MATERIALS AND METHODS ----- The outer surface protein A (OspA) vaccine against Lyme disease: efficacy in the rhesus monkey [Vaccine 1997 Volume 15 Number 17/18, pages 1872-1887] ----- Animal care and housing Animals were cared for and housed as described previously (15). Challenge infection and xenodiagnosis Ixodes scapularis nymphs infected with the B31 strain of B. burgdorferi were used to deliver a challenge infection. For this purpose, ten ticks were placed in each of two capsules. The capsules were already attached to both scapular areas of each monkey by a procedure described previously (15). For xenodiagnosis; performed 4 weeks after the beginning of the IS protocol, 20 uninfected nymphs were enclosed in each capsule and both capsules were placed on each of the five animals involved in the immune suppression experiment. Ticks were left on the animals for 5 days, both for the challenge infection and the xenodiagnosis. Xenodiagnosis ticks were analysed by DFA 3 weeks after removal from the capsule. As a positive control for the xenodiaganosis procedure, a rhesus macaque was infected with B. burgdorferi and subsequently xenodiagnosed as above. Detection of spirochaetes in ticks by DFA DFA was performed as described previously (21). Negative preparations were scanned completely under the fluorescent microscope; DFA-positive specimens were scanned only until spirochaetes were observed. Western blotting The procedure used was described previously (22) but B. burgdorferi strain B31 (4th passage) was used instead of strain JD1. Sodium dodecyl sulphate (SDS) extracts of whole B. burgdorferi B31 were electrophoresed on SDS polyacrylamide gels (15% acrylamide). Serum samples from all animals were incubated with the nitrocellulose blot at a final concentration of 1:50. Antibodies that bind to OspA also appear to bind to other components of greater and lesser molecular mass, which span the whole length of the gel (4). To avoid these confusing bands, serum samples were preincubated with just enough recombinant non-lipidated OspA to permit the detection of OspA as a 31 kDa band (166 µg per ml OspA for serum samples from animals vaccinated with lipidated OspA, and 33 µg per ml for all other vaccinated animals). To verify that the preincubation of serum with OspA did not inhibit binding of antibody to any other B. burgdorferi antigens, blots of whole B. burgdorferi antigens were incubated with serum obtained from control animals at 15 week PC with and without added OspA (166 µg per ml). No differences were observed in the antigen patterns obtained with and without added OspA (not shown). PCR Ante-mortem biopsy samples were collected aseptically. Necropsy instruments such as forceps, scissors and blades were flamed before being used to collect samples. Skin was sterilised with 70% ethanol. Samples from different tissues/organs were kept separately in sterile cryogenic vials with 0.5 ml TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH7.4) in each vial. All samples were kept at -70 degrees C until use. Before performing the PCR, samples were thawed and a sterile needle was used to move the sample out from the vial to a weighing boat, to be cut into approximately 20 mg pieces with a sterile blade. Blade, needle and weighing boat were discarded after processing each sample. Each 20mg piece was processed for PCR as described previously (23). The readout of this protocol is a Southern blot. Hence, whenever reference is made in the text to a positive PCR reaction, a positive Southern blot is implied and amplicons rated as positive are therefore specific for the amplified DNA fragment. From each of the skin biopsy samples collected before the challenge infection and on weeks 1-4 PC, only one aliquot of lysate was amplified. From each tissue lysate of samples collected post-mortem from animals that were part of the immune suppression experiment, two aliquots were amplified, and three from all of the tissue lysates of tissues collected post-mortem from all of the remaining animals. Between four and seven negative control tubes (containing no tissue lysate) were interspersed and processed in parallel with each of ten tubes with lysate subjected to the PCR. In vitro cultivation of skin biopsy samples The procedure employed was described previously (15). However, two changes were introduced, namely, BSK-H (Sigma, Chemical Co., St. Louis, Missouri) was used instead of BSK-II, and tissue samples were incubated at a larger ratio of tissue to medium volumes. Thus, whereas in previous experiments, samples were incubated at a ratio of 1:1000-1:3000 (15), in the present experiments this ratio was typically 1:100. Histopathological and immunohistochemical analyses of skin biopsy samples and organ tissues collected post-mortem Skin punch biopsies (8mm in diameter) were taken from sites adjacent to the tick feeding areas and fixed in Streck Tissue Fixative, a non- formalin