Interleukin (IL)-6 directs the differentiation of IL-4-producing CD4+ T cells.

Interleukin (IL)-4 is the most potent factor that causes naive CD4+ T cells to differentiate to the T helper cell (Th) 2 phenotype, while IL-12 and interferon gamma trigger the differentiation of Th1 cells. However, the source of the initial polarizing IL-4 remains unclear. Here, we show that IL-6, probably secreted by antigen-presenting cells, is able to polarize naive CD4+ T cells to effector Th2 cells by inducing the initial production of IL-4 in CD4+ T cells. These results show that the nature of the cytokine (IL-12 or IL-6), which is produced by antigen-presenting cells in response to a particular pathogen, is a key factor in determining the nature of the immune response.

Borrelia burgdorferi OspA is an arthropod-specific transmission-blocking Lyme disease vaccine.

Borrelia burgdorferi, the spirochetal agent of Lyme disease, is transmitted by Ixodes ticks. A vaccine based on B. burgdorferi outer surface protein (Osp) A protects mice from spirochete infection. Here we report on the expression of OspA on spirochetes inside engorging ticks and relate OspA expression to antispirochetal immunity. Spirochetes in the gut of unfed nymphal ticks were stained by an OspA antibody, whereas in feeding ticks, the majority of spirochetes in the gut and salivary glands did not stain with the antibody. Thus, OspA was not expressed on most spirochetes during transmission from the vector to the vertebrate host. To examine the mechanism of protection afforded by OspA antibody, mice were passively immunized with OspA antibody at different times relative to tick attachment. When OspA antibody was administered to mice before or at the time of tick attachment, spirochetal development events in the vector, such as growth and salivary gland invasion, were blocked and the mice were protected from B. burgdorferi infection. When OspA antibody was administered to mice 48 h after tick attachment, spirochetes persisted in the nymphs and the mice were not protected despite the presence of circulating antibodies in the host as well as in the tick blood meal. Thus, OspA immunity appears to be effective only during a narrow window time at the beginning of the blood meal when antibodies bind to OspA-expressing spirochetes in the tick gut and block transmission from the vector to the host.

Protection against antigenically variable Borrelia burgdorferi conferred by recombinant vaccines.

Due to local variation in the antigenicity of the agent of Lyme disease (Borrelia burgdorferi), a vaccine derived from any one isolate of this spirochete may fail to protect against the heterogeneous population of organisms that may be present in an enzootic focus. Accordingly, we determined whether antigenically variable spirochetes delivered by naturally infected ticks, collected from a site where transmission is intense, may fail to infect mice actively immunized with recombinant glutathione transferase outer surface fusion proteins A or B (OspA and OspB). Virtually all mice vaccinated by either immunogen appeared not to become infected, as determined by culture or histopathology of their tissues. We conclude that Osp vaccination of mice effectively prevents infection by the agent of Lyme disease in a simulated natural cycle of transmission.

Vaccination against Lyme disease caused by diverse Borrelia burgdorferi.

Diversity and mutations in the genes for outer surface proteins (Osps) A and B of Borrelia burgdorferi sensu lato (B. burgdorferi), the spirochetal agent of Lyme disease, suggests that a monovalent OspA or OspB vaccine may not provide protection against antigenically variable naturally occurring B. burgdorferi. We now show that OspA or OspB immunizations protect mice from tick-borne infection with heterogeneous B. burgdorferi from different geographic regions. This result is in distinct contrast to in vitro killing analyses and in vivo protection studies using syringe injections of B. burgdorferi as the challenge inoculum. Evaluations of vaccine efficacy against Lyme disease and other vector-borne infections should use the natural mode of transmission and not be predicated on classification systems or assays that do not rely upon the vector to transmit infection.

Borrelia miyamotoi infection leads to cross-reactive antibodies to the C6 peptide in mice and men.

