[Show abstract][Hide abstract] ABSTRACT: The ank gene of the agent of human granulocytic ehrlichiosis (HGE) codes for a protein with a predicted molecular size of 131.2 kDa that is recognized by serum from both dogs and humans infected with granulocytic ehrlichiae. As part of an effort to assess the phylogenetic relatedness of granulocytic ehrlichiae from different geographic regions and in different host species, the ank gene was PCR amplified and sequenced from a variety of sources. These included 10 blood specimens from patients with confirmed human granulocytic ehrlichiosis (three from New York, four from Wisconsin, two from Slovenia, and one from Sweden). Also examined was a canine granulocytic ehrlichia sample obtained from Minnesota, Ehrlichia equi from California, Ehrlichia phagocytophila from Sweden, and the granulocytic ehrlichia isolate USG3. The sequences showed a high level of homology (>95.5% identity), with the lowest homology occurring between a New York HGE agent and the Swedish E. phagocytophila. Several 3-bp deletions and a variable number of 51- and 81-bp direct repeats were noted. Although the North American HGE sequences showed the highest conservation (>98.1% identity), phylogenetic analyses indicated that these samples represent two separate clades, one including the three New York HGE samples and the USG3 strain and another with the Wisconsin HGE and Minnesota canine sequences. Two of the New York samples and the USG3 strain showed 100% identity over the entire 3,696-bp product. Likewise, three of the Wisconsin human samples and the Minnesota dog sample were identical (3,693 bp). Whereas phylogenetic analysis showed that the E. equi sequence was most closely related to the Upper Midwest samples, analysis of the repeat structures showed it to be more similar to the European samples. Overall, the genetic analysis based on the ank gene showed that the granulocytic ehrlichiae are closely related, appear to infect multiple species, and can be grouped into at least three different clades, two North American and one European.
Full-text · Article · Aug 2000 · Journal of Clinical Microbiology
[Show abstract][Hide abstract] ABSTRACT: Unlike the elderly, healthy middle aged adults are at relatively low risk of acquiring serious pneumococcal disease. An explanation that has been proposed is that people in this age group have significant amounts of serum antibody (primarily IgG2) that react with any pneumococcal capsular polysaccharide serotypes. The level of antibody can be as high as several hundred micrograms per milliliter of blood for some serotypes. A significant component of this reactivity is directed toward the conserved C-polysaccharide depletion. Even after C-polysaccharide depletion, which is included as a routine part of the assay to determine antibody levels, resting antibody levels in a normal healthy adult population can vary widely. We have analyzed the reactivity of serum from 76 people to 16 pneumococcal capsular polysaccharide serotypes. The antibody reactivities to 13 of 16 serotypes are highly correlated with one another. Depletion of serum with C-polysaccharide and purified capsular polysaccharide inhibited antibody binding to type specific capsular polysaccharide. Cross-serotype inhibition of antibody binding was also observed. This indicates that there are materials contained within the pneumococcal polysaccharides that contribute to the cross-reactivity of serum antibodies in people that have not been vaccinated with the pneumococcal vaccine.
[Show abstract][Hide abstract] ABSTRACT: Western blot analysis of proteins from a cell culture isolate (USG3) of the human granulocytic ehrlichiosis (HGE) agent has identified a number of immunoreactive proteins, including major antigenic proteins of 43 and 45 kDa. Peptides derived from the 43- and 45-kDa proteins were sequenced, and degenerate PCR primers based on these sequences were used to amplify DNA from USG3. Sequencing of a 550-bp PCR product revealed that it encodes a protein homologous to the MSP-2 proteins of Anaplasma marginale. Concurrently, an expression library made from USG3 genomic DNA was screened with granulocytic Ehrlichia (GE)-positive immune sera. Analysis of two clones showed that they contain one partial and three full-length highly related genes, suggesting that they are part of a multigene family. Amino acid alignment showed conserved amino- and carboxy-terminal regions which flank a variable region. The conserved regions of these proteins are also homologous to the MSP-2 proteins of A. marginale; thus, they were designated GE MSP-2A (45 kDa), MSP-2B (34 kDa), and MSP-2C (38 kDa). The PCR fragment obtained as a result of peptide sequencing was completely contained within the msp-2A clone, and all of the sequenced peptides were found in the GE MSP-2 proteins. Recombinant MSP-2B protein and an MSP-2A fusion protein were expressed in Escherichia coli and reacted with human sera positive for the HGE agent by immunofluorescence assay. These data suggest that the 43- and 45-kDa proteins of the HGE agent are encoded by members of the GE MSP-2 multigene family.
