WO1999061048A1

WO1999061048A1 - CORRELATIVE PROTECTION USING OspA ANTIBODY TITERS

Info

Publication number: WO1999061048A1
Application number: PCT/US1999/011158
Authority: WO
Inventors: Dennis Parenti; Yves Lobet; Marc Gillet
Original assignee: Smithkline Beecham Corporation; Smithkline Beecham Biologicals
Priority date: 1998-05-22
Filing date: 1999-05-20
Publication date: 1999-12-02
Also published as: AU4193399A; CA2332979A1; EP1089756A4; EP1089756A1

Abstract

Methods of protecting human beings from infection by $(B. burgdorferi) are disclosed, which comprises internally administering lipoprotein OspA, or a combination of lipoprotein OspAs using specified dosing schedules.

Description

CORRELATIVE PROTECTION USING OSPA ANTIBODY TITERS

Field of the Invention This invention relates to methods for protecting human beings from infection by B. burgdorferi. More particularly, this invention relates to a method for establishing alternative dosing schedules/protocols for protection against Lyme disease.

Background of the Invention

This invention resides in the discovery that anti-OspA antibody titer is a useful surrogate marker for protection against infection by Borrelia burgdorferi sensu lato, the causative agent of Lyme Disease. Specifically, this invention resides in the discovery that if a human is vaccinated with an OspA lipoprotein containing vaccine using a dosing schedule that has been predetermined to produce an IgG anti- OspA antibody titer of at least 1000 El.U/ml, preferably at least 1200 El.U/ml and more preferably at least 1400 El.U/ml or an LA-2 monoclonal antibody anti-OspA titer of at least 1000 El.U/ml, preferably at least 1200 El.U/ml, and more preferably at least 1400 El.U/ml, in a majority of a vaccinated population, there is an acceptably high likelihood that the person will be protected against disease caused by infection with B. burgdorferi sensu lato.

Lyme disease in humans is a chronic progressive disease caused by spirochete B. burgdorferi, and in particular 3 species are now associated with Lyme disease in humans, B. burgdorferi sensu stricto, B. garinii and B. afzelii. Collectively, they, along with other Borrelia species, are commonly referred to as B. burgdorferi sensu lato and which are transmitted to humans mainly by Ixodes ticks. The spirochete attacks many organs, notably the skin, heart, liver, central and peripheral nervous system, kidneys as well as the musculoskeletal system.

Lyme disease itself is the most common vector borne infection in the USA and has been reported in every continent except Antarctica.

A number of groups have isolated and proposed the major surface protein (OspA) of B. burgdorferi, as being a potential vaccine candidate for use against Lyme disease. It has been shown that immunization with the outer surface protein, lipoprotein OspA, is protective against direct inoculation of spirochetes in tick-challenge models (Fikrig et al., Infect Immun, 60:657-661 (1992) and Gern et al., Immunol Lttr, 39:249-258 (1994)). Furthermore, two recombinant subunit vaccines have been reported in development for human use (see, Steere et al., N Eng J Med, 339:209-215 (1998) and Segal et al., N Eng J Med, 339:216-222 (1998)). B. burgdorferi sensu stricto strain ZS7 OspA lipoprotein has recently received regulatory review at the U.S. Food and Drug Administration as a vaccine against Lyme disease. The current dosing schedule for the vaccine is 30 ug at 0, 1 and 12 months. Prior to this invention, to improve on the dosing schedule of an OspA vaccine would be a long, costly and labor intensive process.

In addition, prior to the present invention, previous methods were poor at: detecting non-responders (individuals who do not achieve an effective level of protection); indicating at what point along the 12 month dosing protocol individuals can expect to achieve protection from B. burgdorferi challenge; providing a fast, reliable and cost effective method to assess newly proposed OspA dosing schedules; and applying all the above to populations (pediatrics, for example) not included in the efficacy trial. Thus there is a need in the art for a fast, reliable and cost effective method to determine the level of protection of an individual or to predict the level of protection of a population in response to a given OspA lipoprotein vaccination schedule against B. burgdorferi.

Summary of the Invention

In one aspect the present invention relates to a method for protecting humans from infection by B. burgdorferi which comprises administering an OspA vaccine using a dosing schedule other than 30ug at 0, 1 and 12 months, such that the anti-

OspA titer after the final dosing is at least 1000 El.U/ml.

In another related aspect, the present invention relates to a method for protecting humans from infection by B. burgdorferi which comprises administering lipoprotein OspA using a dosing schedule that results in anti-OspA titer after the primary dosing schedule of at least 1000 El.U/ml followed by boosting with OspA such that the anti-OspA titer after boosting is at least 1000 El.U/ml.

In yet a further related aspect, the present invention relates to a method for determining the dosing schedule for a lipoprotein OspA-containing vaccine for protecting humans from Lyme Disease which comprises testing a variety of dosing schedules on a population of test subjects and selecting a schedule, or schedules, that produces an anti-OspA titer of at least 1000 El.U/ml in at least 80% of the test subjects.

In a further related aspect, the present invention relates to a method for determining the booster dosing schedule for a lipoprotein OspA-containing vaccine for protecting humans from Lyme Disease which comprises testing a variety of dosing schedules on a population of test subjects previously vaccinated with an

OspA-containing vaccine and selecting a schedule, or schedules, that produces an anti-OspA titer of at least 1400 El.U/ml in at least 80% of the test subjects. In yet a further related aspect, the present invention relates to a method for protecting human beings from infection by B. burgdorferi which comprises: (1) administering a lipoprotein OspA-containing vaccine; (2) at least 6 months thereafter, checking the anti-OspA titer of the patient; and (3) if said titer is below 1000 El.U/ml, administering a booster dose.

