Description and Characterization of a Novel Live-Attenuated Tri-Segmented Machupo Virus in Guinea Pigs Amélie D

Zaza et al. Virology Journal (2018) 15:99 https://doi.org/10.1186/s12985-018-1009-4 SHORT REPORT Open Access Description and characterization of a novel live-attenuated tri-segmented Machupo virus in Guinea pigs Amélie D. Zaza1,2*, Cécile H. Herbreteau1 and Christophe N. Peyrefitte2,3 Abstract Background: Machupo virus (MACV) is a member of the Mammarenavirus genus, Arenaviridae family and is the etiologic agent of Bolivian hemorrhagic fever, which causes small outbreaks or sporadic cases. Several other arenaviruses in South America Junín virus (JUNV) in Argentina, Guanarito in Venezuela, Sabiá in Brazil and Chapare in Bolivia, also are responsible for human hemorrhagic fevers. Among these arenaviruses, JUNV caused thousands of human cases until 1991, when the live attenuated Candid #1 vaccine, was used. Other than Candid #1 vaccine, few other therapeutic or prophylactic treatments exist. Therefore, new strategies for production of safe countermeasures with broad spectrum activity are needed. Findings: We tested a tri-segmented MACV, a potential vaccine candidate with several mutations, (r3MACV). In cell culture, r3MACV showed a 2-log reduction in infectious virus particle production and the MACV inhibition of INF-1β was removed from the construct and produced by infected cells. Furthermore, in an animal experiment, r3MACV was able to protect 50% of guinea pigs from a simultaneous lethal JUNV challenge. Protected animals didn’t display clinical symptoms nor were virus particles found in peripheral blood (day 14) or in organs (day 28 post-inoculation). The r3MACV provided a higher protection than the Candid #1 vaccine. Conclusions: The r3MACV provides a potential countermeasure against two South America arenaviruses responsible of human hemorrhagic fever. Keywords: Vaccine candidate development, Therapeutic, Mammarenaviruses, Machupo virus, Junín virus, Candid #1, Tri-segmented virus, Reverse genetic approaches Findings number [3]. Furthermore, three others mammarenaviruses Machupo virus (MACV) belongs to the Mammarena- have caused HHF outbreaks in South America and include virus genus of the Arenaviridae family and is the etio- Guanarito (GTOV), Chapare (CHAPV) and Sabiá (SABV) logic agent of Bolivian Hemorrhagic Fever (BHF) [1], viruses [4–7]. Post-exposure passive transfer of human discovered in 1959. From 1962 to 1964, it caused 515 plasma-derived antibodies was used for humans and also human cases, 114 of which were fatal [1]. Then, 40 years non-human primates (NHPs) challenged by JUNV and later, from 2006through 2008, 200 human cases were MACV [3, 8]. However, this treatment induced a reported including 12 deaths [2]. Similarly, the 1950s, late-neurological syndrome [8]. Although Ribavirin reduced another arenavirus, Junín virus (JUNV), was responsible lethality in humans, administration at very early stage of in- for cases of human hemorrhagic fever (HHF) in fection was necessary [9]duetoitssideeffects[3]. There- Argentina [3]. Since 1991, the use of the Candid #1 vaccine, fore, alternative strategies with a broad spectrum activity a live-attenuated vaccine against JUNV, decreased the case are needed. Here, we report the use of the reverse genetic strategy to design a potential vaccine candidate targeting * Correspondence: [email protected] MACV and JUNV. 1Fab’entech, 24 rue Jean Baldassini Bat B 69007, Lyon, France Mammarenaviruses are enveloped viruses with a 2Unité de virologie, Institut de Recherche Biomédicale des Armées, 1 place Valérie André, 91220 Brétigny-sur-Orge, France single-stranded RNA genome composed of two segments Full list of author information is available at the end of the article [9]. Each segment encodes two ambisense genes, separated © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zaza et al. Virology Journal (2018) 15:99 Page 2 of 7 by a hairpin intergenic region (Fig. 1a). The L segment con- polymerase sequence. This mutation resulted in an tains the genes of the small ring finger Z protein and of the 8-fold decrease in polymerase activity (mini-genome RNA-dependent RNA polymerase L protein. In the S seg- assay, Additional file 1). MACV and JUNV are closely ment are found the genes of the viral nucleoprotein (NP) genetically related, and, indeed, Candid #1, protected and of the glycoprotein complex (GPC). It was previously guinea pigs and primates from a lethal MACV challenge demonstrated that the bi-segmented genome of arena- [14]. Since SABV and CHAPV are genomically closely viruses could be modified into a tri-segmented genome, by related [6], r3MACVwas designed to target MACV, the duplication of the S segment [10]. To select and main- JUNV and each these additional South America arena- tain tri-segmented viruses, the NP gene was removed from