Research ArticleEMERGING INFECTIONS

A single-shot Lassa vaccine induces long-term immunity and protects cynomolgus monkeys against heterologous strains

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Science Translational Medicine  09 Jun 2021:
Vol. 13, Issue 597, eabf6348
DOI: 10.1126/scitranslmed.abf6348
  • Fig. 1 Outline of the experiments.

    (A) Scheme presenting the time of immunization, sampling, challenge, and necropsy for the four groups of the cross-protection study. Prime immunizations are indicated by blue arrows. Black arrows indicate sampling, and purple arrows indicate the time of challenge. Red arrows point to the day of necropsy, and the number of animals euthanized is indicated. VB, MeV-NP immunization, Benin lineage VII challenge; VN, MeV-NP immunization, Nigeria lineage II challenge; CB, MeV immunization, Benin lineage VII challenge; CN, MeV immunization, Nigeria lineage II challenge. (B) Scheme presenting the time of immunization, sampling, challenge, and necropsy for the four groups of the long-term protection study. Prime immunizations are indicated by a blue arrow, and boost immunizations are indicated by an orange arrow. Black arrows indicate sampling, and purple arrows indicate the time of challenge. Red arrows point to the day of necropsy, and the number of monkeys euthanized is indicated. VJ-P, MeV-NP immunization, Josiah lineage IV challenge, prime only; VJ-PB, MeV-NP immunization, Josiah lineage IV challenge, prime-boost; CJ, VJ-P, prime-boost.

  • Fig. 2 Antibody responses induced after immunization of cynomolgus monkeys.

    (A) Detection of LASV-specific IgM against lineage IV (top), lineage VII (middle), and lineage II (bottom) in VB, CB, VN, and CN monkeys by ELISA. (B) Detection of LASV-specific IgG against lineage IV (top), lineage VII (middle), and lineage II (bottom) in VB, CB, VN, and CN monkeys by ELISA. (C) Detection of NP lineage IV (top)– and GP lineage IV (bottom)–specific IgG in VB and VN monkeys by ELISA. (D) Detection of NP lineage II (top)– and GP lineage II (bottom)–specific IgG in VB and VN monkeys by ELISA. For (A) to (D), each point represents the means ± SEM of three samples. (E) Detection of LASV-specific IgM against lineage IV in VJ-P, VJ-PB, and CJ monkeys by ELISA. (F) Detection of NP lineage IV, GP lineage IV, and LASV lineage IV IgG in VJ-P, VJ-PB, and CJ monkeys by ELISA. Each point represents the means ± SEM of four samples for VJ-P and VJ-PB and three samples for the CJ controls. Statistical significance: *P ≤ 0.05, Mann-Whitney test.

  • Fig. 3 T cell responses induced after immunization of cynomolgus monkeys.

    (A and B) Quantification of CD8+ (A) and CD4+ (B) T cells specific for LASV GPC (top) and NP (bottom) peptides. The percentage of T cells producing TNF-α or IFN-γ in response to peptide stimulation among total CD8+ or CD4+ T cells is presented according to the time after immunization after subtraction of the respective value measured for unstimulated T cells that represent the background T cell response. Each bar represents the means ± SEM of six monkeys for LASV Josiah–derived peptides and three monkeys for LASV Benin– and LASV Nigeria–derived peptides. The dashed lines represent the percentage of T cells from control monkeys producing cytokines in response to LASV peptides before challenge (mean of 10 samples) (C) Pie chart representation of the proportion of each subpopulation of cytokine-positive CD8+ and CD4+ T cells responding to GPC or NP stimulation. (D) Quantification of CD8+ (top) and CD4+ (bottom) T cells specific for LASV GPC peptides in primed monkeys (left) or primed-boosted monkeys (right). (E) Quantification of CD8+ (top) and CD4+ (bottom) T cells specific for LASV NP peptides in primed monkeys (left) or primed-boosted monkeys (right). For (C) and (D), the percentage of T cells producing TNF-α or IFN-γ in response to peptide stimulation among total CD8+ or CD4+ T cells is presented according to the time after immunization after subtraction of the respective value measured for unstimulated T cells. Each bar represents the means ± SEM of eight samples. Statistical significance: *P ≤ 0.05, **P ≤ 0.01, one-way ANOVA. The dashed lines represent the percentage of T cells from control monkeys producing cytokines in response to LASV peptides before challenge (mean of eight samples) (F) Pie chart representation of the proportion of each subpopulation of cytokine-positive CD8+ and CD4+ T cells responding to GPC or NP stimulation. (G to I) LASV-specific T cell responses were evaluated in PBMCs isolated from immunized monkeys at 317 days after immunization. The proportion of GrzB or perforin-expressing CD8+ and CD4+ T cells (G), CD137-expressing CD8+ and CD4+ T cells (H), or HLA-DR–expressing CD4+ T cells (I) in response to LASV Josiah GPC– and LASV Josiah NP–derived peptides is shown. Data are presented as the percentage of cells expressing the indicated marker in response to peptide stimulation minus the percentage of expressing cells in response to mock stimulation for CJ (gray bars), VJ-P (red bars), and VJ-PB (blue bars). Each bar represents the means ± SEM of three (CJ) or four samples (VJ-P and VJ-PB) of PBMCs obtained 317 days after immunization.