Detailed Description of the Invention

It is herein reported, for the first time, the discovery that humans who are vaccinated with an OspA lipoprotein containing vaccine which uses a dosing schedule that has been predetermined to produce an IgG anti-OspA antibody titer of at least 1000 El.U/ml, preferably at least 1200 El.U/ml and more preferably at least 1400 El.U/ml or a LA-2 monoclonal antibody anti-OspA titer of at least 1000 El.U/ml, preferably at least 1200 El.U/ml, and more preferably at least 1400 El.U/ml, in a majority of a vaccinated population, in at least 90% of the population, there is an acceptably high likelihood that the person will be protected against disease caused by infection with B. burgdorferi sensu lato . Preferably, the correlation between antibody titer and the level of protection (i.e., % humans protected from B. burgdorferi challenge one month after the primary dosing schedule, i.e., the second or third dose) is at least about 70%, preferably at least about 80%, more preferably at least about 90% of the vaccinated population will achieve a protective antibody titer against disease caused by infection with B. burgdorferiβensu lato .

The difficulties encountered using OspA lipoprotein as an vaccine, such as optimizing an OspA vaccination schedule, determining an OspA booster schedule or detecting non-responders (individuals who do not achieve an effective level of protection), are overcome by the current invention by using an IgG anti-OspA antibody titer of at least 1000 El.U/ml, preferably at least 1200 El.U/ml, more preferably at least 1400 El.U/ml, or an LA-2 monoclonal antibody (See Kramer et al. Immunobiol. 181: 357 to 366 (1990) and US Patent 5,780,030) -OspA titer of at least 1000 El.U/ml, preferably at least 1200 El.U/ml, more preferably at least 1400 El.U/ml, as a surrogate marker of protection against disease caused by infection with B. burgdorferi sensu lato .

In utilizing the present invention the following improved dosing schedules, which are expected to provide an IgG anti-OspA antibody titer of at least 1000 El.U/ml, preferably at least 1200 El.U/ml, more preferably at least 1400 El.U/ml or an LA-2 anti-OspA antibody titer of at least 1000 El.U/ml, preferably at least 1200 El.U/ml, more preferably at least 1400 El.U/ml in humans, were discovered by internally administering (intramuscular, subcutaneous, intravenous, oral, mucosal, transdermal, etc.) an OspA lipoprotein (or gene encoding an OspA lipoprotein) at selected intervals.

Internally administering 30 ug OspA lipoprotein at 0, 1 and another dose selected from 2 to 11 months. Internally administering 30 ug of OspA lipoprotein at 0, 1 and 6 months.

Internally administering 30 ug of OspA lipoprotein at 0, 1 and 2 months.

Internally administering 3 doses within a 30 day period, for example 30 ug of OspA lipoprotein at 0, 7 and 28 days.

Internally administering 60 ug of OspA lipoprotein at 0 and 1 month.

Internally administering 60 ug of OspA lipoprotein at 0 and 2 months.

Internally administering 90 ug of OspA lipoprotein at 0 and 1 month. Internally administering 120 ug of OspA lipoprotein at 0 and 1 month.

In utilizing the present invention the following boosting schedules, which are expected to provide an IgG anti-OspA antibody titer of at least 1000 El.U/ml, preferably at least 1200 El.U/ml, more preferably at least 1400 El.U/ml or an LA-2 anti-OspA antibody titer of at least 1000 El.U/ml, preferably at least 1200 El.U/ml in humans, more preferably at least 1400 El.U/ml, were discovered.

Internally administering 30 ug of OspA lipoprotein at 0, 1 and another dose selected from 2 to 12 months followed by boosting with 30 ug at 12 months.

Internally administering 30 ug of OspA lipoprotein at 0, 1 and another dose selected from 2 to 12 months followed by boosting with 30 ug at 24 months.

Internally administering 30 ug of OspA lipoprotein at 0, 1 and another dose selected from 2 to 12 months followed by boosting with 30 ug at 36 months.

Internally administering 30 ug of OspA lipoprotein at 0, 1 and another dose selected from 2 to 12 months followed by boosting with 30 ug at least once per each 12 month period or as soon as the anti-OspA titer of a vaccinee falls to about 400 El.U/ml or less.

In utilizing the present invention the following dosing schedules, which are expected to provide an IgG anti-OspA antibody titer of at least 1000 El.U/ml, preferably at least 1200 El.U/ml, more preferably at least 1400 El.U/ml, or an LA-2 anti-OspA antibody titer of at least 1000 El.U/ml, preferably at least 1200 El.U/ml, more preferably at least 1400 El.U/ml, in specified subgroups of humans, were discovered.

Internally administering 30 ug of OspA lipoprotein at 0, 1 and another dose selected from 2 to 11 months to a human patient who is over 60 years old.

Internally administering 30 ug of OspA lipoprotein at 0, 1 and another dose selected from 2 to 11 months to a human patient who is from 15 to 60 years old.

Internally administering 30 ug of OspA lipoprotein at 0, 1 and another dose selected from 2 to 11 months to a human patient who is from 2 to 5 years old.

Internally administering 30 ug of OspA lipoprotein at 0, 1 and another dose selected from 2 to 1 1 months to a human patient who is under 2 years old. Internally administering 30 ug of OspA lipoprotein at 0, 1 month to a human patient who is from 2 to 5 years old.

Internally administering 15 ug of OspA lipoprotein at 0, 1 and 2 months to a human patient who is from 2 to 5 years old.

Internally administering 15 ug of OspA lipoprotein at 0 and 1 month to a human patient who is from 2 to 5 years old.

