Recombinant adenovirus expressing the haemagglutinin of peste des petits ruminants virus (PPRV) protects goats against challenge with pathogenic virus; a DIVA vaccine for PPR
© Herbert et al.; licensee BioMed Central Ltd. 2014
Received: 8 November 2013
Accepted: 17 February 2014
Published: 26 February 2014
Peste des petits ruminants virus (PPRV) is a morbillivirus that can cause severe disease in sheep and goats, characterised by pyrexia, pneumo-enteritis, and gastritis. The socio-economic burden of the disease is increasing in underdeveloped countries, with poor livestock keepers being affected the most. Current vaccines consist of cell-culture attenuated strains of PPRV, which induce a similar antibody profile to that induced by natural infection. Generation of a vaccine that enables differentiation of infected from vaccinated animals (DIVA) would benefit PPR control and eradication programmes, particularly in the later stages of an eradication campaign and for countries where the disease is not endemic. In order to create a vaccine that would enable infected animals to be distinguished from vaccinated ones (DIVA vaccine), we have evaluated the immunogenicity of recombinant fowlpox (FP) and replication-defective recombinant human adenovirus 5 (Ad), expressing PPRV F and H proteins, in goats. The Ad constructs induced higher levels of virus-specific and neutralising antibodies, and primed greater numbers of CD8+ T cells than the FP-vectored vaccines. Importantly, a single dose of Ad-H, with or without the addition of Ad expressing ovine granulocyte macrophage colony-stimulating factor and/or ovine interleukin-2, not only induced strong antibody and cell-mediated immunity but also completely protected goats against challenge with virulent PPRV, 4 months after vaccination. Replication-defective Ad-H therefore offers the possibility of an effective DIVA vaccine.
Peste des petits ruminants virus (PPRV) causes a devastating disease in goats with mortality rates reaching 70% and higher depending on the virus isolate and health of the animals. The virus is widespread throughout Africa, Asia and the Middle East. Clinical signs of disease include leukopenia, pyrexia, congestion of mucosal surfaces, severe ocular and nasal discharge, necrotic stomatitis, diarrhoea and suppression of the immune system often leading to co-infections. Currently, live attenuated PPRV vaccines are available and can protect animals from subsequent infection. However, these vaccines are not thermostable, requiring a cold chain for delivery to the field which is an added issue, as countries most affected by the disease are hot and often have limited infrastructure. While work is in progress in other labs to improve the thermostability of lyophilised PPRV preparations, development of an intrinsically more thermotolerant vaccine, such as poxvirus- or adenovirus-vectored vaccines would be beneficial. Vaccinated animals produce high levels of neutralizing antibodies against the haemaglutinin (H) and fusion (F) proteins as well as non-neutralizing antibodies against the nucleocapsid protein (N), similar to that seen in animals that have recovered from natural infection . These vaccines do not allow infected-recovered animals to be distinguished from vaccinated animals. A vaccine that allows differentiation of infected from vaccinated animals (DIVA) would be of value in PPRV control programmes as well as a PPRV eradication campaign.
Previous studies have suggested that protective immunity against PPRV could be elicited by expression of just the viral glycoproteins. Recombinant vaccinia virus expressing F and H proteins of rinderpest virus (RPV), which is a close relative of PPRV, protected goats against PPRV challenge, although it did not induce PPRV-specific neutralising antibodies . Similarly, recombinant capripox viruses expressing F and H proteins from RPV , or PPRV H or F have been shown to protect goats against PPR . We have sought to evaluate two alternative vectors for expression of the PPRV H and F glycoproteins, fowlpox virus (FP) and replication-defective human adenovirus type 5 (Ad). Recombinant FP-based vaccines have been proven to be effective when used in mammals, despite their inability to replicate in mammalian cells [5, 6]. Replication-defective adenovirus vectors have been shown to be a promising platform for delivery of vaccine antigens in a number of species. Although many conventional vaccines are based on induction of protective antibodies, it is clear that, for many pathogens, induction of CD8+ T-cell responses are critical for rapid clearance of the pathogen . Vaccination with Ad vectors have been shown to elicit better CD8+ T-cell responses compared with poxvirus vectors . The CD8+ T-cell response elicited by Ad5 is predominantly an effector memory phenotype . Ad5 induces a CD8+ T-cell response with a protracted contraction phase and sustained memory population [10–12]. Ad-based vaccines have shown promise as a single dose vaccine in mice against respiratory syncytial virus , Mycobacterium tuberculosis and measles virus .
