; Lane 3, serotype 6B amplified with PI and P2; Lane 4, negative control amplified with P3 and P2; Lane 5, negative control amplified with PI and P2; Lane 6, serotype 16F (ATCC# 6316) amplified with P3 and P2; Lane 7, serotype 16F (ATCC# 6316) amplified with PI and P2. Figure 3. Amplification of 16F clinical isolates using oligonucleotide primers PI and P2. Agarose gel electrophoresis of PCR amplified products from S pneumoniae serotype 16F clinical isolates. Lane 1. DNA markers; Lanes 2-1 1. amplified clinical strains.
  • universal designates a process or relates to a composition that is effective for carrying out the objectives of the present invention as they relate to at least a portion of the psaA gene of Streptococcus pneumoniae or at least a portion of the PsaA gene product encoded by the gene or portion thereof, regardless of the serotype or strain of S. pneumoniae. within the limits of the serotypes or strains disclosed herein.
  • 90 serotypes or strains are known.
  • primer set PI and P2 are universal since they amplify each of the ninety known serotypes of Streptococcus pneumoniae.
  • telomere sequence a nucleic acid sequence such as an oligonucleotide sequence
  • telomere sequence a nucleic acid sequence that hybridizes, under conditions used in given experimental circumstances, to the target but does not hybridize under those circumstances to sequences that are not target sequences.
  • specific amplification relates to the ability of a primer pair to promote the amplification of a segment of a nucleic acid originating from any of the 90 serotypes of Streptococcus pneumoniae, and the inability of the primer pair to promote the amplification of any segment of a nucleic acid originating from another species.
  • Nucieotide sequences that are specific for a particular target include sequences every one of whose bases is complementary to the corresponding base on the target.
  • nucleic acid sequence for a target sequence also encompasses nucleic acids and oligonucleotides in which a small number of nucleotides may not be complementary to the corresponding nucieotide of the target sequence.
  • sequences are still “specific” for the target sequence, as used herein, as long as the extent of deviation from complementarity remains functionally of little consequence, since they still hybridize to the target.
  • such a sequence is “specific" for the target sequence as long as it hybridizes effectively to the target sequence but does not hybridize to non-target sequences, under the conditions used in given experimental circumstances.
  • Deviation from complete complementarity may underlie a weaker extent of hybridization of a primer to the target sequence under given experimental conditions. Under such circumstances, the yield of the amplicon produced when such a primer is used may be poor. Nevertheless a finding that an amplicon has been produced, regardless of its amount, is sufficient for the purposes of the present invention to conclude that the target sequence is present.
  • PCR polymerase chain reaction
  • Other amplification techniques can be used to detect the presence of Streptococcus pneumoniae in a sample, such as ligase chain reaction (LCR), the self-sustained sequence replication (3SR) system, the transcription-based amplification system (TAS), and the RNA replication system based on Q ⁇ replicase.
  • LCR ligase chain reaction
  • 3SR self-sustained sequence replication
  • TAS transcription-based amplification system
  • Q ⁇ replicase Q ⁇ replicase
  • the amplification procedure used in the present invention is PCR.
  • the terms "polymerase chain reaction” or "PCR” relate to a procedure whereby a limited segment of a nucleic acid molecule, which frequently is a desired or targeted segment, is amplified repetitively to produce a large amount of DNA molecules which consist only of the segment.
  • PCR and the other amplification procedures are described in operational detail in, for example, "Molecular Cloning: A Laboratory Manual", 2nd Ed. , Sambrook, Fritsch and Maniatis, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989; “Current Protocols in Molecular Biology” , Ausubel et al. , John Wiley and Sons, New York 1987 (updated quarterly); and in U.S.
  • Patent 4,965,188 Additional guidance concerning PCR and related methods can be found in, for example, U.S. Patents 4,683,202; 5,578,467; 5,545,522; and 5,624,833. The references cited in this paragraph are specifically incorporated by reference
  • primer or "oligonucleotide primer” relates to an oligonucleotide that includes a specific or desired nucieotide sequence which is complementary to a particular sequence on one of the strands of a DNA duplex.
