Effect of dose and route of immunisation on the immune response induced in cattle by heterologous Bacille Calmette–Guerin priming and recombinant adenoviral vector boosting

Veterinary Immunology and Immunopathology 158 (2014) 208–213

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Research paper

Effect of dose and route of immunisation on the immune response induced in cattle by heterologous Bacille Calmette–Guerin priming and recombinant adenoviral vector boosting G. Dean a , D. Clifford a , S. Gilbert b , H. McShane b , R.G. Hewinson a , H.M. Vordermeier a , B. Villarreal-Ramos a,∗ a b

Bovine TB, AHVLA—Weybridge, New Haw, Addlestone, Surrey KT15 3NB, UK The Jenner Institute Old Road Campus Research Building Oxford University, Roosevelt Drive, Oxford OX3 7DQ, UK

a r t i c l e

i n f o

Article history: Received 4 September 2013 Received in revised form 19 December 2013 Accepted 21 January 2014

Keywords: Adenovirus Mycobacteria Vaccine Route of immunisation Tuberculosis

a b s t r a c t BCG is used experimentally as a vaccine against tuberculosis (TB), induced by Mycobacterium bovis, in cattle (bTB). However, the efficacy of BCG is variable in humans, cattle and guinea pigs. An adenoviral vector expressing Antigen 85A (Ad5Ag85A) has enhanced protection against TB in mice when used in combination with BCG for prime-boost experiments. However, the route of immunisation affects the degree of protection seen. This work examines the immunogenicity of a new vectored vaccine (Ad5-TBF) that expresses Ag85A, Rv0287, Rv0288 and Rv0251c to explore the effects of dose of adenoviral boost and route of inoculation on immunogenicity. We found that 2 × 109 infectious units (iu) delivered intradermally conferred the most consistent and strongest responses of the different regimes tested. Crown Copyright © 2014 Published by Elsevier B.V. All rights reserved.

1. Introduction Infection with Mycobacterium bovis (M. bovis), the agent responsible for bovine tuberculosis (bTB), in the UK cattle herd is an increasing problem. This increase is thought to be due in part to the presence of a wildlife reservoir and it has implications for both animal and human health and welfare (Vordermeier et al., 2006). It is thought that, in the presence of a wildlife reservoir, the current UK test and slaughter policy alone will not be sufficient for the control of bTB (Krebs and Group, 1997). Vaccination has been proposed as

∗ Corresponding author. Tel.: +44 1932 359529; fax: +44 1932 357260. E-mail address: [email protected] (B. Villarreal-Ramos).

a measure that could help control bTB (Krebs and Group, 1997). BCG is a live attenuated strain of M. bovis that was isolated by Calmette and Guerin in the early 1900s from a case of bTB mastitis and it has been used as a vaccine against TB in humans since 1921 (Calmette, 1931). It has also been used, on an experimental basis, as a vaccine in cattle. The protection afforded by BCG has been shown to be variable in both humans and cattle (Griffin et al., 2001). Furthermore, vaccination with BCG interferes with the current diagnosis for bTB and its use in the field is currently prohibited by both domestic and European legislation. Nevertheless, on an experimental basis, BCG is the standard by which all other vaccines are judged. A serotype 5, replication deficient adenovirus (Ad) modified to express the mycobacterial antigen Ag85A

0165-2427/$ – see front matter. Crown Copyright © 2014 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetimm.2014.01.010

