Recombinant Outer Surface Protein Research Articles

OspA immunization decreases transmission of Borrelia burgdorferi spirochetes from infected Peromyscus leucopus mice to larval Ixodes scapularis ticks.

Recombinant outer surface protein A (OspA) vaccination of wild animal reservoirs has potential application for reducing Borrelia burgdorferi transmission in nature and subsequent risk of human infection. As a major reservoir host, the white-footed mouse (Peromyscus leucopus) is a candidate for a vaccination program designed to reduce infection prevalence in vector ticks. In this study we characterized the effect of various levels of immunization with recombinant OspA-glutathione transferase fusion protein on transmission dynamics from infected P. leucopus to larval ticks. Control mice were vaccinated with glutathione transferase alone. All mice were experimentally infected with B. burgdorferi before vaccination. The immune responses of the immunized mice were assessed by enzyme-linked immunosorbent assay for antibodies to OspA. Transmission of B. burgdorferi from infected mice was determined by xenodiagnosis with uninfected larval ticks. Spirochetes in ticks were counted by direct immunofluorescence assay. The concentration of antibody to OspA increased with each OspA vaccination but most markedly after the first and second vaccinations. In comparison with control mice, there was reduced transmission by OspA-vaccinated mice to uninfected ticks. One, two, or three doses of OspA reduced infection prevalence in xenodiagnostic ticks by 48%, 92%, or 99% and the numbers of spirochetes per tick by 84%, 98%, or 99%, respectively. This study suggests that vaccination of P. leucopus with OspA could reduce transmission to the tick vector in nature despite prior infection of the reservoir host.

Modulation of lymphocyte proliferative responses by a canine Lyme disease vaccine of recombinant outer surface protein A (OspA)

The modulation of human lymphocyte proliferative responses was demonstrated with a recombinant outer surface protein A (OspA) vaccine preparation for the prevention of Borrelia burgdorferi infection. After exposure to either the unaltered vaccine preparation or OspA prepared in saline, normal lymphocyte responses to the mitogens concanavalin A, phytohemagglutinin-M or pokeweed mitogen, or the antigen BCG were consistently reduced. Whole cell extracts of B. burgdorferi also modulated immune responses but required a much greater quantity of protein than needed for the OspA preparation. The magnitude of modulation was directly dependent on the quantity of OspA. OspA interferes with the response of lymphocytes to proliferative stimuli including a blocking of cell cycle phase progression. Future studies designed to delete the particular region or component of the OspA molecule responsible for this effect may lead to improved vaccine preparations.

Detection of multiple reactive protein species by immunoblotting after recombinant outer surface protein A lyme disease vaccination.

Laboratory confirmation of the diagnosis of Lyme disease is based on the detection of an immune response to Borrelia burgdorferi. The serodiagnosis of B. burgdorferi infection is complex and may be further confounded by the immune response to the recombinant outer surface protein A (OspA) Lyme disease vaccine. To describe how the serological response to the recombinant OspA Lyme disease vaccine affects testing for antibody to B. burgdorferi, 240 specimens from 80 study subjects were obtained at defined intervals after recombinant OspA Lyme disease vaccination. Samples were tested by indirect enzyme-linked immunosorbent assay (ELISA), antibody capture enzyme immunoassay (EIA), and Western blotting (WB). After recombinant OspA Lyme disease vaccination, ELISA for 98% of the study subjects revealed reactivity. WB with use of OspA-containing B. burgdorferi strains as sources of antigens demonstrated multiple bands. Results of testing with a US Food and Drug Administration-approved WB kit showed homogeneous reactivity in the molecular weight region >30 kDa. Testing with OspA-free strains completely eliminated all vaccine-associated reactivity by both antibody capture EIA and WB.

Modulation of lymphocyte proliferative responses by a canine Lyme disease vaccine of recombinant outer surface protein A (OspA)

The modulation of human lymphocyte proliferative responses was demonstrated with a recombinant outer surface protein A (OspA) vaccine preparation for the prevention of Borrelia burgdorferi infection. After exposure to either the unaltered vaccine preparation or OspA prepared in saline, normal lymphocyte responses to the mitogens concanavalin A, phytohemagglutinin-M or pokeweed mitogen, or the antigen BCG were consistently reduced. Whole cell extracts of B. burgdorferi also modulated immune responses but required a much greater quantity of protein than needed for the OspA preparation. The magnitude of modulation was directly dependent on the quantity of OspA. OspA interferes with the response of lymphocytes to proliferative stimuli including a blocking of cell cycle phase progression. Future studies designed to delete the particular region or component of the OspA molecule responsible for this effect may lead to improved vaccine preparations.

