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Safety and Immunogenicity of an rVSV Lassa Fever Vaccine Candidate. BACKGROUND: No vaccine is currently available for Lassa fever, a viral hemorrhagic disease that is estimated to cause thousands of deaths each year in western Africa. A replication-competent recombinant vesicular stomatitis virus-vectored vaccine encoding a Lassa virus (LASV) glycoprotein complex, rVSVΔG-LASV-GPC, has been developed, but data on its safety and immunogenicity are limited. METHODS: In this phase 1, double-blind trial conducted in the United States and Liberia, we randomly assigned healthy adults (18 to 50 years of age) to receive rVSVΔG-LASV-GPC or placebo intramuscularly. Participants received a single vaccine dose of 2×104 plaque-forming units (PFU), 2×105 PFU, 2×106 PFU, or 2×107 PFU or placebo or received two vaccine doses of 2×107 PFU or placebo, within a window of 6 to 20 weeks. The side-effect profile was assessed according to the incidence of solicited and unsolicited adverse events (primary end point). Because Lassa fever can cause sensorineural hearing loss, hearing acuity was measured before and after the injection. Secondary end points were levels of binding antibodies against LASV glycoprotein, neutralizing antibodies, and vaccine vector-derived viral RNA and PFU in plasma, urine, and saliva. RESULTS: A total of 114 adults were enrolled. No serious vaccine-related adverse events were reported. The vaccine caused minimal local reactions and dose-dependent, mild-to-severe early-onset systemic reactogenicity events that were transient. No hearing loss was detected. All doses induced robust long-lasting cellular and humoral (binding and neutralizing) responses that cross-reacted against common LASV lineages. No infectious vaccine virus particles were found in plasma, urine, or saliva. CONCLUSIONS: The rVSVΔG-LASV-GPC vaccine resulted in transient local and systemic reactogenicity events but no hearing loss or serious adverse events. The vaccine had immunogenicity over a wide dose range in healthy adults in the United States and Liberia. (Funded by the Coalition for Epidemic Preparedness Innovations and the National Institute of Allergy and Infectious Diseases; ClinicalTrials.gov number, NCT04794218; Pan African Clinical Trials Registry number, PACTR2021106625781067.).

Seasonal acute viral hemorrhagic Lassa fever, caused by at least seven LASV phylogenetic lineages, has pandemic potential.1-4 There have been recent Nigerian outbreaks,5,6 and cases of European7 and US8 introduction; additionally, endemicity has been established in West Africa.9 No vaccines are approved for Lassa fever to alleviate health and economic burdens or to prepare for pandemics. A consortium led by International Aids Vaccine Initiative (IAVI) is advancing a Lassa fever vaccine based on replication-competent recombinant vesicular stomatitis virus (rVSV) vector with VSV envelope glycoprotein gene exchanged for LASV glycoprotein complex (LASV-GPC; rVSVΔG-LASV-GPC).10 Based on the same approach, Merck developed a rVSVΔG Zaire Ebola virus (ERVEBO) chimera, approved by FDA, EMA, and African countries.11-14 Since 2016, more than 300,000 ERVEBO doses have been administered during Ebola outbreaks15 and prophylactically for frontline workers.13,16 rVSVΔG-LASV-GPC, developed at the Public Health Agency of Canada10,17 demonstrated 100% efficacy in non-human primates and other models.18-20 A single rVSV∆G-LASV-GPC administration completely protected cynomolgus macaques from lethal LASV.21 This first-in-human study of rVSVΔG-LASV-GPC was designed to assess safety and immunogenicity in healthy US and Liberian adults.

This randomized, double-blind, placebo-controlled, dose-escalation, phase 1 study evaluated the safety, tolerability, and immunogenicity of rVSVΔG-LASV-GPC. Its manufacture is described in the supplementary appendix. Healthy participants, aged 18-50 years, from 3 US and 1 Liberian sites were enrolled in eight groups and randomized (4:1) to intramuscular rVSV∆G-LASV-GPC or placebo (matched diluent). In the Dose-Escalation phase, 10 US participants per group (Groups 1-3), were randomized within groups to receive a single dose 2×104, 2×105 or 2×106 PFU, respectively or placebo. Twenty-two Group 4 participants received 2×107 PFU or placebo and were invited, per protocol amendment, to receive a second active dose or placebo between 6 and 20 weeks post-first immunization. Eleven participants opted to receive a second dose identical to the first, without unblinding (Group 4B). The assignment into Group 4A occurred for participants declining second dose or to balance the overall group sizes between Group 4A and 4B. There was no randomization/blinding between Group 4A and 4B schedules; randomization/blinding pertained only to active dose versus placebo. The authors vouch for the accuracy and completeness of data and data analyses, along with the conduct of the trial according to the protocol (available at NEJM.org).