Full-text · Article · Sep 1998 · Infection and Immunity
[Show abstract][Hide abstract] ABSTRACT: As an initial attempt to investigate the possible role of outer surface protein A (OspA) escape mutants of Borrelia burgdorferi in decreasing the efficacy of the OspA vaccine, mutants of the HB19 strain of B. burgdorferi sensu stricto were selected in vitro from an uncloned, low-passage-number isolate. The antiserum used for selection was obtained from rhesus monkeys that had been given a vaccine of the same formulation and dose, and by the same route of administration, as that given to humans in several trials. All of the mutants selected in liquid medium and subsequently cloned twice in solid medium expressed a single abundant protein of 28 to 34 kDa instead of both OspA and OspB. Depending on the mutant, this protein reacted strongly, weakly, or not detectably with the anti-OspA antibody used for selection. Analysis of the ospAB locus of each of four representatives from these three groups of mutants by PCR with oligonucleotide primers that hybridize to flanking regions of the ospAB operon, and of the corresponding phenotype with monoclonal antibodies that bind to the amino or carboxyl terminus of the OspA or OspB polypeptide, indicated that in all cases a deletion within the operon had occurred. Spirochetes from the four mutant strains chosen for further analysis could be killed in antibody-dependent, complement-mediated killing assays with the selecting anti-OspA antibody, despite their resistance to killing with this antibody in the absence of complement. Complement-mediated killing occurred at an antibody concentration higher than that required to kill wild-type spirochetes. If anti-OspA antibody acts only within the tick, where complement is probably ineffective due to tick-derived decomplementing factors, then OspA escape mutants, if infectious, could seriously diminish the efficacy of OspA vaccines. On the other hand, if the killing of B. burgdorferi with anti-OspA antibody also takes place within the human host, then our results indicate that chimeric/deletion escape mutants will be killed as well.
Preview · Article · Jul 1998 · Infection and Immunity
[Show abstract][Hide abstract] ABSTRACT: Granulocytic Ehrlichia was isolated from canine blood obtained from animals challenged with field-collected Ixodes scapularis and propagated in HL60 cells. PCR primers specific for the 16S ribosomal DNA (rDNA) of the Ehrlichia genogroup comprising E. equi, E. phagocytophila, and the agent of human granulocytic ehrlichiosis (HGE) amplified DNA from extracts of these cells. Sequence analysis of this amplified DNA revealed that it is identical to the 16S rDNA sequence of the HGE agent. A genomic library was constructed with DNA from granulocytic Ehrlichia and screened with pooled sera from tick-challenged, granulocytic Ehrlichia-infected dogs. Several clones were isolated and sequenced. Three complete genes encoding proteins with apparent molecular masses of 100, 130, and 160 kDa were found. The recombinant proteins reacted with convalescent-phase sera from dogs and human patients recovering from HGE. This approach will be useful for identifying candidate diagnostic and vaccine antigens for granulocytic ehrlichiosis and aid in the classification of genogroup members.