The Lyme disease vaccine: conception, development, and implementation.

In the past 20 years, remarkable strides have been made toward understanding and preventing Lyme disease in humans. In December 1998, the U.S. Food and Drug Administration approved a recombinant outer surface protein A vaccine against Lyme disease (LYMErix, SmithKline Beecham, Philadelphia, Pennsylvania). The vaccine, which is derived from a lipidated outer surface protein of the causative spirochete Borrelia burgdorferi, is important because it may decrease the morbidity and financial costs associated with Lyme disease. Its mechanism is unique because it works inside the tick vector itself, preventing the human from becoming infected.

Vaccination with feline immunodeficiency virus induces CD4 epitope masking by soluble factors

Soluble factors are important effector mechanisms to control for lentiviral replication. Vaccination of cats with recombinant outer surface proteins (SU) of the FIV envelope protein in combination with complete Freund adjuvant (CFA) and rabies nucleocapsid (NC) protein led to significantly reduced viral loads [Leutenegger, C.M., Hofmann-Lehmann, R., Holznagel, E., Cuisinier, A.M., Wolfensberger, C., Duquesne, V., Cronier, J., Allenspach, K., Aubert, A., Ossent, P., Lutz, H., 1998. AIDS Res. Hum. Retroviruses, 14(3) 275–283]. Lymphocytes from vaccinated and non-vaccinated cats were stained with two monoclonal antibodies, Fel7 and CAT30A, directed against the feline CD4 antigen. Peripheral blood lymphocytes from cats vaccinated with the SU glycoprotein, CFA and rabies NC protein showed a significantly reduced number of cells after staining with CAT30A, while the number in Fel7 positive lymphocytes remained unchanged. This decreased CAT30A fluorescent staining could be reproduced in vitro by pre-incubating FIV-negative lymphocytes with immune sera from cats in which reduced CAT30A staining was detected. Neither experimental infection nor vaccination with the unglycosylated SU protein alone resulted in this epitope masking. Furthermore, this masking phenomenon was negatively correlated with a decreased susceptibility to activation-induced cell death (AICD). These findings will be discussed based on the current knowledge of CD8 + T-cell antiviral factors and their involvement in lentiviral infection and/or replication.

Safety and immunogenicity profile of a recombinant outer-surface protein a Lyme disease vaccine: Clinical trial of a 3-dose schedule at 0, 1, and 2 months

Objectives This study compared the tolerability of a Lyme disease vaccine administered intramuscularly at 0 and 1 months with that of a vaccine administered at 0, 1, and 2 months to determine (1) whether adding a third dose of vaccine 1 month after the second would affect the safety profile, and (2) whether a shortened vaccination schedule of 0, 1, and 2 months would provide an immune response similar to that obtained with vaccine administered at 0, 1, and 12 months. Background An efficacy trial of a Lyme disease vaccine had demonstrated safety and efficacy against definite (clinically manifested and laboratory-confirmed) Lyme disease after 3 doses at 0, 1, and 12 months and resulted in 90% of subjects having titers ≥1400 enzyme-linked immunosorbent assay units (EL.U)/mL (the proposed seroprotective level for 1 tick season). Methods This multicenter, open-label, prospective, randomized study assessed the safety and efficacy of different doses of a recombinant outer-surface protein A (OspA) vaccine in 956 volunteers aged 17 to 72 years from 3 Lyme disease-endemic sites. Blood samples were collected at months 0, 2, 3, 12, and 13 to assess total immunoglobulin-G anti-OspA titers. Results Most adverse events were transient and mild to moderate. The geometric mean antibody titer increased 2.8-fold from month 2 (1786 EL.U/mL to 4842 EL.U/mL), and ~90% of the volunteers had a titer ≥1400 and 99% had a titer ≥400 EL.U/mL (the minimum seroprotective level at any given time) after the third dose. An antibody kinetics model predicts that protection would last for a typical tick-transmission season. Conclusions In volunteers aged 17 to 72 years, 3 doses of vaccine administered in 2 months was well tolerated, more immunogenic than 2 doses, and provided a higher probability of protection before exposure or travel to Lyme disease-endemic areas.