fixative, for 1 week, according to the manufacturer's instructions (Streck Tissue Fixative, Omaha, NB [sic]) and then processed using a standardised paraffin embedding technique. Tissues were sectioned at 3-6 µm thickness. For immunohistochemistry, the primary antibodies used were either a monoclonal antibody (mAb) directed to a B. burgdorferi 7.5 kDa lipoprotein (24) or an anti-OspA mAb (LA31) (25). Bound antibody was revealed with anti-mouse IgG labelled with alkaline phosphatase, and fast-red as chromogen (Kirkegaard & Perry Laboratories, Gaithersburg, Maryland). Samples of organs collected post-mortem were processed in the same way. Morphometric quantification of cells that bind the anti-OspA mAb LA31 Tissue. The areas of full cross-sections of both right and left sections of the heart, septum and each of the lung lobes were measured. The variations in areas measured reflect differences in tissue size. All tissue sections measured were 3-4 µm thick paraffin sections. They were immunostained as above, using the LA31 anti-OspA mAb and cells that stained red were counted. Controls. A pellet of cultured B. burgdorferi (B31) was fixed by the same procedure employed for the tissue sections and used as a positive control. Negative controls were: (1) a section of the heart or lungs from an uninfected animal, incubated with the LA31 mAb; (2) a section of the heart or lungs from the animal being studied, incubated with the second antibody but not the LA31 mAb; (3) a 'positive control' slide incubated with the second antibody but not the LA31 mAb. No cells were stained on negative control slides. Measurements. All measurements were made using a computer-based morphometric device (Bioscan, Optimas Corporation, Edmonds, WA). The system was calibrated with a stage micrometer. Area was determined using a 1.2x objective, and the numbers of stained cells were determined with a 10x and/or a 20x objective. Silver staining of brain sections Brain samples collected post-mortem and fixed as described above (histopathology section) were processed using a modified form of the original Steiner silver-staining method. The modified Steiner procedure has been described elsewhere (26). Immune suppression protocol The IS protocol consisted of a combination of surgical splenectomy and administration of cyclophosphamide (CP) and prednisone sodium phosphate (PSP). Three doses had been considered: low (CP 2.6 mg per kg; PSP 4.1 mg per kg), medium (CP 6.5 mg per kg; PSP 8.3 mg per kg), and high (CP 10.6 mg per kg; PSP 12 mg per kg). The high dose was chosen after assessing the level of IS elicited in three rhesus monkeys, each of which received a low, medium or high dose. Administration of both drugs began 1 week after splenectomy and continued for a total of 5 weeks. CP was given every other day by orogastric intubation while the animal was anaesthetised. PSP was given by intramuscular injection once every day. Bronchoalveolar lavage and urine collection in immune suppressed animals Bronchoalveolar lavage and urine sample collection began 1 week after the first dose of immune suppressive drugs and continued on a weekly basis until sacrifice. For bronchoalveolar lavage, animals were preanaesthetised with glycopyrolate and acepromazine and anaesthesia induced with ketamine HCl. A fibreoptic bronchoscope was manipulated into the trachea after applying local anaesthetic (lidocaine) to the larynx. After directing the scope into the right primary bronchus, a volume of 20 ml of warmed normal saline was instilled through the bronchoscope. The saline was then collected by aspiration and the animal was given O2 by face mask for 5 min before being returned to its cage. Urine was collected by cystocentesis. Nerve conduction studies Peripheral nerve conduction velocity and amplitude of the response to stimulation was determined in both motor and sensory ulnar and median nerves and the tibial motor nerve (one side only), 1 week before and 1 month after initiation of the IS protocol. Possible influence of IS on nerve conduction properties was ruled out by performing NCS once before and twice after the initiation of drug administration on the three animals participating in the trail IS study. The method employed for NCS was described previously (19). Statistical analyses The frequency of appearance of PCR amplicons in organs collected post-mortem from control and vaccinated animals was compared by a nonparametric X² [chi-square] test. The mean numbers of monocyte-type cells that stained with the anti-OspA mAb LA31 in the heart and lungs of control and vaccinated animals were compared using Kruskal-Wallis ANOVA by ranks or MANOVA, as appropriate. ----- See complete document at: The outer surface protein A (OspA) vaccine against Lyme disease: efficacy in the rhesus monkey [Vaccine 1997 Volume 15 Number 17/18, pages 1872-1887] http://www.geocities.com/HotSprings/Oasis/6455/rhesus-index.html