Methods and compositions for pertussis diagnosis

Compositions and methods for the detection and diagnosis of Bordetella pertussis are disclosed. Provided are antibodies, or antigen binding fragment thereof, specific for tracheal colonization factor A (TcfA). Also provided are compositions comprising an anti-TcfA antibody of the instant invention and a carrier; and methods for inhibiting, treating, and/or preventing pertussis and/or a B. pertussis infection in a subject in need thereof are provided, comprising administering an anti-TcfA antibody of the instant invention to the subject.

Incorporated herein by reference in its entirety is the Sequence Listing being concurrently submitted via EFS-Web as a text file named SegList.txt, created Nov. 18, 2020, and having a size of 70,928 bytes.

FIELD OF THE INVENTION

This invention relates generally to the field of pertussis. Specifically, the invention provides novel compositions and methods for the early diagnosis of pertussis.

BACKGROUND OF THE INVENTION

Pertussis is a respiratory disease caused by the gram-negative bacteriumBordetella pertussis. It is airborne, highly contagious, and responsible for an annual 18.4 million illnesses and 254,000 deaths worldwide (Warfel, et al. (2012) J. Infect. Dis., 206(6):902-6; Mertsola, et al. (1983) J. Pediatr., 103(3):359-63; Wirsing von Konig, et al. (1998) Eur. J. Pediatr., 157(5):391-4). Globally, pertussis is one of the leading causes of death for children under 5 years old (Black, et al. (2010) Lancet 375(9730):1969-87).

Pertussis incidence in the United States has been increasing since the early 1980s (Black, S. (1997) Pediatr. Infect. Dis. J., 16(4 Suppl):S85-9; Crowcroft, et al. (2006) Lancet 367(9526):1926-36). Despite high vaccine coverage, there were still over 48,000 cases reported in the U.S. in 2012, which is the highest number since 1955 (Centers for Disease Control and Prevention (2013) MMWR Morb. Mortal Wkly. Rep., 62(33):669-82; Centers for Disease Control and Prevention (1980) MMWR Morb. Mortal Wkly. Rep., 28(54)). Moreover, reported cases represent a large underestimate of pertussis infections (Cherry, et al. (2005) Pediatr. Infect. Dis. J., 24(5 Suppl):S25-34; van den Brink, et al. (2014) BMC Infect. Dis., 14:526). Unfortunately neither vaccination nor previous infection provide life-long immunity to pertussis (Wendelboe, et al. (2005) Pediatr. Infect. Dis. J., 24(5 Suppl):S58-61). In particular, vaccine-induced immunity wanes after 4-12 years (Wendelboe, et al. (2005) Pediatr. Infect. Dis. J., 24(5 Suppl):S58-61), leaving many children and adults vulnerable to infection as well as household infants who are too young to have yet received the vaccine.

However, prompt diagnosis of early pertussis is challenging because its symptoms are non-specific and because there are no assays that can rapidly diagnose pertussis at the point-of-care (POC). For example, bacterial culture, while being suitable for early diagnosis, is a very slow assay that requires 5-7 days at a site not at the point of care. Serological tests, while sensitive, cannot be used to detect early disease because patient antibodies are required. Various PCR or other DNA amplification based assays such as RT-PCR, helicase-dependent amplification (HDA) (e.g., AmpliVue®BordetellaAssay (Quidel, San Diego, Calif.)), nested multiplex PCR (e.g., FilmArray® Respiratory Panel (BioFire, Salt Lake City, Utah)), and loop mediated isothermal amplification (LAMP) (e.g., Illumigene® Pertussis DNA Amplification Assay (Meridian Bioscience, London, England)) are available, but have many drawbacks. Indeed, these assays: 1) can be very expensive; 2) are not point of care assays as they are generally performed in a hospital or off-site lab; and 3) do not report on the antibiotic susceptibility of theB. pertussis. In view of the foregoing, it is clear that improved methods for early diagnosis of pertussis are needed.

SUMMARY OF THE INVENTION

In accordance with one aspect of the instant invention, antibodies or antigen binding fragment thereof specific for tracheal colonization factor A (TcfA) are provided. In a particular embodiment, the anti-TcfA antibody or fragment thereof specifically binds amino acids 140-160, amino acids 229-240, amino acids 288-304, amino acids 286-321, amino acids 289-324, amino acids 305-323, amino acids 322-330, or amino acids 337-345 of TcfA. In a particular embodiment, the anti-TcfA antibody or fragment thereof specifically binds amino acids 140-150, amino acids 148-159, amino acids 151-156, amino acids 151-159, amino acids 229-240, amino acids 289-300, amino acids 305-312, amino acids 286-321, amino acids 289-324, amino acids 289-294, amino acids 292-300, amino acids 307-315, amino acids 310-315, amino acids 313-321, amino acids 322-330, or amino acids 337-345 of TcfA. The anti-TcfA antibodies of the instant invention may be conjugated to a detectable label such as a gold nanoparticle. Composition comprising an anti-TcfA antibody of the instant invention and a carrier are also provided.

In accordance with another aspect of the instant invention, methods of detectingBordetella pertussisin a sample are provided. The methods comprise contacting the sample with an anti-TcfA antibody. Generally, the sample is a biological sample obtained from a subject. In a particular embodiment, the biological sample is a nasopharyngeal swab, aspirate, or wash.

In accordance with another aspect of the instant invention, methods for inhibiting, treating, and/or preventing pertussis and/or aB. pertussisinfection in a subject in need thereof are provided. The methods comprise administering an anti-TcfA antibody of the instant invention to the subject. The method may further comprise administering antibiotics to the subject.

In accordance with yet another aspect of the instant invention, immunoassays for detectingB. pertussisare provided. The immunoassays comprise at least one anti-TcfA antibody of the instant invention. In a particular embodiment, the immunoassay is a lateral flow immunoassay test strip. In a particular embodiment, the immunoassay comprises a conjugated antibody which specifically binds amino acids 139-150 or amino acids 151-156 of TcfA. In a particular embodiment, the immunoassay comprises a test line antibody which specifically binds amino acids 289-324 of TcfA, amino acids 289-294 of TcfA, amino acids 292-300 of TcfA, and/or amino acids 322-330 of TcfA. In a particular embodiment, the immunoassay comprises a test line antibody which specifically binds amino acids 289-324, 229-240, 289-300, and/or 304-312 of TcfA (e.g., the same epitope as 13E11) and a test line antibody which specifically binds amino acids 288-304 or 292-300 (e.g., the same epitope as 14D12), particularly with a conjugated antibody which specifically binds amino acids 140-160 or 139-150 of TcfA (e.g., the same epitope as 10B1). In accordance with another aspect of the instant invention, methods of detectingBordetella pertussisin a sample using the immunoassays are provided.

DETAILED DESCRIPTION OF THE INVENTION

Herein, a point-of-care, lateral flow immunoassay (LFI) diagnostic for early pertussis that enables immediate treatment initiation (e.g., during the patient's initial clinic visit) is provided. This point-of-care assay for detection of early pertussis will improve patient care and public health. It is well established that disease severity and duration can be reduced with pertussis if patients receive antibiotic treatment early (Tiwari, et al. (2005) MMWR Recomm. Rep., 54(RR-14):1-16; von Konig, C. H. (2005) Pediatr. Infect. Dis. J., 24(5 Suppl):S66-8; Mattoo, et al. (2005) Clin. Microbiol. Rev., 18(2):326-82; Hewlet, et al. (2005) N. Engl. J. Med., 352(12):1215-22). For infants, early diagnosis would also save lives. Currently, infants require more doctor visits to reach a pertussis diagnosis than do older patients (Lee, et al. (2000) Arch. Fam. Med., 9(10):989-96). Infants also have the highest risk of mortality and severe neurological complications (Tanaka, et al. (2003) JAMA 290(22):2968-75). For infants, early diagnosis would enable not only earlier treatment with antibiotics, but also key supportive care for dehydration and malnutrition (Crowcroft, et al. (2006) Lancet 367(9526):1926-36; Hewlet, et al. (2005) N. Engl. J. Med., 352(12):1215-22).

Early diagnosis can change the course of an outbreak because patients are most infectious from the start of nonspecific symptoms until three weeks after paroxysmal cough onset (Tiwari, et al. (2005) MMWR Recomm. Rep., 54(RR-14):1-16). Antibiotic treatment eliminates culturable bacteria from the nasopharynx (Bergquist, et al. (1987) Pediatr. Infect. Dis. J., 6(5):458-61), which decreases the patients' infectious period and limits transmission (Wirsing von Konig, et al. (1998) Eur. J. Pediatr., 157(5):391-4). Moreover, once patients are diagnosed, their close contacts can receive prophylactic antibiotics (Tiwari, et al. (2005) MMWR Recomm. Rep., 54(RR-14):1-16; von Konig, C. H. (2005) Pediatr. Infect. Dis. J., 24(5 Suppl):S66-8). Thus, early diagnosis would reduce the size of pertussis outbreaks.

Minimizing outbreak size through early diagnosis would also reduce the economic burden of pertussis. More than $17 billion were spent on pertussis costs (direct and indirect) from 2001-2010 in the U.S. (Purdy, et al. (2004) Clin. Infect. Dis., 39(1):20-8). Preventing infant cases is particularly important because 70% of infants with pertussis become hospitalized (Tanaka, et al. (2003) JAMA 290(22):2968-75) and each infant hospital stay costs an average of $10,000, excluding outpatient direct and societal indirect costs (O'Brien, et al. (2005) BMC Infect. Dis., 5:57).