Conformational nature of the Borrelia burgdorferi B31 outer surface protein C protective epitope.

Active immunization with Escherichia coli-expressed recombinant outer surface protein C (OspC) of Borrelia burgdorferi has been demonstrated to confer protection against a tick-transmitted infection on laboratory animals. A previous study in this laboratory showed that OspC antibody raised against a denatured immunogen isolated from B. burgdorferi cells failed to provide protective immunity. Therefore, to determine whether the protective epitope of the recombinant antigen was sensitive to denaturation, recombinant OspC preparations were subjected to heat and chemical treatments prior to animal immunization. Following seroconversion to OspC, the animals were challenged with an infectious dose of B. burgdorferi B31 by tick bite. Whereas mice immunized with a soluble, nondenatured form continued to show protection rates close to 100%, mice that had been immunized with denatured antigen were not protected. Furthermore, mice that were immunized with an insoluble (rather than a soluble), nondenatured form of the recombinant OspC showed a protection rate of only 40%. Protective epitope localization experiments showed that either the amino or the carboxy end of the recombinant protein was required to react with a protective OspC-specific monoclonal antibody. The data from these experiments demonstrate that a conformational organization of the protein is essential for the protective capability of the strain B31 OspC immunogen.

Cardiac myocytes of hearts from patients with end-stage dilated cardiomyopathy do not contain Borrelia burgdorferi DNA

Objective To determine if end-stage dilated cardiomyopathy (DCM) is associated with the presence of Lyme disease causing spirochete Borrelia burgdorferi in the myocardium, we used nested polymerase chain reaction to detect B burgdorferi DNA in myocardial samples from explanted hearts of patients with end-stage DCM. Patients originated from endemic areas for Lyme disease (Bavaria, Lower Saxony, Germany). Methods and Results This was a retrospective study. Polymerase chain reaction was used to detect the specific B burgdorferi recombinant outer surface protein A (OspA) gene in myocardial tissue from 68 patients with end-stage DCM who had undergone heart transplantation. The clinical history of Lyme disease, the presence of Borrelia burgdorferi OspA, and antibodies against OspA in myocardial tissue and serum were investigated. B burgdorferi DNA was not detected in any of the 68 human hearts. Immunoglobulin G antibodies against specific B burgdorferi antigens were observed in 3 (12.5%) of 24 patients. In contrast, 4 hearts from rats experimentally infected with B burgdorferi were all positive for OspA DNA as measured by polymerase chain reaction. Conclusion Our data show that cardiac myocytes of hearts obtained from subjects with end-stage DCM did not contain B burgdorferi DNA as investigated by polymerase chain reaction. However, B burgdorferi shows a high affinity for myocardial tissue as shown by the animal studies, indicating that myocardial infections are nevertheless possible. (Am Heart J 1999;138:269-72.)

Lyme disease prevention and vaccine prophylaxis.

To provide a comprehensive review of the epidemiology, diagnosis, and prevention of Lyme disease with a focus on the Lyme disease vaccine. A computerized search of MEDLINE (January 1996-December 1998) was used to identify articles regarding Lyme disease, Borrelia burgdorferi, epidemiology, prevention, and vaccine. Lyme disease is a condition caused by infection with B. burgdorferi. The organism is carried by certain species of Ixodes ticks and is the most common tick-borne disease in the US. In patients with clinical manifestations of Lyme disease, various pharmacotherapeutic approaches have proven effective in treatment of the clinical features. Prevention strategies exist; however, their application is sometimes difficult. A vaccine for the prevention of Lyme disease is available, and another is being considered for approval. The recombinant outer surface protein A (OspA) vaccines to prevent Lyme disease are immunogenic and have an acceptable adverse effect profile. These vaccines are highly efficacious for the prevention of Lyme disease. Lyme disease is the most common tick-borne disease in the US. The infection, caused by B. burgdorferi, results in dermatologic, neurologic, cardiovascular, and musculoskeletal manifestations. Until recently, tick bite prevention strategies were the only means of decreasing the risk of acquiring the infection. The OspA vaccines are efficacious for the prevention of infection. Although universal immunization with these vaccines is unlikely, the availability of effective vaccines represents an important tool for the prevention of Lyme disease in endemic regions of the US.

