Vaccination of horses with Lyme vaccines for dogs induces short ...

17 downloads 168 Views 1MB Size Report
Horse. OspA. a b s t r a c t. Borrelia burgdorferi can induce Lyme disease. Approved .... A total of 47 Icelandic horses of the specific-pathogen-free herd at Cornell ...
Vaccine xxx (2017) xxx–xxx

Contents lists available at ScienceDirect

Vaccine journal homepage: www.elsevier.com/locate/vaccine

Vaccination of horses with Lyme vaccines for dogs induces short-lasting antibody responses Cassandra Guarino a, Sanda Asbie a, Jennifer Rohde a, Amy Glaser a, Bettina Wagner a,⇑ a

Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA

a r t i c l e

i n f o

Article history: Received 3 March 2017 Received in revised form 15 May 2017 Accepted 15 June 2017 Available online xxxx Keywords: Lyme disease Borrelia burgdorferi Lyme vaccine Horse OspA

a b s t r a c t 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. Ó 2017 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction Lyme disease is induced by Borrelia burgdorferi, a spirochete that is transmitted to humans, dogs, horses, and other mammals by Ixodes ticks [1–3]. Controversial opinions exist about the occurrence of Lyme disease in horses. Clinical signs associated with Lyme disease include lameness and behavioral changes which are rather non-specific and can have different causes. Experimental infection inducing the disease in horses has not yet been reported. High seroprevalence in clinically healthy horses in endemic areas further adds to the controversy [2]. Nevertheless, accumulating evidence supports Lyme disease in horses infected with B. burgdorferi. Most commonly reported clinical signs are sporadic lameness and diverse orthopedic problems, often associated with shifting or multiple leg lameness, muscle tenderness, hyperesthesia, chronically poor performance, swollen joints, or arthritis [4,5]. Less ⇑ Corresponding author.

frequently described are cases of Borrelia-associated pseudolymphoma [6], uveitis [7], and neurologic signs including ataxia, depression, head tilt and encephalitis [8–10]. The diagnosis of Lyme disease in horses is based on clinical signs compatible with the disease in the absence of other causes, potential exposure to ticks, and positive serological testing for antibodies to B. burgdorferi [5]. Response to treatment alone is not sufficient to confirm diagnosis due to the anti-inflammatory effects of commonly used antibiotics [11], but follow-up antibody testing post-treatment and decline of B. burgdorferi-specific antibodies can support the diagnosis [5]. Antibody testing for B. burgdorferi has improved in recent years, with a quantitative Lyme Multiplex assay becoming available. The Lyme Multiplex assay can distinguish between early and chronic infection stages, via antibodies against outer surface protein C (OspC) and OspF, as well as measure responses to vaccination by quantification of OspA antibodies [12,13]. In addition, OspF of B. burgdorferi is the preferred antigen for confirmation of chronic infection in horses because antibody

E-mail address: [email protected] (B. Wagner). http://dx.doi.org/10.1016/j.vaccine.2017.06.052 0264-410X/Ó 2017 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article in press as: Guarino C et al. Vaccination of horses with Lyme vaccines for dogs induces short-lasting antibody responses. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.06.052

2

C. Guarino et al. / Vaccine xxx (2017) xxx–xxx

responses to OspF are more widely distributed than those to C6, thereby allowing improved quantification [12]. Currently, prevention of Lyme disease is primarily focused on decreasing risk of exposure to ticks. However, vector control methods are typically not sufficient on their own, leading veterinarians to utilize Lyme vaccines [5]. Approved Lyme vaccines for horses are not currently available, despite evidence that OspA antibodies may protect horses from infection [14]. Several Lyme vaccines are currently approved in the US for use in dogs, some of which have been given off-label to horses. Most of the canine vaccines are wholebacterin vaccines. One is composed of recombinant OspA only, which is known to be self-adjuvanting [15] and was shown to be safe and efficacious in dogs [16]. The serologic response to successful Lyme vaccination is typically characterized by high values for antibodies to the OspA antigen. High amounts of OspA are expressed when B. burgdorferi is cultured in vitro [17,18], leading to bacterin-based vaccines containing this immunogenic protein. Anti-OspA antibodies have been shown to protect from infection by inhibiting transmission of B. burgdorferi from the tick vector to mammalian hosts, as frequently described in humans [19,20], laboratory rodents [21,22], dogs [23,24], and also in horses [14]. The OspA protein of B. burgdorferi is essential for colonization and survival of the bacteria within the tick midgut [25,26]. During transmission to the mammalian host, OspA becomes downregulated in the bacteria, followed by swift up-regulation of OspC [27]. Bacterial strains that produce elevated levels of OspC in culture have been developed for use in vaccines [28]. The OspC protein plays a role in bacterial invasion of the tick salivary gland and transmission of bacteria is assumed to be partially inhibited if the tick blood meal contains anti-OspC antibodies [29,30]. There is some evidence that antibodies against OspC may also have bacteriocidal effects [31,32], but to the author’s knowledge, there is no direct evidence that anti-OspC antibodies offer protection from B. burgdorferi infection in horses. The goal of this work was to evaluate the serologic response in horses to vaccination with Lyme vaccines for dogs. We first evaluated diagnostic samples submitted by veterinarians in the field, and then investigated antibody responses induced by these vaccines after a series of experimental vaccinations in a controlled herd of horses. 2. Materials and methods

