Mario T. Philipp - US grants

The broad, long-term goal that is envisaged is the development of a "second generation" of surface-antigen-based vaccines for the prevention of Lyme disease in humans. Towards this aim, the objective of this proposal is the identification and cloning of surface proteins of Borrelia burgdroferi that are antigenically conserved and whose conserved epitopes are targeted by antibody-dependent complement-mediated lysis (ADCL) in primate hosts. An initial assessment of whether the cloned antigens are subjected to antigenic variation will be made as well. In making this the proposal's objective, it is recognized that, although very significant progress has been made towards providing the develop a second generation of vaccines based on more conserved and non-variant surface antigens. This necessity arises because OspA is antigenically polymorphic, such that recombinant OspA derived from one strain of B. burgodorferi may not protect against a challenge infection with spirochetes of a different strain and, moreover, OspA is the subject of an as yet unidentified mechanism of antigenic variation. The strategy to be employed unfolds from the following results: using serum from rhesus monkeys infected with B. burgdorferi strain JD-1, an antibody reagent that specifically recognizes 4 surface antigens of this spirochete was generated. These antigens are neither OspA nor OspB. They were named P1-4, and it was demonstrated that they are the target of (ADCL) of B. burgdorferi, i.e. the expressed by strain B-31 and, moreover, that at least one isomorph of these molecules appears to be present also on the taxonomically distant strain IP-90, considered by some authors to belong to a different species altogether (Borreli garinii). It is planned to extend this survey of strains killed by anti- P1-4 antibodies to a larger number of isolates, encompassing 10 isolates from 3 different genospecies, and thus generate an order of priority for the cloning of P1-4, such that only those most conserved antigens will be cloned. Cloning of the selected subpopulation of P1-4, will be done with the aid of the anti-P1-4 antibody reagent mentioned above, and whole-DNA libraries made in the bacteriophage expression vector gammagt11. After the antigens are subcloned, overexpressed and purified, likely in the form of fusion proteins with the Escherichia coli maltose binding protein, rhesus monkeys will be immunized with the individual recombinant antigens and monospecific antisera will be generated. These sera will be used initially for two purposes: 1) to confirm that all strains expressing the cloned (conserved) surface antigens can be killed by ADCL in vitro, and 2) to assess whether the chosen antigens undergo antigenic variation, by quantifying the frequency of appearance of escape variants in vitro, under selective pressure induced by the monospecific antibodies. At this point an informed decision will be made on whether it is merited to solicit the large amount of money required to assess two or more surface antigens, individually or in combinations, as a comprehensive vaccine against Lyme disease in nonhuman primates. This is a desired next step to which the research described in this proposal should lead us.

We report on an antigen (Mr 39,500) that is expressed in vitro by spirochetes of Borrelia garinii strain IP90 and apparently only in vivo by spirochetes of the B. burgdorferi sensu stricto strain JD1. Antibody against this 39.5 kDa antigen kills IP90 spirochetes in vitro by antibody-dependent, complement-mediated killing (ADCK) regardless of whether the antibody was obtained by 1) affinity purification using as immunoabsorbant native P39.5 antigen of IP90 spirochetes (as separated on a Western blot) and as source of antibody the antiserum generated during an infection of rhesus monkeys with JD1 spirochetes or, 2) by immunizing mice with recombinant P39.5 from IP90 (rP39.5). The mouse anti-rP39.5 antibody does not kill JD1 spirochetes in vitro by ADCK. A segment of 1190 bp of the coding region of the p39.5 gene of IP90 was sequenced. It encompasses a single open reading frame that encodes a putative 37.7 kDa protein. Both ends of the gene have not been sequenced as yet. The sequenced fragment is composed of mostly hydrophilic domains that contain several internally repeated regions; the corresponding deduced amino acid sequence is up to 22% identical to members of the Vmp family of outer surface lipoproteins of Borrelia hermsii. The native P39.5 was fully extracted into the detergent phase in a Triton-X114 extraction experiment. Therefore, P39.5 is probably a lipoprotein. The internal repeats of P39 indicate that this protein, like OspA, may undergo homologous recombinations in Borrelia which may lead to mutants expressing shortened, partially-deleted versions of the gene. While this could be a cause of concern for a vaccine antigen, we reported last year that OspA deletion mutants that are resistant to antibody alone (escape mutants) can be killed with antibody plus complement. Hence, deletion mutants of vaccine antigens that, like P39.5 and unlike OspA, are expressed in vivo, will be killed upon infection of the vertebrate host.

Objectives Assess infection of the central nervous system (CNS) by B. burgdorferi (Bb) in the rhesus monkey. Results Five age-matched male rhesus macaques were needle-inoculated with a neurotropic strain of B. burgdorferi (NT1). One animal was left uninfected, as a control. All animals, except the control, were verified to be infected by recovering Bb, and amplifying Bb DNA, from skin biopsy samples. Infection was monitored longitudinally by determining the titer of anti-P39 antibodies and number of bands detected on Western blots. CNS infection was monitored by determining, cerebral spinal fluid (CSF) pleocytosis, presence of Bb DNA in CSF, (by nested PCR) and production of intrathecal anti-Bb antibodies. CNS disease was monitored by measuring somatosensory evoked potentials (SEP) recorded from both upper and lower extremities immediately following infection, and longitudinally over a period of nine months. The level of anti-P39 antibodies and the number of antigen bands on IgG West ern blots increased until week 10 post-infection (PI), and then remained essentially unchanged until the end of the study (90 weeks PI). This suggest that the animals remained infected throughout the study period. CSF pleocytosis, and presence of B. burgdorferi DNA in the CSF were observed in all animals at different periods within the first 20 weeks PI. Intrathecal anti-Bb antibody ptoduction was also oberved in some animals within the 90 week study period. Two animals showed significant changes which are consistent with abnormalities in the central somatosensory system, one animal by weeks 11 and 35 PI, and the other at week 35 PI. Peripheral nerve function was found to be normal in the nerves that were selected to determine SEP, thus indicating that these changes were based in the central somatosensory pathway. Our study contributes solid and conclusive evidence that infection of the rhesus monkey with B. burgdorferi causes not only infection and disease of the peripheral nervous sy stem, as we have already demonstrated, but also of the CNS. Future directions A thorough survey of brain pathology in these animals is underway. FUNDING CDC Cooperative Agreement (U50/CCU606604-07) and Base grant. PUBLICATIONS Roberts, E. D., Bohm, Jr., R. P., Lowrie, Jr., R. C., Habicht, G., Piesman, J. and Philipp, M. T. 1998. Pathogenesis of Lyme Neuroborreliosis in the Rhesus Monkey the Acute and Chronic Phases of Disease in the Peripheral Nervous System. J. Infect. Dis. 178, 722-732.

Previously, we had identified non-OspA-OspB surface proteins of Borrelia burgdorferi that are targeted by the antibody-dependent complement-mediated killing mechanism. Here we demonstrate by Western blot that one of these proteins, P35, is upregulated at the onset of stationary phase in vitro. Northern analysis revealed that the upregulation of P35 is at the level of transcription. In addition, the expression of an open reading frame (ORF) located downstream of the p35 gene was found to be regulated in the same fashion as P35. This ORF encodes a 7.5 kDa lipoprotein. The transcriptional start sites for both of these genes were determined, to aid in the identification of their putative promoter regions. Additional sequencing of the 5' flanking region of the p35 gene revealed a region of dyad symmetry 52 bp upstream of the transcription start site. Southern analysis demonstrated that the expression of these genes was not due to a cell-density-dependent rearrangement in the genome of B. burgdorferi. These findings provide an in vitro model for studying mechanisms of gene regulation in B. burgdorferi.

Cell-density-dependent expression of p35, a lipoprotein of B. burgdorferi has been described by us. Here we further assess this phenomenon by investigating whether other antigens of B. burgdorferi, including some well-characterized ones, are similarly regulated. Low passage spirochetes (strain JD1) were inoculated into fresh BSK-H medium at a concentration of 105 cells/ml and allowed to multiply and reach stationary phase (approximately 108 cells/ml) over an 8-day period. Cells were harvested at various times along the growth curve and processed for Western and Northern blot analyses. To verify the reversibility of the phenomenon of cell-density-dependent expression, cells in stationary phase were again diluted into fresh medium at 105 cells/ml and allowed to undergo another two cycles of growth; during the second cycle cells were sampled in the mid-log phase (log-revertant). The expression of BmpD (P37) and P66, as assessed by Western blot analysis, increased significantly at the onset of stationary phase, as compared to their expression in mid-log phase. Northern blotting revealed a corresponding spike in the steady state level of bmpD transcript at the onset of the stationary phase. Northern blotting is yet to be performed for the p66 mRNA. In contrast, the expression of BmpA (P39), OspA, OspB, OspC, p72, p93 and flagellin all remained unchanged throughout growth of the spirochetes in culture. Western blots developed with serum from monkeys with an acute B. burgdorferi infection revealed two additional antigens, 16 kDa and 26 kDa, whose expression steadily increased during growth in culture. These findings provide additional candidate genes whose expression can be investigated in vitro in an otherwise intractable organism that cycles between arthropod and mammalian hosts.

