Quantitative and Standardized Pseudovirus Neutralization Assay for COVID-19.

COVID-19 is a global pandemic caused by the highly infectious SARS-CoV-2 virus. Efforts to combat SARS-CoV-2 infection include mass vaccination and development of monoclonal and convalescent plasma therapeutics that require precise measurements of correlative, functional neutralizing antibodies that prevent virus infection. Developing rapid, safe, easy-to-use, and high-quality neutralization assays are essential for the success of the massive effort. Here, we developed a vesicular stomatitis virus-based neutralization assay that was capable of quantifying varying degrees of neutralization in patient serum samples. This assay has two detection readouts, flow cytometry and live cell imaging. The two readout methods produced consistent values of all 50% neutralization titers, further enhancing measurement confidence on the assay. Moreover, the use of available reference standards such as the World Health Organization International Standard (NIBSC code 20/136) enables quantification and standardization of the pseudovirus neutralization assay with neutralizing antibody titers measured in International Units/mL. Quantitative and standardized neutralization assays are critical for reliable efficacy evaluation and comparison of numerous vaccines and therapeutics.

Monoclonal Antibodies as SARS-CoV-2 Serology Standards: Experimental Validation and Broader Implications for Correlates of Protection.

COVID-19 has highlighted challenges in the measurement quality and comparability of serological binding and neutralization assays. Due to many different assay formats and reagents, these measurements are known to be highly variable with large uncertainties. The development of the WHO international standard (WHO IS) and other pool standards have facilitated assay comparability through normalization to a common material but does not provide assay harmonization nor uncertainty quantification. In this paper, we present the results from an interlaboratory study that led to the development of (1) a novel hierarchy of data analyses based on the thermodynamics of antibody binding and (2) a modeling framework that quantifies the probability of neutralization potential for a given binding measurement. Importantly, we introduced a precise, mathematical definition of harmonization that separates the sources of quantitative uncertainties, some of which can be corrected to enable, for the first time, assay comparability. Both the theory and experimental data confirmed that mAbs and WHO IS performed identically as a primary standard for establishing traceability and bridging across different assay platforms. The metrological anchoring of complex serological binding and neuralization assays and fast turn-around production of an mAb reference control can enable the unprecedented comparability and traceability of serological binding assay results for new variants of SARS-CoV-2 and immune responses to other viruses.

Development of a Cell-Based SARS-CoV-2 Pseudovirus Neutralization Assay Using Imaging and Flow Cytometry Analysis.

COVID-19 is an ongoing, global pandemic caused by the novel, highly infectious SARS-CoV-2 virus. Efforts to mitigate the effects of SARS-CoV-2, such as mass vaccination and development of monoclonal therapeutics, require precise measurements of correlative, functional neutralizing antibodies that block virus infection. The development of rapid, safe, and easy-to-use neutralization assays is essential for faster diagnosis and treatment. Here, we developed a vesicular stomatitis virus (VSV)-based neutralization assay with two readout methods, imaging and flow cytometry, that were capable of quantifying varying degrees of neutralization in patient serum samples. We tested two different spike-pseudoviruses and conducted a time-course assay at multiple multiplicities of infection (MOIs) to optimize the assay workflow. The results of this assay correlate with the results of previously developed serology and surrogate neutralization assays. The two pseudovirus readout methods produced similar values of 50% neutralization titer values. Harvest-free in situ readouts for live-cell imaging and high-throughput analysis results for flow cytometry can provide unique capabilities for fast evaluation of neutralization, which is critical for the mitigation of future pandemics.

The Obligate Intracellular Bacterial Pathogen Anaplasma phagocytophilum Exploits Host Cell Multivesicular Body Biogenesis for Proliferation and Dissemination.

