Introduction

In New Zealand, the common brushtail possum (Trichosurusvulpecula) has reached an estimated population of 70 millionand causes irreparable damage to native forests. In addition,possums are a major vector of bovine tuberculosis for domes-tic and feral animals.1 Considerable emphasis is now beingplaced on developing strategies for the biological control ofpossums. The suggested implementation of a vaccinationstrategy for biological control will require the induction of anappropriate immune response. High persistent levels of anti-body will be required for a vaccine to inhibit reproduction,lactation or induce infertility of pouch young by transfer ofcolostral derived antibody. In order to rationally develop andimplement methods for vaccination of possums it is essentialto increase our understanding of basic immunology in theseanimals and, in particular, the role of cytokines in regulatingimmune responses.

The successful use of a vaccine requires both a suitabledelivery system and adjuvants capable of stimulating theappropriate immune responses. Cytokines administeredduring the immunization period have been demonstrated to

be effective immunological adjuvants in several species andmodel systems.2–6 Cytokines have the potential to bothenhance the immune response to the vaccine antigen and toalter the type of immune response generated. Recently,Rothel et al. demonstrated that incorporation of recombinantovine TNF-α in aqueous or Al(OH)3 vaccine formulationenhanced antibody responses to a cestode antigen.7 We havepreviously cloned TNF-α from possums,8 and now haveproduced bioactive recombinant possum TNF-α in the yeastPichia pastoris. The present paper studies some of the prop-erties of P. pastoris-expressed TNF-α, including the physio-logical effects on possums and the adjuvant effect of TNF-αon antibody responses to the model antigen, keyhole limpethaemocyanin (KLH).

Materials and Methods

Construction of expression plasmid for TNF-α

Possum alveolar macrophages were isolated and cultured for 6 h inthe presence of LPS as previously described.8 The isolation of totalRNA and reverse transcription into cDNA were as described.9 Aplasmid for expression of TNF-α in the yeast P. pastoris wasconstructed by isolating the cDNA encoding the mature part ofpossum TNF-α (amino acids 78–233) using polymerase chain reac-tion (PCR) primers 5′-TATCTCGAGAAAAGAGTCAGATCTTT-

Immunology and Cell Biology (1999) 77, 28–33

Research Article

Physiological effects and adjuvanticity of recombinant brushtailpossum TNF-α

DN WEDLOCK, LP GOH, AR McCARTHY, RG MIDWINTER, NA PARLANE and BM BUDDLE

AgResearch, Wallaceville Animal Research Centre, Upper Hutt, New Zealand

Summary The present paper describes the physiological properties of recombinant possum TNF-α and an adju-vant effect on antibody responses to the model protein antigen, keyhole limpet haemocyanin (KLH). For thesestudies recombinant possum TNF-α was produced in the yeast Pichia pastoris. The recombinant cytokine wassecreted into the culture medium and purified by gel filtration. Possum TNF-α produced in this expression systemwas N-glycosylated and bioactive in two different assays. In a murine fibroblast L929 cytotoxicity assay, thepossum TNF-α had lower specific activity compared to human TNF-α, while in a possum-specific assay, possumTNF-α enhanced the proliferation of PHA-stimulated possum thymocytes and was more active than human TNF-α. The physiological effect of the recombinant possum TNF-α was investigated in groups of possums administereddoses of 6, 30 or 150 µg of cytokine. For each dose, TNF-α caused profound effects on the numbers of circulatingleucocytes characterized by a three-to-four-fold increase in neutrophil numbers at 6–24 h after injection and aninitial sharp decrease in lymphocyte numbers. The efficacy of TNF-α as an immunological adjuvant was deter-mined in possums administered KLH (125 µg) in an aqueous or Al(OH)3-based formulation with or without addedrecombinant TNF-α (150 µg). Serum antibody responses to KLH were monitored by ELISA. The TNF-α stimu-lated two-fold and four-fold increases in antibody levels in aqueous and Al(OH)3-based vaccine formulations,respectively. The strongest antibody responses were observed in the group of possums that received KLH formu-lated in Al(OH)3 with addition of TNF-α.

