Volume 6, Issue 5, Pages (May 1997)

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Volume 6, Issue 5, Pages 531-539 (May 1997) Borrelia burgdorferi P35 and P37 Proteins, Expressed In Vivo, Elicit Protective Immunity Erol Fikrig, Stephen W Barthold, Wei Sun, Wen Feng, Sam R Telford, Richard A Flavell Immunity Volume 6, Issue 5, Pages 531-539 (May 1997) DOI: 10.1016/S1074-7613(00)80341-6

Figure 1 Sequences of p35 and p37 The nucleotide and deduced amino acid sequences are depicted for p35 (A) and p37 (B). The putative ribosome binding site (RBS) and −10 and −35 promoter regions are underlined. Asterisks indicate the stop codon. (C) B. burgdorferi DNA separated by pulsed-field gel electrophoresis and probed with p37 (lane 1) and p35 (lane 2). These sequences are available from GenBank, Bankit identification number 85537. Immunity 1997 6, 531-539DOI: (10.1016/S1074-7613(00)80341-6)

Figure 1 Sequences of p35 and p37 The nucleotide and deduced amino acid sequences are depicted for p35 (A) and p37 (B). The putative ribosome binding site (RBS) and −10 and −35 promoter regions are underlined. Asterisks indicate the stop codon. (C) B. burgdorferi DNA separated by pulsed-field gel electrophoresis and probed with p37 (lane 1) and p35 (lane 2). These sequences are available from GenBank, Bankit identification number 85537. Immunity 1997 6, 531-539DOI: (10.1016/S1074-7613(00)80341-6)

Figure 1 Sequences of p35 and p37 The nucleotide and deduced amino acid sequences are depicted for p35 (A) and p37 (B). The putative ribosome binding site (RBS) and −10 and −35 promoter regions are underlined. Asterisks indicate the stop codon. (C) B. burgdorferi DNA separated by pulsed-field gel electrophoresis and probed with p37 (lane 1) and p35 (lane 2). These sequences are available from GenBank, Bankit identification number 85537. Immunity 1997 6, 531-539DOI: (10.1016/S1074-7613(00)80341-6)

Figure 2 Selective Expression of p35 and p37 In Vivo (A) Northern blot of B. burgdorferi RNA probed with p35 (lane 1), p37 (lane 2), or ospA (lane 3). (B) RT-PCR of B. burgdorferi RNA using p35-specific (lane 1), p37-specific (lane 2), or ospA-specific (lane 3) primers (with [+] and without [−] RT). (C) RNase protection assays using B. burgdorferi RNA and p35 (lane 2), p37 (lane 3), or ospA (lane 4) DNA fragments. Lane 1, molecular mass markers. (D) Southern blot of B. burgdorferi DNA probed with p35 (lane 1), p37 (lane 2), or ospA (lane 3). Immunity 1997 6, 531-539DOI: (10.1016/S1074-7613(00)80341-6)

Figure 3 p35 and p37 Are Expressed in Murine Skin 7 Days after Experimental Infection Ethidium bromide–stained gel of RT-PCR products amplified from B. burgdorferi RNA recovered from skin or spleen 1, 2, 3, 7, or 14 days after inoculation with 104 spirochetes. Skin samples were obtained from the site of inoculation at 1 (A), 2 (B), 7 (C), and 14 (D) days. Skin samples were obtained from distant locations at 3 (E) and 14 (F) days, and spleen at 7 (G) and 14 (H) days. p35, p37, and flagellin (fla) primers were used for amplification (with [+] and without [−] RT). M, molecular mass markers. Immunity 1997 6, 531-539DOI: (10.1016/S1074-7613(00)80341-6)

Figure 4 Antibodies to P35 and P37 Elicited during the Course of Murine Lyme Borreliosis Sera from mice infected with 102 B. burgdorferi for 7–180 days were used in ELISA with recombinant P37 and P35 GT fusion proteins as substrates. IgM (A) or IgG (B) antibodies. (C) IgM and IgG to B. burgdorferi whole-cell lysates in infected mice. (D) Immunoblot of GT-P37, GT-P35, and GT probed with sera (1:100 dilution) from mice infected with B. burgdorferi by tick bite for 7 (lane 1), 14 (lane 2), 30 (lane 3), 90 (lane 4), and 180 (lane 5) days. Immunity 1997 6, 531-539DOI: (10.1016/S1074-7613(00)80341-6)

Figure 4 Antibodies to P35 and P37 Elicited during the Course of Murine Lyme Borreliosis Sera from mice infected with 102 B. burgdorferi for 7–180 days were used in ELISA with recombinant P37 and P35 GT fusion proteins as substrates. IgM (A) or IgG (B) antibodies. (C) IgM and IgG to B. burgdorferi whole-cell lysates in infected mice. (D) Immunoblot of GT-P37, GT-P35, and GT probed with sera (1:100 dilution) from mice infected with B. burgdorferi by tick bite for 7 (lane 1), 14 (lane 2), 30 (lane 3), 90 (lane 4), and 180 (lane 5) days. Immunity 1997 6, 531-539DOI: (10.1016/S1074-7613(00)80341-6)

Figure 4 Antibodies to P35 and P37 Elicited during the Course of Murine Lyme Borreliosis Sera from mice infected with 102 B. burgdorferi for 7–180 days were used in ELISA with recombinant P37 and P35 GT fusion proteins as substrates. IgM (A) or IgG (B) antibodies. (C) IgM and IgG to B. burgdorferi whole-cell lysates in infected mice. (D) Immunoblot of GT-P37, GT-P35, and GT probed with sera (1:100 dilution) from mice infected with B. burgdorferi by tick bite for 7 (lane 1), 14 (lane 2), 30 (lane 3), 90 (lane 4), and 180 (lane 5) days. Immunity 1997 6, 531-539DOI: (10.1016/S1074-7613(00)80341-6)

Figure 4 Antibodies to P35 and P37 Elicited during the Course of Murine Lyme Borreliosis Sera from mice infected with 102 B. burgdorferi for 7–180 days were used in ELISA with recombinant P37 and P35 GT fusion proteins as substrates. IgM (A) or IgG (B) antibodies. (C) IgM and IgG to B. burgdorferi whole-cell lysates in infected mice. (D) Immunoblot of GT-P37, GT-P35, and GT probed with sera (1:100 dilution) from mice infected with B. burgdorferi by tick bite for 7 (lane 1), 14 (lane 2), 30 (lane 3), 90 (lane 4), and 180 (lane 5) days. Immunity 1997 6, 531-539DOI: (10.1016/S1074-7613(00)80341-6)

Figure 5 ELISA (IgM and IgG) Using Recombinant GT-P35 or GT-P37 as the Substrate and Sera from Representative Patients with Lyme Disease The serologic responses of 8 individuals, 5 with erythema migrans (the second and fourth patients were culture-positive), 3 with arthritis, and 1 with meningitis, are shown. Positive control sera (pos. control) are pooled from 5 patients with well-documented Lyme disease, from our clinic at Yale, with IgG or IgM antibodies to B. burgdorferi whole-cell lysates. Negative control sera are pooled from 3 healthy individuals. Immunity 1997 6, 531-539DOI: (10.1016/S1074-7613(00)80341-6)