Abstract
Pathogenic New World arenaviruses (NWAs) cause haemorrhagic fevers and can have high mortality rates, as shown in outbreaks in South America. Neutralizing antibodies (Abs) are critical for protection from NWAs. Having shown that the MOPEVAC vaccine, based on a hyperattenuated arenavirus, induces neutralizing Abs against Lassa fever, we hypothesized that expression of NWA glycoproteins in this platform might protect against NWAs. Cynomolgus monkeys immunized with MOPEVACMAC, targeting Machupo virus, prevented the lethality of this virus and induced partially NWA cross-reactive neutralizing Abs. We then developed the pentavalent MOPEVACNEW vaccine, expressing glycoproteins from all pathogenic South American NWAs. Immunization of cynomolgus monkeys with MOPEVACNEW induced neutralizing Abs against five NWAs, strong innate followed by adaptive immune responses as detected by transcriptomics and provided sterile protection against Machupo virus and the genetically distant Guanarito virus. MOPEVACNEW may thus be efficient to protect against existing and potentially emerging NWAs.
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Data availability
The datasets from the RNA sequencing generated during the current study are not publicly available due to current ongoing analyses but are available from the corresponding author upon reasonable request. S. Baize is the corresponding author for any request or correspondence (sylvain.baize@pasteur.fr). Data are available in public open access repositories. For the transcriptomic analyses, they are available on Zenodo (https://zenodo.org/record/7229439).
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Acknowledgements
We thank S. Godard, B. Labrosse, C. Leculier, S. Mely, E. Moissonnier, S. Mundweiler, D. Pannetier, A. Pocquet, H. Theoule and D. Thomas (P4 INSERM–Jean Merieux, US003, INSERM) for BSL4 management during these experiments. We also thank G. Fourcaud, B. Lafoux and K. Noy for their logistical help. We thank M. Caroll and R. Hewson (Public Health England Porton Down), S. Günther (BNI) and T.G. Ksiazek (Centers for Disease Control and Prevention (CDC)) for providing the Machupo, Guanarito and Junin viruses and T. G. Ksiazek, P. E. Rollin and P. Jahrling (Special Pathogens Branch, CDC) for the polyclonal anti-MACV Abs. Ab KL-AV-2A1 was a kind gift of F. Krammer (Department of Microbiology, Icahn School of Medicine at Mount Sinai). We thank C. Gerke and M.-A. Dillies for their support in vaccine development and bioinformatics and the Grand Projet Fédérateur de Vaccinologie of the Institut Pasteur that funded this project (grant obtained by S. Baize).
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Contributions
S.R. managed and performed the experiments, analysed the results and wrote the publication. X.C. performed the reverse genetics experiments to rescue the vaccine candidates. X.C., C.P., V.B.-C., A.J., M. Mateo, C.G., J.H. and S. Baize performed the experiments on samples during the animal experiments. L.F. and P.-H.M. were in charge of the animal experiments in the BSL2 facility. C.P., V.B.-C. and L.A. were responsible for the neutralization assays. A.J. performed the ELISA experiments and M. Mateo realized the viral titrations in organs. E.P. and N.P. computed all transcriptomic data and performed the related analyses. A.V., S. Barron, A.D., O.L., O.J. and M. Moroso managed the animals in the BSL4 facility. M. Dirheimer was the referent veterinarian of this study. M. Daniau and C.L.-L. performed the sequencing for the transcriptomic analyses. H.R. and C.C. managed the BSL4 team. S. Baize supervised the entire project.
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The authors declare no competing interests. The MOPEVAC vaccine platform described in this study is protected by US patent 62/245,631; the authors listed as co-inventors are S.R., S. Baize, X.C., M. Mateo and A.J.
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Extended data
Extended Data Fig. 1 GPs expression by MOPEVAC viruses.
a. VeroE6 cells were infected at a moi of 0.001 and cellular RNAs were extracted at day 0 and day 3 post-infection. The ratio of GPC expression of day 3 relative to day 0 was calculated and represented for each MOPEVAC virus. b. Expression of GP2 detected by KL-AV-2A149 antibody. GP2 protein expression was detected by western blot from 105 ffu of MOPEVAC viruses, in a single experiment. The antibody has been described to detect JUNV, GTOV and MACV but its binding on CHAV and SABV was not known. These results show the expression by the different MOPEVAC viruses of the GP2 proteins of all NWA except the one of SABV, probably because of a lack of cross-reactivity of the antibody.
Extended Data Fig. 2 Real-time recording of body temperature after challenge.
Recording systems were implanted in the CMs to evaluate the body temperature throughout the protocol. A number were defective. We thus obtained data for seven CMs: the three controls, three prime only vaccinated animals, and one prime boost. The recording was stopped unintentionally for a small period for five animals, this is clearly visible in the graphs.
Extended Data Fig. 3 Hematological parameters and viral loads in the organs at the day of necropsy.
a. Cell counts and hemoglobin concentrations in whole blood were measured at each sampling. b. Viral RNA was quantified by RT-qPCR from crushed organs or cells. RT-qPCR-positive samples were evaluated for infectious virus titers. Li: liver, mLN: mesenteric lymph node, iLN: inguinal lymph node, Ki: kidney, Lu: lung, Bl: bladder, AG: adrenal gland, Br: brain, Ce: cerebellum, Sp: spleen, Spleno: splenocytes.
