Christopher G. Earnhart

Demonstration of OspC Type Diversity in Invasive Human Lyme Disease Isolates and Identification of Previously Uncharacterized Epitopes That Define the Specificity of the OspC Murine Antibody Response

ABSTRACT Outer surface protein C (OspC) of the Lyme disease spirochetes is an important virulence factor that has potential utility for vaccine development. Of the 21 OspC types that have been identified, it has been postulated that types A, B, I, and K are specifically associated with invasive infections. Through an analysis of isolates collected from patients in Maryland we found that OspC types C, D, and N are also associated with invasive infections. This observation suggests that there is greater diversity in the group of OspC types associated with invasive infection than has been previously suggested. Detailed knowledge of the antigenic structure of OspC is essential for vaccine development. To determine if the antibody response to OspC is type specific, recombinant proteins of several different OspC types were immunoblotted and screened with sera from mice infected with isolates having known OspC types. These analyses revealed a high degree of specificity in the antibody response and suggested that the immunodominant epitopes of OspC reside in the variable domains of the protein. To localize these epitopes, OspC fragments were generated and screened with serum collected from infected mice. These analyses led to identification of previously uncharacterized epitopes that define the type specificity of the OspC antibody response. These analyses provide important insight into the antigenic structure of OspC and also provide a basis for understanding the variable nature of the antibody response to this important virulence factor of the Lyme disease spirochetes.

An Octavalent Lyme Disease Vaccine Induces Antibodies That Recognize All Incorporated OspC Type-Specific Sequences

Lyme disease is the most common vector-borne disease in North America and Europe and, if untreated, has significant arthritic, cardiac, dermatological and neurological sequelae. There is no currently available human Lyme disease vaccine. Outer surface protein C, because of its antigenicity, protective ability, and expression characteristics has emerged as a promising second generation vaccine candidate; however, significant sequence heterogeneity has impeded its development. Analyses of OspC sequences have revealed the existence of stable phylogenetic clusters or types, and the type-defining sequence variation within OspC occurs in defined domains of the protein. Recent data indicating that immunodominant, and potentially protective OspC epitopes are located in these hypervariable regions has allowed development of a tetravalent, epitope-based, chimeric vaccine. In this report, we have extended that previously described tetravalent construct to include four additional OspC types. We demonstrate that the construct is highly immunogenic, and elicits type-specific antibodies that recognize each of the eight incorporated OspC type-specific epitopes. Antibody raised to the octavalent construct readily binds to the surface of strains expressing each component OspC type, indicating that the incorporated epitopes are presented on the surface of intact cells. In addition, the construct elicits antibody isotypes associated with complement-dependent bactericidal activity. These results represent an important step forward in the design of a broadly protective polyvalent OspC-based Lyme disease vaccine.

OspC phylogenetic analyses support the feasibility of a broadly protective polyvalent chimeric Lyme disease vaccine.

ABSTRACT Using available Borrelia outer surface protein C (OspC) sequences, a phylogenetic analysis was undertaken to delineate the number of antigenic domains required for inclusion in a broadly protective, chimeric, OspC-based Lyme disease vaccine. The data indicate that approximately 34 would be required and that an OspC-based vaccinogen is feasible.

Identification of residues within ligand-binding domain 1 (LBD1) of the Borrelia burgdorferi OspC protein required for function in the mammalian environment.

Borrelia burgdorferi outer surface protein C (ospC) is required for the establishment of infection in mammals. However, its precise function remains controversial. The biologically active form of OspC appears to be a homodimer. Alpha helix 1 and 1′ of the apposing monomers form a solvent‐accessible pocket at the dimeric interface that presents a putative ligand‐binding domain (LBD1). Here we employ site‐directed and allelic‐exchange mutagenesis to test the hypothesis that LBD1 is a determinant of OspC function in the mammalian environment. Substitution of residues K60, E61 and E63 which line LBD1 resulted in the loss of infectivity or influenced dissemination. Analyses of the corresponding recombinant proteins demonstrated that the loss of function was not due to structural perturbation, impaired dimer formation or the loss of plasminogen binding. This study is the first to assess the involvement of individual residues and domains of OspC in its in vivo function. The data support the hypothesis that OspC interacts with a mammalian derived ligand that is critical for survival during early infection. These results shed new light on the structure–functions relationships of OspC and challenge existing hypotheses regarding OspC function in mammals.

Analysis of antibody response in humans to the type a OspC loop 5 domain and assessment of the potential utility of the loop 5 epitope in lyme disease vaccine development

ABSTRACT The OspC protein of Borrelia burgdorferi is an immunodominant antigen. Here we demonstrate that the loop 5 domain of type A OspC is surface exposed, elicits bactericidal antibody in mice, and is antigenic in humans. The data suggest that loop 5 may be suitable for inclusion in a polyvalent, chimeric OspC vaccinogen.