G. Dean et al. / Veterinary Immunology and Immunopathology 158 (2014) 208–213

(Ad5Ag85A) (Wang et al., 2004) has been used as a vaccine against TB, induced by M. tuberculosis (Mtb), in mouse models both alone and as a boost in combination with BCG priming. Wang and collaborators (Wang et al., 2004) showed that a single intranasal (i.n.), but not intramuscular (i.m.), immunisation with Ad5Ag85A provided improved protection against airway Mtb. challenge compared with cutaneous BCG vaccination. Similarly, Xing et al. (2009) and Santosuosso et al. (2006) showed in mice that Ad5Ag85A was protective against pulmonary Mtb. challenge when administered i.n. but not by the i.m. route. It was also highly effective in enhancing protection when used as a boost following parenteral BCG immunisation. Vordermeier and co-workers have also used a heterologous prime-boost vaccination schedule in cattle where subcutaneous BCG vaccination was followed by boosting with intradermal (i.d.) Ad5Ag85A. In terms of protection, this regime showed a degree of promise in that, compared with BCG vaccination, fewer animals presented with visible lesions after infection with M. bovis (Vordermeier et al., 2009) It has previously been shown that cattle infected with M. bovis, followed by treatment with isoniazid are better protected against a subsequent M. bovis challenge compared to naïve controls (Dean et al., 2008). This experiment showed that antigens Rv0288 and Rv0251 are recognised earlier in re-challenged animals than in challenged naïve controls, which suggested that these antigens may play a role in protective immunity. Accordingly, in the current experiments to determine immunogenicity, an Ad5-TBF construct, incorporating the mycobacterial antigens Ag85A, TB10.4 (Rv0288) and its partner TB9.8 (Rv0287) (Lightbody et al., 2008) and Acr2 (Rv0251c) was constructed and the immunogenicity evaluated in cattle. The aim was to generate a vector expressing these antigens with the aim of making a comparison, in future studies, of the protective efficacy between this novel vector and Ad5Ag85A. The aim of this current work was to elucidate the correlation between the dose of adenoviral vector used and immunogenicity. Further, in view of earlier vaccine challenge experiments on small animal models (Santosuosso et al., 2006; Xing and Lichty, 2006; Xing et al., 2009) that indicated that protection can be influenced by route of immunisation, we also aimed to determine whether route of administration had any effect on the response induced. The results of this work will inform the selection of the dose and route of administration of adenoviral boost for future experiments that will consider the effects of prime-boost vaccination on protection against experimental infection with M. bovis. 200

400

600

Ag85A

800

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2. Materials and methods 2.1. Cattle The study protocol was approved by the AHVLA Animal Use Ethics Committee (UK Home Office PCD number 70/6905) and performed in accordance with the UK Animal (Scientific Procedures) Act 1986. Male Holstein-Friesian and Aberdeen Angus cross cattle of 4–6 months of age were purchased from farms that had been known to be free of bTB for at least 5 years. Thirty two animals were divided into eight groups of four animals each and inoculated as described below. 2.2. BCG and Ad5-TBF The BCG vaccine strain used for priming was the human vaccine M. bovis BCG Danish 1331, prepared as per manufacturer’s instructions (SSI, Denmark). The replication deficient Ad5 vector was used to prepare the Ad5-TBF construct encoding for Ag85A, Rv0287, Rv0288 and Rv0251c. The four antigens were expressed as a single fusion protein following the human tPA leader sequence, under the control of the CMV IE promoter, followed by the bovine growth hormone transcription termination sequence. The expression cassette was inserted at the E1 site of human Ad5, which had also had the E3 region deleted (Fig. 1). 2.3. Inoculation All 32 cattle were inoculated with ca. 106 cfu of BCG SSI subcutaneously as previously described (Vordermeier et al., 2004). After eight weeks, animals were divided into eight groups of four; each to be boosted or not with different regimes of the Ad5-TBF construct. Groups 1, 3 and 5 were boosted i.d.; groups 2, 4 and 6 were boosted i.m.; group 7 was boosted both i.d. and i.m.; group 8 remained as BCG control. The dose for groups 1 and 2 was 2 × 109 iu; for groups 3 and 4 the dose was 1 × 109 iu; and for groups 5 and 6 the dose was 5 × 108 iu; for group 7 the dose was 1 × 109 iu via each route, i.e. a total of 2 × 109 iu. A summary of the groups with the different routes and inoculation doses is presented in Table 1. 2.4. Evaluation of immune responses 2.4.1. IFN assays Immune responses induced by vaccination and boosting were monitored through the measurement of secretion of IFN␥ after in vitro stimulation of whole blood cultures by ELISA. The frequency of precursors of IFN␥ secreting cells after in vitro stimulation of peripheral blood mononuclear cells (PBMC) was determined using the ELISpot assay. 1000

1200

RV0287

1400

1600

Rv0251

1800

2000

Rv0288

Fig. 1. Schematic diagram showing the order in which the proteins were inserted into the vector Ad5. Antigens were expressed inserted into Ad5 as indicated in materials and methods. Numbers in the ruler refer to nucleotides.