If such recombinant viruses are to be useful as field vaccines against PPR, it will be important that they are effective after a single dose, since the main cost in large scale vaccination campaigns is taken up by distribution and administration of vaccines. We have therefore investigated the possible adjuvant properties of virus-vectored delivery of cytokines, granulocyte macrophage colony stimulating factor (GMCSF) and interleukin-2 (IL-2). GMSCF is a cytokine important for recruitment, activation and maturation of antigen-presenting cells . In a number of DNA vaccine studies, GMCSF has been shown to have adjuvant properties [17–21]. Furthermore, GMCSF expressed by recombinant FP has been shown to enhance CTL responses in mice compared to vaccination with antigen and recombinant GMCSF protein . However, other groups have found that GMCSF did not enhance CD8+ T-cell or antibody responses [23–25]. IL-2 is involved in activation and recruitment of immune effector cells such as T cells, NK cells, B cells and antigen-presenting cells [26, 27]. Co-delivery of IL-2 with DNA vaccination has been shown to enhance antibody responses and increase protection in a variety of animal species [28–30]. Furthermore, IL-2 in combination with GMCSF has been shown to enhance CTL and memory responses [31, 32] and act in combination to have an increased adjuvant effect [33, 34]. In a number of other animal diseases, addition of IL-2 and GMCSF to vaccines has shown promise as potential adjuvants [35, 36]. We have investigated FP and Ad recombinants as vaccine vectors to deliver PPRV surface glycoproteins to small ruminants. The addition of vectors expressing ovine IL-2 and GMCSF was also investigated. We have shown that a single dose of Ad expressing PPRV H glycoprotein is sufficient to protect goats from PPRV challenge and that the addition of IL-2 may contribute to induction of sterile immunity.
Materials and methods
Recombinant FP virus and recombinant Ad virus expressing ovine IL-2, ovine GMCSF, PPRV H or PPRV F protein, as well as control adenovirus constructs expressing GFP (Ad-GFP)  or an irrelevant antigen (Ag85) (Ad-85)  were produced by the Vector Core Facility, Jenner Institute, Oxford, using Fowlpox 9  and E1, E3 deleted human Adenovirus type 5 (Virapower, Life Technologies) vectors. PPRV F and H coding sequences were derived from the attenuated Nigeria75/1 PPRV vaccine strain  and have been previously published . The plasmids OvIL-2/pGEM-T-Easy and OvGM-CSF/pGEM-T-Easy were the gift of Gary Entrican, Moredun Institute, Edinburgh. Titres of recombinant FP virus stocks ranged from 2 × 108 to 1 × 109 PFU/mL, and titres of recombinant Ad virus stocks ranged from 1 × 1010 to 2 × 1011 IU/mL.
Human embryonic kidney (HEK 293) cells were obtained from ECACC (European Collection of Cell Cultures, catalogue No: 85120602) and cultured in D-MEM containing 10% foetal calf serum (FCS), 100 units/mL penicillin, 100 units/mL streptomycin and 50 μg/mL Nystatin (DMEM complete). Chicken embryonic fibroblast cells (CEFs) were prepared by the Microbiological Services department at the Pirbright Institute, Compton site, from 9 day-old Rhode Island Red embryos obtained from the poultry production unit at the institute.
PPRV F and H specific peptides were synthesised by Mimotopes, Ltd. Peptides were designed against the whole protein sequence of each protein and consisted of 15mer peptides, overlapping by 10 amino acids. Peptides were dissolved in DMSO.