DESCRIPTION (Adapted from the applicant's abstract): This project, presented by an outstanding group of investigators that includes Drs. M. T. Philipp as P.I. and M. S. Klempner, is designed to complement and expand a human study already in course directed to probe the efficacy of an antibiotic regimen designed to treat chronic Lyme disease. The investigators propose to study in rhesus monkeys the efficacy of ceftriaxone and doxycycline to eliminate B. burgdorferi for tissues, especially from the central nervous system. The protocol is two-tiered. In the first tier, 6 monkeys will be tick infected with neurotropic B. burgdorferi NT1 to confirm the neurotropism of the strain. One monkey will be left uninfected, as a control. In the second tier, 24 animals will be tick infected with strain NT1, with four remaining uninfected. The animals will be divided into two groups (12 + 2) and one will be treated with antibiotics and the other given placebo. The ability of the antibiotic to clear up tissues from infection with B. burgdorferi NT1 will be ascertained by a variety of serially applied procedures that will include: culture and PCR of skin biopsies; PCR and RT-PCR applied to CSF; immunological detection of bacterial antigens in urine, and qualitated and quantitated analysis of anti-borrelial antibodies paired sera and CSF, anti p39 (BmpA) antibody in serum and levels of matrix metalloproteinases in samples from nervous tissues. The analysis will also include postmortem histological examination, culture and PCR studies in tissues from many organs.

The broad, long-term goal that is envisaged is the development of a "second generation" of surface-antigen-based vaccines for the prevention of Lyme disease in humans. Towards this aim, the objective of this proposal is the identification and cloning of surface proteins of Borrelia burgdroferi that are antigenically conserved and whose conserved epitopes are targeted by antibody-dependent complement-mediated lysis (ADCL) in primate hosts. An initial assessment of whether the cloned antigens are subjected to antigenic variation will be made as well. In making this the proposal's objective, it is recognized that, although very significant progress has been made towards providing the develop a second generation of vaccines based on more conserved and non-variant surface antigens. This necessity arises because OspA is antigenically polymorphic, such that recombinant OspA derived from one strain of B. burgodorferi may not protect against a challenge infection with spirochetes of a different strain and, moreover, OspA is the subject of an as yet unidentified mechanism of antigenic variation. The strategy to be employed unfolds from the following results: using serum from rhesus monkeys infected with B. burgdorferi strain JD-1, an antibody reagent that specifically recognizes 4 surface antigens of this spirochete was generated. These antigens are neither OspA nor OspB. They were named P1-4, and it was demonstrated that they are the target of (ADCL) of B. burgdorferi, i.e. the expressed by strain B-31 and, moreover, that at least one isomorph of these molecules appears to be present also on the taxonomically distant strain IP-90, considered by some authors to belong to a different species altogether (Borreli garinii). It is planned to extend this survey of strains killed by anti- P1-4 antibodies to a larger number of isolates, encompassing 10 isolates from 3 different genospecies, and thus generate an order of priority for the cloning of P1-4, such that only those most conserved antigens will be cloned. Cloning of the selected subpopulation of P1-4, will be done with the aid of the anti-P1-4 antibody reagent mentioned above, and whole-DNA libraries made in the bacteriophage expression vector gammagt11. After the antigens are subcloned, overexpressed and purified, likely in the form of fusion proteins with the Escherichia coli maltose binding protein, rhesus monkeys will be immunized with the individual recombinant antigens and monospecific antisera will be generated. These sera will be used initially for two purposes: 1) to confirm that all strains expressing the cloned (conserved) surface antigens can be killed by ADCL in vitro, and 2) to assess whether the chosen antigens undergo antigenic variation, by quantifying the frequency of appearance of escape variants in vitro, under selective pressure induced by the monospecific antibodies. At this point an informed decision will be made on whether it is merited to solicit the large amount of money required to assess two or more surface antigens, individually or in combinations, as a comprehensive vaccine against Lyme disease in nonhuman primates. This is a desired next step to which the research described in this proposal should lead us.

Background and Objectives We have shown that human monocytes but not T or B cells, when stimulated with heat-killed B. burgdorferi, induced at the mRNA level, IL-1 , IL-6 and IL-10 cytokine gene transcripts. We also showed that the monocytic cell line THP-1 responded to heat-killed B. burgdorferi and lipidated OspA (L--OspA) by producing IL-6 and IL-10 in a dose dependent fashion. The objectives of this research were to assess whether CD14 is involved as a receptor for B. burgdorferi lipoproteins. THP-1 cells were incubated with increasing concentrations of monoclonal antibodies (MAbs) that bind to CD14. This was done in order to assess if MAb binding would inhibit the production of IL-6 and IL-10 by the THP-1 cells after exposing them to lipoproteins. LPS was used as a positive control. The MAbs were MY4 and 60bca, which bind to the portion of CD14 which interacts with LPS, and 26ic, which also binds to CD14 but not at the receptor's active site for LPS binding. The stimulants employed were LPS, L-OspA and also Pam3Cys-Ser-Lys4-OH, a derivative that contains only the lipid moiety of such lipoproteins attached to a hexapeptide (Pam3Cys). Results Incubation with increasing concentrations of MAbs 60bca and MY4 resulted in an increased inhibition of the production of IL-10 or IL-6 by the THP-1 cells, when the cells were incubated either with the L-OspA or Pam3Cys, whereas incubation with increasing concentrations of the 26ic antibody resulted in no inhibition of IL-10 or IL-6 production. Production of IL-6 or IL-10 by LPS was equally inhibited by the MY4 and 60bca MAbs but not by the 26ic MAb. These results show that the THP-1 cell line could be used as a model for monocytes that are contained in PBMC of humans and nonhuman primates and, more importantly, that CD14, the receptor for LPS, is probably also the receptor for spirochetal lipoproteins. Future directions To definitively identify and purify the receptor for spirochetal

Objectives To develop reagents for the improved diagnostic serology of Lyme disease. Results A segment (P7-1) of the variable domain of VlsE, the variant surface antigen of the spirochete Borrelia burgdorferi sensu lato was cloned and sequenced. and a conserved, immunodominant region of 26 amino acids (IR6) was identified. A 26-mer peptide (C6) was synthesized on the basis of the IR6 sequence and a peptide ELISA was developed for serodiagnosis of Lyme disease. Ten rhesus monkeys were infected with Borrelia burgdorferi strains JD1 or B31 by either tick or needle inoculation. Blood samples were periodically collected for up to 3 years post-infection (PI) until the animals were sacrificed, and serum samples were assessed for antibody responses to C6. Anti-C6 antibody was detectable in 6 monkeys as early as week 2 and in the remaining animals between weeks 3 and 5 PI. Antibody persisted at high levels for as long as serum samples were monitored. Forty-one serum samples from patients the majority of which had culture-confirmed, acute Lyme borreliosis, were obtained from the Centers for Disease Control and Prevention (CDC) and assessed with the C6 ELISA. Sensitivity was 85% (35/41), whereas with a commercially available Lyme disease ELISA it was 78% (32/41), and 49% (20/41) with a commercially available IgG Lyme immunoblot, 51% (21/41) with an IgM immunoblot, and 78% (32/41) by the combination of both IgG and IgM immunoblots. Of a group of 99 randomly collected serum samples from hospital patients in Louisiana, where Lyme disease is not endemic, only 2 were positive with the C6 ELISA. In addition, serum samples from 9/9 relapsing fever patients and 12/12 syphilitic patients also had no detectable anti-C6 antibodies. On the basis of the small number of serum samples tested thus far, the C6 peptide-based ELISA permits sensitive, specific and simple serodiagnosis of Lyme disease.. Future directions To extend our survey of crossreactivity; samples from patients with autoim mune diseases will be tested. FUNDING PUBLICATIONS None

Objectives and Background The objective of this research is to increase our understanding of the mechanisms that regulate the expression of B. burgdorferi lipoproteins, a type of molecule that plays a key role in Lyme disease pathogenesis. Last year we reported on the identification of two lipoproteins of Borrelia burgdorferi, P35 and P7.5, whose expression in vitro was transcriptionally regulated in a cell-density- or growth-phase-dependent manner. At low cell density (mid log phase) these proteins were not expressed whereas at higher cell density (late log to stationary phase) they were expressed. Sequencing of the 5' flanking region of the p35 gene revealed a region of dyad symmetry 52 bp upstream of the transcription start site. Results Using an electrophoretic mobility shift assay we have identified a DNA binding protein that binds to a DNA fragment that contains the region of dyad symmetry mentioned above. We have constructed a total DNA library of B. burgdorferi strain B31 in the lambda Zap II bacteriophage vector and are currently screening it in order to clone the DNA binding protein. Future directions To explore the role of the cloned DNA binding protein in the regulation of lipoprotein expression.