vaccination study. Lyme disease has not been reported in Iceland [35]. At the time of importation, horses were thus considered Lyme-free and immunologically naïve to B. burgdorferi. After release from importation quarantine, horses were kept on pasture all year long. The pasture area was surrounded by woods and frequently visited by wildlife. Grass hay was fed ad libitum. Horses were annually vaccinated against rabies, tetanus, West Nile Virus, Eastern and Western Encephalitis, and were regularly dewormed. Horses were clinically healthy throughout the studies. A summary of age, gender and vaccination groups of the horses in the four experimental vaccine trials is shown in Table 1. 2.2. Experimental vaccination approach and blood sample collection Unless stated otherwise, vaccinations were performed intramuscularly (IM) into the pectoral muscle using 20-gauge needles. Blood samples for serum collection were obtained by jugular venipuncture with a vacutainer collection system without coagulant. If horses were vaccinated on a specific day, blood samples were taken directly before vaccination. Serum was harvested from clotted blood by centrifugation at 700g for 10 min and was stored at 20 °C until serological analysis. All animal procedures for this study were carried out in accordance with the recommendation in the Guide for the Care and Use of Laboratory Animals of the National Institute of Health. The animal protocol was approved by the Institutional Animal Care and Use Committee, Cornell University (protocol #2011-0011). 2.2.1. Vaccination trial #1 – vaccine comparison Lyme naïve horses in the 1st experimental Lyme vaccination trial received their first Lyme vaccine dose in the quarantine barn immediately before they were released to the pasture in April 2013. Initially, we compared the serologic response of B. burgdorferi naïve horses to the three US commercial canine Lyme vaccines available in 2013 (Supplementary Table 1): Duramune LymeÒ (lot#: 522108A), RecombitekÒ Lyme (lot#: 42180A), and NobivacÒ Lyme (lot#: 02161126). Seven horses were assigned to each group (Table 1). One animal from the NobivacÒ Lyme group had to be removed from the trial due to reasons unrelated to this study and was not included in the analysis. Animals received a 1 mL dose of vaccine on days 0, 25, and 108. An additional 9 horses were not vaccinated and monitored as a control group.

2.1. Horses and horse environment 2.1.1. Diagnostic samples Serum samples from horses at multiple locations in the US were submitted by veterinarians for Lyme Multiplex testing to the Animal Health Diagnostic Center (AHDC) at Cornell University between July 2011 and July 2014. Additional information on vaccination status was obtained from the respective equine practice when the submission form indicated ‘vaccination against Lyme disease’, including date and manufacturer of the most recent vaccine administered. Horses were included in this study if the most recent vaccination was at least one and less than ten months prior to sample submission. This resulted in 65 samples from horses vaccinated with Duramune LymeÒ (Boehringer Ingelheim Vetmedica, Inc., St. Joseph, MO), 105 samples from horses vaccinated with RecombitekÒ Lyme (Merial Inc., Duluth, GA). Pre-vaccination serology and infection history was not available for these animals. 2.1.2. Horses for experimental vaccination trials A total of 47 Icelandic horses of the specific-pathogen-free herd at Cornell University were enrolled in this study [33,34]. All horses were kept at this facility before and during the experimental

2.2.2. Vaccination trial #2 – dose comparison Some practitioners double the dose of the dog vaccine when used in horses. Trial 2 evaluated the impact of dose on the magnitude and duration of antibody responses to vaccination. Yearlings and two-year-old horses received their first Lyme vaccination in April 2014. Prior to this vaccination, they were kept on pasture with potential exposure to B. burgdorferi infected ticks for one or two years, respectively. NobivacÒ Lyme vaccine (lot#: 02161130 for the initial vaccination [day 0]; lot#: 02161131 for the boost [day 24]) was used for vaccination. The 27 foals were randomly assigned into two age-matched groups. Foals received 1 mL (n = 13) or 2 mL (n = 14) of vaccine per injection. An additional 5 horses were vaccinated and monitored as a control group. 2.2.3. Vaccination trial #3 – impact of previous Lyme vaccination In 2015, one year following vaccination trial #2, 18 of the horses were vaccinated with 1 mL RecombitekÒ Lyme (lot#: 42198) on day 0 and day 28 to analyze if previous vaccination impacted magnitude and duration of serologic responses to vaccination. Another 9 horses vaccinated with NobivacÒ Lyme the previous year were not vaccinated and monitored as a control group.

Please cite this article in press as: Guarino C et al. Vaccination of horses with Lyme vaccines for dogs induces short-lasting antibody responses. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.06.052

3

C. Guarino et al. / Vaccine xxx (2017) xxx–xxx Table 1 Experimental vaccination trials. Overview about the experimental vaccination trials of horses with Lyme vaccines for dogs. Trial

1

2 3 4

Vaccination

Horses

Vaccine

Doses

Vol per dose (ml)

Route

N per group

Median age (range)

Gender (mares, geldings)

Previously vaccinated

RecombitekÒ Lyme NobivacÒ Lyme DuramuneÒ Lyme NobivacÒ Lyme

3

1 ml

i.m.

i.m.

2

1 ml 2 ml 1 ml

1

1 ml

s.c. i.m.

2 2 2 1 1 2 2 3 3 3

3, 2, 2, 7, 8, 8, 7, 2, 4, 7,

no

2

7 6 7 13 14 18 9 7 7 8

RecombitekÒ Lyme Not vaccinated RecombitekÒ Lyme

i.m.

