Maternal Transfer of Vaccine-Induced Anti-OspA Antibodies in Peromyscus spp Protects Pups from Tick-Transmitted Borrelia burgdorferi

Maternal Transfer of Oral Vaccine Induced Anti-OspA Antibodies Protects Peromyscus spp Pups from Tick-Transmitted Borrelia burgdorferi | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Maternal Transfer of Oral Vaccine Induced Anti-OspA Antibodies Protects Peromyscus spp Pups from Tick-Transmitted Borrelia burgdorferi Jose F. Azevedo , Greg Joyner , View ORCID Profile Suman Kundu , Kamalika Samanta , View ORCID Profile Maria Gomes-Solecki doi: https://doi.org/10.1101/2025.02.24.639966 Jose F. Azevedo 1 University of Tennessee Health Science Cen ter, Department of Microbiology, Immunology, and Biochemistry Find this author on Google Scholar Find this author on PubMed Search for this author on this site Greg Joyner 1 University of Tennessee Health Science Cen ter, Department of Microbiology, Immunology, and Biochemistry Find this author on Google Scholar Find this author on PubMed Search for this author on this site Suman Kundu 1 University of Tennessee Health Science Cen ter, Department of Microbiology, Immunology, and Biochemistry Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Suman Kundu Kamalika Samanta 1 University of Tennessee Health Science Cen ter, Department of Microbiology, Immunology, and Biochemistry Find this author on Google Scholar Find this author on PubMed Search for this author on this site Maria Gomes-Solecki 1 University of Tennessee Health Science Cen ter, Department of Microbiology, Immunology, and Biochemistry Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Maria Gomes-Solecki For correspondence: mgomesso{at}uthsc.edu Abstract Full Text Info/History Metrics Preview PDF Abstract The efficacy and duration of passive immunity protection depends on maternal antibody levels and transfer efficiency. We investigated whether oral vaccination of Peromyscus leucopus dams with recombinant OspA-expressing E. coli could induce maternal transfer of anti-OspA antibodies and protect pups from Borrelia burgdorferi challenge. Dams were vaccinated until breeding pairs were created (i), until parturition (ii), and until pups were 2 weeks old (iii). Pups were challenged with nymphal I. scapularis -transmitted B. burgdorferi at ∼ 4 weeks of age. Anti-OspA IgG were quantified in dams and pups, and anti -B. burgdorferi IgG were quantified in pups. B. burgdorferi burden was assessed by flaB qPCR in pups’ tissues ∼ 4 weeks after tick challenge and viability of B. burgdorferi was assessed by culture of heart tissue. P. leucopus pups born to dams vaccinated until breeding had low serologic anti-OspA antibody and were not protected from tick transmitted B. burgdorferi infection. However, when dams’ vaccination extended until parturition and until pups were two weeks old, significant anti-OspA antibody transfer and protection from B. burgdorferi infection occurred. This was evidenced by absence of antibody to B. burgdorferi PepVF, absence of B. burgdorferi flaB DNA in heart and bladder tissues, and absence of flaB in culture from heart tissues from pups euthanized >9 weeks after birth. We show that transfer of anti-OspA antibodies from vaccinated P. leucopus dams to offspring prevents tick transmission and infection dynamics of B. burgdorferi in the major reservoir host of this spirochete in the United States. Importance This study contributes to our understanding of how interventions based in reservoir targeted OspA vaccines designed to block transmission of B. burgdorferi from infected I. scapularis ticks may disrupt the enzootic cycle of this spirochete and reduce incidence of Lyme disease. Introduction Lyme disease, caused by Borrelia burgdorferi , is the most prevalent vector-borne disease in North America ( 1 ). The white-footed mouse ( Peromyscus leucopus ) serves as the primary reservoir host ( 2 ), maintaining B. burgdorferi in enzootic cycles and facilitating transmission to Ixodes ticks that subsequently infect humans and other mammals ( 3 ). Unlike humans, reservoir hosts do not develop significant disease following B. burgdorferi infection, yet they serve as a crucial source of spirochete acquisition for larval Ixodid ticks ( 4 ). Thus, interrupting transmission of B. burgdorferi ( 5 ) has been proposed as a strategy for controlling Lyme disease ( 6 , 7 ). Outer surface protein A (OspA) is a major target for Lyme disease vaccine development ( 8 ) due to its essential role in B. burgdorferi persistence within the tick vector ( 9 ). OspA facilitates spirochete adherence to the tick midgut, allowing the bacterium to survive between blood meals ( 10 ). During tick feeding, B. burgdorferi downregulates OspA while upregulating OspC ( 11 ), which enables migration to the salivary glands and subsequent transmission to the host ( 12 – 14 ). Vaccines targeting OspA aim to