Objectives Borrelia burgdorferi, the spirochete that causes Lyme disease, expresses a surface lipoprotein, VlsE, which undergoes antigenic variation (Zhang et al., Cell. 89:275-285, Infect. Immun. 66:3698-3704). Variation takes place via a unidirectional recombination mechanism whereby vlsE exchanges portions of a central ?cassette? segment of the molecule with 15 similar cassettes located upstream from the vlsE locus (Ibid.). The N- and C- terminal domains of VlsE, each roughly 1/3 of the molecule?s length, as well as several regions within the cassette, remain invariant (Ibid.). Nothing is known about the functional or antigenic properties of these invariant regions. We set out to determine these properties, as such invariant regions could be potential diagnostic reagents or vaccinogens. Results We cloned and sequenced a cassette segment of the vls locus of the IP90 strain of Borrelia garinii, expressed it in Escherichia coli and purified the corresponding polypeptide (P7-1). We an alyzed the antigenicity of the invariant regions (IRs) of P7-1 using standard algorithms, and determined the percent identity of their amino acid sequences with the corresponding IRs of published VlsE cassettes of B. burgdorferi ss strains B31 and 297. One IR, namely IR6, showed the highest level of identity among Borrelia species and strains, and exhibited a stretch of 7 amino acids which was predicted to be antigenic by the Hopp-Woods algorithm. We synthesized a peptide, C6, which encompassed the aa sequence of IR6, and assessed its antigenicity in B. burgdorferi-infected mice, monkeys and humans. We also performed antigenic competition experiments, to determine what proportion of B. burgdorferi-infected mouse, monkey and human antibody responses to P7-1 was directed to C6. External exposure of IR6 on the VlsE molecule was determined by immunoprecipitation of detergent-solubilized, native VlsE of IP90 with rabbit antisera raised against C6, and exposure of IR6 on the spirochetal surf ace was assessed by immunofluorescence and antibody-dependent, complement-mediated killing assays with the anti-C6 antibody. The results indicate that IR6 is exposed on the VlsE ?surface? but not on the surface of the spirochete; it is immunodominant and may thus contribute to focus the antibody response onto a conserved section of VlsE. Future directions Extend this antigenic analysis to the additional 5 invariant regions of VlsE. FUNDING Grant from SmithKline Beecham Biologicals and Base grant. PUBLICATIONS None

Objectives To understand the role of OspA in the immunobiology of Borrelia burgdorferi. Results 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, could 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 efficiency 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 spirochete strain. The relative inefficiency of the complement independent killing mechanism by anti-Osp A antibody may explain why the OspA vaccine efficacy is critically dependent on antibody titer. Future directions to further explore the role of OspA in tick transmission by investigating efficiency of transmission of B. burgdorferi mutants that express truncated forms of OspA. FUNDING Grant U50/CCU606604 from the Centers for Disease Control and Prevention, AI35027 from the NIAID, and the Base grant. PUBLICATIONS Nowling, J., and Philipp, M. T. 1999. Killing of Borrelia burgdorferi by antibody to the OspA vaccine is inefficient in the absence of complement. Infect. Immun. 67, 443-445. Soli, M., Bantar, C., Indest, K., Gu, Y., Ramamoorthy, R. Coughlin, R. and Philipp, M. T. 1998. Borrelia burgdorferi escape mutants that survive in the presence of antiserum to the OspA vaccine are killed when complement is also present. Infect. Immun. 66, 2540-2546. Philipp, M. T. Studies on OspA, a source of new paradigms in Lyme disease Research. 1998. Trends Microbiol., 6, 44-47.

Objectives and Background The objective of this research is to increase our understanding of the mechanisms that regulate the expression of B. burgdorferi lipoproteins, a type of molecule that plays a key role in Lyme disease pathogenesis. We have already 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. Sole 4, R. D. Gilmore, B. J. B. Johnson, and M. T. Philipp, Infect. Immun. 65:1165?1171, 1997). Results We further assessed 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. Future directions To further assess mechanisms of regulation of lipoprotein gene expression. FUNDING Base grant and NIH-NIAID F32 AI09980 PUBLICATIONS Ramamoorthy, R. and Philipp, M.T. 1998. Differential expression of Borrelia burgdorferi proteins during growth in vitro . Infection and Immunity. 66 5119?5124.

Parvovirus infection is responsible for erythema infectiosum (Fifth?s disease) in children and aplastic crisis, hemolytic anemias, and fetal death, as well as a rheumatic syndrome in adults. It is also reported to be responsible for anemias, fevers, pneumonitis, and aplastic crisis in AIDS patients. The human B19 parvovirus is most commonly implicated. Little is known about B19 due to its difficult in vitro propagation. Histologic evidence was shown in the bone marrow of a severely anemic rhesus monkey with advanced SIV immunodeficiency disease and suggested parvovirus infection in the rhesus as well. Bone marrow and other tissues from this animal were collected and cryopreserved. A pilot study was conducted to determine whether we could reisolate virus as well as reproduce the disease, and whether immunodeficient animal would more readily progress to disease, and if SIV is a cofactor of disease. We developed a molecular PCR assay for diagnosis of infection. Six animals were inoculated intravenously with the bone marrow preparation. 2of 3 naive animals not only developed progressive SIV immunodeficiency disease, but also severe anemia coincident with high reticulocytes and Parvovirus viremia the time of necropsy. The 3 previously SIV-infected animals, however, had only one animal progress to SIV disease, but none became anemic. A second study was then conducted with six additional SIV infected animals. Only two of six became infected and viremic. One animal progressed to disease, and Parvovirus was documented at necropsy, however, again no anemia was apparent. We are following the other animal for signs of SIV disease and/or anemia. This resistance to disease progression in the previously infected SIV animals was observed in both studies. It may be due to a lack of severe immunodeficiency from the original SIV. These animals were relatively stable, having all been infected with SIV for 1.5years or greater. In addition, they appear resistance to superinfection with the second SIV strain in the bone marrow inoculum, which had a more rapid disease course in the naive animals. FUNDING Base Grant, Venture Research. PUBLICATIONS None

Backround and Objectives The preferred antibiotic treatment of chronic Lyme disease, and that of chronic neuroborreliosis in particular, is still not fully defined. In an effort to develop improved and more reliable remedies for chronic Lyme disease, an eclectic treatment regimen has been designed. It includes two antibiotics that are highly effective against both acute and chronic Lyme disease ceftriaxone (2 g, IV, once a day, 30 days) and doxycycline (100 mg/kg, bid, per os, 60 days). The goal of this project is to assess the efficacy of this combination regimen, originally devised for humans, in rhesus macaques. More specifically, to monitor the elimination of a Borrelia burgdorferi infection from all organs commonly targeted by this spirochete, and especially from the central nervous system. Results Six rhesus macaques were inoculated with a neurotropic strain of B. burgdorferi (strain NT1) by exposure to infected ticks, and one control animal was exposed to the bite of uninfect ed ticks. All animals were confirmed to be infected by cultivating spirochetes, and amplifying spirochetal DNA by PCR, from skin biopsy samples. Animals were monitored over a six-month period. Unlike previous experiments, in which animals had been infected by needle inoculation with the NT1 strain, no evidence of infection in the central nervous system was obtained. Success in infecting the CNS may thus depend on the inoculum dose, which is likely higher when animals are infected with 108 spirochetes by needle inoculation than by exposure to the bites of ticks. Future Directions The antibiotic efficacy trial will now begin, but needle-inoculated animals will be employed. FUNDING NIH-NIAID RO1 AI42352-01 and Base grant. PUBLICATIONS None.