Not vaccinated

2.2.4. Vaccination trial #4 – influence of injection route Finally, the influence of injection site, subcutaneous (SC) vs. IM, on the immune response was compared in previously vaccinated horses. In 2016, a single 1 mL dose of RecombitekÒ Lyme (lot#: 42203) was either injected IM into the pectoral muscle (n = 7), or SC into the subcutis on top of the pectoral muscle (n = 7) on day 0. All vaccinated horses received two doses of 1 mL RecombitekÒ Lyme IM in the previous year. Eight horses from the previous year’s control group continued to be non-vaccinated and monitored. 2.3. Tick monitoring, collection and PCR The horses were examined for reasons unrelated to this study (allergy scoring) every week in spring, summer and fall, and 2–3 times per month in the winter. They were touched and examined at the face, submandibular space, chest, belly, and other body areas during the allergy scoring. As soon as the first tick was found, the horses were systematically checked for ticks by thoroughly running hands around the ears, nose, chin, along the submandibular region, down the neck, along the chest, and between the front legs and the girth area. Although single ticks may have been missed occasionally by this procedure, we have examined the horses regularly throughout the years of the experimental Lyme vaccination studies described here to identify the major tick activity times and to estimate the percentages of B. burgdorferi infected ticks in their environment. The times when ticks were collected from the horses were in spring (April/May) and fall (October/November). Ticks collected from horses were stored in 70% ethanol at 4 °C until PCR analysis. All collected ticks identified as Ixodes scapularis were analyzed for B. burgdorferi DNA by PCR at the AHDC, Cornell University. 2.4. Serological detection of antibodies against B. burgdorferi All serum samples were measured for antibodies to OspA, OspC and OspF by the Lyme Multiplex assay at the AHDC, Cornell University, as described previously [13]. 2.5. Statistical analysis The comparison of antibody values between the different vaccine groups was performed using repeated-measures ANOVA with

(1–10) (1–8) (1–8) (1–2) (1–2) (2–3) (2–3) (3–4) (3–4) (3–4)

4 4 5 6 6 10 2 5 3 1

no yes (trial 2) yes (trials 2 & 3) yes (trial 2)

Tukey’s multiple comparison tests and the GraphPad Prism program version 6. P-values (p < 0.05) were considered significant. 3. Results and discussion 3.1. OspA antibodies in diagnostic samples from vaccinated horses Veterinarians interested in monitoring vaccine responses or concerned about possible infection with B. burgdorferi routinely submit samples for the Lyme Multiplex assay. Samples from horses vaccinated between one and nine months prior to sample submission were analyzed (Table 2). The most common dog vaccines administered to horses to protect against Lyme disease were RecombitekÒ Lyme, followed by Duramune LymeÒ. OspA antibody values of similar magnitude were observed in both groups of vaccinated horses 1–3 months post-vaccination, but less than 65% of all vaccinated horses demonstrated OspA antibody values > 2000 MFI, the positive cut-off value of the assay, in this time-frame. Horses vaccinated with RecombitekÒ Lyme had higher OspA antibody values 4–6 months post-vaccination than horses vaccinated with Duramune LymeÒ (p = 0.0196). Nevertheless, the differences in OspA antibody response should not be exclusively interpreted as response to vaccination. Pre-vaccination OspA antibody values and possible infection history were missing for these horses. Experimental infection in dogs has shown that OspA antibodies can be transiently induced after infection [36]. This supports that the highly immunogenic OspA is not completely down-regulated from the bacterial surface at the time of infecting the mammalian host. In addition, OspA antibodies have been observed in nonvaccinated horses by practitioners in Lyme endemic areas. Although this information has not yet been systematically investigated or reported, the manifestation of an OspA antibody response in a non-vaccinated horse could be a result of frequent exposure to infected ticks and residual OspA on the spirochete surface, which provides a repeated stimulation of the immune response. For the sample set evaluated here, the majority of horses no longer had OspA antibody values in the positive range by 7–9 months postvaccination (Table 2). This data is in stark contrast to a similar data set collected from dog diagnostic samples, where > 97% (43/44) of dogs developed antibody values > 2000 MFI 1–3 months post-vaccination, and > 75% (21/25) maintained OspA antibody

Table 2 Horse diagnostic submissions. Serum antibodies against B. burgdorferi OspA (MFI) of horses by months after vaccination with commercial Lyme vaccines for dogs. MFI > 2000 is the Equine Lyme Multiplex assay positive cut-off value for antibodies against OspA. Months post vaccination

Whole bacterin vaccine (Duramune LymeÒ)

Recombinant OspA vaccine (RecombitekÒ Lyme)

N

Median anti-OspA value (range)

Samples with antiOspA > 2000

N

Median anti-OspA value (range)

Samples with antiOspA > 2000

1–3 4–6 7–9

18 30 17

1563 (453–10733) 469 (54–4685) 514 (97–5864)

9/18 4/30 1/17

51 38 16

3197 (230–25976) 2385 (402–23808) 1582 (415–16816)

33/51 22/38 7/16

Please cite this article in press as: Guarino C et al. Vaccination of horses with Lyme vaccines for dogs induces short-lasting antibody responses. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.06.052

