Combination immunotherapy with vaccine and oncolytic HSV virotherapy is time dependent.

PURPOSE: Oncolytic virotherapy or immunovirotherapy is a strategy that utilizes viruses to selectively infect and kill tumor cells while also stimulating an immune response against the tumor. Early clinical trials in both pediatric and adult patients using oncolytic herpes simplex viruses (oHSVs) have demonstrated safety and promising efficacy; however, combinatorial strategies designed to enhance oncolysis while also promoting durable T cell responses for sustaining disease remission are likely required. We hypothesized that combining the direct tumor cell killing and innate immune stimulation by oHSV with a vaccine that promotes T cell mediated immunity may lead to more durable tumor regression. EXPERIMENTAL DESIGN: To this end, we investigated the preclinical efficacy and potential synergy of combining oHSV with a self-assembling nanoparticle vaccine co-delivering peptide antigens and Toll-like receptor-7 and -8 agonists (TLR-7/8a) (referred to as SNAPvax™), that induces robust tumor specific T cell immunity. We then assessed how timing of the treatments (i.e., vaccine before or after oHSV) impacts T cell responses, viral replication, and preclinical efficacy. RESULTS: The sequence of treatments was critical, as survival was significantly enhanced when the SNAPvax™ vaccine was given prior to oHSV. Increased clinical efficacy was associated with reduced tumour volume and increases in virus replication and tumor antigen specific CD8+ T cells. CONCLUSIONS: These findings substantiate the criticality of combination immunotherapy timing and provide preclinical support for combining SNAPvax with oHSV as a promising treatment approach for both pediatric and adult tumors.

Structural basis for self-discrimination by neoantigen-specific TCRs.

T cell receptors (TCR) are pivotal in mediating tumour cell cytolysis via recognition of mutation-derived tumour neoantigens (neoAgs) presented by major histocompatibility class-I (MHC-I). Understanding the factors governing the emergence of neoAg from somatic mutations is a major focus of current research. However, the structural and cellular determinants controlling TCR recognition of neoAgs remain poorly understood. This study describes the multi-level analysis of a model neoAg from the B16F10 murine melanoma, H2-Db/Hsf2 p.K72N68-76, as well as its cognate TCR 47BE7. Through cellular, molecular and structural studies we demonstrate that the p.K72N mutation enhances H2-Db binding, thereby improving cell surface presentation and stabilizing the TCR 47BE7 epitope. Furthermore, TCR 47BE7 exhibited high functional avidity and selectivity, attributable to a broad, stringent, binding interface enabling recognition of native B16F10 despite low antigen density. Our findings provide insight into the generation of anchor-residue modified neoAg, and emphasize the value of molecular and structural investigations of neoAg in diverse MHC-I contexts for advancing the understanding of neoAg immunogenicity.

mRNA vaccination of rabbits alters the fecundity, but not the attachment, of adult Ixodes scapularis.

19ISP is a nucleoside-modified mRNA-lipid nanoparticle vaccine that targets 19 Ixodes scapularis proteins. We demonstrate that adult I. scapularis have impaired fecundity when allowed to engorge on 19ISP-immunized rabbits. 19ISP, therefore, has the potential to interrupt the tick reproductive cycle, without triggering some of the other effects associated with acquired tick resistance. This may lead to the development of new strategies to reduce I. scapularis populations in endemic areas.

Bulk and single-nucleus RNA sequencing highlight immune pathways induced in individuals during an Ixodes scapularis tick bite.

Ticks are hematophagous arthropods that use a complex mixture of salivary proteins to evade host defenses while taking a blood meal. Little is known about the immunological and physiological consequences of tick feeding on humans. Here, we performed the first bulk and single-nucleus RNA sequencing (snRNA-seq) of skin and blood of four persons presenting with naturally acquired, attached Ixodes scapularis ticks. Pathways and individual genes associated with innate and adaptive immunity were identified based on bulk RNA sequencing, including interleukin-17 signaling and platelet activation pathways at the site of tick attachment or in peripheral blood. snRNA-seq further revealed that the Hippo signaling, cell adhesion, and axon guidance pathways were involved in the response to an I. scapularis bite in humans. Features of the host response in these individuals also overlapped with that of laboratory guinea pigs exposed to I. scapularis and which acquired resistance to ticks. These findings offer novel insights for the development of new biomarkers for I. scapularis exposure and anti-tick vaccines for human use.