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Table 1 Immunisation regimens for the different groups according to route (intradermal, intramuscular or both) and dose (2 × 109 , 1 × 109 or 5 × 108 ) of inoculation. Route of boosting immunisation Vaccine dose (iu)

a

2 × 109 1 × 109 5 × 108 0

BCG control

Intradermal (id)

Intramuscular (im)

Group 1 Group 3 Group 5

Group 2 Group 4 Group 6

Intradermal and intramuscular Group 7a

Group 8

Group 7 received 1 × 109 pfu through each of the intradermal and intramuscular routes, to receive a total of 2 × 109 pfu.

To measure secretion of IFN␥ in whole blood, peripheral blood was collected as previously described (Lightbody et al., 1998; Villarreal-Ramos et al., 2006) and incubated with medium alone (negative control [NC]), M. bovis PPD (PPD-B) (10 ␮g/ml) (VLA-Weybridge, UK), Rv0287 (10 ␮g/ml), Rv0288 (5 ␮g/ml) (Proteix, Prague, Czech Republic), Ag85A protein (10 ␮g/ml) (Lionex, Germany) or pokeweed mitogen (PWM) Sigma-Aldrich, UK) (5 ␮g/ml) and incubated at 37 ◦ C in an atmosphere of 5% CO2 and 95% humidity. After overnight incubation, blood was centrifuged at 300 × g for 10 min and plasma harvested and stored at −80 ◦ C until use. Concentration of IFN␥ was determined using the BovigamTM assay (Prionics AG, Switzerland); results are first corrected for background by subtracting baseline values and then expressed as mean O.D. readings ± standard error of the mean. Evaluation of the ability of recombinant Rv0251c to stimulate T-cells in pilot experiments proved too variable and therefore it was not used in the experiments described in this manuscript; the reasons for this variability are unknown.

To determine the frequency of precursors, 96-well flat membrane-bottomed plates (Immobilon-P polyvinylidene difluoride membranes; Millipore, Ireland) were prepared by coating overnight at 4 ◦ C with a bovine IFN␥ specific monoclonal antibody (Mabtech, Stockholm, Sweden) and the assay conducted in accordance with the manufacturers’ instructions, as previously described (Vordermeier et al., 2004). 2 × 105 PBMC were added to each well in 200 ␮l of RPMI-1640 medium containing Glutamax-1 (Life Technologies, Paisley, UK), 10% heat-inactivated fetal calf serum (FCS), 5 × 10−5 M 2-mercaptoethanol, 100 U/ml penicillin and 100 ␮g/ml streptomycin. PMBC were stimulated for 24 h with the same concentration of antigen as that used for stimulation of whole blood. Results were corrected for background by subtracting baseline (unstimulated) values and adjusted to express numbers of precursors per million cells. All data were tested for statistical significance using Graphpad Instat 3, the Kruskal-Wallis Test (Nonparametric ANOVA) and Dunn’s Multiple Comparisons Post Test.

Fig. 2. Antigen specific IFN␥ secretion, measured by ELISA, following boosting of BCG-vaccinated cattle at week 8 with Ad5-TBF encoding for Ag85A, Rv0287, Rv0288 and Rv0251c. Ad5-TBF was inoculated i.d. (shaded bars, groups 1, 3 and 5), i.m. (open bars, groups 2, 4 and 6), or both i.d. and i.m. (horizontally hatched bar, group 7) and BCG control (diagonally hatched bar, group 8). Ad5-TBF was given at doses of 2 × 109 (groups 1, 2 and 7), 1 × 109 (groups 3 and 4) or 5 × 108 (groups 5 and 6) iu. Antigen-specific whole blood IFN␥ secretion was evaluated at the weeks indicated on the x axis. The dotted line in the y axis indicates the highest response observed against each antigen induced by vaccination with BCG alone at any pre-boosting time point. Data are presented as antigen O.D. 450 nm minus PBS O.D 450 nm.