Full-text · Article · Apr 1998 · Infection and Immunity
[Show abstract][Hide abstract] ABSTRACT: The antigenic variation associated with Human Immunodeficiency Virus type-1 (HIV-1) envelope proteins could limit their utility in vaccines if the immune responses induced are specific for immunodominant variable epitopes. We evaluated the ability of experimental subunit vaccines, containing recombinant forms of the envelope glycoprotein (rgp120) from two HIV-1 variants, to induce immune responses capable of recognizing unrelated HIV-1 variants. A vaccine formulation based on HIV-1IIIB/LAI rgp120 and supplemented with saponin adjuvant (QS-21) induced neutralizing antibodies specific for the HIV-1IIIB/LAI variant. This antibody response was presumably specific for the variable principle neutralizing determinant (PND) of the third variable region of gp120, the V-3 region. This formulation induced cytotoxic T-lymphocytes (CTL) specific for the dominant V-3 epitope but also to an additional unidentified epitope outside of this region. The CTL specific for this second epitope also recognized gp120 from the HIV-1MN and HIV-1RF variants in a "cross-reactive" manner. A second vaccine formulation based on HIV-1MN rgp120 and QS-21 adjuvant induced neutralizing antibodies that were again variant-specific but also CTL that recognized all three HIV-1 variants in a cross-reactive manner. These data demonstrate that CTL capable of recognizing different HIV-1 variants, which are presumed to be specific for a conserved HIV-1 gp120 epitope, can be induced using subunit vaccines with the appropriate adjuvant while variant-specific antibody responses are produced. These findings support further evaluation of this vaccine format.
[Show abstract][Hide abstract] ABSTRACT: An indirect immunofluorescence assay for the detection of human antibodies to the agent of human granulocytic ehrlichiosis (HGE) was developed and standardized. Antigen was prepared from a human promyelocytic leukemia cell line (HL-60) infected with a tick-derived isolate of the HGE agent (USG3). Suitable antigen presentation and preservation of cellular morphology were obtained when infected cells were applied and cultured on the slide, excess medium was removed, and cells were fixed with acetone. Use of a buffer containing bovine serum albumin and goat serum reduced background fluorescence, and use of an immunoglobulin G (gamma-specific) conjugate reduced nonspecific binding. The assay readily detected specific antibody from HGE patients and did not detect antibody from healthy individuals. No significant reactivity was noted in sera from patients with high titers of antibodies to other rickettsial species. We were able to identify antibodies reactive to USG3 antigen in samples from areas where HGE is endemic that had tested negative to other rickettsial agents. Animal sera reactive against Ehrlichia equi or Ehrlichia phagocytophila bound to the HGE antigen, indicating that the assay may be useful for veterinary use. Comparability between two different laboratories was assessed by using coded human sera exchanged between laboratories. Results from the two laboratories were similar, indicating that the assay can be easily integrated into use for routine testing for HGE. The assay was then compared to an assay using horse neutrophils infected with ehrlichiae. The two assays gave comparable results, indicating that the cell culture-derived antigen can be used for testing samples that have been previously tested with E. equi as an antigen. The new assay offers several advantages over other immunofluorescence methods that use animal-derived antigen and is suitable for use in testing for human antibodies to the HGE agent.
[Show abstract][Hide abstract] ABSTRACT: A new indirect fluorescent-antibody (IFA) assay with antigen produced in vitro in the human promyelocytic leukemia cell line HL60 was used to identify the first recognized case of human granulocytic ehrlichiosis in Rhode Island. This IFA assay was used to detect granulocytic ehrlichiae in white-footed mice and in a dog inhabiting the area surrounding the patient's residence. Host-seeking Ixodes scapularis ticks found in the same habitat also were infected. I. scapularis ticks collected from other locations were fed on dogs and New Zealand White rabbits to assess the competency of these species as hosts of granulocytotropic Ehrlichia. Tick-induced infections of dogs were confirmed by serologic testing, tissue culture isolation, and PCR amplification, whereas several rabbits seroconverted but were PCR and culture negative. PCR amplification of the 16S rRNA gene and DNA sequencing of the PCR products or culture isolation was used to confirm granulocytic Ehrlichia infections in humans, dogs, white-footed mice, and ticks.