  • Fig. 4 Clinical monitoring of immunized monkeys after an LASV challenge.

    (A) Clinical scores of individual monkeys from groups VB, CB, VN, and CN after challenge with LASV Benin (VB and CB) or LASV Nigeria (VN and CN) in the cross-protection experiment. (B) Monitoring of body temperature by rectal measurement during the course of LASV Benin and LASV Nigeria infections. Individual data are presented for each monkey. (C) Clinical scores of individual monkeys from groups VJ-P, VJ-PB, and CJ after the challenge with LASV Josiah in the long-term experiment. (D) Monitoring of body temperature by real-time measurement during the course of LASV Josiah infection. Individual data are presented for each monkey. (E) Analysis of biological parameters during the course of LASV infection. Each point represents the means ± SEM of three (VB, VN, CB, CN, and CJ) or four samples (VJ-P and VJ-PB) for the ALT, AST, and CRP concentrations, except for individualized points representing the values of one monkey. For statistical purposes, data collected from monkeys euthanized on day 14 were compared to data collected on day 15 for the other monkeys and data collected at the end of the experiment (days 28 to 30) were compared with each other. (F) ALT (top) and AST (bottom) concentrations in control monkeys at day 12 after challenge. Statistical significance: *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001, one-way ANOVA.

  • Fig. 5 LASV replication in cynomolgus monkeys.

    (A) Quantification (in FFU per milliliter) of LASV infectious particles in plasma (top), nasal swabs (middle), or oral swabs (bottom) according to the time after challenge with LASV Benin (VB and CB) or LASV Nigeria (VN and CN) in the cross-protection study. (B) Quantification (in FFU per milliliter) of LASV infectious particles in plasma (top), nasal swabs (middle), or oral swabs (bottom) according to the time after challenge with LASV Josiah in the long-term experiment. (C) Quantification of viral load by RT–quantitative PCR in the plasma (top), nasal swabs (middle), or oral swabs (bottom) according to the time after challenge with LASV Benin (VB and CB) or LASV Nigeria (VN and CN) in the cross-protection study. (D) Quantification of viral load by RT–quantitative PCR in the plasma (top), nasal swabs (middle), or oral swabs (bottom) according to the time after challenge LASV Josiah in the long-term experiment. Each point represents the means ± SEM of three (VB, VN, CB, CN, and CJ) or four samples (VJ-P and VJ-PB), except for individualized points representing the values of one monkey. For statistical purposes, data collected from monkeys euthanized on day 14 were compared to data collected on day 15 for the other monkeys, and data collected at the end of the experiment (days 28 to 30) were compared with each other. Statistical significance: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001. For (A) and (C), an unpaired t test or a Mann-Whitney test was performed after testing normality. For (B) and (D), a one-way ANOVA or a Kruskal-Wallis test was performed after testing normality.

  • Fig. 6 Analysis of the antibody responses induced after LASV infection.

