A rhesus macaque model of Streptococcus pneumoniae carriage.

BACKGROUND: Nasopharyngeal colonization by Streptococcus pneumoniae precedes pneumococcal disease. Elucidation of procedures to prevent or eradicate nasopharyngeal carriage in a model akin to the human would help to diminish the incidence of both pneumonia and invasive pneumococcal disease. METHODS: We conducted a survey of the nasopharynx of infant rhesus macaques from our breeding colony, in search of natural carriers of S. pneumoniae. We also attempted experimental induction of colonization, by nasopharyngeal instillation of a human S. pneumoniae strain (19F). RESULTS: None of 158 colony animals surveyed carried S. pneumoniae in the nasopharynx. Colonization was induced in eight of eight infant rhesus by nasopharyngeal instillation and lasted 2weeks in 100% of the animals and 7weeks in more than 60%. CONCLUSION: Rhesus macaques are probably not natural carriers of S. pneumoniae. The high rate and duration of colonization obtained in our experiments indicates that the rhesus macaque will serve as a human-like carriage model.

Animal models of Lyme disease: pathogenesis and immunoprophylaxis.

Valuable insights into the pathogenesis and immunoprophylaxis of Lyme disease are beginning to emerge from studies in animal models. This review highlights two animal models: the mouse, which has allowed us to investigate the role of both the immune response and spirochete phenotype in determining the outcome of the disease; and the Rhesus monkey, which manifests signs of nerve involvement, in addition to showing erythema migrans and arthritis.

Borrelia burgdorferi possesses a collagenolytic activity.

Lyme disease is a multisystemic disorder caused by Borrelia burgdorferi, an invasive spirochete. B. burgdorferi has a predilection for collagenous tissue and one major clinical manifestation of the disease is arthritis. We have identified a collagenolytic activity in B. burgdorferi detergent lysates using iodinated gelatin as well as iodinated pepsinized human collagen types II and IV as protein substrates. In addition, we describe several proteolytic activities in B. burgdorferi with molecular masses greater than 200 kDa on sodium dodecyl sulfate polyacrylamide gels containing copolymerized gelatin. We propose that the collagenolytic activity of B. burgdorferi has a role in invasion, in the pathogenesis of Lyme arthritis, and perhaps also in other manifestations of Lyme borreliosis.

DNA-Binding proteins possibly involved in regulation of the post-logarithmic-phase expression of lipoprotein P35 in Borrelia burgdorferi.

Previously, we have shown that the transcription of p35, a lipoprotein gene of Borrelia burgdorferi, is upregulated or initiated during the post-logarithmic bacterial growth phase in vitro. To identify potential regulatory factors, we examined the formation of DNA-protein complexes by electromobility shift assay, using a 157-bp DNA fragment that spans the p35 promoter region and cell-free extracts of spirochetes harvested from both logarithmic and stationary growth phases. The binding properties of the extracts with the promoter region of the flaB gene, a constitutively expressed, growth-phase-independent gene, were also compared. The results from these experiments demonstrate that B. burgdorferi stationary-phase cell-free extracts have a growth-phase-dependent DNA binding protein that interacts specifically with the p35 promoter region. We show, in addition, that a segment from the 157-bp p35 promoter region which contains both a T-rich stretch and an inverted repeat is able to compete off the stationary-phase-specific complex when the segment is present in molar excess.

Epitope mapping of the immunodominant invariable region of Borrelia burgdorferi VlsE in three host species.

VlsE, the variable surface antigen of Borrelia burgdorferi, contains a 26-amino-acid-long immunodominant invariable region, IR(6). In the present study, three overlapping 14-mer peptides reproducing the sequence of IR(6) were used as peptide-based enzyme-linked immunosorbent assay antigens to map this invariable region in infected monkeys, mice, and human Lyme disease patients. Antibodies of the two primate species appeared to recognize IR(6) as a single antigenic determinant, while mouse antibodies recognized multiple epitopes within this region.

Killing of Borrelia burgdorferi by antibody elicited by OspA vaccine is inefficient in the absence of complement.

