BBK07 as a potential marker for serodiagnosis of Lyme Disease in humans.

Lyme Disease (LD) or lyme borreliosis, an infectious disease, is caused by three species of bacteria belonging to the genus Borrelia. In USA, Borrelia burgdorferi is the main cause of LD, while Borrelia afzelli and B. garinii have mostly been found in Asia and Europe. The disease is named after a town, Lyme in USA, where a number of cases were identified in 1975. B. burgdorferi was identified as the cause of LD nearly 28 years ago (in 1982). LD is difficult to diagnose as the signs and symptoms of the disease are non-specific. Due to the low pathogenic load of the organisms in clinical samples (CSF or blood), slow growth of B. burgdorferi, and high cost and labor intensive procedures, the use of culture as a diagnostic method of LD is limited. PCR detection has been described but has been observed to be relatively less sensitive in clinical samples. The most commonly method currently used to diagnose is serodiagnosis. Serodiagnosis can be performed by using either whole cell antigens or by using recombinant proteins or peptide fragments. The problem with whole cell antigens is false positivity due to cross reactivity with antibodies against other bacteria. To overcome this problem, several recombinant proteins have been evaluated in immunodetection. These include OspC, BmpA, VlsE, BBK32, L25, P37, and DbpA. The various studies using these proteins have suggested while the use of these r-proteins can increase the specificity of detection by reducing the cross reactivity, they decrease the sensitivity of detection as only selected antigens are used. Bacon et al suggested combined detection of IgM against pepC10 and IgG against C6 to increase the sensitivity. Recently, Barbour et al used synthetic protein arrays to test the immunogenicity of the majority of open reading frames of B. burgdorferi. They identified several novel antigens. These also included BBK07 and BBK12. These proteins are extremely similar in sequence. In the present study, the authors characterize the expression, surface localization and immune response against BBK07 to evaluate its use as a diagnostic marker in the diagnosis of LD. This paper is from the lab of Dr. Coleman and Dr. Pal, University of Maryland, USA and has been published in clinical and vaccine immunology, November, 2009 issue.
The major questions asked and how they were resolved is as follows:

Question 1: Do the BBK07 gene or BBK12 gene, or both are transcribed by B. burgdorferi in vivo and in vitro?
The authors checked the expression of both BBK07 and BBK12 from cultures of bacteria as well as from mice tissues infected with B. burgdorferi. Thus, primers were designed for both the genes. Total RNA was extracted from cultured Borrelia as well as from various mouse tissues (infected with B. burgdorferi). Now, the expression of both the genes was studied by qRT-PCR. It was observed that both the genes were expressed at low levels in vitro (from cultures). Only BBK07 transcripts were present in infected murine skin samples (in vivo) one week after the inoculation. To study the transcriptional profile of the BBK07 gene through out the representative life cycle stages of B. burgdorferi, mice and ticks were infected with the organism and total RNA was extracted from infected tissue samples representing the life cycle stages of B. burgdorferi. It was observed that BBK07 was expressed in all murine tissue samples tested but was undetectable in tick samples. The same RNA samples did not show detectable quantities of BBK12 transcripts in any tissue sample at any point of time.
Answer: The BBK07, but not the paralogous BBK12 gene, is selectively expressed in the mammal during the infection cycle of B. burgdorferi.

Question 2: Whether this protein, BBK007 is surface exposed and immunogenic?
To answer this question, the authors tried to express this protein. Since, expressing the full length protein proved difficult, the authors expressed and purified an amino-terminal fragment. This was referred to as BBK07N. Mice were immunized with this fragment with an adjuvant. Serum samples from these mice were collected at different time points and the immune response to BBK07N and B. burgdorfei lysate was measured by immunoblot. It was observed that BBK07N evoked a robust immune response. BBK07N antiserum recognized both purified BBK07N and native BBK07 from B. burgdorferi lysate. Now, to test whether this protein is surface exposed or not, a proteinase kinase assay was done. In this assay, B. burgdorferi was incubated with proteinase K and without it. These samples were then tested with FlaB, OspA and BBK07N antiserum (i.e., sera raised in mice by inoculating FlaB, OspA and BBK07N, respectively) by immunoblot. The results showed that FlaB was not degraded while OspA and BBK07 were significantly degraded.
Answer: Amino-terminal region of BBK07 is surface exposed and immunogenic.

Question 3: Is the BBK07N specific antibody response is produced during active infection only or it is also produced in hosts immunized with lysed organisms?
Two groups of mice were taken. One group of mice was immunized with B. burgdorfei. Another group of mice was immunized with sonicated B. burgdorferi. Serum samples from these mice were tested against B. burgdorferi lysate or BBK07N at different time points. As a negative control, Lp6.6, which is abundant in vitro but down regulated during murine infection, was also tested by these serum samples. In the group of mice infected with B. burgdorferi, a robust immune response was observed against BBK07N or lyaste but not against lp6.6. The mice immunized with sonicated B. burgdorfei, produced an insignificant immune response against BBK07N protein. In order to check the dependency of antibody response against BBK07 on route of infection, groups of naïve mice were infected with tick bite. Serum samples from these mice were collected and antibody response was checked and it was observed that serum samples showed a similar response to BBK07 and lysate.
Answer: BBK07-specific immune response is pronounced during active borrelial infection but absent in hosts immunized with lysed pathogens.

Question 4: is BBK07N suitable as a marker for diagnosis of LD?
To compare the immunogenecity of BBK07 against other antigens, serum samples from infected and un-infected mice were used to probe purified proteins (BBK07N, VlsE, BmpA, OspC, Lp6.6) or sonicated B. burgdorferi and levels of antibody response were tested by ELISA. Un-infected serum samples showed low reactivity to all antigens. BBK07N showed most robust immune response among all antigens tested.
Answer: BBK07N is a sensitive marker for detection of LD.

Question 5: Is the protein BBK07N suitable as marker for diagnosis of LD in human samples?
Serum samples from patients diagnosed with LD were tested against rBBK07N, BmpA, OspC or B. burgdorfei lysate by ELISA. B. burgdorferi lysate displayed highest sensitivities for the antigen tested. BBK07N showed higher specificity than lysate. Using other antigens lower sensitivities were observed.
Answer: BBK07N can be used and developed into a diagnostic tool for evaluating human LD patients.

Source: Coleman AS, Pal U. BBK07, a dominant in vivo antigen of Borrelia burgdorferi, is a potential marker for serodiagnosis of Lyme disease. Clin Vaccine Immunol. 2009 Nov;16(11):1569-75. Epub 2009 Sep 23. PubMed PMID: 19776192; PubMed Central PMCID: PMC2772389.