OBJECTIVES: Borrelia miyamotoi is a relapsing fever Borrelia, transmitted by hard (Ixodes) ticks, which are also the main vector for Borrelia burgdorferi. A widely used test for serodiagnosis of Lyme borreliosis is an enzyme immunoassay (EIA) based on the C6 peptide of the B. burgdorferi sl VlsE protein. We set out to study C6 reactivity upon infection with B. miyamotoi in a large well-characterized set of B. miyamotoi disease (BMD) patient sera and in experimental murine infection. METHODS: We performed in silico analyses, comparing the C6-peptide to immunodominant B. miyamotoi variable large proteins (Vlps). Next, we determined C6 reactivity in sera from mice infected with B. miyamotoi and in a unique longitudinal set of 191 sera from 46 BMD patients. RESULTS: In silico analyses revealed similarity of the C6 peptide to domains within B. miyamotoi Vlps. Cross-reactivity against the C6 peptide was confirmed in 21 out of 24 mice experimentally infected with B. miyamotoi. Moreover, 35 out of 46 BMD patients had a C6 EIA Lyme index higher than 1.1 (positive). Interestingly, 27 out of 37 patients with a C6 EIA Lyme index higher than 0.9 (equivocal) were negative when tested for specific B. burgdorferi sl antibodies using a commercially available immunoblot. CONCLUSIONS: We show that infection with B. miyamotoi leads to cross-reactive antibodies to the C6 peptide. Since BMD and Lyme borreliosis are found in the same geographical locations, caution should be used when relying solely on C6 reactivity testing. We propose that a positive C6 EIA with negative immunoblot, especially in patients with fever several weeks after a tick bite, warrants further testing for B. miyamotoi.

Protection of mice against the Lyme disease agent by immunizing with recombinant OspA.

Lyme borreliosis is a tick-borne illness caused by Borrelia burgdorferi. The gene for outer surface protein A (OspA) from B. burgdorferi strain N40 was cloned into an expression vector and expressed in Escherichia coli. C3H/HeJ mice actively immunized with live transformed E. coli or purified recombinant OspA protein produced antibodies to OspA and were protected from challenge with several strains of B. burgdorferi. Recombinant OspA is a candidate for a vaccine for Lyme borreliosis.

Lyme borreliosis in transgenic mice tolerant to Borrelia burgdorferi OspA or B.

The evolution of Lyme borreliosis in transgenic mice tolerant to Borrelia burgdorferi outer surface proteins (Osps) A or B was assessed to investigate the role of immunity to OspA or B in infection and pathogenesis of Lyme disease. Antibodies to OspA or B protect immunocompetent C3H/HeJ or C.B.17 severe combined immunodeficient (scid) mice from challenge with B. burgdorferi. Moreover, arthritis in infected C3H mice resolves with the rise of high titers of B. burgdorferi specific antibodies, including OspA and B, whereas disease persists in scid mice--suggesting that the regression of arthritis may be due to the development of borreliacidal OspA or B antibodies. To evaluate the course of Lyme borreliosis in OspA or B tolerant mice we developed transgenic mice that expressed OspA or B under control of the major histocompatibility complex (MHC) class I promoter. Mice carrying OspA or B transgenes on a C3H/HeJ (C3H, disease-susceptible) or C57BL/6 (B6, disease-resistant) background, immunized with OspA or B, did not mount a humoral or cellular immune response to OspA or B, respectively, but responded normally to other B. burgdorferi antigens. The evolution of Lyme borreliosis, including infection and the development of arthritis and carditis, was similar in transgenic and nontransgenic littermates suggesting that an OspA or B immune response is not singularly involved in either the genesis or regression of Lyme disease in C3H or B6 mice.

Effect of anti-interleukin 12 treatment on murine lyme borreliosis.