  • primer When the primer is caused to hybridize to the specific sequence in a DNA duplex to which it is complementary, it may serve as the priming position, or the initiation position, for the action of a primer-dependent DNA polymerase activity.
  • the primer once hybridized, acts to define one end of the operation of the transcription activity of the polymerase on the duplex.
  • a specific pair of primers is employed, wherein each hybridizes to one of the strands such that transcription, which proceeds from the 5'- end to the 3'- end, is in the direction leading to the site of hybridization of the second primer on the opposite strand of the duplex.
  • the amplified DNA produced is a segment having a defined length whose ends are defined by the sites to which the primers hybridize.
  • a "vector” relates to a nucleic acid which functions to incorporate a particular nucleic acid segment, such as a sequence including a particular gene, into a cell. In most cases, the cell does not naturally contain the gene, so that the particular gene being incorporated is a heterologous gene.
  • a vector may include additional functional elements that direct and regulate transcription of the inserted gene or fragment.
  • the regulatory or control sequence is operably positioned with respect to the protein-encoding sequence such that, when the vector is introduced into a suitable host cell and the regulatory sequence is triggered, the protein encoded by the gene is expressed.
  • Regulatory or control sequences may include, by way of non-limiting example, a promoter, regions upstream or downstream of the promoter such as enhancers that may regulate the transcriptional activity of the promoter, and an origin of replication.
  • a vector may additionally include appropriate restriction sites, antibiotic resistance, or other markers for selection of vector containing cells, RNA splice junctions, a transcription termination region, and so forth.
  • the invention provides oligonucleotide primers which, when used in specified pairs in a nucleic acid amplification protocol applied to a nucleic acid originating from any of the 90 serotypes or strains of Streptococcus pneumoniae, promote the preparation of an amplicon having approximately the same size, in terms of number of bases contained therein, regardless of the serotype or strain subjected to the amplification protocol.
  • the primers were designed based on the nucieotide sequence of a nucleic acid comprising the psaA gene from strain 6B of Streptococcus pneumoniae , as disclosed in U.S. Patent 5,854,416 (incorporated herein by reference in its entirety) .
  • primers employed in the present invention i.e., P3, having the sequence AGGATCTAATGAAAAAATTAG (SEQ ID NO:3; priming forward sense replication)
  • the remaining two primers i.e. , the universal primers
  • the primer pair PI and P2 is universal and specific for the amplification of a segment of the nucleic acid from all 90 serotypes or strains of Streptococcus pneumoniae that includes at least a portion of the psaA gene.
  • the present invention relates in an important aspect to carrying out a diagnostic assay for the presence of Streptococcus pneumoniae in a biological samples.
  • a diagnostic assay for the presence of Streptococcus pneumoniae in a biological samples.
  • the diagnostic assay employs specific primer pairs formulated from the oligonucleotide primers disclosed herein to amplify at least a portion of the psaA gene of any serotype of Streptococcus pneumoniae that may be present in a biological sample.
  • the primer pair is composed of oligonucleotides that include the sequences of SEQ ID NO: l (forward amplification) and SEQ ID NO: 2 (reverse amplification). Amplicons provided by the use of this primer pair contain approximately 838 bases, and provide a universal and specific assay for Streptococcus pneumoniae .
  • primer pairs composed of oligonucleotides that include the sequences of SEQ ID NO: l (forward amplification) and SEQ ID NO:2 (reverse amplification) as well as including the sequences of SEQ ID NO: 3 (forward amplification) and SEQ ID NO: 2 (reverse amplification), providing amplicons having approximately 838 and 867 bases, respectively, may be used.
  • the presence of amplicons after carrying out an amplification procedure permits the operator to conclude that the biological sample contains a serotype of Streptococcus pneumoniae.
  • the methods available for detecting the presence of the amplicon include the following procedures, by way of nonlimiting example.
  • the size of the amplicon may be determined by a procedure such as gel electrophoresis or comparable size-determining method to ensure its size is commensurate with the anticipated product.
  • the presence of the amplicon may be determined using conventional techniques and procedures.
  • the primers can be radioactively labeled such that the amplification product containing these labeled primers are detectable by the radioactive decay of the label.