Key words: adjuvant, antibody, cytokine, Pichia pastoris, possum, TNF-α, yeast.

Correspondence: DN Wedlock, AgResearch, Wallaceville AnimalResearch Centre, PO Box 40063, Upper Hutt, New Zealand.

Received 28 July 1998; accepted 6 October 1998.

GCAGAATG-3′ (sense) and 5′-TAGAATTCTCTCAAAGGG-CAATGGC-3′ (antisense). The XhoI and EcoRI sites included at the5′ prime end of these primers are underlined. The expressionplasmid, pPICTNF, was constructed by ligation of the cDNA into theXhoI and EcoRI sites of the expression vector pPIC9 (InvitrogenCorporation, Carlsbad, CA, USA), which placed the mature TNF-αprotein in frame with the yeast alpha-mating factor (MF-α) signalsequence.

Production of recombinant TNF-α

P. pastoris strain GS115 (Invitrogen) was transformed with SalIdigested pPICTNF by the LiCl method.10 Transformants wereselected on minimal dextrose (MD) medium (13.4 g/L yeast nitrogenbase (YNB), 0.4 mg/L biotin, 20 g/L D-glucose) and screened forproduction and secretion of product in small-scale culture. Strainswere grown at 30°C for 48 h in well aerated 10 mL buffered minimalglycerol-complex (BMGY) medium (20 g/L peptone, 10 g/L yeastextract, 13.4 g/L YNB, 0.4 mg/L biotin, 100 mmol/L phosphatebuffer (pH 6.0), 1% v/v glycerol). The cells were harvested by cen-trifugation and re-suspended in 2 mL buffered minimal methanol-complex (BMMY) medium (same as BMGY except 0.5% methanolreplaced the glycerol) to induce expression. At 72 h after induction,culture supernatants were analysed by SDS-PAGE for the presence ofsecreted product. One strain that strongly expressed TNF-α wasselected for larger scale production. Culture supernatant from a 100 mL BMMY culture of this strain was concentrated 15-fold in astirred cell concentrator using a YM10 diaflo ultrafiltration mem-brane (Amicon Inc., Beverly, MA, USA). The concentrated culturemedium was fractionated on Sephadex G-75 (Amersham PharmaciaBiotech, Uppsala, Sweden) with 100 mmol/L NH4HCO3 (pH 7.8)buffer. Fractions were analysed by SDS-PAGE and those containingpurified TNF-α were pooled, and concentrated. The purifiedcytokine preparation was freeze-dried and stored at –70°C andreconstituted in PBS prior to use. The protein concentration wasdetermined using bicinchoninic acid (BCA; Pierce, Rockford, IL,USA). The amount of endotoxin present in the purified TNF-α was< 0.1 EU/µg protein as determined by the limulus amoebocyte lysateassay (E-Toxate; Sigma Chemical Co., St Louis, MO. USA).

To control for any possible in vivo effects of yeast-derivedproteins that may have contaminated the purified recombinant TNF-α, a preparation was produced from a P. pastoris strain trans-formed with the pPIC9 vector without an inserted gene. The strainwas cultured in 100 mL of BMMY for 72 h as described for TNF-αand the culture supernatant concentrated 7.5-fold using a stirred cell concentrator with an YM10 diaflo ultrafiltration membrane(Amicon). The crude preparation was dialysed against PBS and theprotein concentration measured with the BCA protein assay.

Assay of TNF-α biological activity

Recombinant TNF-α was assayed in two bioassays. Cytotoxic activ-ity was measured using murine fibroblast L929 cells,11 as describedpreviously.8 The ability of TNF-α to stimulate the proliferation ofpossum thymocytes in the presence of submitogenic concentrationsof lectin was determined as described.8 Briefly, possum thymocyteswere isolated from the thymus of a 5-month-old possum, and theassay performed in quadruplicate in 96-well plates with 5 × 105 cellsin 200-µL RPMI-1460 supplemented with antibiotics, glutamine,10% FCS and 25 µg/mL phytohaemagglutinin (PHA-P, Sigma).Following incubation at 37°C for 48 h,3H-thymidine (37 kBq/well)was added. After a further 18 h of incubation, the incorporation oflabel was measured following harvesting of the cells.