The effect of anti-interleukin (IL-12 treatment on Lyme borreliosis in C3H/HeN (C3H) mice was assessed because other studies have implicated CD4+ T cell helper (Th) type 1 responses in the genesis of disease caused by Borrelia burgdorferi. Infection of inbred mice with B. burgdorferi results in varying degrees of arthritis: BALB/c mice develop mild disease and C3H mice develop severe arthritis that is most pronounced 2-4 wk after infection. Since IL-12 is a major inducer of Th1 responses, we blocked this cytokine in vivo in B. burgdorferi infected C3H mice, and evaluated the effects of treatment on the development of arthritis at the peak of acute joint inflammation (14 d) and in the resolution phase (60 d) of disease. As expected, intraperitoneal administration of an anti-IL-12 monoclonal antibody (mAb) to C3H mice resulted in a decrease in both IFN-gamma and B. burgdorferi-specific IgG2a in serum, indicative of diminished Th1 responses. No IL-4 production was detected in serum of anti-IL-12 mAb treated or control mice. IgG1 and IgG2b levels did not increase in B. burgdorferi infected mice treated with anti-IL-12 mAb compared with controls suggesting that Th2 responses were not affected. Nevertheless, CD4+ T cells from both control and anti-IL-12 mAb treated mice had similar in vitro responses to B. burgdorferi antigens. Treatment with anti-IL-12 mAb produced a significant reduction in peak arthritis severity, and an increase in the number of spirochetes in ear tissue. These data show that treatment of B. burgdorferi infected mice with anti-IL-12 mAb results in a reduction of the Th1 and/or innate immune responses in vivo and a reduction in the severity of acute murine Lyme arthritis.

The agent of Human Granulocytic Ehrlichiosis resides in an endosomal compartment.

The composition of cytoplasmic vacuoles containing the agent of Human Granulocytic Ehrlichiosis (HGE) was studied to investigate how this pathogen exists within infected host cells. Electron microscopy demonstrated that the HGE organism resides in a membrane-bound compartment within HL-60 cells: early forms of the HGE agent have a round reticular appearance while later structures are small and dense. Vacuoles containing HGE bacteria incorporated endocytosed colloidal gold particles, suggesting that they are part of the endocytic pathway. Antibodies directed to the mannose-6-phosphate receptor labeled vacuole membranes. Antibodies to the transferrin receptor and to the lysosomal membrane glycoprotein LAMP 1 did not. Moreover, 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine, which normally accumulates in compartments with low pH, was not present inside these vacuoles. These results suggest that vacuoles containing the agent of HGE fail to mature into phagolysosomes. We conclude that the agent of HGE appears to enter and modify part of the endocytic pathway.

Temporal pattern of Borrelia burgdorferi p21 expression in ticks and the mammalian host.

The temporal synthesis of the P21 protein of Borrelia burgdorferi and the development of the humoral response to this antigen was assessed in infected mice. p21 is a member of the ospE-F gene family and its protein, P21, has been shown to be expressed by B. burgdorferi within infected mice but not by spirochetes cultured in vitro. P21 was not detected on B. burgdorferi in unfed or engorged Ixodes dammini (also known as I. scapularis) ticks, further supporting the postulate that P21 synthesis is specific for the mammalian host. In B. burgdorferi-infected mice, ospE mRNA and OspE antibodies were observed at 7 d, whereas p21 mRNA and P21-specific antibodies were detected at 21-28 d, suggesting that p21 is expressed later than ospE. Moreover, ospA mRNA was not discernible until day 14, indicating that ospA, like p21, is not expressed in the early stages of tick-transmitted murine Lyme borreliosis. Because p21 is expressed during infection in mice, we assessed the human humoral response to P21. 28% (34 of 122) of the patients with either early- or late-stage Lyme disease, and 33% (11 of 33) of the individuals with Lyme arthritis had P21 antibodies, suggesting that a P21 response may serve, at least partially, as a marker of infection. Active immunization with recombinant P21 did not protect C3H mice from tick-borne B. burgdorferi infection, and passive transfer of P21 antiserum to infected mice did not alter the course of disease. These data suggest that the antigenic structure of B. burgdorferi changes during the early stages of murine infection.

Immunization against the agent of human granulocytic ehrlichiosis in a murine model.