Characterisation of the Ad and FP constructs
For analysis of the expression of PPRV proteins, HEK 293 cells were infected with Ad-F, Ad-H or Ad-GMCSF in 6 well plates for several days until cpe was starting to show. Cells were lysed in SDS-PAGE sample buffer and western blot analysis carried out using an anti-PPRV F monoclonal antibody designed in our laboratory or rabbit polyclonal antibody raised to purified PPRV H (the kind gift of Prof M.S. Shaila, Indian Institute of Science, Bangalore). Parallel samples were analysed using Vero-SLAM cells infected with PPRV. For assays of cytokine expression, HEK 293 cells were infected at a multiplicity of infection (MOI) of 10 in 6 well plates. Virus was allowed to adsorb for 2 h before replacing the inoculum with 2 mL of fresh medium and culturing overnight at 37 °C. The tissue culture supernatants (SNs) were harvested from each well and cell lysates (CLs) were prepared by scraping the cells into 500 μL of sterile water. Chicken embryonic fibroblasts (CEF) were infected with recombinant FP at a MOI of 5 in 6 well plates for 2 h before replacing the inoculum with fresh medium and leaving the cells overnight at 37 °C. CLs and SNs were harvested and stored at -20 °C before analysis of the functional activity of IL-2 and GMCSF produced from infected cells.
GMCSF activity was analysed by measuring the ability of SNs and CLs from Ad- or FP-infected cells to induce proliferation of bone marrow cells. Bone marrow cells were prepared from a fresh goat metacarpal bone. Tissue was rotated in PBS containing 5 mM EDTA at room temperature for one hour to extract cells. The cell suspension was passed first through sterile muslin and then a 70 μm cell strainer before centrifugation for 8 min at 500 × g at 4 °C to pellet cells. Contaminating red cells were lysed in ammonium chloride lysis buffer (0.8% NH4Cl, 0.1 mM EDTA) and the bone marrow cells washed three times in PBS before re-suspending in RPMI/10 containing 10% goat serum (GS), 5 × 10-5 M 2-mercaptoethanol, 100 units/mL penicillin, 100 units/mL streptomycin and 50 μg/mL Nystatin (RPMI complete). Duplicate, 2-fold dilutions of CLs or SNs were incubated with 1 × 105 bone marrow cells per microtitre well. Plates were incubated for 6 days at 37 °C with 5% CO2 and then labelled overnight with [3H]-thymidine. The lymphocyte proliferative responses were expressed as the ratio of cpm in cultures stimulated with CLs or SNs from virus-infected cells to that of cultures stimulated with CLs and SNs from non-infected cells and expressed as a stimulation index (S.I.). The S.I. for CLs and SNs was combined.
IL-2 activity was analysed by measuring the ability of SNs and CLs from Ad- or FP-infected cells to induce the proliferation of peripheral blood lymphocytes. Duplicate, 2-fold dilutions of CLs or SNs were incubated with heparinised goat blood for 6 days before labelling over-night with [3H]-thymidine. The lymphocyte proliferative responses were expressed as the ratio of cpm in cultures stimulated with CLs or SNs from virus-infected cells to that of cultures stimulated with CLs and SNs from non-infected cells and expressed as the S.I.
Goats and experimental design
Male goats, aged between 6 months and 1 year were sourced locally. All were of European breeds, but were of mixed breeds. All animal studies were carried out in accordance with UK Home Office regulations and under the supervision of the local Ethical Committee. Animals were vaccinated intra-muscularly in the left shoulder with vaccine made up to 1 mL with sterile PBS. The doses of recombinant virus vectors used to vaccinate goats were similar to those that had previously been used in man and goats [39, 42].
One animal was vaccinated with a mixture of 1 × 109 IU Ad-F and 1 × 109 IU Ad-H and the other goat was vaccinated with a mixture of 1 × 108 PFU FP-F and 1 × 108 PFU FP-H. Blood was taken weekly for sera and preparation of peripheral blood mononuclear cells (PBMC). Animals were given an homologous boost, 5 weeks later, and were killed 8 weeks post vaccination by intravenous pentobarbitone overdose. At post mortem examination, pre-scapular lymph nodes (PLN) were removed for analysis of H- and F-specific T-cell responses.
