A parainfluenza virus 5 (PIV5)-vectored intranasal vaccine for Lyme disease provides long-lasting protection against tick transmitted Borrelia burgdorferi in mice

Lyme disease (LD) is the most prevalent vector borne disease in North America and Europe and its geographic range continues to expand. Strategies for disease control are necessary to effectively reduce incidence of LD including development of safe vaccines for human use. Parainfluenza virus 5 (PIV5) vector has an excellent safety record in animals and PIV5-vectored COVID-19 and RSV vaccines are currently under clinical development. We constructed PIV5-vectored LD vaccine candidates expressing OspA from B. burgdorferi sensu stricto (OspAB31) and a chimeric protein containing sequences from B. burgdorferi and B. afzelii (OspABPBPk). Immunogenicity and vaccine efficacy were analyzed in C3H-HeN mice after prime-boost intranasal (IN) vaccination with PIV5-OspAB31 and PIV5-OspABPBPk, subcutaneous (SC) vaccination with rOspAB31+Alum as well as the respective controls. Mice vaccinated with either PIV5-AB31 or PIV5-ABPBPk intranasally had high endpoint titers of serum antibody against OspA antigen beyond 1 year post vaccination, similar to levels detected in mice vaccinated SC with rOspAB31. Flowcytometric analysis of spleen cells at 9-months post-immunization demonstrated that immunization with the intranasal PIV5 vaccine candidates led to an overall increase in the number of memory B cells, cytotoxic T and cytotoxic effector T cells compared to SC groups. Borreliacidal activity measured by neutralization assay was maintained up to 18 months post-immunization, with the response greater in intranasal PIV5-delivered OspA vaccines, than that induced by SC rOspAB31. Challenge with infected ticks (10-19 strains of B. burgdorferi) performed at 4-, 9- or 15-months post-immunization showed increased breakthrough infections in mice vaccinated with SC rOspAB31 compared to IN PIV5-AB31 or IN PIV5-ABPBPk at 9- and 15-months, as determined by qPCR of B. burgdorferi in tissues, culture of B. burgdorferi from tissues, and antibodies against B. burgdorferi protein VIsE. These data demonstrate that intranasal PIV5-based immunization is superior to parenteral immunization with the same recombinant protein and provides long-lasting protection (> 1 year) against Lyme disease.


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Lyme disease (LD) is a tick-borne illness caused by the spirochete Borrelia burgdorferi sensu 46 lato (Bbsl), (Borreliella genus novum under consideration 1 ). In humans, it is a progressive 47 illness with a wide range of common clinical manifestations gradually developing from early to 48 late stage. Late disseminated infection is associated with permanent damage to the nervous 49 and musculoskeletal systems 2 3 4 5 . LD is the most prevalent vector borne disease throughout 50 the Northeast, Mid-Atlantic and Midwest regions of the U.S. where the disease is endemic and 51 its range continues to expand 6 . Analysis of anonymous insurance claims data from 2010 -2018 7 52 led the CDC to estimate that ~ 476,000 people were diagnosed and treated for LD annually 53 during that period. This new estimate further highlights the importance of LD as a public health 54 concern. The most effective way to avoid this debilitating disease is to avoid tick-infested areas.

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Additional disease control strategies are necessary to effectively reduce incidence of Lyme 56 disease including development of safe vaccines for human use.

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Outer surface protein A (OspA) is the only immunogen proven to provide 76-92% protection 8,9 58 against tick-transmitted B. burgdorferi in fully vaccinated human subjects. After a 20-year hiatus, 59 a re-engineered vaccine based on the C-terminus sequence of OspA from several Bbsl

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Viruses: The PIV5-AB31 and PIV5-ABPBPk plasmids, encoding the full-length genome of PIV5 and 80 a Borrelia burgdorferi strain B31 OspA protein and well as BPBPk protein gene sequences 21 81 inserted between the PIV5 SH and HN genes was constructed as previously 14 .Virus rescue 82 was performed as described 14 . Briefly, the PIV5-AB31 and PIV5-ABPBPk plasmids and four helper 83 plasmids-pPIV5-NP, pPIV5-P, pPIV5-L, and pT7-polymerase, encoding the NP, P, and L 84 proteins and T7 RNA polymerase, respectively-were co-transfected into BHK21 cells at 90% 85 confluence in 6-cm plates using JetPrime (Polyplus). Recovery of the virus is indicated by 86 syncytia formation. The virus was then plaque-purified as a single plaque from BHK21 cells. The 87 full-length genomes of the plaque-purified single clone of PIV5-AB31 and PIV5-ABPBPk viruses 88 were sequenced as described before 14 . Viruses were grown in MDBK cells for 5 to 7 days using 89 DMEM containing 2% fetal bovine serum (FBS). Media were collected and pelleted at 3000 rpm 90 to remove cell debris by using a Sorvall tabletop centrifuge for 10 min. Virus supernatant was 91 supplemented with 10% sucrose-phosphate-glutamate buffer, snap-frozen in liquid nitrogen, 92 and stored at −80°C immediately after collection.

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Western blot: Immunoblotting was performed on Vero cells in 6-well plates that were infected

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Immunization, immunogenicity and vaccine efficacy analysis

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All experiments were performed in accordance with protocols approved by the IACUC at the 102 University of Georgia (A2023 01-021-Y1-A0) and at the University of Tennessee Health Science 103 Center (19-103). A graphic representation on the animal experimental design is shown in Figure   104 1.

