ORIGINAL PAPER Immunological evaluation of a DNA cocktail vaccine with co-delivery of calcium phosphate nanoparticles (CaPNs) against the Toxoplasma gondii RH strain in BALB/c mice Mohammad Taghi Rahimi 1,2 & Shahabeddin Sarvi 2,3 & Mahdi Sharif 2,3 & Saeid Abediankenari 4 & Ehsan Ahmadpour 5 & Reza Valadan 4,6 & Mahdi Fasihi- Ramandie 7 & Seyed-Abdollah Hosseini 2,8 & Ahmad Daryani 2,3 Received: 31 May 2016 /Accepted: 8 November 2016 /Published online: 2 December 2016 # Springer-Verlag Berlin Heidelberg 2016 Abstract Many recent studies have been conducted to eval- uate protective immunity mediated by DNA vaccines against toxoplasmosis. Cocktail DNA vaccines showed better im- mune responses compared to single vaccines. The objective of the current study was to evaluate the protective efficacy of rhomboid 4 (ROM4) and cocktail DNA vaccines (ROM4 + GRA14) of the Toxoplasma gondii RH strain with or without coated calcium phosphate nanoparticles (CaPNs) as the adju- vant to improve the immunogenicity against the T. gondii RH strain in BALB/c mice. Cocktail DNA vaccines of pcROM4 + pcGRA14 of the T. gondii RH strain were constructed. CaPNs were synthesized and the cocktail DNA vaccine was coated with the adjuvant of CaPNs. Immunogenicity and the protec- tive effects of cocktail DNA vaccines with or without CaPNs against lethal challenge were evaluated in BALB/c mice. pcROM4 and cocktail DNA vaccine coated with CaPNs sig- nificantly enhanced cellular and humoral immune responses against Toxoplasma compared to pcROM4 and cocktail DNA vaccine without CaPNs (p < 0.05). These findings indicate that the survival time of immunized mice after challenge with the RH strain of T. gondii was increased compared to that of controls and the DNA vaccine provided significant protection in mice (p < 0.05). The CaPN-based cocktail DNA vaccine of pcROM4 + pcGRA14 showed the longest survival time com- pared to the other groups. Co-immunization with CaPN-based cocktail DNA vaccine (pcROM4 + pcGRA14) boosted im- mune responses and increased the protective efficacy against acute toxoplasmosis in BALB/c mice compared to both single gene and bivalent DNA vaccine without nano-adjuvants. Keywords Toxoplasma gondii . Rhomboid 4 . Dense granule 14 . Immune response . DNA vaccine . Adjuvant Introduction Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan that can infect a wide vari- ety of mammals including humans. Although toxoplasmosis is typically asymptomatic in immunocompetent people, in congenital cases it may cause microcephaly, hydrocephalus, blindness, mental retardation, spontaneous abortion, and death of fetuses (Elmore et al. 2010; Petersen et al. 2012). In immu- nocompromised individuals, recrudescence of latent toxoplas- mosis may cause encephalitis or even lethal damage (Cenci- Goga et al. 2011; Pereira-Chioccola et al. 2009). Toxoplasma * Ahmad Daryani daryanii@yahoo.com 1 School of Medicine, Shahroud University of Medical Sciences, Shahroud, Iran 2 Toxoplasmosis Research Center (TRC), Mazandaran University of Medical Sciences, Sari, Iran 3 Parasitology and Mycology Department, Toxoplasmosis Research Center (TRC), Sari Medical School, Mazandaran University of Medical Sciences, P.O. Box 48175-166518th km of Khazar Abad Road, Sari, Mazandaran Province, Iran 4 Immunology Department, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran 5 Infectious and Tropical Diseases Research Center, Tabriz University of Medical Sciences, Tabriz, Iran 6 Molecular and Cell Biology Research Center (MCBRC), Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Mazandaran, Iran 7 Molecular Biology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran 8 Student Research Committee, Mazandaran University of Medical Sciences, Sari, Iran Parasitol Res (2017) 116:609–616 DOI 10.1007/s00436-016-5325-6