Animal experiments

Brushtail possums were trapped in urban areas around Upper Hutt.They were housed in individual cages and fed and watered asdescribed previously.12 Possums were anaesthetized for vaccinationand bleeding as described.12

The in vivo effect of recombinant possum TNF-α was studied ingroups of possums (three per group) injected intravenously witheither PBS (0.5 mL) or a dose of 6, 30 or 150 µg TNF-α in 0.5 mLPBS. Samples of blood were collected from the jugular vein at the time of administration and at 2, 6, 24 and 72 h after injection with3-mL vacutainers containing 7.5 mg EDTA (Becton-Dickinson,Rutherford, NY, USA). Rectal temperatures were monitored atsimilar time points after injection. As a further control, a group ofthree possums were injected intravenously with a dose of 250 µg of yeast-derived proteins prepared from a culture of P. pastoristransformed with the pPIC9 vector.

To determine the adjuvant effect of TNF-α, groups of possums(three animals per group) were inoculated intramuscularly witheither 125 µg KLH in PBS, 125 µg KLH formulated in Al(OH)3

(0.6 mg), 125 µg KLH and TNF-α (150 µg) in PBS or 125 µg KLHformulated with Al(OH)3 with added TNF-α (150 µg). The possumswere re-vaccinated by the same route 18 days after the first vaccina-tion. Sera were collected at the time of the first immunization and at 2, 4.5 and 7.5 weeks after the first immunization.

Haematology

Total and differential leucocytes were determined on fresh EDTAblood. Blood (0.1 mL) was diluted into 1.9 mL 0.1 N HCl and totalleucocytes counted in a haemocytometer (Improved Neubauerchamber). Cell numbers were expressed as cells per mL. Differentialcell counts (neutrophils and lymphocytes) were determined on bloodfilms fixed in methanol (100%) for 10 min and stained with Giemsastain (10% v/v in water) for 10 min. The smears were rinsed withdistilled water, air-dried and 100 cells counted under oil immersionmicroscopy.

Serology

Measurement of antibodies to KLH in sera was determined byELISA. Flat-bottom 96-well plates (Maxisorb, Nunc, Copenhagen,Denmark) were coated with 50 mL of KLH (Sigma, 5 µg/mL) in 50 mmol/L carbonate buffer (pH 9.6) for 16 h at 4°C. The plateswere washed four times in PBST (PBS containing 0.05% Tween 20(Sigma)) and non-specific binding blocked by the addition of PBSTwith 0.1% BSA, and incubated at room temperature for 60 min. Theplates were washed four times with PBST and 50 µL of two-foldserial dilutions of possum sera in PBST with 0.05% BSA added tothe wells. The plates were incubated at room temperature for 60min, washed in PBST and re-incubated with 50 mL of a 1:4000dilution of sheep anti-possum Ig conjugated to horse-radish perox-idase13 for 60 min. Plates were washed and incubated with 3,3′,5,5′tetra methyl benzidine (TMB) substrate for 10 min, the reactionsstopped by the addition of 25 µL of 0.5 mol/L H2SO4 and theabsorbance read at 450 nm in a microtitre plate reader. A positiveserum titrated in two-fold serial dilutions and a negative serum wereincluded on each plate as controls. The antibody titre of each serumwas expressed as the log2 of the reciprocal of the highest dilutionthat gave an absorbance value greater than a background value,defined as the mean value for the animals prior to immunization +3 SD.

Biological properties of possum TNF-α 29

Statistical analyses

Analyses of the leucocyte counts were undertaken using A N OVA onloge transformed data, while the analysis of antibody responses usedA N OVA on log2 transformed data.

Results

Production of recombinant TNF-α

Recombinant possum TNF-α was expressed in the methylo-trophic yeast P. pastoris in a secreted form and purified fromthe culture supernatant by size fractionation. The purificationprocedure was effective at removing high- and low-molecular-weight contaminating yeast-derived proteins and also mediaconstituents. Analysis of the TNF-α on SDS-PAGE underreducing conditions revealed the presence of two proteinbands; a major band at ∼ 20 kDa and a minor band at 25 kDa(data not shown).