In accordance with one aspect of the instant invention, anti-tracheal colonization factor A (TcfA) antibodies and fragments thereof are provided. The anti-TcfA antibodies may be monoclonal or polyclonal. In a particular embodiment, the antibody or fragment thereof is immunologically specific for TcfA ofBordetella pertussis(e.g., Tohama I strain). Amino acid and nucleotide sequences of TcfA are provided in GenBank Accession No. NP_879974 and Gene ID: 2666888.FIG.6provides an amino acid sequence for TcfA (SEQ ID NO: 1) and certain anti-TcfA antibody epitopes. The anti-TcfA antibodies or fragments thereof may recognize a linear epitope or a conformational epitope, particularly a linear epitope. In a particular embodiment, the anti-TcfA antibody or fragment thereof recognizes a linear epitope. In a particular embodiment, the anti-TcfA antibody or fragment thereof is immunologically specific for a polypeptide comprising amino acids 140-160, amino acids 288-304, amino acids 305-323, amino acids 322-330, or amino acids 337-345 of TcfA. In a particular embodiment, the anti-TcfA antibody or fragment thereof is immunologically specific for a polypeptide comprising amino acids 139-150, amino acids 148-159, amino acids 151-156, amino acids 151-159, amino acids 229-240, amino acids 289-300, amino acids 304-312, amino acids 286-321, amino acids 289-324, amino acids 289-294, amino acids 292-300, amino acids 307-315, amino acids 310-315, amino acids 313-321, amino acids 322-330, or amino acids 337-345 of TcfA. The above epitopes may be longer or shorter than the above identified sequences by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids, particularly 1, 2, 3, 4, or 5 amino acids, at the N-terminus and/or C-terminus of the sequence. In another embodiment, the above epitopes have at least 90%, 95%, 97%, 99%, or 100% homology or identity with the sequence provided inFIG.6(SEQ ID NO: 1). Antibodies which bind the same epitope as an antibody provided herein are also encompassed by the instant invention.

In a particular embodiment, the anti-TcfA antibody or fragment thereof is immunologically specific for amino acids 288-304 or 292-300 of TcfA. In a particular embodiment, the anti-TcfA antibody is 14D12 (as depicted inFIG.11A), optionally wherein the signal peptides removed, or a fragment thereof. In a particular embodiment, the anti-TcfA antibody comprises a heavy chain comprising SEQ ID NO: 19 and/or a light chain comprising SEQ ID NO: 21. In a particular embodiment, the anti-TcfA antibody comprises a heavy chain comprising: EVMLVESGGALVKPGGSLKLSCAASGITFSNYAMSWIRQTPEKRLEWV ASISSGGSYIYYSDSVKGRFTISRDNARNTLNLQMSSLRSEDTAMYYCVRGAH GNFDYWGQGTTLTVSS (SEQ ID NO: 22) and/or a light chain comprising: DIVLTQSPASLAVSLGQRATISCRTSETVDYDGDSYMNWYQQKSGQP PKLLISGASNVESGVPARFSGSGSGTDFSLNIIIPVEEDDITMYFCQQNRKLPYT FGSGTKLEMK (SEQ ID NO: 23). In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises one, two, three, four, five, or all six complementarity determining regions (CDRs) of 14D12 (e.g., as determined by IMGT, Chothia, Kabat, Martin (e.g., enhanced Chothia) or AHo numbering scheme, particularly Kabat). In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises one, two, three, four, five, or all six CDRs depicted inFIG.11A. In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises a heavy chain comprising one, two, or all three CDRs depicted in the heavy chain provided inFIG.11A. In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises a light chain comprising one, two, or all three CDRs depicted in the light chain provided inFIG.11A. Ina particular embodiment, the anti-TcfA antibody or fragment thereof comprises one, two, three, four, five, or all six of: NYAMS (SEQ ID NO: 24), SISSGGSYIYYSDSVKG (SEQ ID NO: 25), GAHGNFDY (SEQ ID NO: 26), RTSETVDYDGDSYMN (SEQ ID NO: 27), GASNVES (SEQ ID NO: 28), and QQNRKLPYT (SEQ ID NO: 29). In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises a heavy chain comprising one, two, or all three of: NYAMS (SEQ ID NO: 24), SISSGGSYIYYSDSVKG (SEQ ID NO: 25), and GAHGNFDY (SEQ ID NO: 26) and/or a light chain comprising one, two, or all three of: RTSETVDYDGDSYMN (SEQ ID NO: 27), GASNVES (SEQ ID NO: 28), and QQNRKLPYT (SEQ ID NO: 29). In another embodiment, the anti-TcfA antibody or fragment thereof comprise an amino acid sequence having at least 90%, 95%, 97%, 99%, or 100% homology or identity with any of the sequences provided above (e.g., any of SEQ ID NOs: 19 and 22-29).

In a particular embodiment, the anti-TcfA antibody or fragment thereof is immunologically specific for amino acids 288-304 or 292-300 of TcfA. In a particular embodiment, the anti-TcfA antibody is 23F8 (as depicted inFIG.11), optionally wherein the signal peptides removed, or a fragment thereof. In a particular embodiment, the anti-TcfA antibody comprises a heavy chain comprising SEQ ID NO: 31 and/or a light chain comprising SEQ ID NO: 33. In a particular embodiment, the anti-TcfA antibody comprises a heavy chain comprising: EVQLVESGGGLVKPGGSRKLSCAASGFTFSDYGMHWVRQAPEKGLEWV AYISSGSRTIYYADTVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCARLGY GYDWYFDVWGTGTTVTVSS (SEQ ID NO: 34) and/or a light chain comprising: DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGRTYLNWLLQRPGQS PKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCWQGTHFPQ TFGGGTKLEIK (SEQ ID NO: 35). In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises one, two, three, four, five, or all six CDRs of 23F8 (e.g., as determined by IMGT, Chothia, Kabat, Martin (e.g., enhanced Chothia) or AHo numbering scheme, particularly Kabat). In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises one, two, three, four, five, or all six CDRs depicted inFIG.11B. In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises a heavy chain comprising one, two, or all three CDRs depicted in the heavy chain provided inFIG.11B. In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises a light chain comprising one, two, or all three CDRs depicted in the light chain provided inFIG.11B. In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises one, two, three, four, five, or all six of: DYGMH (SEQ ID NO: 36), YISSGSRTIYYADTVKG (SEQ ID NO: 37), LGYGYDWYFDV (SEQ ID NO: 38), KSSQSLLDSDGRTYLN (SEQ ID NO: 39), LVSKLDS (SEQ ID NO: 40), and WQGTHFPQT (SEQ ID NO: 41). In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises a heavy chain comprising one, two, or all three of: DYGMH (SEQ ID NO: 36), YISSGSRTIYYADTVKG (SEQ ID NO: 37), and LGYGYDWYFDV (SEQ ID NO: 38) and/or a light chain comprising one, two, or all three of: KSSQSLLDSDGRTYLN (SEQ ID NO: 39), LVSKLDS (SEQ ID NO: 40), and WQGTHFPQT (SEQ ID NO: 41). In another embodiment, the anti-TcfA antibody or fragment thereof comprise an amino acid sequence having at least 90%, 95%, 97%, 99%, or 100% homology or identity with any of the sequences provided above (e.g., any of SEQ ID NOs: 31 and 33-41).

In a particular embodiment, the anti-TcfA antibody or fragment thereof is immunologically specific for amino acids 337-345 of TcfA. In a particular embodiment, the anti-TcfA antibody is 18B2 (as depicted inFIG.11C), optionally wherein the signal peptides removed, or a fragment thereof. In a particular embodiment, the anti-TcfA antibody comprises a heavy chain comprising SEQ ID NO: 43 and/or a light chain comprising SEQ ID NO: 45. In a particular embodiment, the anti-TcfA antibody comprises a heavy chain comprising: QVQLQQSGAELVRPGTSVKMSCKAAGYTFTNYWIGWVKQRPGHGLEWI GDIYPGGVYTNYNENFKGKATLTADTSSSTAHMQLSSLTSEDSAIYYCVRGG KYGNFFAMDYWGQGTSVTVSS (SEQ ID NO: 46) and/or a light chain comprising: DIVITQDELSNPVTSGESVSISCRSSKSLLYKDGKTYLNWFLQRPGQ SPQLLIYLMSTRASGVSDRFSGSGSGTDFTLEISRVKAEDVGVYYCQQLVEYP FTFGSGTKLEIK (SEQ ID NO: 47). In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises one, two, three, four, five, or all six CDRs of 18B2 (e.g., as determined by IMGT, Chothia, Kabat, Martin (e.g., enhanced Chothia) or AHo numbering scheme, particularly Kabat). In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises one, two, three, four, five, or all six CDRs depicted inFIG.11C. In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises a heavy chain comprising one, two, or all three CDRs depicted in the heavy chain provided inFIG.11C. In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises a light chain comprising one, two, or all three CDRs depicted in the light chain provided inFIG.11C. In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises one, two, three, four, five, or all six of: NYWIG (SEQ ID NO: 48), DIYPGGVYTNYNENFKG (SEQ ID NO: 49), GGKYGNFFAMDY (SEQ ID NO: 50), RSSKSLLYKDGKTYLN (SEQ ID NO: 51), LMSTRAS (SEQ ID NO: 52), and QQLVEYPFT (SEQ ID NO: 53). In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises a heavy chain comprising one, two, or all three of: NYWIG (SEQ ID NO: 48), DIYPGGVYTNYNENFKG (SEQ ID NO: 49), and GGKYGNFFAMDY (SEQ ID NO: 50) and/or a light chain comprising one, two, or all three of: RSSKSLLYKDGKTYLN (SEQ ID NO: 51), LMSTRAS (SEQ ID NO: 52), and QQLVEYPFT (SEQ ID NO: 53). In another embodiment, the anti-TcfA antibody or fragment thereof comprise an amino acid sequence having at least 90%, 95%, 97%, 99%, or 100% homology or identity with any of the sequences provided above (e.g., any of SEQ ID NOs: 43 and 45-53).