Intravenous heterologous prime-boost vaccination activates innate and adaptive immunity to promote tumor regression.

Therapeutic neoantigen cancer vaccines have limited clinical efficacy to date. Here, we identify a heterologous prime-boost vaccination strategy using a self-assembling peptide nanoparticle TLR-7/8 agonist (SNP) vaccine prime and a chimp adenovirus (ChAdOx1) vaccine boost that elicits potent CD8 T cells and tumor regression. ChAdOx1 administered intravenously (i.v.) had 4-fold higher antigen-specific CD8 T cell responses than mice boosted by the intramuscular (i.m.) route. In the therapeutic MC38 tumor model, i.v. heterologous prime-boost vaccination enhances regression compared with ChAdOx1 alone. Remarkably, i.v. boosting with a ChAdOx1 vector encoding an irrelevant antigen also mediates tumor regression, which is dependent on type I IFN signaling. Single-cell RNA sequencing of the tumor myeloid compartment shows that i.v. ChAdOx1 reduces the frequency of immunosuppressive Chil3 monocytes and activates cross-presenting type 1 conventional dendritic cells (cDC1s). The dual effect of i.v. ChAdOx1 vaccination enhancing CD8 T cells and modulating the TME represents a translatable paradigm for enhancing anti-tumor immunity in humans.

Evaluation of the in vitro acaricidal effect of five organic compounds on the cattle fever tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).

The cattle fever tick, Rhipicephalus (Boophilus) microplus, is the most economically important tick worldwide. Infestations with this tick can lead to direct damage and cattle mortality due to the transmission of potentially deadly pathogens. Management of this tick species has been focused on the use of synthetical acaricides; however, the emergence of acaricide resistance to single or multiple active ingredients has resulted in a need for novel acaricide compounds. Among potential avenues for the discovery of novel acaricides are plant-derived compounds. The efficacy of five organic compounds (nootkatone, Stop the Bites®, BioUD®, lavender oil, and cedarwood oil) was evaluated using larval immersion tests (LITs), repellency assays, and adult immersion tests (AITs). The results from the LITs indicate that three of the organic compounds (NootkaShield™, Stop the Bites, BioUD) led to significant mortalities at low concentrations (0.2, 0.02, and 0.08%, respectively). By comparison, lavender and cedar oil led to around 90% mortality at 10 and 1% concentrations, respectively. Similarly, NootkaShield, Stop the Bites, and BioUD had strong repellent properties with over 90% repellency at the two highest concentrations tested. Using the FAO 2004 guidelines, we evaluated the effectiveness of these organic compounds at reducing the fecundity of R. (B.) microplus and show that Nootkatone, Stop the Bites, and BioUD may significantly decrease tick populations (Drummond's index > 90% at concentrations of 5%), highlighting their potential as alternatives to synthetic acaricides for the control of cattle fever ticks.

Structural Basis for Self-Discrimination by Neoantigen-Specific TCRs.

Physical interactions between T cell receptors (TCRs) and mutation-derived tumour neoantigens (neoAg) presented by major histocompatibility class-I (MHC-I) enable sensitive and specific cytolysis of tumour cells. Adoptive transfer of neoAg-reactive T cells in patients is correlated with response to immunotherapy; however, the structural and cellular mechanisms of neoAg recognition remain poorly understood. We have identified multiple cognate neoAg:TCRs from B16F10, a common murine implantable tumour model of melanoma. We identified a high affinity TCR targeting H2-Db-restricted Hsf2K72N that conferred specific recognition of B16F10 in vitro and in vivo. Structural characterization of the peptide-MHC (pMHC) binary and pMHC:TCR ternary complexes yielded high-resolution crystal structures, revealing the formation of a solvent-exposed hydrophobic arch in H2-Db that enables multiple intermolecular contacts between pMHC and the TCR. These features of structural stability strikingly mimic that of a previously published influenza peptide-H2-Db complex and its corresponding TCR, suggesting that there are shared structural motifs between neoantigens and viral peptides that explain their shared immunogenicity.

Systemic vaccination induces CD8+ T cells and remodels the tumor microenvironment.