Extended Data Fig. 4 Body temperature before and after challenge in the MOPEVACNEW experiment.
Intraperitoneal implants recorded the body temperature throughout the experiment at 15-min intervals. a. Post immunization period in vaccinated CMs. All received the same vaccine, but the color indicates the virus used for the challenge. b. Body temperature of vaccinated and control animals after challenge.
Extended Data Fig. 5 Gating strategy for determination of IgG fixation on GPs.
The gates used to quantify the cells expressing or not GPs that fixed IgG from plasma are presented. FSC / SSC was used to gate cells, then singlets were determined using SSC / SSC-W and live cells were gated: Live Dead negative cells. The cells that fixed IgG and the secondary anti-IgG FITC were defined with the gate ‘Positive’. Three conditions of the same plasma sample are presented for comparison: empty vector, cells expressing GPs of MOPV and cells expressing GPS of JUNV.
Extended Data Fig. 6 Viral loads in organs and immune-preserved compartments.
a. Viral RNA was quantified by RT-qPCR from crushed organs or cells. RT-qPCR-positive samples were evaluated for infectious virus titers. Grey: GTOV-infected controls. Red: MACV-infected controls. b. Viral RNA was quantified from cerebrospinal fluid (CSF) and eye vitreous humor and infectious virus titration was also performed. Li: liver, mLN: mesenteric lymph node, iLN: inguinal lymph node, Ki: kidney, Lu: lung, Bl: bladder, AG: adrenal gland, Br: brain, Ce: cerebellum, LI: large intestine, SI: small intestine, Ov: ovary, Pa: pancreas, Th: thymus, Sp: spleen, Spleno: splenocytes.
Extended Data Fig. 7 Hematological and biochemical parameters after challenge in the MOPEVACNEW experiment.
a. Cell counts and hemoglobin concentrations were measured at each sampling after challenge. b. Biochemical parameters were assayed in plasma at each sampling. C-reactive protein (CRP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and plasmatic albumin levels are presented.
Extended Data Fig. 8 Gating strategy for flow cytometry analysis.
The gates used to quantify IFNγ-producing and CD154-expressing T cells are presented for an unstimulated sample (a) and for the same sample stimulated with staphylococcus-enterotoxin A (SEA), as a positive control (b). FSCint/FSCtof was used to select singlets (singlets gate). Then, dead cells are excluded using live-dead staining (live gate). Lymphocytes were selected using FSCint/SSCint parameters (LC gate). Then, CD4+ and CD8+ T cells were selected using CD3/CD4 and CD3/CD8 staining (CD4+ and CD8+ gates). Finally, the percentage of IFNγ-producing and CD154-expressing CD4+ and CD8+ T cells is determined using a quadrant in the IFNγ/CD154 dotplot. c. A similar strategy was applied for CD137 and GrzB detection.
Extended Data Fig. 9 Activation of T cells in response to peptide stimulation.
a. PBMCs sampled at days 14 and 24 post-prime and day 19 post-boost were stimulated with overlapping peptides covering MACV NP and GP and LASV strain Josiah NP. SEA was used as a positive control. After an overnight incubation, the cells were stained with conjugated antibodies and analyzed by flow cytometry for the expression of CD154, CD137, GrzB and IFNγ. Expression values represent the difference between stimulated and non-stimulated cells. Light blue dots represent animals vaccinated with a prime-boost strategy (n = 4, except for J19 boost where SEA n = 2, NP LASV n = 3) and black dots the control animals (n = 3). The dots were not separated when the expression values were close to 0. b. After challenge, peptide stimulation was performed on whole blood. GPC and NP specific T cell responses were evaluated. The difference from the non-stimulated condition is represented (Ctrl: n = 3, Vacc: n = 4). The SEA control at day 0 is presented for comparison.
Supplementary information
Supplementary Tables
Supplementary Table 1 Evolution of vaccine candidate genomes during serial passages in Vero E6 cells. The consensus genome sequences of the 5 vaccine candidates were determined at passages 2, 5 and 10 and compared to the initial sequences (P2). The changes in codon sequences and the position of the mutation in the genome were indicated. The amino acid changes are indicated for non-synonymous mutations whereas synonymous mutations are coloured green. The passage after which the mutation has been detected is indicated by the presence of a coloured box. Supplementary Table 2 Parameters used to establish the clinical score after challenge and their respective values. Supplementary Table 3 Exact P values corresponding to Fig. 6. For each condition the comparison was made with the day 0 time point.
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Reynard, S., Carnec, X., Picard, C. et al. A MOPEVAC multivalent vaccine induces sterile protection against New World arenaviruses in non-human primates. Nat Microbiol 8, 64–76 (2023). https://doi.org/10.1038/s41564-022-01281-y
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DOI: https://doi.org/10.1038/s41564-022-01281-y
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