Regions where HIV infection is prominent frequently overlap malaria endemic regions. However, the impact of coinfection with HIV and malaria is not clear. We inoculated a monkey with Plasmodium knowlesi. 57 days after SIV inoculation, a time when the SIV infection had already caused a decrease in the percentage of CD4+ cells. Maximum parasitemia (11.6%) occurred 9 days after malaria inoculation andwas treated by chloroquine. After treatment the hematocrit recovered steadily through day 26, when malaria re-emerged and a single chloroquine treatment was given. The parasitemia and accompanying decrease in hematocrit rapidly resolved. Malaria re-emerged on day 46 and was again treated. After this third recrudescence and treatment, the parasite did not reappear, so the monkey was reinoculated with malaria. Nine days later the parasite was detected but did not require treatment, remaining a chronic low level infestation. The coinfection with malaria did not affect the progress of SI V infection. The percentage of cells expressing CD4, CD8, CD2, or CD20 did not differ in the malaria-infected monkey compared to other monkeys inoculated with SIV at the same time. No conclusions can be drawn based on 1 monkey, but the feasibility of the SIV/malaria coinfection has been demonstrated. An additional SIV-infected monkey was recently inoculated with malaria 327 days after SIV, a point when the percentage of CD4+ cells had declined markedly. A naive control monkey was inoculated at the same time. It will be interesting to see if the SIV-infected monkey inoculated with malaria at a much later stage of SIV infection will still be able to contain the parasite after 3 treatments, and to see if the ability to control the parasite differs in the control monkey. FUNDING NIH-N01-AI-65310 PUBLICATIONS NONE

A large RSV vaccine study using a Praxis Biologics, formalin-inactivated (FI) RSV vaccine was conducted. Twenty-one seronegative rhesus were divided into seven groups of three monkeys each. Four groups (Gp) received IM injections of the FI-RSV vaccine, ranging from high (250ul) to low (50ul of a 1:25 dilution), in five-fold dilutions, while Gp 5 received the Praxis prepared FI-sham vaccine. An independently prepared, Prince FI-RSV vaccine was used to inoculate Gp6, IM. Finally, Gp 7 was intranasally infected with live RSV. Twenty-one days later, a booster vaccine was administered to the first six groups. Finally, on day 56 post inoculation, a live RSV challenge was administered to all 21 animals. The high dose vaccine group had the greatest antibody response to vaccine and the lowest level of virus in both the upper and lower respiratory tract, and indicated a dose effect of the vaccine in these animals. As expected, the reinfection group was protected on challenge. Flow cytometry of bronchoalveolar samples showed a three-four fold increase in the B lymphocyte population in the lungs of the FI-RSV vaccine recipients following RSV challenge, compared with the sham vaccine and the reinfection groups. Conversely, these two latter groups demonstrated an increase in T cells, specificially CD8+ T cells. On day 8 post challenge, the animals were necropsied. Sections from each of the lung lobes were harvested and scored for lesions, by histopathology. Both Praxis and Prince vaccines resulted in increased lung scores over that of the reinfection group, suggesting a distinct immunopotentiation. These animals had a neutrophilic infiltration, predominately of eosinophils, along with some elevation in IL4 and IL5 levels, suggesting their role in the pathology of the disease. The lung scores, eosinophils and cytokine levels of the control FI-sham vaccine animals, however, were as high as the experimental animals and suggested the vaccine was not a true sham as thought. The preparation of this control is under investigation. FUNDING NIH RO1 AI 37197-04, $135,800, UAB subcontract PUBLICATIONS None

Objectives Assess infection of the central nervous system (CNS) by B. burgdorferi (Bb) in the rhesus monkey. Results Five age-matched male rhesus macaques were needle-inoculated with a neurotropic strain of B. burgdorferi (NT1). One animal was left uninfected, as a control. All animals, except the control, were verified to be infected by recovering Bb, and amplifying Bb DNA, from skin biopsy samples. Infection was monitored longitudinally by determining the titer of anti-P39 antibodies and number of bands detected on Western blots. CNS infection was monitored by determining, cerebral spinal fluid (CSF) pleocytosis, presence of Bb DNA in CSF, (by nested PCR) and production of intrathecal anti-Bb antibodies. CNS disease was monitored by measuring somatosensory evoked potentials (SEP) recorded from both upper and lower extremities immediately following infection, and longitudinally over a period of nine months. The level of anti-P39 antibodies and the number of antigen bands on IgG West ern blots increased until week 10 post-infection (PI), and then remained essentially unchanged until the end of the study (90 weeks PI). This suggest that the animals remained infected throughout the study period. CSF pleocytosis, and presence of B. burgdorferi DNA in the CSF were observed in all animals at different periods within the first 20 weeks PI. Intrathecal anti-Bb antibody ptoduction was also oberved in some animals within the 90 week study period. Two animals showed significant changes which are consistent with abnormalities in the central somatosensory system, one animal by weeks 11 and 35 PI, and the other at week 35 PI. Peripheral nerve function was found to be normal in the nerves that were selected to determine SEP, thus indicating that these changes were based in the central somatosensory pathway. Our study contributes solid and conclusive evidence that infection of the rhesus monkey with B. burgdorferi causes not only infection and disease of the peripheral nervous sy stem, as we have already demonstrated, but also of the CNS. Future directions A thorough survey of brain pathology in these animals is underway. FUNDING CDC Cooperative Agreement (U50/CCU606604-07) and Base grant. PUBLICATIONS Roberts, E. D., Bohm, Jr., R. P., Lowrie, Jr., R. C., Habicht, G., Piesman, J. and Philipp, M. T. 1998. Pathogenesis of Lyme Neuroborreliosis in the Rhesus Monkey the Acute and Chronic Phases of Disease in the Peripheral Nervous System. J. Infect. Dis. 178, 722-732.

DESCRIPTION (provided by applicant): It would be immensely useful for the management of Lyme disease (LD) treatment to have available a test for cure. Such a test could be employed not only to ascertain if treatment of acute LD was successful, thereby preventing the transition to the chronic, more intractable form of the disease, but also to distinguish among the possible etiologies of the so-called post-treatment LD syndrome. The PI and coworkers recently developed a sensitive and specific enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of LD. The test is based on the detection of antibody (Ab) to an immunodominant, invariable region (lR) of the lipoprotein VIsE. VIsE is the molecule that undergoes antigenic variation in Borrelia hurgdorfen (the etiologic agent of LD). A peptide (C6) representing the invariable region 6 (IR6) of VIsE serves as antigen. It is hypothesized that, because the spirochete should not simultaneously express on its surface more than one (or a few) VIsE variant(s) at any time, the VIsE lipoprotein must be rapidly turned over and degraded by the spirochete as new variants are progressively expressed. As a consequence of this postulated intrinsic instability, VIsE should be scarce on dead or dying spirochetes, and secondary Ab responses to the C6 peptide should decline in unison with the infection's demise, following antibiotic treatment. It is further hypothesized that the decline in titer of the C6 Ab as a function of time after treatment may serve as a test for Lyme disease cure. Preliminary results indicate that the C6 ELISA titer in cured patients falls by a factor greater or equal than 4 whereas for treatment-resistant patients the fall is by a factor <4. This is similar to the VDRL test used to diagnose syphilis cure.The broad, long-term objective of this project is to assess both retrospectively and prospectively the ability of the C6 ELISA to serve as a test for LD cure. In this proposal the C6 test will be assessed retrospectively by achieving three specific aims: Specific Aim 1: To assess retrospectively the C6 ELISA as a test for cure in patients with acute LD. Serial serum samples from patients with either erythema migrans ( n = 90) and/or culture-confirmed infection ( n = 156) will have been collected at presentation and at 6 and 12 months thereafter. The samples will be titrated for anti-C6 Ab. Specific Aim 2: To assess retrospectively the C6 ELISA as a test for cure in patients with chronic LD and post-treatment LD syndrome. Same as for SAl, but with patients with chronic LD (n = 150) and post-treatment LD syndrome (n = 60). Specific Aim 3: To assess the C6 ELISA as a test for cure in animal models of LD. Cure of LD will be assessed objectively (by culture and PCR) both in rhesus monkeys (chronic LD) and in mice (acute LD). Correlation between LD cure and anti-C6 Ab titers will be evaluated.

communicable disease transmission; Lyme disease; Ixodes; Primates; animal colony;