elicit antibodies that neutralize spirochetes within the tick gut, preventing their transmission before they reach the mammalian host ( 15 , 16 ). These vaccines have been explored in multiple contexts, including human vaccination ( 17 , 18 ), direct vaccination of P. leucopus ( 19 ), and oral bait delivered reservoir-targeted vaccines ( 6 ). Beyond direct vaccination, maternal transfer of antibody provides an alternative strategy to protect neonates during the critical early stages of life ( 20 ). In the context of ecology of vector-borne pathogens, this temporary immunity reduces early-life susceptibility to infection ( 21 ). Previous studies have demonstrated that oral vaccination of dams with OspA results in anti-OspA IgG in offspring ( 22 ), though the degree to which this protects against B. burgdorferi transmission remains unknown. In this study, we addressed this critical gap by investigating the extent of maternal antibody transfer in P. leucopus and assessing whether vaccination of dams with recombinant OspA-expressing E. coli confers protection to neonates. We tested whether vaccination of dams until breeding pairs were created, until parturition and until the pups reached 2 weeks of age influences antibody transfer and protection against tick-mediated B. burgdorferi challenge. Our findings have implications for reservoir-targeted vaccination strategies aimed at reducing Lyme disease risk by breaking the transmission cycle of B. burgdorferi . Materials and Methods Ethics Statement This study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the US National Institutes of Health. The protocol was approved by the University of Tennessee Health Science Center Institutional Animal Care and Use Committee (IACUC), protocol #22-0400. Peromyscus leucopus were sourced from the Peromyscus Genetic Stock Center (PGSC) at the University of South Carolina. Vaccine Preparation Recombinant E. coli BL21(DE3)pLysS encoding B. burgdorferi OspA ( E. coli -OspA) was produced as previously described ( 23 ). Briefly, E. coli -OspA was grown in Terrific Broth (TBY) at 37°C until OD600 ∼0.8, induced with 1 mM IPTG for 3 h, transferred to filter-top flasks and inactivated with 1% β-propiolactone (BPL, 98%, AlfaAesar), incubated at room temperature at 100 rpm for 24h, and harvested by centrifugation at 2000×g at 4°C. The bacterial biomass was resuspended in phosphate-buffered saline (PBS) and sprayed onto feed pellets. Pellets (RTV pellets) were allowed to dry between application layers. Inactivation was tested by inoculation of BPL-treated culture in TBY (1:200) and incubation overnight at 37°C, shaking at 250 rpm. The following day, culture inactivation was confirmed by checking turbidity and recording OD 600 =0. Vaccination Strategy Dams (n=9) received RTV pellets daily for 5 consecutive days per week, with a 2-day rest period over the weekend in which they received regular mouse chow. Control dams received uncoated pellets. Dams consumed RTV pellets continuously as per vaccination schedule up to 36 weeks ( Fig 1 ) and were assigned to three experimental groups based on breeding milestones. The level of IgG to OspA was analyzed in dams’ blood before selecting animals with high antibody in serum to create breeding pairs. Download figure Open in new tab Figure 1. Experimental design. A) Oral vaccination schedule of adult female Peromyscus leucopus : mice were allowed to eat RTV pellets on vaccination weeks; pellets were exchanged for regular feed during rest weeks; control mice received uncoated pellets; B) Timeline for breeding of dams: dams were bled to evaluate serologic antibody to OspA and three experimental groups were set as 1 st , 2 nd and 3 rd Breeding based on duration of vaccination and breeding milestones; C) Birth of pups, challenge with B. burgdorferi infected I. scapularis ticks and collection of samples. Breeding and Litter Management Breeding pairs were established based on dams having anti-OspA titers (OD450>0.8) in serum diluted 1:100 assessed by ELISA. Each vaccinated female was cohoused with an unvaccinated male for 3–4 weeks, after which pregnant females were single-housed until parturition ( Fig 1 ). The first group (Breeding 1) was created with 5 dams vaccinated for 12 weeks prior to breeding pairs were formed. The second group, Breeding 2, was created with 4 dams vaccinated for 24 weeks until parturition. The third group, Breeding 3, was created with the dams who maintained high levels of antibodies to OspA and remained on the vaccination regimen for 36 weeks, ensuring that immunization continued until their pups reached two weeks of age. Dams that lost the ability to reproduce or had low levels of serologic antibody to OspA were euthanized. A group of pups (n=7) born after the 3 rd breeding were euthanized immediately after they dropped at parturition and blood was collected. Pups remained with their dams