oup 4A and 4B. There was no randomization/blinding between Group 4A and 4B schedules; randomization/blinding pertained only to active dose versus placebo. The authors vouch for the accuracy and completeness of data and data analyses, along with the conduct of the trial according to the protocol (available at NEJM.org). In the Dose-Expansion phase, 60 Liberian participants were randomized to single dose 2×105 (Group 5), 2×106 (Group 6) or 2×107 PFU (Group 7) or placebo. An additional US participant was enrolled (Group 6) and administered a vaccine identical to Group 3. To facilitate data reporting of US vs. Liberian study populations, data from this single Group 6 US-based participant are reported with Group 3. Participants were followed for 12 months post-vaccination. Randomization, blinding, US FDA and the Liberia Medicine and Health Regulatory Authority Investigational New Drug authorizations, Institutional Review, eligibility criteria and enrollment are detailed in the supplementary appendix.

In the Dose-Expansion phase, 60 Liberian participants were randomized to single dose 2×105 (Group 5), 2×106 (Group 6) or 2×107 PFU (Group 7) or placebo. An additional US participant was enrolled (Group 6) and administered a vaccine identical to Group 3. To facilitate data reporting of US vs. Liberian study populations, data from this single Group 6 US-based participant are reported with Group 3. Participants were followed for 12 months post-vaccination. Randomization, blinding, US FDA and the Liberia Medicine and Health Regulatory Authority Investigational New Drug authorizations, Institutional Review, eligibility criteria and enrollment are detailed in the supplementary appendix. The primary objective was to evaluate rVSVΔG-LASV-GPC dose level safety and tolerability. Participants completed solicited local and systemic reactogenicity symptom diaries for 14 days post-vaccination; unsolicited events were recorded for 28 days and serious adverse events (SAEs) and adverse events of special interest (AESIs) (new hearing loss ≥30 decibels) for study duration. Since LF is associated with sensorineural hearing loss,22 participants were assessed before and 1-, 3- and 12-months post-vaccination by pure tone audiometry. Blood samples were collected for safety assessment (CBC, AST, ALT and creatinine). Details of assessments are provided in the supplementary appendix.

y duration. Since LF is associated with sensorineural hearing loss,22 participants were assessed before and 1-, 3- and 12-months post-vaccination by pure tone audiometry. Blood samples were collected for safety assessment (CBC, AST, ALT and creatinine). Details of assessments are provided in the supplementary appendix. Secondary objectives included assessment of LASV-GPC-specific antibody responses and rVSVΔG-LASV-GPC plasma, urine and saliva distribution. These assessments were performed in persons who received at least one vaccine or placebo and contributed both a pre-vaccination sample and at least one post-vaccination serum sample with valid results. Blood was collected at baseline, days 8, 29, 85, 169, 253 and 337 for immunogenicity assessments. Additional Group 4B samples were collected before and two and four weeks post-second dose. Blood, saliva and urine samples were collected pre-vaccination and days 4, 8, 15 and 29 for rVSVΔG-LASV-GPC distribution analyses. Anti-LASV-GPC serum IgG antibodies were quantified by ELISA using a standard curve (first International Standard of Anti-LASV Antibodies [National Institute for Biological Standards and Control, 20/202]). Neutralizing serum antibodies were assessed using lentivirus luciferase reporters pseudotyped with LASV-GPC lineages II, III or IV. ReLASV® Pan-Lassa NP IgG/IgM ELISA Kit (Zalgen Labs) was used following manufacturer’s instructions to screen Liberian participants for potential previous exposure to LASV.

Anti-LASV-GPC serum IgG antibodies were quantified by ELISA using a standard curve (first International Standard of Anti-LASV Antibodies [National Institute for Biological Standards and Control, 20/202]). Neutralizing serum antibodies were assessed using lentivirus luciferase reporters pseudotyped with LASV-GPC lineages II, III or IV. ReLASV® Pan-Lassa NP IgG/IgM ELISA Kit (Zalgen Labs) was used following manufacturer’s instructions to screen Liberian participants for potential previous exposure to LASV. Plasma, urine and saliva rVSVΔG-LASV-GPC RNA was detected by reverse transcription quantitative PCR (RT-qPCR) specific for the VSV nucleocapsid N gene. RT-qPCR positive samples were tested for replication-competent rVSVΔG-LASV-GPC by Vero cell plaque assay. Cryopreserved peripheral blood mononuclear cells (PBMC) were assessed for LASV-specific T-cells by IFNγ ELISpot assay using overlapping peptides matched to LASV-GPC.