DESCRIPTION (provided by applicant): The broad, long-term objective of this proposal is to understand the pathogenesis of Lyme neuroborreliosis of the central nervous system (CNS). Inflammation in the CNS is thought to play a primary role in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD) and the AIDS-dementia complex (ADC). Lyme borreliosis (LB), caused by the spirochete Borrelia burgdorferi, is primarily an inflammatory disease, and neuroborreliosis, i.e. LB of the nervous system, is a mild neurodegenerative disease. The central hypothesis to be explored in this grant proposal is that 1) B. burgdorferi spirochetes that have accessed the CNS can cause inflammation and 2) this inflammatory response may lead to neural injury, and ultimately loss of neural cells. Limited loss of these cells is viewed as a determinant of the neurologic impairment seen in Lyme neuroborreliosis. The secondary hypothesis to be examined is that spirochetal lipoproteins can induce, in part or in toto, the inflammatory effects of B. burgdorferi. More specifically, the cytokines IL-6, TNF-alpha, and IL-1beta, which have been implicated as possible effectors/mediators of neurodegeneration in AD and ADC, are elicited in the CNS by B. burgdorferi or its lipoproteins, and can mediate glial/neuronal loss. A corollary of our secondary hypotheses is that the innate immune response, in the form of a response to pathogen-associated molecular patterns (in this case lipoproteins) exerts via toll-like receptors (TLR) some or all of the inflammatory effects of B. burgdorferi. The following specific aims are proposed, using the rhesus macaque, the only animal model of LB of the CNS: SA-1: Experiments in vivo. SA-1a: assessment of inflammation and glial/neuronal loss in rhesus after intracerebral stereotaxic inoculations with live spirochetes and lipoproteins. Local cytokine secretion will be assessed by confocal microscopy using a novel procedure whereby cytokines are stained intracellularly with appropriate antibodies in freshly fixed brain slices treated ex vivo with Brefeldin A. The cell phenotype also will be identified, TLR expression determined, and glial/neuronal apoptosis verified in situ by the TUNEL assay. Spirochetal antigens will be localized with anti-lipoprotein antibodies. Neurons will be counted using image analysis software, and numbers compared with control animals; SA-1b: as with SA-1a, but following natural infections (by tick) of rhesus with B. burgdorferi; SA-1c: Archival fixed samples of Bb-infected rhesus brain and meninges will be assessed for inflammatory infiltrates and glial/neuronal apoptosis. Neurons will be counted differentially as above. SA-2: Experiments in vitro. SA-2a will assess the production of IL-1beta, TNF-alpha and IL-6 in single-cell-type and mixed primary cultures of rhesus glia stimulated with live B. burgdorferi and lipoprotein. The human neuroblastoma cell lines SK-N-SH and SH-SY5Y also will be employed, alone and in combination with rhesus glia, as well as rhesus neuronal-glial primary cultures; SA-2b will assess the role of these cytokines in eliciting glial/neuronal apoptosis; SA-2c will assess the expression and role of TLR in mediating cytokine production and apoptosis in the different cell combinations.

immunologic assay /test; diagnosis design /evaluation; Lyme disease; Primates; communicable disease diagnosis; animal colony; membrane proteins; bacterial proteins; bacterial antigens; antibody;

microorganism immunology; host organism interaction; chronic disease /disorder; Lyme disease; Borrelia; Primates; animal colony;

vaccine evaluation; toll like receptor; bacterial vaccines; membrane proteins; bacterial proteins; Lyme disease; chemical structure function; animal colony; Primates; lipids; bacterial antigens; gene mutation; genetically modified animals; laboratory mouse;

bacterial vaccines; membrane proteins; bacterial proteins; enzyme linked immunosorbent assay; communicable disease diagnosis; Primates; animal colony; diagnosis design /evaluation; bacterial antigens; recombinant proteins; dogs;

nonhuman therapy evaluation; disease /disorder model; chronic disease /disorder; antibiotics; Primates; animal colony; Lyme disease; Borrelia; Macaca mulatta;

diagnosis design /evaluation; diagnostic tests; borreliosis; Borrelia; Primates; animal colony;

tissue /cell culture; nervous system infection; borreliosis; Primates; animal colony;

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Lyme neuroborreliosis is likely caused by inflammatory effects of the tick-borne spirochete Borrelia burgdorferi on the nervous system. Microglia, the resident macrophage cells within the central nervous system (CNS), are important in initiating an immune response to microbial products. In addition, astrocytes, the major CNS glial cell type, also can contribute to brain inflammation. TLRs (Toll-Like Receptors) are used by glial cells to recognize pathogen-associated molecular patterns (PAMPs), mediate innate responses, and initiate an acquired immune response. Here we hypothesize that because of their PAMP specificities, TLR 1, 2, 5, and 9 may be involved in the pathogenesis of Lyme neuroborreliosis. Previous reports have shown that the rhesus monkey is the only animal model to exhibit signs of Lyme neuroborreliosis. Therefore, we used primary cultures of rhesus astrocytes and microglia to determine the role of TLRs in mediating pro-inflammatory responses to B. burgdorferi. Results of experiments involving quantification of TLR transcript by RT-PCR and microscopic observation of expression of TLR protein indicate that microglia and astrocytes respond to B. burgdorferi through TLR1/2 and TLR5. In addition, we observed that phagocytosis of B. burgdorferi by microglia enhances not only the expression of TLR1, 2, and 5, but also that of TLR4. Taken together, our data provide proof of the concept that astrocyte and microglial TLR 1, 2, and 5 are involved in the in vivo response of primate glial cells to B. burgdorferi. The pro-inflammatory molecules elicited by these TLR-mediated responses could be a significant factor in the pathogenesis of Lyme neuroborreliosis. A manuscript based on these results was published in the journal Infection and Immunity.

3-10C; 4H-Cyclopent(f)oxacyclotridecin-4-one,1,6,7,8,9,11a-beta,12,13,14,14a-alpha-decahydro-1-beta-13-alpha-dihydroxy-6-beta-methyl-; AMCF-I; ANGIE2; Apopain; Apoptosis; Apoptosis Pathway; Ascotoxin; Astrocytes; Astrocytus; Astroglia; B cell differentiation factor; B cell stimulating factor 2; B-Cell Differentiation Factor-2; B-Cell Stimulatory Factor-2; B. burgdorferi; B.burgdorferi; BCA1; BCDF; BLC; BLR1L; BRM; BSF-2; BSF2; BSF2 (B cell stimulating factor 2); Biological Response Modifiers; Biomodulators; Body Tissues; Borrelia burgdorferi; Borrelia burgdorferi sensu stricto; Brain; Brefeldin A; CASP-3; CASP3; COX-2; COX2; CPP-32; CPP32; CPP32 protein; CPP32B; CPP32beta; CRISP; CXCL13; CXCL13 gene; CXCL8; Caspase 3, Apoptosis-Related Cysteine Protease; Cell Death, Programmed; Cells; Central Nervous System; Cessation of life; Cognitive deficits; Computer Retrieval of Information on Scientific Projects Database; Confocal Microscopy; Cyanein; Cysteine Protease CPP32; Cytokines, Chemotactic; Death; Decumbin; Differentiation Factor, B-Cell; Encephalon; Encephalons; Environment; Funding; GCP-1; GCP1; Gene Products, RNA; Genes; Glia; Glial Cells; Grant; HPGF; Hepatocyte-Stimulating Factor; Homologous Chemotactic Cytokines; Hortega cell; Hybridoma Growth Factor; IFN-beta 2; IFNB2; IL-6; IL-8; IL6 Protein; IL8; IL8 gene; Immune Mediators; Immune Mediators/Modulators; Immune Regulators; Immunofluorescence; Immunofluorescence Immunologic; Immunologic, Immunofluorescence; In Situ; Inflammatory; Institution; Intercrines; Interleukin 6 (Interferon, Beta 2); Interleukin-6; Investigators; K60; Kolliker's reticulum; LECT; LUCT; LYNAP; Life; Lyme Disease Spirochete; Lyme Neuroborreliosis; MDNCF; MGI-2; MONAP; Macaca mulatta; Microarray Analysis; Microarray-Based Analysis; Microglia; Microscopy, Confocal; Modeling; Myeloid Differentiation-Inducing Protein; NAF; NIH; National Institutes of Health; National Institutes of Health (U.S.); Nerve Cells; Nerve Unit; Nervous System Diseases; Nervous System Lyme Borreliosis; Nervous System Lyme Disease; Nervous System, Brain; Nervous System, CNS; Neural Cell; Neuraxis; Neuroborreliosis, Borrelia burgdorferi; Neurocyte; Neuroglia; Neuroglial Cells; Neurologic Disorders; Neurological Disorders; Neurons; Non-neuronal cell; Oligodendrocytes; Oligodendrocytus; Oligodendroglia; Oligodendroglia Cell; Order Spirochaetales; PARP Cleavage Protease; PGG/HS; PGHS-2; PHS-2; PTGS2; PTGS2 gene; Pathogenesis; Plasmacytoma Growth Factor; Protein Export; Protein Export Pathway; RNA; RNA, Non-Polyadenylated; Research; Research Personnel; Research Resources; Researchers; Resources; Rhesus; Rhesus Macaque; Rhesus Monkey; Ribonucleic Acid; SCA-1; SCYB13; SCYB8; SIS cytokines; SREBP Cleavage Activity 1; Slice; Source; Spirochaetales; Spirochetes; Staining method; Stainings; Stains; Synergisidin; TSG-1; TUNEL Assay; TdT-Mediated dUTP Nick End Labeling Assay; Testing; Tissue Extracts; Tissues; Transcript; United States National Institutes of Health; Yama; Yama protein; apoptosis of neuronal cells; b-ENAP; base; brain tissue; caspase-3; chemoattractant cytokine; chemokine; cysteine protease P32; cytokine; gitter cell; hCOX-2; in vivo Model; interferon beta 2; lyme spirochete; mesoglia; microarray technology; microglial cell; microgliocyte; nerve cement; nervous system disorder; neurological disease; neuron apoptosis; neuronal; neuronal apoptosis; perivascular glial cell; programmed cell death of neuronal cells by apoptosis; programmed cell death of neurons by apoptosis; programmed cell death, neuronal cells; programmed cell death, neurons

