Abstract: NS4B is one of the nonstructural proteins of classical swine fever virus (CSFV), the etiological agent of a severe, highly lethal disease of swine. Protein domain analysis of the predicted amino acid sequence of the NS4B protein of highly pathogenic CSFV strain Brescia (BICv) identified a putative Toll/interleukin-1 receptor (TIR)-like domain. This TIR-like motif harbors two conserved domains, box 1 and box 2, also observed in other members of the TIR superfamily, including Toll-like receptors (TLRs). Mutations within the BICv NS4B box 2 domain (V2566A, G2567A, I2568A) produced recombinant virus NS4B.VGIv, with an altered phenotype displaying enhanced transcriptional activation of TLR-7-induced genes in swine macrophages, including a significant sustained accumulation of interleukin-6 (IL-6) mRNA. Transfection of swine macrophages with the wild-type NS4B gene partially blocked the TLR-7-activating effect of imiquimod (R837), while transfection with the NS4B gene harboring mutations in either of the putative boxes displayed decreased blocking activity. NS4B.VGIv showed an attenuated phenotype in swine, displaying reduced replication in the oronasal cavity and limited spread from the inoculation site to secondary target organs. Furthermore, the level and duration of IL-6 production in the tonsils of pigs intranasally inoculated with NS4B.VGIv were significantly higher than those for animals infected with BICv. The peak of IL-6 production in infected animals paralleled the ability of animals infected with NS4B.VGIv to resist challenge with virulent BICv. Interestingly, treatment of peripheral blood mononuclear cell cultures with recombinant porcine IL-6 results in a significant decrease in BICv replication.
Abstract: The induction of innate immune responses by toll-like receptor (TLR) agonists is the subject of intense investigation. In large part, this reflects the potential of such compounds to be effective vaccine adjuvants. For that reason, we analyzed the activation of innate cells in swine by TLR7 and TLR8 agonists. These agonists activated porcine NK cells by increasing gamma interferon (IFN-gamma) expression and perforin storage. The activation of porcine NK cells was mediated by accessory cells, since their depletion resulted in reduced cytotoxicity toward target cells. Accessory cells were stimulated to produce interleukin 12 (IL-12), IL-15, IL-18, and IFN-alpha after treatment with TLR7 or TLR8 agonists. Neutralization of these cytokines reduced but did not completely inhibit the induction of NK cell cytotoxicity. Direct stimulation of NK cells with TLR7 or TLR8 agonists resulted in minimal cytotoxicity but levels of IFN-gamma equivalent to those detected in the presence of accessory cells. Porcine NK cells express both TLR7 and TLR8 mRNAs, and treatment with these TLR agonists induced higher mRNA expression levels of TRAIL and IL-15Ralpha, which may contribute to the activity of NK cells. These data indicate that TLR7 and TLR8 agonists indirectly or directly activate porcine NK cells but that optimum levels of activation require cytokine secretion by accessory cells activated by these compounds. Interestingly, NK cells activated by TLR7 or TLR8 agonists were cytotoxic against foot-and-mouth disease virus (FMDV)-infected cells in vitro, indicating that these TLR agonists may be beneficial as adjuvants to stimulate the innate immunity against FMDV.
Abstract: Natural killer (NK) cells play a vital role in innate response against viral infections and cellular transformation. In vivo modulation of their response may enhance their antiviral function. Here we describe the phenotype of porcine NK cells, test potential proinflammatory cytokines for activation of these cells and assess the capability of porcine NK cells to kill virus-infected or tumor cells in vitro. The CD2(+)/CD8(+)/CD3(-) cell compartment contained porcine NK cells, which at the resting stage were minimally cytotoxic toward foot-and-mouth disease virus (FMDV)-infected porcine cells or tumor cell lines. Direct stimulation of NK cells with proinflammatory cytokines induced efficient lysis of FMDV-infected cells with interleukin (IL)-2 or IL-15 showing the highest stimulatory capacity. Lower levels of NK cell activation were induced by IL-12, IL-18, or interferon (IFN)-alpha, however, IL-12 and IL-18 synergistically activated NK cells. Combinations of IL-15 and IL-12 or IL-15 and IL-18 did not further increase the porcine NK cell lytic capability over IL-15 alone. Natural killer cells expressed IFN-gamma regardless of the cytokine used for stimulation while expression of perforin increased modestly. The enhancement of porcine NK cell activity by proinflammatory cytokines offers a promising tool for development of antiviral approaches against virus infection.