A Lyme disease vaccine, based on the Borrelia burgdorferi lipoprotein OspA, has recently undergone phase III trials in humans. The results of one of these trials indicate that vaccine efficacy positively correlates with anti-OspA antibody titer. Spirochete killing within the tick vector midgut, upon which vaccine efficacy appears to depend, may occur chiefly via a mechanism that involves antibody alone, as it has been reported that complement is degraded by tick saliva decomplementing factors. We compared the in vitro killing efficiencies of anti-OspA antibody elicited in rhesus monkeys by the OspA vaccine, in the presence and in the absence of monkey complement. Killing in the absence of complement was between 14 and 3,800 times less efficient than with complement present, depending on the spirochete strain. The relative inefficiency of the complement-independent killing mechanism by anti-OspA antibody may explain why OspA vaccine efficacy is critically dependent on antibody titer.

Differential expression of Borrelia burgdorferi proteins during growth in vitro.

In an earlier paper we described the transcriptionally regulated differential levels of expression of two lipoproteins of Borrelia burgdorferi, P35 and P7.5, during growth of the spirochetes in culture from logarithmic phase to stationary phase (K. J. Indest, R. Ramamoorthy, M. Solé, R. D. Gilmore, B. J. B. Johnson, and M. T. Philipp, Infect. Immun. 65:1165-1171, 1997). Here we further assess this phenomenon by investigating whether the expression of other antigens of B. burgdorferi, including some well-characterized ones, are also regulated in a growth-phase-dependent manner in vitro. These studies revealed 13 additional antigens, including OspC, BmpD, and GroEL, that were upregulated 2- to 66-fold and a 28-kDa protein that was downregulated 2- to 10-fold, during the interval between the logarithmic- and stationary-growth phases. Unlike with these in vitro-regulated proteins, the levels of expression of OspA, OspB, P72, flagellin, and BmpA remained unchanged throughout growth of the spirochetes in culture. Furthermore, ospAB, bmpAB, groEL, and fla all exhibited similar mRNA profiles, which is consistent with the constitutive expression of these genes. By contrast, the mRNA and protein profiles of ospC and bmpD indicated regulated expression of these genes. While bmpD exhibited a spike in mRNA expression in early stationary phase, ospC maintained a relatively higher level of mRNA throughout culture. These findings demonstrate that there are additional genes besides P7.5 and P35 whose regulated expression can be investigated in vitro and which may thus serve as models to facilitate the study of regulatory mechanisms in an organism that cycles between an arthropod and a vertebrate host.

Analysis of antibody response to invariable regions of VlsE, the variable surface antigen of Borrelia burgdorferi.

VlsE, the variable surface antigen of Borrelia burgdorferi, consists of two invariable domains at the amino and carboxyl termini and one central variable domain. The latter contains six invariable regions, IR(1) to IR(6), and six variable regions. In the present study, the antigenicity of all of the invariable regions in B. burgdorferi-infected monkeys, humans, and mice was assessed by peptide-based enzyme-linked immunosorbent assays. Only one invariable region, IR(6), was antigenic in all animals of the three host species. IR(2) and IR(4) were also antigenic in mice.

C-terminal invariable domain of VlsE may not serve as target for protective immune response against Borrelia burgdorferi.

VlsE, the variable surface antigen of the Lyme disease spirochete, Borrelia burgdorferi, contains two invariable domains, at the amino and carboxyl termini, respectively, which collectively account for approximately one-half of the entire molecule's length and remain unchanged during antigenic variation. It is not known if these two invariable domains are exposed at the surface of either the antigen or the spirochete. If they are exposed at the spirochete's surface, they may elicit a protective immune response against B. burgdorferi and serve as vaccine candidates. In this study, a 51-mer synthetic peptide that reproduced the entire sequence of the C-terminal invariable domain of VlsE was conjugated to the carrier keyhole limpet hemocyanin and used to immunize mice. Generated mouse antibody was able to immunoprecipitate native VlsE extracted from cultured B. burgdorferi B31 spirochetes, indicating that the C-terminal invariable domain was exposed at the antigen's surface. However, this domain was inaccessible to antibody binding at the surface of cultured intact spirochetes, as demonstrated by both an immunofluorescence experiment and an in vitro killing assay. Mouse antibody to the C-terminal invariable domain was not able to confer protection against B. burgdorferi infection, indicating that this domain was unlikely exposed at the spirochete's surface in vivo. We concluded that the C-terminal invariable domain was exposed at the antigen's surface but not at the surface of either cultured or in vivo spirochetes and thus cannot elicit protection against B. burgdorferi infection.