The agent of human granulocytic ehrlichiosis (HGE) is a newly recognized tick-borne pathogen that resides within polymorphonuclear leukocytes. C3H/HeN mice can become infected with the agent of HGE (designated aoHGE) by syringe inoculation or tick-borne infection and develop transient neutropenia. They thereby partially mimic human disease and provide a model in which to study immunity to this microorganism. Mice vaccinated with lysates of purified aoHGE, or administered aoHGE antisera, were partially protected from both syringe- and tick-transmitted challenge with aoHGE. These data suggest that antibodies are sufficient to provide substantial, but not complete, immunity against aoHGE.

Attachment of Borrelia burgdorferi within Ixodes scapularis mediated by outer surface protein A.

Borrelia burgdorferi outer surface protein (Osp) A has been used as a Lyme disease vaccine that blocks transmission: OspA antibodies of immune hosts enter ticks during blood feeding and destroy spirochetes before transmission to the host can occur. B. burgdorferi produce OspA in the gut of unfed Ixodes scapularis ticks, and many spirochetes repress OspA production during the feeding process. This preferential expression suggests that OspA may have an important function in the vector. Here we show that OspA mediates spirochete attachment to the tick gut by binding to an I. scapularis protein. The binding domains reside in the central region and COOH-terminus of OspA. OspA also binds to itself, suggesting that spirochete-spirochete interactions may further facilitate adherence in the gut. OspA-mediated attachment in the tick provides a possible mechanism for how stage-specific protein expression can contribute to pathogenesis during the B. burgdorferi natural cycle.

Identification of Salp15 homologues in Ixodes ricinus ticks.

The 15-kDa Ixodes scapularis salivary gland protein Salp15 protects Borrelia burgdorferi sensu stricto from antibody-mediated killing and facilitates infection of the mammalian host. In addition, Salp 15 has been shown to inhibit T-cell activation. We determined whether Ixodes ricinus, the major vector for Lyme borreliosis in Western Europe, also express salp15-related genes. We show that engorged I. ricinus express salp15 and we have identified three Salp15 homologues within these ticks by reverse transcriptase-polymerase chain reaction (RT-PCR). One of the predicted proteins showed 80% similarity to I. scapularis Salp15, evenly distributed over the entire amino acid sequence, whereas the two other predicted proteins showed approximately 60% similarity, mainly confined to the signal sequence and C-terminus. Comparison of the DNA and protein sequences with those deposited in several databases indicates that these proteins are part of a Salp15 family of which members are conserved among different Ixodes species, all capable of transmitting B. burgdorferi sensu lato. This suggests that these Salp15 homologues could also play a role in the transmission of diverse Borrelia species and in inhibition of T-cell activation.

Characterization of two genes, p11 and p5, on the Borrelia burgdorferi 49-kilo base linear plasmid.

A putative operon encoding two Borrelia burgdorferi N40 genes, p11 and p5 was cloned and localized to the 49 kilobase linear plasmid. p11 encodes an 11 kDa protein and p5 encodes a 5 kDa protein. The first 88 nucleotides of p11 have 81% identity with orf5 on a circular plasmid from Borrelia afzelii strain Ip21, suggesting that homologues of these genes may be present in different regions of the B. burgdorferi genome.

Detection of antibodies to Borrelia burgdorferi in naturally infected horses in the USA by enzyme-linked immunosorbent assay using whole-cell and recombinant antigens.

Blood samples were collected from 98 horses suspected of having borreliosis or granulocytic ehrlichiosis in Connecticut and New York State, USA during 1985, 1995, and 1996. Serum antibodies to Borrelia burgdorferi were detected by an enzyme-linked immunosorbent assay (ELISA), based on whole-cell and recombinant antigens, in 82 (84%) horses. Of the 181 sera tested, 59% were positive, using whole-cell antigens, compared to 48% with protein (p)37 and 35% with VlsE antigens. An ELISA containing either of these fusion proteins can be used as an adjunct to general screening by an ELISA or immunoblotting in animals not vaccinated for this disease.

Heterogeneity of Lyme disease spirochaetes within individual vector ticks.