Another aspect of the claimed invention provides for a sustained IgG anti- OspA antibody titer of at least 1000 El.U/ml, preferably at least 1200 El.U/ml or an LA-2 anti-OspA antibody titer of at least 1000 El.U/ml, preferably at least 1200 El.U/ml, for at least 6 months in humans using dosing schedules specified herein. Another aspect of the claimed invention provides for a sustained IgG anti-

OspA antibody titer of at least 1000 El.U/ml, preferably at least 1200 El.U/ml or an LA-2 anti-OspA antibody titer of at least 1000 El.U/ml, preferably at least 1200 El.U/ml, for at least 8 months in humans using dosing schedules specified herein. Another aspect of the claimed invention provides for a sustained IgG anti- OspA antibody titer of at least 1000 El.U/ml, preferably at least 1200 El.U/ml or an LA-2 anti-OspA antibody titer of at least 1000 El.U/ml, preferably at least 1200 El.U/ml, for at least 10 months in humans using dosing schedules specified herein. Another aspect of the claimed invention provides a method for determining the dosing schedule for an OspA-containing vaccine for protecting humans from Lyme Disease which comprises testing a variety of dosing schedules on a population of test subjects and selecting a schedule or the schedules that produces an anti-OspA IgG titer or an LA-2 anti-OspA antibody titer of at least 1000 El.U/ml, preferably at least 1200 El.U/ml, more preferably at least 1400 El.U/ml, in at least 80% of the test patients, preferably in at least 90% of the test patients. Another aspect of the claimed invention provides a method for determining the booster dosing schedule for an OspA-containing vaccine for protecting humans from Lyme Disease which comprises testing a variety of dosing schedules on a population of test subjects previously vaccinated with an OspA-containing vaccine and selecting a schedule or the schedules that produce an anti-OspA IgG titer or an LA-2 anti-OspA antibody titer of at least 1000 El.U/ml, preferably at least 1200 El.U/ml, more preferably at least 1400 El.U/ml, in at least 80% of the test patients, preferably in at least 90% of the test patients one month after administration of the booster dose. Another aspect of the claimed invention provides a method for protecting human beings from infection by B. burgdorferi which comprises internally administering OspA using a dosing schedule that results in anti-OspA titer after the primary dosing schedule of at least 1000 El.U/ml, preferably at least 1200 El.U/ml, more preferably at least 1400 El.U/ml. followed by boosting with OspA such that the anti-OspA titer after boosting is at least 1000 El.U/ml, preferably at least 1200 El.U/ml, more preferably at least 1400 El.U/ml.

Another aspect of the claimed invention provides a method for protecting human beings from infection by B. burgdorferi which comprises (1) internally administering an OspA-containing vaccine, (2) at least 6 months thereafter, checking the anti-OspA IgG titer or the LA-2 anti-OspA antibody titer of the patient and (3) if the titer is below 1000 El.U/ml, preferably below 400 El.U/ml, administering a booster dose.

Another aspect of the claimed invention provides a method for determining an OspA vaccine dosing schedule which comprises evaluating an anti-OspA antibody titer one month after a primary immunization schedule of an OspA lipoprotein and correlating said anti-OspA antibody titer with the level of protection from B. burgdorferi challenge in a vaccinated population.

Preferred OspAs for use herein include strains ZS7, B31, N40, 297, ACA-1 and ZQ 1. Particularly preferred is ZS7. Also preferred is B31. All of the aforementioned OspAs are well known in the art.

In addition, OspA may be administered as a chimeric molecule, either "fused" to a non-OspA protein to further enhance or elicit an immune response, or as a fusion to another OspA protein (or protective fragments therein) from a different strain. As used herein the term "anti-OspA titer" and derivatives thereof, refers to one or both of the IgG anti-OspA titer or the LA-2 anti-OspA antibody titer.

All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as though fully set forth. The difficulties encountered using OspA as a vaccine, as described above, are over come by the current invention as depicted in the Experimental data presented below. This Experimental data is not intended to limit the scope of the invention as defined hereinabove and as claimed below.

Examples

Example 1

A human clinical study was performed using the OspA lipoprotein from B. burgdorferi strain ZS7. Approximately one half of the test patients received a placebo and the other half received the OspA lipoprotein from B. burgdorferi strain ZS7 as the antigen (30ug purified OspA lipoprotein adsorbed onto 0.5mg Al as Al(OH)₃). For the purposes of this experiment, the antigen was administered intramuscularly using a dosing schedule of 30ug at 0, 1 and 12 months. The anti- OspA IgG titer was determined at month 2 (one month after the second dose) and month 13 (one month after the third dose) so that test subjects received and had time to respond immunologically to the 30ug doses of OspA lipoprotein given.

The test patients resumed normal activities for a period of twenty months (from time 0). At month 12 and at month 20 test patients were evaluated for antibody titer and for the contraction of Lyme disease.

Incidents of Lyme disease were determined by the presentation of any of the following four (1.1 to 1.4) well known clinical manifestations plus at least one laboratory confirmation (A, B or C).

Category 1.1 Erythema Migrans (EM) (an expanding red skin lesion, often with partial central clearing).

1.2 Neurologic manifestations (meningitis, cranial neuritis).

1.3 Musculoskeletal manifestations (arthritis -joint swelling).

1.4 Cardiovascular manifestations (atrioventicular block). Laboratory confirmation of Lyme disease includes: A. Positive culture for B. burgdorferi after skin biopsy,

B. Positive PCR result for B. burgdorferi in skin biopsy, cerebrospinal fluid or joint fluid,

C. Seroconversion as determined by a Western blot test (detection of antibodies to B. burgdorferi).

The results of the Lyme disease evaluation are indicated in Tables 1 and 2. Table 1: Attack Rates of Lyme Disease and Vaccine Efficacy By Category During Year 1 (VE1ATP) After Two Doses

AR: Attack rate.

VE: Vaccine efficacy.

*Chi-square test done between attack rates in the vaccine and placebo groups.

Table 2: Attack Rates of Lyme Disease and Vaccine Efficacy by Category During the Second Year - After the Third Dose

AR: Attack rate.

VE: Vaccine efficacy.

*Chi-square test done between attack rates in the vaccine and placebo groups.

The percentage of vaccinees in the subset of subjects who had repeated blood sampling and had a titer above a given cut-off is presented in Table 3. Table 3: Percentage of vaccinees having IgG titers (E U/ml) above a given cutoff

In view of the above data three statistical methods (Discriminant Analysis,

Linear Logistic Regression and Non-parametric Logistic Regression) are used to determine the relationship between titer levels of the IgG anti-OspA antibody and prevention of confirmed Category 1.1 Lyme disease.