Four goats were vaccinated with a mixture of 1 × 109 IU Ad-F and 1 × 109 IFU Ad-H and four goats were vaccinated with a mixture of 1 × 108 PFU FP-F and 1 × 108 PFU FP-H. Two animals from each group were also vaccinated with a mixture of 1 × 109 IU Ad-IL-2 and 1 × 109 IU Ad-GMCSF or a mixture of 1 × 108 PFU FP-IL-2 and 1 × 108 PFU FP-GMCSF respectively. Blood was taken weekly for sera and preparation of PBMC, and animals were killed 12 weeks post vaccination by intravenous pentobarbitone overdose. At post mortem examination, PLN were removed.
Vaccine groups for challenge study
1 × 109 IU
1 × 109 IU
1 × 109 IU
2 × 109 IU
1 × 109 IU
1 × 109 IU
1 × 109 IU
1 × 109 IU
1 × 109 IU
1 × 109 IU
1 × 109 IU
1 × 109 IU
1 × 109 IU
1 × 109 IU
≤ Initial + 0.1
Initial + 0.1 to ≤ initial + 2
> Iinitial + 2
1 or 2 vesicles in gums
Nasal swabs were washed in medium (DMEM containing 10% FCS, 100 units/mL penicillin, 100 units/mL streptomycin) to extract virus from the swabs and then serial dilutions were plated with VDS cells in at least quadruplicate, starting with a 1 in 10 dilution, for determination of virus titre by TCID50. Alternatively, a 1 in 6 dilution of the sample was used to inoculate VDS cells for virus isolation (VI). Infected cells were cultured for up to 7 days at 37 °C with 5% CO2. Wells were scored for the presence of cpe.
Blood samples and swabs taken after PPRV challenge were analysed by reverse transcription-real time PCR  to determine the level of viraemia and virus in nasal secretions. RNA was extracted from EDTA blood or swabs, reverse transcribed and amplified with SuperScript® III Platinum® One-Step qRT-PCR w/ROX Kit, (Invitrogen™). Primer 5’-3’sequences were, PPRVFOR:- AGAGTTCAATATGTTRTTAGCCTCCAT;
probe: FAM-CACCGGAYACKGCAGCTGACTCAGAA – Tamra.
Primers were used at 10 pmol/μL and the probe at 5 pmol/μL. The thermal profile was 50 °C for 30 min, 95 °C for 10 min, then 45 cycles of 95 °C for 15 s followed by 60 °C for 1 min.
Isolation of PBMCs and lymph node cells
PBMC were prepared from heparinised venous blood by centrifugation at 1200 g over Histopaque 1083 (Sigma-Aldrich, Inc.) for 45 min at 20 °C. Cells were washed three times in PBS before re-suspension in RPMI/10 complete medium. Pieces of lymph node were pushed gently through a sterile, metal tea strainer into a petri dish containing PBS, 200 units/mL penicillin, 200 units/mL streptomycin and 100 μg/mL Nystatin. The cell suspension in PBS was further filtered through a 100 μm cell strainer, and cells were purified on histopaque gradients as described above for PBMCs and then cultured in RPMI/10 complete medium. Spare cells were also frozen in FCS with 10% DMSO.
Serum antibodies specific for the PPRV H glycoprotein were analysed by competition ELISA (cELISA) using a PPR Antibody ELISA kit (BDSL). Antibodies specific for the PPRV N protein were detected by cELISA using the ID Screen PPR Competition kit (ID Vet).
PPRV neutralising antibodies were analysed essentially as described in the OIE manual, chapter 2.7.11, section 3a. Sera was heat inactivated at 56 °C for 30 min and serially diluted two-fold in quadruplicate. PPRV Nigeria 75/1 was diluted in media to give 100-150 infectious units/50 μL and incubated with the diluted serum at 37 °C in 5% CO2 for one hour. Vero-dog-SLAM (VDS) cells were added to the virus/serum mixture at 1 × 105 cells per microtitre well. Plates were incubated for one week and then scored for the presence or absence of cytopathic effect. The neutralising titre was the reciprocal of the highest dilution that completely blocked CPE in 50% of infected wells. Neutralising titres were calculated using the Spearman-Kärber equation to determine TCID50.