until 24–25 days of age, at which point they underwent nymphal tick challenge. Blood Collection Blood samples were collected via submandibular vein bleed. Dams were sampled at day 0 and before each breeding. Pups were sampled on the day they were born, at day 24 (pre-challenge) and day ∼71 (terminal bleed). Samples were allowed to clot for 30 min at room temperature before centrifugation at 2000×g for 10 min at 4°C. Serum was aliquoted and stored at −80°C until analysis. Tissue Collection and Processing At necropsy on d71 (∼9-10 week old pups), heart and bladder tissues were harvested, transported on dry ice, and stored at −80°C. For organ culture experiments, freshly excised heart tissue was immediately placed into Barbour-Stoenner-Kelly H (BSK-H) medium supplemented with 6% rabbit serum (Sigma-Aldrich, St. Louis, MO) and incubated at 34°C for 3 weeks (on the 7 th day of culture, the tissue was removed to avoid putrefaction). On day 21 of culture supernatants were analyzed for motile B. burgdorferi detection using Petroff-Hausser chamber (dark-field microscopy, 200× magnification) and for DNA quantification via flaB qPCR. Tick Challenge and Monitoring of B. burgdorferi Infection To assess susceptibility to B. burgdorferi infection, pups were challenged with infected nymphal I. scapularis ticks at ∼ 4 weeks of age. Each pup was exposed to 5–7 infected nymphs, which were allowed to feed for 72 hours in a controlled containment chamber. Flat nymphal ticks were carefully transferred from storage vials using fine forceps and placed at the base of the neck and upper back of gently restrained pups. After placement, pups were monitored to ensure proper tick attachment before being returned to their cages. After 72 hours, all engorged ticks naturally detached and were collected from the bottom of the cages. Detached ticks were stored at −20°C until further analysis for B. burgdorferi colonization via dark-field microscopy. The infected nymphs used in this study were generated by feeding larval I. scapularis (Oklahoma Tick Laboratory) on a mouse infected with a multi-strain culture of B. burgdorferi sensu stricto. This B. burgdorferi culture was recovered from heart and bladder tissues of C3H/HeN mice infected with nymphal ticks collected in Massachusetts in 2020. To maintain infection across generations, the multi-strain B. burgdorferi inoculum was first introduced into C3H mice, after which successive mice were infected through tick-borne transmission. Larvae were allowed to feed on infected mice, acquire B. burgdorferi , and molt into infected nymphs, which were then used to challenge mice in a cycle that mimics natural enzootic transmission of B. burgdorferi . Detection of Anti-OspA and Anti-PepVF IgG by ELISA Serum antibody responses to OspA and to B. burgdorferi pepVF were assessed using an enzyme-linked immunosorbent assay (ELISA). PepVF contains two peptides from B. burgdorferi VlsE and FlaB proteins in tandem, separated by a 3aa glycine linker, and is used in our lab to determine B. burgdorferi infection ( 24 ). Presence of antibody to PepVF indicates exposure to B. burgdorferi . Purified recombinant OspA (1 μg/mL) or recombinant PepVF (1 μg/mL) was adsorbed onto Nunc MaxiSorp plates (Thermo Fisher Scientific, Waltham, MA) by overnight incubation at 4°C. Following antigen coating, the plates were blocked with 5% bovine serum albumin (BSA) in PBS-Tween (0.05%) for 1 hour at room temperature to prevent nonspecific binding. Serum samples were diluted 1:100 in blocking buffer and incubated on the plates for 2 hours at 37°C. To detect antigen-specific IgG, a goat anti-mouse IgG antibody conjugated to horseradish peroxidase (HRP) (1:6000; Jackson ImmunoResearch, West Grove, PA) was added, followed by a 1-hour incubation at 37°C. After washing, plates were developed using TMB substrate (Sigma-Aldrich) for 15 minutes before stopping the reaction with 1N H₂SO₄. Optical density (OD) was measured at 450 nm and blanked against the control using a SpectraMax Plus ELISA reader (Molecular Devices, San Jose, CA). For Cutoff determination the positivity threshold was defined as mean + 3 SD of negative controls. Molecular Detection of B. burgdorferi DNA by qPCR Total DNA was extracted from heart and bladder tissues and heart culture media using the DNeasy Blood & Tissue Kit (Qiagen, Germantown, MD) according to the manufacturer’s protocol. qPCR was performed to quantify B. burgdorferi flaB gene copies per mg of tissue or per mL of culture media. Quantitative PCR (qPCR) was performed to quantify B. burgdorferi flaB gene copies in DNA extracted from heart and bladder tissues, as well as culture supernatants. Reactions were carried out in a 25 μL volume using 2X TaqMan Master Mix (Thermo Fisher Scientific), with final primer and probe concentrations of 200 nM. Each reaction contained 5 μL of DNA template, and all samples were run in