Antibody response to the vaccine was evaluated by measuring total IgG anti- OspA. Total IgG anti-OspA antibodies were measured by ELISA as described by Van Hoecke et al. (Vaccine, 14: 1620-1626 (1996) and incorporated by reference herein). Total IgG anti-OspA titers were calculated relative to the reference using four-parameter interpolation logistics as described by (Karpinski et al., J. Immun. Meth., 103: 189-194 (1987) and incorporated by reference herein). A geometric mean titer (GMT) was obtained using the log-transformation of individual titers and taking the anti-log of the mean of these transformed values on seropositive volunteers. GMTs of antibodies and their confidence limits (95% C.I.) were calculated on pre and post-vaccination blood samples. IgG anti-OspA antibody titers greater than or equal to the assay cut-off of 20 El.U/ml (ELISA units / ml) were considered seropositive.

Statistical analysis on the data obtained from human clinical trials conducted using the OspA lipoprotein from B. burgdorferi strain ZS7, such as the 50% vaccine efficacy rate observed in Table 1, the 78% vaccine efficacy rate observed in Table 2 and the cutoff titers observed in Table 3, was conducted to determine whether vaccine efficacy could be predicted based on an anti-OspA titer. The data from human clinical trials conducted using an OspA antibody established that a protective titer is established at an anti-OspA titer, preferably an IgG anti-OspA titer, or an LA- 2 anti-OspA antibody titer, of at least 1000 El.U/ml, preferably at least 1200 El.U/ml, more preferably at least 1400 El.U/ml, in at least 80% of the test patients, preferably in at least 90% of the test patients.

Three doses of lipoprotein OspA administered on a 0, 1, 12 month schedule was well tolerated and provided protective efficacy of 76% against definite Lyme disease due to B. burgdorferi and 100% against asymptomatic infection. An IgG anti-OspA titer of > 1200 El.U/ml before tick season provided protection for approximately 95% of the population. The majority of the vaccinees who contracted Lyme disease after dose 2 had IgG anti-OspA antibody titers < 400 El.U/ml at the onset of the disease. (Titers of 400 El.U/ml are thought to be the minimum titer sufficient to seroprotect at any given point in time.)

Example 2

Evaluation of an OspA vaccine when administered on a 0, 1, 6 month schedule. Eight hundred volunteers (396 males, 404 female, aged between 14 and 54 years at the time of first vaccination) were randomly allocated into a vaccination schedule of 0, 1, 6 months (group I) or 0, 1, 12 months (group II). The majority of adverse events reported were mild to moderate in intensity and transient.

Almost all subjects (> 99%) had seroconverted for IgG anti-OspA antibodies after 2 doses of lipoprotein OspA and this increased to 100% one month after the third dose irrespective of schedule. GMTs one month post third dose were 7205 and 10659 El.U/ml for groups I and II, respectively. 92% of group I vaccinees had a titer >1200 El.U/ml which is equivalent to that observed of group II vaccinees (93-95% after dose 3).

Example 3 Evaluation of an OspA vaccine when administered on a 0, 1, 2 month schedule. In Example 1, the OspA vaccine demonstrated 80% efficacy against clinical and laboratory confirmed Lyme disease following 3 doses when administered at 0, 1 and 12 months. Efficacy against asymptomatic infection was 100%. For this evaluation, a randomized, multicenter, open label study was done with 956 individuals (17 to 72 years of age) comparing the reactogenicity and immunogenicity of lipoprotein OspA on a schedule of 0, 1, 2 months or 0, 1, 12 months. Serum samples were obtained 1 month after the second and third doses and the immune response elicited by lipoprotein OspA was assessed. The immunogenicity data from 0, 1 dose verses 0, 1 , 2 doses were compared to responses obtained from Example 1. The 0, 1, 2 dosing demonstrated that vaccine was safe and well tolerated. Most local and general reactions were mild to moderate in severity and self-limited.

The distribution of titers was very similar. The results demonstrated that equivalent immune response were elicited in this study, after 2 and 3 doses at 0, 1, 2 (total IgG anti-OspA GMTs were 1770 and 4787 El.U/ml) compared to Example 1 (IgG anti-OspA GMTs were approximately 967 and 5060 El.U/ml) for 2 and 3 doses at a 0, 1, 12 month dosing schedule.

Three doses of lipoprotein OspA, whether on a 0, 1, 2 or 0, 1, 12 schedule provides a similar antibody response and should provide protection against Lyme disease equal to that observed in Example 1.

Example 4

Evaluation of an OspA vaccine when administered on an accelerated schedule. An open label study was conducted to evaluate the reactogenicity and immunogenicity of lipoprotein OspA on a schedule of 0, 7 and 28 days. Serum samples were obtained 1 month after the third dose and the immune response elicited by lipoprotein OspA was assessed using ELISA to determine anti-OspA antibodies. Thirty-five subjects received doses intramuscularly at 0, 7 and 28 days. A similar response was elicited 1 month after the third dose with regards to geometric mean antibody titer (total IgG anti-OspA GMTs were 4889 El.U/ml) as compared to Example 1 (IgG anti-OspA GMTs were 5060 El.U/ml for 3 doses). The OspA vaccine was safe and well tolerated despite administering 3 doses within 4 weeks. Most local and general reactions were mild to moderate in severity and self-limited.

Example 5

OspA vaccine profile in children aged 5-15 years. 250 children were recruited and allocated to 2 groups either receiving 15 or 30ug of Lipoprotein OspA in a 0, 1, 2 month schedule. Most reported symptoms were local (injection site pain, headache, malaise) and the incidence of all symptoms was comparable in both groups. Both doses elicited a satisfactory immune response, indicated by 99-100% seroconversion after the second dose for both IgG and LA-2 equivalent anti-OspA antibodies with higher titers for the higher antigen dose. The third dose of vaccine increased GMTs 2.5 fold. Both vaccine doses were safe and well tolerated and elicited a satisfactory immune response.

Example 6 For individuals who contracted Lyme disease, a follow-up analysis was conducted to see whether this was due to strain variability (lack of cross-protection) or whether individuals did not develop protective levels of anti-OspA antibodies. B. burgdorferi strains isolated from Lyme disease patients which occurred in the human clinical trial mentioned in Example 1 were analyzed as follows: (1) to determine the sequence of the OspA gene isolate, and (2) to evaluate the susceptibility of isolates to OspA specific antibodies in an in vitro bactericidal assay.