In a particular embodiment, the anti-TcfA antibody or fragment thereof is immunologically specific for amino acids 305-323 or 307-315 of TcfA. In a particular embodiment, the anti-TcfA antibody is 20F4 (as depicted inFIG.11D), optionally wherein the signal peptides removed, or a fragment thereof. In a particular embodiment, the anti-TcfA antibody comprises a heavy chain comprising SEQ ID NO: 55 and/or a light chain comprising SEQ ID NO: 57. In a particular embodiment, the anti-TcfA antibody comprises a heavy chain comprising: QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWM GWINTYTGEPTYADDFKGRFAFSLETSATTAYLQINNLKNEDTATYFCARAA TGYFDYWGQGTTLTVSS (SEQ ID NO: 58) and/or a light chain comprising: DIVMSQSPSSLAVSAGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPG QSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYNE YTFGGGTKLEIK (SEQ ID NO: 59). In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises one, two, three, four, five, or all six CDRs of 20F4 (e.g., as determined by IMGT, Chothia, Kabat, Martin (e.g., enhanced Chothia) or AHo numbering scheme, particularly Kabat). In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises one, two, three, four, five, or all six CDRs depicted inFIG.11D. In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises a heavy chain comprising one, two, or all three CDRs depicted in the heavy chain provided inFIG.11D. In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises a light chain comprising one, two, or all three CDRs depicted in the light chain provided inFIG.11D. In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises one, two, three, four, five, or all six of: NYGMN (SEQ ID NO: 60), WINTYTGEPTYADDFKG (SEQ ID NO: 61), AATGYFDY (SEQ ID NO: 62), KSSQSLLYSSNQKNYLA (SEQ ID NO: 63), WASTRES (SEQ ID NO: 64), and QQYYNEYT (SEQ ID NO: 65). In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises a heavy chain comprising one, two, or all three of: NYGMN (SEQ ID NO: 60), WINTYTGEPTYADDFKG (SEQ ID NO: 61), and AATGYFDY (SEQ ID NO: 62) and/or a light chain comprising one, two, or all three of: KSSQSLLYSSNQKNYLA (SEQ ID NO: 63), WASTRES (SEQ ID NO: 64), and QQYYNEYT (SEQ ID NO: 65). In another embodiment, the anti-TcfA antibody or fragment thereof comprise an amino acid sequence having at least 90%, 95%, 97%, 99%, or 100% homology or identity with any of the sequences provided above (e.g., any of SEQ ID NOs: 55 and 57-65).

In a particular embodiment, the anti-TcfA antibody or fragment thereof is immunologically specific for amino acids 305-323 or 313-321 of TcfA. In a particular embodiment, the anti-TcfA antibody is 14G11 (as depicted inFIG.11E), optionally wherein the signal peptides removed, or a fragment thereof. In a particular embodiment, the anti-TcfA antibody comprises a heavy chain comprising SEQ ID NO: 67 and/or a light chain comprising SEQ ID NO: 69. In a particular embodiment, the anti-TcfA antibody comprises a heavy chain comprising: EVKLVESGGGLVQPGGSLSLSCAASGFTFTDYYMSWVRQPPGKALEWL GFIRNKANGYTTEYSASVKGRFTISRDNSQSILYLQMNALRAEDSATYYCARY RRDYYGSLNYYTMD YWGQGTSVTVSS (SEQ ID NO: 70) and/or a light chain comprising: DIQMTQSPASLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQLL VYNAKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYCQNHYGIPLTFGA GTKLELK (SEQ ID NO: 71). In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises one, two, three, four, five, or all six CDRs of 14G11 (e.g., as determined by IMGT, Chothia, Kabat, Martin (e.g., enhanced Chothia) or AHo numbering scheme, particularly Kabat). In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises one, two, three, four, five, or all six CDRs depicted inFIG.11E. In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises a heavy chain comprising one, two, or all three CDRs depicted in the heavy chain provided inFIG.11E. In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises a light chain comprising one, two, or all three CDRs depicted in the light chain provided inFIG.11E. In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises one, two, three, four, five, or all six of: DYYMS (SEQ ID NO: 72), FIRNKANGYTTEYSASVKG (SEQ ID NO: 73), YRRDYYGSLNYYTMDY (SEQ ID NO: 74), RASENIYSYLA (SEQ ID NO: 75), NAKTLAE (SEQ ID NO: 76), and QNHYGIPLT (SEQ ID NO: 77). In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises a heavy chain comprising one, two, or all three of: DYYMS (SEQ ID NO: 72), FIRNKANGYTTEYSASVKG (SEQ ID NO: 73), and YRRDYYGSLNYYTMDY (SEQ ID NO: 74) and/or a light chain comprising one, two, or all three of: RASENIYSYLA (SEQ ID NO: 75), NAKTLAE (SEQ ID NO: 76), and QNHYGIPLT (SEQ ID NO: 77). In another embodiment, the anti-TcfA antibody or fragment thereof comprise an amino acid sequence having at least 90%, 95%, 97%, 99%, or 100% homology or identity with any of the sequences provided above (e.g., any of SEQ ID NOs: 67 and 69-77).

In a particular embodiment, the anti-TcfA antibody or fragment thereof is immunologically specific for amino acids 288-323, 229-240, 289-300, and/or 304-312 of TcfA. In a particular embodiment, the anti-TcfA antibody is 13E11 (as depicted inFIG.11F), optionally wherein the signal peptides removed, or a fragment thereof. In a particular embodiment, the anti-TcfA antibody comprises a heavy chain comprising SEQ ID NO: 79 and/or a light chain comprising SEQ ID NO: 81. In a particular embodiment, the anti-TcfA antibody comprises a heavy chain comprising: EVQLVESGGGLVKPGGSRKLSCAASGFTFSDYGMHWVRQAPEKGLEWV AYISSGSSTIYYADTVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCARPRS GRYFDYWGQGTTLTVSS (SEQ ID NO: 82) and/or a light chain comprising: DVMMTQTPLTLSVTIGQPASISCKSSQSLLDSNGNTYLHWLLQRPGQS PKILIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCLQGTHFPYT FGGGTKLEIK (SEQ ID NO: 83). In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises one, two, three, four, five, or all six CDRs of 13E11 (e.g., as determined by IMGT, Chothia, Kabat, Martin (e.g., enhanced Chothia) or AHo numbering scheme, particularly Kabat). In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises one, two, three, four, five, or all six CDRs depicted inFIG.11F. In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises a heavy chain comprising one, two, or all three CDRs depicted in the heavy chain provided inFIG.11F. In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises a light chain comprising one, two, or all three CDRs depicted in the light chain provided inFIG.11F. In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises one, two, three, four, five, or all six of: DYGMH (SEQ ID NO: 84), YISSGSSTIYYADTVKG (SEQ ID NO: 85), PRSGRYFDY (SEQ ID NO: 86), KSSQSLLDSNGNTYLH (SEQ ID NO: 87), LVSKLDS (SEQ ID NO: 88), and LQGTHFPYT (SEQ ID NO: 89). In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises a heavy chain comprising one, two, or all three of: DYGMH (SEQ ID NO: 84), YISSGSSTIYYADTVKG (SEQ ID NO: 85), and PRSGRYFDY (SEQ ID NO: 86) and/or a light chain comprising one, two, or all three of: KSSQSLLDSNGNTYLH (SEQ ID NO: 87), LVSKLDS (SEQ ID NO: 88), and LQGTHFPYT (SEQ ID NO: 89). In another embodiment, the anti-TcfA antibody or fragment thereof comprise an amino acid sequence having at least 90%, 95%, 97%, 99%, or 100% homology or identity with any of the sequences provided above (e.g., any of SEQ ID NOs: 79 and 81-89).