Transcriptional regulation in spirochetes.

Spirochetes belong to a widely diverse family of bacteria. Several species in this family can cause a variety of illnesses including syphilis and Lyme disease. Despite the fact that the complete genome sequence of two species, Borrelia burgdorferi and Treponema pallidum, have been deciphered, much remains to be understood about spirochetal gene regulation. In this review we focus on the environmental transitions that spirochetes undergo during their life cycles and the mechanisms of transcriptional regulation that might possibly mediate spirochetal adaptations to such changes.

Cryptic and exposed invariable regions of VlsE, the variable surface antigen of Borrelia burgdorferi sl.

Borrelia burgdorferi, the Lyme disease spirochete, possesses a surface protein, VlsE, which undergoes antigenic variation. VlsE contains two invariable domains and a variable one that includes six variable and six invariable regions (IRs). Five of the IRs are conserved among strains and genospecies of B. burgdorferi sensu lato. IR(6) is conserved, immunodominant, and exposed at the VlsE surface but not at the spirochete surface, as assessed in vitro. In the present study, the remaining conserved IRs (IR(2) to IR(5)) were investigated. Antisera to synthetic peptides based on each of the IR(2) to IR(5) sequences were produced in rabbits. Antipeptide antibody titers were similarly high in all antisera. Native VlsE was immunoprecipitable with antibodies to IR(2), IR(4), and IR(5) but not to IR(3), indicating that the first three sequences were exposed at the VlsE surface. However, negative surface immunofluorescence and in vitro antibody-mediated killing results indicated that none of the IRs were accessible to antibody at the spirochetal surface in vitro.

Borrelia burgdorferi antigens that are targeted by antibody-dependent, complement-mediated killing in the rhesus monkey.

We identified surface antigens of Borrelia burgdorferi that are targeted by antibody-dependent, complement-mediated killing (ADCK) in the rhesus monkey. For this purpose, we had available serum samples from three animals infected with B. burgdorferi JD1 by needle inoculation and from two monkeys that were infected with the same B. burgdorferi strain by Ixodes scapularis tick bite. Sera from monkeys from the first group contained antibodies to OspA and OspB detectable by Western blot (immunoblot) using whole B. burgdorferi antigens, whereas serum samples from animals in the second group did not. The targeting of OspA and OspB by functional antibodies was demonstrated directly by showing that ADCK was partially inhibited when antibodies were preincubated with an excess of soluble recombinant OspA or OspB. Simultaneous addition of OspA and OspB did not result in an additive inhibitory effect on ADCK, a result that suggests that the epitopes on OspA and that on OspB targeted by antibody in this mechanism are the same, or at least cross-reacting. The targeting of non-OspA, non-OspB surface antigens was inferred from the fact that sera from tick-inoculated animals, which did not contain detectable anti-OspA or anti-OspB antibodies, were able to effect ADCK. This killing effect was not inhibitable by the addition of recombinant OspA or OspB or both proteins together. We also showed that both immunoglobulin G and M antibodies participate in the ADCK mechanism in the rhesus monkey. Rhesus complement does not kill B. burgdorferi in vitro in the absence of antibody, and antibody alone is effective in killing only at serum dilutions lower than 1:15. However, such "complement-independent" antibodies were not present in all bleeds. Two main conclusions may be drawn from the analysis of our results. First, both OspA and OspB are targeted by the ADCK mechanism in the rhesus monkey. Second, one or more B. burgdorferi surface antigens that are neither OspA nor OspB also participate in ADCK.

Dirofilaria immitis: ultrastructural localization, molecular characterization, and analysis of the expression of p27, a small heat shock protein homolog of nematodes.

