Protection against tick-transmitted Lyme disease in dogs vaccinated with a multiantigenic vaccine

In an effort to develop a safe and effective vaccine for the prevention of Lyme borreliosis that addresses concerns raised over currently available vaccines, dogs were vaccinated twice with a multiantigenic preparation of Borrelia burgdorferi, strain N40, on days 0 and 20 of the experiment. About 70 and 154 days after the first immunization, dogs were challenged by exposing them to field-collected Ixodes scapularis ticks harboring B. burgdorferi. Vaccinated dogs were completely protected from infection by all criteria utilized to assess infection, developed high-titer anti-B. burgdorferi serum antibodies and growth inhibitory activity which persisted for over 200 days, and did not demonstrate any untoward consequence of vaccination. Serum absorption experiments revealed that borreliacidal and most likely protective antibodies in dogs receiving the multiantigenic preparation were not only elicited against the OspA antigen, but were also produced against additional yet to be determined targets on B. burgdorferi organisms. These data demonstrate that a multiantigenic vaccine is effective in preventing Lyme disease transmitted via the natural vector.     

Development of a baited oral vaccine for use in reservoir-targeted strategies against Lyme disease

Lyme disease is a major human health problem which continues to increase in incidence and geographic distribution. As a vector-borne zoonotic disease, Lyme disease may be amenable to reservoir targeted strategies for control. We have previously reported that a vaccinia virus (VV) based vaccine expressing outer surface protein A (OspA) of Borrelia burgdorferi, the causative agent of Lyme disease, protects inbred strains of laboratory mice against infection by feeding ticks and clears the ticks of infection when administered by gavage. Here we extend these studies to develop an effective bait formulation for delivery of the VV based vaccine and test its characteristics under simulated environmental conditions. We show that this vaccine is efficacious in decreasing acquisition of B. burgdorferi by uninfected larval ticks as well as in decreasing transmission from infected ticks to its natural reservoir, Peromyscus leucopus, when fed to mice in oral baits. Using live, in vivo imaging techniques, we describe the distribution of vaccinia virus infection after ingestion of the baited vaccines and establish the use of in vivo imaging technology for optimization of bait delivery. In summary, a VV based OspA vaccine is stable in an oral bait preparation and provides protection against infection for both the natural reservoir and the tick vector of Lyme disease.     

Vaccination against lyme disease with recombinant Borrelia burgdorferi outer-surface protein A (rOspA) in horses.

Eight 1-year-old ponies were vaccinated with recombinant OspA (ospA gene derived from B. burgdorferi B31) with adjuvant (aluminium hydroxide). Four ponies were used as non-vaccinated controls with adjuvant. One hundred and twelve days after the first vaccination, the vaccinated and non-vaccinated ponies were challenged by exposure to B. burgdorferi-infected adults tick (Ixodes scapularis) collected from Westchester County, New York (tick infection rate >/=60%). Protection from infection was evaluated by culture for B. burgdorferi from three monthly skin biopsies taken near the site of tick bites. B. burgdorferi was not isolated from any of the vaccinated ponies. In contrast, three of four control ponies challenged by tick exposure were skin culture positive. At the time of tick exposure, vaccinated ponies had antibody to B. burgdorferi demonstrable by KELA (kinetic-ELISA), western blot and a serum growth inhibition assay. Antibodies in the challenge control ponies were only detectable by two to three months after tick exposure and remained at intermediate levels until termination of the study. By western blot analysis, antibodies to OspA first appeared in the sera of vaccinated ponies three weeks after the first vaccination. The absence of additional bands, known to develop when the animal is infected, suggests that infection was blocked after tick exposure of vaccinated ponies. Results from this study show that vaccination with recombinant OspA protected ponies against infection after experimental challenge with B. burgdorferi-infected ticks.     