G. Dean et al. / Veterinary Immunology and Immunopathology 158 (2014) 208–213

3. Results 3.1. Immune responses Fig. 2 shows the specific immune responses induced by boosting with the different Ad5-TBF regimes indicated in materials and methods. Overall it appeared that levels of specific IFN␥ produced were dependent on the dosage of Ad5-TBF used for the heterologous boost at week 8. Group 1, boosted i.d. with 2 × 109 iu Ad5-TBF, gave the largest in vitro responses to PPD-B, Rv0287, Rv0288, Ag85A stimulation at week 9 of the experiment, one week after boosting; these responses persisted at week 10 and dropped at week 11. Responses at weeks 9 and 10 were significantly higher than those seen at week 8 ( < 0.001 for PPD-B, Rv0287, Rv0288, Ag85A at week 9 and  < 0.05 for PPD-B and Rv0288,  < 0.001 for Rv0287 and  < .01 for Ag85A at week 10). Lower doses of vaccine induced lower and less sustainable responses, OD readings for group 3, boosted i.d. with 1 × 109 iu Ad5-TBF, were lower than for group 1 at week 9 although these responses were still significantly higher than for week 8 ( < 0.001 for PPD-B, Rv0287 and Ag85A and  < 0.01 for Rv0288). At week 10 responses to Rv0287 and Rv0288 were significantly reduced relative to group 1 ( < 0.05 (Rv0287) and  < 0.01 (Rv0288). Group 5, boosted i.d. with 5 × 108 iu Ad5-TBF, showed lower specific responses for all four antigens with highest levels being seen at week 10. These levels of response were maintained at week 11 ( < 0.01 for PPD-B at weeks 10 and 11 and  < 0.05 at week 9,  < 0.001 at week 10 and  < 0.01 at week 11 for Ag85A).

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Responses induced by i.m. boosting with Ad5-TBF were less clear cut. Specific IFN␥ responses to Ag85A were lower in all three groups compared with i.d. boosting with the highest dose. Responses in group 2, boosted i.m. with 2 × 109 iu Ad5-TBF, at week 9 for all four antigens were significantly lower than in group 1 ( < 0.0001,  < 0.01,  < 0.0001 and  < 0.001, respectively) However, there were no significant differences between these two groups at week 10, suggesting that speed of response, but not peak response, was affected by the route of immunisation. It was also notable that, within the i.m. inoculated animals, the highest specific IFN␥ responses were not always observed in group 2. Groups 2 and 4 responded differently to PPD-B ( < 0.05) whereas groups 4 and 6 responded differently to Rv0287 and Ag85A ( < 0.05). It was possible that a combination of i.d. and i.m. would induce better responses than a single route of immunisation. Accordingly, we selected the highest dose to test this possibility. Responses in group 7, immunised simultaneously with 1 × 109 iu Ad5-TBF both i.d. and i.m. indicate that this is not the case. Responses observed in this group were lower than those seen in group 1. Fig. 3 shows the frequency of IFN␥ precursors after boosting. There were significant differences in responses at week 9 between the different dose regimes. Group 1, boosted with 2 × 109 iu Ad5-TBF i.d., had an increased frequency of precursors in response to PPD-B and Rv0288 compared with group 3, boosted with 1 × 109 iu Ad5TBF i.d., ( < 0.05). There were also significant differences between groups 1 and 5 in response to Rv0288 ( < 0.05). Boosting with 2 × 109 iu Ad5-TBF i.d. (group 1) at week 8 significantly increased the frequency of precursors

Fig. 3. Antigen specific IFN␥ ELISpot response following boosting of BCG vaccinated cattle at week 8 with Ad5-TBF encoding for Ag85A, Rv0287, Rv0288 and Rv0251c. Ad5-TBF was inoculated as described in Fig. 2, i.d. (shaded bars), i.m. (open bars), or both i.d. and i.m. (horizontally hatched bar) and BCG control (diagonally hatched bar). Ad5-TBF was given at doses of 2 × 109 , 1 × 109 or 5 × 108 iu. IFN␥ ELISpot was evaluated at the weeks indicated on the x axis. The dotted line in the y axis indicates the highest response observed against each antigen induced by vaccination with BCG alone at any pre-boosting time point.