PBMCs were stimulated with either peptide pools, at final concentration of 10 μg/mL, or with Ad or FP viruses. Stimulation with Ad was at a MOI of 100 virus particles and FP at a MOI of 0.1 PFU. Concanavalin A was used as a positive control at a final concentration of 25 μg/mL. All assays were performed in triplicate. The lymphocyte proliferative responses were expressed as the ratio of counts incorporated in cultures stimulated with peptide to counts incorporated in cultures with media containing DMSO at the same concentration as the peptides, or the ratio of counts in cultures stimulated with Ad or FP expressing PPRV antigens to counts in cells stimulated with a corresponding virus expressing an irrelevant antigen. Data is expressed as the S.I., and an S.I. greater than 5 was considered to be significant.
PBMCs and lymphocytes from the PLN were stimulated with peptide pools, or Ad or FP viruses as described above for 24 h and brefeldin A was added for the last 4 h of culture. Live cells were stained using live/dead aqua (Molecular probes®), and monoclonal antibodies (mAbs) to surface markers CD4 conjugated to allophycocanin (APC) (clone 44.38, MCA2213A647 from Serotec) and CD8 conjugated to R-Phycoerythrin (RPE) (clone CC63, MCA837 from Serotec). Cells were permeabilised with BD FACS permeabilisation buffer and stained with mAb to IFNγ (clone CC327, MCA2334 from Serotec). Cells were analysed using the LSR Fortessa (BD Biosciences). The DIVA software was used to acquire the data and FCS Express 3 (De Novo Software) or FlowJo (Tree Star Inc.) used for analysis. The PPRV-specific response was calculated as the percentage of IFNγ+ -producing cells after stimulation with Ad or FP constructs expressing the PPRV protein minus the percentage of such cells after stimulation with Ad or FP expressing an irrelevant antigen.
At day 0, 4 and 7 pc with PPRV, heparinised blood was stained with antibodies for surface markers CD4, CD8, using antibodies described above, annexin V antibody conjugated to FITC (Aposcreen kit, SouthernBiotech), CD14 (clone CCG33) antibody conjugated to R-PE and antibody to WC1, a marker for γδ T cells (clone 197), conjugated to APC using a Zenon® kit (Life Technologies). Red blood cells were lysed using BD FACS lysis solution. Cells were fixed in 4% PFA and permeabilised as above. Intracellular virus was detected using the anti-PPRV H monoclonal antibody C77 conjugated to Alexa Fluor-405 using a Zenon® kit (Life Technologies).
Characterisation of Ad and FP vaccine vectors expressing PPR glycoproteins, ovine IL-2 or ovine GMCSF
The production of biologically active GMCSF by the recombinant vaccine vectors was confirmed by the finding that CLs and SNs from cells infected with Ad-GMCSF (Figure 1B) or FP-GMSCF (data not shown), but not from mock-infected, Ad-H or FP-H-infected cells, induced the proliferation of goat bone marrow cells. The production of biologically active IL-2 was confirmed by the finding that SNs from Ad-IL-2 (Figure 1C) or FP-IL-2-infected cells (data not shown), but not mock-, Ad-H or FP-H infected cells induced proliferation of goat lymphocytes.