  • the primers can be labeled to contain other detectable moieties, such as biotin. When biotin is used, it is further labeled and detected by means of a labeled form of avidin or streptavidin.
  • amplified nucleic acid can be stained and visualized (e.g. , ethidium bromide staining).
  • labeled probes designed to hybridize with sequences unique to the amplified product, can be used to detect the presence of the amplified product.
  • the diagnostic assays disclosed herein provide the advantage, heretofore unknown in the field of diagnosing Streptococcus pneumoniae infection, of specifically detecting all of the 90 serotypes of Streptococcus pneumoniae currently known using a single assay with a single set of reagents.
  • These reagent sets can include primers PI and P2, or primers PI , P2, and P3 employed as the primer pairs specified herein. This detection proceeds with high sensitivity and great economy.
  • These assays therefore represent a significant advance over the currently available diagnostic assays for Streptococcus pneumoniae which rely on capsular polysaccharide antigen combinations, and which frequently provide false negative determinations.
  • the present invention relates to nucleic acids containing the amplicon obtained when a nucleic acid from a serotype of Streptococcus pneumoniae is contacted with a pair of oligonucleotide primers that prime the universal amplification of at least a portion of the psaA gene of a serotype of Streptococcus pneumoniae and the Streptococcus pneumoniae so primed is amplified to provide the amplicon.
  • amplicons may be produced using the primer pair (i.e.
  • PI and P2 composed of oligonucleotides that include the sequences of SEQ ID NOT (forward amplification) and SEQ ID NO:2 (reverse amplification) providing amplicons containing approximately 838 bases.
  • another assay can be carried out using the primer pair (i.e., P3 and P2) composed of oligonucleotides that include the sequences of SEQ ID NO: 3 (forward amplification) and SEQ ID NO: 2 (reverse amplification), providing amplicons predicted to have approximately 867 bases.
  • amplicons have beneficial applications in systems that may be used to express the polypeptides encoded by them.
  • polypeptides containing at least a portion of the PsaA surface protein, are useful as antigens and immunogens related to infection by Streptococcus pneumoniae. They additionally are useful when incorporated into nucleic acid vectors that may be used directly in a host as an immunogen- producing nucleic acid.
  • the preferred methods and assays employ the universal primer pair (i.e., PI and P2) alone.
  • the universal primer pair i.e., PI and P2
  • at least one specific sample of a serotype i.e. , a sample of serotype 16F obtained from ATCC
  • a characteristic amplicon could, however, be obtained from this sample using the second primer pair P3/P2.
  • the second prier pair which is designed to be used in conjunction with the universal primer pair, could also be used.
  • the amplicon is introduced into a vector, such as a plasmid or virion, in such a way that it is operably linked to control elements in the nucleic acid of the vector.
  • the control elements promote the expression of the polypeptide when the nucleic acid is introduced into a suitable host cell and the cell is cultivated under conditions that promote expression of the protein.
  • the polypeptide synthesized includes at least a portion of the PsaA protein of a serotype of Streptococcus pneumoniae encoded by the gene.
  • Procedures to be used in preparing such vectors, in transducing bacteria or transfecting eukaryotic cells with such vectors, and expressing the PsaA or portion thereof using the recombinant cells so produced, are known to workers of skill in molecular biology and clinical microbiology, and are disclosed, for example, in Sambrook et al. and Ausubel et al.
  • the invention additionally relates to the PsaA polypeptide comprising an amino acid sequence encoded by the nucleic acid amplicons obtained when nucleic acids from various serotypes of Streptococcus pneumoniae are contacted with a pair of oligonucleotide primers that prime the universal amplification of at least a portion of the psaA gene of a serotype of Streptococcus pneumoniae and the Streptococcus pneumoniae so primed is amplified to provide the amplicon.
  • This polypeptide is useful, for example, as a probe in enzyme immunoassay intended to detect the presence of an ⁇ -Streptococcus pneumoniae antibodies in a biological sample, as well as an antigen or immunogen to stimulate an immune response or a protective immune response, respectively, in a mammal to which the polypeptide is administered.