Abstract: Emergency vaccination as part of the control strategies against foot-and-mouth disease virus (FMDV) has the potential to limit virus spread and reduce large-scale culling. To reduce the time between vaccination and the onset of immunity, immunostimulatory CpG was tested for its capacity to promote early protection against FMDV challenge in pigs. To this end, CpG 2142, an efficient inducer of alpha interferon, was injected intramuscularly. Increased transcription of Mx1, OAS, and IRF-7 was identified as a sensitive measurement of CpG-induced innate immunity, with increased levels detectable to at least 4 days after injection of CpG formulated with Emulsigen. Despite this, CpG combined with an FMD vaccine did not promote protection. Pigs vaccinated 2 days before challenge had disease development, which was at least as acute as that of unvaccinated controls. All pigs vaccinated 7 days before challenge were protected without a noticeable effect of CpG. In summary, our results demonstrate the caution required when translating findings from mouse models to natural hosts of FMDV.
Abstract: Natural killer (NK) cells provide one of the initial barriers of cellular host defense against pathogens, in particular intracellular pathogens. The role of these cells in foot-and-mouth disease virus (FMDV) infection is unknown. Previously, we characterized the phenotype and function of NK cells from swine (F. N. Toka et al., J. Interferon Cytokine Res. 29:179-192, 2009). In the present study, we report the analysis of NK cells isolated from animals infected with FMDV and tested ex vivo and show that NK-dependent cytotoxic activity against tumor cells as targets was impaired. More relevantly to this infection, the killing of target cells infected with FMDV also was inhibited. Further, the proportion of NK cells capable of producing gamma interferon and storing perforin was reduced. Peripheral blood mononuclear cells isolated from infected animals are not productively infected, but virus exposure in vivo resulted in the significant induction of NKp30 and Toll-like receptor 3 expression and the moderate activation of SOCS3 and interleukin-15 receptor mRNA. However, there was little alteration of mRNA expression from a number of other receptor genes in these cells, including SH2D1B and NKG2A (inhibitory) as well as NKp80, NKp46, and NKG2D (activating). These data indicate that this virus infection influences the ability of NK cells to recognize and eliminate FMDV-infected cells. In addition, a reduction in NK cell cytotoxicity coincided with the increase in virus titers, indicating the virus blocking of NK cell-associated innate responses, albeit temporarily. These effects likely culminate in brief but effective viral immune evasion, allowing the virus to replicate and disseminate within the host.
Abstract: Viruses have evolved multiple mechanisms to evade the innate immune response, particularly the actions of interferons (IFNs). We have previously reported that exposure of dendritic cells (DCs) to foot-and-mouth disease virus (FMDV) in vitro yields no infection and induces a strong type I IFN (IFN-alpha and IFN-beta) response, indicating that DCs may play a critical role in the innate response to the virus. In vivo, FMDV induces lymphopenia and reduced T-cell proliferative responses to mitogen, viral effects that may contribute to evasion of early immune responses. In this study we analyzed the in vivo effects of FMDV infection on the IFN-alpha response of two populations of dendritic cells. During the acute phase of infection of swine, production of IFN-alpha from monocyte-derived DCs (MoDCs) and skin-derived DCs (skin DCs) is inhibited. This effect occurs concurrently with rising viral titers in the blood; however, these cells are not productively infected. Interestingly, there are no changes in the capability of these DCs to take up particles and process antigens, indicating that antigen-presenting cell function is normal. These data indicate that inhibition of the IFN-alpha response of dendritic cell populations from blood and skin by FMDV enhances viral pathogenesis in infected animals.