用聚合酶链方法对北京林区莱姆病疫源地及莱姆病病原体基因型的探索性研究

目的：进一步明确北京林区是否存在莱姆病的自然疫源地及其分布，方法基于莱姆病螺旋体外膜蛋白A基因建立半巢式聚合酶链式反应(polymerase chain reactio,PCR)方法，对从北京6个林区采集的蜱和鼠进行检测和基因分型，选择阳性标准本进行克隆和序列测定，与已知序进行同源性比较，间接免疫荧光法检测抗莱姆病螺旋体IgG抗体，从长角血蜱中分离莱姆病螺旋体。结果：从门头沟区东灵山采集的标本中检测到莱姆病螺旋体DNA片段，3只游离全沟硬蜱1只检测阳性，57只寄全沟硬蜱若蜱中1只检测阳性；119只野鼠中9只检测阳性，其中8只，B.garinii阳性，1只B.afzelii阳性。50份野鼠血清有5份莱姆病螺旋体IgG抗体阳性，采集采的160只长角血蜱（20只／组）。未分离到莱姆病螺旋体菌株。结论：北京门头沟区东灵山可能存在莱姆病的自然疫源地，包括两个基因型，全沟硬蜱可能是莱姆病的传播媒体，野鼠可能是贮存宿主。     

伯氏疏螺旋体传播过程中病原体、媒介、宿主间的相互作用

莱姆病是一种蜱媒传播的人兽共患病，在北半球广泛流行。其病原体是伯氏疏螺旋体，具有极为复杂的传播机制和过程。在过去20多年中，‘科学家们对此进行了广泛而深入的探索。本综述拟对近5年这方面的研究进展，尤其是Fikrig实验室的工作进行总结。内容主要包括：证明伯氏疏螺旋体外膜蛋白OspA是一种粘附素（adhesion），对螺旋体在硬蜱中肠（midgut）定居至关重要，并发现硬蜱中肠肠腔表面蛋白TROSPA是OspA的受体；发现蜱唾液腺蛋白Salp15协助伯氏疏螺旋体感染哺乳类动物；发现伯氏疏螺旋体膜蛋白bmpA和bmpB介导螺旋体在关节的定居及关节炎的发生。这些发现有助于我们深入了解莱姆病的传播特征和致病机制。     

Immunogenicity of the Lyme disease antigen OspA,particleized by cobalt porphyrin-phospholipid liposomes

Outer surface protein A (OspA) is a Borrelia lipoprotein and an established Lyme disease vaccine target. Admixing non-lipidated, recombinant B. burgdorferi OspA with liposomes containing cobalt porphyrin-phospholipid (CoPoP) resulted in rapid, particulate surface display of the conformationally intact antigen. Particleization was serum-stable and led to enhanced antigen uptake in murine macrophages in vitro. Mouse immunization using CoPoP liposomes that also contained a synthetic monophosphoryl lipid A (PHAD) elicited a Th1-biased OspA antibody response with higher IgG production compared to other vaccine adjuvants. Antibodies were reactive with intact B. burgdorferi spirochetes and Borrelia lysates, and induced complement-mediated borreliacidal activity in vitro. One year after initial immunization, mice maintained high levels of circulating borreliacidal antibodies capable of blocking B. burgdorferi transmission from infected ticks to human blood in a feeding chamber.     

Identification of a defined linear epitope in the OspA protein of the Lyme disease spirochetes that elicits bactericidal antibody responses: Implications for vaccine development

The lipoprotein OspA is produced by the Lyme disease spirochetes primarily in unfed ticks. OspA production is down-regulated by the blood meal and it is not produced in mammals except for possible transient production during late stage infection in patients with Lyme arthritis. Vaccination with OspA elicits antibody (Ab) that can target spirochetes in the tick midgut during feeding and inhibit transmission to mammals. OspA was the primary component of the human LYMErix™ vaccine. LYMErix™ was available from 1998 to 2002 but then pulled from the market due to declining sales as a result of unsubstantiated concerns about vaccination induced adverse events and poor efficacy. It was postulated that a segment of OspA that shares sequence similarity with a region in human LFA-1 and may trigger putative autoimmune events. While evidence supporting such a link has not been demonstrated, most efforts to move forward with OspA as a vaccine component have sought to eliminate this region of concern. Here we identify an OspA linear epitope localized within OspA amino acid residues 221–240 (OspA_221–240) that lacks the OspA region suggested to elicit autoimmunity. A peptide consisting of residues 221–240 was immunogenic in mice. Ab raised against OspA_221–240 peptide surface labeled B. burgdorferi in IFAs and displayed potent Ab mediated-complement dependent bactericidal activity. BLAST analyses identified several variants of OspA_221–240 and a closely related sequence in OspB. It is our hypothesis that integration of the OspA_221–240 epitope into a multivalent-OspC based chimeric epitope based vaccine antigen (chimeritope) could result in a subunit vaccine that protects against Lyme disease through synergistic mechanisms.     