VACCINATION AS A MODALITY TO PREVENT LYME DISEASE: A Status Report

Recombinant outer surface protein A (OspA) of Borrelia burgdorferi is a highly protective immunogen for prevention of Lyme disease in experimental animals. Humoral immunity is sufficient for protection. The principal mechanism of action is prevention of transmission of the spirochete from tick to host. A recombinant OspA vaccine has been licensed for use in dogs. The recent licensure of an OspA vaccine for humans resulted from a critical analysis of recently completed efficacy studies.

Analysis of lipidation of recombinant Lyme vaccine protein (rOspA) by electrospray mass spectroscopy

Lipidation at the N-terminal of recombinant outer surface protein A (rOspA) is an important determinant of its immunogenicity. Lipidation patterns of rOspA can be sensitive to processing environments and storage conditions. In order to assure product consistency and stability, it is essential to characterize and monitor lipidation patterns of rOspA through its life-cycle. Electrospray mass spectroscopy combined with maximum entropy calculation was employed to analyze the lipidation of rOspA. The results revealed that more than 90% of protein is a tri-lipidated rOspA and the remainder is di-lipidated. It was demonstrated that the method is both sensitive and quantitative and has the potential to be used for routine quality control and stability testing.

Specific issues in the design and implementation of an efficacy trial for a Lyme disease vaccine.

Lyme disease is an emerging infection that has now become the most commonly reported vector-borne disease in the United States. In the 20 years since its initial description, scientific and technological advances have led to candidate vaccines for the prevention of Lyme disease. Recombinant outer surface protein A (OspA) vaccines have been successful in protecting mice in tick-challenge experiments. A candidate OspA vaccine has been found to be safe and immunogenic in phase I and II studies. This article describes some of the lessons that were learned and some of the unique obstacles encountered in the design and implementation of a large phase III efficacy field trial. Pivotal trials of vaccines for Lyme disease can be a major investment of time and resources for subjects, investigators, and sponsors. If properly conducted, they also present unique opportunities to expand our knowledge of the disease.

Oral delivery of purified lipoprotein OspA protects mice from systemic infection with Borrelia burgdorferi

The lipoprotein outer surface protein A (OspA) of the Lyme disease agent, Borrelia burgdorferi, has provided protection to mice and other animals against systemic infection when delivered orally as a recombinant protein in Escherichia coli, bacille Calmette-Guerin or Salmonella typhimurium. In the present study purified recombinant strain B31 OspA or outer surface protein D (OspD), another lipoprotein of B. burgdorferi, were administered either subcutaneously (s.c.) or orally without cell carrier or adjuvant to mice. In comparison to the OspD preparation, the OspA protein was 256-fold more resistant to trypsin. Whereas OspA in the suspension was in regular complexes of 17–25 nm in size, OspD formed amorphous globules of different sizes. Animals received a primary immunization and at least one booster. Mice immunized s.c. with either OspA or OspD had detectable antibodies to B. burgdorferi by enzyme-linked immunosorbent assay (ELISA), growth inhibition assay (GIA) and immunoblot. Delivered orally, OspA but not OspD elicited a specific antibody response, including IgA, as determined by these assays. The geometric mean titre of sera from mice who received 4 μg of OspA orally on days 1, 2, 4, 21 and 22 was 1470 by Ig ELISA, 320 by IgA ELISA and 128 by GIA. In infectious challenge experiments with B. burgdorferi strain Sh2-2–82 (OspA +OspD −) inoculated intradermally at 100 × the ID 50, all eight mice immunized with the 4 μg dose of OspA were protected; none of the mice immunized with the 4 μg dose of OspD were protected ( P<0.001 by Fisher exact test). These studies indicate that the lipoprotein OspA provides protection against systemic B. burgdorferi infection when delivered orally as a purified protein.

Treatment and prevention of Lyme disease, with emphasis on antimicrobial therapy for neuroborreliosis and vaccination.

Antibiotic therapy is recommended for all forms of neuroborreliosis. Although stage 2 neuroborreliosis will usually resolve without any treatment, antibiotic therapy has been associated with faster resolution of symptoms and may prevent additional non-neurologic disease manifestations. Ceftriaxone is the most convenient parenteral agent for stage 2 and 3 neuroborreliosis because of its once-daily dosage. Available data indicate that a 2-4-week treatment course is adequate for most patients. Patients with isolated seventh nerve palsy may be treated with an oral agent (for example, doxycycline). Recombinant outer surface protein A of Borrelia burgdorferi is a highly protective immunogen for prevention of Lyme disease in experimental animals. Humoral immunity is sufficient for protection. A recombinant OspA vaccine has been licensed for prevention of Lyme disease in dogs. Licensure of an OspA vaccine for humans will depend on a critical analysis of the results of recently completed efficacy studies.

