ABSTRACT
Abstract The treatment with hyperimmune sera constitute the only specific and effective therapy available against snakebite envenomation, most common in developing countries. Serum quality is an important factor on patient recovery time and in the incidence of death and permanent disability. To date, most sera consist of pepsin digested IgG antibodies harvested from hyperimmune animals. The use of animal derived enzymes, such as pepsin, to digest IgG, constitute a source of adventitious agents and contaminants, such as porcine circovirus. The present study aims to evaluate the use of the plant derived enzymes bromelain and ficin, as an alternative to pepsin. To this purpose, horse serum immunized against Bothrops venoms was purified with caprylic acid and digested with bromelain or ficin. SDS-PAGE results evidence the formation of F(ab)'2 fragments and suggest that a digestion time superior to 8 hours may be required to completely digest the antibodies with bromelain or ficin. F(ab)'2 fragments obtained by digestion with either bromelain or ficin digestion preserved the ability to recognize Bothrops sp. venom in western blotting assays. Therefore, both enzymes are suitable for use in large-scale production, minimizing contamination risks and increasing safety and efficiency of serotherapy treatments.
ABSTRACT
O Plasmodium vivax é a espécie mais comum de parasita causador da malária humana encontrada fora da África, com maior endemicidade na Ásia, América Central e do Sul e Oceania. Embora o Plasmodium falciparum cause a maioria do número de mortes, o P. vivax pode levar à malária grave e resultar em morbimortalidade significativa. O desenvolvimento de uma vacina protetora será um passo importante para a eliminação da malária. Recentemente, uma formulação contendo as três variantes alélicas da proteína circumsporozoíta de P. vivax (PvCSP - All epitopes) induziu proteção parcial em camundongos após desafio com esporozoíto híbrido Plasmodium berghei (Pb), no qual as repetições centrais do PbCSP foram substituídas por repetições PvCSP-VK210 (esporozoítos Pb/Pv). No presente estudo, a proteína quimérica PvCSP contendo as variantes alélicas (VK210, VK247 e P. vivax-like) fusionadas com a proteína de nucleocapsídeo do vírus da caxumba (formando partículas semelhantes a nucleocapsídeos ou do inglês, NLP - Núcleo Like Particles) na ausência (NLP-CSPR) ou na presença do domínio C-terminal (CT) conservado da PvCSP (NLP-CSPCT). Para a realização do estudo selecionamos os adjuvantes Poly (I:C), um RNA sintético de dupla fita, agonista do receptor Toll do tipo 3 (TLR3) ou o adjuvante Montanide ISA 720, uma emulação óleo em agua. Para obter uma forte resposta imune, a levedura Pichia pastoris foi usada para expressar as proteínas recombinantes na forma de NLPs. Camundongos foram imunizados com cada uma das proteínas recombinantes em combinação com os adjuvantes citados. Embora ambas as NLPs tenham sido capazes de gerar uma forte resposta imune, com altos níveis de títulos e longevidade, apenas a formulação contendo a proteína NLP-CSPCT na presença do adjuvante Poly (I:C) foi selecionada para ser explorada em experimentos futuros. Esta proteína em combinação com o adjuvante Poly (I:C) induziu alta frequência de células secretoras de anticorpos específicas para o antígeno homólogo nos dias 5 e 30, no baço e na medula óssea, respectivamente. Altos títulos de IgG contra as 3 variantes de PvCSP foram detectados nos soros. Posteriormente camundongos imunizados com NLP-CSPCT foram desafiados com esporozoítos Pb/Pv e a parasitemia no 5º dia demonstrou proteção estéril em 30% dos camundongos desafiados. Portanto, a formulação vacinal gerada neste estudo tem potencial para ser explorada no desenvolvimento de uma vacina universal contra a malária causada por P. vivax
Plasmodium vivax is the most common species of human malaria parasite found outside Africa, with high endemicity in Asia, Central and South America, and Oceania. Although Plasmodium falciparum causes the majority of deaths, P. vivax can lead to severe malaria and result in significant morbidity and mortality. The development of a protective vaccine will be a major step toward malaria elimination. Recently, a formulation containing the three allelic variants of the P. vivax circumsporozoite protein (PvCSP--All epitopes) showed partial protection in mice after a challenge with the hybrid Plasmodium berghei (Pb) sporozoite, in which the PbCSP central repeats were replaced by the VK210 PvCSP repeats (Pb/Pv sporozoite). In the present study, the chimeric PvCSP allelic variants (VK210, VK247, and P. vivax-like) were fused with the mumps virus nucleocapsid protein (assembling into nucleo like particles - NLP) in the absence (NLP-CSPR) or presence of the conserved C-terminal (CT) domain of PvCSP (NLP-CSPCT). To carry out the study, we selected the adjuvants Poly (I:C), a synthetic double-stranded RNA, Toll-like receptor 3 (TLR3) agonist or Montanide ISA 720 adjuvant, an oil-water emulation. To elicit stronger immune response, Pichia pastoris yeast was used to produce the NLPs. Mice were immunized with each recombinant protein in combination with above. Although both NLPs were able to generate stronger immune response, with high antibodies titer levels and longevity, formulation containing NLP-CSPCT in the presence of Poly (I:C) was selected to be explored in future experiments. NLP-CSPCT with Poly (I:C) adjuvant presented a high frequency of antigen-specific antibody-secreting cells (ASCs) on days 5 and 30, respectively, in the spleen and bone marrow. Moreover, high IgG titers against all PvCSP variants were detected in the sera. Later, immunized mice with NLP-CSPCT were challenged with Pb/Pv sporozoites. Sterile protection was observed in 30% of the challenged mice. Therefore, this vaccine formulation use has the potential to be a good candidate for the development of a universal vaccine against P. vivax malaria.