Risk of Lyme disease: perceptions of residents of a Lone Star tick-infested community.

BACKGROUND: Lone Star ticks (Amblyomma americanum) have been suggested as a vector of the agent of Lyme disease (Borrelia burgdorferi sensu lato) in the USA, based on associations with an infection manifesting mainly as erythema migrans. In laboratory experiments, however, they failed to transmit B. burgdorferi sensu stricto. METHODS: In this study, carried out from 1994 to 1996, we determined the seroprevalences of B. burgdorferi (1.2%), Ehrlichia chaffeensis (7%), E. phagocytophila (0%), Rickettsia rickettsii (0%), R. typhi (0%), Coxiella burneti (0%), Francisella tularensis (0%), and Babesia microti (0%) by standard serological methods for 325 residents (97% of the total population) of Gibson Island, coastal Maryland, USA, where 15% of the residents reported having had Lyme disease within a recent 5-year span. FINDINGS: Of the 167 seronegative individuals who were followed up prospectively for 235 person-years of observation, only 2 (0.85%) seroconverted for B. burgdorferi. Of 1556 ticks submitted from residents, 95% were identified as Lone Star ticks; only 3% were deer ticks (Ixodes dammini), the main American vector of Lyme disease. B. burgdorferi s.s. infected 20% of host-seeking immature deer ticks, and borreliae ("B. lonestari") were detected in 1-2% of Lone Star ticks. Erythema migrans was noted in 65% of self-reports of Lyme disease, but many such reports indicated that the rash was present while the tick was still attached, suggesting a reaction to the bite itself rather than true Lyme disease. Sera from individuals reporting Lyme disease generally failed to react to B. burgdorferi or any other pathogen antigens. CONCLUSION: The residents of Gibson Island had an exaggerated perception of the risk of Lyme disease because they were intensely infested with an aggressively human-biting and irritating nonvector tick. In addition, a Lyme disease mimic of undescribed etiology (named Masters' disease) seems to be associated with Lone Star ticks, and may confound Lyme disease surveillance. The epidemiological and entomological approach used in this study might fruitfully be applied wherever newly emergent tickborne zoonoses have been discovered.     

Oral vaccine that breaks the transmission cycle of the Lyme disease spirochete can be delivered via bait

Borrelia burgdorferi causes Lyme disease, a potentially debilitating human disease for which no vaccine is currently available. We developed an oral bait delivery system for an anti-B. burgdorferi vaccine based in OspA. Mice were immunized orally via gavage and bait feeding. Challenge was performed via Ixodes scapularis field nymphs carrying multiple B. burgdorferi strains. Vaccination protected 89% of the mice and the systemic immune response was skewed toward IgG2a/2b production. Moreover, this oral vaccine reduced the pathogen in the tick vector by eight-fold. We conclude that this oral vaccine induces a protective systemic immune response against a variety of infectious B. burgdorferi strains found in nature and therefore it can eliminate this zoonotic pathogen from its major host reservoirs. Because we observed elimination of the spirochete from the tick vector, a broad delivery of this oral vaccine to wildlife reservoirs is likely to disrupt the transmission cycle of this pathogen.     