In a particular embodiment, the anti-TcfA antibody or fragment thereof is immunologically specific for amino acids 140-160 or 139-150 of TcfA. In a particular embodiment, the anti-TcfA antibody is 10B1 (as depicted inFIG.11G), optionally wherein the signal peptides removed, or a fragment thereof. In a particular embodiment, the anti-TcfA antibody comprises a heavy chain comprising SEQ ID NO: 91 and/or a light chain comprising SEQ ID NO: 93. In a particular embodiment, the anti-TcfA antibody comprises a heavy chain comprising: EVQLQQSGAELVKPGASVKLSCTASGFNIKDTYIHWVKQRPEQGLEWI GRIDPANGNTIYASKFQGKAPITAVTSSNTAYMQFSSLTSGDTAVYYCTAMD YWGQGTSVTVSS (SEQ ID NO: 94) and/or a light chain comprising: DVVMTQTPLTLSVTIGQPASISCKSSQSLLHSNGKTYLNWLLQRPGQS PKLLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCLQATHFPHT FGSGTKLEIK (SEQ ID NO: 95). In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises one, two, three, four, five, or all six CDRs of 10B1 (e.g., as determined by IMGT, Chothia, Kabat, Martin (e.g., enhanced Chothia) or AHo numbering scheme, particularly Kabat). In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises one, two, three, four, five, or all six CDRs depicted inFIG.11G. In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises a heavy chain comprising one, two, or all three CDRs depicted in the heavy chain provided inFIG.11G. In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises a light chain comprising one, two, or all three CDRs depicted in the light chain provided inFIG.11G. In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises one, two, three, four, five, or all six of: DTYIH (SEQ ID NO: 96), RIDPANGNTIYASKFQG (SEQ ID NO: 97), MDY (SEQ ID NO: 98), KSSQSLLHSNGKTYLN (SEQ ID NO: 99), LVSKLDS (SEQ ID NO: 100), and LQATHFPHT (SEQ ID NO: 101). In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises a heavy chain comprising one, two, or all three of: DTYIH (SEQ ID NO: 96), RIDPANGNTIYASKFQG (SEQ ID NO: 97), and MDY (SEQ ID NO: 98) and/or a light chain comprising one, two, or all three of: KSSQSLLHSNGKTYLN (SEQ ID NO: 99), LVSKLDS (SEQ ID NO: 100), and LQATHFPHT (SEQ ID NO: 101). In another embodiment, the anti-TcfA antibody or fragment thereof comprise an amino acid sequence having at least 90%, 95%, 97%, 99%, or 100% homology or identity with any of the sequences provided above (e.g., any of SEQ ID NOs: 91 and 93-101).

In a particular embodiment, the anti-TcfA antibody or fragment thereof is immunologically specific for amino acids 140-160 or 139-150 of TcfA. In a particular embodiment, the anti-TcfA antibody is 7E11 (as depicted inFIG.11H), optionally wherein the signal peptides removed, or a fragment thereof. In a particular embodiment, the anti-TcfA antibody comprises a heavy chain comprising SEQ ID NO: 103 and/or a light chain comprising SEQ ID NO: 105. In a particular embodiment, the anti-TcfA antibody comprises a heavy chain comprising: EVQLQQSGAELVKPGASVKLSCTASGFNIKDTYIHWVKQRPEQGLEWI GRIDPANGNIIYASKFQGEATITADTSSNTAYMQLSSLTSGDTAVYYCSAMDY WGQGTSVTVSS (SEQ ID NO: 106) and/or a light chain comprising: DVVMTQTPLTLSLTIGQPASISCKSSQSLLHSNGKTYLNWLLQRPGQS PKLLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCLQATHFPHT FGSGTKLEIK (SEQ ID NO: 107). In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises one, two, three, four, five, or all six CDRs of 7E11 (e.g., as determined by IMGT, Chothia, Kabat, Martin (e.g., enhanced Chothia) or AHo numbering scheme, particularly Kabat). In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises one, two, three, four, five, or all six CDRs depicted inFIG.11H. In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises a heavy chain comprising one, two, or all three CDRs depicted in the heavy chain provided inFIG.11H. In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises a light chain comprising one, two, or all three CDRs depicted in the light chain provided inFIG.11H. In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises one, two, three, four, five, or all six of: DTYIH (SEQ ID NO: 108), RIDPANGNIIYASKFQG (SEQ ID NO: 109), MDY (SEQ ID NO: 110), KSSQSLLHSNGKTYLN (SEQ ID NO: 111), LVSKLDS (SEQ ID NO: 112), and LQATHFPHT (SEQ ID NO: 113). In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises a heavy chain comprising one, two, or all three of: DTYIH (SEQ ID NO: 108), RIDPANGNIIYASKFQG (SEQ ID NO: 109), and MDY (SEQ ID NO: 110) and/or a light chain comprising one, two, or all three of: KSSQSLLHSNGKTYLN (SEQ ID NO: 111), LVSKLDS (SEQ ID NO: 112), and LQATHFPHT (SEQ ID NO: 113). In another embodiment, the anti-TcfA antibody or fragment thereof comprise an amino acid sequence having at least 90%, 95%, 97%, 99%, or 100% homology or identity with any of the sequences provided above (e.g., any of SEQ ID NOs: 103 and 105-113).

In a particular embodiment, the anti-TcfA antibody or fragment thereof is immunologically specific for amino acids 140-160 or 151-156 of TcfA. In a particular embodiment, the anti-TcfA antibody is 3E6 (as depicted inFIG.11I), optionally wherein the signal peptides removed, or a fragment thereof. In a particular embodiment, the anti-TcfA antibody comprises a heavy chain comprising SEQ ID NO: 115 and/or a light chain comprising SEQ ID NO: 117. In a particular embodiment, the anti-TcfA antibody comprises a heavy chain comprising: EVKLVESGGGLVQPGGSLRLSCATSGFTFTDYYMSWVRQPPGKALEWM GFIRNKAKGYTTDYSASVKGRFTISRDDSQSILYLQMNTLRPEDSATYYCARN YDYSMDYWGQGTSVTVSS (SEQ ID NO: 118) and/or a light chain comprising: DIQLTQSPASLSASVGETVTITCRASDNIIKYLAWYQQKQGKSPQRL VYNAKTLADGVPSRFNGSGSGTQYSLKINSLQPEDFGIYYCQHFWSTPLTFGA GTKLELK (SEQ ID NO: 119). In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises one, two, three, four, five, or all six CDRs of 3E6 (e.g., as determined by IMGT, Chothia, Kabat, Martin (e.g., enhanced Chothia) or AHo numbering scheme, particularly Kabat). In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises one, two, three, four, five, or all six CDRs depicted inFIG.11I. In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises a heavy chain comprising one, two, or all three CDRs depicted in the heavy chain provided inFIG.11I. In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises a light chain comprising one, two, or all three CDRs depicted in the light chain provided inFIG.11I. In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises one, two, three, four, five, or all six of: DYYMS (SEQ ID NO: 120), FIRNKAKGYTTDYSASVKG (SEQ ID NO: 121), NYDYSMDY (SEQ ID NO: 122), RASDNIHKYLA (SEQ ID NO: 123), NAKTLAD (SEQ ID NO: 124), and QHFWSTPLT (SEQ ID NO: 125). In a particular embodiment, the anti-TcfA antibody or fragment thereof comprises a heavy chain comprising one, two, or all three of: DYYMS (SEQ ID NO: 120), FIRNKAKGYTTDYSASVKG (SEQ ID NO: 121), and NYDYSMDY (SEQ ID NO: 122) and/or a light chain comprising one, two, or all three of: RASDNIHKYLA (SEQ ID NO: 123), NAKTLAD (SEQ ID NO: 124), and QHFWSTPLT (SEQ ID NO: 125).

In another embodiment, the anti-TcfA antibody or fragment thereof comprise an amino acid sequence having at least 90%, 95%, 97%, 99%, or 100% homology or identity with any of the sequences provided above (e.g., any of SEQ ID NOs: 115 and 117-125).

Compositions comprising an anti-TcfA antibody or fragment thereof and a carrier such as a pharmaceutically acceptable carrier are also encompassed by the instant invention. In a particular embodiment, the composition comprises at least one anti-TcfA antibody or antibody fragment and at least one carrier (e.g., a pharmaceutically acceptable carrier).

Nucleic acid molecules encoding an anti-TcfA antibody or fragment thereof are encompassed by the instant invention. Examples of nucleic acid molecules encoding anti-TcfA antibodies are provided inFIG.11. In a particular embodiment, the nucleic acid molecule encoding the anti-TcfA antibody or fragment thereof comprise a nucleotide sequence having at least 90%, 95%, 97%, 99%, or 100% homology or identity with any of the sequences provided above (e.g., any of SEQ ID NOs: 18, 20, 30, 32, 42, 44, 54, 56, 66, 68, 78, 80, 90, 92, 102, 104, 114, and 116) or a nucleotide sequence encoding any of the amino sequences provided above. In a particular embodiment, the nucleic acid molecules of the instant invention are contained within a vector, particularly an expression vector. The instant invention also encompasses cells comprising and, optionally, expressing a nucleic acid molecule of the instant invention (e.g., hybridomas that secrete monoclonal anti-TcfA antibodies).

The antibody may be a synthetic or modified antibody (e.g., a recombinantly generated antibody; a chimeric antibody; a bispecific antibody; a humanized antibody; a camelid antibody; and the like). In a particular embodiment of the instant invention, the antibody is a monoclonal antibody.

The antibodies of the instant invention may be an antibody fragment. In a particular embodiment, the antibody fragment is an antigen binding fragment of the antibody. Antibody fragments include, without limitation, immunoglobulin fragments including, without limitation: single domain (Dab; e.g., single variable light or heavy chain domain), Fab, Fab′, F(ab′)2, and F(v); and fusions (e.g., via a linker) of these immunoglobulin fragments including, without limitation: scFv, scFv2, scFv-Fc, minibody, diabody, triabody, and tetrabody. The antibody may also be a protein (e.g., a fusion protein) comprising at least one antibody or antibody fragment.