Plasma, urine and saliva rVSVΔG-LASV-GPC RNA was detected by reverse transcription quantitative PCR (RT-qPCR) specific for the VSV nucleocapsid N gene. RT-qPCR positive samples were tested for replication-competent rVSVΔG-LASV-GPC by Vero cell plaque assay. Cryopreserved peripheral blood mononuclear cells (PBMC) were assessed for LASV-specific T-cells by IFNγ ELISpot assay using overlapping peptides matched to LASV-GPC. This study was not powered for formal hypothesis testing and analyses were not adjusted for multiplicity. For each safety endpoint, the proportion of participants with an event was calculated by dose. The frequency and proportions of participants with LASV-GPC binding and neutralizing antibody responses were calculated by dose and time. Binding and neutralizing antibody levels were summarized by geometric mean (GM) and 95% confidence interval (CI). Antibody seroconversion was defined as: antibody response above assay cut-off in participants with negative baseline results, or at least 4-fold increase from baseline in baseline positive participants. Post-hoc correlation analyses used Spearman’s rank test.

The primary objective was to evaluate rVSVΔG-LASV-GPC dose level safety and tolerability. Participants completed solicited local and systemic reactogenicity symptom diaries for 14 days post-vaccination; unsolicited events were recorded for 28 days and serious adverse events (SAEs) and adverse events of special interest (AESIs) (new hearing loss ≥30 decibels) for study duration. Since LF is associated with sensorineural hearing loss,22 participants were assessed before and 1-, 3- and 12-months post-vaccination by pure tone audiometry. Blood samples were collected for safety assessment (CBC, AST, ALT and creatinine). Details of assessments are provided in the supplementary appendix. Secondary objectives included assessment of LASV-GPC-specific antibody responses and rVSVΔG-LASV-GPC plasma, urine and saliva distribution. These assessments were performed in persons who received at least one vaccine or placebo and contributed both a pre-vaccination sample and at least one post-vaccination serum sample with valid results. Blood was collected at baseline, days 8, 29, 85, 169, 253 and 337 for immunogenicity assessments. Additional Group 4B samples were collected before and two and four weeks post-second dose. Blood, saliva and urine samples were collected pre-vaccination and days 4, 8, 15 and 29 for rVSVΔG-LASV-GPC distribution analyses.

Anti-LASV-GPC serum IgG antibodies were quantified by ELISA using a standard curve (first International Standard of Anti-LASV Antibodies [National Institute for Biological Standards and Control, 20/202]). Neutralizing serum antibodies were assessed using lentivirus luciferase reporters pseudotyped with LASV-GPC lineages II, III or IV.

Plasma, urine and saliva rVSVΔG-LASV-GPC RNA was detected by reverse transcription quantitative PCR (RT-qPCR) specific for the VSV nucleocapsid N gene. RT-qPCR positive samples were tested for replication-competent rVSVΔG-LASV-GPC by Vero cell plaque assay.

This study was not powered for formal hypothesis testing and analyses were not adjusted for multiplicity. For each safety endpoint, the proportion of participants with an event was calculated by dose. The frequency and proportions of participants with LASV-GPC binding and neutralizing antibody responses were calculated by dose and time. Binding and neutralizing antibody levels were summarized by geometric mean (GM) and 95% confidence interval (CI). Antibody seroconversion was defined as: antibody response above assay cut-off in participants with negative baseline results, or at least 4-fold increase from baseline in baseline positive participants. Post-hoc correlation analyses used Spearman’s rank test.

The study was conducted from July 2021 to December 2023. Of 80 US volunteers screened, 53 (31 female, 22 male) were enrolled, median age 26 years (range: 18-49) (Table 1, Figure S1A). Forty four (83.0%) completed the trial, and 9 (17.0%) terminated early; 7 (13.2%) were lost to follow-up despite efforts to contact them. Of 175 Liberian volunteers screened, 61 (32 female, 29 male) were enrolled, median age 24 years (range: 18-43) (Table 1, Figure S1B). Most exclusions (45/175) were due to baseline hearing impairment ≥26 dB. One participant, found ineligible post-randomization, did not receive study vaccine; 60 completed the study. Tables S1 and S2 summarize participant demographics and characteristics. The representativeness of the enrolled individuals is summarized in Table S3. At least one local reactogenicity event, Grade 1 or 2, was reported by 67.4% (29/43) of US and 31.3% (15/48) of Liberian vaccinees, and 40.0% (4/10) of US and 0% (0/12) Liberian placebo recipients. (Figures 1, and S2, S3 and S4). Administration site tenderness (65.1% [28/43] US; 22.9% [11/48] Liberia) and pain (44.2% [19/43] US; 18.8% [9/48] Liberia) were the most vaccinee-reported local events.