Evaluation of Venezuelan Equine Encephalitis (VEE) replicon-based Outer surface protein A (OspA) vaccines in a tick challenge mouse model of Lyme disease

Venezuelan Equine Encephalitis (VEE) virus replicon particles (VRPs) encoding Borrelia burgdorferi Outer surface protein A (OspA) were evaluated for their ability to induce an immune response and provide protection from tick-borne spirochetes. VRPs expressing ospA that accumulated intracellularly (VRP OspA) or that was secreted from host cells (VRP tPA-OspA) were tested. Both VRP OspA and VRP tPA-OspA expressed ospA in immunized mice. Mice vaccinated with VRPs expressing secreted OspA produced significant amounts of anti-OspA antibodies, whereas VRPs expressing intracellular OspA were less immunogenic. The VRP method of delivery induced a Th1 type immune response unlike the recombinant OspA protein in Freund's adjuvant, which induced a mixed (Th1 and Th2) immune response. The VRP tPA-OspA construct induced an immune response that reduced the bacterial load in feeding Ixodes scapularis and blocked transmission to the host. These results indicate that VRPs are capable of providing protection against tick-borne B. burgdorferi, and potentially can be used for developing improved vaccines against Lyme disease.     

The outer surface protein A (OspA) vaccine against Lyme disease: efficacy in the rhesus monkey

The efficacy of an outer surface protein A (OspA) vaccine in three different formulations was investigated in the rhesus monkey. The challenge infection was administered using Ixodes scapularis ticks that were infected with the B31 strain of Borrelia burgdorferi. Protection was assessed against both infection and disease, by a variety of procedures. Some of the animals were radically immune suppressed, as an attempt to reveal any putative low level infection in the vaccinated animals. The significant difference found between the spirochaetal infection rates of ticks that had fed on vaccinated vs. control monkeys, lack of seroconversion in the vaccinated animals, and the absence of spirochaetal DNA in the skin of vaccinated animals in the weeks following the challenge, indicate that vaccinated monkeys were protected against tick challenge. The post-mortem immunohistochemical and polymerase chain reaction analyses, however, suggest that these monkeys may have undergone a low-level infection that was transient.     

Vaccination with meningococcal outer membrane vesicles carrying Borrelia OspA protects against experimental Lyme borreliosis

Currently there is no human vaccine against Lyme borreliosis, and most research focuses on recombinant protein vaccines, as such a vaccine has been proven to be successful in the past. The expression of recombinant antigens in meningococcal Outer Membrane Vesicles (OMVs), with the OMV functioning both as adjuvant and delivery vehicle, greatly enhances their potential. Immunization studies in mice have shown that OMV-based vaccines can protect against various pathogens and an OMV-based meningococcal vaccine is approved and available for human use. Because of its surface localization in Borrelia and the detailed knowledge regarding its immunogenicity and structure, OspA was chosen as a suitable lipoprotein to be tested as an OMV-based vaccine against Lyme borreliosis. We have previously shown that the OMV-OspA vaccine was immunogenic in mice and here we assessed the efficacy of OMV-OspA.We generated a second-generation OMV-OspA vaccine and vaccinated C3H/HeN mice with (EDTA extracted) meningococcal OMVs expressing OspA from B. burgdorferi strain B31. The adjuvant effect of empty OMVs on recombinant OspA was tested as well. We subsequently challenged mice with a subcutaneous injection of B. burgdorferi.Average antibody end-point titers against the OspA-OMV construct were high, although lower compared to the antibodies raised against recombinant OspA. Interestingly, antibody titers between recombinant OspA adjuvanted with aluminum hydroxide and recombinant OspA with OMV as adjuvant were comparable. Finally, qPCR and culture data show that both the OspA-OMV and the vaccine based on recombinant OspA with OMV as adjuvant provided significant, yet partial protection, against Borrelia infection.OMV-based vaccines using Borrelia (lipo)proteins are an easy and feasible vaccination method protecting against B. burgdorferi infection and could be a promising strategy in humans.     