duplicate to ensure reproducibility. The primer and probe set used for TaqMan-based detection of the flaB gene was as follows: Forward primer: 5’-AAGCAATCTAGGTCTCAAGC-3’; Reverse primer: 5’-GCTTCAGCCTGGCCATAAATAG-3’; Probe: 5’-FAM-AGATGTGGTAGACCCGAAGCCGAG-TAMRA-3’. Amplification was performed using a QuantStudio 7 Real-Time PCR System (Applied Biosystems) under the following cycling conditions: an initial denaturation at 95°C for 10 minutes, followed by 45 cycles of 95°C for 15 seconds and 60°C for 1 minute. Fluorescence was measured at each cycle, and flaB copy numbers were determined by comparison to a standard curve generated from known concentrations of B. burgdorferi genomic DNA Statistical Analysis Statistical analyses were conducted using GraphPad Prism 9.0 (GraphPad Software, San Diego, CA). To assess the distribution of the data, the Shapiro-Wilk test was applied to determine normality. For group comparisons, an unpaired t-test was used when data followed a normal distribution, while the Mann-Whitney U test was applied to non-normally distributed data. A significance threshold of p < 0.05 was used for all statistical tests. Results Transfer of Anti-OspA Antibodies from Dams to Pups Pups were born to dams undergoing a continuous vaccination schedule. To determine the extent of maternal antibody transfer, serum anti-OspA IgG titers were measured in pups when they reached D24 (pre-challenge). Pups born from dams vaccinated until breeding pairs were formed are grouped under 1 st breeding. Pups born from dams vaccinated until parturition are grouped under 2 nd breeding. Pups born from dams vaccinated until the pups were two weeks old are grouped under 3 rd breeding. The results are shown in Fig 2 . Anti-OspA IgG was significantly increased in D24 pups born from vaccinated dams during the 1 st , the 2 nd and the 3 rd breeding, in contrast to pups born to control unvaccinated dams. Nevertheless, we note that pups born from the 1st breeding of vaccinated dams had an average anti-OspA IgG OD 450 ∼ 0.271 (SD± 0.094); pups born from the 2nd breeding of vaccinated dams had an average anti-OspA IgG OD 450 ∼0.809 (SD± 0.073); and pups born from the 3rd breeding of vaccinated dams had an average anti-OspA IgG OD 450 ∼1.532 (SD± 0.591). The data shows that continued vaccination of the dams past parturition maintained sufficient levels of protective antibody (OD450>0.8) in blood. Download figure Open in new tab Figure 2. Transfer of OspA antibodies from dams to pups. ELISA quantification of pre-challenge anti-OspA IgG in sera from D24 pups born to vaccinated dams. Optical density (OD) at 450nm (blanked) is shown for each group. Each point represents an individual pup, with lines indicating group means ± SD. Statistical analysis by Mann-Whitney test *p<0.05, ** p<0.005, *** p<0.0005. Seroconversion to B. burgdorferi in Tick Challenged Pups To assess whether pups were protected or infected with B. burgdorferi after tick challenge, anti-PepVF IgG was measured in serum samples collected at euthanasia (∼day 71), at least 3 weeks after the last day of challenge ( Fig 3 ). Pups (n=8) from the 1 st Breeding of vaccinated dams had equivalent anti-PepVF IgG as control pups (n=10), confirming infection with tick-transmitted B. burgdorferi in both groups (p = 0.7642) ( Fig 3A ). Although 2/3 pups from the 2 nd Breeding of vaccinated dams did not have anti-PepVF antibodies ( Fig 3B , circle), overall differences with the control group (n=5) were not significant (p = 0.9432). Strikingly, pups (n=6) from the 3 rd Breeding of vaccinated dams ( Fig 3C ) had anti-PepVF IgG levels below the cutoff in contrast to the control group (n=6), p = 0.0147, indicating absence of infection. These results suggest that maternal vaccination that extended until and beyond parturition conferred anti-OspA passive immunity to pups. Download figure Open in new tab Figure 3. Seroconversion to B. burgdorferi in tick challenged pups. (A–C) ELISA quantification of anti-PepVF IgG in terminal sera from pups born to vaccinated dams. Pups were bled ∼ 4 weeks post tick challenge. Optical density (OD) at 450 nm (blanked) is shown for each group. Each data point represents an individual pup, with lines indicating group means ± SD. Statistics by Unpaired t test: ns, not significant, * p 4 weeks after tick challenge (∼ day 71) and performed qPCR targeting the B. burgdorferi flaB gene ( Fig 4 ). In pups from the 1 st Breeding, there are no differences in flaB loads in heart tissues from vaccinated and control groups; in pups from the 2 nd Breeding, the same 2 mice (2/3) in the vaccinated group that did not seroconvert to B. burgdorferi , also did not have flaB in heart tissues, whereas 5/5 mice in the control group had flaB DNA in heart tissues. Differences between the groups are not significant (p=0.3393). However, in pups from the 3 rd Breeding, no detectable flaB copies