29/43) of US and 31.3% (15/48) of Liberian vaccinees, and 40.0% (4/10) of US and 0% (0/12) Liberian placebo recipients. (Figures 1, and S2, S3 and S4). Administration site tenderness (65.1% [28/43] US; 22.9% [11/48] Liberia) and pain (44.2% [19/43] US; 18.8% [9/48] Liberia) were the most vaccinee-reported local events. At least one systemic reactogenicity event, Grades 1 to 3, was reported by 88.4% (38/43) of US and 52.1% (25/48) of Liberian vaccinees and 80.0% (8/10) of US and 41.7% (5/12) of Liberian placebo recipients. The most common events were malaise, headache, myalgia and chills (Figure 1, Table S4). Eleven (25.6%) US participants, all in Groups 4A and B, reported Grade 3 events. The most common was malaise, typically reported for 2-3 days with concurrent Grade 3 systemic events including myalgia, chills, headache, arthralgia and/or fever, resolving within 1-5 days. In Group 4B participants, reactogenicity appeared milder following the second dose than following the first (Figure S4). Two (4.2%) Liberian vaccinees reported Grade 3 systemic events, abdominal pain/diarrhea and fever, for 1-2 days. Laboratory abnormalities grade 2 or higher, not associated with clinical manifestations, occurred in 1 US and 3 Liberian vaccinees (Table S5).

following the second dose than following the first (Figure S4). Two (4.2%) Liberian vaccinees reported Grade 3 systemic events, abdominal pain/diarrhea and fever, for 1-2 days. Laboratory abnormalities grade 2 or higher, not associated with clinical manifestations, occurred in 1 US and 3 Liberian vaccinees (Table S5). Unsolicited AEs were reported by 51.2% (22/43) of US and 27.1% (13/48) of Liberian vaccinees (versus 20.0% (2/20) and 25% (3/12) of placebo recipients, respectively, through day 28 (Tables S6-S7). Most unsolicited events were Grades 1 or 2; Grade 3 unsolicited AEs were reported by 4.7% (2/43) US vaccinees and no Liberian vaccinees, versus 10% (1/10) and 16.7% (2/12) of respective placebo recipients. Among Grade 3 AEs, one was considered probably vaccine-related-elevated AST at Day 14 in a Group 4A participant lasting 5 days. Two other unsolicited AEs were assessed as definitely vaccine-related: Grade 1 lymphadenopathy in a Group 4A participant at day 4 lasting 3 days and Grade 1 sinus tachycardia in a Group 4B participant 1-day post-dose 1, resolving next day without treatment. There were no reports of arthritis or rashes. There were no reported AESIs, vaccine-associated SAEs and no discontinuations due to AEs.

Unsolicited AEs were reported by 51.2% (22/43) of US and 27.1% (13/48) of Liberian vaccinees (versus 20.0% (2/20) and 25% (3/12) of placebo recipients, respectively, through day 28 (Tables S6-S7). Most unsolicited events were Grades 1 or 2; Grade 3 unsolicited AEs were reported by 4.7% (2/43) US vaccinees and no Liberian vaccinees, versus 10% (1/10) and 16.7% (2/12) of respective placebo recipients. Among Grade 3 AEs, one was considered probably vaccine-related-elevated AST at Day 14 in a Group 4A participant lasting 5 days. Two other unsolicited AEs were assessed as definitely vaccine-related: Grade 1 lymphadenopathy in a Group 4A participant at day 4 lasting 3 days and Grade 1 sinus tachycardia in a Group 4B participant 1-day post-dose 1, resolving next day without treatment. There were no reports of arthritis or rashes. There were no reported AESIs, vaccine-associated SAEs and no discontinuations due to AEs. Among US vaccinees, serum LASV GPC-specific IgG antibody responses were detected relative to baseline in 42/43 (97.7%), with 37/42 (88.1%) responding by day 29 (Figures 2A and S5). IgG levels typically peaked at day 85 in single-dose US vaccinees, with Geometric Means of 2258.4 (95% CI, 1177.7-4330.7), 1108.5 (95% CI, 539.5-2277.6), 3506.5 (95% CI, 1833.2-6707.3) and 2056.1 (95% CI, 808.7-5227.5) IU/mL in Groups 1, 2, 3 and 4A, respectively, compared to 1.9 (95% CI, 0.4-8.3) for placebo. In the two-dose group (4B), geometric mean was 1495.8 (95% CI, 782.5-2859.5) pre-second dose, with 1531.7 (95% CI, 915.3-2563.5) IU/mL observed 28 days post-second vaccination. At month 12, these antibody responses were detected in 31/36 (86.1%) vaccinees.

A, respectively, compared to 1.9 (95% CI, 0.4-8.3) for placebo. In the two-dose group (4B), geometric mean was 1495.8 (95% CI, 782.5-2859.5) pre-second dose, with 1531.7 (95% CI, 915.3-2563.5) IU/mL observed 28 days post-second vaccination. At month 12, these antibody responses were detected in 31/36 (86.1%) vaccinees. Among Liberian vaccinees, serum LASV GPC-specific IgG antibody responses were detected in 44/48 (91.7%) vaccinees (Figures 2B and S5). Most (27/48, 56.3%) generated responses by day 29, with 42/48 (87.5%) responding by day 85. Available month 12 test results demonstrated 30/48 (62.5%) vaccinee responses relative to baseline. Peak binding IgG levels, typically at day 85, were geometric means of 1400.8 (95% CI, 952.5-2060.0), 622.4 (95% CI, 145.1-2669.0) and 886.3 (95% CI, 528.4-1487.0) IU/mL in Groups 5, 6 and 7 respectively, compared to 28.6 (95% CI, 4.2-195.9) for placebo. Vaccine-induced antibodies exhibited multiple LASV lineage cross-reactivity (Figure S6).