Full-text · Article · May 1997 · Journal of Clinical Microbiology
[Show abstract][Hide abstract] ABSTRACT: A subunit canine Lyme disease vaccine formulated with recombinant lipidated Osp A and OspB and saponin QS21 was assessed for safety, protective efficacy, and immunogenicity. Ten normal beagles were subcutaneously vaccinated twice at age 12 and 16 weeks, respectively. Three months after the second vaccination, the vaccinates and another 10 nonvaccinated control beagles were challenged by feeding ticks on each dog for 5 days using eight field-collected adult female and six adult male Ixodes scapularis infected with Lyme disease spirochetes per dog. Adverse reactions associated with the vaccinations were limited to injection site swellings which occurred within the first 48 h and resolved within a week. The local reaction was independent of vaccination times and tick challenge. On the basis of typical clinical signs, xenodiagnosis, and diagnostic immunoblotting, all 10 controls were infected; five developed lameness and three of them experienced at least two to three episodes of limping during a 10-month monitoring period. In contrast, eight of ten vaccinates were protected and two infected vaccinates, as judged by xenodiagnosis, were asymptomatic. None of the protected vaccinates developed antibodies to diagnostic spirochetal antigens other than OspA and OspB. In contrast, most controls produced antibodies to borrelial antigens, but not to OspA and OspB. Antibody production in vaccinates receiving a third vaccination 10 months postchallenge was greatly boosted; the geometric mean antibody titer was significantly higher (P < 0.0001) than that tested prechallenge. Thus, the subunit canine Lyme disease vaccine was safe and protective and elicited immunological memory. Vaccinated dogs were serologically distinguishable from those naturally exposed.
[Show abstract][Hide abstract] ABSTRACT: Conjugate vaccines were prepared with S. aureus type 8 capsular polysaccharide (CP) using three carrier proteins: Pseudomonas aeruginosa exotoxin A (ETA), a non-toxic recombinant ETA (rEPA), and diphtheria toxoid (DTd). Adipic acid dihydrazide (ADH) or N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) was used as a spacer to link the CP to carrier protein. All conjugates gave a high immune response with a boost after the second immunization. Conjugates prepared with ADH gave higher antibody titers than conjugates prepared with SPDP. IgG1 was the primary subclass elicited by all conjugates regardless of the carrier protein or the conjugation method used to prepare the vaccines. The non-immunogenic CP and the conjugates were formulated with either monophosphoryl lipid A (MPL), QS21, or in Novasomes and evaluated in mice. While the adjuvants failed to improve the immunogenicity of the nonconjugated CP, a more than fivefold increase in the antibody levels was observed when these adjuvants were used with the conjugates. Significant rises in IgG2b and IgG3 were observed with all formulations. The enhancement of the immunogenicity and the IgG subclass shift, as seen with some adjuvants, may prove to be important in immunocompromised patients.
[Show abstract][Hide abstract] ABSTRACT: The neutralizing epitopes of the major outer surface proteins A and B (OspA and OspB) of Borrelia burgdorferi B31 were investigated by epitope mapping using overlapping synthetic peptides, encompassing full-length OspA and OspB, and antiborrelial monoclonal antibodies (MAbs). OspA MAb N4B12 and OspB MAbs N5G5, W7C2, and P4D1 displayed a complement-independent antiborrelial activity, and complement failed to enhance the antiborrelial activity, as measured by a sensitive colorimetric assay. A combination of N4B12 with N5G5 displayed a higher antiborrelial activity than did the MAbs individually. OspA MAbs B3G11 and L3B5, however, exhibited a significant antiborrelial activity only in the presence of complement. Epitope mapping showed that B3G11 bound to one OspA synthetic peptide with the sequence of amino acids 247 to 256 (QYDSNGTKLE) and produced more than sixfold-higher reactivity than with other sequences, as measured by an enzyme-linked immunosorbent assay. OspB MAb N5G5 bound to an OspB peptide with the sequence of amino acids 211 to 220 (TLKREIEKDG), yielding at least threefold-higher reactivity than with other sequences. These two peptide sequences were found to contain neutralizing epitopes. Other MAbs had weak binding activities with the synthetic peptides, and their specific epitopes remain to be further analyzed. Thus, this study demonstrated both complement-independent and complement-dependent antiborrelial MAbs and identified the linear epitopes on OspA and OspB capable of inducing neutralizing antibody responses.