were found in bladder tissues from the vaccinated group (0/6), in contrast to the pups from the control group that were all positive (6/6) for B. burgdorferi flaB (p=0.0022). Absence of B. burgdorferi flaB DNA in pups from the 2 nd and 3 rd Breeding correlate with absence of PepVF seroconversion, further confirming the protective effect of maternal antibody transfer when dams were vaccinated until and beyond parturition. Download figure Open in new tab Figure 4. Burden of B. burgdorferi in tissues from tick challenged pups. (A–C) qPCR quantification of the flaB gene in heart and bladder tissues collected from pups ∼ 4 weeks post tick challenge. Each data point represents an individual pup, with lines indicating group means. Statistics by Mann Whitney test: ns, not significant, **p<0.005. Viability of B. burgdorferi in Tissues from Tick Challenged Pups To further assess B. burgdorferi persistence, heart tissues from pups born from 3 rd Breeding dams were cultured in BSK-H medium at 34°C for 21 days, and live spirochetes were counted using a Petroff-Hausser chamber under a dark field microscope ( Figure 5A ). Only pups (4/5) from unvaccinated control dams had detectable motile B. burgdorferi in culture, confirming that live spirochetes had successfully colonized cardiac tissue after tick challenge. The control group exhibited a range of 10 5 -10 6 spirochetes per mL of culture, while pups from vaccinated dams had no visible live bacteria, p=0.0152. qPCR analysis of heart culture supernatants ( Figure 5B ) confirmed that high flaB copy numbers (mean 10 5 ) were detected in control heart cultures (5/5), whereas no B. burgdorferi DNA was present in pups from vaccinated dams (0/5), p=0.0022. This indicates that dams that received vaccine until 2 weeks post-parturition, produced pups that were protected from cardiac colonization by B. burgdorferi . Download figure Open in new tab Figure 5. Viability of B. burgdorferi in tissues from tick challenged pups. Quantification of B. burgdorferi in cultures of heart tissue from pups at ∼ 4 weeks post-challenge: (A) Enumeration of motile B. burgdorferi using a Petroff-Hausser chamber under a dark field microscope; (B) qPCR quantification of B. burgdorferi flaB in heart culture media. Each data point represents an individual pup, with lines indicating group means ± SD. Statistics by Mann Whitney test, *p<0.05 and ** p<0.005. Absence of OspA IgG in Serum from Pups Euthanized After Birth To determine whether maternal antibodies were transferred in utero, OspA ELISA was performed on serum from pups euthanized immediately after they dropped at birth in comparison to the respective dam ( Fig 6 ). In contrast to the respective dam, no anti-OspA IgG was detected in newborns from the vaccinated dam, confirming that placental transfer of antibodies was absent. Download figure Open in new tab Figure 6. Absence of OspA IgG in serum from pups euthanized after birth. ELISA quantification of anti-OspA IgG in serum from pups euthanized immediately after birth from dams vaccinated until two weeks parturition (3 rd Breeding). Optical density (OD) at 450 nm (blanked) is shown. Each data point represents an individual pup, with lines indicating group means. The cutoff value (dashed line) represents the mean + 3 SD of negative controls. Anti-OspA IgG depletion from vaccinated dam serum after lactation To investigate whether maternal antibody depletion occurred due to antibody transfer to offspring, serum anti-OspA IgG were measured in dams used for the 3 rd breeding at two time points (11Jun and 14Oct) and in the respective litters, on the latter timepoint, before tick challenge ( Fig 7 ). We found that the reduction in OspA antibody measured between the two time points in the dams corresponds to the increase in OspA antibody measured in the respective litters in the latter timepoint: ∼0.7OD 450 for Dam 1 ( Fig 7A ) and ∼1.8OD 450 for Dam 2 ( Fig 7B ). These findings suggest that transfer of antibody from dam to pups may be dependent on the efficiency of the pups’ suckling. Download figure Open in new tab Figure 7. Anti-OspA IgG depletion from vaccinated dam serum after pup suckling. ELISA quantification of anti-OspA IgG in serum from two dams used in the 3rd breeding and the respective litters. Sera from dams were collected at two time-points ∼ 4 months apart. Sera from the respective litters were collected on the latter time point, before tick challenge. Each data point represents an individual pup, with lines indicating group means ± SD. Discussion Our study demonstrates that vaccination of Peromyscus leucopus dams against B. burgdorferi lead to transfer of anti-OspA IgG to offspring and that, the extent of transfer and protective efficacy depends on maintenance of maternal immunization. Pups born to dams vaccinated until parturition and until two weeks post-parturition exhibited significantly