Therapeutic cancer vaccines are designed to increase tumor-specific T cell immunity. However, suppressive mechanisms within the tumor microenvironment (TME) may limit T cell function. Here, we assessed how the route of vaccination alters intratumoral myeloid cells. Using a self-assembling nanoparticle vaccine that links tumor antigen peptides to a Toll-like receptor 7/8 agonist (SNP-7/8a), we treated tumor-bearing mice subcutaneously (SNP-SC) or intravenously (SNP-IV). Both routes generated antigen-specific CD8+ T cells that infiltrated tumors. However, only SNP-IV mediated tumor regression, dependent on systemic type I interferon at the time of boost. Single-cell RNA-sequencing revealed that intratumoral monocytes expressing an immunoregulatory gene signature (Chil3, Anxa2, Wfdc17) were reduced after SNP-IV boost. In humans, the Chil3+ monocyte gene signature is enriched in CD16- monocytes and associated with worse outcomes. Our results show that the generation of tumor-specific CD8+ T cells combined with remodeling of the TME is a promising approach for tumor immunotherapy.

Immunization of guinea pigs with cement extract induces resistance against Ixodes scapularis ticks.

As hematophagous parasites, many tick species are important vectors of medical and veterinary disease agents. Proteins found in tick saliva and midgut have been used with some success in immunizations of animal hosts against feeding ticks, and whole saliva has been used effectively in this capacity against Ixodes scapularis, the primary vector of tickborne pathogens in the United States. Tick saliva is a complex substance containing hundreds of proteins, and the identification of specific protective antigens is ongoing. We performed a series of experiments immunizing guinea pigs with extracts prepared from midgut or attachment cement collected from adult female I. scapularis followed by challenge with nymphs of the same species. Midgut extract did not induce protective immunity, while immunization with cement extract resulted in partial protection of hosts as evidenced by premature tick detachment and 34-41% reduction in tick engorgement weights. Proteomic characterization of I. scapularis cement was performed, demonstrating that the cement extract was compositionally different from tick saliva, and vitellogenin-like lipoproteins were the most abundant proteins in cement extract (>40%). Cement was also heavily enriched with lysozymes and defensins, including those originating from both the mammalian host as well as ticks. These results demonstrate that I. scapularis cement contains immunogenic components capable of stimulating host resistance against tick feeding. Because the cement is present at the tick-host interface for an extended period of time during the feeding process, these antigens present auspicious candidates for further evaluation and potential inclusion in an anti-tick vaccine.

CD55 Facilitates Immune Evasion by Borrelia crocidurae, an Agent of Relapsing Fever.

Relapsing fever, caused by diverse Borrelia spirochetes, is prevalent in many parts of the world and causes significant morbidity and mortality. To investigate the pathoetiology of relapsing fever, we performed a high-throughput screen of Borrelia-binding host factors using a library of human extracellular and secretory proteins and identified CD55 as a novel host binding partner of Borrelia crocidurae and Borrelia persica, two agents of relapsing fever in Africa and Eurasia. CD55 is present on the surface of erythrocytes, carries the Cromer blood group antigens, and protects cells from complement-mediated lysis. Using flow cytometry, we confirmed that both human and murine CD55 bound to B. crocidurae and B. persica. Given the expression of CD55 on erythrocytes, we investigated the role of CD55 in pathological B. crocidurae-induced erythrocyte aggregation (rosettes), which enables spirochete immune evasion. We showed that rosette formation was partially dependent on host cell CD55 expression. Pharmacologically, soluble recombinant CD55 inhibited erythrocyte rosette formation. Finally, CD55-deficient mice infected with B. crocidurae had a lower pathogen load and elevated proinflammatory cytokine and complement factor C5a levels. In summary, our results indicate that CD55 is a host factor that is manipulated by the causative agents of relapsing fever for immune evasion. IMPORTANCE Borrelia species are causative agents of Lyme disease and relapsing fever infections in humans. B. crocidurae causes one of the most prevalent relapsing fever infections in parts of West Africa. In the endemic regions, B. crocidurae is present in ~17% of the ticks and ~11% of the rodents that serve as reservoirs. In Senegal, ~7% of patients with acute febrile illness were found to be infected with B. crocidurae. There is little information on host-pathogen interactions and how B. crocidurae manipulates host immunity. In this study, we used a high-throughput screen to identify host proteins that interact with relapsing fever-causing Borrelia species. We identified CD55 as one of the host proteins that bind to B. crocidurae and B. persica, the two causes of relapsing fever in Africa and Eurasia. We show that the interaction of B. crocidurae with CD55, present on the surface of erythrocytes, is key to immune evasion and successful infection in vivo. Our study further shows the role of CD55 in complement regulation, regulation of inflammatory cytokine levels, and innate immunity during relapsing fever infection. Overall, this study sheds light on host-pathogen interactions during relapsing fever infection in vivo.