Preview · Article · Jul 1995 · Infection and Immunity
[Show abstract][Hide abstract] ABSTRACT: A vaccine consisting of purified Escherichia coli-expressed recombinant full-length outer surface proteins A (OspA) and B (OspB) and the saponin adjuvant QS21 was evaluated
for protection against Borrelia burgdorferi infection. Eleven beagles were vaccinated twice and then challenged with 10 field-collected adult female Ixodes scapularis. Xenodiagnosis revealed that all 11 nonvaccinated control dogs and 2 of 10 vaccinated dogs were infected with B. burgdorferi. Six of 11 control dogs also developed fever (0.75 ± 0.38°C) and were lethargic. One of the control dogs also developed a
limp. Both of the infected vaccinated dogs were asymptomatic. Thus, the vaccine prevented tick-vectored infection and associated
symptoms of Lyme disease.
No preview · Article · May 1995 · The Journal of Infectious Diseases
[Show abstract][Hide abstract] ABSTRACT: Canine antibody responses to Lyme disease subunit vaccines and to natural borrelial infection were investigated. Vaccines
were formulated with QS21 and outer surface proteins A (OspA) and B (OspB) derived from Borrelia burgdorferi B31. Vaccines containing QS21 and the lipoproteins gave 4-fold higher IgG 1 and 8-fold higher IgG2 antibody responses than
without QS21. Antisera to lipidated OspA or OspB vaccines containing QS21 had high antiborrelial activity against isolates
B31 and CA-2-87, similar to those with a vaccine containing both OspA and OspB. Only the combination vaccine induced antiborrelial
activity against heterologous isolates 24008 Fr and Borrelia garinii G25. Nonlipidated OspA- and OspB-based vaccines with QS21 elicited lower antibody and antiborrelial activity than did lipidated
OspA and OspB vaccines; 49% of naturally exposed dogs had low titers to OspA or OspB. Thus, vaccines using lipidated OspA,
OspB, and QS21 could induce higher antiborrelial activity than did natural exposure.
No preview · Article · May 1995 · The Journal of Infectious Diseases
[Show abstract][Hide abstract] ABSTRACT: QS-21, a purified Quillaja saponaria saponin immunologic adjuvant, contains two functional groups that we hypothesized to be involved in the adjuvant mechanism of action through charge or Schiff base interaction with a cellular target. Derivatives, prepared by modification of these sites, were prepared and tested for their ability to augment the immunogenicity of the antigen ovalbumin (OVA) in C57BL/6 mice. QS-21 derivatives that were modified at the carboxyl group on an anionic sugar, glucuronic acid, retained adjuvant activity for antibody stimulation, inducing relative increases in antibody titers similar to those induced by QS-21, although the minimum adjuvant dose required for this stimulation was increased several fold relative to the dose of unmodified QS-21. One of these derivatives also retained significant activity for induction of OVA-specific cytotoxic T-lymphocytes. In contrast, QS-21 derivatives modified at an aldehyde on the triterpene did not show adjuvant activity for antibody stimulation or for induction of cytotoxic T-lymphocytes, suggesting that this functional group may be involved in the adjuvant mechanism.
[Show abstract][Hide abstract] ABSTRACT: Three types of experimental vaccines containing O-side-chain polysaccharide from the enterotoxigenic strain Escherichia coli 018 were evaluated. The immunogenicity of free O-polysaccharide (PS), a polysaccharide-diphtheria toxoid conjugate (PS-conj), and detoxified lipopolysaccharide (dLPS) was tested in female ICR mice, either alone or in combination with QS-21, a purified saponin adjuvant derived from the bark of the tree Quillaja saponaria Molina. Both the number of individual mice responding and the titres of O-polysaccharide specific antibodies in pools of sera were increased by the addition of QS-21. The immune response to both O-specific polysaccharide and carrier was primarily IgM and IgG1. The addition of QS-21 not only increased the level of IgG1, but also had a significant adjuvant effect on antigen-specific IgG2a, IgG2b and IgG3.