Abstract: SUMMARY: The interface between successful pathogens and their hosts is often a tenuous balance. In acute viral infections, this balance involves induction and inhibition of innate responses. Foot-and-mouth disease virus (FMDV) is considered one of the most contagious viruses known and is characterized by rapid induction of clinical disease in cloven hoofed animals exposed to infection. Viral shedding is extensive before the equally rapid resolution of acute disease. This positive strand RNA virus is an extremely successful pathogen, due in part to the ability to interrupt the innate immune response. Previous reviews have described the inhibition of cellular innate responses in the infected cell both in vitro and in vivo. Here, we present a review of virus inhibition of cells that are a source of antiviral function in swine. Particularly in the case of dendritic cells and natural killer cells, the virus has evolved mechanisms to interrupt the normal function of these important mediators of innate function, even though these cells are not infected by the virus. Understanding how this virus subverts the innate response will provide valuable information for the development of rapidly acting biotherapeutics to use in response to an outbreak of FMDV.
Abstract: Langerhans cells (LCs) are resident dendritic cells (DCs) of skin and mucosal epithelium. The standard for identifying skin DCs as LCs is expression of langerin (CD207), a surface protein that mediates Birbeck granule (BG) formation upon internalization. Reports of BGs in porcine skin DC are contradictory, due to lack of langerin detection. Here, we present the sequence of porcine langerin/CD207, showing that the predicted porcine protein shares 75%/86% amino acid identity/similarity with human. Langerin mRNA was detected in porcine skin DCs by PCR and langerin protein was detected in both isolated skin DCs and skin sections by immunostaining. Approximately, 50-70% of skin DCs expressed langerin, demonstrating that the majority of porcine skin DCs are LCs. The full length sequence combined with the identification of antibodies reactive with porcine langerin, facilitates the study of LCs in swine, and advances the use of swine for studying skin diseases and infectious disease processes involving skin.
Abstract: Dendritic cells (DCs) are a critical aspect of innate immune responses in addition to initiating adaptive immunity. In vitro generation of monocyte derived dendritic cells (MoDC) by culturing cells in IL-4 and granulocyte/macrophage colony stimulating factor (GM-CSF) has been reported for multiple species including swine. However, IL-4 is not a prominent cytokine detected in the periphery of common breeds of swine such as Yorkshire pigs. In this study, we report the generation and characterization of porcine MoDC in vitro using porcine IL-13 and porcine GM-CSF. These cells have the predicted expression of Class II MHC and T cell costimulatory molecules, phagocytic capacity and the ability to process and present antigen. Critically, porcine IL-13/GM-CSF MoDC have the unique ability to stimulate a primary mixed lymphocyte response in vitro. The type I interferon response of these MoDC to poly I:C (TLR3 ligand), LPS (TLR4 ligand) and CpG (TLR9 ligand) was tested. Of these TLR agonists, LPS or CpG did not stimulate induction of type I interferons, but a strong response was observed to poly I:C. This analysis shows that the generation of MoDCs in IL-13 yields cells of equivalent phenotype and function as IL-4 generated DC. However, for swine, in vitro generation of MoDC in IL-13 is likely to induce a more physiological cell population to study given expression of IL-4 is lacking in the periphery of these animals.
Abstract: Inoculation of vesicular stomatitis New Jersey virus (VSNJV) by skin scarification of the coronary-band in cattle, a natural host of VSNJV, resulted in vesicular lesions and 6-8 log(10) TCID(50) increase in skin virus titers over a 72 h period. Virus infection was restricted to the lesion sites and lymph nodes draining those areas but no virus or viral RNA was found in the blood or in 20 other organs and tissues sampled at necropsy. Scarification of flank skin did not result in lesions or a significant increase in viral titer indicating that viral clinical infection is restricted to skin inoculation at sites where lesions naturally occur. Viral antigens co-localized primarily with keratinocytes in the coronary band, suggesting these cells are the primary site of viral replication. Viral antigen also co-localized with few MHC-II positive cells, but no co-localization was observed in cells positive for macrophage markers. Although granulocyte infiltration was observed in lesions, little viral antigen co-localized with these cells. This is the first detailed description of VSNJV tissue distribution and infected cell characterization in a natural host. The pathogenesis model shown herein could be useful for in-vivo tracking of virus infection and local immune responses.