viruses responsible of HHF. one of the two S segments, and the GPC gene was removed To replicate the pattern of ther3LCMV construct, re- from the second S segment, so that two genes of interest verse genetic systems were designed using synthesized couldbeinsertedintotheseemptyloci(Fig.1b). Prior Z, GPC, NP, NP D380A and L genes as well as S and L studies demonstrated efficacy using this approach for Old backbones (Eurofins Genomics, France) based on pub- World Lymphocytic choriomeningitis virus (LCMV) or lished sequences (GenBank acc # AY619643 and the New World JUNV [10, 11]. They constructed a AY619642). The S and L backbones contained all the tri-segmented JUNV (r3JUNV) or LCMV (r3LCMV), the non-coding regions of the related viral segment, and the latter of which showed high attenuation vs WT and exhib- genes loci were replaced by BsmBI (GPC and L loci), ited protection against LCMV challenge when used as a BbsI (NP locus) or SapI (Z locus) restriction sites (Fig. vaccine candidate in mice [10]. 1). These sites were designed to further eliminate all the In these studies, we used the reverse genetic system non-viral sequences after the enzymatic restriction step. applied to the MACV Carvallo strain genome. As illus- Restriction sites required to clone the genes into the trated in Fig. 1 we generated three S segments, a backbones or the pCAGGs plasmids were either wild-type one along with two genetically modified S seg- included during the gene synthesis, or added by PCR ments, one harboring the CHAPV strain 810,419 GPC using primers with 5′ extensions using the PCR gene (acc # EU260463.1) into the MACV GPC gene Extender system (5PRIME) or the Q5 High-Fidelity PCR locus, and the second displaying GTOV strain Kit (New England Biolabs), following manufacturer’s INH-95551 GPC gene (acc # AY129247.1) into the recommendations [10]. The E166G polymerase mutation MACV NP gene locus. This virus was named r3MACV was inserted into the expression plasmids using the Q5 (Fig. 1b). Thirty three (33) amino acids were deleted site-directed mutagenesis kit (New England Biolabs). from the C-termini of the two GPC sequences, required Amplified products were then purified using the for production of infectious viral particles [12], and QIAquick PCR Purification kit (Qiagen) and digested with avoid any competition with the MACV wt GPC. Such a the corresponding enzyme (BsmBI, BbsI or SapI, New competition might have changed the virus cellular England Biolabs). Restriction products were gel purified tropism or led to the loss of one of the two modified S using agarose (Thermo Fisher) and the QIAquick Gel segment. Additional modifications aimed to stimulate Extraction Kit (Qiagen) then ligated using the Quick the adaptive immune response, by insertion of mutation Ligation Kit (New England Biolabs). Ligated products (D380A in NP gene), shown to prevent the MACV NP were transformed into the NEB 10-β or NEB 5-α F’Iq IFN type-I inhibition [13]. Finally, glutamic acid was Competent E. coli (New England Biolabs). The bacterial replaced by glycine at position166 (E166G) in the clones were screened using the PCR (PCR Master Mix, Fig. 1 Schematic representation of MACV wt and r3MACV genomic constructs. Arenaviruses have a bisegmented negative single-stranded RNA genome. a For the MACV wt, the S segment encodes the viral glycoprotein precursor (GPC) and the nucleoprotein (NP), whereas L segment encodes the viral polymerase (L) and the small RING finger protein Z. b For the r3MACV, one S segment encodes the MACV GPC and the GTOV GPCΔ33, and the other encodes the CHAPV GPCΔ33 and the MACV NP, and the L segment is unchanged than the wt genome. Mutations inserted in r3MACV genome were noted Zaza et al. Virology Journal (2018) 15:99 Page 3 of 7 Thermo Fisher) following the manufacturer’s instructions. triplicate. All results of r3MACV sequencing were The positive clones were grown in 50 ml LB broth similar. Sequencing revealed three changes between (Thermo Fisher), then the plasmids were extracted using r3MACV and MACV wt in the L-segment. Two changes the Plasmid Plus Midi Kit (Qiagen). Two vectors, kindly resulted in false sense mutation (T➔C) at position 1733 provided by JC de la Torre, were used: the pCAGGs vector (leucine ➔ proline) and at position 6598 (serine ➔ pro- to express the viral proteins [15], and the pol I vector to line) in the polymerase. We also identified a 35 nt dele- produce the viral RNA segments [16]. The r3MACV tion in the intergenic region (on position 417 to 452). genomic sequences are shown in Additional file 2. Sequencing revealed one change under one S segment of After the construction and production of the MACV r3MACV: surprisingly, we found an 1124 nt deletion in wt and r3MACV, using conditions previously described the C-termini of the CHAPV GPCΔ33.

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