[Show abstract][Hide abstract] ABSTRACT: Dogs become infected with Borrelia burgdorferi after being bitten by infected adult ticks. However, it is not known whether dogs are competent reservoirs of the organism, that is, it is not known whether infected dogs can subsequently transmit the bacterium to feeding immature ticks. To determine reservoir competence of dogs, 11 Beagles were experimentally infected by means of challenge exposure to infected adult deer ticks (Ixodes scapularis). Three weeks later, larval ticks were allowed to feed on the dogs. Engorged larvae were collected, allowed to molt to the nymph stage, and tested, by means of a direct fluorescent antibody assay, to detect the presence of B burgdorferi organisms. Overall, 78% of immature ticks tested were found to have become infected. We concluded that dogs might serve to increase human risk of exposure to B burgdorferi-infected ticks and, therefore, should be protected from exposure to infected ticks as well as immature ticks seeking a blood meal.
Full-text · Article · Aug 1994 · Journal of the American Veterinary Medical Association
[Show abstract][Hide abstract] ABSTRACT: The in vivo and in vitro accessory cell requirements of class I major histocompatability complex (MHC) antigen-restricted cytotoxic T-lymphocyte (CTL) responses were determined using cell-depletion experiments coupled with active immunizations using ovalbumin (OVA) as the immunogen and saponin adjuvant (QS-21). To paralyze macrophage activity in vivo, C57BL/6 mice were treated with particulate silica or carrageenan. In vivo depletion of helper T-lymphocytes was accomplished by treatment with GK1.5 rat monoclonal antibody, which is specific for the murine CD4 antigen, and by genetic depletion of class II MHC antigens. Following treatments, the mice were immunized with formulations containing OVA alone or mixed with QS-21 saponin adjuvant, which induces MHC class I antigen-restricted CTL responses. In vivo treatment to paralyze macrophages abrogated these CTL responses but not antigen-specific antibody or lymphocyte proliferative responses. Depletion of CD4+ T-lymphocytes had no effect on CTL responses but significantly reduced proliferation and antibody responses. In vitro depletion and reconstitution experiments were done to compare the contributions of different antigen-presenting cells (APC), specifically dendritic cells (DC) and macrophages. Again, the requirement for macrophages was absolute but there was no indication that DC were involved. These data suggest that antigen processing and presentation functions are critical to the induction of CTL and that they are a function of macrophages but that CD4+ helper T-lymphocyte functions are not required.
No preview · Article · May 1994 · Cellular Immunology
[Show abstract][Hide abstract] ABSTRACT: Six murine hybridoma cell lines producing monoclonal antibodies (MAbs) specific for Toxin B of Clostridium difficile have been generated from toxin-immunized female RBF/DnJ mice. All six antibodies were reactive in Western blots with a > 200-kD protein in the supernatants of the toxigenic strain 10463 and were unreactive with similarly prepared material from the nontoxigenic strain 2037. Polyclonal antisera from rabbits immunized with Toxin B reacted on Western blots primarily with Toxin B, a 40-kD and a 55-kD band with a minor set of triplet bands at approximately 100 kD. None of the MAbs reacted in a direct EIA with purified Toxin A from C. difficile but two MAbs reacted weakly with a trypsin-sensitive band (> 200 kD) in Western blots of C. sordellii. Polyclonal antisera developed against Toxin B reacted strongly with supernatants from C. sordellii, C. bifermentans, and the nontoxigenic strain 2037. Toxin B-specific antisera was unreactive with supernatants from C. perfringens or purified Toxin A from C. difficile in direct EIA. Toxin B-specific MAbs linked to an affinity column were able to deplete bacterial supernatant of cytotoxigenic activity.
[Show abstract][Hide abstract] ABSTRACT: A simple, colorimetric borreliacidal assay was developed for the determination of borroliacidal activity of immune serum to Borrelia burgdorferi. This microtiter assay was capable of quantitatively detecting the borreliacidal activity of mouse antisera to the outer surface protein A or lysates of Borrelia burgdorferi strain B31. The borreliacidal activity was shown to be specific and dose-dependent. Normal mouse serum was not borreliacidal. The absorbance changes due to the borrelial growth in the presence of phenol red in the assay correlated with [3H]thymidine incorporation by the spirochetes R = 0.977). This colorimetric assay had the advantages of reliability and convenience, and could handle large number of serum samples. The borreliacidal assay should prove valuable in both research and clinical laboratories.
No preview · Article · Mar 1993 · Journal of Microbiological Methods