Sample collection. A 2 mm punch biopsy was taken from the advancing edge of a suspected EM lesion, following local disinfection and anesthesia. Disinfection of the skin was performed using Betadine® solution (povidone-iodine 10%) that was cleaned after approximately 2 minutes with a 70% alcohol solution which was allowed to dry and evaporate before the biopsy was performed. Preservative-free 1 % lidocaine single use vial for injection was used for local anesthesia.

Specimen processing and culture. Intact skin biopsy specimens were placed directly into a 15 ml tube of BSK medium which had been previously brought to room temperature. The specimens were then shipped on the same day to Dr. Allen Steere's lab at the New England Medical Center, Boston, MA, USA. Special precautions were taken to avoid exogenous contamination. The samples were opened in a separate laboratory not exposed to B. burgdorferi. Skin samples for culture were placed directly in an incubator at 33°C in that laboratory for two weeks. The samples were inspected visually each day for gross contamination. When any contamination was found, ciprofloxacin and rifampin were added at a concentration of 0.4 and 40 μg/ml, respectively. If this did not work, the sample was filtered with a 0.22 micron filter. After 2 weeks, the biopsies were removed from the culture tube and taken to a different lab for the extraction of DNA for PCR detection of the organism. The cultures were then examined by darkfield microscopy for motile spirochetes weekly for one month. The identity of all isolates were confirmed by reactivity with a monoclonal antibody to OspA and by reactivity with PCR primers that are specific for B. burgdorferi. Isolates were then frozen in 30% glycerol and sent to SmithKline Beecham Biologicals, Rixensart, Belgium for further manipulations. For sequencing of the OspA gene, samples were prepared as follows. An aliquot (0.5 ml) of the suspension of bacteria frozen in 30% glycerol were used to inoculate 14.5 ml of BSK medium. Bacteria were cultured at 35°C to reach a concentration of 40 x 10⁶ to 60 x 10⁶ spirochetes/ml. Cells were harvested and washed twice with the same volume of PBS by centrifugation (3500 rpm for 15 minutes at 25°C). The pellet was resuspended in PBS at a concentration of 5 x 10 to 10 x 10 spirochetes/ml. In case of contamination, culture was performed in BSK medium containing 50 μg/ml rifampicin, 20 μg/ml phosphomycin, 2.5 μg/ml amphotericin B, 230 μg/ml cystein and 150 μg/ml DL-DTT. When this condition did not allow to culture B. burgdorferi, spirochetes contained in 1 ml of the suspension frozen in 30% glycerol were harvested, and washed as described above. In any case, B. burgdorferi were inactivated by incubating the suspension at 60°C for 1 hour and then stored at 4°C before PCR amplification of the OspA gene.

PCR and sequencing. OspA gene PCR amplification was carried out at Eurogentec, Seraing, Belgium with dead bacteria as template using standard amplification protocol in a Perkin-Elmer 9700 thermocycler (Perkin-Elmer, Northwalk, CT, USA). Amplification forward and reverse primer sequences were 5'- ATGAAAAAATATTTATTGGGAATA-3' and 5' - CATAAATTCTCCTTATTTTAAAGC -3", respectively; they allowed the amplification of the full gene sequence. Ten μl of template cells (10⁴cells/μl) were mixed with 65.5 μl of water, denatured at 95°C for 5 min and mixed with 24.5 μl of the PCR cocktail (20 μM of primers, 1.5 mM MgCl₂ and 2.5 units of Goldstar DNA polymerase (Eurogentec, Seraing, Belgium). The cycling temperature profile was 1 min at 95°C, 1 min at 50°C, and 1 min at 72°C for 30 cycles followed by an extra extension step at 72°C for 10 minutes. The PCR fragment was purified on a Qiagen column and following the manufacturer protocol (Qiagen, Dusseldorf, Germany).

The fragment was sequenced using the BigDye Terminator technology and following the manufacturer protocol (Applied BioSystems, a division of Perking-Elmer, Foster City, CA, USA). Internal primers were designed and synthesized by Eurogentec (Seraing, Belgium). The full length of the gene was sequenced by the primer walking method; the primers were designed in such a way that the all gene was sequenced at least twice. Briefly, 50 ng of PCR fragment and 3.2 pmoles of sequencing primer were mixed in a 20 μl total volume reaction with 8 μl of the BigDye premix solution. Twenty five cycles of sequencing reactions were performed in a thermocycler (model 9700, Perking-Elmer, Northwalk, CT, USA) following the recommended cycling profile: 1 min at 95°C, 1 minute at 50°C and 4 min at 60°C. The sequencing reaction products were purified by ethanol precipitation, washed with ethanol 70% and resuspended in 2 μl of loading buffer as described by the manufacturer. One half microliter was loaded on an 5% long Ranger (FMC, Rockland, ME, USA) 36 cm long slab gel, resolved on an ABI377 instrument (Applied BioSystems, a division of Perking-Elmer, Foster City, CA, USA) and analyzed with the Analysis software version 3.0 (Applied BioSystems, a division of Perkin-Elmer, Foster City, CA, USA). Contig assembly was performed using the Sequencher package version 3.0 (Gene Codes, Ann Harbor, MI, USA). Deduced amino acid sequences were analyzed at SmithKline Beecham Biologicals by alignment with MegAlign (Dnastar Inc. Madison, Wisconsin) using the Custal method to determine their level identity.