  • the invention relates to a method of stimulating an immune response against serotypes of Streptococcus pneumoniae in a host comprising administering to the host an immunogenically effective amount of a polypeptide comprising an amino acid sequence encoded by a nucleic acid amplicon obtained when a serotype of Streptococcus pneumoniae is contacted with a pair of oligonucleotide primers that prime the universal amplification of at least a portion of the psaA gene of a serotype of Streptococcus pneumoniae and the Streptococcus pneumoniae so primed is amplified to provide the amplicon.
  • This invention additionally relates to using the amplicons obtained as described above, when incorporated into suitable vectors, as nucleic acid vaccines that elicit protection against Streptococcus pneumoniae infection in a mammal.
  • the invention discloses a method of stimulating an immune response against serotypes of Streptococcus pneumoniae in a mammal in which an amount of a nucleic acid comprising a nucleic acid amplicon obtained when a nucleic acid from a serotype of Streptococcus pneumoniae is contacted with a pair of oligonucleotide primers that prime the universal amplification of at least a portion of the psaA gene of a serotype of Streptococcus pneumoniae and the Streptococcus pneumoniae so primed is amplified to provide the amplicon, is administered to the host.
  • the vector including the amplicon is appropriately structured such that when incorporated into the cells of a host organism, such as a human or other mammal intended to be vaccinated, the vector interacts with the naturally occurring components of the cell such that the vector promotes the expression of the PsaA polypeptide or portion thereof encoded by the amplicon.
  • the amount of the nucleic acid administered is effective to express an immunogenic amount of at least a portion of the PsaA protein encoded by the amplicon in the cells of the mammal.
  • PCR Bacterial strains were grown for isolation on Trypticase soy agar plates supplemented with 5 % defibrinated sheep blood for 12 h at 37°C in CQ. For PCR amplification, approximately 5 CFUs were placed directly into a PCR reaction mixture and allowed to lyse in the thermocycler. If this method failed to produce an amplified product, whole cells were boiled in 200 ⁇ L of filtered water for 10 minutes, followed by a cooling time on ice for at least 5 minutes. An aliquot of boiled lysate was then used in the PCR reaction mixture.
  • Primers The sequences of the primers used to amplify psaA were as follows: 5'CTTTCTGCAATCATTCTTG3' (PI; SEQ ID NO: 1) as the forward primer and 5 OCCTTCTTTACCTTGTTCTGC3 ' (P2; SEQ ID NO: 2) as the reverse primer. These primers, as well as primer P3 (SEQ ID NO: 3), were designed from the nucleic acid sequence data obtained from serotype 6B (GenBank # U53509; Sampson et al. 1997). All primers were prepared at the Biotechnology Core Facility at CDC.
  • the PCR reaction mixture (100 ⁇ L) contained 100 ng of each primer (PI and P2), 2.0 ⁇ L of 10 mM dNTPS (Boehringer Mannheim, Indianapolis. IN), 10.0 ⁇ L 10 mM MgCl 2 . 50.0 ⁇ L of PCR Master Mix (Boehringer Mannheim), approximately 5 CFUs of bacteria, or 10 ⁇ L of boiled lysate.
  • Amplification was performed in a Thermal Cycler 480 (Perkin-Elmer, Norwalk, Conn.) for 35 cycles, (95°C, 0.5 min; 52°C, 0.5 min; 72°C, 2.0 min for denaturing, annealing, and extension, respectively), with final extension at 72°C for 8.0 min.
  • Negative controls contained the PCR mixture without the template DNA.
  • the positive control contained serotype 6B DNA as template DNA.
  • 16F was common to all strains of serotype 16F, 10 clinical strains of serotype 16F from the United States (provided by The Streptococcal Reference Laboratory (CDC, Atlanta, GA)) and 10 clinical 16F isolates from Brazil (provided by Drs. Maria Christina de C Brandileone and Claudio Sacchi, Instituto Adolfo Lutz, Brazil) were tested with the primers. All 20 clinical strains were amplified using the primers PI and P2 (see Fig. 3, which shows the results for the 10 samples obtained in the United States). This indicates that the lack of amplification of the one ATCC isolate of 16F, when primed with P1/P2, identified in Example 1 was peculiar to that strain of 16F and not to all 16F serotypes.
