key: cord-0260123-wkt5oe5t
authors: O’Connell, Kathryn; Nair, Nisha; Samanta, Kamalika; Azevedo, Jose F.; Brown, Grant D.; Petersen, Christine A.; Gomes-Solecki, Maria
title: Maternal transfer of neutralizing antibodies to OspA after oral vaccination of the rodent reservoir
date: 2021-01-27
journal: bioRxiv
DOI: 10.1101/2021.01.27.428441
sha: 38a91891d2de581ec92f30c7a8e61e3931e111b7
doc_id: 260123
cord_uid: wkt5oe5t

Lyme Disease presents unique challenges for public health efforts. We hypothesized that transfer of protective antibodies between mothers and offspring should occur after oral vaccination of C3H-HeN mice with E. coli overexpressing OspA. We present new evidence for maternal transfer of vaccine induced neutralizing anti-OspA IgG antibodies to mouse pups through ingestion of colostrum. Protective levels of OspA antibodies in pups were present from 2-5 weeks after birth and they persisted in some mice until 9 weeks of age. This was corroborated by detection of neutralizing antibodies in the serum of all pups at 2-3 weeks after birth and in some mice at 9 weeks of age. A clear association was found between robust antibody responses in mothers and the length of antibody persistence in the respective pups using a novel longitudinal Bayesian model. These factors are likely to impact the enzootic cycle of B. burgdorferi when reservoir targeted OspA-based vaccination interventions are implemented.

evidence of this type of protection against Borrelia burgdorferi infection. We hypothesized 71 that maternal transfer of neutralizing anti-OspA antibodies occurred after vaccination of 72 female mice. We immunized mice orally with the same vaccine master stock previously 73 tested 12,13 , and evaluated transfer of OspA-specific antibodies to offspring via 74 transplacental and transmammary transmission. Furthermore, we developed a novel 75 Bayesian longitudinal model to jointly evaluate mother and pup immunity, allowing a 76 robust exploration of the potential mechanisms for maternal transfer of antibody based on 77 the time at which we detect antibodies in pups paired with the mother's immune state. vaccine group and one group of 6 females was assigned to placebo. Females that received 119 oral vaccine (EcA) were labeled as mothers M1 to M10 and females that received oral placebo 120 (Ec, Ctrl) were labelled as mothers M11 to M16. A timeline diagram depicting vaccination, 121 breeding and collection of samples is shown in Fig. 1 .

Immunization schedule: 6-week old female mice received 1 vaccine dose (~10 9 E. coli cells) 124 daily by oral gavage for 2 full work-weeks (10 doses); mice rested for 1 week and on the 125 following week they received the 1 st boost (1 dose/day for 5 days) and after resting another 126 week they received a 2 nd boost (1 dose/day for 5 days) for a total of 20 vaccine doses per 127 mouse.

Breeding and birth: 2 weeks after receiving the last vaccine dose (8-week vaccination 130 schedule), pairs of females were co-housed with an age matched male for about 3-4 weeks.

After breeding each female was single housed until parturition. Parturition occurred over a 132 span of ~6 weeks due to differences in successful mating between pairs. Females started 133 giving birth to pups ~11-17 weeks after the first vaccine dose. Pups were followed for 17 134 weeks.

Collection of blood for serology and neutralization assays: blood was collected from the 137 mothers before immunization on D0, before the first boost on D21, before the second 138 boost on D35, post-vaccination before breeding ~D54, post-vaccination post-birth on 139 D133/154 (when the pups ~7 weeks old) and post-vaccination at termination ~ D203/218 140 (when the pups were ~17 weeks old). The pups were split into two cohorts. The first 141 cohort of 44 pups (Cohort A) was sacrificed for collection of blood on the day of birth Presence of neutralizing anti-OspA antibody was determined in serum from mothers and 168 pups as previously described 13 . We used serum from mothers collected after vaccination 169 and before breeding (~D54). The serum from pups was collected on the day of birth (0-170 24h), at 2-3 weeks after birth and at 9 weeks after birth. Briefly, a 10 7 /mL multi-strain Rather than considering group differences and treating pup and mother analyses 178 separately, we introduced a Bayesian model to jointly consider anti-OspA antibody levels 179 in mothers and her pups. Specifically, we introduced a pair of auto-correlated, log-scale 180 time-series with forcing parameters related to oral vaccine dose-timing (mothers) and 181 birth/weaning processes (pups). Parameters were estimated separately between vaccine 182 and placebo groups. For experimental group , mother , and time index , we modeled 183 the outcome as an auto-regressive Gaussian time series (Eq. 1). 196 . ~(0,10), 2 ~gamma(1,1), ρ k ~beta(1,1), and ~(0,1).

The model was implemented as a reproducible R-markdown document and is referenced 198 along with the requisite data sets in Table S1 . The analysis presents a novel approach to 199 joint modeling of mechanistically-related longitudinal data sets and allows us to make full 

A total of 105 C3H -HeN pups were born to the 16 mothers: 59 pups were born to 210 mothers vaccinated with E. coli overexpressing OspA (EcA), and 46 pups were born 211 to mothers that received E. coli placebo control (Ec, Ctrl) with an average litter size of 5.9

for EcA and 5.75 for Ctrl. Insufficient blood was collected from 2 pups born to control 213 mothers, and as such, those pups were not included in further analysis.

Kinetics of OspA IgG in serum from orally vaccinated mothers over 7 months 216 We measured anti-OspA IgG in serum from orally vaccinated C3H-HeN mothers and 217 placebo controls by ELISA (Fig. 2) . As expected, the prevalence of mice with protective OspA-specific antibody were not significant between pups born to EcA and Ctrl mothers.