Immune responses induced in goats vaccinated with Ad or FP expressing PPRV glycoproteins
Effect of virus-vectored ovine IL-2 and GMCSF on immune responses induced in goats vaccinated with Ad or FP constructs expressing PPRV glycoproteins
From week 2 post-vaccination to week 12, sera from Ad-vaccinated animals showed a significantly greater inhibition of binding of an H-specific mAb to PPRV antigen than sera from FP-vaccinated animals (p < 0.0001; 2-way ANOVA) (Figure 3C). Furthermore, the average percentage inhibition by sera from the Ad-vaccinated animals that had also been given Ad-IL-2 and Ad-GMCSF, was significantly greater than the average of the two goats vaccinated with Ad-F and Ad-H only (p < 0.05), from week 4 post vaccination to week 12. PPRV-specific neutralising antibodies were not detected in any FP-vaccinated animals (Figure 3D). In contrast, all Ad-vaccinated animals developed neutralising antibodies from week 2 post vaccination, which reached a peak between 2 to 5 weeks after vaccination, and then gradually declined, although 3 out of 4 goats still had neutralising antibody titres > log10 1.0 at 12 weeks after a single vaccination with Ad-vectored vaccines. One goat, which was also vaccinated with Ad-IL-2 plus Ad-GMCSF, had very high levels of neutralising antibodies which peaked 5 weeks after vaccination and remained high at 12 weeks post vaccination.
Adenovirus vaccination protects against challenge with PPRV
PPRV H-specific IFNγ-producing CD8+ cells were detected in all Ad-vaccinated goats 3 weeks after vaccination and the proportion of these cells was highest in animals also vaccinated with Ad-GMCSF (Figure 4B). The proportion of H-specific CD8+ IFNγ+ cells at the time of PPRV challenge (week 15) was low and similar to that seen prior to vaccination. However one week after challenge, an increase in H-specific IFNγ producing CD8+ cells was observed in all the vaccinated animals except the Ad-H + Ad-GMCSF group. In contrast, H-specific IFNγ-producing CD4+ T-cells were seen only at very low levels throughout the study, although there was a slight increase in the proportion of CD4+ IFNγ+ cells in Ad-H + Ad-IL-2 vaccinated animals one week after challenge (Figure 4C). As seen previously, Ad vaccination was more effective at priming CD8+ cells than CD4+ cells.
In order to assess the potential of Ad-H as a DIVA vaccine, we analysed the development of antibodies to the PPRV nucleocapsid (N) protein in vaccinated goats before and after challenge with PPRV. As expected, none of the animals had N-specific antibodies before challenge. However, N-specific antibodies were detected from day 7 pc in the controls (Figure 5C). N-specific antibodies were slower to develop in the Ad5-H vaccine groups and were first detected at days 11 and 14 pc. Analysis of the level of N-specific antibodies suggested that they were significantly lower in goats vaccinated with Ad-H plus Ad-IL-2 (P < 0.001). Ad-GMCSF did not appear to have an effect on induction of N-specific antibodies after challenge.
PPRV in the nasopharynx 7 days post challenge (pc)
Mean PPRV titre in nasopharynx (log10TCDI50/mL)b
Ad-GFP + Ad-IL-2 + Ad-GMCSF
3.88 +/- 0.84 (4/4)c
Ad-H + Ad-GFP
< 0.1 (0/3)
Ad-H + Ad-IL-2
< 0.1 (0/3)
Ad-H + Ad-GMCSF
< 0.1 (0/3)
Ad-H + Ad-IL-2 + Ad-GMCSF
< 0.1 (0/3)
In light of the recent success of the rinderpest eradication campaign, PPRV would be an excellent candidate for eradication [44, 45] and moves have already been made towards this. The availability of a DIVA vaccine would facilitate PPRV sero-surveillance programmes and speed up the steps leading to disease eradication . In countries newly affected by PPRV, where sporadic outbreaks of disease occur and where the disease is not endemic, a DIVA vaccine would be of value to prevent animal movement restrictions being imposed on countries which cannot prove that animals have been vaccinated and not infected. Since the virus has only 6 genes, all of which are essential for growth, creating a DIVA version of the current live PPRV vaccines would require expressing an extra protein from the viral genome (positively marked vaccine). The alternative is to express one or two viral proteins from an alternative virus vector, thereby eliciting immune protection while not inducing the complete repertoire of antibodies induced following natural infection or vaccination with a live, attenuated PPRV vaccine. This was done successfully for RPV using vaccinia or capripox virus as the vaccine vectors [47–50]. However, these constructs were never used in the field, in part because they did not offer the same duration of protection as the existing vaccine and because the rinderpest eradication campaign was completed without an explicit requirement for a DIVA vaccine. MVA expressing PPRV F and H proteins have been shown to protect goats against subsequent challenge with virulent PPR but two doses of vaccine were given prior to challenge , which would not be practical for a small ruminant vaccination programme. Capripox virus vectors expressing PPRV glycoproteins have been developed [4, 52]. However, in one case, the ability of the vaccine to protect against PPRV was not investigated , while in the other, although protection against PPRV was proven , the vaccine has not been used in the field. The positive aspects of using a recombinant capripox are that it would simultaneously vaccinate against two serious sheep/goat diseases; in addition, capripox-based vaccines would benefit from the intrinsic thermotolerance of poxviruses. However, recombinant capripox vaccines may not be suitable as PPRV DIVA vaccines, as vaccinated animals did not all give good antibody responses, possibly due to pre-existing vector immunity. This is important as it is the comparative antibody response that is likely to be used as the DIVA test, with infected animals having anti-PPRV N and anti-PPRV H antibodies, while the vaccinees will only have anti-PPRV H antibodies. There are existing, well established and validated commercially available cELISAs for both anti-N and anti-H antibodies, making this an attractive DIVA test.