In vitro biological activity of recombinant TNF-α

The biological activity of recombinant possum TNF-α wasdetermined in two separate in vitro assays. The cytolyticactivity of TNF-α against L929 cells is shown in Table 1. Forcomparison the activity of a glutathione-S-transferase(GST)/TNF-α fusion protein8 and a recombinant humanTNF-α (Sigma) was also determined. Both the possum TNF-α expressed in P. pastoris and TNF-α as a GST fusionprotein8 showed cytotoxic activity. The specific activity of the yeast-derived possum TNF-α and the GST TNF-α fusionprotein was ~ 220-fold lower than the recombinant humanTNF-α. Both forms of possum TNF-α were active in apossum-specific thymocyte proliferation assay when titratedto a concentration of 0.1 µg/mL, while at a similar concen-tration the human TNF-α was less active than the possumTNF-α (Table 1).

Physiological effect of possum TNF-α in vivo

Administration of TNF-α intravenously to possums elevatedbody temperature with a peak effect at 24 h after injection.The group that received the higher dose of 150 µg showed amean temperature increase of 2.7°C at 24 h, which thenreturned to a pre-injection value by 72 h after injection.Animals injected with the lower doses developed a smalltemperature increase averaging 0.7 and 1.5°C for the 30- and6-µg groups, respectively. No significant change in tempera-ture was seen in the group that was injected with PBS orgiven a high dose of 250 µg of yeast-derived proteins. Severalanimals in the TNF-α groups showed signs of disorientationand uncoordination in the first 24 h after injection, althoughthese effects were not dose-related. No changes in animalbehaviour were seen in the group administered PBS or yeast-derived proteins.

Intravenous administration of TNF-α had a marked effecton the total number of leucocytes, neutrophils and lympho-cytes found in the peripheral blood. Possums injected with150 µg recombinant TNF-α showed a mean decrease of 58%in numbers of leucocytes at 2 h, followed by a mean increaseof 1.9-fold at 24 h after injection (Fig. 1). Doses of 6 and 30 µg of TNF-α induced a similar pattern of changes inleucocyte counts but the changes were of smaller magnitude.The mean total leucocyte counts in the group injected withPBS showed a decrease at 6 h but had returned to normalvalues by 24 h. Possums injected with the 150-µg dose ofTNF-α showed a mean decrease of 38% in neutrophilnumbers at 2 h after injection. There was a marked increasein circulating neutrophil numbers, peaking at 6 or 24 h afterinjection, in animals administered with TNF-α at each of thethree doses. Mean neutrophil numbers had increased 3.2, 3.9 and 3.9-fold at 24 h after injection, compared to pre-injection, for the groups injected with 6, 30 or 150 µg TNF-α,respectively. In comparison, mean neutrophil counts in thegroup that was administered PBS remained relativelyunaltered over the 72-h period. Neutrophil numbers in thegroups given either 6 or 150 µg of TNF-α were significantlyhigher (P < 0.05) than in the PBS group at 24 h afterinjection, while in the group that was administered 30 µg of TNF-α, neutrophil counts were significantly higher (P < 0.05) at 6 h. The mean lymphocyte counts in all groupsthat received either TNF-α or PBS showed an initial sharpdecrease at 2 or 6 h after injection, followed by a slow returnto pre-injection values. Mean lymphocyte counts at 6 h afterinjection were decreased 3.7, 4.8, 4.1 and 1.8-fold in thegroups administered with a dose of 6, 30, 150 µg TNF-α orPBS, respectively. Lymphocyte counts in each of the TNF-αgroups were significantly lower (P < 0.05) than in the PBSgroup at 2, 6 and 24 h after injection. For the yeast proteincontrol group, there were minimal changes in total leucocyte,neutrophil and lymphocyte counts after injection with 250 µgof yeast protein. In the period when TNF-α induced maximalresponses (6–24 h), the counts for the yeast control groupwere between those for the PBS and 6 µg TNF-α groups.