Abstract: The adjuvant effect of porcine interferon alpha (pIFN-alpha) was examined in swine vaccinated with a replication-defective adenovirus containing foot-and-mouth disease virus (FMDV) A24 capsid and 3C proteinase coding regions (Ad5-A24). Groups of swine were inoculated with either high or low doses of Ad5-A24 in the presence or absence of Ad5-pIFNalpha or with a control Ad5 and challenged by intradermal inoculation in the heel bulb with FMDV at 42 days post-vaccination. After challenge all control animals developed viremia and lesions. Animals receiving low-dose Ad5-A24 had similar clinical disease, but only three of five animals developed viremia, while addition of IFN resulted in a delayed onset of lesions in three animals and only one animal had detectable viremia. Animals vaccinated with high-dose Ad5-A24 had no viremia, significantly fewer lesions and delayed onset of disease compared to the control and low-dose vaccine groups. Four of five pigs vaccinated with high-dose Ad5-A24 plus IFN were completely protected from disease and only one animal had a lesion which was restricted to the site of challenge. Thus, pIFN-alpha enhances the long-term level of protection induced by the Ad5-FMD vaccine, supporting its use as a potential adjuvant in FMD vaccination strategies.
Abstract: Recent outbreaks of foot-and-mouth disease virus (FMDV) demonstrate that this highly contagious viral infection of cloven hoofed animals continues to be a significant economic problem worldwide. Debate about the most effective way to respond to outbreaks of FMDV in disease free countries continues to center on the use of vaccines. In this report, we present data showing that a commercially available, standard dose vaccine formulation can fully protect cattle against direct challenge with the virus in as little as 7 days with no carrier transmission to naïve animals. Cattle challenged 4 days after vaccination have reduced disease severity, no detectable virus in blood and little virus shedding from nasal secretions. These significant effects at 4 days post vaccination, confirmed in two separate trials, support the value of using currently available vaccines as a first line of defense against foot-and-mouth disease (FMD) outbreaks.
Abstract: The role of dendritic cells (DC) in the initiation of immune responses against foot-and-mouth disease virus (FMDV) is poorly understood. We analyzed the innate response of freshly isolated swine skin DC to the virus and show a rapid induction of beta interferon (IFN-beta) mRNA but not IFN-alpha mRNA. However, these DC secreted both IFN-alpha and IFN-beta proteins in response to live virus but not killed virus. Furthermore, the surface expression of swine major histocompatibility complex class II (SLA II) or CD80/CD86 molecules and antigen processing functions were not affected by FMDV exposure. Given the demonstrated sensitivity of FMDV to IFN-alpha/beta, there was no productive or nonproductive infection of these cells. Finally, freshly isolated skin DC constitutively expressed intracellular IFN-alpha protein in the absence of stimulation, with no detectable secretion of the cytokine until virus exposure. In situ analysis of these DC showed that these cells express and store IFN-alpha in uninfected animals. This is the first demonstration of the constitutive expression of IFN-alpha in resident, tissue-derived DC and indicates that skin DC can play an important role in the innate immune response of swine to viral infections.
Abstract: We have previously demonstrated that swine vaccinated with one dose of a replication-defective human adenovirus type 5 (Ad5) vector containing the capsid and 3C proteinase coding regions of foot-and-mouth disease virus (FMDV) were protected when challenged 7 days later with homologous virus. In the current study, we have extended this approach to cattle, the most economically important animals susceptible to FMD. Five cattle were vaccinated with the Ad5-FMDV subunit vaccine and these animals and 2 co-housed control animals were challenged intradermolingually 7 days later. Both control animals developed typical signs of FMD including fever and vesicular lesions on all 4 feet. All 5 vaccinated animals were protected against disseminated disease.