Bactericidal assay. Susceptibility of clinical isolates to OspA-specific antibodies was measured at SmithKline Beecham Biologicals. The assay is a modification of that described by M.K. Aydintug, Y. Gu and M. Philipp (Infect. Immun. 1994, 62: 4929-4937). Briefly, frozen samples of β. burgdorferi were quickly thawed at 37°C and cultured for 3 days in BSK medium to reach the mid-log phase. The phase culture is centrifuged (8000 g for 20 min) and the pellet is resuspended in BSK medium to have around 10⁷ spirochetes/ml. The bactericidal assay was performed in 96-well tissue culture plates: 2.5 10⁵ spirochetes in 25 μl of BSK medium were added to each well containing 50 μl of heat inactivated (56°C , 30 min) serum sample serially diluted in the same medium. The plates were incubated 20 min at 35°C before the addition of 25 μl of complement (normal guinea pig serum). After 5 hours of incubation at 35°C, the total numbers of dead (non-motile) and live (motile) bacteria in each well were determined by dark field microscopy (400 x) on 5 μl aliquots mounted on glass slides. The bactericidal titer is given by the serum dilution corresponding to 50% killing (immobilization) of spirochetes.

Results. A total of 99 isolates were sequenced (22 from vaccinees, 69 from placebos). Deduced amino acid sequences of OspA genes were found to be highly conserved. As shown in Table 4, OspA sequences of the clinical isolates varied at only 3 positions, defining 5 groups of isolates. Most of them (97 out of 99) belong to groups 1 to 3, those groups being defined by 100% amino acid identity to the known strains N40, 297 or B31, respectively. The 2 remaining isolates define groups 4 and 5, differing by one or two residues from groups 1, 2 and 3; no identical sequence to those 2 isolates was found in public databases. Group 1, 2 and 3 variants were encountered in both vaccinee and placebo group, while group 4 and 5 were only in the placebo group. When tested in a bactericidal assay with the serum of a vaccinated individual, similar bactericidal titers were measured against clinical isolates from placebos and vaccinees (Table 5). The higher values of bactericidal titers observed with B31 and ZS7 strains compared to clinical isolates could reflect different level of expression of OspA resulting, e.g. from adaptation of the former strains to in vitro conditions.

Table 4. OspA variants among clinical isolates: all OspA deduced amino acid sequences are identical except at position indicated in the table.

Reference OspA = B. burgdorferi strain ZS7

Table 5. Bactericidal titers.

Strains Clinical isolate's Bactericidal titers*

Groups⁰

Vaccinee group

8271 2 150

1339 3 180

Placebo ϊroup

10361 1 95

1835 2 81

8211 3 86

590 5 80

Control

B31 - 285

ZS7 - 760

° as defined in Table 4.

*with human serum from patient 11838 Lyme 008 month 13. Conclusions. Analysis of the deduced amino acid sequences of the OspA genes from the clinical isolates shows that OspA variants which were found in the vaccinee group were also found in the placebo group; hence, the only clinical isolates for which no identical homolog was found in public databases occurred in the placebo group. When tested in a bacteridal assay, isolates from vaccinees and placebos showed equivalent susceptibility to the serum of a vaccine recipient. These findings, combined with Example 1 , confirm that the most likely explanation of the appearance of breakthrough cases among vaccinees is that infection occurred in those individuals who did not develop protective anti-OspA antibody titers.

While the preferred embodiments of the invention are illustrated by the above, it is to be understood that the invention is not limited to the precise instructions herein disclosed and that the right to all modifications coming within the scope of the following claims is reserved.

Claims

What is claimed is:

1. A method for protecting humans from infection by B. burgdorferi which comprises administering an OspA vaccine using a dosing schedule other than 30ug at 0, 1 and 12 months, such that the anti-OspA titer after the final dosing is at least 1000 El.U/ml.

2. The method of claim 1 wherein the dosing schedule is 30 ug at 0, 1, 2-11 months.

3. The method of claim 2 wherein the dosing schedule is 30 ug at 0, 1, 6 months.

4. The method of claim 2 wherein the dosing schedule is 30 ug at 0, 1, 2 months.

5. The method of claim 1 wherein the dosing schedule is 30 ug at 0, 7, 28 days.

6. The method of claim 1 wherein the dosing schedule is 60 ug at 0, 1 month.

7. The method of claim 1 wherein the dosing schedule is 60 ug at 0, 2 months.

8. The method of claim 1 wherein the anti-OspA titer after the final dosing is at least 1200 El.U/ml.

9. The method of claim 1 wherein the anti-OspA titer after the final dosing is at least 1400 El.U/ml.

10. A method for protecting humans from infection by B. burgdorferi which comprises administering lipoprotein OspA using a dosing schedule that results in anti-OspA titer after the primary dosing schedule of at least 1000 El.U/ml followed by boosting with OspA such that the anti-OspA titer after boosting is at least 1000 El.U/ml.

11. The method of claim 10 wherein the primary dosing schedule is 30 ug at 0, 1, 2 or 0, 1, 6 or 0, 1, 12 months followed by boosting at 30 ug at 12 months.

12. The method of claim 10 wherein the primary dosing schedule is 30 ug at 0, 1, 2 or 0, 1, 6 or 0, 1, 12 months followed by boosting at 30 ug at 24 months.

13. The method of claim 10 wherein the primary dosing schedule is 30 ug at 0, 1, 2 or 0, 1, 6 or 0, 1, 12 months followed by boosting at 30 ug at 36 months.

14. The method of claim 10 wherein boosting is repeated at least as frequently as once per each 12 month period or when the anti-OspA titer of a vaccinee is 400 El.U/ml or less.

15. The method of claim 10 wherein the anti-OspA titer after boosting is at least 1200 El.U/ml.

16. The method of claim 10 wherein the anti-OspA titer after boosting is at least 1400 El.U/ml.

17. The method of claim 1 wherein the patient is over 60 years old.

18. The method of claim 1 wherein the patient is from 15 and 60 years old.

19. The method of claim 1 wherein the patient is from 5 to 15 years old.

20. The method of claim 1 wherein the patient is from 2 to 5 years old.

21. The method of claim 1 wherein the patient is under 2 years old.

22. The method of claim 19 wherein the dosing schedule is 30 ug at 0, 1 month.

23. The method of claim 19 wherein the dosing schedule is 30 ug at 0, 1, 2 months.

24. The method of claim 19 wherein the dosing schedule is 15 ug at 0, 1,

2 months.