  • ATCC serotype 16F may be a genetic variant which may have acquired mutations within the gene through recombination with other species.
  • Example 3 Amplification of isolates of serotype 16F of Streptococcus pneumoniae.
  • the specificity of the primers used in the PCR assay was tested using various bacteria, including heterologous upper respiratory bacteria.
  • the Streptococcal Reference Laboratory (CDC, Atlanta, GA) provided clinical isolates of the heterologous species, Streptococcus mitis, S. oralis, S. parasanguinis, S. sanguinis, S. crista, S. gordonii, S. vestibularis, S. salivarius, various ⁇ -hemolytic strains of Streptococcus pyogenes.
  • heterologous genera include
  • Staphylococcus aureus Enterococcus faecalis, Corynebacterium minutissimum, Corynebacterium pseudodiphtheriticum, Corynebacterium xerosis, Corynebacterium pseudotuberculosis, Staphylococcus epidermidis, Klebsiella pneumoniae, Escherichia coli, Moraxella catarrhalis, and Haemophilus influenzae were also obtained from the Streptococcal Reference Laboratory. Eleven multidrug-resistant strains of S. pneumoniae were provided by the Pneumococcal Molecular Epidemiology Network.
  • Mycobacterium fortuitum, Norcardia farcinica, and Rhodococcus equi were provided by the Actinomycetes Reference Laboratory, Meningitis and Special Pathogens Branch, CDC; Chlamydia pneumoniae, Pseudomonas aeruginosa. and Mycoplasma pneumoniae were provided by the Respiratory Diseases Laboratory, Respiratory Diseases Branch, CDC.
  • primer pair P1/P2 is specific for the universal amplification of the Streptococcus pneumoniae psaA gene. It is further concluded that false positive results derived from other common bacterial pathogens in a PCR diagnostic assay using these primers will be very unlikely.
  • Example 4 Sensitivity of the amplification procedure.
  • the sensitivity of the PCR was evaluated by using pneumococcal DNA as a target in 5-fold dilutions. The sensitivity was about 140 fg as determined by visualization of the 838-bp band in agarose gels (not shown).
  • Example 5 Detection of Streptococcus pneumoniae in clinical samples.
  • Nasopharyngeal secretions were collected in a blinded manner from children under 5 years of age attending a clinic or emergency room in the United States, China, or Israel. These specimens had no identifiers and were unlinked. The secretions were inoculated into STGG transport medium for transfer to the Centers for Disease Control and Prevention. For the present assessment, the samples were first enriched as follows. A 10- ⁇ L aliquot of the specimen was introduced into 2.0 mL of Todd-Hewitt broth and the suspension was incubated in a tightly capped test tube for 3.5 hours in a 37 °C water bath. The suspension was then centrifuged at 14,000 x g for ten minutes in a microcentrifuge (Eppendorf model 5415C).
  • the pellet was washed by resuspending it in 100 ⁇ L of ultra-filtered water, and the washed suspension was centrifuged again. The final pellet was resuspended in 50 ⁇ L of filtered water and boiled for 10 minutes. After boiling, the suspension was cooled on ice for at least 5 minutes, then used in the psaA-PCR reaction mixture as described in Example 1. In addition, the samples were cultured in conventional fashion to provide diagnostic standard results.
  • the enrichment step described above was incorporated into the procedure prior to conducting the PCR assay in order to improve sensitivity, since the specimen size was small (20 ⁇ L). By so doing, it was found that positive results could be obtained without purification of DNA template.
  • Three of four specimens were determined to be positive for S. pneumoniae by culture. Of these three specimens, two were amplified using the psaA-PCR of the invention. These specimens contained more than J> 25 colonies per 0.1 mL. The third specimen, determined by culture to have fewer than 25 organisms per 0.1 mL, was not detected by psaA-PCR. The fourth specimen was negative by both culture and psaA-PCR.