Evaluation of the safety, reactogenicity and immunogenicity of three recombinant outer surface protein (OspA) lyme vaccines in healthy adults

The safety, reactogenicity and immunogenicity of three candidate Lyme vaccines based on recombinant outer surface protein (OspA) presented in either lipidated or unlipidated forms, were assessed in 300 seronegative volunteers. Subjects received three doses of one of the three formulations at monthly intervals and were evaluated for antibody levels and the presence of symptoms after each dose. All formulations proved to be safe, the majority of local reactions being reported as mild, and all general symptoms were perceived to be either mild or moderate in intensity. No subject refused a subsequent vaccine dose. All subjects were tested for both anti-OspA IgG and LA-2 equivalent antibodies up until day 84. All three vaccines induced an immune response, but subjects who received lipoprotein OspA had the highest anti-OspA IgG and LA-2 equivalent GMTs after each dose and this was also true for the subset of subjects tested on day 180. The lipoprotein OspA group also had the largest number of subjects who remained seropositive for anti-OspA IgG antibodies. As the lipoprotein formulation produced the strongest immune response, with symptoms which were acceptable to all the vaccinees, we suggest further development of this vaccine.

Characterization of the protective borreliacidal antibody response in humans and hamsters after vaccination with a Borrelia burgdorferi outer surface protein A vaccine.

Significant borreliacidal antibody was induced in volunteers and hamsters 60 days after primary and secondary vaccination with high concentrations of recombinant outer surface protein A (rOspA). However, the borreliacidal antibody response waned rapidly. Only 1 person had detectable cidal activity 180 days after vaccination. Similarly, the borreliacidal antibody response waned rapidly in hamsters by week 10 of vaccination. By contrast, the total anti-rOspA antibody response remained elevated in volunteers and hamsters. When isolates of Borrelia burgdorferi sensu lato were incubated in sera from vaccinated humans or hamsters, only the vaccine-specific isolate was killed. These results were confirmed by challenging rOspA-vaccinated hamsters with different isolates of B. burgdorferi sensu lato. The results showed that monitoring total rOspA antibody is inappropriate for evaluating the efficacy of an rOspA vaccine. The rOspA vaccine must be improved to yield comprehensive protection and maintain sustained levels of protective borreliacidal antibodies.

The recombinant outer surface protein A (lipOspA) of Borrelia burgdorferi: a Lyme disease vaccine.

The recombinant outer surface protein A (lipOspA) of Borrelia burgdorferi: a Lyme disease vaccine.

Outer surface protein C (OspC), but not P39, is a protective immunogen against a tick-transmitted Borrelia burgdorferi challenge: evidence for a conformational protective epitope in OspC

Outbred mice were immunized with the soluble fraction of a crude Escherichia coli lysate containing either recombinant outer surface protein C (OspC or P39 of Borrelia burgdorferi B31 (low passage). Following seroconversion, the mice were challenged with an infectious dose of B. burgdorferi B31 via the natural transmission mode of tick bite. Three mice immunized with P39 were not protected; however, all 12 of the recombinant OspC-immunized mice were protected from infection as assayed by culture and serology. Although OspC has been shown to be a protective immunogen against challenge with in vitro-cultured borrelia administered by needle, this study is the first to demonstrate OspC effectiveness against tick-borne spirochetes. Following feeding, all ticks still harbored B. burgdorferi, suggesting that the mechanism of protection is not linked to destruction of the infectious spirochete within the tick. In a separate experiment, groups of four mice were immunized with protein fractions from B. burgdorferi B31 purified by preparative gel electrophoresis in an attempt to identify potential protective antigens. Many of these mice developed high-titer-antibody responses against OspC, but curiously the mice were susceptible to B. burgdorferi infection via tick bite. These results suggest that the protective epitope(s) on OspC is heat sensitive/conformational, a finding which has implications in vaccine development.

Lyme disease vaccine.

Subunit vaccines consisting of single recombinant outer surface proteins (Osp) of Borrelia 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 Lyme Borrelia 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.