Oral vaccination with vaccinia virus expressing the tick antigen subolesin inhibits tick feeding and transmission of Borrelia burgdorferi

Immunization with the Ixodes scapularis protein, subolesin, has previously been shown to protect hosts against tick infestation and to decrease acquisition of Anaplsma marginale and Babesia bigemina. Here we report the efficacy of subolesin, a conserved tick protein that can act as a regulator of gene expression, expressed from vaccinia virus for use as an orally delivered reservoir - targeted vaccine for prevention of tick infestation and acquisition/transmission of Borrelia burgdorferi to its tick and mouse hosts. We cloned subolesin into vaccinia virus and showed that it is expressed from mammalian cells infected with the recombinant virus in vitro. We then vaccinated mice by oral gavage. A single dose of the vaccine was sufficient for mice to generate antibody response to subolesin. Vaccination with the subolesin expressing vaccinia virus inhibited tick infestation by 52% compared to control vaccination with vaccinia virus and reduced uptake of B. burgdorferi among the surviving ticks that fed to repletion by 34%. There was a reduction in transmission of B. burgdorferi to uninfected vaccinated mice of 40% compared to controls. These results suggest that subolesin has potential as a component of a reservoir targeted vaccine to decrease B. burgdorferi, Babesia and Anaplasma species infections in their natural hosts.     

Modulatory effect of cattle on risk for lyme disease

To determine the effect of cattle on the risk for Lyme disease, we compared the prevalence of spirochete infection in questing vector ticks collected from a pasture with low-intensity cattle grazing with the prevalence in those collected from a site on which no cattle grazed. The presence of cattle limited the prevalence of Borrelia burgdorferi s.l., but not B. miyamotoi, in vector ticks. The reintroduction of traditional, nonintensive agriculture in central Europe may help reduce risk for Lyme disease.     

Epidemiological and aetiological evidence for transmission of Lyme disease by adult Ixodes persulcatus in an endemic area in China

Involvement of adult Ixodes persulcatus ticks in the transmission of Lyme disease in Hailin County, Heilongjiang Province, China, is reported. In 1986 from April through August adult I. persulcatus was the dominant tick in this endemic area with an infection rate of 43% for the Lyme disease spirochaete, Borrelia burgdorferi. The incidence of Lyme disease cases presenting the symptom of erythema chronicum migrans (ECM) within this area was correlated with the seasonal abundance of adult I. persulcatus and the number of people bitten by ticks. The frequency of ECM formation in all age groups varied and was associated with the frequency of tick bites. In addition, a strain of B. burgdorferi was isolated from a pool of six female I. persulcatus collected from this area. We demonstrate that the seasonal abundance of adult I. persulcatus and its frequent attachment to humans result in the spring and summer transmission of Lyme disease in this endemic area. The role of immature I. persulcatus in Lyme disease transmission is apparently minimal.     

Vaccination of horses with Lyme vaccines for dogs induces short-lasting antibody responses

Borrelia burgdorferi can induce Lyme disease. Approved Lyme vaccines for horses are currently not available. In an effort to protect horses, veterinarians are using Lyme vaccines licensed for dogs. However, data to assess the response of horses to, or determine the efficacy of this off-label vaccine use are missing. Here, antibodies against outer surface protein A (OspA), OspC, and OspF were quantified in diagnostic serum submissions from horses with a history of vaccination with canine Lyme vaccines. The results suggested that many horses respond with low and often short-lasting antibody responses. Subsequently, four experimental vaccination trials were performed. First, we investigated antibody responses to three canine vaccines in B. burgdorferi-naïve horses. One killed bacterin vaccine induced antibodies against OspC. OspA antibodies were low for all three vaccines and lasted less than 16 weeks. The second trial tested the impact of the vaccine dose using the OspA/OspC inducing bacterin vaccine in horses. A 2 mL dose produced higher OspA and OspC antibody values than a 1 mL dose. However, the antibody response again quickly declined, independent of dose. Third, the horses were vaccinated with 2 doses of a recombinant OspA vaccine. Previous vaccination and/or environmental exposure enhanced the magnitude and longevity of the OspA antibody response to about 20 weeks. Last, the influence of intramuscular versus subcutaneous vaccine administration was investigated for the recombinant OspA vaccine. OspA antibody responses were not influenced by injection route. The current work highlights that commercial Lyme vaccines for dogs induce only transient antibody responses in horses which can also be of low magnitude. Protection from infection with B. burgdorferi should not be automatically assumed after vaccinating horses with Lyme vaccines for dogs.     

