Immunisation with a Multivalent, Subunit Vaccine Reduces Patent Infection in a Natural Bovine Model of Onchocerciasis during Intense Field Exposure

Human onchocerciasis, caused by the filarial nematode Onchocerca volvulus, is controlled almost exclusively by the drug ivermectin, which prevents pathology by targeting the microfilariae. However, this reliance on a single control tool has led to interest in vaccination as a potentially complementary strategy. Here, we describe the results of a trial in West Africa to evaluate a multivalent, subunit vaccine for onchocerciasis in the naturally evolved host-parasite relationship of Onchocerca ochengi in cattle. Naïve calves, reared in fly-proof accommodation, were immunised with eight recombinant antigens of O. ochengi, administered separately with either Freund's adjuvant or alum. The selected antigens were orthologues of O. volvulus recombinant proteins that had previously been shown to confer protection against filarial larvae in rodent models and, in some cases, were recognised by serum antibodies from putatively immune humans. The vaccine was highly immunogenic, eliciting a mixed IgG isotype response. Four weeks after the final immunisation, vaccinated and adjuvant-treated control calves were exposed to natural parasite transmission by the blackfly vectors in an area of Cameroon hyperendemic for O. ochengi. After 22 months, all the control animals had patent infections (i.e., microfilaridermia), compared with only 58% of vaccinated cattle (P = 0.015). This study indicates that vaccination to prevent patent infection may be an achievable goal in onchocerciasis, reducing both the pathology and transmissibility of the infection. The cattle model has also demonstrated its utility for preclinical vaccine discovery, although much research will be required to achieve the requisite target product profile of a clinical candidate

Validation of B-cell epitopes in the mosaic BVDV antigens.

<p>Authenticity of the adenovirus-expressed novel BVDV mosaic antigens was confirmed by immunocytometric analysis using E2-specific neutralizing monoclonal antibodies 26A and 348 (both neutralize BVDV-1 & 2); bovine anti-BVDV hyper-immune serum (generated by immunizing steers multiple times with BVDV-1 & 2 vaccines followed by boosting with killed diverse BVDV-1 & 2 strains and then challenged with wild type BVDV-1 & 2 strains (The sera have high BVDV-1 & 2 neutralizing titers [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170425#pone.0170425.ref059" target="_blank">59</a>]); and goat anti-BVDV polyclonal serum generated against multiple wild-type BVDV-1 & 2 strains. A) HEK-293A cells expressing N^proE2^1-3; B) HEK-293A cells expressing NS^2-31; C) HEK-293A cells expressing NS^2-32; and D) HEK-293A cells expressing luciferase.</p

Virus isolation from calves on day 7 and day 10 post-challenge.

<p>Virus isolation from calves on day 7 and day 10 post-challenge.</p

Clinical manifestations post-challenge.

<p>A) Mean rectal temperature fluctuation; and B) Mean change ratios of white blood cell counts in the vaccinated and negative control groups post-challenge. Asterisks denote statistically significant differences as compared to the negative controls. *P<0.05; **P<0.01 and ***P<0.001.</p

Persistence of replication-incompetent adenovirus in cattle.

<p>Viable recombinant adenovirus inoculated intradermally is only recoverable within three days. Presence of adenovirus rescued from tissue samples of four steers at defined time points was tracked by immunocytometric analysis of HEK-293A cells. Representative data from one steer is shown: A, D, G, J, and M are positive controls at 24 hr., 48 hr., 72 hr., day 7, and day 21, respectively. B, E, H, and K, are skin biopsies taken from the inoculation sites on the neck of the steers at 24 hr., 48 hr., 72 hr., and day 7, respectively, whereas C, F, I, and L, are cognate control skin biopsies taken concurrently from the flank. N and O are draining lymph node and spleen samples, respectively, collected three weeks post-inoculation.</p

Validation of mosaic antigens using BVDV-specific T-cells.

<p>Authenticity of T-cell epitopes in the mosaic BVDV antigens was validated by proliferation assay using PBMCs from a BVDV-1 & 2 hyper-immune steer [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170425#pone.0170425.ref059" target="_blank">59</a>]. The data shown is minus background counts from negative control (media alone) treatment. The asterisks denote a statistically significant difference (P<0.01) between the proliferation induced by the N^proE2^1-3, NS^2-31 and the NS^2-32 antigens and both whole killed viruses BVDV-1b and BVDV-2. This outcome is representative of assays conducted using PBMCs from other BVDV immune steers.</p

Mosaic BVDV vaccine induced BVDV-1 specific neutralizing antibodies.

<p>Serum neutralization assays were used to evaluate BVDV-1-specific neutralization titers at A) One-week post-boost; and B) one-week pre-challenge against five BVDV type 1 strains. Mean group titers are represented by the bars. Statistically significant differences between the groups are denoted by asterisks. *P<0.05; **P<0.01.</p

Immunization timeline.

<p>On day -228 pre-challenge, cattle in the treatment group were vaccinated with a cocktail of the recombinant adenoviruses expressing mosaic BVDV antigens (AdBVDV), whereas positive control cattle received a commercial MLV BVDV vaccine. Negative control cattle were inoculated with the recombinant AdLuc. On day -149 pre-challenge, the cattle were boosted with the respective priming inoculum and dose (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170425#pone.0170425.t001" target="_blank">Table 1</a>). On day 0, all the cattle were challenged by intranasal delivery of a BVDV-1373 using an atomizer. Blood samples were collected on selected days (0, 3, 6, 10, 12, 13 and 15), whereas clinical observations and rectal temperatures were monitored and recorded daily from days 1–15 post-challenge.</p