However, a breakdown of data by birth mother showed there was one litter of pups born 240 to M9 that clearly had anti-OspA antibodies in serum (Fig. 3B ). In the second cohort of autocorrelation, were estimated separately ( Table 1) . We estimate a 99% probability that 265 the effect of maternal vaccination on presence of anti-OspA antibodies in the pups was 266 higher in EcA group than Ec (Fig. 4) . In addition, the EcA group exhibited higher we observe unambiguous differences between vaccine-induced OspA-specific antibodies 275 and unmodified cross-reactive antibodies in the control group. In addition, we find 276 evidence (probability ≈ 1) that autocorrelation was higher in EcA group pups than in 277 control group pups, and that the decay of antibody levels was slower (probability 0.85). had levels of OspA-specific antibodies OD450>0.8 before breeding on D54 ( Fig. 2A) . We 289 did not include M1 because this mother did not develop antibodies to OspA. We were 290 able to keep one mother from each group used to generate the two cohorts of mice (A 291 and B) that maintained highest levels of antibody to OspA on ~D210 (M3 and M9), to test 292 independently. We ran an immunoblot of a multi-strain culture used for the neutralization 293 assay against an anti-OspA monoclonal antibody and show that OspA is expressed by B.

294 burgdorferi (Bb) in culture (Fig S1) . We found that serum from mothers M6 to M10 that (M4) (Fig 5C) . Serum from pups born to M3 still had enough anti-OspA antibody to 305 neutralize motile Bb in culture by 1 Log10 9 weeks after birth (Fig 5F) .

In this study we demonstrate that specific antibodies from immunized mouse mothers 310 were transferred to offspring born to mice orally vaccinated with E. coli overexpressing 311

OspA. Immunoglobulin G specific to OspA was passed v i a t r a n s m a m m a r y 312 t r a n s m i s s i o n to pups through ingestion of colostrum and milk during the lactation 313 period, shortly after birth lasting 3 weeks. Although likely, we were unable to conclude 314 with all confidence that antibodies are not transferred via the placenta. We found a strong gavage with a live culture of E. coli overexpressing OspA (Fig 2A) . In our previous studies anti-OspA immunity differed considerably between the mothers (Fig 2B) . This is also 347 consistent with our previous findings using the same vaccine formulation and schedule 12 .

Nevertheless, vaccination of P. leucopus using a baited formula rather than oral gavage 349 and a different schedule of immunization 13 it is reasonable to expect that oral bait to vaccinated mothers we found a clear difference in antibody levels at birth versus 2+ weeks after birth (Fig 3) . After birth, antibodies to OspA remained detectable in the pups 357 for about 2 months. Furthermore, the robustness of the maternal immune response to 358 OspA directly correlated with the amount of antibody transferred to offspring. We 359 observed a positive association between high levels of antibodies in the birth mother and 360 the length of time these antibodies persisted in the offspring serum; for example, the 361 mother with the highest level of anti-OspA antibodies produced offspring that had 362 persistent antibodies 9 weeks after birth, indicating a more durable antibody response.

Another interesting finding was that pups from larger litters had less total antibody to 364 OspA in serum which strongly suggests a dilution effect between mother and offspring. In 365 other words, the mother produces a fixed amount of antibody to OspA at the peak of her 366 immune response and this is split between the offspring depending on time of suckling. At 367 birth, the pups born to 4/5 mothers did not have antibody to OspA. However, one mother 368 (M9) produced pups that had antibodies to OspA on the day of birth. Since most of the 369 pups were born overnight, M9 may have been the first mother to give birth and her pups 370 may have begun nursing much sooner than the pups born to the other mothers. Although 371 the data is highly suggestive of absence of transplacental transfer, we cannot reach an 372 unequivocal conclusion, given that the mothers that produced the Cohort A pups also 373 developed less overall antibody to OspA after vaccination. This difference could be 374 explained by the longer time these mothers took to breed and produce offspring.

Dual modelling of mothers and their corresponding pups, we observed systematic 376 differences in antibody profiles. In addition to the unambiguous detection of immunization 377 effects in mothers and the presence of maternal transfer of antibodies to pups (Fig 4) , we Fig. 2 . Kinetics of OspA-speci c antibodies in orally vaccinated and placebo mothers over ~7 months. We measured anti-OspA IgG by ELISA in serum from 10 mothers orally vaccinated E. coli expressing OspA (EcA, M1-M10) and 6 non-vaccinated placebo controls (Ec, M11-M16). Mice were bled before (D0) and after immunization (D21, D35), before breeding (D54) after the pups were born ~D140 (7 wk old pups) and ~D210 (17 wk old pups). A, Comparison of anti-OspA IgG (OD450>0.8) between EcA and placebo Ec groups. Di erences between EcA and Ec groups are statistically signi cant by unpaired t test with Welch's correction, D21 p=0.0038, D35 p= 0.0260, D54 p=0.0003, D140 p=0.0147, D210 p=0.0488. B, Longitudinal log scale trajectory of antibody response over time, each line represents one mother, M5 was not bled on D140. 

Dualistic Role as an Antigen-adjuvant 478

Delivery Vehicle for Oral Immunization

 433 The following authors declare potential conflicts of interest: MGS (grants from federal 434 agencies, employment, patents and consultant), CAP (grants from federal agencies, 435 employment, and consultant). All other authors declare no conflicts.