Anaplasma phagocytophilum is the etiologic agent of the emerging infection, granulocytic anaplasmosis. This obligate intracellular bacterium lives in a host cell-derived vacuole that receives membrane traffic from multiple organelles to fuel its proliferation and from which it must ultimately exit to disseminate infection. Understanding of these essential pathogenic mechanisms has remained poor. Multivesicular bodies (MVBs) are late endosomal compartments that receive biomolecules from other organelles and encapsulate them into intralumenal vesicles (ILVs) using endosomal sorting complexes required for transport (ESCRT) machinery and ESCRT-independent machinery. Association of the ESCRT-independent protein, ALIX, directs MVBs to the plasma membrane where they release ILVs as exosomes. We report that the A. phagocytophilum vacuole (ApV) is acidified and enriched in lysobisphosphatidic acid, a lipid that is abundant in MVBs. ESCRT-0 and ESCRT-III components along with ALIX localize to the ApV membrane. siRNA-mediated inactivation of ESCRT-0 and ALIX together impairs A. phagocytophilum proliferation and infectious progeny production. RNA silencing of ESCRT-III, which regulates ILV scission, pronouncedly reduces ILV formation in ApVs and halts infection by arresting bacterial growth. Rab27a and its effector Munc13-4, which drive MVB trafficking to the plasma membrane and subsequent exosome release, localize to the ApV. Treatment with Nexinhib20, a small molecule inhibitor that specifically targets Rab27a to block MVB exocytosis, abrogates A. phagocytophilum infectious progeny release. Thus, A. phagocytophilum exploits MVB biogenesis and exosome release to benefit each major stage of its intracellular infection cycle: intravacuolar growth, conversion to the infectious form, and exit from the host cell. IMPORTANCE Anaplasma phagocytophilum causes granulocytic anaplasmosis, a globally emerging zoonosis that can be severe, even fatal, and for which antibiotic treatment options are limited. A. phagocytophilum lives in an endosomal-like compartment that interfaces with multiple organelles and from which it must ultimately exit to spread within the host. How the bacterium accomplishes these tasks is poorly understood. Multivesicular bodies (MVBs) are intermediates in the endolysosomal pathway that package biomolecular cargo from other organelles as intralumenal vesicles for release at the plasma membrane as exosomes. We discovered that A. phagocytophilum exploits MVB biogenesis and trafficking to benefit all aspects of its intracellular infection cycle: proliferation, conversion to its infectious form, and release of infectious progeny. The ability of a small molecule inhibitor of MVB exocytosis to impede A. phagocytophilum dissemination indicates the potential of this pathway as a novel host-directed therapeutic target for granulocytic anaplasmosis.

An In Vitro Model of the Efficacy of Breast Implant Irrigant Solutions Against Gram-Negative Infections.

BACKGROUND: In implant-based breast surgery, infections remain a clinically challenging complication. Surgeons often prophylactically address this risk by irrigating the implant at the time of placement. However, there remain few data on the ideal irrigant for gram-negative species. METHODS: The authors assessed the relative efficacy of 10% povidone-iodine, triple-antibiotic solution, Prontosan, Clorpactin, and normal saline (negative control) against 3 gram-negative bacterial backgrounds: Escherichia coli , Pseudomonas aeruginosa , and Proteus species. A laboratory-adapted strain and a clinical isolate were selected for each group of bacteria. Sterile, smooth implant discs were immersed in each irrigant solution and then incubated in suspensions of each bacterial strain overnight at 37°C. Each disc was then rinsed and sonicated to displace biofilm-forming bacteria from the implant surface. The displaced bacteria were enumerated by plating, and normalized values were calculated for the bacterial counts of each irrigant. RESULTS: Povidone-iodine resulted in the greatest reduction of bacterial load for all 6 strains by a factor of 10 1 to 10 6 . Prontosan had a lesser, yet significant reduction in all bacterial strains. Triple-antibiotic solution demonstrated the greatest reduction in one Proteus species strain, and Clorpactin reduced bacterial counts in only half of the bacterial strains. When comparing laboratory strains to clinical isolates, significant differences were seen in each bacterial species in at least 2 irrigant solutions. CONCLUSIONS: Povidone-iodine has been proven the most effective at reducing bacterial contamination of E. coli, P. aeruginosa , and Proteus species in both laboratory-adapted strains and clinical isolates. CLINICAL RELEVANCE: This study proves that povidone-iodine is the most effective at preventing gram-negative infections in breast implant surgery.

A Novel Lipid-Based MALDI-TOF Assay for the Rapid Detection of Colistin-Resistant Enterobacter Species.