Abstract: Foot and mouth disease virus (FMDV) is a picornavirus that causes an acute vesicular disease of cloven-hoofed animals. This virus continues to be a threat to livestock worldwide with outbreaks causing severe economic losses. The present study shows an analysis of immune system phenotype and function during the acute phase of FMDV infection in swine. In the first days of infection, a significant lymphopenia is observed that involves all T cell subsets, CD4(+), CD8(+), and CD4(+)/CD8(+). This marked lymphopenia is not a result of active infection of PBMC with the virus. Further, the response of residual peripheral blood T cells to the mitogen, Concanavalin A (ConA) is significantly reduced and occasionally eliminated. Animals usually resolve clinical signs of disease and develop antigen specific T cell responses to the virus and recover ConA reactivity. These characteristics of acute phase infection likely play an important role in viral pathogenesis, propagation and shedding of viral particles and may be targeted as a way of improving vaccine formulations.
Abstract: A pilot study was carried out in cattle to determine the immunogenicity of a synthetic consensus peptide comprising the G-H loop region of foot-and-mouth disease virus (FMDV) type-O VP1 and a non-VP1 T-helper (Th) epitope. Cattle vaccinated intramuscularly either once (n = 5) or twice (n = 4) with 50 microg of the peptide preparation at a 21-day interval developed antibodies to the peptide as determined by ELISA with the exception of one steer that received a single dose. However, neutralizing antibody titers against FMDV type-O were modest and all animals presented with clinical FMD signs upon challenge 21 days after the last vaccination. In contrast, four of the five animals inoculated with an inactivated FMD type-O commercially prepared vaccine developed neutralizing antibodies and were fully protected against clinical disease following virus challenge 21 days post-vaccination (dpv). Nucleotide sequence comparison of the VP1 region between the challenge virus and RT-PCR products recovered from a lesion of the peptide-vaccinated animal with the highest neutralizing antibody titer 5 days post-challenge (dpc) showed no evidence for selection of a neutralization-resistant mutant. We conclude that although the synthetic peptide induced an antibody response in cattle, it failed to confer protection against FMDV challenge.
Abstract: A low-density cell population was isolated from skin explants of pigs and characterized as a highly enriched dendritic cell (DC) population based on phenotypical and functional properties. The skin-derived DCs were identified by their characteristic ultrastructural properties as well as by consistent co-expression of the CD1 and SWC3a antigens that clearly differentiate them from other porcine leukocytes. These cells exhibit higher expression of porcine MHC class II (SLAII) and CD80/86 antigens as compared to macrophage/monocyte cells. They consistently expressed the S100 beta antigen at high levels and did not express the lymphoid markers CD3, CD4 or CD8. Within this population of skin-derived DCs there was variable expression of CD11c, CD14 and CD16. Functional characterization of this DC population revealed that they are efficient in uptake and processing of soluble protein antigens and in endocytosis of small (0.02 microm) but not large (2 microm) polystyrene beads. Further, these cells were efficient inducers of primary allogeneic responses and in stimulating antigen-specific and mitogen-induced proliferation and IFN gamma responses in autologous lymphocytes. This study provides important information to further characterize the cutaneous DCs and develop models to analyze the role of these cells in immune responses in vivo.
Abstract: We have generated a panel of IgG monoclonal antibodies (MAbs) directed against Borrelia burgdorferi strain B31 antigens, using a method whereby mice were primed with organisms naturally inoculated by Ixodes scapularis nymphal ticks. Western blot analysis showed that these MAbs recognized several B. burgdorferi B31 antigens, including the complement inhibitor factor H-binding proteins ErpA/I/N and ErpC. Two other MAbs were specific for the RevA protein, and have enabled characterization of that previously unknown protein. The data presented here suggest that the production of MAbs from animals infected by tick-bite is a potentially useful tool for the identification of novel proteins synthesized by B. burgdorferi during mammalian infection.