higher anti-OspA IgG levels than those from dams vaccinated until breeding pairs were created. After challenge with B. burgdorferi infected I. scapularis ticks, pups born to vaccinated dams that maintained high systemic anti-OspA IgG had no anti- B. burgdorferi antibody in serum, no B. burgdorferi DNA in bladder or heart tissues and no motile B. burgdorferi in heart tissue. Furthermore, we detected no serologic anti-OspA IgG in offspring from vaccinated dams, euthanized immediately after birth, before suckling. The data indicates that anti-OspA antibody is transferred from P. leucopus dams to pups via lactation and that it protects against tick-transmitted B. burgdorferi . Vaccination of P. leucopus dams until the breeding pairs were created (1 st Breeding) did not protect P. leucopus pups from tick-mediated infection due to absence of sufficient levels of anti-OspA antibodies (OD 450 <0.8, Fig 2 ) in pups born ∼ 10 weeks after the dams were vaccinated. This was surprising given our previous studies in P. leucopus ( 25 ) and in C3H/HeN mice ( 22 ) in which we vaccinated mice with the reservoir targeted vaccine (RTV). In the first study, we vaccinated P. leucopus with 20-30 units of live RTV for 1-4 months and found that mice maintained anti-OspA antibody for up to a year ( 25 ). In the second study, we found that C3H/HeN dams vaccinated with 20 units of live RTV over a period of 8 weeks, produced pups 33-79 days after vaccination that maintained protective titers of OspA antibody from 2-9 weeks after birth ( 22 ). The major difference between the studies is that in the current study we used RTV inactivated with BPL, whereas in the former studies we used live RTV. This change was intentional to evaluate a vaccine that mimics the product currently licensed by USDA, which is inactivated with BPL ( 26 ). This data indicates that production of anti-OspA antibody by P. leucopus requires persistent vaccination, if the vaccine formulation is inactivated likely because it does not colonize the mouse gut. Previous studies show that transfer of maternal IgG provides passive immunity but does not necessarily confer sterilizing protection against vector-borne pathogens ( 20 , 21 ). The degree of IgG transfer and its impact on early-life immunity can vary depending on maternal antibody titers, the mechanism of transfer (transplacental vs. lactogenic), the duration of exposure ( 27 ) and neutralizing capability of the antibody ( 25 ). In our study, the neutralizing capability of anti-OspA antibody transferred from P. leucopus dams to its pups is evidenced by the absence of B. burgdorferi antibody in serum of pups (2/3 in Breeding 2 and 6/6 in Breeding 3, Fig 3 ), as well as absence of DNA in heart and bladder tissues (the same mice 2/3 in Breeding 2, and 6/6 in Breeding 3, Fig 4 ) and complete absence of motile spirochetes in cultures from heart tissue (6/6 in Breeding 3, Fig 5 ). All pups from breeding 1, born to vaccinated and control dams, had antibodies to B. burgdorferi antigens after tick challenge ( Fig 3 ) but we did not detect B. burgdorferi DNA in heart tissue of these mice ( Fig 4 ). We perform two independent tests (antibody to B. burgdorferi in serum and B. burgdorferi DNA in tissues) and we accept one positive result as evidence of B. burgdorferi dissemination ( 24 ). This method offsets risks incurring from tracking all datapoints in experiments that can last up to 1 year. Thus, for Breeding 1, we consider all mice in this experimental group to be infected and that this was associated with insufficient production of anti-OspA antibody by the respective vaccinated dams. Final efficacy of our intervention is determined by culture of B. burgdorferi from tissues of tick-challenged mice. Fig 5 shows conclusively that pups born to dams vaccinated until 2 weeks post-parturition did not have viable B. burgdorferi in cultures from heart tissue. This shows that for tick-transmitted B. burgdorferi, if P. leucopus is persistently vaccinated and produces sufficient anti-OspA antibody to be transferred to pups, the offspring is protected to at least 4 weeks post parturition. In addition, we show that immediately after birth, pups do not have anti-OspA antibodies in serum ( Fig 6 ). Thus, we infer that P. leucopus pups acquire OspA antibodies during suckling. This observation aligns with our previous studies in C3H mice demonstrating that anti-OspA IgG transfer via milk is a primary mechanism of passive immunity ( 22 ). A potential question may arise from differences in Fig 2 data showing significant increase of anti-OspA IgG in pups euthanized on the day of birth from dams vaccinated until parturition in Breeding 2 and data in Fig 6 showing absence of anti-OspA IgG in pups euthanized immediately after birth in Breeding 3. This is explained by the fact that pups from the 2 nd breeding were born overnight and were euthanized in the