Enterobacter species are classified as high-priority pathogens due to high prevalence of multidrug resistance from persistent antibiotic use. For Enterobacter infections caused by multidrug-resistant isolates, colistin (polymyxin E), a last-resort antibiotic, is a potential treatment option. Treatment with colistin has been shown to lead to emergence of polymyxin resistance. The primary mechanism for colistin resistance is modification of terminal phosphate moieties of lipid A, leading to decreased membrane electronegativity and reducing colistin binding affinity. Detection of these modifications, including the addition of phosphoethanolamine and 4-amino-4-deoxy-l-arabinose (Ara4N), can be used for prediction of colistin resistance using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The objective of this study was to identify lipid A markers for colistin resistance in Enterobacter species and Klebsiella aerogenes (formerly Enterobacter aerogenes). Using a collection of Enterobacter and Klebsiella aerogenes clinical isolates, broth MICs for colistin were determined initially. Subsequently, killing assays were carried out to determine how the concentration of colistin at which there is approximately 50% survival (kill50) equates to their MICs. Finally, lipid A analysis was conducted via MALDI-TOF MS using the novel rapid extraction method, termed fast lipid analysis technique (FLAT), to correlate MIC and killing efficacy with predictive lipid A modifications. Sensitivity and specificity of the MS assay compared to MIC interpretation were 100% and 53.4%, respectively. A receiver operator characteristic (ROC) demonstrated that MS was highly correlated with killing, with area under the curve of 0.97. This analysis demonstrated the potential utility of MALDI-TOF MS as a rapid diagnostic platform of colistin resistance in Enterobacter species. IMPORTANCE In this study, we develop a novel method for identifying colistin resistance in Enterobacter species and Klebsiella aerogenes without performing antimicrobial susceptibility testing. Typically, susceptibility testing requires an additional 24 to 48 h, while the MS assay described in this study allows for resistant identifications in under 1 h after initial culture. Identification using MALDI-TOF MS would save time and prevent inappropriate use of colistin. MALDI-TOF MS is an easy-to-use, readily available, robust diagnostic tool in clinical laboratories. Furthermore, this study highlights limitations of polymyxin susceptibility testing. Use of a killing assay best captures how colistin treats infection and is shown to be highly correlated with our MS assay; thus, the MS assay in this study effectively predicts how colistin would treat a patient's infection. Use of MALDI-TOF MS for accurate and early identification of antimicrobial resistance can improve antimicrobial stewardship and patient outcomes.

High-resolution crystal structure of the Borreliella burgdorferi PlzA protein in complex with c-di-GMP: new insights into the interaction of c-di-GMP with the novel xPilZ domain.

In the tick-borne pathogens, Borreliella burgdorferi and Borrelia hermsii, c-di-GMP is produced by a single diguanylate cyclase (Rrp1). In these pathogens, the Plz proteins (PlzA, B and C) are the only c-di-GMP receptors identified to date and PlzA is the sole c-di-GMP receptor found in all Borreliella isolates. Bioinformatic analyses suggest that PlzA has a unique PilZN3-PilZ architecture with the relatively uncommon xPilZ domain. Here, we present the crystal structure of PlzA in complex with c-di-GMP (1.6 Å resolution). This is the first structure of a xPilz domain in complex with c-di-GMP to be determined. PlzA has a two-domain structure, where each domain comprises topologically equivalent PilZ domains with minimal sequence identity but remarkable structural similarity. The c-di-GMP binding site is formed by the linker connecting the two domains. While the structure of apo PlzA could not be determined, previous fluorescence resonance energy transfer data suggest that apo and holo forms of the protein are structurally distinct. The information obtained from this study will facilitate ongoing efforts to identify the molecular mechanisms of PlzA-mediated regulation in ticks and mammals.

Rapid identification of mcr-1-positive Escherichia coli from patient urine using a novel lipid-based MALDI-TOF-MS assay.

Mobilized colistin resistance (mcr) genes confer resistance to colistin, a last-resort antibiotic for multidrug-resistant Gram-negative infections. In this case report, we describe a novel lipid-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) diagnostic used to rapidly identify an mcr-1-positive Escherichia coli directly from a patient with a urinary tract infection without the need for ex vivo growth.

Ehrlichia chaffeensis EplA Interaction With Host Cell Protein Disulfide Isomerase Promotes Infection.