Effect of recombinant TNF-α on antibody responses toKLH

An experiment was conducted to investigate the adjuvanteffect of TNF-α on the antibody responses of possums to the

DN Wedlock et al.30

Table 1 Cytotoxic activity of recombinant TNF-α and enhance-ment of proliferation of PHA-stimulated possum thymocytes

* Cytolytic activity was assayed on murine L929 fibroblasts. Thetitre was the reciprocal of a two-fold serial dilution that gave 50%cell lysis and specific activity expressed as titre/µg protein.

† Enhancement of proliferation of possum thymocytes by recom-binant TNF-α expressed in P. pastoris (present study), GST/TNF-αfusion protein8 and human TNF-α (Sigma). The activity shown iswhen each TNF-α preparation was added to thymocyte culture at aconcentration of 0.1 µg/mL. Proliferation was measured by incorpo-ration of 3H-thymidine and activity expressed as c.p.m. (mean±SEMof triplicate assays) after subtraction of background counts from cellsincubated with PHA alone (mean 3323 c.p.m.).

Type of TNF-α Expression host Cytolytic Proliferationactivity* of thymocytes†

Possum P. pastoris 2.9 × 102 1589 ± 323E. coli 4.2 × 101 2912 ± 660

Human Yeast 6.4 × 104 417 ± 172

model antigen KLH. Serum antibody levels were determinedin possums that either received KLH in an aqueous form(PBS) or KLH formulated in Al(OH)3. Mean group antibodyresponses are shown in Fig. 2. Possums immunized withKLH in PBS with added TNF-α had two-fold higher antibodytitres than the group that received KLH in PBS alone,although the differences were not statistically significant. Themean antibody levels in the Al(OH)3 plus TNF-α group at 4.5 weeks and 7.5 weeks after the first immunization were,respectively, two-fold and four-fold higher than the meanantibody levels in the Al(OH)3 group without added TNF-α.The difference between the levels in the Al(OH)3 with andwithout TNF-α groups was statistically significant (P < 0.05)

at 7.5 weeks. The addition of Al(OH)3 to the vaccine prepa-rations induced a significant increase (P < 0.01) in antibodytitres at 2, 4.5 and 7.5 weeks compared to the aqueous prepa-rations. No signs of lethargy or other adverse clinical signswere observed in the animals injected with TNF-α during thecourse of the experiment.

Discussion

In the present study the effects of recombinant possum TNF-α on possum physiology and humoral immuneresponses were studied. For these purposes bioactive TNF-αwas produced in the methylotrophic yeast P. pastoris, anexpression host which has a number of advantages overEscherichia coli including absence of endotoxin and secre-tion of the recombinant protein into the culture medium. The recombinant TNF-α produced for the present study waspurified from the culture medium by gel filtration and shownto contain negligible levels of endotoxin. The TNF-α wasbioactive in two different bioassays and showed speciesspecificity because the activity on L929 cells was 220-foldless compared to human TNF-α. The 25-kDa molecular formof the TNF-α was susceptible to digestion by glycosidicenzymes that removed N-linked carbohydrates (data notshown), suggesting that possum TNF-α expressed in yeasthas N-linked glycosylation. At present it is not knownwhether the native possum TNF-α is glycosylated and whatrole the carbohydrate has in the localization, secretion andbiological function of this cytokine.

Tumour necrosis factor-α caused profound biologicaleffects in vivo. Injection of doses of 6–150 µg TNF-α wasassociated with transient perturbations in the total and dif-ferential leucocyte counts of possums. A transient decrease in

Biological properties of possum TNF-α 31

Figure 1 Mean (a) total leucocyte, (b) neutrophil and (c) lym-phocyte counts in the peripheral blood of groups of possums(three per group) injected intravenously with (C) 6, (n) 30 and (▫) 150 µg recombinant possum TNF-α or (n) PBS.