25. The method of claim 20 wherein the dosing schedule is 15 ug at 0, 1, 2 months.

26. The method of claim 20 wherein the dosing schedule is 15 ug at 0, 1, month.

27. The method of claim 1 wherein the anti-OspA titer of at least 1000

El.U/ml is sustained for at least 6 months.

28. The method of claim 1 wherein the anti-OspA titer of at least 1000 El.U/ml is sustained for at least 8 months.

29. The method of claim 1 wherein the anti-OspA titer of at least 1000 El.U/ml is sustained for at least 10 months.

30. A method for determining the dosing schedule for a lipoprotein OspA-containing vaccine for protecting humans from Lyme Disease which comprises testing a variety of dosing schedules on a population of test subjects and selecting a schedule, or schedules, that produces an anti-OspA titer of at least 1000 El.U/ml in at least 80% of the test subjects.

31. The method of claim 30 wherein the anti-OspA titer is at least 1200

El.U/ml in at least 80% of the test subjects.

32. The method of claim 30 wherein the anti-OspA titer is at least 1400 El.U/ml in at least 80% of the test subjects.

33. A method for determining the booster dosing schedule for a lipoprotein OspA-containing vaccine for protecting humans from Lyme Disease which comprises testing a variety of dosing schedules on a population of test subjects previously vaccinated with an OspA-containing vaccine and selecting a schedule, or schedules, that produces an anti-OspA titer of at least 1400 El.U/ml in at least 80% of the test subjects.

34. A method for protecting human beings from infection by B. burgdorferi which comprises: (1) administering a lipoprotein OspA-containing vaccine; (2) at least 6 months thereafter, checking the anti-OspA titer of the patient; and (3) if said titer is below 1000 El.U/ml, administering a booster dose.

35. The method of claim 34 wherein said booster dose is administered if the anti-OspA titer of the patient is at or below 400 El.U/ml.

36. A method of determining an OspA vaccine dosing schedule which comprises evaluating an anti-OspA antibody titer one month after a primary immunization schedule of an OspA lipoprotein and correlating said anti-OspA antibody titer with the level of protection from B. burgdorferi challenge in a vaccinated population.

37. The use of lipoprotein OspA in the manufacture of a vaccine composition for the prevention of Lyme disease in humans, which vaccine composition is administered using a dosing schedule other than 30ug at 0, 1 and 12 months, such that the anti-OspA titer after the final dosing is at least 1000 El.U/ml.

38. A use according to claim 37 wherein the dosing schedule is 30 ug at 0, 1, 2-11 months.

39. A use according to claim 38 wherein the dosing schedule is 30 ug at 0, 1, 6 months.

40. A use according to claim 38 wherein the dosing schedule is 30 ug at 0, 1, 2 months.

41. A use according to claim 37 wherein the dosing schedule is 30 ug at 0, 7, 28 days.

42. A use according to claim 37 wherein the dosing schedule is 60 ug at

0, 1 month.

43. A use according to claim 37 wherein the dosing schedule is 60 ug at 0, 2 months.

44. A use according to claim 37 wherein the dosing schedule is 90 ug at 0 and 1 month.

45. A use according to claim 37 wherein the dosing schedule is 120 ug at 0 and 1 month.

46. A use according to claim 37 wherein the anti-OspA titer after the final dosing is at least 1200 El.U/ml.

47. A use according to claim 37 wherein the anti-OspA titer after the final dosing is at least 1400 El.U/ml.

48. The use of lipoprotein OspA in the manufacture of a vaccine composition for the prevention of Lyme disease in humans, which vaccine composition is administered using a dosing schedule that results in anti-OspA titer after the primary dosing schedule of at least 1000 El.U/ml followed by boosting with OspA such that the anti-OspA titer after boosting is at least 1000 El.U/ml.

49. A use according to claim 48 wherein the primary dosing schedule is

30 ug at 0, 1, 2 or 0, 1, 6 or 0, 1, 12 months followed by boosting at 30 ug at 12 months.

50. A use according to claim 48 wherein the primary dosing schedule is 30 ug at 0, 1, 2 or 0, 1, 6 or 0, 1, 12 months followed by boosting at 30 ug at 24 months.

51. A use according to claim 48 wherein the primary dosing schedule is 30 ug at 0, 1, 2 or 0, 1, 6 or 0, 1, 12 months followed by boosting at 30 ug at 36 months.

52. A use according to claim 48 wherein boosting is repeated at least as frequently as once per each 12 month period or when the anti-OspA titer of a vaccinee is 400 El.U/ml or less.

53. A use according to claim 48 wherein the anti-OspA titer after boosting is at least 1200 El.U/ml.

54. A use according to claim 48 wherein the anti-OspA titer after boosting is at least 1400 El.U/ml.

55. A use according to claim 37 wherein the patient is over 60 years old.

56. A use according to claim 37 wherein the patient is from 15 and 60 years old.

57. A use according to claim 37 wherein the patient is from 5 to 15 years old.

58. A use according to claim 37 wherein the patient is from 2 to 5 years old.

59. A use according to claim 37 wherein the patient is under 2 years old.

60. A use according to claim 57 wherein the dosing schedule is 30 ug at 0, 1 month.

61. A use according to claim 57 wherein the dosing schedule is 30 ug at 0, 1, 2 months.

62. A use according to claim 57 wherein the dosing schedule is 15 ug at 0, 1, 2 months.

63. A use according to claim 58 wherein the dosing schedule is 15 ug at

0, 1, 2 months.

64. A use according to claim 58 wherein the dosing schedule is 15 ug at 0, 1, month.

65. A use according to claim 37 wherein the anti-OspA titer of at least 1000 El.U/ml is sustained for at least 6 months.

66. A use according to claim 37 wherein the anti-OspA titer of at least 1000 El.U/ml is sustained for at least 8 months.

67. A use according to claim 37 wherein the anti-OspA titer of at least 1000 El.U/ml is sustained for at least 10 months.

68. The use of lipoprotein OspA in the manufacture of a vaccine composition for the prevention of Lyme disease in humans, which vaccine composition is administered using a dosing schedule on a population of test subjects that produces an anti-OspA titer of at least 1000 El.U/ml in at least 80% of the test subjects.