Ehrlichia chaffeensis is an obligate intracellular bacterium that invades monocytes to cause the emerging and potentially severe disease, monocytic ehrlichiosis. Ehrlichial invasion of host cells, a process that is essential for the bacterium's survival and pathogenesis, is incompletely understood. In this study, we identified ECH_0377, henceforth designated as EplA (E. chaffeensis PDI ligand A) as an E. chaffeensis adhesin that interacts with host cell protein disulfide isomerase (PDI) to mediate bacterial entry into host cells. EplA is an outer membrane protein that E. chaffeensis expresses during growth in THP-1 monocytic cells. Canine sera confirmed to be positive for exposure to Ehrlichia spp. recognized recombinant EplA, indicating that it is expressed during infection in vivo. EplA antiserum inhibited the bacterium's ability to infect monocytic cells. The EplA-PDI interaction was confirmed via co-immunoprecipitation. Treating host cell surfaces with antibodies that inhibit PDI and/or thioredoxin-1 thiol reductase activity impaired E. chaffeensis infection. Chemical reduction of host cell surfaces, but not bacterial surfaces with tris(2-carboxyethyl)phosphine (TCEP) restored ehrlichial infectivity in the presence of the PDI-neutralizing antibody. Antisera specific for EplA C-terminal residues 95-104 (EplA95-104) or outer membrane protein A amino acids 53-68 (OmpA53-68) reduced E. chaffeensis infection of THP-1 cells. Notably, TCEP rescued ehrlichial infectivity of bacteria that had been treated with anti-EplA95-104, but not anti-EcOmpA53-68. These results demonstrate that EplA contributes to E. chaffeensis infection of monocytic cells by engaging PDI and exploiting the enzyme's reduction of host cell surface disulfide bonds in an EplA C-terminus-dependent manner and identify EplA95-104 and EcOmpA53-68 as novel ehrlichial receptor binding domains.

Serologic Evidence for the Exposure of Eastern Coyotes (Canis latrans) in Pennsylvania to the Tick-Borne Pathogens Borreliella burgdorferi and Anaplasma phagocytophilum.

Lyme disease and anaplasmosis are tick-borne bacterial diseases caused by Borreliella and Anaplasma species, respectively. A comprehensive analysis of the exposure of eastern coyotes (Canis latrans) in the northeastern United States to tick-borne pathogens has not been conducted. In this report, we assess the serological status of 128 eastern coyotes harvested in Pennsylvania in 2015 and 2017 for antibodies to Borreliella burgdorferi and Anaplasma phagocytophilum Immunoblot and dot blot approaches were employed to test each plasma sample by using cell lysates and recombinant proteins as detection antigens. The results demonstrate high seropositivity incidences of 64.8% and 72.7% for B. burgdorferi and A. phagocytophilum, respectively. Antibodies to both pathogens were detected in 51.5% of the plasma samples, indicating high potential for coinfection. Antibodies to the B. burgdorferi proteins DbpB, VlsE, DbpA, BBA36, and OspF (BBO39) were detected in 67.2, 63.3, 56.2, 51.6, and 48.4% of the plasma samples, respectively. Antibodies to the A. phagocytophilum P44 and P130 proteins were detected in 72.7 and 60.9% of the plasma samples, respectively.IMPORTANCE The incidence of Lyme disease (Borreliella burgdorferi) and anaplasmosis (Anaplasma phagocytophilum) are increasing in North America and Europe. The causative agents of these debilitating tick-transmitted infections are maintained in nature in an enzootic cycle involving Ixodes ticks and diverse mammals and birds. It has been postulated that predators directly or indirectly influence the dynamics of the enzootic cycle and disease incidence. Here, we demonstrate high seropositivity of eastern coyotes for B. burgdorferi and A. phagocytophilum As coyotes become established in urban and suburban environments, interactions with humans, companion animals, and urban/suburban wildlife will increase. Knowledge of the pathogens that these highly adaptable predators are exposed to or carry, and their potential to influence or participate in enzootic cycles, is central to efforts to reduce the risk of tick-borne diseases in humans and companion animals.

Immunization against Anaplasma phagocytophilum Adhesin Binding Domains Confers Protection against Infection in the Mouse Model.