Among Liberian vaccinees, serum LASV GPC-specific IgG antibody responses were detected in 44/48 (91.7%) vaccinees (Figures 2B and S5). Most (27/48, 56.3%) generated responses by day 29, with 42/48 (87.5%) responding by day 85. Available month 12 test results demonstrated 30/48 (62.5%) vaccinee responses relative to baseline. Peak binding IgG levels, typically at day 85, were geometric means of 1400.8 (95% CI, 952.5-2060.0), 622.4 (95% CI, 145.1-2669.0) and 886.3 (95% CI, 528.4-1487.0) IU/mL in Groups 5, 6 and 7 respectively, compared to 28.6 (95% CI, 4.2-195.9) for placebo. Vaccine-induced antibodies exhibited multiple LASV lineage cross-reactivity (Figure S6). Serum neutralizing antibody responses developed in 42/43 (97.7%) US vaccinees (Figures 2C and S7), most within 3 months (32/43, 74.4%). At month 12, 34/36 (94.4%) vaccinees demonstrated responses relative to baseline. One vaccinee without binding and neutralizing antibody responses post-vaccination was lost to follow up after day 8. Peak geometric mean neutralizing antibody ID50 values were 152.6 (95% CI, 96.4-241.5) in Group 1, 114.1 (95% CI, 51.6-252.3) Group 2, 119.8 (95% CI, 72.6-197.7) Group 3, 107.7 (95% CI, 49.8-233.3) Group 4A, 174.3 (95% CI, 135.4-224.5) Group 4B and 11.2 (95% CI, 9.5-13.2) in placebo.

n was lost to follow up after day 8. Peak geometric mean neutralizing antibody ID50 values were 152.6 (95% CI, 96.4-241.5) in Group 1, 114.1 (95% CI, 51.6-252.3) Group 2, 119.8 (95% CI, 72.6-197.7) Group 3, 107.7 (95% CI, 49.8-233.3) Group 4A, 174.3 (95% CI, 135.4-224.5) Group 4B and 11.2 (95% CI, 9.5-13.2) in placebo. Serum neutralizing activity was observed in 36/48 (75.0%) Liberian vaccinees (Figures 2D and S7), most responding within 3 months (35/48, 72.9%). Peak geometric mean neutralizing antibody ID50 values were 129.0 (95% CI, 101.8-163.4) in Group 5, 159.0 (95% CI, 89.5-282.3) Group 6, 92.8 (95% CI, 68.0-126.5) Group 7 and 27.9 (95% CI, 15.2-51.2) in placebo. Like LASV GPC-specific IgG responses, neutralizing antibodies were not boosted post-second immunization (Group 4B) (Figures 2E, 2F, S5, and S7). Vaccine-induced antibodies exhibited multiple LASV lineage pseudo-virus cross-neutralization (Figure S8). Among Liberian participants, 4 of 60 (6.7%) had detectable LASV NP-specific IgG antibodies at screening, indicating previous LASV exposure. Two (1 placebo, 1 Group 5) had pre-existing anti-LASV neutralizing antibodies; the Group 5 vaccinee did not generate LASV-GPC-specific antibody responses post-vaccination (Figure S9).

Among Liberian participants, 4 of 60 (6.7%) had detectable LASV NP-specific IgG antibodies at screening, indicating previous LASV exposure. Two (1 placebo, 1 Group 5) had pre-existing anti-LASV neutralizing antibodies; the Group 5 vaccinee did not generate LASV-GPC-specific antibody responses post-vaccination (Figure S9). All of 31 US study vaccinees with PBMC samples exhibited vaccine-specific IFN-γ ELISpot T-cell responses by day 29 (Figure 3A, Table S8). Twenty (64.5%) vaccinees responded to all 5 GPC pools on day 85, indicating multiple LASV-GPC epitope recognition (Figure 3B, Table S8). Responses in Groups 4A and 4B appeared similar on days 169, 252 and 337 (Figure S10). Overall, serum LASV GPC-specific IgG, neutralizing antibody and IFNγ responses demonstrated similar kinetics and magnitude across all dose groups. Positive associations among binding, neutralizing antibody and IFNγ responses were observed (Figure S11).