A comparison of horizontal and transovarial transmission efficiency of Borrelia miyamotoi by Ixodes scapularis.

Borrelia miyamotoi is a relapsing fever spirochete carried by Ixodes spp. ticks throughout the northern hemisphere. The pathogen is acquired either transovarially (vertically) or horizontally through blood-feeding and passed transtadially across life stages. Despite these complementary modes of transmission, infection prevalence of ticks with B. miyamotoi is typically low (<5%) in natural settings and the relative contributions of the two transmission modes have not been studied extensively. Horizontal transmission of B. miyamotoi (strain CT13-2396 or wild type strain) was initiated using infected Ixodes scapularis larvae or nymphs to expose rodents, which included both the immunocompetent CD-1 laboratory mouse (Mus musculus) and a natural reservoir host, the white-footed mouse (Peromyscus. leucopus), to simulate natural enzootic transmission. Transovarial transmission was evaluated using I. scapularis exposed to B. miyamotoi as either larvae or nymphs feeding on immunocompromised SCID mice (M. musculus) and subsequently fed as females on New Zealand white rabbits. Larvae from infected females were qPCR-tested individually to assess transovarial transmission rates. Tissue tropism of B. miyamotoi in infected ticks was demonstrated using in situ hybridization. Between 1 and 12% of ticks were positive (post-molt) for B. miyamotoi after feeding on groups of CD-1 mice or P. leucopus with evidence of infection, indicating that horizontal transmission was inefficient, regardless of whether infected larvae or nymphs were used to challenge the mice. Transovarial transmission occurred in 7 of 10 egg clutches from infected females. Filial infection prevalence in larvae ranged from 3 to 100% (median 71%). Both larval infection prevalence and spirochete load were highly correlated with maternal spirochete load. Spirochetes were disseminated throughout the tissues of all three stages of unfed ticks, including the salivary glands and female ovarian tissue. The results indicate that while multiple transmission routes contribute to enzootic maintenance of B. miyamotoi, transovarial transmission is likely to be the primary source of infected ticks and therefore risk assessment and tick control strategies should target adult female ticks.

Tick immunity using mRNA, DNA and protein-based Salp14 delivery strategies.

Guinea pigs exposed to multiple infestations with Ixodes scapularis ticks develop acquired resistance to ticks, which is also known as tick immunity. The I. scapularis salivary components that contribute to tick immunity are likely multifactorial. An anticoagulant that inhibits factor Xa, named Salp14, is present in tick saliva and is associated with partial tick immunity. A tick bite naturally releases tick saliva proteins into the vertebrate host for several days, which suggests that the mode of antigen delivery may influence the genesis of tick immunity. We therefore utilized Salp14 as a model antigen to examine tick immunity using mRNA lipid nanoparticles (LNPs), plasmid DNA, or recombinant protein platforms. salp14 containing mRNA-LNPs vaccination elicited erythema at the tick bite site after tick challenge that occurred earlier, and that was more pronounced, compared with DNA or protein immunizations. Humoral and cellular responses associated with tick immunity were directed towards a 25 amino acid region of Salp14 at the carboxy terminus of the protein, as determined by antibody responses and skin-testing assays. This study demonstrates that the model of antigen delivery, also known as the vaccine platform, can influence the genesis of tick immunity in guinea pigs. mRNA-LNPs may be useful in helping to elicit erythema at the tick bite site, one of the most important early hallmarks of acquired tick resistance. mRNA-LNPs containing tick genes is a useful platform for the development of vaccines that can potentially prevent selected tick-borne diseases.

mRNA vaccination induces tick resistance and prevents transmission of the Lyme disease agent.

