Keskin, Sercan; Pavel, Shaikh Terkis Islam; Sak, Rabia; Bahadori, Fatemeh; Aslan, Ahmet Furkan; Aktaş, Münir; Karakeçili, Faruk; Kalkan, Ahmet; Özdarendeli, Aykut; Doymaz, Mehmet Ziya

The Crimean-Congo Hemorrhagic Fever Virus (Orthonairovirus haemorrhagiae) causes a hemorrhagic fever with mortality rates reaching up to 40%. For years, this virus has maintained its position among the top priority pathogens identified by the World Health Organization (WHO). This is due to its endemic presence across a vast region—from Africa and Spain to the Balkans, the Middle East, and throughout Asia—its potential for human-to-human transmission, and the lack of an effective and approved vaccine or treatment. Therefore, the development of an effective vaccine against CCHFV is of critical importance. Building on the success of mRNA-based vaccines during the Coronavirus Disease 2019 (COVID-19) pandemic, this study reports the development of a messenger ribonucleic acid (mRNA) vaccine candidate expressing the nucleocapsid protein (NP) of CCHFV. The CCHFV NP in vitro transcript (IVT) was designed with pseudouridine (Ψ) nucleoside modification. As part of the preclinical characterization of the IVT vaccine candidate, the biochemical and immunological properties of NP were confirmed in Huh-7 cells transfected with IVT NP-ΨmRNA. Afterwards, the efficacy of IVT NP-ΨmRNA immunization was evaluated in immunocompetent BALB/c and transiently immunosuppressed (IS) C57BL/6 mice. In CCHFV challenge studies, IS C57BL/6 mice were used. IS C57BL/6 mice were immunized intramuscularly with 2 doses of IVT NP- ΨmRNA, either naked or encapsulated in Poly(lactic-co-glycolic acid) (PLGA) nanoparticles, administered 14 days apart. High levels of CCHFV NP-specific humoral (IgM and IgG) and cellular (cytokine and lymphoproliferative) responses were demonstrated in BALB/c mice immunized with IVT NP- ΨmRNA. In challenge experiments, 100% survival was observed with both the naked and PLGA-encapsulated IVT NP-ΨmRNA immunizations. These findings demonstrate 100% survival following lethal CCHFV challenge under the experimental conditions tested in this mouse model and support the potential of NP-encoding pseudouridine-modified mRNA vaccines. Additionally, 100% survival was observed in mice immunized with inactivated CCHFV, whereas only 20% survival was detected in the unmodified IVT NP-mRNA vaccinated animals. In the protected mice, viral clearance was observed in the spleen, liver tissues, and blood on day 14 post-challenge. This study demonstrates that NP, the most abundant protein of the virus, is capable of providing significant survival benefits in the tested mouse model. Furthermore, our report represents a significant step in identifying a potential vaccine candidate and provides a solid foundation for further preclinical studies necessary to support future clinical development.

Kalaycioglu, Atila T.; Baykal, Atakan; Guldemir, Dilek; Bakkaloglu, Zekiye; Korukluoglu, Gulay; Coskun, Aslihan; Torunoglu, Mehmet Ali; Ertek, Mustafa; Durmaz, Riza

Genetic characterization of measles viruses (MVs) combined with acquisition of epidemiologic information is essential for measles surveillance programs used in determining transmission pathways. This study describes the molecular characterization of 26 MV strains (3 from 2010, 23 from 2011) obtained from urine or throat swabs harvested from patients in Turkey. MV RNA samples (n=26) were subjected to sequence analysis of 450 nucleotides comprising the most variable C-terminal region of the nucleoprotein (N) gene. Phylogenetic analysis revealed 20 strains from 2011 belonged to genotype D9, 3 to D4, 2 strains from 2010 to genotype D4 and 1 to genotype B3. This study represents the first report describing the involvement of MV genotype D9 in an outbreak in Turkey. The sequence of the majority of genotype D9 strains was identical to those identified in Russia, Malaysia, Japan, and the UK. Despite lack of sufficient epidemiologic information, the presence of variants observed following phylogenetic analysis suggested that exposure to genotype D9 might have occurred due to importation more than once. Phylogenetic analysis of five genotype D4 strains revealed the presence of four variants. Epidemiological information and phylogenetic analysis suggested that three genotype D4 strains and one genotype B3 strain were associated with importation. This study suggests the presence of pockets of unimmunized individuals making Turkey susceptible to outbreaks. Continuing molecular surveillance of measles strains in Turkey is essential as a means of acquiring epidemiologic information to define viral transmission patterns and determine the effectiveness of measles vaccination programs designed to eliminate this virus. J. Med. Virol. 85:2128-2135, 2013.