The antibodies of the instant invention may be further modified. For example, the antibodies may be humanized. In a particular embodiment, the antibodies (or a portion thereof) are inserted into the backbone of an antibody or antibody fragment construct (e.g., an antibody framework), particularly a human construct/framework. For example, the variable light domain and/or variable heavy domain of the antibodies of the instant invention or the CDRs contained therein may be inserted into another antibody construct or framework, particularly human. Methods for recombinantly producing antibodies are well-known in the art. Indeed, commercial vectors for certain antibody and antibody fragment constructs are available.

The antibodies of the instant invention may also be conjugated/linked to other components. For example, the antibodies may be operably linked (e.g., covalently linked, optionally, through a linker) to at least one detectable agent, or imaging agent, contrast agent. Detectable agents include, without limitation, colloidal gold or gold nanoparticles, fluorescent probe, colored latex particles, colored cellulose nanobeads, horseradish peroxidase, and europium (Eu) nanoparticles. The antibodies of the instant invention may also comprise at least one purification tag (e.g., a His-tag). In a particular embodiment, the antibodies of the instant invention are conjugated to biotin or streptavidin (or analog thereof).

The antibody molecules of the invention may be prepared using a variety of methods known in the art. Polyclonal and monoclonal antibodies may be prepared as described in Current Protocols in Molecular Biology, Ausubel et al. eds. Antibodies may be prepared by chemical cross-linking, hybrid hybridoma techniques and by expression of recombinant antibody fragments expressed in host cells, such as bacteria or yeast cells. In one embodiment of the invention, the antibody molecules are produced by expression of recombinant antibody or antibody fragments in host cells. The nucleic acid molecules encoding the antibody may be inserted into expression vectors and introduced into host cells. The resulting antibody molecules are then isolated and purified from the expression system. The antibodies optionally comprise a purification tag by which the antibody can be purified.

The purity of the antibody molecules of the invention may be assessed using standard methods known to those of skill in the art, including, but not limited to, ELISA, immunohistochemistry, ion-exchange chromatography, affinity chromatography, immobilized metal affinity chromatography (IMAC), size exclusion chromatography, polyacrylamide gel electrophoresis (PAGE), western blotting, surface plasmon resonance and mass spectroscopy.

In accordance with another aspect of the instant invention, immunoassays for detectingB. pertussisare provided. In a particular embodiment, the immunoassay provides a rapid, point of care assay to detectB. pertussisduring early disease. The immunoassays use at least one of the anti-TcfA antibodies of the instant invention. In a particular embodiment, the immunoassay can be performed at the point-of-care without the need for expensive equipment or the need for specialized equipment or off-site equipment to analyze the data. In a particular embodiment, the immunoassay is carried out using a sample capture device, such as a lateral flow device, more particularly a lateral flow test strip, that allows detection of the TcfA biomarker. In a particular embodiment, the immunoassay is performed on a lateral flow test strip.

The immunoassay of the instant invention has multiple advantages over existing diagnostics. For example, the immunoassay can be performed at the point of care. Other non-limiting advantages include low cost (e.g., no specialized equipment required), ease of use (e.g., no specialized user expertise required), and rapid results (e.g., less than about 40 minutes, less than about 30 minutes, less than about 20 minutes, or less than about 15 minutes). The immunoassay of the instant invention is also convenient for single samples and there is no need or advantage to batch samples.

Lateral flow immunoassays (LFI or LFIA) are simple tests for rapid detection of the presence or absence of a target analyte in a sample. Generally, lateral flow test strips comprise a matrix or material through which a mobile phase (e.g., a liquid sample) can flow through by capillary action to a test site where a detectable signal is generated to indicate the presence or absence of the target analyte. A lateral flow immunoassay may be used in a vertical or a horizontal orientation or in an orientation between vertical and horizontal.

A test strip is an article of manufacture that includes one or more zones, such as, for example, one or more of the following in any appropriate configurations: sample application area or sample pad, absorbent pad or wicking pad, test site, and conjugation pad. The different zones of the test strip may be made of the same material or different material. In a particular embodiment, the test site comprises nitrocellulose, nylon, polyester or polyester composite, matrix membranes (e.g., FUSION 5 (GE Healthcare Life Sciences, Pittsburgh, Pa.)), glass fiber membranes (e.g., Standard 14 or 17), or PVDF. In a particular embodiment, the test and control lines are on nitrocellulose. In a particular embodiment, the conjugate antibody is on a matrix membrane such as FUSION 5. In a particular embodiment, the sample application area or sample pad is a glass fiber membrane. The different zones may be joined by abutting and/or overlapping. The test strip may further comprise a backing to provide rigidity to the test strip (e.g., a supporting non-interactive material such as polyester).

Generally, the lateral flow immunoassay test strip of the present invention comprises a flow path from an upstream sample application area or sample pad to a test site or capture zone. The test site comprises an area (e.g., a line or stripe) of immobilized anti-TcfA antibodies (e.g., one or more anti-TcfA antibodies (either same or different epitopes)) and, optionally, a control area (e.g., a line or stripe) of immobilized control antibodies (e.g., anti-IgG antibodies, anti-species antibodies (e.g., anti-chicken IgG antibodies (e.g., from donkey or goat)). If the conjugated antibodies are biotin-labeled, then the control area (e.g., a line or stripe) may comprise streptavidin (or analogs thereof) instead of or in conjunction with the control antibodies. Alternatively, if the conjugated antibodies are labeled with streptavidin (or analogs thereof), then the control area (e.g., a line or stripe) may comprise biotin instead of or in conjunction with the control antibodies. When present, the control line is preferably further downstream than the test line. Downstream of the test site is generally an absorbent pad or wicking pad to promote capillary action and movement of the fluid from the sample application area or sample pad. Downstream of the sample application area or sample pad and upstream to the test site is a conjugation pad. The conjugation pad comprises anti-TcfA antibodies (e.g., one or more anti-TcfA antibodies (either same or different epitopes)) conjugated to a detectable agent (e.g., colloidal gold or gold nanoparticles, fluorescent probe, colored latex particles, colored cellulose nanobeads, horseradish peroxidase, and europium (Eu) nanoparticles). In a particular embodiment, the detectable agent generates a direct signal that can be observed by a human (e.g., color from gold colloidal). While other detectable agents require additional steps to produce a signal (e.g., an enzyme to produce detectable product upon reaction with suitable substrate (e.g., horseradish peroxidase); a secondary antibody, etc.), these detectable agents are less preferred.

The assay run length of the lateral flow immunoassay test strip will generally be less than 100 mm in length (e.g., including sample pad, conjugate pad, nitrocellulose, and wicking pad). In a particular embodiment, the assay run length is less than about 80 mm, less than about 70 mm, or less than about 60 mm in length. The test site need only be long enough (e.g., at least about 10 mm) to be able to visualize and differentiate the test line and the control line, when present.

Generally, the anti-TcfA antibody of the conjugation pad binds a different epitope than the anti-TcfA antibody of the test site. In a particular embodiment, the anti-TcfA antibody of the conjugation pad (e.g., the antibody conjugated to a detectable agent such as gold) binds amino acids 140-160 of TcfA. In a particular embodiment, the anti-TcfA antibody of the conjugation pad is 7E11, 10B1, or 3E6 (particularly 10B1) or an anti-TcfA antibody which is a fragment or homolog of 7E11, 10B1, or 3E6 as described hereinabove (e.g., an antibody which binds the same epitope or an antibody comprising all 6 CDRs of 7E11, 10B1, or 3E6). In a particular embodiment, the anti-TcfA antibody of the test site binds amino acids 288-304, amino acids 305-323, and/or amino acids 322-330 of TcfA. In a particular embodiment, the anti-TcfA antibody of the test site is 13E11, 14D12, 23F8, 19F9, 14D9, or 25E3 (particularly 13E11 and/or 14D12) or an anti-TcfA antibody which is a fragment or homolog of 13E11, 14D12, 23F8, 19F9, 14D9, or 25E3 as described hereinabove (e.g., an antibody which binds the same epitope or an antibody comprising all 6 CDRs of 13E11, 14D12, 23F8, 19F9, 14D9, or 25E3). In a particular embodiment, the anti-TcfA antibody of the conjugation pad is 101 or an anti-TcfA antibody which is a fragment or homolog of 10B1 as described hereinabove (e.g., an antibody which binds the same epitope as 101 or an antibody comprising all 6 CDRs of 10B1) and the test site comprises 1) 13E11 or an anti-TcfA antibody which is a fragment or homolog of 13E11 as described hereinabove (e.g., an antibody which binds the same epitope as 13E11 or an antibody comprising all 6 CDRs of 13E11), and 2) 14D12 or an anti-TcfA antibody which is a fragment or homolog of 14D12 as described hereinabove (e.g., an antibody which binds the same epitope as 14D12 or an antibody comprising all 6 CDRs of 14D12).