Abstract: OBJECTIVE: To assess human skin biopsy specimens from erythema migrans lesions for the presence of infection with multiple strains of the Lyme disease spirochete, Borrelia burgdorferi. DESIGN: Skin biopsy specimens were obtained prospectively from patients with erythema migrans. To determine allelic differences and strain identification of B burgdorferi, the biopsy specimens were analyzed by cold single-strand conformation polymorphism of an amplified fragment of the outer surface protein C (ospC) gene. Further single-strand conformation polymorphism patterns of amplified ospC genes from culture isolates were compared with polymerase chain reaction products obtained directly from erythema migrans biopsy specimens. SETTING: A private dermatology office and a university medical center outpatient department. PATIENTS: Sixteen patients presenting with erythema migrans. RESULTS: Two of the 16 patients in this cohort were infected with 2 B burgdorferi sensu stricto strains, as evidenced by 2 ospC alleles in their skin biopsy results. CONCLUSION: This is the first documented description of the existence of more than a single strain of B burgdorferi sensu stricto in a human specimen.
Abstract: Lyme disease begins at the site of a tick bite, producing a primary infection with spread of the organism to secondary sites occurring early in the course of infection. A major outer surface protein expressed by the spirochete early in infection is outer surface protein C (OspC). In Borrelia burgdorferi sensu stricto, OspC is highly variable. Based on sequence divergence, alleles of ospC can be divided into 21 major groups. To assess whether strain differences defined by ospC group are linked to invasiveness and pathogenicity, we compared the frequency distributions of major ospC groups from ticks, from the primary erythema migrans skin lesion, and from secondary sites, principally from blood and spinal fluid. The frequency distribution of ospC groups from ticks is significantly different from that from primary sites, which in turn is significantly different from that from secondary sites. The major groups A, B, I, and K had higher frequencies in the primary sites than in ticks and were the only groups found in secondary sites. We define three categories of major ospC groups: one that is common in ticks but very rarely if ever causes human disease, a second that causes only local infection at the tick bite site, and a third that causes systemic disease. The finding that all systemic B. burgdorferi sensu stricto infections are associated with four ospC groups has importance in the diagnosis, treatment, and prevention of Lyme disease.
Abstract: In recent years, the utility of serum-based diagnostic testing for Lyme disease has improved substantially; however, recovery by culture of the bacterium from skin biopsies of suspected patients is still the only definitive laboratory test. Reinfection of patients has been assumed to occur but as yet has not been documented by serial isolates from the same person. We present a case of culture-confirmed reinfection of a patient in Menominee County, Michigan. Borrelia burgdorferi was isolated from the skin punch biopsy specimens during each episode of erythema migrans (EM) and was subjected to molecular strain typing, genetic analysis of two outer surface protein genes, protein profile analysis, and serum antibody response testing. Results show that these isolates are distinct strains of the bacterium and that the two episodes of EM were caused by independent infections. This report describes the documented, culture-confirmed reinfection of a human by two different strains of B. burgdorferi.
Abstract: This report describes the vector competence of 3 ixodid tick species, Ixodes scapularis (Say), I. spinipalpis (Nuttall), and Dermacentor andersoni (Stiles), for Borrelia burgdorferi in Colorado. The study was based on preliminary field work performed in 6 Colorado counties, where rodents and ticks were collected and assayed for the presence of B. burgdorferi. Four of the 6 counties produced 52 rodent and 39 I. spinipalpis isolates of B. burgdorferi. Two B. burgdorferi isolates were tested under laboratory conditions and found to be infective to Imperial Cancer Research Fund (ICRF) outbred mice. The 1st, a low-passage strain originating from New York (B-31, passage 6) was used as a control, and the 2nd was isolated from ear tissue of a Neotoma mexicana (Baird) (Mexican wood rat) that was trapped in Colorado. Larvae of I. scapularis, I. spinipalpis. and D. andersoni were fed on infected mice and cultured in Barbour-Stoner-Kelly media to assay for infection at 1, 2, 3, and 4 wk after repletion. The infection rates in replete larvae. were 75, 69, and 8.5%, respectively, whereas transstadial nymphal infection rates were 80, 75, and 0%, respectively. Both I. scapularis and I. spinipalpis were shown to be competent vectors that acquired the infection from the host reservoir mice and subsequently transmitted it to naive mice. Given that I. scapularis are not found in Colorado, I. spinipalpis are restricted to the nests and burrows of rodents, and because of the semiarid environment in Colorado, the risk of human contact with B. burgdorferi appears to be low.