69. A use according to claim 68 wherein the anti-OspA titer is at least 1200 El.U/ml in at least 80% of the test subjects.

70. A use according to claim 68 wherein the anti-OspA titer is at least 1400 El.U/ml in at least 80% of the test subjects.

71. A process for determining the booster dosing schedule for a lipoprotein OspA-containing vaccine for protecting humans from Lyme Disease which comprises testing a variety of dosing schedules on a population of test subjects previously vaccinated with an OspA-containing vaccine and selecting a schedule, or schedules, that produces an anti-OspA titer of at least 1400 El.U/ml in at least 80% of the test subjects.

72. A process for protecting human beings from infection by B. burgdorferi which comprises: (1) administering a lipoprotein OspA-containing vaccine; (2) at least 6 months thereafter, checking the anti-OspA titer of the patient; and (3) if said titer is below 1000 El.U/ml, administering a booster dose.

73. The process of claim 72 wherein said booster dose is administered if the anti-OspA titer of the patient is at or below 400 El.U/ml.

74. A process of determining an OspA vaccine dosing schedule which comprises evaluating an anti-OspA antibody titer one month after a primary immunization schedule of an OspA lipoprotein and correlating said anti-OspA antibody titer with the level of protection from B. burgdorferi challenge in a vaccinated population.

75. A pharmaceutical composition comprising lipoprotein OspA for use in the manufacture of a vaccine composition for the prevention of Lyme disease in humans, which vaccine composition is administered using a dosing schedule other than 30ug at 0, 1 and 12 months, such that the anti-OspA titer after the final dosing is at least 1000 El.U/ml.

76. A composition according to claim 75 wherein the dosing schedule is 30 ug at 0, 1, 2-11 months.

77. A composition according to claim 76 wherein the dosing schedule is 30 ug at 0, 1, 6 months.

78. A composition according to claim 76 wherein the dosing schedule is 30 ug at 0, 1, 2 months.

79. A composition according to claim 75 wherein the dosing schedule is 30 ug at 0, 7, 28 days.

80. A composition according to claim 75 wherein the dosing schedule is 60 ug at 0, 1 month.

81. A composition according to claim 75 wherein the dosing schedule is 60 ug at 0, 2 months.

82. A composition according to claim 75 wherein the dosing schedule is

90 ug at 0 and 1 month.

83. A composition according to claim 75 wherein the dosing schedule is 120 ug at 0 and 1 month.

84. A composition according to claim 75 wherein the anti-OspA titer after the final dosing is at least 1200 El.U/ml.

85. A composition according to claim 75 wherein the anti-OspA titer after the final dosing is at least 1400 El.U/ml.

86. A pharmaceutical composition comprising lipoprotein OspA for use in the manufacture of a vaccine composition for the prevention of Lyme disease in humans, which vaccine composition is administered using a dosing schedule that results in anti-OspA titer after the primary dosing schedule of at least 1000 El.U/ml followed by boosting with OspA such that the anti-OspA titer after boosting is at least 1000 El.U/ml.

87. A composition according to claim 86 wherein the primary dosing schedule is 30 ug at 0, 1, 2 or 0, 1, 6 or 0, 1, 12 months followed by boosting at 30 ug at 12 months.

88. A composition according to claim 86 wherein the primary dosing schedule is 30 ug at 0, 1, 2 or 0, 1, 6 or 0, 1, 12 months followed by boosting at 30 ug at 24 months.

89. A composition according to claim 86 wherein the primary dosing schedule is 30 ug at 0, 1, 2 or 0, 1, 6 or 0, 1, 12 months followed by boosting at 30 ug at 36 months.

90. A composition according to claim 86 wherein boosting is repeated at least as frequently as once per each 12 month period or when the anti-OspA titer of a vaccinee is 400 El.U/ml or less.

91. A composition according to claim 86 wherein the anti-OspA titer after boosting is at least 1200 El.U/ml.

92. A use according to claim 86 wherein the anti-OspA titer after boosting is at least 1400 El.U/ml.

93. A composition according to claim 75 wherein the patient is over 60 years old.

94. A composition according to claim 75 wherein the patient is from 15 and 60 years old.

95. A composition according to claim 75 wherein the patient is from 5 to 15 years old.

96. A composition according to claim 75 wherein the patient is from 2 to 5 years old.

97. A composition according to claim 75 wherein the patient is under 2 years old.

98. A composition according to claim 95 wherein the dosing schedule is 30 ug at 0, 1 month.

99. A composition according to claim 95 wherein the dosing schedule is

30 ug at 0, 1, 2 months.

100. A composition according to claim 95 wherein the dosing schedule is 15 ug at O, 1, 2 months.

101. A composition according to claim 96 wherein the dosing schedule is 15 ug at O, 1, 2 months.

102. A composition according to claim 96 wherein the dosing schedule is 15 ug at O, 1, month.

103. A composition according to claim 75 wherein the anti-OspA titer of at least 1000 El.U/ml is sustained for at least 6 months.

104. A composition according to claim 75 wherein the anti-OspA titer of at least 1000 El.U/ml is sustained for at least 8 months.

105. A composition according to claim 75 wherein the anti-OspA titer of at least 1000 El.U/ml is sustained for at least 10 months.

106. A pharmaceutical composition comprising lipoprotein OspA for use in the manufacture of a vaccine composition for the prevention of Lyme disease in humans, which vaccine composition is administered using a dosing schedule on a population of test subjects that produces an anti-OspA titer of at least 1000 El.U/ml in at least 80% of the test subjects.

107. A composition according to claim 106 wherein the anti-OspA titer is at least 1200 El.U/ml in at least 80% of the test subjects.

108. A composition according to claim 106 wherein the anti-OspA titer is at least 1400 El.U/ml in at least 80% of the test subjects.

109. The method of claim 1 wherein the dosing schedule is 90 ug at 0 and

1 month.

110. The method of claim 1 wherein the dosing schedule is 120 ug at 0 and 1 month.