In general, a fluid sample (e.g., by dipping or spotting) is applied to the sample application area or sample pad. In a particular embodiment, the sample is a biological sample obtained from a subject. Biological samples obtained from the subject to be used in the immunoassay of the instant invention include, without limitation: nasopharyngeal swabs, aspirates, and washes. The cells in the biological sample may be lysed prior to analysis. The obtained biological sample may be diluted, purified, concentrated, filtered, dissolved, suspended or otherwise manipulated prior to assay to optimize the immunoassay results. In a particular embodiment, the biological sample is diluted in a carrier or buffer (e.g., a dilution and/or extraction buffer). In a particular embodiment, biological sample is diluted in phosphate buffered saline (PBS). In a particular embodiment, biological sample is diluted in PBS comprising a surfactant (e.g., 0.05% to 2.0%, particularly, 0.1%, 0.25%, 0.5%, 0.75%, or 1.0%), such as an anionic surfactant. Examples of surfactants include, without limitation, alkyl sulfates, alkyl ether sulfates, alkyl ether phosphates, ammonium lauryl sulfate, sodium dodecyl sulfate (SDS), sodium lauryl ether sulfate, and sodium myreth sulfate.

The biological sample for testing can be from any subject. The immunoassay will be particularly useful for the rapid diagnosis of infants (e.g., in pediatrician offices, urgent care clinics, small hospital emergency departments, or medical clinics) with early pertussis. Infants have higher bacterial loads than children or adults (Eby, et al., Infect. Immun. (2013) 81(5):1390-8; Nakamura et al. (2011) Clin. Microbiol. Infect. (2011) 17(3):365-70; Tenenbaum et al., Eur. J. Clin. Microbiol. Infect. Dis. (2012) 31(11):3173-82). This higher load can facilitate detection ofB. pertussisantigens. Nonetheless, the immunoassay of the instant invention can also be used on older patients (e.g., children and adults).

Methods for detectingB. pertussisare provided using an immunoassay are also encompassed by the instant invention. In a particular embodiment, the method comprises obtaining a sample (e.g., a biological sample (e.g., from a subject)), diluting the sample in a carrier or buffer, and applying the diluted sample to the immunoassay (e.g., LFI). In a particular embodiment, the method further comprises diagnosing a subject as having aB. pertussisinfection based on the presence of a positive result from the immunoassay.

The immunoassays of the instant invention may further comprise assays, particularly other point of care assays. In a particular embodiment, the pertussis diagnostic technology of the instant invention is combined or multiplexed with other respiratory diseases or disorders. In a particular embodiment, the immunoassays of the instant invention further comprise an assay for respiratory syncytial virus (RSV). Many RSV-positive infants are also pertussis-positive (generally 8-16%) and most infants with pertussis are co-infected with RSV (approximately 67-74%) (Nuolivirta, et al., Pediatr. Infect. Dis. J. (2010) 29(11):1013-5; Cosnes-Lambe, et al., Eur. J. Pediatr. (2008) 167(9):1017-9). There is no difference in cough symptoms at hospital admission between infants infected only with RSV vs. infants coinfected with RSV and pertussis (Nuolivirta, et al., Pediatr. Infect. Dis. J. (2010) 29(11):1013-5; Crowcroft, et al., Arch. Dis. Child. (2003) 88(9):802-6). But importantly, detecting co-infection is clinically relevant as infants with RSV andB. pertussisco-infections need antibiotics whereas infants infected solely with RSV do not antibiotics (Bronchiolitis AaoPSoDaMo, Pediatrics (2006) 118(4):1774-93). Thus, the instant invention also encompasses immunoassays, particularly LFIs, comprising the anti-TcfA antibodies of the instant invention in a first test line and RSV detecting agents in a second test line (e.g., antibodies against RSV viral fusion protein (see, e.g., QuickVue® RSV Test (Quidel, San Diego Calif.)) and/or RSV nucleotprotein (see, e.g., ImmunoCard STAT!® RSV (Meidian Biosciences, Cincinnati, Ohio)). These LFIs allow for rapid diagnosis ofB. pertussisand/or RSV infections that could otherwise remain undiagnosed and untreated, particularly in infants.

Generally, the immunoassays of the instant invention will be used as a diagnostic for pertussis. Significantly, the immunoassays of the instant invention can also be used to monitor the therapy of a subject with pertussis. For example, after diagnosis with pertussis and administration of an appropriate therapy (e.g., antibiotics), the immunoassays of the instant invention can be used to monitor the amount ofB. pertussisand the clearance of the bacteria.

While the present invention has been described with regard to pertussis diagnosis and detectingB. pertussis, the gene for TcfA is also found inB. parapertussis, B. holmesii, andB. bronchiseptica. Notably,B. parapertussisandB. holmesiiinfect humans andB. bronchisepticais the causative agent for kennel cough in animals. Thus, in certain embodiments, the anti-TcfA antibodies can also be used to detect the presence ofB. parapertussis, B. holmesii, andB. bronchiseptica. Indeed, as seen in Example 2,B. holmesiiwas detected with one of the five LFIs tested.

In a particular embodiment, the immunoassay of the instant invention is specific for diagnosis and detectingB. pertussis. For example, the immunoassay may be specific for diagnosis and detectingB. pertussiswhile not significantly detectingB. parapertussis, B. holmesii, and/orB. bronchiseptica.

In accordance with another aspect of the instant invention, compositions and methods for the inhibition, treatment, and/or prevention of pertussis and/or aB. pertussisinfection are provided. The methods comprise administering an anti-TcfA antibody or fragment thereof of the instant invention to a subject in need thereof. The anti-TcfA antibodies may be administered in a composition further comprising a pharmaceutically acceptable carrier. The composition may further comprise at least one other therapeutic agent againstB. pertussis(e.g., antibiotics). Alternatively, the other therapeutic agent may be contained within a separate composition(s) with at least one pharmaceutically acceptable carrier. The composition(s) comprising at least one anti-TcfA antibody and/or the composition(s) comprising at least one other therapeutic agent may be contained within a kit.

TcfA has been shown to be required for efficient colonization of the trachea in a mouse model of pertussis (Finn, et al., Mol. Microbiol. (1995) 16(4):625-634). Furthermore, the heterologous expression of TcfA inE. colienables the engineeredE. colito bind human lung epithelial cells, thereby suggesting a role for TcfA for cellular binding and infection (Gasperini, et al., Mol. Cell Proteomics (2018) 17(2):205-215). Notably, passive immunity has been demonstrated with humanized mouse anti-pertussis toxin mAbs when administered to infected baboons. The anti-pertussis toxin mAbs were capable of blunting the post-infection white blood cell increase and increasing the speed by whichB. pertussisbacteria was cleared (Nguyen, et al., Sci. Transl. Med. (2015) 7(316):316ra195). Based on the requirement for TcfA for efficient colonization, blocking TcfA with an antibody of the instant invention will provide therapeutic effects by inhibiting the ability ofB. pertussisto bind and infect cells.

The compositions of the present invention can be administered by any suitable route, for example, by injection (e.g., for local or systemic administration), intravenous, oral, pulmonary, nasal or other modes of administration. In a particular embodiment, the compositions are administered orally or by inhalation. The compositions comprising the antibodies of the invention may be conveniently formulated for administration with an acceptable medium such as water, buffered saline, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), dimethyl sulfoxide (DMSO), oils, detergents, suspending agents or suitable mixtures thereof. Selection of a suitable pharmaceutical preparation depends upon the method of administration chosen. The concentration of the antibodies in the chosen medium may be varied and the medium may be chosen based on the desired route of administration of the pharmaceutical preparation. Except insofar as any conventional media or agent is incompatible with the agents to be administered, its use in the pharmaceutical preparation is contemplated. The pharmaceutical composition of the present invention can be prepared, for example, in liquid form, or can be in dried powder form (e.g., lyophilized).

Pharmaceutical compositions containing agents of the present invention as the active ingredient in intimate admixture with a pharmaceutical carrier can be prepared according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration. In preparing the antibody in oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like in the case of oral liquid preparations (such as, for example, suspensions, elixirs and solutions); or carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations (such as, for example, powders, capsules and tablets).

A pharmaceutical preparation of the invention may be formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to a physically discrete unit of the pharmaceutical preparation appropriate for the patient undergoing treatment. Each dosage should contain a quantity of active ingredient calculated to produce the desired effect in association with the selected pharmaceutical carrier. Procedures for determining the appropriate dosage unit are well known to those skilled in the art. Dosage units may be proportionately increased or decreased based on the weight of the patient. Appropriate concentrations for alleviation of a particular pathological condition may be determined by dosage concentration curve calculations, as known in the art.

The dose and dosage regimen of the antibodies according to the invention that is suitable for administration to a particular patient may be determined by a physician considering the patient's age, sex, weight, general medical condition, and the specific condition and severity thereof for which the agent is being administered. The physician may also consider the route of administration of the antibodies, the pharmaceutical carrier with which the antibodies may be combined, and the antibodies' biological activity. The appropriate dosage unit for the administration of the agents of the invention may be determined by evaluating the toxicity of the agents in animal models. Appropriate dosage unit may also be determined by assessing the efficacy of the agents in combination with other standard drugs.

The compositions comprising the agents of the instant invention may be administered at appropriate intervals, for example, at least once a day or more until the pathological symptoms are reduced or alleviated, after which the dosage may be reduced to a maintenance level. The appropriate interval in a particular case would normally depend on the condition of the patient.