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Abstract

La présente invention concerne des amorces oligonucléotidiques et des techniques de diagnostic utilisant ces amorces dans lesquelles des paires d'amorces particulières amorcent l'amplification universelle d'un amplicon spécifique à Streptococcus pneumoniae parmi tous les 90 sérotypes connus. Cet amplicon comprend au moins une partie du gène psaA de Streptococcus pneumoniae. Dans ces techniques, l'amplification d'un échantillon biologique utilisant les paires d'amorces de l'invention ne fournit un amplicon que si cet échantillon contient un sérotype de Streptococcus pneumoniae. Cette invention concerne aussi un acide nucléique isolé comprenant un amplicon d'acide nucléique obtenu par l'utilisation d'une paire d'amorces oligonucléotidiques de l'invention. Un autre aspect de cette invention concerne un polypeptide comprenant une séquence d'acides aminés codée par un amplicon d'acide nucléique obtenu par l'utilisation d'amorces oligonucléotidiques. En outre, des techniques de stimulation de réponse immunitaire contre un sérotype de Streptococcus pneumoniae chez un mammifère consistent soit à administrer un polypeptide codé par un amplicon d'acide nucléique obtenu par l'utilisation de la paire d'amorces oligonucléotidiques de l'invention, soit à administrer un acide nucléique contenant un amplicon d'acide nucléique obtenu par l'utilisation de la paire d'amorces oligonucléotidiques de l'invention.
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US7674769B2 (en) * 2001-10-15 2010-03-09 Novartis Vaccines And Diagnostics, Inc. Treatment of severe pneumonia by administration of tissue factor pathway inhibitor (TFPI)
WO2006104486A1 (fr) * 2005-03-25 2006-10-05 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Sevices, Centers For Disease Control And Prevention Mise au point de la reaction en chaine de la polymerase en temps reel pour la detection d'adn pneumococcique et diagnostic de maladie pneumococcique
JP2008536491A (ja) * 2005-03-25 2008-09-11 アメリカ合衆国 肺炎球菌ｄｎａ検出用リアルタイムｐｃｒアッセイの開発および肺炎球菌疾患の診断
US7476733B2 (en) 2005-03-25 2009-01-13 The United States Of America As Represented By The Department Of Health And Human Services Development of a real-time PCR assay for detection of pneumococcal DNA and diagnosis of pneumococccal disease
AU2005330019B2 (en) * 2005-03-25 2011-07-28 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Sevices, Centers For Disease Control And Prevention Development of a real-time PCR assay for detection of pneumococcal DNA and diagnosis of Pneumococcal Disease
JP4891985B2 (ja) * 2005-03-25 2012-03-07 アメリカ合衆国 肺炎球菌ｄｎａ検出用リアルタイムｐｃｒアッセイの開発および肺炎球菌疾患の診断
JP2020533438A (ja) * 2017-09-07 2020-11-19 メルク・シャープ・アンド・ドーム・コーポレーションＭｅｒｃｋ Ｓｈａｒｐ ＆ Ｄｏｈｍｅ Ｃｏｒｐ． 肺炎球菌多糖体および免疫原性多糖体−キャリアタンパク質コンジュゲートでのその使用
EP3678653A4 (fr) * 2017-09-07 2021-05-26 Merck Sharp & Dohme Corp. Polysaccharides antipneumococciques et leur utilisation dans des conjugués immunogènes polysaccharide-protéine porteuse
US11395849B2 (en) 2017-09-07 2022-07-26 Merck Sharp & Dohme Llc Pneumococcal polysaccharides and their use in immunogenic polysaccharide-carrier protein conjugates
JP7293201B2 (ja) 2017-09-07 2023-06-19 メルク・シャープ・アンド・ドーム・エルエルシー 肺炎球菌多糖体および免疫原性多糖体－キャリアタンパク質コンジュゲートでのその使用
US11759511B2 (en) 2017-09-07 2023-09-19 Merck Sharp & Dohme Llc Pneumococcal polysaccharides and their use in immunogenic polysaccharide-carrier protein conjugates
US11759510B2 (en) 2017-09-07 2023-09-19 Merck, Sharp & Dohme LLC Pneumococcal polysaccharides and their use in immunogenic polysaccharide-carrier protein conjugates

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