Lyme disease vaccine

Subunit vaccines consisting of single recombinant outer surface proteins (Osp) ofBorrelia burgdorferi have been highly successful in protecting mice against challenge by borrelial strains closely related to the strain from which the immunogen was derived. Humoral immunity is sufficient for protection. A dual mode of action for these vaccines has been suggested because serum factors ingested by the tick during the blood meal may begin to reduce the spirochete inoculum prior to transmission to the host. At present two different recombinant OspA vaccine preparations (monovalent) are being evaluated in humans in large-scale phase III efficacy trials in the United States. Local discomfort at the intramuscular injection site has been the principal adverse effect seen to date with these vaccines, but further data on safety are being collected. The greater heterogeneity of OspA among LymeBorrelia in Europe implies that a vaccine preparation containing multiple antigens (multivalent) may be necessary there, although this is also a concern in the United States. Subunit-Vakzinen, die aus einzelnen rekombinanten Oberflächenproteinen (Osp) vonBorrelia burgdorferi bestehen, führten bei Mäusen zu einem sehr wirksamen Schutz gegen die Infektion mit Borrelienstämmen, die nahe Verwandtschaft mit dem für den Impfstoff verwendeten Stamm haben. Humorale Immunität erwies sich als ausreichend protektiv. Man hat angenommen, daß diese Impfstoffe eine doppelte Wirkung haben, da Serumfaktoren, die die Zecken während des Blutsaugens aufnehmen, das Spirochäteninokulum vor Übertragung auf den Wirt zu reduzieren vermögen. Derzeit werden zwei verschiedene rekombinante (monovalente) OspA-Impfstoffe in großen Phase-III-Studien in den Vereinigten Staaten erprobt. Lokale Beschwerden an der intramuskulären Injektionsstelle sind derzeit die wichtigsten Nebenwirkungen, die mit diesen Impfstoffen beobachtet werden, doch werden weitere Daten zur Frage der Sicherheit erhoben. Lyme-Borrelien haben in Europa eine ausgeprägtere Heterogenität, deshalb müßte eine Vakzine dort mehrere Antigene enthalten (multivalente Vakzine). Dieses Problem trifft allerdings auch für die USA zu.SummarySubunit vaccines consisting of single recombinant outer surface proteins (Osp) ofBorrelia burgdorferi have been highly successful in protecting mice against challenge by borrelial strains closely related to the strain from which the immunogen was derived. Humoral immunity is sufficient for protection. A dual mode of action for these vaccines has been suggested because serum factors ingested by the tick during the blood meal may begin to reduce the spirochete inoculum prior to transmission to the host. At present two different recombinant OspA vaccine preparations (monovalent) are being evaluated in humans in large-scale phase III efficacy trials in the United States. Local discomfort at the intramuscular injection site has been the principal adverse effect seen to date with these vaccines, but further data on safety are being collected. The greater heterogeneity of OspA among LymeBorrelia in Europe implies that a vaccine preparation containing multiple antigens (multivalent) may be necessary there, although this is also a concern in the United States.ZusammenfassungSubunit-Vakzinen, die aus einzelnen rekombinanten Oberflächenproteinen (Osp) vonBorrelia burgdorferi bestehen, führten bei Mäusen zu einem sehr wirksamen Schutz gegen die Infektion mit Borrelienstämmen, die nahe Verwandtschaft mit dem für den Impfstoff verwendeten Stamm haben. Humorale Immunität erwies sich als ausreichend protektiv. Man hat angenommen, daß diese Impfstoffe eine doppelte Wirkung haben, da Serumfaktoren, die die Zecken während des Blutsaugens aufnehmen, das Spirochäteninokulum vor Übertragung auf den Wirt zu reduzieren vermögen. Derzeit werden zwei verschiedene rekombinante (monovalente) OspA-Impfstoffe in großen Phase-III-Studien in den Vereinigten Staaten erprobt. Lokale Beschwerden an der intramuskulären Injektionsstelle sind derzeit die wichtigsten Nebenwirkungen, die mit diesen Impfstoffen beobachtet werden, doch werden weitere Daten zur Frage der Sicherheit erhoben. Lyme-Borrelien haben in Europa eine ausgeprägtere Heterogenität, deshalb müßte eine Vakzine dort mehrere Antigene enthalten (multivalente Vakzine). Dieses Problem trifft allerdings auch für die USA zu.

Identification of Borrelia burgdorferi ospC genotypes in canine tissue following tick infestation: Implications for Lyme disease vaccine and diagnostic assay design

In endemic regions, Lyme disease is a potential health threat to dogs. Canine Lyme disease manifests with arthritis-induced lameness, anorexia, fever, lethargy, lymphadenopathy and, in some cases, fatal glomerulonephritis. A recent study revealed that the regional mean for the percentage of seropositive dogs in the north-east of the USA is 11.6%. The outer surface protein C (OspC) of Lyme disease spirochetes is an important virulence factor required for the establishment of infection in mammals. It is a leading candidate in human and canine Lyme disease vaccine development efforts. Over 30 distinct ospC phyletic types have been defined. It has been hypothesized that ospC genotype may influence mammalian host range. In this study, Ixodes scapularis ticks collected from the field in Rhode Island were assessed for infection with B. burgdorferi. Ticks were fed on purpose bred beagles to repletion and infection of the dogs was assessed through serology and PCR. Tissue biopsies (n=2) were collected from each dog 49 days post-tick infestation (dpi) and the ospC genotype of the infecting strains determined by direct PCR of DNA extracted from tissue or by PCR after cultivation of spirochetes from biopsy samples. The dominant ospC types associated with B. burgdorferi canine infections differed from those associated with human infection, indicating a relationship between ospC sequence and preferred host range. Knowledge of the most common ospC genotypes associated specifically with infection of dogs will facilitate the rational design of OspC-based canine Lyme disease vaccines and diagnostic assays.