All of 31 US study vaccinees with PBMC samples exhibited vaccine-specific IFN-γ ELISpot T-cell responses by day 29 (Figure 3A, Table S8). Twenty (64.5%) vaccinees responded to all 5 GPC pools on day 85, indicating multiple LASV-GPC epitope recognition (Figure 3B, Table S8). Responses in Groups 4A and 4B appeared similar on days 169, 252 and 337 (Figure S10). Overall, serum LASV GPC-specific IgG, neutralizing antibody and IFNγ responses demonstrated similar kinetics and magnitude across all dose groups. Positive associations among binding, neutralizing antibody and IFNγ responses were observed (Figure S11). Vector-derived RNA was measured in plasma, saliva or urine following the first dose in US participants (Figures S12 and 13; Table S9). By day 29, vector-derived plasma RNA was detected in 12.5% Group 1, 62.5% Group 2, 100% Group 3, 88.9% Group 4A and 100% Group 4B participants; it was not detected in any placebo group participants. Levels appeared highest on day 4 and fell below the lower quantification limit (500 copies/mL) by day 29. Peak antibody responses appeared to track weakly with plasma vaccine RNA levels (Figure S14). Data were insufficient to analyze associations between plasma RNA and clinical symptoms. Vector-derived saliva RNA was detected in 12.5% Group 1, 37.5% Group 2, 87.5% Group 3, 33.3% Group 4A and 66.7% Group 4B participants. Saliva vector RNA was detected in one placebo participant, a finding that was presumed to reflect sample contamination. Saliva vector-derived RNA levels were highest on day 15; 48% of participants had detectable saliva RNA levels on day 29. Vector RNA was undetectable in urine. No infectious vaccine virus was recovered by plaque assay from any RNA-positive samples.

The study was conducted from July 2021 to December 2023. Of 80 US volunteers screened, 53 (31 female, 22 male) were enrolled, median age 26 years (range: 18-49) (Table 1, Figure S1A). Forty four (83.0%) completed the trial, and 9 (17.0%) terminated early; 7 (13.2%) were lost to follow-up despite efforts to contact them. Of 175 Liberian volunteers screened, 61 (32 female, 29 male) were enrolled, median age 24 years (range: 18-43) (Table 1, Figure S1B). Most exclusions (45/175) were due to baseline hearing impairment ≥26 dB. One participant, found ineligible post-randomization, did not receive study vaccine; 60 completed the study. Tables S1 and S2 summarize participant demographics and characteristics. The representativeness of the enrolled individuals is summarized in Table S3.

At least one local reactogenicity event, Grade 1 or 2, was reported by 67.4% (29/43) of US and 31.3% (15/48) of Liberian vaccinees, and 40.0% (4/10) of US and 0% (0/12) Liberian placebo recipients. (Figures 1, and S2, S3 and S4). Administration site tenderness (65.1% [28/43] US; 22.9% [11/48] Liberia) and pain (44.2% [19/43] US; 18.8% [9/48] Liberia) were the most vaccinee-reported local events. At least one systemic reactogenicity event, Grades 1 to 3, was reported by 88.4% (38/43) of US and 52.1% (25/48) of Liberian vaccinees and 80.0% (8/10) of US and 41.7% (5/12) of Liberian placebo recipients. The most common events were malaise, headache, myalgia and chills (Figure 1, Table S4). Eleven (25.6%) US participants, all in Groups 4A and B, reported Grade 3 events. The most common was malaise, typically reported for 2-3 days with concurrent Grade 3 systemic events including myalgia, chills, headache, arthralgia and/or fever, resolving within 1-5 days. In Group 4B participants, reactogenicity appeared milder following the second dose than following the first (Figure S4). Two (4.2%) Liberian vaccinees reported Grade 3 systemic events, abdominal pain/diarrhea and fever, for 1-2 days. Laboratory abnormalities grade 2 or higher, not associated with clinical manifestations, occurred in 1 US and 3 Liberian vaccinees (Table S5).

Among US vaccinees, serum LASV GPC-specific IgG antibody responses were detected relative to baseline in 42/43 (97.7%), with 37/42 (88.1%) responding by day 29 (Figures 2A and S5). IgG levels typically peaked at day 85 in single-dose US vaccinees, with Geometric Means of 2258.4 (95% CI, 1177.7-4330.7), 1108.5 (95% CI, 539.5-2277.6), 3506.5 (95% CI, 1833.2-6707.3) and 2056.1 (95% CI, 808.7-5227.5) IU/mL in Groups 1, 2, 3 and 4A, respectively, compared to 1.9 (95% CI, 0.4-8.3) for placebo. In the two-dose group (4B), geometric mean was 1495.8 (95% CI, 782.5-2859.5) pre-second dose, with 1531.7 (95% CI, 915.3-2563.5) IU/mL observed 28 days post-second vaccination. At month 12, these antibody responses were detected in 31/36 (86.1%) vaccinees. Among Liberian vaccinees, serum LASV GPC-specific IgG antibody responses were detected in 44/48 (91.7%) vaccinees (Figures 2B and S5). Most (27/48, 56.3%) generated responses by day 29, with 42/48 (87.5%) responding by day 85. Available month 12 test results demonstrated 30/48 (62.5%) vaccinee responses relative to baseline. Peak binding IgG levels, typically at day 85, were geometric means of 1400.8 (95% CI, 952.5-2060.0), 622.4 (95% CI, 145.1-2669.0) and 886.3 (95% CI, 528.4-1487.0) IU/mL in Groups 5, 6 and 7 respectively, compared to 28.6 (95% CI, 4.2-195.9) for placebo. Vaccine-induced antibodies exhibited multiple LASV lineage cross-reactivity (Figure S6).