Definitions

The following definitions are provided to facilitate an understanding of the present invention:

“Pharmaceutically acceptable” indicates approval by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

A “carrier” refers to, for example, a diluent, adjuvant, excipient, auxiliary agent or vehicle with which an active agent of the present invention is administered. Pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers. Suitable pharmaceutical carriers are described, for example, in “Remington's Pharmaceutical Sciences” by E. W. Martin.

An “antibody” or “antibody molecule” is any immunoglobulin, including antibodies and fragments thereof, that binds to a specific antigen. As used herein, antibody or antibody molecule contemplates intact immunoglobulin molecules, immunologically active portions of an immunoglobulin molecule (e.g., antigen-binding fragment), and fusions of immunologically active portions of an immunoglobulin molecule.

As used herein, the term “immunologically specific” refers to proteins/polypeptides, particularly antibodies, that bind to one or more epitopes of a protein or compound of interest, but which do not substantially recognize and bind other molecules in a sample containing a mixed population of antigenic biological molecules.

As used herein, “diagnose” refers to detecting and identifying a disease or disorder in a subject. The term may also encompass assessing or evaluating the disease or disorder status (progression, regression, stabilization, response to treatment, etc.) in a patient known to have the disease or disorder.

As used herein, the term “prognosis” refers to providing information regarding the impact of the presence of a disease or disorder (e.g., as determined by the diagnostic methods of the present invention) on a subject's future health (e.g., expected morbidity or mortality). In other words, the term “prognosis” refers to providing a prediction of the probable course and outcome of a disease/disorder and/or the likelihood of recovery from the disease/disorder.

As used herein, the term “subject” refers to an animal, particularly a mammal, particularly a human.

A “therapeutically effective amount” of a compound or a pharmaceutical composition refers to an amount effective to prevent, inhibit, treat, or lessen the symptoms of a particular disorder or disease. The treatment of a disease or disorder herein may refer to curing, relieving, and/or preventing the disease or disorder, the symptom(s) of it, or the predisposition towards it.

As used herein, the term “therapeutic agent” refers to a chemical compound or biological molecule including, without limitation, nucleic acids, peptides, proteins, and antibodies that can be used to treat a condition, disease, or disorder or reduce the symptoms of the condition, disease, or disorder.

The term “isolated” refers to the separation of a compound from other components present during its production or from its natural environment. “Isolated” is not meant to exclude artificial or synthetic mixtures with other compounds or materials, or the presence of impurities that do not substantially interfere with the fundamental activity, and that may be present, for example, due to incomplete purification, or the addition of stabilizers.

As used herein, a “biological sample” refers to a sample of biological material obtained from a subject, particularly a human subject, including a tissue, a tissue sample, cell(s), and a biological fluid (e.g., blood (e.g., whole blood), serum, plasma, urine, sweat, tears, saliva, mucosal secretions, sputum, CSF).

The following examples are provided to illustrate various embodiments of the present invention. The examples are not intended to limit the invention in any way.

A bioinformatics-based strategy was used to identify tracheal colonization factor A (TcfA) as a novel biomarker forB. pertussisinfection. Epitope-specific polyclonal antibodies (pAbs) that recognize the cell-associated and secreted isoforms of TcfA were developed. Specifically, polyclonal antibodies were generated against amino acids 140-160, 288-304, or 305-323 of TcfA.

The specificity of the anti-TcfA antibodies was tested. Specifically, the anti-TcfA pAbs were incorporated into immunoassays, particularly in ELISA and LFI formats. Cultures of bacterial species potentially found in the nasopharynx (Bordetella pertussis, Moraxella catarrhalis, Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Haemophilus influenza, andEscherichia coli) were washed from culture plates with phosphate buffered saline (PBS), normalized to an OD600of 1.0, and lysed in 1% sodium dodecyl sulfate (SDS) in PBS for 1 hour at 65° C. The lysates were then tested by antigen-capture ELISA with anti-TcfA pAbs. As seen inFIG.1, an antigen-capture ELISA with the anti-TcfA pAbs does not detect (i.e., cross-react) with any of the other tested bacteria. In addition to those shown inFIG.1, the anti-TcfA pAb-based antigen-capture ELISA did not cross-react withEnterobacter aerogenes, Enterococcus faecalis, Staphylococcus epidermidis, Streptococcus mutans, Streptococcus mitis, andCorynebacterium pseudodiptherium. Similarly, a strong ability to detectB. pertussislysates while showing no detectable cross-reactivity with the above bacteria was observed with the LFI immunoassay. Notably, when the pAbs are used individually in Western blot with lysates of these bacteria, some of them do react with a very limited number of protein bands in only some of the other bacterial species. However, antigen-capture ELISA and LFI require both the capture and detector antibodies to bind the same target. Therefore, it is unlikely that the same cross-reactive protein would be picked up by both the capture and detector pAb since the cross-reactivity in Western blot was so limited. Consequently, neither the antigen-capture ELISA nor the LFI resulted in the detection of any of the other listed bacterial species.

Despite demonstrating specificity forB. pertussis, the anti-TcfA pAb-based immunoassay was still able to recognize multiple strains ofB. pertussis. Specifically, material from twoB. pertussisstrains—Tohama and 165—were tested. Both the Tohama and 165 strains were detected by all anti-TcfA pAb combinations tested by LFI. This result is likely due to the conserved epitopes of the anti-TcfA antibodies. Indeed, the peptide sequences of all 3 of the targeted TcfA epitopes are conserved in >99% (n=436) of clinical isolates from the U.S. and 8 other countries (van Amersfoorth, et al., J. Clin. Microbiol. (2005) 43(6):2837-43; Packard, et al., J. Med. Microbiol. (2004) 53(Pt 5):355-65; van Loo, et al., J. Clin. Microbiol. (2002) 40(6):1994-2001).

TcfA has a cell-associated and a secreted isoform (Finn, et al., Mol. Microbiol. (1995) 16(4):625-34), and the anti-TcfA pAbs described herein are reactive with both isoforms. As seen inFIG.2, both isoforms were detectable in a Western blot assay as two bands of the appropriate molecular weight are clearly present in cellular lysates. Notably, only the secreted form is observed in 0.2 m-filteredB. pertussisconditioned liquid medium (Stainer-Scholte medium). As with the Western blot analysis, both isoforms were detectable using a LFI assay. The secreted isoform was detected on 0.2 m-filteredB. pertussisconditioned liquid medium (Stainer-Scholte medium) and the cell-associated isoform was detected on intact, formaldehyde-inactivated cells. Thus, sample preparation for analysis by LFI does not require cell lysis. However, it was determined that detergent lysis ofB. pertussiscells increased the sensitivity of the anti-TcfA pAb-based LFI. Lysis in 0.1% SDS in PBS, pH 7.4 at room temperature for as little as 5 minutes provided an enhanced signal compared to unlysed samples, but more signal was seen after 60 minutes of lysis or when the lysis was performed at 65° C. rather than room temperature.

The limit of detection of with one of the anti-TcfA pAbs was tested. Specifically, an LFI was performed withB. pertussislysates prepared by detergent lysis for 60 minutes at 65° C. with 0.1% SDS in PBS. The lowest serial dilution scored as positive by 3 of 3 blinded reviewers contained material from 1.7×105colony forming units (CFU) (FIG.3A). A quantitative limit of detection analysis was also performed. The ESE-Quant lateral flow reader (Qiagen, Germantown, Md.) and LF Studio software were used to quantify the test line intensity of multiple dilutions tested in replicates over three days. Background-adjusted data were fit to a linear curve on a semi-log plot with an R2value of 0.85 (FIG.3B). The calculated limit of detection was 1.6×105CFU, which fits well with the limit of detection determined by reviewers visually. The limit of detection by antigen-capture ELISA was determined to be 3.3×104CFU. Notably, both the LFI and ELISA limits of detection are well below the bacterial burden found in infant nasopharynx washes (107to 1010CFU/ml) or swabs (106CFU) (Eby, et al., Infect. Immun. (2013) 81(5):1390-8; Nakamura, et a., Clin. Microbiol. Infect. (2011) 17(3):365-70; Tenenbaum, et al., Eur. J. Clin. Microbiol. Infect. Dis. (2012) 31(11):3173-82).

Although pAbs can be used in commercial LFIs, monoclonal antibodies (mAbs) are preferred for commercialization because of their superior affinity (i.e., better LFI sensitivity), lot-to-lot reproducibility, and their more efficient, reliable, and economic large-scale production. Thus, a library of anti-TcfA mAbs was developed and the mAbs were incorporated into an advanced mAb-based LFI diagnostic to achieve enhanced analytical sensitivity and better commercialization potential.

Generating a library of mAbs against TcfA required an unusually large number of immunization and hybridoma screening strategies. Ultimately, a large library of mAb-secreting hybridomas was generated and the mAbs showed robust reactivity in ELISA with immobilized antigen (recombinant TcfA, endogenous TcfA including formaldehyde-inactivatedB. pertussiscells and/or TcfA peptides). Thirty-six mAbs were advanced for large-scale hybridoma culture and mAb purification via Protein A affinity chromatography.