4

C. Guarino et al. / Vaccine xxx (2017) xxx–xxx

values > 2000 MFI 7–9 months post-vaccination (Supplementary Table 2). OspC and OspF antibody values were not analyzed in this sample set based on the unknown exposure and treatment status of the horses. The OspA antibody results corresponded to data from vaccination trials using rOspA expressed from E. coli showing that dogs maintained protective OspA antibodies 6-months after vaccination [37]. In contrast, vaccinated ponies (n = 7) developed OspA antibodies which began to decline prior to a booster dose administered on day 82 [14]. The differences in magnitude and duration of the OspA antibody response after Lyme vaccination observed here in diagnostic sample submissions from horses (Table 2) and dogs (Supplementary Table 2) can have several reasons: (1) the vaccine history and OspA antibody values prior to the last vaccination was unknown for these horses and dogs. Dogs may have obtained higher overall numbers of annual Lyme vaccinations than horses, resulting in the observed differences between the species; (2) the optimal vaccination dose for horses is not yet established. The vaccine dose was not available and may have been suboptimal for horses included in this dataset; (3) many other confounders, such as age, breed, risk of infection with B. burgdorferi or previous infection/treatment data were not available for the animals contributing to these samples and have the potential to influence the response to vaccination; (4) horses may respond to the available Lyme vaccines for dogs with a broader range and sometimes lower immune response than dogs. This could be due to differences in major histocompatibility complexes and antigen presentation of the OspA antigen in these two species. In summary, the analysis of diagnostic samples from vaccinated horses suggested that Lyme vaccines for dogs can result in shortlasting OspA antibody responses of varying magnitude in horses. We initiated a series of experimental vaccination trials in a controlled group of horses to further investigate antibody induction by available Lyme vaccines. 3.2. Antibody response in B. Burgdorferi naïve horses A first experimental vaccine trial tested three available Lyme vaccines for dogs in horses without previous exposure to B. burgdorferi. During this trial in 2013, horses had a low-moderate risk of exposure to B. burgdorferi with 4.3% PCR positive ticks collected from the horses (Supplementary Table 3). After an initial vaccination followed by one boost, horses developed overall low OspA antibody values, with mean OspA antibody values dropping below 2000 MFI in less than 16 weeks, independent of the vaccine used (Fig. 1A). Only 3/7, 4/7 and 5/6 horses, respectively, developed OspA antibody values > 2000 MFI following the first booster dose of Duramune LymeÒ, RecombitekÒ Lyme, and NobivacÒ Lyme. A trend towards higher OspA antibody values was observed in response to NobivacÒ Lyme vaccination (Fig. 1A). Horses vaccinated with NobivacÒ Lyme also developed significantly higher OspC antibody values following both booster vaccinations on days 25 and 108 (Fig. 1B). Horses from the other two groups did not mount an OspC antibody response. The third vaccination on day 108 was performed because of overall low antibody responses to the first two vaccine doses. It has been reported that a third vaccination dose in dogs resulted in significantly increased OspA antibodies values compared to the tradition two dose series [24]. In the horses vaccinated here, the OspA antibody response was of similar magnitude after the 2nd and 3rd vaccine administrations. Only the mean OspC antibody response was elevated following the 3rd dose of NobivacÒ Lyme vaccine (Fig. 1B). Following the third dose, only NobivacÒ Lyme vaccinated horses trended towards longer duration OspA immune response (Fig. 1A). However, by 24 weeks following the 3rd dose, mean OspA antibody values dropped again below 2000 MFI.

Fig. 1. Antibody response to vaccination in naïve horses. Horses were vaccinated with 1 mL RecombitekÒ Lyme (n = 7), NobivacÒ Lyme (n = 6), or DuramuneÒ Lyme (n = 7) on day 0, 25, and 108. Antibodies to B. burgdorferi OspA (A), OspC (B), OspF (C) were measured by Lyme Multiplex assay1. The horizontal dotted lines show the positive cut-off value for each bead assay. Arrows point to the time of vaccination. * = p < 0.05; *** = p < 0.001, **** = p < 0.0001.

Elevated OspF antibody values, indicative of infection with B. burgdorferi, were not detected for the duration of the vaccination trial, with the exception of two horses in the RecombitekÒ Lyme vaccine group on days 61–108 and day 137 post-vaccination, respectively (Fig. 1C). These OspF responses were likely related to subclinical infection with B. burgdorferi. Overall, the OspA antibody responses after experimental vaccination of horses with commercial Lyme vaccines for dogs confirmed that these vaccines induce short-lasting antibody responses

Please cite this article in press as: Guarino C et al. Vaccination of horses with Lyme vaccines for dogs induces short-lasting antibody responses. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.06.052

C. Guarino et al. / Vaccine xxx (2017) xxx–xxx

5

in B burgdorferi naïve horses. OspA antibody responses were low to almost not detectable in individual horses, while others mounted OspA antibodies as expected after vaccination. It should be noted that all horses in the experimental Lyme vaccine trials were Icelandic horses. This could be seen as strength because it removes breed as a possible confounding difference. However, it could also be speculated that these horses represent a group of individuals that is genetically similar and that the unique breed of the horse may have contributed to the low vaccine response observe here. Based on our previous observations, it is rather unlikely that the OspA antibody responses of these horses were reduced because of their breed. Icelandic horses responded to equine herpesvirus type I (EHV-1) infection [34] and EHV or West Nile virus vaccination with high antibody responses similar to other breeds [33,38]. Nevertheless, one possible explanation for the low OspA response in these horses is that the Lyme vaccine dose of 1 mL used in this trial was optimized for dogs and is potentially too low for horses. 3.3. Influence of vaccine dose on antibody responses Of the three vaccines tested, NobivacÒ Lyme produced the most notable antibody response in horses. This vaccine was tested again in previously non-vaccinated horses to investigate whether doubling of the vaccine dose increases the antibody response. Both OspA antibody values (Fig. 2A) and OspC antibody values (Fig. 2B) were significantly increased in animals receiving 2 mL instead of 1 mL doses per vaccination (p < 0.01 and p < 0.0001, respectively). Nevertheless, the duration of antibody response after vaccination with both doses was again short and comparable to that observed in trial 1 with this vaccine. A total of 12/14 horses that received 2 mL doses developed OspA antibody values > 2000 MFI at 8-weeks post-vaccination, compared to only 8/13 horses that received 1 mL doses. By 18-weeks post-vaccination, 8/14 horses that received the 2 mL doses maintained OspA antibody values > 2000 MFI, compared to only 1/13 that received the 1 mL doses. By 7 months post-vaccination, only 2/14 horses that received the 2 mL doses maintained OspA antibody values > 2000 MFI. These results confirmed that doubling the dose of the canine Lyme vaccine for horses resulted in an enhanced magnitude but still short-term duration of antibody responses. Unlike the first vaccination trial, where horses were naïve to B. burgdorferi exposure, the animals in this trial experienced 1– 2 years in an endemic environment, and >21% of the 55 ticks collected from horses during this trial were PCR positive for B. burgdorferi DNA (Supplementary Table 3). Interestingly, unlike in naïve horses, where only OspA and OspC antibody values increased in response to NobivacÒ Lyme vaccine, in this trial, OspF antibody values were also elevated following the booster dose (Fig. 2C). Experimental infection studies in dogs revealed that OspF antibodies can be detected with the Lyme Multiplex assay by 5 weeks post infection and afterwards [36]. A similar experimental approach to identify the onset of OspF antibodies in infected horses has not yet been performed, but it can be expected that these will be detectable at approximately the same time as in dogs. Although natural infection could not be completely ruled-out in our vaccinated horses developing OspF antibodies, no ticks were found on horses in the herd in the months preceding the initial and booster vaccination. In addition, a group of non-vaccinated horses did not develop OspF antibody value increases in the same time-period (Supplementary Fig. 1B). These findings suggest that the wholebacterin-based NobivacÒ Lyme vaccine, which contains many antigens of B. burgdorferi, can induce low and short-lasting antibodies against OspF and other antigens, including C6 (data not shown). Previous studies refuting this claim were performed in B. burgdorferi naïve dogs [39]. It is known that cultured killed bacteria