morning. Thus, for pups born from the 3 rd breeding and to confirm if antibody was transferred via lactation, as we showed previously for C3H mice ( 22 ), we euthanized the pups immediately after they dropped to prevent any potential suckling time. The decline in maternal anti-OspA IgG over time, particularly during lactation ( Fig 7 ), suggests a depletion of circulating IgG that could have impacted the duration of passive protection provided to pups. This observation is consistent with findings in other models where maternal antibody levels decrease postnatally as antibodies are transferred to offspring ( 20 ). From a reservoir-targeted vaccine intervention perspective, our findings suggest that if maternal anti-OspA antibodies reduce tick colonization efficiency or delay early-stage infection in P. leucopus , this may have broader ecological implications by disrupting pathogen transmission cycles. Further studies are needed to assess the effect of passively transferred anti-OspA antibody in reduction of nymphal infection prevalence. Acknowledgments Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Awards Number R01AI139267 (MGS), R43AI155211 (MGS) and R44AI167605 (MGS). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. License: CC-BY-ND Funding NIH-NIAID, , R01AI139267 , R43AI155211 , R44AI167605 Footnotes The text and the figures have been revised according to the reviewers recommendations. No supplemental material is included. References 1. ↵ Marques AR . 2010 . Lyme disease: a review . Curr Allergy Asthma Rep 10 : 13 – 20 . OpenUrl CrossRef PubMed Web of Science 2. ↵ Levine JF , Wilson ML , Spielman A . 1985 . Mice as reservoirs of the Lyme disease spirochete . Am J Trop Med Hyg 34 : 355 – 60 . OpenUrl Abstract / FREE Full Text 3. ↵ Donahue JG , Piesman J , Spielman A . 1987 . Reservoir competence of white-footed mice for Lyme disease spirochetes . Am J Trop Med Hyg 36 : 92 – 6 . OpenUrl Abstract / FREE Full Text 4. ↵ Mather TN , Mather ME . 1990 . Intrinsic competence of three ixodid ticks (Acari) as vectors of the Lyme disease spirochete . J Med Entomol 27 : 646 – 50 . OpenUrl CrossRef PubMed 5. ↵ Tsao J , Barbour AG , Luke CJ , Fikrig E , Fish D . 2001 . OspA immunization decreases transmission of Borrelia burgdorferi spirochetes from infected Peromyscus leucopus mice to larval Ixodes scapularis ticks . Vector Borne Zoonotic Dis 1 : 65 – 74 . OpenUrl CrossRef PubMed 6. ↵ Richer LM , Brisson D , Melo R , Ostfeld RS , Zeidner N , Gomes-Solecki M . 2014 . Reservoir targeted vaccine against Borrelia burgdorferi: a new strategy to prevent Lyme disease transmission . J Infect Dis 209 : 1972 – 80 . OpenUrl CrossRef PubMed 7. ↵ Gomes-Solecki M . 2014 . Blocking pathogen transmission at the source: reservoir targeted OspA-based vaccines against Borrelia burgdorferi . Front Cell Infect Microbiol 4 : 136 . OpenUrl PubMed 8. ↵ Schaible UE , Kramer MD , Eichmann K , Modolell M , Museteanu C , Simon MM . 1990 . 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 U S A 87 : 3768 – 72 . OpenUrl Abstract / FREE Full Text 9. ↵ Schwan TG , Piesman J , Golde WT , Dolan MC , Rosa PA . 1995 . Induction of an outer surface protein on Borrelia burgdorferi during tick feeding . Proc Natl Acad Sci U S A 92 : 2909 – 13 . OpenUrl Abstract / FREE Full Text 10. ↵ de Silva AM , Telford SR , 3rd . , Brunet LR , Barthold SW , Fikrig E. 1996 . Borrelia burgdorferi OspA is an arthropod-specific transmission-blocking Lyme disease vaccine . J Exp Med 183 : 271 – 5 . OpenUrl Abstract / FREE Full Text 11. ↵ Stevenson B , Schwan TG , Rosa PA . 1995 . Temperature-related differential expression of antigens in the Lyme disease spirochete, Borrelia burgdorferi . Infect Immun 63 : 4535 – 9 . OpenUrl Abstract / FREE Full Text 12. ↵ Schwan TG , Piesman J . 2000 . Temporal changes in outer surface proteins A and C of the lyme disease-associated spirochete, Borrelia burgdorferi, during the chain of infection in ticks and mice . J Clin Microbiol 38 : 382 – 8 . OpenUrl Abstract / FREE Full Text 13. Pal U , Yang X , Chen M , Bockenstedt LK , Anderson JF , Flavell RA , Norgard MV , Fikrig E . 2004 . OspC facilitates Borrelia burgdorferi invasion of Ixodes scapularis salivary glands . J Clin Invest 113 : 220 – 30 . OpenUrl CrossRef PubMed Web of Science 14. ↵ Grimm D , Tilly K , Byram R , Stewart PE , Krum JG , Bueschel DM , Schwan TG , Policastro PF , Elias AF , Rosa PA . 2004 . Outer-surface protein C of the Lyme disease spirochete: a protein induced in ticks for infection of mammals . Proc Natl Acad Sci U S A 101 : 3142 – 7 . OpenUrl Abstract / FREE Full Text 15. ↵ Fikrig E , Barthold SW , Kantor FS , Flavell RA . 1990 . Protection of mice against the Lyme disease agent by immunizing with recombinant OspA . Science 250 : 553 – 6 . OpenUrl Abstract / FREE Full Text 16. ↵ Fikrig E , Barthold SW , Kantor FS , Flavell RA . 