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We and others have postulated that inflammation plays a role in the pathogenesis of Lyme neuroborreliosis. Borrelia burgdorferi (Bb) is the bacterial spirochete that causes Lyme disease and is known to induce the production of inflammatory mediators in glial cells. In experiments where Bb was co-cultured in vitro with microglial cells isolated from primary rhesus cortex, we observed robust expression and release of the inflammatory cytokines/chemokines IL-6 and 8, MIP-1[unreadable], MIP-1[unreadable], RANTES and MCP-1 but we detected no concomitant induction of microglial apoptosis. SH-SY5Y (SY) neurons independently co-cultured with Bb expressed negligible amounts of inflammatory molecules and were also highly resistant to apoptosis. In contrast, when microglia were combined with SY cells and then co-cultivated with Bb, significant increases in apoptosis consistently occurred. Confocal microscopy images of the mixed cultures stained for apoptosis by the TUNEL assay and with cell specific markers suggested that it was predominantly the neuronal type cells that were dying in response to stimulation with Bb. Microarray analysis demonstrated that in addition to the inflammatory mediators secreted by microglia in response to Bb, increased expression of TLR2 and NFKB1 transcripts were also observed and may additionally contribute to a neurotoxic environment. Taken together, these findings indicate that Bb is not directly toxic to neurons;rather, neurons become impaired/die in the inflammatory surroundings generated by microglial cells through a bystander effect. This neuronal impairment may eventually contribute to the neurocognitive symptoms seen in neuroborreliosis.

Animals; Antibodies; Antibody Formation; Antibody Production; Antibody Response; Antigen Variation; Antigenic Determinants; Antigenic Variability; Antigenic Variation; B. burgdorferi; B.burgdorferi; Binding Determinants; Body Tissues; Borrelia burgdorferi; Borrelia burgdorferi sensu stricto; CRISP; Characteristics; Computer Retrieval of Information on Scientific Projects Database; DNA Recombination; DNA recombination (naturally occurring); Epitopes; Funding; Genetic Recombination; Grant; Immune; Infection; Institution; Investigators; Lyme Borreliosis; Lyme Disease; Lyme Disease Spirochete; Mammalia; Mammals; Mammals, General; Mammals, Mice; Mammals, Rabbits; Mice; Monitor; Murine; Mus; NIH; National Institutes of Health; National Institutes of Health (U.S.); Order Spirochaetales; Oryctolagus cuniculus; Peptides; Permissivenesses; Rabbit, Domestic; Rabbits; Recombination; Recombination, Genetic; Recovery; Research; Research Personnel; Research Resources; Researchers; Resources; Source; Spirochaetales; Spirochetes; Time; Tissues; United States National Institutes of Health; Week; antibody biosynthesis; immunoglobulin biosynthesis; lyme spirochete; pathogen; permissiveness; response

2(3H)-Furanone, 3-ethyldihydro-4-((1-methyl-1H-imidazol-5-yl)methyl)-, (3S-cis)-; 2,7-Naphthalenedisulfonic acid, 3,3'-((3,3'-dimethyl(1,1'-biphenyl)-4,4'-diyl)bis(azo))bis(5-amino-4-hydroxy-), tetrasodium salt; 3-10C; AMCF-I; Antibodies; B cell differentiation factor; B cell stimulating factor 2; B-Cell Differentiation Factor-2; B-Cell Stimulatory Factor-2; B. burgdorferi; B.burgdorferi; BCDF; BSF-2; BSF2; BSF2 (B cell stimulating factor 2); Benzamine Blue; Black-legged Tick; Blood monocyte; Body Tissues; Borrelia burgdorferi; Borrelia burgdorferi sensu stricto; C34H28N6O14S4; CRISP; CXCL8; Cachectin; Cachectin-Tumor Necrosis Factor; Cells; Chromatography, High Performance Liquid; Chromatography, High Pressure Liquid; Chromatography, High Speed Liquid; Computer Retrieval of Information on Scientific Projects Database; Cytokines, Chemotactic; Detection; Differentiation Factor, B-Cell; ELISA; Enzyme-Linked Immunosorbent Assay; Funding; GCP-1; GCP1; Grant; HPGF; HPLC; Harvest; Head and Neck, Saliva; Hepatocyte-Stimulating Factor; High Pressure Liquid Chromatography; Homologous Chemotactic Cytokines; Hour; Human; Human, General; Hybridoma Growth Factor; I. scapularis; IFN-beta 2; IFNB2; IL-6; IL-8; IL6 Protein; IL8; IL8 gene; INFLM; Immune; In Vitro; Infection; Inflammation; Inflammatory; Inflammatory Response; Injury; Institution; Intercrines; Interleukin 6 (Interferon, Beta 2); Interleukin-6; Investigators; Ixodes scapularis; Ixodid ticks; Ixodida; K60; LECT; LUCT; LYNAP; Lead; Life; Lyme Disease Spirochete; MDNCF; MGI-2; MONAP; Man (Taxonomy); Man, Modern; Marrow monocyte; Mediator; Mediator of Activation; Mediator of activation protein; Methods; Myeloid Differentiation-Inducing Protein; NAF; NIH; National Institutes of Health; National Institutes of Health (U.S.); Pb element; Pilocarpine; Plasmacytoma Growth Factor; Process; Production; Research; Research Personnel; Research Resources; Researchers; Resources; SCYB8; SIS cytokines; Saliva; Site; Source; Staining method; Stainings; Stains; TNF; TNF Alpha; TNF protein, human; TNF superfamily, member 2 protein, human; TNF-2 protein, human; TNF-alpha; TNFA; TNFSF2 protein, human; TSG-1; Ticks; Tissues; Transmission; Trypan Blue; Tumor Necrosis Factor; Tumor Necrosis Factor-alpha; Tumor Necrosis Factor-alpha (macrophage-derived); United States National Institutes of Health; b-ENAP; chemoattractant cytokine; chemokine; concept; density; feeding; heavy metal Pb; heavy metal lead; human TNF protein; interferon beta 2; lyme spirochete; monocyte; response; transmission process; tumor necrosis factor, human; tumor necrosis factor-2 protein, human; tumor necrosis factor-alpha promoter allele-2 protein, human