Abstract: The response to recombinant vaccines for Lyme disease was studied to determine serum antibody levels effective in protecting against tick-transmitted infection. Data presented here demonstrate a significant correlation between antibody to an epitope on outer surface protein A (OspA) and protection against infection with Borrelia burgdorferi in canines and mice. A competitive enzyme-linked immunosorbent assay was developed to measure antibody to a site on OspA, defined by monoclonal antibody LA-2. Comparison of LA-2 titers against infection of canines and mice following vaccination and challenge established a predicted value for LA-2 titers. The statistical relationship between serum antibody levels and protection was calculated by logistic regression analysis. The statistical model predicted that an LA-2 titer of 0.32 microg equivalents (eq) per ml correlated to an 80% predicted probability of protection for both mice and dogs. This value was used to classify mice and dogs as to their protected status at the time of tick exposure. The LA-2 cutoff titer (0.32 microg eq/ml) correctly classified all dogs (n = 13) and mice (n = 44) that failed to become infected. By contrast, 20 of 22 dogs and 28 of 31 mice with titers of less than 0.32 microg eq/ml became infected. On the basis of these results, we conclude that an LA-2 titer is a reliable indicator of immune status for estimating immune protection following use of OspA-based vaccines for B. burgdorferi sensu stricto.
Abstract: The ability of naturally infected and cured mice to resist reinfection with tick-transmitted Borrelia burgdorferi was tested over a 1-year period. All of the mice were resistant to reinfection when they were challenged at 1.5 months after cure. The majority of animals were resistant to reinfection for up to 10.5 months after cure, but this resistance was lost at 1 year after cure. Both protected and unprotected animals showed a diverse array of antibodies on Western immunoblots. Protection was not associated with the killing of spirochetes in ticks, and naturally infected mice produced no antibodies to outer surface protein A (OSP A). The titers to whole Borrelia sonicate and OSP C, however, remained high throughout the 1-year study period. The levels of borreliacidal antibodies were highest in the 1.5 month-after-cure group. Natural immunity to reinfection with B. burgdorferi is limited in time, is complex, and may involve both humoral and cellular components.
Abstract: Development of a vaccine for the Lyme disease spirochete, Borrelia burgdorferi, has focused on the bacterial lipoprotein, major outer surface protein A (OspA). With few exceptions, testing of OspA vaccines in animal models has involved challenge with needle inoculation of cultured spirochetes. Recombinant OspA proteins from two OspA divergent strains of B. burgdorferi were tested for their vaccine potential in three different strains of mice challenged with laboratory reared ticks with a high rate of B. burgdorferi infection. All formulations of the B. burgdorferi sensu stricto derived OspA vaccine protected all strains of mice when challenged by ticks infected with an OspA homologous strain of the spirochete, whereas heterologous OspA from B. afzelii did not protect. Furthermore, ticks feeding on protected mice had reduced OspA levels compared to unvaccinated controls.
Abstract: Lyme disease spirochetes, Borrelia burgdorferi sensu lato, are maintained in zoonotic cycles involving ticks and small mammals. In unfed ticks, the spirochetes produce one outer surface protein, OspA, but not OspC. During infection in mammals, immunological data suggest that the spirochetes have changed their surface, now expressing OspC but little or no OspA. We find by in vitro growth experiments that this change is regulated in part by temperature; OspC is produced by spirochetes at 32-37 degrees C but not at 24 degrees C. Furthermore, spirochetes in the midgut of ticks that have fully engorged on mice now have OspC on their surface. Thus two environmental cues, an increase in temperature and tick feeding, trigger a major alteration of the spirochetal outer membrane. This rapid synthesis of OspC by spirochetes during tick feeding may play an essential role in the capacity of these bacteria to successfully infect mammalian hosts, including humans, when transmitted by ticks.