Fig. 2. Influence of vaccine dose on antibody response to NobivacÒ Lyme. Horses were vaccinated with 1 mL (n = 13) or 2 mL (n = 14) of NobivacÒ Lyme on day 0 and 24. Antibodies to B. burgdorferi OspA (A), OspC (B), OspF (C) were measured by Lyme Multiplex assay. The gray bars indicate the time frame when ticks were found on the horses, September/October/early Nov. The horizontal dotted lines show the positive cut-off value for each bead assay. Arrows indicate the time of vaccination. ** = p < 0.01, **** = p < 0.0001.

express all of these proteins and this fact has been used for many years for Lyme Western blot diagnostics to determine serological responses to various antigens of B. burgdorferi [40,41]. Consequently, vaccination with whole-bacterin Lyme vaccines can interfere with the ability to diagnose both acute and chronic infections, unlike vaccination with the OspA-based RecombitekÒ Lyme vaccine. 3.4. Effect of previous vaccination on OspA antibody duration Horses vaccinated with NobivacÒ Lyme in the previous year were vaccinated with two doses of RecombitekÒ Lyme to evaluate the effect of previous vaccination on the OspA antibody response.

Please cite this article in press as: Guarino C et al. Vaccination of horses with Lyme vaccines for dogs induces short-lasting antibody responses. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.06.052

6

C. Guarino et al. / Vaccine xxx (2017) xxx–xxx

The RecombitekÒ Lyme vaccination resulted in an earlier onset of OspA antibodies, with 15/18 horses developing OspA antibody values > 2000 MFI prior to the booster dose (Fig. 3A). Additionally, there was a trend towards increased longevity, with 7/18 horses maintaining OspA antibody values > 2000 MFI at >18 weeks postvaccination (Fig. 3A). Compared to RecombitekÒ Lyme vaccination in naïve horses, both intensity and longevity of the response were increased (Supplementary Table 4). However, by 24 weeks postvaccination, OspA antibody values were again < 2000 MFI in all horses (Fig. 3A). Antibodies against OspC were not observed in this trial (Fig. 3B). However, 2 out of 9 horses in the non-vaccinated control group developed elevated OspF antibody values indicative of infection with B. burgdorferi in late fall after a period of potential exposure to infected ticks (Fig. 3C). None of the horses in the vaccinated group showed increased OspF antibodies, suggesting that OspA antibodies mounted in the vaccinated group resulted in protection from infection.

3.5. Influence of injection route on antibody response A single dose annual booster vaccination with RecombitekÒ Lyme in the following year produced a short-lived OspA response

Fig. 4. Influence of injection route on response to REKOMBITEKÒ Lyme. Previously vaccinated horses were administered a single 1 mL dose of RecombitekÒ Lyme the following year IM (n = 7) or SC (n = 7) on day 0. An additional group of horses was monitored as a control (n = 8). Antibodies to B. burgdorferi OspA (A), OspC (B), OspF (C) were measured by Lyme Multiplex assay. The horizontal dotted lines show the positive cut-off value for each bead assay. Animals were vaccinated on day 0.

Fig. 3. Antibody response to RecombitekÒ Lyme in previously vaccinated animals. Horses vaccinated with NobivacÒ Lyme in the previous year were vaccinated with 1 mL RecombitekÒ Lyme on day 0 and 28 (n = 18), or not vaccinated and monitored as a control group (n = 9). Antibody values against antigens OspA (A), OspC (B), and OspF (C) were measured by Lyme Multiplex assay. The gray bars indicate the time frame when ticks were found on the horses, Sept/Oct/early Nov. The horizontal dotted lines show the positive cut-off value for each bead assay. Arrows indicate the time of vaccination. * = p < 0.05, ** = p < 0.01, **** = p < 0.0001.

in all animals, regardless of SC or IM injection route. Only 2/14 horses maintained OspA antibody values > 2000 MFI at 16 weeks post-vaccination (Fig. 4A). Four out of 7 and 5/7 animals, respectively, developed OspA antibody values > 2000 MFI 19 days after SC and IM injection. The magnitude of the peak OspA antibody response was similar after SC or IM vaccination (Fig. 4A). OspC or OspF antibodies were not elevated during the trial (Fig. 4B/C). SC versus IM injection route were previously reported to impact the immune response to a vaccine [42]. More frequently, as also seen here, these injection routes resulted in equivalent immune response to vaccination [43–45]. The horses vaccinated here had received two doses of the same vaccine, RecombitekÒ Lyme, in the previous year. The annual boost given during the injection route trial resulted in a faster declining OspA antibody response of lower magnitude (Fig. 4A) compared to the OspA response observed in the previous year (Fig. 3A). This suggested that annual revaccination intervals may not be sufficient for many horses to maintain OspA antibodies for more than 3– 4 months post vaccination. 4. Conclusions Lyme vaccination has the potential to protect horses from infection with B. burgdorferi and is especially desirable in