2-dimensional; 3-D; 3-Dimensional; Apoptosis; Apoptosis Pathway; Apoptotic; CRISP; Cell Culture System; Cell Death, Programmed; Cell Line; Cell Lines, Strains; Cell division; CellLine; Cells; Central Nervous System; Computer Retrieval of Information on Scientific Projects Database; Condition; Coupled; Drops; FLR; Failure (biologic function); Funding; Genes; Grant; In Vitro; Institution; Investigators; Messenger RNA; Microarray Analysis; Microarray-Based Analysis; N-myc Proto-Oncogenes; NIH; National Institutes of Health; National Institutes of Health (U.S.); Nerve Cells; Nerve Unit; Nervous System, CNS; Neural Cell; Neuraxis; Neurocyte; Neuronal Dysfunction; Neurons; Pathogenesis; Phenotype; Proteins; Proto-Oncogenes, N-myc; RNA, Messenger; RNA-Binding Proteins; Rate; Research; Research Personnel; Research Resources; Researchers; Resources; Source; Standards; Standards of Weights and Measures; Thapsigargin; United States National Institutes of Health; cultured cell line; failure; gene product; mRNA; microarray technology; neuronal; pro-apoptotic protein; two-dimensional

American; Antibodies; Antibody Formation; Antibody Production; Antibody Response; Antigenic Determinants; Assay; Binding; Binding (Molecular Function); Binding Determinants; Bioassay; Biologic Assays; Biological Assay; Blood Serum; CRISP; Computer Retrieval of Information on Scientific Projects Database; Detection; Diagnosis; Disease; Disorder; ELISA; Enzyme-Linked Immunosorbent Assay; Epitopes; Funding; Gamma Globulin, 19S; Gamma Globulin, 7S; Grant; IgG; IgM; Immunoglobulin G; Immunoglobulin M; Immunoglobulin V; Immunoglobulin Variable Region; Individual; Institution; Investigators; Length; Lyme Borreliosis; Lyme Disease; Molecular; Molecular Interaction; NIH; National Institutes of Health; National Institutes of Health (U.S.); Patients; Peptides; Research; Research Personnel; Research Resources; Research Specimen; Researchers; Resources; Series; Serum; Source; Specificity; Specimen; Surface; Testing; United States National Institutes of Health; Variable Region; Variable Region, Ig; antibody biosynthesis; base; disease/disorder; immunoglobulin biosynthesis; response

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The goal of this proposal is to establish a nonhuman primate model of persistent infection and disease caused by bacteria of the genus Moraxella. The broad, long-term objective is to utilize such a model in the study of pathogenesis and immunoprophylaxis of disease caused by Moraxella catarrhalis. This bacterium is emerging as a major etiologic agent of diseases of the respiratory tract and middle ear but there are no animal models of Moraxella infection that are adequate to study disease pathogenesis or immunoprophylaxis. Our first goal was to determine whether a human isolate of M. catarrhalis could, at the very least, colonize the nasopharynx (NP) of rhesus macaques and, at best, elicit epistaxis and/or otitis media OM. Two animals were given a nasopharyngeal inoculation with human strain KSA, and 6 animals with the human strain 7169. Only one of the animals (given M. catarrhalis strain 7169) sustained detectable colonization for 2 weeks. This animal also exhibited epistaxis. It appears, therefore, that M. catarrhalis is largely human-host specific. We now plan to give 4 of the above animals an inoculation with rhesus Moraxella isolate CI24. We would monitor these animals serially for colonization, epistaxis, and OM. The expectation is that inoculation with these bacteria, which were originally isolated from a rhesus macaque with epistaxis, will fulfill Koch's postulates by colonizing the NP of, and causing epistaxis in the majority of the animals. We will also monitor the incidence of OM. If the rhesus Moraxella causes OM in addition to epistaxis, OM being an aspect of this infection hitherto not explored, then this bacterium, which is not M. catarrhalis, would emerge as a pathogen of significant veterinary importance in colony rhesus. The next step would be to evaluate rhesus Moraxella not only as model for the human pathogen but also as a target for a vaccine for veterinary use.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Tetracyclines moderate inflammatory responses of various etiologies. We hypothesized that tetracyclines exerted, in addition to antimicrobial function, control over inflammation elicited by Borrelia burgdorferi. To model systemic effects we used the human monocytic cell line THP-1, and for effects in the central nervous system we employed rhesus monkey brain astrocytes and microglia. Cells were stimulated with live or sonicated B. burgdorferi, or the OspA lipoprotein, in the presence of increasing concentrations of doxycycline or minocycline. Both antibiotics significantly reduced the production of TNF-alpha, IL-6, and IL-8 in a dose-dependent manner in all cell types. Microarray analyses of the effect of doxycycline on gene transcription in spirochete-stimulated monocytes revealed that the NF[unreadable]B and IKK-alpha genes were down-regulated. Functionally, phosphorylation of I[unreadable]B-alpha and binding of NF[unreadable]B to target DNA were both reduced in these cells. Our results prove the principle that tetracyclines may have a dual therapeutic effect in Lyme disease.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Lyme neuroborreliosis is caused by the spirochete B. burgdorferi, and is often manifest by neurocognitive deficits. As a possible mechanism for Lyme neuroborreliosis we hypothesized that B. burgdorferi induces in the central nervous system the production of inflammatory mediators, with concomitant neuronal and/or glial apoptosis. To test our hypothesis we set up an ex vivo model consisting of freshly collected slices from brain cortex of l rhesus macaques, and allowed live B. burgdorferi to penetrate the tissue. Numerous transcripts of genes that regulate inflammation as well as both oligodendrocyte and neuronal apoptosis were significantly perturbed, as assessed by DNA microarray analysis. Transcriptional fold-increases of 7.43 (p = 0.005) for the cytokine TNF-alpha and 2.31 (p = 0.016) for the chemokine IL-8 were also detected by real-time-PCR array analysis. The immune mediators IL-6, IL-8, IL-1beta, COX-2, and CXCL13 were visualized in glial cells in situ by immunofluorescence staining and confocal microscopy. Concomitantly, significant proportions of both oligodendrocytes and neurons undergoing apoptosis were present in spirochete-stimulated tissues. IL-6 production by astrocytes, and oligodendrocyte apoptosis were also detected, albeit at lower levels, in rhesus macaques that had received in vivo intraparenchymal stereotaxic inoculations of live B. burgdorferi. These results provide proof of concept for our hypothesis. A manuscript including these results was published in the American Journal of Pathology.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The Diagnostic Parasitology Core Lab is the first line of defense against parasitic diseases in the animal colony and provides diagnostic services to clinical veterinarians and researchers when parasitic infections are suspected. From 5/1/10-4/30/11, the lab examined the following: 2629 stools, 1 blood, and 11 skin samples from the monkey colony and 21 stools, 18 perianals, and 3 pelage samples from the rodent colony, giving a total of 2683 samples from the Primate Center. Of these, 79 stools and 1 skin sample were done for in-house research and 95 stools and 10 skin samples were done for outside collaboration. Additionally, samples from both uptown and downtown New Orleans rodent colonies consisted of 188 fecals and 547 pelage slides for a total of 735 samples delivered from outside colonies. The record keeping system for parasitology results has now become completely paperless, with information on laboratory results forwarded to clinical veterinarians through the Blacksmith program at the end of each workday. The laboratory now bills for all services rendered and sends a statement every month to projects for which parasitology diagnostic work was performed.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Members of the Moraxella genus are gram-negative, aerobic bacteria that have been isolated from a variety of mammalian hosts. During a survey of bacteria that colonize the nasopharynx of rhesus macaques, isolates of the Moraxella genus, identified as M. catarrhalis, were observed in the majority of animals with demonstrated epistaxis, or "bloody nose syndrome." Several isolates from this survey were cryopreserved at the Tulane National Primate Research Center. One isolate was also obtained from an epistatic cynomolgus macaque at the U.S. Army Medical Research Institute of Infectious Diseases. Based on differences between the colony and gram stain morphology of rhesus isolates compared to that of human M. catarrhalis isolates, we hypothesized that the nonhuman primate Moraxella might be unique. These isolates were reassessed by multiple parameters. Despite differences in cell and colony morphology, the rhesus isolates, by each biochemical test, were identical to M. catarrhalis and not other human Moraxella subspecies. Biochemical analysis of the cynomolgus isolate by Vitek 2 compact (St Louis, Mo) revealed that it belonged to a Moraxella group, but could not differentiate between species. However, sequencing of the 16S ribosomal RNA gene from four representative rhesus isolates and the cynomolgus isolate showed closest homology to Moraxella lincolnii, a human respiratory tract inhabitant, with 90.16% identity. In order to examine rhesus macaques as potential hosts for M. catarrhalis, a total of eight animals were inoculated with human isolates of this strain. Only one of the animals was colonized and showed disease, whereas four of four macaques became epistatic after inoculation with the rhesus Moraxella isolate. Thus, this nasopharyngeal Moraxella species is a previously uncharacterized, distinct pathogen of nonhuman primates. It is not M. catarrhalis.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Pneumococcal infection causes an estimated 40,000 deaths annually in the US. Colonization of the nasopharynx by S. pneumoniae always precedes pneumococcal disease. Therefore, elucidation of procedures to prevent or eradicate nasopharyngeal (NP) carriage would help diminish the incidence of both pneumonia and invasive disease. We thus endeavored to develop a rhesus monkey model of S. pneumoniae carriage, to complement and improve upon the existing rodent models, with an animal whose NP anatomy and epithelial lining is more akin to the human. We first surveyed the nasopharynx of infant rhesus macaques from the Center's breeding colony, in search of animals that were natural carriers. A total of 158 rhesus of a median age of 1.23 years (range 0.59 [unreadable]1.99) were surveyed. No S. pneumoniae colonies were isolated from any of the animals. Thus rhesus are not likely natural carriers of S. pneumoniae. Second, we attempted to induce carriage experimentally in infants (n = 8), by NP instillation of a human S. pneumoniae strain (19F). This was done both in antibiotic pre-treated and untreated animals. We also searched for pneumonia and disseminated infection secondary to colonization, and whether these forms of infection could be facilitated by splenectomy. The rate of NP colonization remained at 100% for 2 weeks post-instillation (PI), and above 60% for 7 weeks. It then oscillated around 30% until week 14, when the study ended;it did not appear to be affected by antibiotic pre-treatment. Four of the animals were splenectomized between weeks 5 and 6 PI. There was no invasive infection or disease in any of the animals, splenectomized or eusplenic, at any time. Blood and BAL cultures were negative throughout, and X-rays, CBC, and serum chemistry analyses were normal at all times. We concluded that the rhesus monkey is a suitable model to study pneumococcal colonization. However, the transition to invasive disease may not be fostered by splenectomy in this model.