Abstract: Natural tick transmission of infection by Borrelia burgdorferi induces a very different serum antibody response than needle inoculation of spirochetes. We present data, obtained by using the mouse model, that show that the OspA response was barely detectable, whereas all animals developed significant anti-P39 titers after exposure to B. burgdorferi-infected ticks.
Abstract: The causative agent of Lyme disease, Borrelia burgdorferi, is transmitted by ticks of the Ixodes ricinus complex. In this study, we report the antibody response of recombinant inbred strains of mice of the H-2, b, d, and k haplotypes, infected with B. burgdorferi as a result of exposure to infected I. dammini. The patterns of antibody response assayed by Western blot analysis indicate significant major histocompatibility complex (MHC) restriction to bacterial antigens within the first 2 mo of infection in mice. Other bacterial antigens induce a significant response across the MHC haplotypes tested when assayed on the same bacterial strain used to transmit the infection, but do not crossreact with the same proteins derived from heterologous strains of B. burgdorferi. No response to outer surface protein A was detected at any time during the 60-d period we analyzed this infection. A third group of bacterial antigens appear to generate a MHC-nonrestricted response, and this lack of restriction is maintained when assaying the crossreactivity of the response with other strains of B. burgdorferi. These proteins may provide more accurate diagnostic probes than those currently in use. Finally, there appears to be a significant difference in the expression of most bacterial antigens when the spirochete is cultured for many passages since the same strain of bacterium isolated from low-passage and high-passage preparations exhibit different banding patterns in Western blots when assayed with the same sera.
Abstract: T cell binding to target cells involves not only the TCR and its MHC-bound ligand, but also a collection of additional proteins on both the T cell and its target. In an attempt to identify new molecules involved in this binding, mAb were raised against APC, and screened for their abilities to inhibit T cell recognition of Ag plus MHC on B cells. Six antibodies were identified that inhibited this reaction and that bound a cell-surface glycoprotein (Lgp55), with core polypeptide Mr 30,000 and a glycosylated Mr of approximately 55,000 depending upon the cell source. The properties of Lgp55 were consistent with it being the mouse homologue of a recently identified human ligand (intercellular adhesion molecule-2) for lymphocyte functional Ag-1 because the proteins are of comparable Mr, and antibody to Lgp55, like anti-lymphocyte functional antigen-1, blocks T cell recognition of Ag presented by B cells, but not of Ag presented by mouse fibroblasts.
Abstract: We have studied the role of the murine lymphocyte function associated antigen-1 (LFA-1) in the major histocompatibility complex (MHC)-restricted responses of a panel of T-cell hybridomas to protein antigens. Monoclonal antibodies to LFA-1 showed a differential blocking effect in these responses that correlated with the overall "sensitivity" of a given hybrid to antigen and MHC as defined by other criteria already reported. This result differs completely from similar experiments in the CTL system where all clones regardless of their overall "avidity" for target cells are very sensitive to the blocking effects of anti-LFA-1. Further, we show that no blocking effects are observed in the response of our hybridomas when Class I or Class II transfected fibroblasts or cultured 3T3 fibroblasts are used as synthetic antigen presenting cells and the result is unaltered by preincubation of such cells with interferon-gamma (IFN-gamma).
Abstract: We show that the LFA-1 molecule on T cells does not play a role in the stimulation of T cell hybridomas by certain targets, namely antigen presented by L cell derivatives or polyvalent anti-receptor antibody. These results suggest that LFA-1 may act by binding to ligands that are not present on all cells. We hope this result will help us and others to establish the true role of LFA-1 in T cell responses.