Please cite this article in press as: Guarino C et al. Vaccination of horses with Lyme vaccines for dogs induces short-lasting antibody responses. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.06.052

C. Guarino et al. / Vaccine xxx (2017) xxx–xxx

Lyme-endemic areas, where animals can be regularly re-infected. OspA antibodies have been identified as correlates for protection from infection with B. burgdorferi in many species, including horses [14,19–24]. Our current results highlight that Lyme vaccines for dogs induce OspA antibodies in horses only transiently and inconsistently. However, the data presented here also indicate that increasing the vaccination dose for horses can enhance the OspA antibody response, and adding another booster vaccination can extend the duration of the OspA antibody response. As long as optimal vaccination doses and intervals are not established for horses, currently available Lyme vaccines for dogs can be suboptimal in inducing OspA antibodies. Although individual horses may respond to vaccination with high OspA antibody responses, many horses mount very low OspA antibody values that are unlikely to offer full protection over the course of a year. Additionally, this work revealed that vaccination with wholebacterin vaccines may interfere with subsequent diagnosis. Particularly in animals with likelihood for previous exposure to B. burgdorferi, whole-bacterin Lyme vaccine may boost pre-existing OspC, OspF and C6 antibody responses. This interference with Lyme testing interpretation can be avoided by using the OspAbased vaccine. To the authors’ knowledge, major adverse reactions to Lyme vaccination in horses have not been reported and were also not observed during the experimental vaccination trial performed here. Based on our current data, OspA antibody responses after Lyme vaccination are short-lasting. It can thus be recommended to vaccinate horses in close proximity to tick season, approximately 4 weeks before ticks are typically abundant in the area. Because low OspA antibody responses have been observed in many horses, a confirmatory testing of OspA antibodies after Lyme vaccination is recommended on an individual horse basis. Future studies are required for further optimizing the vaccination dose and intervals for horses with commercial Lyme vaccines for dogs and also to identify the OspA antibody values correlated with protection from infection with B. burgdorferi. 5. Contribution of authors BW planned and performed the vaccination approach and performed the data analysis. CG drafted the manuscript with contributions from BW and assisted with data analysis. SA and JR were instrumental in gathering diagnostic submission data. AG directed the tick analysis and B. burgdorferi PCR. All authors contributed to the preparation of the article and approved the final version prior to submission. 6. Funding sources The experimental canine Lyme vaccine approach in horses was supported by Hatch Fund #NYC-478401 from the National Institute of Food and Agriculture, U.S. Department of Agriculture. The horses used for the project were further supported by the Harry M. Zweig Memorial Fund for Equine Research at Cornell University. 7. Conflict of interest BW is the inventor on a patent entitled ‘Methods for Diagnosing Lyme Disease’, Patent #: US 8,946,393 B2 that uses technology described in this manuscript. Acknowledgments The authors thank Alison Keggan, Michael Zarzosa Gillian Perkins, Elizabeth M. Moore, Christina Watts, Christiane Schnabel,

7

Fahad Raza, Larry Carlisle and Brooke Burke for assisting with sampling and vaccination of horses, Heather Freer, Alison Keggan and Christina Watts for sample processing, Renee Anderson for tick identification, Diane Kilts for coordinating the serological measurements and data organization, and Alicia Rollins, Jesse Brucie, Michelle Wemette, Aziza Solomon for performing the Lyme Multiplex assay.

Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.vaccine.2017.06. 052. References [1] Steere AC. Lyme disease. N Engl J Med 2001;345:115–25. http://dx.doi.org/ 10.1056/NEJM200107123450207. [2] Divers TJ. Equine lyme disease. J Equine Vet Sci 2013;33:488–92. http://dx.doi. org/10.1016/j.jevs.2013.03.187. [3] Littman MP, Goldstein RE, Labato MA, Lappin MR, Moore GE. ACVIM small animal consensus statement on lyme disease in dogs: diagnosis, treatment, and prevention. J Vet.Int Med 2006;20:422–34. http://dx.doi.org/10.1111/ j.1939-1676.2006.tb02880.x. [4] Parker JL, White KK. Lyme borreliosis in cattle and horses: a review of the literature. Cornell Vet 1992;82:253–74. [5] Divers TJ, Mair TS, Chang Y-F. Lyme disease in horses. In: Mair TS, Hutchinson RE, editors. Infect. Dis. Horse, Equine Veterinary Journal Ltd., Fordham [England]; 2009. pp. 286–2. [6] Sears KP, Divers TJ, Neff RT, Miller Jr WH, McDonough SP. A case of Borreliaassociated cutaneous pseudolymphoma in a horse. Vet Dermatol 2012;23:153–6. http://dx.doi.org/10.1111/j.1365-3164.2011.01013.x. [7] Priest HL, Irby NL, Schlafer DH, Divers TJ, Wagner B, Glaser AL, Chang Y-F, Smith MC. Diagnosis of Borrelia-associated uveitis in two horses. Vet Ophthalmol 2012;15:398–405. http://dx.doi.org/10.1111/j.14635224.2012.01000.x. [8] James FM, Engiles JB, Beech J. Meningitis, cranial neuritis, and radiculoneuritis associated with Borrelia burgdorferi infection in a horse. J Am Vet Med Assoc 2010;237:1180–5. http://dx.doi.org/10.2460/javma.237.10.1180. [9] Imai DM, Barr BC, Daft B, Bertone JJ, Feng S, Hodzic E, Johnston JM, Olsen KJ, Barthold SW. Lyme neuroborreliosis in 2 horses. Vet Pathol Online 2011;48:1151–7. http://dx.doi.org/10.1177/0300985811398246. [10] Johnstone Lk, Engiles Jb, Aceto H, Buechner-Maxwell V, Divers T, Gardner R, et al. Retrospective evaluation of horses diagnosed with neuroborreliosis on postmortem examination: 16 cases (2004–2015), J Vet Int Med 30 (2016) 1305–12. doi: 10.1111/jvim.14369. [11] del Castillo JRE. Tetracyclines. In: Medicine SGD, DACVIMessor, Large Animal Internal, J.F.P. VetMB, essor, P.M.D.D. Pharmacology MS, DACVIM, DACVCPessor, Veterinary Clinical (Eds.), Antimicrob. Ther. Vet. Med., John Wiley & Sons, Inc; 2013. p. 257–68. [12] Wagner B, Goodman LB, Rollins A, Freer HS. Antibodies to OspC, OspF and C6 antigens as indicators for infection with Borrelia burgdorferi in horses. Equine Vet J 2013;45:533–7. http://dx.doi.org/10.1111/evj.12033. [13] Wagner B, Freer H, Rollins A, Erb HN, Lu Z, Gröhn Y. Development of a multiplex assay for the detection of antibodies to Borrelia burgdorferi in horses and its validation using Bayesian and conventional statistical methods. Vet Immunol Immunopathol 2011;144:374–81. http://dx.doi.org/10.1016/j. vetimm.2011.08.005. [14] Chang Y-F, Novosol V, McDonough SP, Chang C-F, Jacobson RH, Divers T, Quimby FW, Shin S, Lein DH. Vaccination against lyme disease with recombinant Borrelia burgdorferi outer-surface protein A (rOspA) in horses. Vaccine 1999;18:540–8. http://dx.doi.org/10.1016/S0264-410X(99)00187-5. [15] Erdile LF, Guy B. OspA lipoprotein of Borrelia burgdorferi is a mucosal immunogen and adjuvant. Vaccine 1997;15:988–96. [16] Ma J, Hine PM, Clough ER, Fish D, Coughlin RT, Beltz GA, Shew MG. Safety, efficacy, and immunogenicity of a recombinant Osp subunit canine Lyme disease vaccine. Vaccine 1996;14:1366–74. http://dx.doi.org/10.1016/S0264410X(96)00045-X. [17] Ramamoorthy R, Philipp MT. Differential expression of borrelia burgdorferi proteins during growth in vitro. Infect Immun 1998;66:5119–24. [18] Alverson J, Bundle SF, Sohaskey CD, Lybecker MC, Samuels DS. Transcriptional regulation of the ospAB and ospC promoters from Borrelia burgdorferi. Mol Microbiol 2003;48:1665–77. http://dx.doi.org/10.1046/j.13652958.2003.03537.x. [19] Poland GA, Jacobson RM. The prevention of Lyme disease with vaccine. Vaccine 2001;19:2303–8. http://dx.doi.org/10.1016/S0264-410X(00)00520-X. [20] Comstedt P, Hanner M, Schüler W, Meinke A, Schlegl R, Lundberg U. Characterization and optimization of a novel vaccine for protection against Lyme borreliosis. Vaccine 2015;33:5982–8. http://dx.doi.org/10.1016/ j.vaccine.2015.07.095.

Please cite this article in press as: Guarino C et al. Vaccination of horses with Lyme vaccines for dogs induces short-lasting antibody responses. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.06.052

8

C. Guarino et al. / Vaccine xxx (2017) xxx–xxx

[21] Fikrig E, Barthold SW, Kantor FS, Flavell RA. Protection of mice against the lyme disease agent by immunizing with recombinant OspA. Science 1990;250:553. [22] Schaible UE, Kramer MD, Eichmann K, Modolell M, Museteanu C, Simon MM. Monoclonal antibodies specific for the outer surface protein A (OspA) of Borrelia burgdorferi prevent Lyme borreliosis in severe combined immunodeficiency (scid) mice. Proc Natl Acad Sci 1990;87:3768–72. http:// dx.doi.org/10.1073/pnas.87.10.3768. [23] Jacobson RH, Chang Y-F, Shin SJ. Lyme disease: laboratory diagnosis of infected and vaccinated symptomatic dogs. Semin Vet Med Surg (Small Anim) 1996;11:172–82. http://dx.doi.org/10.1016/S1096-2867(96)80030-2. [24] Töpfer KH, Straubinger RK. Characterization of the humoral immune response in dogs after vaccination against the Lyme borreliosis agent: a study with five commercial vaccines using two different vaccination schedules. Vaccine 2007;25:314–26. http://dx.doi.org/10.1016/j.vaccine.2006.07.031. [25] Pal U, de Silva AM, Montgomery RR, Fish D, Anguita J, Anderson JF, Lobet Y, Fikrig E. Attachment of Borrelia burgdorferi within Ixodes scapularis mediated by outer surface protein A. J Clin Invest 2000;106:561–9. http://dx.doi.org/ 10.1172/JCI9427. [26] Yang XF, Pal U, Alani SM, Fikrig E, Norgard MV. Essential role for OspA/B in the life cycle of the lyme disease spirochete. J Exp Med 2004;199:641–8. http://dx. doi.org/10.1084/jem.20031960. [27] Pal U, Fikrig E. Adaptation of Borrelia burgdorferi in the vector and vertebrate host. Microbes Infect 2003;5:659–66. http://dx.doi.org/10.1016/S1286-4579 (03)00097-2. [28] LaFleur RL, Dant JC, Wasmoen TL, Callister SM, Jobe DA, Lovrich SD, Warner TF, Abdelmagid O, Schell RF. Bacterin that induces anti-OspA and anti-OspC borreliacidal antibodies provides a high level of protection against canine Lyme disease. Clin Vaccine Immunol CVI 2009;16:253–9. http://dx.doi.org/ 10.1128/CVI.00373-08. [29] Pal U, Yang X, Chen M, Bockenstedt LK, Anderson JF, Flavell RA, Norgard MV, Fikrig E. OspC facilitates Borrelia burgdorferi invasion of Ixodes scapularis salivary glands. J Clin Invest 2004;113:220–30. http://dx.doi.org/10.1172/ JCI19894. [30] Gilmore RD, Piesman J. Inhibition of Borrelia burgdorferi migration from the midgut to the salivary glands following feeding by ticks on OspC-immunized mice. Infect Immun 2000;68:411–4. [31] Earnhart CG, Marconi RT. Construction and analysis of variants of a polyvalent Lyme disease vaccine: approaches for improving the immune response to chimeric vaccinogens. Vaccine 2007;25:3419–27. http://dx.doi.org/10.1016/ j.vaccine.2006.12.051. [32] Earnhart CG, Buckles EL, Marconi RT. Development of an OspC-based tetravalent, recombinant, chimeric vaccinogen that elicits bactericidal antibody against diverse Lyme disease spirochete strains. Vaccine 2007;25:466–80. http://dx.doi.org/10.1016/j.vaccine.2006.07.052. [33] Wagner B, Goodman LB, Babasyan S, Freer H, Torsteinsdóttir S, Svansson V, Björnsdóttir S, Perkins GA. Antibody and cellular immune responses of naïve mares to repeated vaccination with an inactivated equine herpesvirus vaccine. Vaccine 2015;33:5588–97. http://dx.doi.org/10.1016/j.vaccine.2015.09.009.