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producing IFN␥ at week 9 for PPD-B, Rv0287 and Rv0288 ( < 0.05,  < 0.001 and  < 0.001 respectively). The responses had waned by week 10 and there was no significant difference in response to PPD-B between week 8 and week 10 or between week 9 and week 10. Group 1 responses to Rv0287 and Rv0288 were significantly lower at week 10 than week 9 ( < 0.05).Groups 3 (1 × 109 iu Ad5TBF i.d.) and five (5 × 108 iu Ad5-TBF i.d.) both showed significant responses to PPD-B at week 9 and week 10 compared with week 8 ( < 0.05 and  < 0.01 respectively at week 9 and  < 0.05 and  < 0.05 respectively at week 10). Week 9 responses were not significantly different from week 10. An increased frequency of IFN␥ precursors was also seen in group 3 in response to Rv0287 at week 9 ( < 0.01). Rv0287 and Ag85A induced no increase in the precursor frequency at weeks 9 or 10 in groups 3 and 5. Antigen specific responses following i.m. boosting, groups 2, 4 and 6 were lower than in the groups boosted i.d. Group 2, boosted i.m. with 2 × 109 iu Ad5-TBF had a significantly lower response to PPD-B than group 1 at week 9 ( < 0.05). However, boosting induced an increase in the response to PPD-B for groups 2, 4 and 6 ( < 0.05,  < 0.001 and  < 0.01, respectively); to Rv0287 for groups 2 and 4 ( < 0.01 and  < 0.01); and to Rv0288 for group 2 ( < 0.05). 4. Discussion In this work, we have evaluated the immunogenicity of an Ad5 vector encoding for the mycobacterial antigens Ag85A, Rv0287, Rv0288 and Rv0251 using a prime-boost vaccination regime in cattle that had previously been vaccinated with BCG. The aim of the work was to determine the optimum dose and route of administration of the boost needed to maximise the immunogenicity of these antigens. We found that 2 × 109 iu Ad5-TBF delivered i.d. conferred the most consistent and strongest responses of the different regimes tested. To our knowledge, this is the first instance of an adenoviral vaccine titration experiment in a prime-boost vaccination regime in cattle. However, few titration experiments of this nature have been carried out in this or other vaccination systems. Steitz et al. (2010), working in a guinea pig model to develop an Ad5 based vaccine against influenza found that 107 viral particles could generate protective antibodies against a highly pathogenic avian influenza virus, H5N1. However, only the subcutaneous route of immunisation induced an effective level of crossreactive antibodies. After i.d. boosting, IFN␥ secretion, was seen generally to peak at week 9 and then to wane rapidly while the responses following i.m. boosting appeared to be slower. with the peak of response being reached, in some groups, at week 10. The specific response to PPD-B did not appear to wane as rapidly as when i.d. boosting was used though generally all responses to i.m. boosting were lower. After boosting, ELISpot data confirmed the findings of the whole blood assay in that a significant increase in the frequency of IFN␥ producers could be seen at week 9, one week after boosting, compared with week 8. Responses to Rv0287 and Rv0288 were significantly lower at week 10 compared with week 9 while there was a trend towards

reduced frequency of IFN␥ producing cells in response to PPD-B and Ag85A. It appears that fewer individual cells are secreting IFN␥, resulting in the reduced responses seen in the ELISA. Whilst the level of IFN␥ responses is not a predictor of protection, given the central role that IFN␥ has in protection against TB and the previous track record of Ad5 constructs it would seem reasonable to select the regimen inducing the highest mycobacteria-specific IFN␥ for further testing in a vaccination and challenge experiment. The variation in immunogenicity caused by route of immunisation in this study has also been seen in small animal model experiments using Ad5Ag85A as the vaccine. Wang et al. (2004) showed that a single i.n., but not i.m., immunisation with Ad5Ag85A provided improved protection against airway Mtb. challenge compared with cutaneous BCG vaccination. This encouraging result was then extrapolated to the more sensitive guinea pig model of pulmonary TB (Xing et al., 2009) where it was found that the viral vector alone conferred little protection while both the i.n. and i.m. routes of booster immunisation, in animals previously primed with BCG, conferred enhanced long-term survival against Mtb. challenge, with the i.n., mucosal, route being the most effective. In conclusion, we have determined that boosting cattle with 2 × 109 iu Ad5-TBF administered i.d. gave the highest and most consistent immunological responses. Further studies will explore the protective efficacy of this vaccination strategy to determine whether the immunological responses described in this work correlate with enhanced protection against challenge with M. bovis.

Acknowledgements The authors would like to thank all members of the Animal Services Unit for their exemplary care of all animals used in these experiments. This project was funded through a DEFRA grant, SE3224. RGH, HMV, HMcS, SG are Jenner Investigators.

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