To determine whether Lyme disease spirochaetes (Borrelia burgdorferi) within vector ticks (lxodes dammini) sampled from enzootic sites comprise single or mixed populations, we compared their reactivity to a polyclonal rabbit immune serum with that to a battery of monoclonal antibodies (mAb) against OspA, OspB and flagellin. More spirochaetes were recognized by the polyclonal antibody than with the mAbs. Spirochaetes from field-sampled ticks reacted poorly to mAbs against OspB. No such differences in reactivity to polyclonal or monoclonal antibodies were observed for the N40 strain of B. burgdorferi from BSK cultures and infected laboratory-reared vector ticks. We conclude that in nature each tick may be infected by an antigenically heterogeneous mixture of spirochaetes.

Characterization of a 30-kDa Borrelia burgdorferi substrate-binding protein homologue.

A Borrelia burgdorferi chromosomal gene encodes a 30-kDa antigen (P30) that has considerable homology with periplasmic substrate-binding proteins of Gram-negative bacteria, and is recognized by antibodies in sera from a subset of patients with Lyme disease and from B. burgdorferi-infected mice. The p30 gene is 801 nucleotides in length and P30 contains 267 amino acids, with a predicted molecular mass of 30 kDa. The P30 amino acid region 36-258 has homology to conserved domains of the oligopeptide permease A of Gram-negative bacteria. Immunofluorescence studies using murine anti-P30 serum suggest that P30 is on the outer surface of B. burgdorferi. P30 expression could be detected in representatives of all 3 subspecies of B. burgdorferi sensu lato, but not in all of the tested strains. Antibodies to P30 were detected in sera of 18 out of 82 patients (22%) with Lyme disease, including individuals with early- or late-stage infection. Although antibodies to P30 are present in the sera of C3H/HeN mice infected with B. burgdorferi for at least 90 days, immunization with recombinant P30 does not protect mice from infection. We conclude that P30 is a putative substrate-binding protein of B. burgdorferi and is immunologically recognized in human and murine Lyme borreliosis.

Spirochetes: vaccines, animal models and diagnostics.

The detailed characterization of various proteins from spirochetes using molecular biology techniques has made possible new approaches to vaccine and diagnostic development that are described in this session. The importance of animal models was emphasized and illustrated.

West Nile virus envelope protein: role in diagnosis and immunity.

The role of antibodies to the West Nile virus envelope (E) protein in serodiagnosis and protection was examined. The E protein was expressed and purified in recombinant form. Antibodies to the E protein were detected in patients with West Nile virus infection. Passive immunization with rabbit anti-E protein sera also partially protected mice from challenge with West Nile virus. The humoral response to the West Nile virus E protein is therefore useful as an aid in the diagnosis and may also play a role in immunity to infection.

Growth and migration of Borrelia burgdorferi in Ixodes ticks during blood feeding.

We have studied the growth of Borrelia burgdorferi in nymphal ticks (Ixodes scapularis) feeding on mice using confocal fluorescence microscopy to follow the distribution of spirochetes. In starved nymphs, the bacteria were only detected in the midgut and each nymph had a mean of 496 spirochetes. Upon attachment of nymphs to the host, the bacteria grew with a doubling time close to 4 hr and reached a mean of 7,848 spirochetes per nymph 15 hr after attachment. During this initial period (36 hr) of rapid growth, the bacteria appeared to be restricted to the gut, but after 48 hr, the spirochetes had disseminated to the salivary glands in the majority of nymphs examined. Thus, a critical event that allows the spirochetes to disseminate and infect the salivary glands takes place 36-48 hr after attachment. A maximum number of 166,575 spirochetes per nymph was noted 72 hr after attachment. Soon after completion of feeding and detachment from the host (96 hr), the mean number of spirochetes decreased to 95,410 per nymph and the spirochetes appeared to be cleared from organs other than the midgut. Thus, dissemination of spirochetes within the vector appears to be a transient phenomenon. These results provide strong evidence in favor of a salivary route of disease transmission while also demonstrating the utility of confocal microscopy to study vector-pathogen interactions in general.