A cDNA fragment encoding the complete coding region of a 27-kDa protein (p27) of Dirofilaria immitis was cloned. Antibody to the recombinant p27 bound to hypodermal tissues of third (L3) and fourth stage larvae (L4) of D. immitis and to both the hypodermis and the cuticle of L3s of Onchocerca volvulus, as visualized by immunoelectronmicroscopy. The deduced amino acid sequence of the central and C-terminal regions of p27 (amino acids S83 to H222) is 18-36% identical to members of the sHsp/alpha-crystallin family of proteins. The homologous region is thought to be responsible for the molecular chaperone activity of members of this family. The p27 cDNA does not encode a hydrophobic signal peptide. At least two homologous yet distinct p27 genes were identified in the D. immitis genome by Southern hybridization using the p27 cDNA as a probe. The p27 transcript was 0.9 kb in length on Northern blots. The expression of p27 in L3s of D. immitis was neither upregulated by heat shock (43 degrees C) nor by incubation at the physiologic temperature of 37 degrees C. Pulse-labeling experiments of both D. immitis and Brugia malayi L3s during the L3-L4 molt in vitro showed that synthesis of p27 is also not upregulated during this developmental phase. However, p27 is expressed constitutively throughout the D. immitis L3-L4 molt and therefore by both larval stages. In addition, both female and male adult worms of this species express p27 constitutively. P27, or an allomorph thereof, was detected in each of nine species representing four nematode superfamilies, thus indicating that this molecule is ubiquitous within the phylum Nematoda. In view of the hypodermal localization of p27, its constitutive expression, and its retention among nematodes, the function of this protein in essential housekeeping roles such as that of molecular chaperone during the molting process is discussed.

Borrelia burgdorferi stimulates the production of interleukin-10 in peripheral blood mononuclear cells from uninfected humans and rhesus monkeys.

Heat-killed Borrelia burgdorferi spirochetes stimulate in vitro production of interleukin-10 (IL-10) at both mRNA and protein levels in peripheral blood mononuclear cells (PBMC) of uninfected rhesus monkeys. A concomitant down-modulation of IL-2 gene transcription was observed. Neither IL-4 nor gamma interferon gene expression was ostensibly affected by B. burgdorferi spirochetes. These phenomena were observed regardless of whether the stimulating spirochetes belonged to the B. burgdorferi sensu stricto, Borrelia afzelii, or Borrelia garinii genospecies, the three main species that cause Lyme disease. B. burgdorferi also induced production of IL-10 in uninfected human PBMC, indicating that this effect might play a role in human Lyme disease. Purified lipidated outer surface protein A (OspA), but not its unlipidated form, induced the production of high levels of IL-10 in uninfected human PBMC. Thus, the lipid moiety is essential in the induction of IL-10 in these PBMC. B. burgdorferi M297, a mutant strain that lacks the plasmid that encodes OspA and OspB, also induced IL-10 gene transcription in PBMC, indicating that this phenomenon is not causally linked exclusively to OspA and its lipid moiety. These results demonstrate that B. burgdorferi can stimulate the production of an antiinflammatory, immunosuppressive cytokine in naive cells and suggest that IL-10 may play a role both in avoidance by the spirochete of deleterious immune responses and in limiting the inflammation that the spirochete is able to induce.

C-terminal invariable domain of VlsE is immunodominant but its antigenicity is scarcely conserved among strains of Lyme disease spirochetes.

VlsE, the variable surface antigen of Borrelia burgdorferi, contains two invariable domains located at the amino and carboxyl terminal ends, respectively, and a central variable domain. In this study, both immunogenicity and antigenic conservation of the C-terminal invariable domain were assessed. Mouse antiserum to a 51-mer synthetic peptide (Ct) which reproduced the entire sequence of the C-terminal invariable domain of VlsE from B. burgdorferi strain B31 was reacted on immunoblots with whole-cell lysates extracted from spirochetes of 12 strains from the B. burgdorferi sensu lato species complex. The antiserum recognized only VlsE from strain B31, indicating that epitopes of this domain differed among these strains. When Ct was used as enzyme-linked immunosorbent assay (ELISA) antigen, all of the seven monkeys and six mice that were infected with B31 spirochetes produced a strong antibody response to this peptide, indicating that the C-terminal invariable domain is immunodominant. None of 12 monkeys and only 11 of 26 mice that were infected with strains other than B31 produced a detectable anti-Ct response, indicating a limited antigenic conservation of this domain among these strains. Twenty-six of 33 dogs that were experimentally infected by tick inoculation were positive by the Ct ELISA, while only 5 of 18 serum samples from dogs clinically diagnosed with Lyme disease contained detectable anti-Ct antibody. Fifty-seven of 64 serum specimens that were collected from American patients with Lyme disease were positive by the Ct ELISA, while only 12 of 21 European samples contained detectable anti-Ct antibody. In contrast, antibody to the more conserved invariable region IR(6) of VlsE was present in all of these dog and human serum samples.

