Toxoplasma gondii: humoral and cellular immune response of BALB/c mice immunized via intranasal route with rTgROP2

Abstract

<jats:p>TgROP2 is an intracellular protein associated with rhoptries of Toxoplama gondii and an antigen component of a candidate vaccine for toxoplasmosis. The purpose of the present study was to evaluate the efficacy of rTgROP2 to stimulate humoral and cellular immune responses in BALB/c mice via intranasal injection. TgROP2 partial coding sequence was (196-561) amplified by PCR from genomic T. gondii RH strain DNA and cloned into the pTrcHis expression vector. Escherichia coli Rosetta 2 cells transformed with pTrcHis-TgROP2 showed high levels (~1 mg.mL-1) of recombinant protein after 4 hours of IPTG induction. Recombinant TgROP2 exhibited an apparent Mr equal to 54 kDa. In order to test immunogenicity of the recombinant protein, 10 BALB/c mice received 10 µg of rROP2 protein + 10 µg of Quil-A via intranasal injection. Doses were administered at days 0, 21, and 42. Three animals were euthanized and used to evaluate cell-ular immune response on day 62. Five (50%) and two (20%) out of ten animals produced IgG (DO mean = 0.307; cut-off = 0.240) and IgA (DO mean = 0.133, cut-off = 0.101), respectively, by ELISA on day 62. The proliferation of splenocytes revealed high stimulation index (SI) when co-cultured with 5, 10 and 15 µg.mL-1 of rTgROP2. These results indicate that intranasal immunization with recombinant protein ROP2 plus Quil-A can elicit both cellular and humoral immune responses in BALB/c mice.</jats:p>

rROP2 from Toxoplasma gondii as a potential vaccine against oocyst shedding in domestic cats

“…This adjuvant was described as being capable of inducing strong Th1 and Th2 responses and moderate CTL responses (COX & COULTER, 1997; PAEPENMÜLLER & MÜLLER-GOYMANN, 2014). Additionally, the Quil-A adjuvant is widely used in animals, has a low cost and a simple design, and is generally safe (COX & COULTER, 1997;GARCIA et al, 2007;IGARASHI et al, 2010;CUNHA et al, 2012).…”

“…For all of these reasons, ROP2 has been used for vaccine studies (UGGLA et al, 1988;VERCAMMEN et al, 2000;LEYVA et al, 2001;DZIADEK et al, 2009;IGARASHI et al, 2010;DZIADEK & BRZOSTEK, 2012).…”

“…The protocols for obtaining of rROP2 were performed as previously described by Igarashi et al (2010).…”

A vaccine formulation combining rhoptry proteins NcROP40 and NcROP2 improves pup survival in a pregnant mouse model of neosporosis

“…Moreover, it has been approved for veterinary use (Sun et al, 2009). In addition, Quil-A has been widely employed in vaccine formulations against other apicomplexan parasites (Garcia et al, 2007;Igarashi et al, 2010;Bitencourt et al, 2013). In the present work, vaccine safety was evaluated in detail to record possible local adverse reactions at the point of inoculation that have been previously reported for Quil-A adjuvant (Sun et al, 2009).…”

Molecular cloning, sequencing, and expression of Eimeria tenella HSP70 partial gene

“…In sequence, the DNA was purified from agarose gel using a QIAquick Gel Extraction Kit (Qiagen Biotechnology Brazil Ltd.). Sequencing reactions were performed using a fluorescent dye-labeled dideoxynucleotide system (BigDye Terminator V3.1 Cycle Sequencing Kit; Applied Biosystems, Foster City, CA, USA) with the same primers mentioned above in pTrcHis2-TOPO to confirm the maintenance of the hsp70 ORF, as described by Igarashi et al (2010), prior to carrying out protein expression on a larger scale. After careful examination of each electropherogram, the consensus sequences were constructed from alignment data using ClustalX (Thompson et al, 1997) and edited by visual inspection using the BioEdit program.…”