Vaccines to combat river blindness: expression, selection and formulation of vaccines against infection with Onchocerca volvulus in a mouse model

Abstract

Human onchocerciasis is a neglected tropical disease caused by Onchocerca volvulus and an important cause of blindness and chronic disability in the developing world. Although mass drug administration of ivermectin has had a profound effect on control of the disease, additional tools are critically needed including the need for a vaccine against onchocerciasis. The objectives of the present study were to: (i) select antigens with known vaccine pedigrees as components of a vaccine; (ii) produce the selected vaccine antigens under controlled conditions, using two expression systems and in one laboratory and (iii) evaluate their vaccine efficacy using a single immunisation protocol in mice. In addition, we tested the hypothesis that joining protective antigens as a fusion protein or in combination, into a multivalent vaccine, would improve the ability of the vaccine to induce protective immunity. Out of eight vaccine candidates tested in this study, Ov-103, Ov-RAL-2 and Ov-CPI-2M were shown to reproducibly induce protective immunity when administered individually, as fusion proteins or in combination. Although there was no increase in the level of protective immunity induced by combining the antigens into one vaccine, these antigens remain strong candidates for inclusion in a vaccine to control onchocerciasis in humans.

Keywords: Mice; Multivalent vaccine; Onchocerca volvulus; Vaccine.

Fig. 1

SDS-PAGE and Coomassie staining of purified recombinant Onchocerca volvulus Ov-103, Ov-RAL-2 and Ov-CPI-2M proteins expressed in Pichia pastoris (yeast) and Escherichia coli. SeeBlue pre-stained proteins (Invitrogen) were used as standard markers. A total of 1 μg was loaded for each recombinant protein.

Fig. 2

SDS-PAGE of purified recombinant fusion proteins and their construct diagrams (A – C). The fusion proteins of three or two protective Onchocerca volvulus (Ov) filarial antigen combinations were constructed as shown in the diagram. The recombinant fusion proteins were expressed in Pichia pastoris (yeast) and Escherichia coli and loaded on SDS-PAGE (each 1 μg, indicated with arrows). SeeBlue pre-stained proteins (Invitrogen) were used as standard markers. 6His, 6-hexahisitidine tag.

Fig. 3

Effect of immunization with a single vaccine antigen expressed by either Escherichia coli or Pichia pastoris on the development of protective immunity to Onchocerca volvulus larvae in mice. (A) Ov-103; (B) Ov-RAL-2; (C) Ov-CPI-2M. Each dot represents larval recovery from an individual animal. Data presented are mean ± S.D. Asterisk represents statistical difference in larval recoveries; P ≤ 0.05.

Fig. 3

Effect of immunization with a single vaccine antigen expressed by either Escherichia coli or Pichia pastoris on the development of protective immunity to Onchocerca volvulus larvae in mice. (A) Ov-103; (B) Ov-RAL-2; (C) Ov-CPI-2M. Each dot represents larval recovery from an individual animal. Data presented are mean ± S.D. Asterisk represents statistical difference in larval recoveries; P ≤ 0.05.

Fig. 3

Effect of immunization with a single vaccine antigen expressed by either Escherichia coli or Pichia pastoris on the development of protective immunity to Onchocerca volvulus larvae in mice. (A) Ov-103; (B) Ov-RAL-2; (C) Ov-CPI-2M. Each dot represents larval recovery from an individual animal. Data presented are mean ± S.D. Asterisk represents statistical difference in larval recoveries; P ≤ 0.05.

Fig. 4

Effect of immunization with fusion antigens on the development of protective immunity to Onchocerca volvulus larvae in mice. (A) Ov-RAL-2/103 fusion protein expressed in Escherichia coli and Pichia pastoris expressed protein. (B) Ov-RAL-2/CPI-2M expressed in E. coli. Each dot represents larval recovery from an individual animal. Data presented are mean ± S.D. Asterisk represents statistical difference in larval recoveries; P ≤ 0.05.

Fig. 4

Effect of immunization with fusion antigens on the development of protective immunity to Onchocerca volvulus larvae in mice. (A) Ov-RAL-2/103 fusion protein expressed in Escherichia coli and Pichia pastoris expressed protein. (B) Ov-RAL-2/CPI-2M expressed in E. coli. Each dot represents larval recovery from an individual animal. Data presented are mean ± S.D. Asterisk represents statistical difference in larval recoveries; P ≤ 0.05.

Fig. 5

Comparative effect of immunization with concurrent injections of Onchocerca volvulus Ov-103 (expressed in Pichia pastoris), Ov-RAL-2 (expressed in Escherichia coli) and Ov-CPI-2M (expressed in E. coli) compared with immunization with the combined fusion antigen Ov-RAL-2/103/CPI-2M (expressed in E. coli). Each dot represents larval recovery from an individual animal. Data presented are mean ± S.D. Asterisk represents statistical difference in larval recoveries; P ≤ 0.05.