1992 . Long-term protection of mice from Lyme disease by vaccination with OspA . Infect Immun 60 : 773 – 7 . OpenUrl Abstract / FREE Full Text 17. ↵ Sigal LH , Zahradnik JM , Lavin P , Patella SJ , Bryant G , Haselby R , Hilton E , Kunkel M , Adler-Klein D , Doherty T , Evans J , Molloy PJ , Seidner AL , Sabetta JR , Simon HJ , Klempner MS , Mays J , Marks D , Malawista SE . 1998 . A vaccine consisting of recombinant Borrelia burgdorferi outer-surface protein A to prevent Lyme disease. Recombinant Outer-Surface Protein A Lyme Disease Vaccine Study Consortium . N Engl J Med 339 : 216 – 22 . OpenUrl CrossRef PubMed Web of Science 18. ↵ Steere AC , Sikand VK , Meurice F , Parenti DL , Fikrig E , Schoen RT , Nowakowski J , Schmid CH , Laukamp S , Buscarino C , Krause DS . 1998 . Vaccination against Lyme disease with recombinant Borrelia burgdorferi outer-surface lipoprotein A with adjuvant. Lyme Disease Vaccine Study Group . N Engl J Med 339 : 209 – 15 . OpenUrl CrossRef PubMed Web of Science 19. ↵ Tsao JI , Wootton JT , Bunikis J , Luna MG , Fish D , Barbour AG . 2004 . An ecological approach to preventing human infection: vaccinating wild mouse reservoirs intervenes in the Lyme disease cycle . Proc Natl Acad Sci U S A 101 : 18159 – 64 . OpenUrl Abstract / FREE Full Text 20. ↵ Hasselquist D , Nilsson JA . 2009 . Maternal transfer of antibodies in vertebrates: trans-generational effects on offspring immunity . Philos Trans R Soc Lond B Biol Sci 364 : 51 – 60 . OpenUrl CrossRef PubMed 21. ↵ Boulinier T , Staszewski V . 2008 . Maternal transfer of antibodies: raising immuno-ecology issues . Trends Ecol Evol 23 : 282 – 8 . OpenUrl CrossRef PubMed Web of Science 22. ↵ Phillip K , Nair N , Samanta K , Azevedo JF , Brown GD , Petersen CA , Gomes-Solecki M . 2021 . Maternal transfer of neutralizing antibodies to B. burgdorferi OspA after oral vaccination of the rodent reservoir . Vaccine 39 : 4320 – 4327 . OpenUrl CrossRef PubMed 23. ↵ Gomes-Solecki M , Richer L . 2018 . Recombinant E. coli Dualistic Role as an Antigen-adjuvant Delivery Vehicle for Oral Immunization . Methods Mol Biol 1690 : 347 – 357 . OpenUrl CrossRef PubMed 24. ↵ Gingerich MC , Nair N , Azevedo JF , Samanta K , Kundu S , He B , Gomes-Solecki M . 2024 . Intranasal vaccine for Lyme disease provides protection against tick transmitted Borrelia burgdorferi beyond one year . NPJ Vaccines 9 : 33 . OpenUrl CrossRef PubMed 25. ↵ Meirelles Richer L , Aroso M , Contente-Cuomo T , Ivanova L , Gomes-Solecki M . 2011 . Reservoir targeted vaccine for lyme borreliosis induces a yearlong, neutralizing antibody response to OspA in white-footed mice . Clin Vaccine Immunol 18 : 1809 – 16 . OpenUrl Abstract / FREE Full Text 26. ↵ Stafford KC , 3rd . , Williams SC , van Oosterwijk JG , Linske MA , Zatechka S , Richer LM , Molaei G , Przybyszewski C , Wikel SK . 2020 . Field evaluation of a novel oral reservoir-targeted vaccine against Borrelia burgdorferi utilizing an inactivated whole-cell bacterial antigen expression vehicle . Exp Appl Acarol 80 : 257 – 268 . OpenUrl CrossRef PubMed 27. ↵ Fouda GG , Martinez DR , Swamy GK , Permar SR . 2018 . The Impact of IgG transplacental transfer on early life immunity . Immunohorizons 2 : 14 – 25 . OpenUrl Abstract / FREE Full Text View the discussion thread. Back to top Previous Next Posted April 23, 2025. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Maternal Transfer of Oral Vaccine Induced Anti-OspA Antibodies Protects Peromyscus spp Pups from Tick-Transmitted Borrelia burgdorferi Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Maternal Transfer of Oral Vaccine Induced Anti-OspA Antibodies Protects Peromyscus spp Pups from Tick-Transmitted Borrelia burgdorferi Jose F. Azevedo , Greg Joyner , Suman Kundu , Kamalika Samanta , Maria Gomes-Solecki bioRxiv 2025.02.24.639966; doi: https://doi.org/10.1101/2025.02.24.639966 Share This Article: Copy Citation Tools Maternal Transfer of Oral Vaccine Induced Anti-OspA Antibodies Protects Peromyscus spp Pups from Tick-Transmitted Borrelia burgdorferi Jose F. Azevedo , Greg Joyner , Suman Kundu , Kamalika Samanta , Maria Gomes-Solecki bioRxiv 2025.02.24.639966; doi: https://doi.org/10.1101/2025.02.24.639966 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Microbiology Subject Areas All Articles Animal Behavior and Cognition (7624) Biochemistry (17651) Bioengineering (13873) Bioinformatics (41887) Biophysics (21424) Cancer Biology (18566) Cell Biology (25465) Clinical Trials (138) Developmental Biology (13365) Ecology (19871) Epidemiology (2067) Evolutionary Biology (24293) Genetics (15591) Genomics (22478) Immunology (17715) Microbiology (40331) Molecular Biology (17150) Neuroscience (88492) Paleontology (666) Pathology (2828) Pharmacology and Toxicology (4817) Physiology (7635) Plant Biology (15114) Scientific Communication and Education (2044) Synthetic Biology (4286) Systems Biology (9817) Zoology (2268)