Anaplasma phagocytophilum causes granulocytic anaplasmosis, a debilitating infection that can be fatal in the immunocompromised. It also afflicts animals, including dogs, horses, and sheep. No granulocytic anaplasmosis vaccine exists. Because A. phagocytophilum is an obligate intracellular bacterium, inhibiting microbe-host cell interactions that facilitate invasion can disrupt infection. The binding domains of A. phagocytophilum adhesins A. phagocytophilum invasion protein A (AipA), A. phagocytophilum surface protein (Asp14), and outer membrane protein A (OmpA) are essential for optimal bacterial entry into host cells, but their relevance to infection in vivo is undefined. In this study, C57BL/6 mice were immunized with a cocktail of keyhole limpet hemocyanin-conjugated peptides corresponding to the AipA, Asp14, and OmpA binding domains in alum followed by challenge with A. phagocytophilum The bacterial peripheral blood burden was pronouncedly reduced in immunized mice compared to controls. Examination of pre- and postchallenge sera from these mice revealed that immunization elicited antibodies against AipA and Asp14 peptides but not OmpA peptide. Nonetheless, pooled sera from pre- and postchallenge groups, but not from control groups, inhibited A. phagocytophilum infection of HL-60 cells. Adhesin domain immunization also elicited interferon gamma (IFN-γ)-producing CD8-positive (CD8+) T cells. A follow-up study confirmed that immunization against only the AipA or Asp14 binding domain was sufficient to reduce the bacterial peripheral blood load in mice following challenge and elicit antibodies that inhibit A. phagocytophilum cellular infection in vitro These data demonstrate that AipA and Asp14 are critical for A. phagocytophilum to productively infect mice, and immunization against their binding domains elicits a protective immune response.

Development and optimization of OspC chimeritope vaccinogens for Lyme disease.

Experimental Outer surface protein (Osp) C based subunit chimeritope vaccinogens for Lyme disease (LD) were assessed for immunogenicity, structure, ability to elicit antibody (Ab) responses to divergent OspC proteins, and bactericidal activity. Chimeritopes are chimeric epitope based proteins that consist of linear epitopes derived from multiple proteins or multiple variants of a protein. An inherent advantage to chimeritope vaccinogens is that they can be constructed to trigger broadly protective Ab responses. Three OspC chimeritope proteins were comparatively assessed: Chv1, Chv2 and Chv3. The Chv proteins possess the same set of 18 linear epitopes derived from 9 OspC type proteins but differ in the physical ordering of epitopes or by the presence or absence of linkers. All Chv proteins were immunogenic in mice and rats eliciting high titer Ab. Immunoblot and enzyme linked immunosorbent assays demonstrated that the Chv proteins elicit IgG that recognizes a diverse array of OspC type proteins. The panel included OspC proteins produced by N. American and European strains of the LD spirochetes. Rat anti-Chv antisera uniformly labeled intact, non-permeabilized Borreliella burgdorferi demonstrating that vaccinal Ab can bind to targets that are naturally presented on the spirochete cell surface. Vaccinal Ab also displayed potent complement dependent-Ab mediated killing activity. This study highlights the ability of OspC chimeritopes to serve as vaccinogens that trigger potentially broadly protective Ab responses. In addition to the current use of an OspC chimeritope in a canine LD vaccine, chimeritopes can serve as key components of human LD subunit vaccines.

Diversity of the Lyme Disease Spirochetes and its Influence on Immune Responses to Infection and Vaccination.

The Lyme disease spirochetes are a highly diverse group of bacteria with unique biological properties. Their ability to cycle between ticks and mammals requires that they adapt to variable and constantly changing environmental conditions. Outer surface protein C is an essential virulence determinant that has received considerable attention in vaccine and diagnostic assay development. Knowledge of OspC diversity, its antigenic determinants, and its production patterns throughout the enzootic cycle, as well as in the laboratory setting, is essential for understanding immune responses induced by infection or vaccination.

Analysis of the antigenic determinants of the OspC protein of the Lyme disease spirochetes: Evidence that the C10 motif is not immunodominant or required to elicit bactericidal antibody responses.