Ixodes scapularis ticks transmit many pathogens that cause human disease, including Borrelia burgdorferi. Acquired resistance to I. scapularis due to repeated tick exposure has the potential to prevent tick-borne infectious diseases, and salivary proteins have been postulated to contribute to this process. We examined the ability of lipid nanoparticle­containing nucleoside-modified mRNAs encoding 19 I. scapularis salivary proteins (19ISP) to enhance the recognition of a tick bite and diminish I. scapularis engorgement on a host and thereby prevent B. burgdorferi infection. Guinea pigs were immunized with a 19ISP mRNA vaccine and subsequently challenged with I. scapularis. Animals administered 19ISP developed erythema at the bite site shortly after ticks began to attach, and these ticks fed poorly, marked by early detachment and decreased engorgement weights. 19ISP immunization also impeded B. burgdorferi transmission in the guinea pigs. The effective induction of local redness early after I. scapularis attachment and the inability of the ticks to take a normal blood meal suggest that 19ISP may be used either alone or in conjunction with traditional pathogen-based vaccines for the prevention of Lyme disease, and potentially other tick-borne infections.

Fab-dimerized glycan-reactive antibodies are a structural category of natural antibodies.

Natural antibodies (Abs) can target host glycans on the surface of pathogens. We studied the evolution of glycan-reactive B cells of rhesus macaques and humans using glycosylated HIV-1 envelope (Env) as a model antigen. 2G12 is a broadly neutralizing Ab (bnAb) that targets a conserved glycan patch on Env of geographically diverse HIV-1 strains using a unique heavy-chain (VH) domain-swapped architecture that results in fragment antigen-binding (Fab) dimerization. Here, we describe HIV-1 Env Fab-dimerized glycan (FDG)-reactive bnAbs without VH-swapped domains from simian-human immunodeficiency virus (SHIV)-infected macaques. FDG Abs also recognized cell-surface glycans on diverse pathogens, including yeast and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike. FDG precursors were expanded by glycan-bearing immunogens in macaques and were abundant in HIV-1-naive humans. Moreover, FDG precursors were predominately mutated IgM+IgD+CD27+, thus suggesting that they originated from a pool of antigen-experienced IgM+ or marginal zone B cells.

Therapeutic cancer vaccines for pediatric malignancies: advances, challenges, and emerging technologies.

Though outcomes for pediatric cancer patients have significantly improved over the past several decades, too many children still experience poor outcomes and survivors suffer lifelong, debilitating late effects after conventional chemotherapy, radiation, and surgical treatment. Consequently, there has been a renewed focus on developing novel targeted therapies to improve survival outcomes. Cancer vaccines are a promising type of immunotherapy that leverage the immune system to mediate targeted, tumor-specific killing through recognition of tumor antigens, thereby minimizing off-target toxicity. As such, cancer vaccines are orthogonal to conventional cancer treatments and can therefore be used alone or in combination with other therapeutic modalities to maximize efficacy. To date, cancer vaccination has remained largely understudied in the pediatric population. In this review, we discuss the different types of tumor antigens and vaccine technologies (dendritic cells, peptides, nucleic acids, and viral vectors) evaluated in clinical trials, with a focus on those used in children. We conclude with perspectives on how advances in combination therapies, tumor antigen (eg, neoantigen) selection, and vaccine platform optimization can be translated into clinical practice to improve outcomes for children with cancer.

Naturally Acquired Resistance to Ixodes scapularis Elicits Partial Immunity against Other Tick Vectors in a Laboratory Host.

In many regions where ticks negatively impact public health or economic production, multiple medically important tick species may have overlapping geographic distribution, and in North America, this includes members of Ixodes, Dermacentor, and Amblyomma genera. Acquired tick resistance is the process by which some animals develop an immune response against feeding ticks after one or more exposures. This form of immunity can restrict the ability of ticks to feed and may inhibit transmission of pathogens. Likewise, many proteins present in tick saliva are conserved among tick species, and prior studies have reported cross-protective host immunity against certain combinations of ticks. In this study, we used a guinea pig model to assess whether host resistance against Ixodes scapularis could confer protection against two other medically important tick vectors, Dermacentor variabilis and Amblyomma americanum. Tick challenges using nymphs were used to induce host resistance against a primary species, followed by additional challenge using a secondary tick species. Tick attachment to hosts and engorgement weights were reduced significantly for D. variabilis and A. americanum feeding on I. scapularis-sensitized hosts. Reciprocally, I. scapularis engorgement weights were reduced to a lesser extent, and attachment was unaffected when feeding on hosts sensitized with either D. variabilis or A. americanum. These results indicate that immunity against I. scapularis could potentially be exploited for use in an anti-tick vaccine targeting multiple tick species and their associated pathogens.

