Determination of the minimum fully protective dose of adenovirus-based DIVA vaccine against peste des petits ruminants virus challenge in East African goats
© Holzer et al. 2016
Received: 5 October 2015
Accepted: 5 January 2016
Published: 21 January 2016
Peste des petits ruminants virus (PPRV) causes an economically important disease of sheep and goats, primarily in developing countries. It is becoming the object of intensive international control efforts. Current vaccines do not allow vaccinated and infected animals to be distinguished (no DIVA capability). We have previously shown that recombinant, replication-defective, adenovirus expressing the PPRV H glycoprotein (AdH) gives full protection against wild type PPRV challenge. We have now tested lower doses of the vaccine, as well as AdH in combination with a similar construct expressing the PPRV F glycoprotein (AdF). We show here that, in a local breed of goat in a country where PPR disease is common (Kenya), as little as 107 pfu of AdH gives significant protection against PPRV challenge, while a vaccine consisting of 108 pfu of each of AdH and AdF gives apparently sterile protection. These findings underline the utility of these constructs as DIVA vaccines for use in PPR control.
Peste des petits ruminants (PPR) is an important disease of sheep and goats which has recently become a major international target for improved control, marked by the adoption in 2014 of a resolution by the World Organisation for Animal Health (OIE) to establish a control programme with a view to eventual eradication of the disease . The disease is caused by a morbillivirus, PPR virus (PPRV), closely related to the human pathogen measles virus (MV), as well as other animal pathogens such as canine distemper virus (CDV) and rinderpest virus (RPV). PPRV is widely distributed through large parts of Africa, the Middle East and Asia and is responsible for significant economic losses, primarily in developing countries [2–5].
Disease control is mostly achieved through the use of clinical or laboratory-based diagnosis coupled with vaccination. All the vaccines currently in use are attenuated strains of PPRV [6, 7]; these vaccines are effective, though they do not provide a DIVA (distinguishing infected from vaccinated animals) capability. These vaccines cause what is essentially a subclinical infection with PPRV, and therefore the antibody signatures of vaccinated and previously-infected animals are identical. Several alternative DIVA vaccines have been proposed based on recombinant viruses [8–13]. We have shown that recombinant replication-defective human adenovirus type 5 (Ad5) expressing the H surface glycoprotein of PPRV can act as a DIVA vaccine, inducing good levels of antibodies and protecting goats from experimental challenge with a pathogenic PPRV 4 months post vaccination . Similar constructs have also been shown to be immunogenic in other studies [10, 11] or, more recently, both immunogenic and protective in sheep . We have carried out an extended study of such recombinant adenovirus constructs in goats in East Africa, an area where PPR is endemic. Using local animals, we have analysed the immunogenicity and protective efficacy of different doses of vaccine, in order to determine the minimum protective dose. We have also compared the protection induced by Ad5-H alone to that induced by a vaccine combining Ad5-H with a similar construct expressing the other PPRV surface glycoprotein, F.
Materials and methods
Cells and viruses
Vero cells expressing the canine version of the morbillivirus receptor SLAM (signalling lymphocyte activation molecule) (vero-dog-SLAM, VDS) were obtained from Dr Paul Duprex, then at Queen’s University Belfast, N. Ireland, and maintained in Dulbecco’s modified Eagle’s medium containing 25 mM HEPES buffer, penicillin (100 U/mL), and streptomycin (100 µg/mL) (DMEM) containing 10% foetal calf serum (FCS). Zeocin was included at 0.1 mg/mL to maintain selection for SLAM expression. PPRV Ivory Coast/89 isolate  and recombinant PPRV rPPRV-GFP  were propagated and titrated in VDS cells. Titres were determined as the 50% tissue culture infectious dose (TCID50), calculated by the method of Spearman and Kärber . Recombinant adenoviruses expressing ovine IL-2 (AdIL-2), PPRV H (AdH) or PPRV F (AdF), as well as the control adenovirus construct expressing GFP (AdGFP) were those previously described .
Amounts of each recombinant adenovirus given to each animal in the experimental groups
107 AdH + 107 AdGFP
108 AdH + 108 AdGFP
107 AdH + 107 AdIL-2
108 AdH + 108 AdIL-2
107 AdH + 107 AdF
108 AdH + 108 AdF
107 AdH + 107 AdF + 107 AdIL-2
108 AdGFP + 108 AdIL-2
Assays for serum antibodies specific for the PPRV H glycoprotein and the PPRV nucleocapsid (N) protein, for PPRV neutralising antibodies were as previously described  Viral RNA was assayed in 6% of the RNA extracted from 100 μL EDTA blood, using reverse transcription-real time PCR as described .
Comparisons between groups were performed using linear mixed models in which animals were taken to be random factors and the other factors as fixed; calculations were performed using the nlme package in R. Multiple comparisons were carried out by using the Tukey corrected 95% confidence intervals as implemented in the R package multcomp, and quoted probability (p) values are from that package.
Previous studies demonstrated that a single dose of 109 pfu of AdH induced complete protection against PPRV, when goats were challenged 4 months after vaccination. Therefore, the present animal study was designed to determine if (i) the AdH vaccine is still protective at 108 or 107 pfu per animal; (ii) it delivers improved protection when combined with AdF (as suggested by ); (iii) there is an adjuvant effect of co-expressing IL-2, as suggested by some data from our previous study . Seven groups, of six animals each, were vaccinated with different amounts and combinations of replication-defective adenovirus constructs as described in “Materials and methods” section, with an eighth group kept as control animals. Six animals of the total 48, from different groups, died of an unspecified respiratory infection between vaccination and challenge, so only partial serology data is available for those animals.