Lyme disease--United States, 2003-2005

Lyme disease is caused by the spirochete Borrelia burgdorferi and is transmitted to humans by the bite of infected blacklegged ticks (Ixodes spp.). Early manifestations of infection include fever, headache, fatigue, and a characteristic skin rash called erythema migrans. Left untreated, late manifestations involving the joints, heart, and nervous system can occur. A Healthy People 2010 objective (14-8) is to reduce the annual incidence of Lyme disease to 9.7 new cases per 100,000 population in 10 reference states where the disease is endemic (Connecticut, Delaware, Maryland, Massachusetts, Minnesota, New Jersey, New York, Pennsylvania, Rhode Island, and Wisconsin). This report summarizes surveillance data for 64,382 Lyme disease cases reported to CDC during 2003-2005, of which 59,770 cases (93%) were reported from the 10 reference states. The average annual rate in these 10 reference states for the 3-year period (29.2 cases per 100,000 population) was approximately three times the Healthy People 2010 target. Persons living in Lyme disease--endemic areas can take steps to reduce their risk for infection, including daily self-examination for ticks, selective use of acaricides and tick repellents, use of landscaping practices that reduce tick populations in yards and play areas, and avoidance of tick-infested areas.     

中俄,中哈边境地区莱姆病自然疫源地调查研究

目的：调查边境地区莱杂姆病和自然疫源地的状况。方法;对血清学、病原学、宿主、媒介,病例和地理景观方面进行调查。结果：用ＩＦＡ和ＥＬＩＳＡ法共检测人畜血清１５０１份,从１９９份人血清中检出抗体阳性１８份,阳性率为９．０５％,并发现莱姆病典型患者１５例;检查马血清２０份,阳性５份,阳性率为２５．００％;检查牛血清３９９份,阳性６９份,阳性率１７．２９％;检查羊血清８８３份,阳性１９９份,阳性率２２．５     

Characterization and optimization of a novel vaccine for protection against Lyme borreliosis

Lyme borreliosis (LB) is the most common vector-borne disease in the northern hemisphere and there is no vaccine available for disease prevention. The majority of LB cases in Europe are caused by four different Borrelia species expressing six different OspA serotypes, whereas in the US only one of these serotypes is present. Immunization with the outer surface protein A (OspA) can prevent infection and the C-terminal part of OspA is sufficient for protection against infection transmitted by Ixodes ticks. Here we show that the order of the stabilized monomeric OspA fragments making up the heterodimers in our LB vaccine does not influence the induced immunogenicity and protection. Using bioinformatics analysis (surface electrostatics), we have designed an improved version of an LB vaccine which has an increased immunogenicity for OspA serotype 3 and an optimized expression and purification profile. The OspA heterodimers were highly purified with low amounts of endotoxin, host cell proteins and host cell DNA. All three proteins were at least 85% triacylated which ensured high immunogenicity. The LB vaccine presented here was designed, produced and characterized to a level which warrants further development as a second generation human LB vaccine.     

Hypersensitivity to ticks and Lyme disease risk

Although residents of Lyme disease-endemic regions describe frequent exposure to ticks, Lyme disease develops in relatively few. To determine whether people who experience cutaneous hypersensitivity against tick bite have fewer episodes of Lyme disease than those who do not, we examined several factors that might restrict the incidence of Lyme disease among residents of Block Island, Rhode Island. Of 1,498 study participants, 27% (95% confidence interval [CI] 23%-31%) reported > or = 1 tick bites, and 17% (95% CI 13%-21%) reported itch associated with tick bite in the previous year. Borrelia burgdorferi infected 23% (95% CI 20%-26%) of 135 nymphal Ixodes scapularis (I. dammini) ticks. The likelihood of Lyme disease infection decreased with >3 reports of tick-associated itch (odds ratio 0.14, 95% CI 0.94-0.03, p = 0.01). Prior exposure to uninfected vector ticks protects residents of disease-endemic sites from Lyme disease.