Despite their successful use in several trials as vectors for human vaccines, the FP-based vaccines elicited very poor antibody responses in goats, as well as poor cell-mediated immune responses. The low level of responses in small ruminants may be due to apoptosis of FP virus-infected antigen presenting cells in these animals, as has been shown recently in cattle . Because of the low level of immune response to the PPRV proteins expressed from the FP vectors, we did not pursue these constructs through to challenge, since they would not be useful DIVA vaccines even if effective in protecting the vaccinated animals from PPRV.
Replication-deficient adenovirus-vectored vaccines induce potent CD8+ and CD4+ T-cell responses as well as high antibody responses , and appear to be safe . Furthermore, the Ad vector also acts as an adjuvant . Adjuvant effects in experimental vaccines have been demonstrated by co-expressing cytokines such as IL-2, IL-12 and GMCSF , presenting other options for vaccine formulation. One of the drawbacks of Ad5-vectored vaccines in humans has been that most people have previously been infected with this virus, and the pre-existing antibodies can inhibit the efficacy of the vaccine . However, vaccines based on Ad5 may be suitable for use in livestock since these animals will not have pre-existing immunity to the vector. Large scale production of Ad viruses can be achieved  and, furthermore, Ad viruses can be made more thermostable and efficacious in a range of formulations that further promote stability [59, 60].
Vaccination of goats with Ad-H, alone or with a similar dose of Ad-F, induced levels of H-specific, neutralising antibodies within 2 to 3 weeks that were comparable to those induced by live, attenuated PPRV vaccines [40, 61]. Furthermore, these antibodies were maintained for several months following vaccination. While this work was in progress, similar Ad constructs were reported , which induced a similar level of neutralising antibody as that described in the present study, following a single dose of replication defective Ad-H. In another recent study, replication competent canine adenovirus expressing PPRV H was also found to be effective at eliciting neutralising antibody in goats . Unfortunately, neither of these studies went on to determine the ability of the Ad-vectored vaccines to protect against virulent PPRV. This is important, as the critical elements of the immune response required for protection against PPRV are not yet known. While the current live attenuated PPRV vaccine induces neutralising antibody, and a titre > 1:10 is used as a marker for competency of preparations of such vaccines, attenuated morbillivirus vaccines also induce cell-mediated immunity [64, 65], which may also be important in protection. We have demonstrated that vaccination with Ad-H, or Ad-H and Ad-F, induced a potent effector memory CD8+ response in goats, although the number of H-specific CD8+ IFNγ+ cells had declined to basal levels by 15 weeks post-vaccination. Further studies are therefore needed to determine the effect of Ad-H vaccination on persistence of central memory CD8+ T cells in goats. No detailed studies have been carried out to establish the mechanisms of protection induced by live, attenuated PPRV vaccines. However, studies on rinderpest showed that induction of neutralising antibodies by vaccination with purified viral proteins did not protect against infection , suggesting that it is not possible to deduce protection based on antibody alone. We therefore analysed the ability of Ad-vectored vaccines to protect against infection with virulent PPRV, and demonstrated that a single dose of Ad expressing the PPRV H protein can protect against PPRV challenge up to 4 months after vaccination. Furthermore, vaccinated goats did not appear to excrete infectious virus from the nasopharynx, suggesting that they may not transmit virus to susceptible, unvaccinated animals. This is the first time that an Ad-vectored vaccine has been shown to protect against virulent PPRV challenge, and shows that the immune responses elicited by the replication-defective vaccine are sufficient to protect the vaccinated animal from infection, with a protective response that is sustained for at least 4 months. Longer term studies will be required to determine the duration of PPRV H-specific serum antibodies induced by Ad-H.