Figure 2 Effect of recombinant TNF-α on antibody responsesto keyhold limpet haemocyanin (KLH). Groups of possums (threeper group) were vaccinated intramuscularly with KLH (125 µg) ineither (¶) an aqueous formulation, (n) aqueous formulation withaddition of TNF-α (150 µg), (n) an Al(OH)3-based formulation,or (▫) an Al(OH)3-based formulation with addition of TNF-α.Animals were re-vaccinated 18 days later. The results are themean ELISA antibody titres (log2) to KLH (±SEM).

circulating leucocyte numbers at 2 h after injection was pri-marily due to a loss of lymphocytes from the peripheralblood, while the increase in leucocyte numbers at 6–24 h afterinjection was due to a marked increase in circulating neu-trophil numbers. The highest dose of TNF-α had the mostinfluence on changes in lymphocytes and neutrophil counts.Rothel et al. reported fluctuations in numbers of circulatingwhite blood cells in sheep after intramuscular injection of100 µg of recombinant ovine TNF-α.7 The cytokine caused anincrease in total leucocyte numbers at 24 h after injection,primarily due to a two–four-fold increase in neutrophilcounts, but did not significantly affect lymphocyte numbers.In a different study, Johnson et al. found that infusion ofovine TNF-α into sheep caused a decrease in numbers of cir-culating neutrophils and lymphocytes for up to 4 h afterinjection.14 In the present study, a decrease in possum lym-phocyte numbers was observed after injection with TNF-α. Atransient decrease in neutrophil numbers of short durationwas also observed but only with the highest dose. Our resultsalso show a number of similarities to those reported byRothel et al. for the effect of ovine IL-1β in sheep.7 In theseanimals administration of cytokine caused a drop in total leu-cocyte numbers within 30 min, primarily due to a loss of neu-trophils, lymphocytes, monocytes and eosinophils from theperipheral blood. This was followed by a marked elevation inwhite blood cell numbers which peaked at 24 h after injec-tion, predominantly due to a four-fold increase in neutrophilnumbers. We have recently shown that LPS in possums alsocauses an elevation of neutrophil numbers and a decrease inlymphocyte counts.15 This similarity between possum TNF-αand LPS activity is not surprising because TNF-α has beenshown to be the major mediator of LPS action.16

In sheep, low doses of ovine TNF-α given intramuscularlycaused only minor increases in temperatures, while a largerdose of 100 µg caused a transient temperature increase of2.0°C and the animals became lethargic.7 In our study thehighest dose of 150 µg TNF-α administered by an intra-venous route caused a transient 2.7°C increase in bodytemperature. Several of the animals became lethargic afteradministration of TNF-α but this effect was not related to thedose. Administration of TNF-α as part of a vaccine formula-tion given intramuscularly did not cause any visible clinicaleffect on the possums.

Our study demonstrated that TNF-α has an adjuvant effecton humoral responses to the model antigen haemocyanin. Anearly study on the adjuvanticity of human TNF-α in miceshowed that this cytokine elevated antibody responses tosheep red blood cells, a T cell-dependent antigen, but did not enhance antibody responses to the T cell-independentantigen, type III pneumococcal polysaccharide.17 Schijns et al. reported that TNF-α increased the immunogenicity ofan inactivated rabies virus vaccine in a mouse model.18 Insheep, administration of recombinant TNF-α enhanced theantibody levels to the recombinant cestode parasite Taeniaovis fusion protein, 45W-GST, between 1.5- and six-fold foran aqueous and between 2.5- and seven-fold for an Al(OH)3-based formulation.7 In the present study the inclusion ofTNF-α in the vaccine stimulated antibody levels two- andfour-fold for aqueous and Al(OH)3-based formulations,respectively. However, the only significant increase inantibody titres as a result of the inclusion of TNF-α was for

the Al(OH)3 formulation, where TNF-α appeared to induce astronger antibody response.

The present study has demonstrated the potential of usinga cytokine in possums to enhance antibody responses to anti-gens which have been selected for biological control of theseanimals. The availability of recombinant possum TNF-α willenable the role of this cytokine as a biological adjuvant to beinvestigated further. Alternatively, the inclusion of the geneencoding TNF-α in a vector expressing an antigenic target, ordevelopment of ways to induce a TNF-α response in ananimal may enhance antibody responses and be pivotal in thesuccess of the vaccine.

Acknowledgements

We thank the staff of the Wallaceville Small Animal Unit forcaring for the possums. The authors thank MAF Policy NewZealand for financial assistance.

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