Serum neutralizing antibody responses developed in 42/43 (97.7%) US vaccinees (Figures 2C and S7), most within 3 months (32/43, 74.4%). At month 12, 34/36 (94.4%) vaccinees demonstrated responses relative to baseline. One vaccinee without binding and neutralizing antibody responses post-vaccination was lost to follow up after day 8. Peak geometric mean neutralizing antibody ID50 values were 152.6 (95% CI, 96.4-241.5) in Group 1, 114.1 (95% CI, 51.6-252.3) Group 2, 119.8 (95% CI, 72.6-197.7) Group 3, 107.7 (95% CI, 49.8-233.3) Group 4A, 174.3 (95% CI, 135.4-224.5) Group 4B and 11.2 (95% CI, 9.5-13.2) in placebo. Serum neutralizing activity was observed in 36/48 (75.0%) Liberian vaccinees (Figures 2D and S7), most responding within 3 months (35/48, 72.9%). Peak geometric mean neutralizing antibody ID50 values were 129.0 (95% CI, 101.8-163.4) in Group 5, 159.0 (95% CI, 89.5-282.3) Group 6, 92.8 (95% CI, 68.0-126.5) Group 7 and 27.9 (95% CI, 15.2-51.2) in placebo. Like LASV GPC-specific IgG responses, neutralizing antibodies were not boosted post-second immunization (Group 4B) (Figures 2E, 2F, S5, and S7). Vaccine-induced antibodies exhibited multiple LASV lineage pseudo-virus cross-neutralization (Figure S8). Among Liberian participants, 4 of 60 (6.7%) had detectable LASV NP-specific IgG antibodies at screening, indicating previous LASV exposure. Two (1 placebo, 1 Group 5) had pre-existing anti-LASV neutralizing antibodies; the Group 5 vaccinee did not generate LASV-GPC-specific antibody responses post-vaccination (Figure S9).

Vector-derived RNA was measured in plasma, saliva or urine following the first dose in US participants (Figures S12 and 13; Table S9). By day 29, vector-derived plasma RNA was detected in 12.5% Group 1, 62.5% Group 2, 100% Group 3, 88.9% Group 4A and 100% Group 4B participants; it was not detected in any placebo group participants. Levels appeared highest on day 4 and fell below the lower quantification limit (500 copies/mL) by day 29. Peak antibody responses appeared to track weakly with plasma vaccine RNA levels (Figure S14). Data were insufficient to analyze associations between plasma RNA and clinical symptoms. Vector-derived saliva RNA was detected in 12.5% Group 1, 37.5% Group 2, 87.5% Group 3, 33.3% Group 4A and 66.7% Group 4B participants. Saliva vector RNA was detected in one placebo participant, a finding that was presumed to reflect sample contamination. Saliva vector-derived RNA levels were highest on day 15; 48% of participants had detectable saliva RNA levels on day 29. Vector RNA was undetectable in urine. No infectious vaccine virus was recovered by plaque assay from any RNA-positive samples.

This first-in-human clinical study tested four rVSVΔG-LASV-GPC doses and single or two-dose regimens. Consistent with preclinical studies conducted during human vaccine candidate development21 and with rVSV∆G-LASV-GPC research vaccine18,19,20, the vaccine had an acceptable safety profile with no vaccine-related SAEs and no cases of hearing loss. Like the similarly constructed ERVEBO vaccine23 and other live-attenuated viral vaccines,24-26 VSV-based vaccines may cause fever, chills, malaise, myalgia, headache, nausea and/or arthralgia. These symptoms were less frequent and milder at 2×104 and 2×105 PFU doses. At 2×107 PFU, about half of US volunteers experienced Grade 3 symptoms on day 1-2, resolving within days. Transient liver enzyme elevation, as reported in one participant, was also reported in two ERVEBO studies.27,28 Reactogenicity appeared lower in Liberian participants, compared to US participants.