DESCRIPTION (provided by applicant): The tachykinin, substance P (SP) mediates a variety of biological effects via high affinity neurokinin-1 receptors (NK-1R), and NK-1R antagonists have been extensively studied for use in the treatment of a variety of disease conditions. We have assembled a compelling body of evidence indicating that SP/NK-1R interactions exacerbate classical inflammation within the central nervous system (CNS). During the previous funding period, we have shown that SP exacerbates the inflammatory responses of isolated murine microglia and astrocytes to clinically relevant bacterial pathogens. Furthermore, we have demonstrated that endogenous SP/NK-1R interactions are required for maximal inflammation and CNS damage in murine models of bacterial meningitis. Most importantly, we have shown that systemic prophylactic or therapeutic administration of an NK-1R antagonist can markedly attenuate bacterially-induced neuroinflammation in our mouse models. For this continuation application, we have established an exciting new collaboration between uniquely qualified researchers to perform a comprehensive preclinical evaluation of the ability of SP to augment classical inflammation in isolated primary human CNS cells and a non-human primate (NHP) model of bacterial meningitis. We will test the hypothesis that inhibition of SP/NK-1R interactions attenuates immune responses of human and NHP microglia and astrocytes to clinically relevant bacterial pathogens thereby limiting inflammatory CNS damage. These experiments build upon our recent rodent studies and their performance will contribute significantly to our understanding of the role played by this neuropeptide in the initiation and/or exacerbation of the immune functions of resident human/NHP CNS cells that occur in response to bacterial infection. As such, these studies will provide essential information in resolving the cellular mechanisms that precipitate classical inflammation within the human/NHP brain during disease states. Furthermore, these studies represent a substantial and possibly final preclinical and translational phase to evaluate the therapeutic potential of NK-1 receptor antagonists in the treatment of classical CNS inflammation prior to human trials.

Abstract ? Office of the Chair, Division of Bacteriology and Parasitology The Office of the Chair of the Division of Bacteriology and Parasitology is the site of administration of the Division. Matters within the Chair's purview include recruiting and evaluating faculty and staff, developing budgets, assigning lab and office space, writing reports, and ensuring efficient functioning of all service cores. In addition, the Chair frequently serves on TNPRC and University committees as well as committees of national and/or international scientific organizations. All of the TNPRC Division Chairs are members of the TNPRC Executive Committee. The Divisional Core leaders report to the Chair, who is himself a Core leader. Among other achievements during this funding period the Office mediated the award of numerous grants and contracts, mostly from the NIH, including 7/R01's, 3/R21's, 1/R33, 7/R01's subcontracts, 2/R21 subcontracts, 2/U19's, 1/UH2, 1/ARRA, 2/LSU COBRE's, 5/Foundation grants, 2/Foundation subcontracts and 1/Howard Hughes grant.

Abstract ? Education and Training A crucial component of the mission of the TNPRC is to educate and train graduate and veterinary students, postdoctoral fellows and veterinarians, undergraduates, and visiting scientists. The TNPRC leadership and faculty strongly believe that it is at National Primate Research Centers where the best training in nonhuman primate research may be provided, and is very much aware of their role in this endeavor. This is evidenced by the learning opportunities that were made available to trainees and students of different levels, and by the numbers of undergraduate and graduate students, postdoctoral, and veterinary fellows that were trained in the current funding period. Faculty and staff have participated, in addition, in the teaching of graduate courses at several departments within and outside Tulane. An important new feature of the TNPRC Education and Training program was the institution of the Mentoring Program. Dr. Cariappa Annaiah was recruited with the mandate to strengthen mentoring activities. He has created a novel and ambitious Mentoring Program. The goal of the Mentoring Program dovetails with the broader educational mission of TNPRC, in that it is intended to facilitate the maturation of academic, scientific, and managerial knowledge and skills in junior faculty, postdoctoral fellows, and graduate students through the time-tested method of mentorship. The Program is viewed as a resource for the mentees, to assist them in meeting TNPRC and Tulane University expectations. It is also an opportunity for senior faculty to share their scientific, and managerial expertise with the next generation of scientists.

Abstract ? Pilot Research Program In accordance with the NPRC guidelines and the FOA, the Tulane National Primate Research Center (TNPRC) Pilot Research Program provides funding to investigators who show promise of developing a career in nonhuman primate biomedical research or to those who wish to add a nonhuman primate component to an existing research program. The Pilot Research Program is also open to investigators with established nonhuman primate research programs who wish to develop substantially new research directions. Research may involve initial model identification, characterization, and development that may follow a case workup of a spontaneously occurring disease, evaluation of improved husbandry or animal management procedures, or more detailed examination of serendipitous laboratory findings. Pilot Research Program support is typically for proposals that do not have sufficient preliminary data or results that are needed to obtain support from normal sources of funding. The overall purpose of the project must complement the objectives of the Center?s research programs, and the projects themselves must have the potential of leading to an extramurally funded grant application. The proposal must be substantially different from the applicant?s funded projects. Investigators must be beyond the postdoctoral rank and based at any non-profit academic or research institution. If the applicant is not a TNPRC Core Staff Scientist, they must secure sponsorship from such a Core Scientist. The latter will assume responsibility for overall management, coordination and reports concerning the project. In general, all major activities related to the approved project must be conducted and carried out on site at the Center. The number of extramural grants that were awarded this funding period, made possible by Pilot Grants was 12. There were 3 RO1 grants, 3 R21 grants, and one grant of each of the following types: R33, COBRE, R56, P20, Foundation, and DoD. The number of papers published during this funding period based on experiments funded with pilot grants was 27.

Abstract ? Diagnostic Parasitology Core The Diagnostic Parasitology (DP) Core provides diagnostic services to clinical veterinarians and core and affiliate investigators when parasitic diseases are suspected in resident nonhuman primates. All newly acquired animals are quarantined before being admitted to the colony and during this quarantine period the Core conducts monthly stool exams and a blood examination for parasites. Animals from the TNPRC breeding colony are examined regularly for parasites as part of the preventive health program. The Core also examines samples from animals entering an experimental protocol, as the presence of parasites may influence clinical results. The Core provides the broadest service, in that it not only assesses animals assigned to specific projects but also colony and quarantine animals. It effectively services all of the investigators that hold animal projects at the TNPRC or that receive samples from nonhuman primates housed at the TNPRC. The Core is run by Chad Massey, MSPH. The overall direction of the Core is provided by Mario Philipp. During the last funding period, the core streamlined the classical techniques that it uses, and achieved an excellent level of efficiency in providing service to the TNPRC and its collaborators.