PPRV-specific serum neutralising antibodies were also analysed (Figure 1B). The two assays gave broadly comparable results, although the cELISA was more sensitive. Again, group 5 showed lower antibody responses during the period prior to challenge than the other groups, although this time there was no statistically significant difference between groups 5 and 3. Group 7 animals showed a significantly higher neutralising antibody response than group 5 (p = 0.034), but were not significantly different to any of the other groups. Group 3 was significantly lower than groups 4 and 6. Notably, inclusion of AdF in the vaccination inoculum either reduced the neutralising antibody response (group 1 vs group 5) or made no difference (group 2 vs group 6; group 3 vs group 7).
Development of antibodies to other PPRV proteins
Number positive for anti-N
antibodies/Number in group
Comparison of the immunogenicity and protective efficacy of different doses of Ad-expressed PPRV vaccines demonstrated that the recombinant adenovirus expressing the PPRV H, previously shown to be protective at a dose of 109 pfu per animal , is also protective at 108 pfu per animal (no detectable viraemia, limited replication of challenge virus). At 109 pfu per animal, in addition to protection from clinical PPRV, we also found complete suppression of viraemia, although all the animals developed anti-N antibodies, showing that this test is an extremely sensitive test for replication of the challenge virus. At the lowest dose of AdH (107 pfu per animal), there was still a strong antibody response, and no viraemia, but more (3 out of 5) of the animals developed anti-N antibodies.
Our data support the suggestion that the combination of AdH and AdF may be more protective than AdH alone, since group 6 restricted the challenge virus better than group 2, although the differences were marginal. However, we did not observe the increased PPRV-neutralising titre seen by Wang et al.  in goats vaccinated with an adenovirus expressing PPRV H and F. A significant difference between these two studies is that, in the present case, we expressed each protein separately, so each had the possibility to fold into its native conformation. In the study by Wang et al. , F and H were expressed as a single fusion protein. Under these circumstances, neither protein is likely to fold normally. It has been shown that such misfolded proteins can be bound directly by MHC class II ; in addition, such a protein will be removed from the ER by the normal quality control system (reviewed in ) and degraded by the ER-associated protein degradation system, rapidly generating F/H protein-derived peptides which can be presented by both class I and class II systems [21, 22]. Such processes may have led to the higher levels of anti-PPRV antibody observed in response to their (F + H) fusion protein compared to AdF or AdH alone.
While it may not have played a role in their reported increased antibody response to Ad(F + H) compared to AdH, Wang et al. also vaccinated goats with two doses of adenovirus-vectored vaccine, whereas we have considered it desirable to have a vaccine that functions after a single dose, since this will be more practical in developing countries where the major cost and effort is in delivering vaccine to the animals.
The overall conclusion was therefore that the vaccine consisting of 108 (AdH + AdF) appears to be completely effective and provides a DIVA vaccine capability when used in conjunction with the existing commercial cELISAs, which recognise antibodies to PPRV H or PPRV N. The protection was complete at 3 months post vaccination and with only a single dose of vaccine. However, a longer term study (at least 1 year) would be advisable to establish the duration of protection and the duration of detectable marker antibody (anti-H antibody).
A very interesting observation was that there appears to be a significant difference in the pathogenicity of the PPRV isolate in UK and E. African goats. Although this isolate (Ivory Coast/89) has been clearly virulent in studies at TPI, the same virus had little effect on the native breed of goats used at ILRI; transient high temperatures were seen in several of the control animals, and none in the vaccinated animals (not shown), but this was not consistent across the group. Mild clinical signs were sometimes observed in different animals, but it was clear that the animals were not being kept isolated from other infectious agents, as six animals died during the time between vaccination and challenge, and the significance of slight nasal discharge for 1 day is limited. The reasons for the lack of severe clinical signs of disease in the control animals, despite clear virus replication, and prolonged viraemia in most cases, are not clear. One possibility is that there is significant and general resistance to PPRV in the breed of goat used or, alternatively, resistance may be due to an interaction of virus and host strain, so that viruses may appear pathogenic or mild depending on what hosts they have recently been in (the challenge strain had been passaged in UK goats). This has implications for surveillance for disease, as virus may be mild or inapparent when first entering a new country or an area with a different breed of goats or sheep.
Studies are necessary in local breeds of goats using local isolates of PPRV as well as other known pathogenic isolates. If some animals are able to support virus growth, and possibly spread, without showing significant clinical signs, this would make control measures based on clinical surveillance alone insufficient.
The authors declare that they have no competing interests.
BH prepared the vaccine inocula, prepared all animal samples during the vaccination period and carried out most of the serology. GT initiated the study, helped design it, and prepared samples from animals during the challenge stage of the study. PRN carried out the real-time PCR tests of post-challenge blood samples. EO carried out all the administration of vaccines and challenge virus to the experimental animals. PT supervised the study at ILRI. SH carried out the N protein cELISA. RH contributed to the study design. MDB helped designed the study, prepared the challenge virus, carried out the pathogenic challenge, prepared samples from animals during the challenge stage and prepared the manuscript. All authors read and approved the final manuscript.
The work was funded by the Bill and Melinda Gates Foundation Grand Challenges Explorations scheme, award number OPP1098823, and the BBSRC Institute Strategic Programme on Livestock Viral Diseases at The Pirbright Institute. The recombinant adenovirus stocks were prepared by the Viral Vector Core Facility, Jenner Institute, Oxford. GT is a Jenner Investigator.
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