As Ixodes ticks spread to new regions, the incidence of Lyme disease (LD) in companion animals and humans will increase. Preventive strategies for LD in canines center on vaccination and tick control (acaricides). Both subunit and bacterin based LD veterinary vaccines are available. Outer surface protein C (OspC), a potent immunogen and dominant early antigen, has been demonstrated to elicit protective antibody (Ab) responses. However, a single OspC protein elicits a relatively narrow range of protection. There are conflicting reports as to whether the immunodominant epitopes of OspC reside within variable or conserved domains. A detailed understanding of the antigenic determinants of OspC is essential for understanding immune responses to this essential virulence factor and vaccinogen. Here, we investigate the contribution of the conserved C-terminal C10 motif in OspC triggered Ab responses. Using a panel of diverse recombinant full length OspC proteins and their corresponding C10 deletion variants (OspCΔC10), we demonstrate that the C10 motif does not significantly contribute to immunization or infection induced Ab responses in rabbits, rats, canines, horses and non-human primates. Furthermore, the C10 motif is not required to trigger potent bactericidal Ab responses. This study provides insight into the antigenic structure of OspC. The results enhance our understanding of immune responses that develop during infection or upon vaccination and have implications for interpretation of LD diagnostic assays that employ OspC.

The Borrelia burgdorferi c-di-GMP Binding Receptors, PlzA and PlzB, Are Functionally Distinct.

Cyclic-di-GMP (c-di-GMP) contributes to the regulation of processes required by the Lyme disease (LD) spirochetes to complete the tick-mammal enzootic cycle. Our understanding of the effector mechanisms of c-di-GMP in the Borrelia is evolving. While most LD spirochete isolates encode a single PilZ domain containing c-di-GMP receptor designated as PlzA, genome analyses have revealed that a subset encode a second PilZ domain protein (PlzB). The c-di-GMP binding potential of PlzB, and its role in LD spirochete biology, have not been investigated. To determine if PlzB binds c-di-GMP, plzB from B. burgdorferi isolate ZS7 was PCR amplified, cloned, and recombinant protein generated. PlzB bound c-di-GMP but not other nucleotides, indicating a specific binding interaction. To determine if PlzA and PlzB are functionally synonymous, a series of allelic-exchange gene deletion and cis-complemented strains were generated in the B. burgdorferi B31 background. B. burgdorferi B31-ΔplzA was competent to infect Ixodes scapularis larvae but not mice when delivered by either needle or tick feeding. B. burgdorferi B31-ΔplzA also displayed an atypical motility phenotype. Complementation in cis of B. burgdorferi B31-ΔplzA with plzA (B31-plzA KI) restored wild-type (wt) phenotype. However, a strain complemented in cis with plzB (B31-plzB KI) did not. The data presented here are consistent with an earlier study that demonstrated that PlzA plays an essential role in spirochete survival in the mammalian environment. We add to our understanding of the c-di-GMP regulatory network by demonstrating that while PlzB binds c-di-GMP, it is not functionally synonymous with PlzA. The absence of plzB from most strains suggests that it is not required for survival. One possibility is that cells that harbor both PlzA and PlzB might have enhanced biological fitness or increased virulence.

Identification of a defined linear epitope in the OspA protein of the Lyme disease spirochetes that elicits bactericidal antibody responses: Implications for vaccine development.

The lipoprotein OspA is produced by the Lyme disease spirochetes primarily in unfed ticks. OspA production is down-regulated by the blood meal and it is not produced in mammals except for possible transient production during late stage infection in patients with Lyme arthritis. Vaccination with OspA elicits antibody (Ab) that can target spirochetes in the tick midgut during feeding and inhibit transmission to mammals. OspA was the primary component of the human LYMErix™ vaccine. LYMErix™ was available from 1998 to 2002 but then pulled from the market due to declining sales as a result of unsubstantiated concerns about vaccination induced adverse events and poor efficacy. It was postulated that a segment of OspA that shares sequence similarity with a region in human LFA-1 and may trigger putative autoimmune events. While evidence supporting such a link has not been demonstrated, most efforts to move forward with OspA as a vaccine component have sought to eliminate this region of concern. Here we identify an OspA linear epitope localized within OspA amino acid residues 221-240 (OspA221-240) that lacks the OspA region suggested to elicit autoimmunity. A peptide consisting of residues 221-240 was immunogenic in mice. Ab raised against OspA221-240 peptide surface labeled B. burgdorferi in IFAs and displayed potent Ab mediated-complement dependent bactericidal activity. BLAST analyses identified several variants of OspA221-240 and a closely related sequence in OspB. It is our hypothesis that integration of the OspA221-240 epitope into a multivalent-OspC based chimeric epitope based vaccine antigen (chimeritope) could result in a subunit vaccine that protects against Lyme disease through synergistic mechanisms.