2×105 PFU doses. At 2×107 PFU, about half of US volunteers experienced Grade 3 symptoms on day 1-2, resolving within days. Transient liver enzyme elevation, as reported in one participant, was also reported in two ERVEBO studies.27,28 Reactogenicity appeared lower in Liberian participants, compared to US participants. Prior studies have shown negligible risk of person-to-person or animal-to-animal infection with rVSVΔG-ZEBOV-GP vaccine virus post-rVSVΔG-ZEBOV-GP vaccination.29 Consistent with findings with rVSVΔG-ZEBOV-GP,23,30 our study of rVSVΔG-LASV-GPC revealed that vector-derived plasma RNA was detected on day 4, but was undetectable on day 29. The time course for detection of saliva RNA differed, with little detected on day 4 and highest levels on days 15 and 29. In rVSVΔG-ZEBOV-GP studies, saliva vaccine RNA appeared later than plasma.27,31,32 No infectious vaccine virus was detected by culture assay, in line with one rVSVΔG-ZEBOV-GP study.30 Although culture is less sensitive than PCR, it may give more accurate representation of infectiousness of biological fluids.

15 and 29. In rVSVΔG-ZEBOV-GP studies, saliva vaccine RNA appeared later than plasma.27,31,32 No infectious vaccine virus was detected by culture assay, in line with one rVSVΔG-ZEBOV-GP study.30 Although culture is less sensitive than PCR, it may give more accurate representation of infectiousness of biological fluids. rVSVΔG-LASV-GPC immunogenicity was assessed over the course of one year. A single dose greater than 2×104 and up to 2×107 PFU elicited robust antibody responses, without an apparent dose response. IgG antibodies binding lineage IV LASV-GPC, predominant in Liberia and Sierra Leone,1 were detected with all doses, usually by day 29, peaking on day 85, with minimal decline one year post-vaccination. Binding IgG to LASV lineages I, II, III, and VII GPCs were also detected. Unlike a measles-vectored Lassa vaccine study,33 we detected homologous serum neutralizing antibodies persisting for a year, and neutralizing activity against LASV lineages II and III, predominant in Nigeria.1 Currently there are no defined correlates of protection against LASV infection, and the immunological thresholds required for vaccine efficacy remain undefined. Although mechanisms of LASV clearance are incompletely understood, T-cells are considered important.34,35 rVSVΔG-LASV-GPC vaccination induced broad, durable T-cell responses in US adults in our study. Second vaccination provided no appreciable benefit to humoral or cellular responses.

rVSVΔG-LASV-GPC immunogenicity was assessed over the course of one year. A single dose greater than 2×104 and up to 2×107 PFU elicited robust antibody responses, without an apparent dose response. IgG antibodies binding lineage IV LASV-GPC, predominant in Liberia and Sierra Leone,1 were detected with all doses, usually by day 29, peaking on day 85, with minimal decline one year post-vaccination. Binding IgG to LASV lineages I, II, III, and VII GPCs were also detected. Unlike a measles-vectored Lassa vaccine study,33 we detected homologous serum neutralizing antibodies persisting for a year, and neutralizing activity against LASV lineages II and III, predominant in Nigeria.1 Currently there are no defined correlates of protection against LASV infection, and the immunological thresholds required for vaccine efficacy remain undefined. Although mechanisms of LASV clearance are incompletely understood, T-cells are considered important.34,35 rVSVΔG-LASV-GPC vaccination induced broad, durable T-cell responses in US adults in our study. Second vaccination provided no appreciable benefit to humoral or cellular responses. All US-based vaccinees with samples beyond day 15 generated LASV-GPC specific IgG binding and neutralizing antibodies, whereas a small proportion of Liberian vaccinees did not. Our study was not designed to compare vaccine responses between US and Liberian cohorts; however, response magnitudes appeared slightly lower in Liberian compared to US cohort. Environmental factors may contribute to differential antibody responses to vaccinations in different study populations.36 For example, pre-existing immunity may influence vaccine responses. Pre-existing LASV NP IgG antibodies were detected in 4 Liberian participants. Additional participants had detectable baseline LASV GPC-specific antibodies. Baseline responses could be related to past LASV exposure, or cross-reactivity to GPCs from other arenaviruses.37,38 Antibody and T cell cross-reactivity to GPCs from LASV and Lymphocytic Choriomeningitis Virus have been previously demonstrated.39,40 Further studies in the LF endemic region are warranted to investigate this cross-reactivity and vaccine responses in previously exposed individuals.

to GPCs from other arenaviruses.37,38 Antibody and T cell cross-reactivity to GPCs from LASV and Lymphocytic Choriomeningitis Virus have been previously demonstrated.39,40 Further studies in the LF endemic region are warranted to investigate this cross-reactivity and vaccine responses in previously exposed individuals. Study limitations warrant comment. This was a Phase 1 study and hypothesis testing was not performed. It included a relatively small sample, among whom several US participants were lost to follow-up and a few participants in Liberia had pre-existing antibodies to LASV. Follow-up Phase 2/3 clinical studies are ongoing (ClinicalTrials.org NCT05868733) or are planned to confirm these findings, expand the safety profile and assess efficacy in LASV-exposed populations. Overall, rVSVΔG-LASV-GPC demonstrated favorable safety and immunogenicity, with a single dose inducing robust cellular and broad long-lasting antibody responses to major lineages. Disclosure forms provided by the authors are available with the full text of this article at NEJM.org.