The reactivity of the mAbs was measured by indirect ELISA with plates coated with various antigen (FIG.4). The immobilized antigens were formaldehyde-inactivatedB. pertussiscells, recombinant TcfA containing a histidine tag, TcfA peptides (amino acids 140-160, 288-304, or 305-323) conjugated to bovine serum albumin (BSA), or control BSA. The reactivity of the mAbs was also tested against clarified, 0.2 μm-filtered supernatant fromB. pertussisTohama I cultures grown in Stainer-Scholte medium or Stainer-Scholte uninocculated medium. As seen inFIG.4, most of the mAb demonstrated a strong but selective reactivity for TcfA.

In order to determine the epitopes recognized by the mAb, purified mAbs were evaluated by indirect ELISA with streptavidin plates loaded with biotinylated peptides. The peptides were 15mers that were offset by 3 amino acids. The minimal overlapping peptide sequence for wells with an OD450greater than 0.75 (and in a series of two or more such adjacent wells) was defined as the minimal linear peptide epitope (Table 1). Notably, mAbs 22B7 and 14D9 reacted with a long series of peptides such that the first peptide in the series did not overlap with the last peptide. For these mAbs, the peptide sequence defined by the entire series of reactive wells is provided. In addition, purified mAbs were evaluated by indirect ELISA with plates coated with the TcfA protein fragments. With the exception of amino acids 40-374, all protein fragments were conjugated to bovine serum albumin.

Anti-TcfA mAbs were evaluated for performance as a detector mAb (i.e., gold conjugate) and as a capture mAb (i.e., test line) in an LFI while holding all other LFI components constant (e.g., nitrocellulose, conjugate pad, sample pad, wicking pad, blocking buffers, chase buffer). A significant number of LFI mAb pair permutations were identified that effectively detected formaldehyde-inactivatedB. pertussiscells (FIG.5). Indeed, most of the ELISA-capable anti-TcfA mAbs evaluated by LFI formed at least 1 functional LFI pair (FIGS.4and5). Notably, many anti-TcfA mAbs had comparable performance in ELISA against immobilized antigen (FIG.4). However, in the LFI, some of the anti-TcfA mAbs worked far better than the others as test line mAbs, other anti-TcfA mAbs worked better as gold conjugate mAbs, and still other anti-TcfA mAbs worked well in both positions (e.g., anti-TcfA mAbs 14D9, 14D12, and 25E3, respectively) (FIG.5). In a further analysis, the anti-TcfA mAbs were binned by their reactivity (or lack thereof) with the three TcfA peptides that were used to generate the polyclonal antibodies. It was found that anti-TcfA mAbs that target the same epitope failed to form functional mAb pairs for LFI (FIG.5). However, when the anti-TcfA mAbs targeted different epitopes, functional mAb pairs for LFI were identified.

Several anti-TcfA mAb combinations for LFIs (FIG.5) were advanced to BSL-2 testing to determine their cross-reactivity with a panel of ten other nasopharyngeal pathogens and to establish a preliminary limit of detection with viableB. pertussiscells. These LFIs were selected based on their strong signal with i) formaldehyde-inactivatedB. pertussiscells (Tohama I strain), ii) detergent lysates ofB. pertussiscells (Tohama I strain), and iii) conditioned media (not formaldehyde-treated) from a second strain ofB. pertussis(strain 165) grown as liquid cultures in Stainer-Scholte medium. Three of the LFIs (14D12 test line+7E11 gold conjugate; 25E3 test line+10B1 gold conjugate; 14D9 test line+7E11 gold conjugate) were found to have no detectable cross-reactivity with viable cell suspensions in PBS ofHaemophilus parainfluenzae, Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pneumoniae, Streptococcus pyogenes, Corynebacterium diphtheriae, Staphylococcus epidermidis, Bordetella parapertussis, Bordetella holmesii, orBordetella bronchiseptica. One LFI had reactivity withH. influenzaandB. holmesii. A different LFI had reactivity withS. epidermidis. One LFI (13E11 and 14D12 test line+10B1 gold conjugate) was subjected to additional cross-reactivity testing, in which cross-reactivity was evaluated with 40 other species of bacteria and fungi potentially found in nasopharyngeal specimens (Table 2). This expanded cross-reactivity testing of Table 2 was performed by testing LFIs in triplicate with samples containing 3.33×107CFU/mL of the indicated pathogen in extraction buffer.

The preliminary limit of detection for five of the LFIs with viableB. pertussiscells was 2×105to 5×105CFU. Two of the three LFIs with no detectable cross-reactivity had preliminary limits of detection of 2×105CFU. These two LFIs are: 1) mAb 14D12 as the test line and mAb 7E11 as the gold conjugate; and 2) mAb 25E3 as the test line and mAb 10B1 as the gold conjugate. The sixth LFI (14D12 and 13E11 test line+10B1 gold conjugate) was subjected to detailed limit of detection testing and was determined to have an analytical sensitivity limit of 3×105CFU/mL (1.8×104CFU per LFI test) (FIG.7). For comparison, the best pAb based LFI (see Example 1) had a final limit of detection of 1.6×105CFU. However, achieving that limit of detection with the pAb based LFI required i) heating theB. pertussiscells in 0.1% SDS at 65° C. for 1 hour, and ii) incorporating an 80 mm assay run length, which necessitated a longer assay run time (optimal run time was 30 minutes, though strong positives were visible sooner). The instant mAb based LFIs have better limit of detection despite their use of viableB. pertussiscells with only a simple 5 minute room temperature incubation in extraction buffer and short development times of 15-20 minutes due to a 60 mm assay run length, of which 25 mm is nitrocellulose.B. pertussisis strictly a human pathogen that lacks an animal or environmental reservoir (Mattoo, et al., Clin. Microbiol. Rev., (2005) 18(2):326-82). However, a baboon model has been developed that is the only animal model that accurately reproduces the full disease course seen in human infection (Warfel, et al., Expert Rev. Vaccines (2014) 13(10):1241-52; Merkel, et al., J. Infect. Dis. (2014) 209 Suppl 1:520-3; Warfel, et al., Infect. Immun. (2012) 80(4):1530-6; Warfel, et al., Proc. Natl. Acad. Sci. (2014) 111(2):787-92). This well-established, clinically relevant model of naturally transmitted infection in baboons can be used to provide nasopharynx samples to study the ability of the LFIs to diagnose various disease stages.

A library ofB. pertussismAbs has been generated. The mAbs bind viable and formaldehyde-inactivatedB. pertussiscells. In addition to use as diagnostic reagents, these mAbs are valuable as therapeutics, prophylactics, and/or research tools (e.g., for fluorescence microscopy on fresh or fixed tissue sections, etc.).

A mAb-based LFI was optimized for sensitive, specific detection ofB. pertussis. The LFI comprised 14D12 and 13E11 in the test line and 10B1 as the conjugated antibody. The LFI detectedB. pertussisantigen in ≤20 minutes with a simple protocol, enabling diagnostic use at the point-of-care.

Nasopharyngeal washes from baboons infected by natural, airborne transmission have greater than or equal to 5×105CFU/mL from approximately Day 15 to Day 33 post-exposure (Warfel, et al., Proc. Natl. Acad. Sci. (2014) 111(2):787-92). The LFI detected antigen from infected baboon nasopharyngeal washes containing as little as 5.5×104CFU/mL (3.3×103CFU per LFI test) (FIG.10). The LFI consistently reported positive results with infected baboon nasopharyngeal washes greater than or equal to 5×105CFU/mL (3×104CFU per LFI test) (FIG.10). No LFI false-positives were observed.

Specifically, as seen inFIGS.9and10, the LFI reacted with nasopharyngeal washes from baboons infected withB. pertussis(strain D420). Individual nasopharyngeal washes from baboons challenged withB. pertussisand containing the listed CFU per LFI testing volume were incubated with extraction buffer for 5 minutes at room temperature. LFI development time was 15 minutes.

As seen inFIG.10, all infected baboon nasopharyngeal washes with greater than or equal to 5×105CFU/mL were positive by LFI (top horizontal reference line), and all infected baboon nasopharyngeal washes with less than or equal to 3.5×104CFU/mL were negative by LFI (bottom horizontal reference line). A total of 30 nasopharyngeal washes from baboons challenged withB. pertussiswere analyzed by LFI. The LFI produced no false-positives with 11 baboon NP washes that had 0 CFU/mL (i.e. the LFI had 100% specificity).

In addition, the LFI has been tested with human patient samples (nasopharyngeal swabs). The LFI was negative with 2 of 2 patient samples that were diagnosed by RT-PCR as negative for pertussis. The LFI was positive with 1 patient sample that was diagnosed by RT-PCR as being positive for pertussis and with a high bacterial burden. The LFI was negative with 1 patient sample that was diagnosed by RT-PCR as positive for pertussis but with a significantly lower bacterial burden. The 1 positive LFI with the 1 patient who was positive by RT-PCR shows proof of concept for detection ofB. pertussisin human nasopharyngeal swab specimens.

Together, the baboon and human results indicate that the LFI can detect infection from two different types of nasopharyngeal specimens: nasopharyngeal washes and nasopharyngeal swabs.

The mAb-based LFI for detection ofB. pertussisantigen allows for early, point-of-care diagnosis of pertussis using minimally-invasive nasopharyngeal specimens. The availability of a rapid and simple test for detection of pertussis will increase early diagnosis, as well as facilitate immediate triage, treatment, and outbreak containment.

Several publications and patent documents are cited in the foregoing specification in order to more fully describe the state of the art to which this invention pertains. The disclosure of each of these citations is incorporated by reference herein.