    (A) Detection of postchallenge LASV lineage–specific IgG in VB, VN, CB, and CN monkeys by ELISA. Each point represents the means ± SEM of three samples except for control monkeys at day 15 or later. Individual points represent the value of one monkey. Statistical significance: *P ≤ 0.05, Kruskal-Wallis test. (B) Detection of postchallenge NP lineage IV– and GPC lineage IV–specific IgG in VB, VN, CB, and CN monkeys by ELISA. Each point represents the means ± SEM of three samples except for control monkeys at day 15 (two monkeys). (C) Detection of postchallenge LASV lineage–specific IgG in CJ, VJ-P, and VJ-PB monkeys by ELISA. Each point represents the means ± SEM of four (VJ-P and VJ-PB) or three samples (CJ). (D) Detection of postchallenge NP lineage IV and GPC lineage IV IgG in CJ, VJ-P, and VJ-PB monkeys by ELISA. Each point represents the means ± SEM of three samples except for control monkeys at day 15 (two monkeys). Statistical significance: *P ≤ 0.05, Kruskal-Wallis test. The red asterisks indicate differences between VJ-P and CJ, and the blue asterisks indicate differences between VJ-PB and CJ. (E and F) Quantification of NAbs in the plasma according to the time after LASV infection in monkeys of the cross-protection study (E) and monkeys of the long-term study (F). Individual data of the monkeys represent the highest dilution of plasma showing more than 50% neutralizing activity. (G and H) Analysis of antibody-dependent cell cytotoxicity induced by immunoglobulins after challenge in monkeys of the cross-protection study (G) and of the long-term study (H). Results are expressed as the percentage of NK cells [defined as CD3 CD20 CD8+ cells among PBMC as previously described (35)] expressing CD107a after culture in the presence of CB, CN, VB, VN, CJ, VJ-P, and VJ-PB plasma in Josiah LASV GPC–coated microplates minus the percentage of CD107a-expressing NK cells when culture was performed with the same plasma in Ebola virus GPC-coated microplates, as negative antigen. For statistical purposes, data collected from monkeys euthanized on day 12 or 14 were compared to data collected on day 15 for the other monkeys and data collected at the end of the experiment (days 28 to 30) were compared with each other. A t test was used to compare control and immunized monkeys for lineages II and VII at 0, 6, and 15 days after infection and to compare VN and VB (G). Statistical significance (P ≤ 0.05) was indicated with a black asterisk and the compared groups of monkeys indicated above (H). A one-way ANOVA test was used for day 0, 6, and 15 to compare CJ, VJ-P, and VJ-PB at each time point and a t test was used to compare only VJ-P and VJ-PB at day 28, and a significant difference (P ≤ 0.05) between CJ and VJ-P is indicated with black asterisks (H).

  • Fig. 7 T cell responses induced in cynomolgus monkeys challenged 1 month after immunization.

    (A to D) Quantification of the proportion of CD8+ (A) and CD4+ (B) T cells specific for LASV GPC peptides derived from Josiah LASV or of CD8+ (C) and CD4+ (D) T cells specific for LASV GPC peptides derived from Benin or Nigeria LASV. (E and F) Quantification of CD8+ T cells specific for LASV NP peptides derived from Josiah LASV (E) or Benin or Nigeria LASV (F). The percentage of T cells producing TNF-α or IFN-γ in response to peptide stimulation among total CD8+ T cells is presented according to the time after challenge after subtraction of the respective value measured for unstimulated T cells. Each bar represents the means ± SEM of six samples for Josiah LASV and three samples for Benin and Nigeria LASV. The proportion of each subpopulation of cytokine-positive T cells is presented in the pie charts. An unpaired t test or a Mann-Whitney test was performed after testing normality to compare Benin LASV– or Nigeria LASV–infected monkeys.

  • Fig. 8 T cell responses induced in cynomolgus monkeys challenged 1 year after immunization.

    (A and B) Quantification of CD8+ T cells specific for LASV GPC (A) or NP (B) peptides derived from Josiah strain. (C and D) Quantification of CD4+ T cells specific for LASV GPC (C) or NP (D) peptides derived from the Josiah strain. The percentage of T cells producing TNF-α or IFN-γ in response to peptide stimulation among total CD8+ T cells is presented according to the time after challenge after subtraction of the respective value measured for unstimulated T cells. Each bar represents the means ± SEM of eight samples. The proportion of each subpopulation of cytokine-positive T cells is presented in the pie charts. Statistical significance: *P ≤ 0.05, one-way ANOVA. (E and F) Percentage of CD4+ T cells expressing CD154 in response to stimulation with Josiah GPC (E) or Josiah NP (F) peptides according to the time after challenge. A one-way ANOVA test was used.

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