In the study by Wang et al. , it was suggested that co-expression of F and H proteins induced higher levels of neutralising antibody than vaccination with Ad expressing either F or H alone. This is a similar finding to that seen in cattle vaccinated with vaccinia virus expressing RPV H, F or both H and F, where it was suggested that the combination of H and F induced stronger protection against RPV . In our studies, expression of H alone (experiment 3) induced neutralising antibody titres at least as high as those seen in animals vaccinated with Ad-F plus Ad-H (experiment 2). This may be because we gave a higher dose of Ad than that used by Wang et al. , and the strength of the response to H alone, coupled with the adjuvant effect of Ad, dominated any co-operative effect of vaccination with Ad H and F together.
This is the first time that the effect of virulent PPRV infection on specific immune cell sub-sets has been analyzed. We have shown that whereas the proportion of circulating WC1+ γ/δ T-cells and CD14+ monocyte/macrophage cells did not change after PPRV infection of control goats, there was a decrease in the proportion of circulating CD4+ cells 4 days after challenge. This decrease may have been due, at least in part, to infection with PPRV, as the proportion of CD4+ cells staining for intracellular H was greater than that of CD8+ cells, 4 days after PPRV challenge. The reduction in CD4+ cells was not observed in any of the Ad-H vaccinated animals. There was a slight increase in the percentage of CD8+ T-cells at 7 days pc in all animals, suggesting induction of CTL responses by PPRV infection. Co-administration of Ad-GMCSF at the time of vaccination appeared to have an effect on infection of CD4+ and CD8+ cells with PPRV. These animals had lower levels of detectable intracellular viral H protein in both the live CD4+ and live CD8+ T cells 4 days after challenge, compared with the other vaccine groups.
The contribution of GMCSF and IL-2 in boosting immune responses to Ad vaccination is not clear from this study. However, the results from the 2nd experiment suggested that the combination of Ad-IL-2 and Ad-GMCSF induced higher H-specific serum antibodies and a greater H-specific CD8+ IFNγ+ response compared with Ad-F and Ad-H alone. The level of N-specific antibodies after challenge with PPRV was significantly lower in the two vaccine groups that received Ad-IL-2 compared with the other groups of goats, suggesting that there was less replication of the challenge virus in these animals and, therefore, that co-administration of Ad-IL-2 induced a more effective protective immune response, even though a significant effect on T cell responses, H-specific or neutralising antibody levels was not seen. It will be interesting to determine if an adjuvant effect of the co-expressed cytokines is more obvious at lower doses of Ad-H/Ad-F. If such studies demonstrate that a co-administered Ad-vectored cytokine has a dose sparing effect on an Ad-vectored PPRV vaccine, then it may be possible to construct a recombinant Ad, which can have an insert of ~7.5 kb, expressing both PPRV H and cytokine.
In conclusion, we have demonstrated that a single vaccination with a recombinant Ad expressing the PPRV H protein induced PPRV-specific neutralising antibodies, primed CD8+ T cells, was safe, and completely protected goats against PPR for at least 4 months.
This research was funded by grant A642 from the BBSRC and DFID. The authors thank the Animal Service team at The Pirbright Institute for all their help. The Vector Core Facility at the Jenner Institute, Oxford, UK, made and propagated the recombinant viral vectored vaccines. GT is a Jenner Institute Investigator.
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