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Immunization: Briefly, groups of 5 to 8-week-old female C3H-HeN mice (Envigo) were 106 anesthetized and intranasally inoculated with 10 6 PFU of PIV5 vector, PIV5-AB31, or PIV5-ABPBPk, 107 or with 20 µg of rOspAB31+Alum subcutaneously. Twenty-one days after prime immunization, the 108 mice were boosted with the same preparations. Blood ( Fig. 1) was collected before tick 109 challenge for determination of anti-OspA antibody on the following days: d17, d86, d100 and 110 d117 for Study 1 (4-month challenge, D117 serum was used for neutralization assays); d17, 111 d88, d103, d118 and monthly thereafter until d270 for Study 2 (9-month challenge, D270 serum 112 was used for neutralization assays). For Study 3 (15-month challenge and 18-month 113 neutralization), groups of mice were bled on d17, d86, d100 and d117 and monthly thereafter 114 until D455 for 15-month challenge; a subset of these mice were not challenged and were kept until 18 months post prime-boost for an additional collection of blood at D533 for analysis of 116 anti-B. burgdorferi neutralization activity.

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Neutralization antibody (nAb) assay: Briefly, blood was collected from groups of vaccinated 128 mice the day before challenge (d117) for the 4-month challenge experiment, and on days 270 129 and 533 from groups of vaccinated mice that were not subjected to challenge for the 9 month

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Data was aligned against the fasta file containing OspC variants provided. SAM files were mined for total read count tables. SAM files were then filtered to obtain only uniquely aligned 167 fragments. Filtered SAM files were mined for unique read count tables. Graphs were created 168 using R 16 .

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Tick challenge: Three colonies of infected I. scapularis were maintained in the laboratory.     (Table S1)

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LSD was used to compare flow cytometric differences within groups (Fig 6). One-way ANOVA 220 followed by Dunnett's multiple comparison test by comparing groups to IN PIV5 group (Fig 8).

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GraphPad Prism was used for the statistical analysis and plotting the graphs.  very low levels of cross-reactive IgG antibodies at some point throughout the study, but the 262 peak titer for these control groups are 3 log10 lower than the peak titers for all vaccine groups.

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These results suggest that immunization with an intranasal, PIV5-based vaccine may lead to 264 longer-lasting protection than a protein-based vaccine given subcutaneously.

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PBS+Alum group. For this study blood was collected on D117 for neutralization assays of B. 270 burgdorferi in culture (Fig. 5) before the mice were challenged (4-month, pre-challenge).

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Because insufficient blood was collected from each mouse, we pooled the serum from each 272 group for the neutralization assay (Fig. 5A). For the longevity studies (Studies 2 and 3) we     (Fig. 7A). Furthermore, heart and bladder tissues were cultured in BSK-H medium to 335 evaluate mobility/viability of B. burgdorferi under a dark field microscope ( Table 1)

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To further confirm B. burgdorferi dissemination after challenge, IgG to B. burgdorferi 350 VlsE protein was quantified in serum from vaccinated mice, by ELISA (Fig. 8). In Study 1 (4-  Table 1.

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Currently, the most effective way to prevent Lyme disease is to avoid I. scapularis tick infested 366 areas. This is unfeasible for those who work outside and for those who enjoy spending quality 367 time outdoors in the Spring and Summer in endemic areas. Thus, there is a pressing need for 368 development and commercialization of effective and acceptable vaccines to control Lyme 369 disease. We developed a novel parainfluenza 5 (PIV5) viral-vectored vaccine for intranasal delivery that provides long-lasting protection against tick-transmitted B. burgdorferi using a 371 prime-boost scheme of immunization.

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In this study, we demonstrate that both PIV5-based vaccines, PIV5-ABPBPk and PIV5-403 AB31, administered in a homologous prime-boost vaccination regimen intranasally, can induce 404 robust humoral IgG immune responses in mice reaching over 5 log10 EPT for serum IgG 405 antibodies at 3 months post-vaccination, with these values moderately decreasing up to 12 406 months post-immunization (Fig. 4). The longevity of the immune response can also be seen in response, but the levels of antibodies decreased more than 10-fold, five months after 420 immunization 10 . A booster dose was administered 5 months after immunization to increase these levels. To accommodate these issues, the VLA15 vaccine undergoing phase 3 clinical 422 trials requires a 3-shot intramuscular vaccination schedule on Day 1, Day 57 and Day 180. Here 423 we show that a mucosal delivered viral-vectored vehicle leverages a non-invasive administration 424 route (intranasal) and generates a rapid, durable and neutralizing IgG response to the 425 immunogen that lasts up to 18 months. We also found that this IgG antibody response was 426 accompanied by increased memory B cells and cytotoxic effector T cell populations that appear 427 to be driven by PIV5 (Fig. 6). PIV5-based vaccines have been shown to induce robust levels of 428 cellular immunity that aid in protection against bacterial and viral pathogens in various animal 429 models 13,32,40 , further validating the use of a live, viral-vectored vaccine platform for vaccine 430 development. Furthermore, longevity of protection was associated with PIV5 vaccinations in 431 challenges performed at 9 and 15 months post-prime vaccination (Fig. 7, Fig. 8 and Table 1         Flat I.s. Nymph