[34] Wagner B, Perkins G, Babasyan S, Freer H, Keggan A, Goodman LB, Glaser A, Torsteinsdóttir S, Svansson V, Björnsdóttir S. Neonatal immunization with a single IL-4/antigen dose induces increased antibody responses after challenge infection with equine herpesvirus type 1 (EHV-1) at weanling age. PLOS ONE. 2017;12:e0169072. http://dx.doi.org/10.1371/journal.pone.0169072. [35] OIE World Animal Health Information System, (n.d.). http://www.oie.int/ wahis_2/public/wahidwild.php/Countryinformation/Animalsituation [accessed January 25, 2017]. [36] Wagner B, Freer H, Rollins A, Garcia-Tapia D, Erb HN, Earnhart C, Marconi R, Meeus P. Antibodies to Borrelia burgdorferi OspA, OspC, OspF, and C6 antigens as markers for early and late infection in dogs. Clin Vaccine Immunol 2012;19:527–35. http://dx.doi.org/10.1128/CVI.05653-11. [37] Chang YF, Appel MJ, Jacobson RH, Shin SJ, Harpending P, Straubinger R, Patrican LA, Mohammed H, Summers BA. Recombinant OspA protects dogs against infection and disease caused by Borrelia burgdorferi. Infect Immun 1995;63:3543–9. [38] Khatibzadeh SM, Gold CB, Keggan AE, Perkins GA, Glaser AL, Dubovi EJ, Wagner B. West Nile virus–specific immunoglobulin isotype responses in vaccinated and infected horses. Am J Vet Res 2014;76:92–100. http://dx.doi.org/10.2460/ ajvr.76.1.92. [39] O’Connor TP, Esty KJ, Hanscom JL, Shields P, Philipp MT. Dogs vaccinated with common lyme disease vaccines do not respond to IR6, the conserved immunodominant region of the VlsE surface protein of Borrelia burgdorferi. Clin Diagn Lab Immunol 2004;11:458–62. http://dx.doi.org/10.1128/ CDLI.11.3.458-462.2004. [40] Ma B, Christen B, Leung D, Vigo-Pelfrey C. Serodiagnosis of Lyme borreliosis by western immunoblot: reactivity of various significant antibodies against Borrelia burgdorferi. J Clin Microbiol 1992;30:370–6. [41] Dressler F, Whalen JA, Reinhardt BN, Steere AC. Western blotting in the serodiagnosis of Lyme disease. J Infect Dis 1993;167:392–400. http://dx.doi. org/10.1093/infdis/167.2.392. [42] Cook IF, Barr I, Hartel G, Pond D, Hampson AW. Reactogenicity and immunogenicity of an inactivated influenza vaccine administered by intramuscular or subcutaneous injection in elderly adults. Vaccine 2006;24:2395–402. http://dx.doi.org/10.1016/j.vaccine.2005.11.057. [43] Hopf S, Garner-Spitzer E, Hofer M, Kundi M, Wiedermann U. Comparable immune responsiveness but increased reactogenicity after subcutaneous versus intramuscular administration of tick borne encephalitis (TBE) vaccine. Vaccine 2016;34:2027–34. http://dx.doi.org/10.1016/j.vaccine.2015.12.057. [44] Diez-Domingo J, Weinke T, Garcia de Lomas J, Meyer CU, Bertrand I, Eymin C, Thomas S, Sadorge C. Comparison of intramuscular and subcutaneous administration of a herpes zoster live-attenuated vaccine in adults aged 50 years: a randomised non-inferiority clinical trial. Vaccine 2015;33:789–95. http://dx.doi.org/10.1016/j.vaccine.2014.12.024. [45] Mark A, Carlsson R-M, Granström M. Subcutaneous versus intramuscular injection for booster DT vaccination of adolescents. Vaccine 1999;17:2067–72. http://dx.doi.org/10.1016/S0264-410X(98)00410-1.

Please cite this article in press as: Guarino C et al. Vaccination of horses with Lyme vaccines for dogs induces short-lasting antibody responses. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.06.052