Early induction of gamma interferon and interleukin-10 production in draining lymph nodes from mice infected with Borrelia burgdorferi.

Lymph node (LN) cells from C3H/HeJ mice (Lyme disease susceptible) infected for 1 week with Borrelia burgdorferi strain JD1 produced higher levels of gamma interferon (IFN-gamma) when stimulated in vitro with B. burgdorferi spirochetes than equivalent cells from B. burgdorferi-infected C57BL/6J mice (disease resistant). The interleukin-10 (IL-10) levels were comparable in the two strains, whereas the IL-4 levels were below detection limits. B. burgdorferi-stimulated LN cells from C57BL/6J mice produced significantly higher levels of IFN-gamma in the presence of neutralizing anti-IL-10 antibody than cells cultured with B. burgdorferi alone. No effect of IL-10 neutralization on IFN-gamma production by LN cells from C3H/HeJ mice was observed. Neutralizing antibody to IFN-gamma had no effect on the production of IL-10 by LN cells from C57BL/6J mice. A slight decrease in IL-10 production was detected in culture supernatants of equivalent cells from C3H/HeJ mice. The differential effect of IL-10 on IFN-gamma production in C57BL/6J and C3H/HeJ mice suggests that IL-10 is probably involved in the regulation of IFN-gamma production by LN cells during infection and may be at the root of the differential susceptibility to Lyme arthritis in these two strains of mice.

Borrelia burgdorferi stimulates in vitro a selective expansion of CD3-CD4-CD8- (triple negative) autoreactive cells in naive rhesus monkey lymphocytes.

Autoreactive cell lines were generated from cell suspensions of freshly isolated naive monkey lymph node (LN) cells and peripheral blood mononuclear cells by cocultivation with freeze-thawed Borrelia burgdorferi spirochetes (Bb/FT). These cells produced interleukin (IL)-6 when cocultured with autologous antigen-presenting cells (APCs) alone without Bb/FT. IL-6 production was not observed when control cell lines were stimulated in the same fashion. CD4+-enriched T cell populations obtained from the LN autoreactive cell line also produced IL-6 when cultivated with APCs alone. When these cells were cultivated further in the presence of APCs, a population of cells whose phenotype was CD56+/-CD4-CD8-CD3- was predominantly selected. These cells both proliferated and produced IL-6 when cocultured with APCs alone. The possible relevance of these cells to Lyme disease pathogenesis remains to be determined.

CD19(+) cells produce IFN-gamma in mice infected with Borrelia burgdorferi.

We have recently shown that production of IFN-gamma and IL-10, but not IL-4 is specifically induced in the lymph nodes of C3H/HeJ (disease susceptible) and C57BL/6J (disease resistant) mice 1 week after infection with Borrelia burgdorferi spirochetes. The present study was conducted to determine the phenotypes of ex vivo lymph node cells obtained from infected mice of both strains at this time point. The percentages of CD3(+), CD4(+), CD8(+), TCRalpha/beta (+) and TCRgamma/delta (+) cells decreased in both strains of mice compared to LN from naive mice. In contrast, there was a threefold increase in the proportion of CD19(+) cells. In view of this expansion of the B cell proportion, we examined the ability of purified CD19(+) cells and CD43(+) cells to produce both IL-10 and IFN-gamma when the cells were restimulated in vitro with B. burgdorferi freeze-thawed spirochetes. As expected, CD43(+) cells were able to produce both cytokines, but not IL-4. Surprisingly, CD19(+) (B) cells also were able to produce IFN-gamma in comparable amounts, in addition to IL-10. Intracellular staining of CD19(+) cells with anti-IFN-gamma antibody confirmed this finding. We discuss this novel phenomenon in terms of its possible underlying mechanisms and its relevance, both in the context of the immunology of Lyme disease and that of other infectious diseases.