Intravenous nanoparticle vaccination generates stem-like TCF1+ neoantigen-specific CD8+ T cells.

Personalized cancer vaccines are a promising approach for inducing T cell immunity to tumor neoantigens. Using a self-assembling nanoparticle vaccine that links neoantigen peptides to a Toll-like receptor 7/8 agonist (SNP-7/8a), we show how the route and dose alter the magnitude and quality of neoantigen-specific CD8+ T cells. Intravenous vaccination (SNP-IV) induced a higher proportion of TCF1+PD-1+CD8+ T cells as compared to subcutaneous immunization (SNP-SC). Single-cell RNA sequencing showed that SNP-IV induced stem-like genes (Tcf7, Slamf6, Xcl1) whereas SNP-SC enriched for effector genes (Gzmb, Klrg1, Cx3cr1). Stem-like cells generated by SNP-IV proliferated and differentiated into effector cells upon checkpoint blockade, leading to superior antitumor response as compared to SNP-SC in a therapeutic model. The duration of antigen presentation by dendritic cells controlled the magnitude and quality of CD8+ T cells. These data demonstrate how to optimize antitumor immunity by modulating vaccine parameters for specific generation of effector or stem-like CD8+ T cells.

Acquired tick resistance: The trail is hot.

Acquired tick resistance is a phenomenon wherein the host elicits an immune response against tick salivary components upon repeated tick infestations. The immune responses, potentially directed against critical salivary components, thwart tick feeding, and the animal becomes resistant to subsequent tick infestations. The development of tick resistance is frequently observed when ticks feed on non-natural hosts, but not on natural hosts. The molecular mechanisms that lead to the development of tick resistance are not fully understood, and both host and tick factors are invoked in this phenomenon. Advances in molecular tools to address the host and the tick are beginning to reveal new insights into this phenomenon and to uncover a deeper understanding of the fundamental biology of tick-host interactions. This review will focus on the expanding understanding of acquired tick resistance and highlight the impact of this understanding on anti-tick vaccine development efforts.

Fractionation of tick saliva reveals proteins associated with the development of acquired resistance to Ixodes scapularis.

Tick-borne diseases pose a global medical problem. As transmission of tick-borne pathogens to their hosts occurs during tick feeding, development of vaccines thwarting this process could potentially prevent transmission of multiple tick-borne pathogens. The idea of tick vaccines is based on the phenomenon of acquired tick immunity, rejection of ticks feeding on hosts which were repeatedly infested by ticks. Recently, we demonstrated that saliva of the blacklegged tick Ixodes scapularis, which is the main vector of tick-borne pathogens in northeast USA, is sufficient for induction of tick immunity in the guinea pig model and that immunity directed against tick glycoproteins is important in this phenomenon. Nevertheless, immunity elicited against individual tick salivary antigens, which have been identified and tested so far, provided only modest tick rejection. We therefore now tested fractions of tick saliva produced by liquid chromatography for their ability to induce tick immunity in the guinea pig model. Immunization with all individual fractions elicited antibodies that reacted with tick saliva, however only some fractions displayed the ability to induce robust protective tick immunity. Mass spectrometry analysis led to identification of 24 proteins present only in saliva fractions which were able to induce tick immunity, suggesting suitable candidates for development of a tick vaccine.

Repeat tick exposure elicits distinct immune responses in guinea pigs and mice.

Ticks deposit salivary proteins into the skin during a bite to mediate acquisition of a blood meal. Acquired resistance to tick bites has been demonstrated to prevent Borrelia burgdorferi sensu lato (s.l.) transmission. However, the mechanism of resistance, as well as the protective antigens, have remained elusive. To address these unknowns, we utilized a guinea pig model of tick resistance and a mouse model of permissiveness. Guinea pigs developed immunity after multiple Ixodes scapularis tick infestations, characterized by rapid tick detachment and impaired feeding. In comparison, mice tolerated at least 6 infestations with no significant impact on feeding. We analyzed the bite sites by RNA-sequencing and histology, identifying several inflammatory pathways in tick immune animals, such as FcεRI signaling and complement activation, and activation of coagulation pathways that could impair local blood flow. Together, these results identify important pathways altered during tick rejection and potential tick proteins that could serve as vaccine candidates.