Introduction
Lassa virus (LASV) is a rodent-borne CDC Tier 1 viral threat agent for which no licensed vaccines or therapeutics are currently available. Lassa fever is the cause of considerable disease burden in the endemic area of West Africa, causing acute and often fatal hemorrhagic fever disease with associated malaise, severe edema, blood loss, and high mortality1–5. Recovery from Lassa fever is also complicated by the development of sensorineural hearing loss in ~30% of survivors, and it does not appear to be linked with severity of disease6–10. In Nigeria, where Lassa is genetically diverse, patients often develop acute kidney injury during acute infection, which has correlated with survival11.
It has been estimated that between 100,000 and 300,000 cases of Lassa fever occur each year, resulting in ~5000 deaths per year5,7,12,13, with mortality rates as high as 69% in hospitalized patients, depending on location14–18. In addition to the endemic disease burden and the long-lasting effects of disease in survivors, imported cases of Lassa fever have been reported in the United States, Europe, and Canada19–27. Nosocomial infections affecting healthcare workers have occurred in nearly every recorded outbreak, indicating that the disease has high potential for person-to-person spread in the outbreak setting28.
Taken together, these factors have led to Lassa fever being categorized as a priority pathogen for countermeasure research and development by the World Health Organization. The development of effective vaccines and vaccine delivery technologies to protect against potential biothreat agents like LASV is of extreme importance. In response, the Coalition for Epidemic Preparedness Innovations (CEPI) has become the largest funder of research in the development and licensure of vaccines against Lassa fever.
Previously, we have reported the development of a DNA-based vaccine candidate against Lassa Fever, INO-4500, that offers complete short-term protective immunity to lethal LASV challenge in guinea pigs and cynomolgus macaques NHPs when delivered with electroporation (EP)29–31. Here, we expand our knowledge on the vaccine regimen and long-term immune responses and protective efficacy of INO-4500 in cynomolgus macaques against lethal LASV challenge. The cynomolgus NHP is a suitable model to study LASV vaccine efficacy, as LASV-infected NHPs develop similar clinical manifestations to those observed in humans32,33. The studies reported herein demonstrate the durability of protection in NHP one year after initial vaccination, the ability of the vaccine to protect against long-term effects of disease and offer insights into potential immune mechanisms associated with protection.
Methods
Nonhuman primate study design
Dose-ranging study (Arm 1)
The dose ranging study was conducted at Rocky Mountain Laboratories (RML) in Hamilton MT. A total of 24 cynomolgus macaques of Mauritius origin (50% male and female, ranging between 2.6 and 3.3 kg) were randomly assigned (stratified by weight and sex) to four groups consisting of six animals per group. The groups were divided between unimmunized control and vaccinated groups that received 0.1, 1, or 2 mg of INO-4500. Immunizations were administered at two time-points, scheduled 4 weeks apart. Immunizations were administered in the dermis above the quadriceps muscle group, followed by intradermal electroporation (ID-EP) using CELLECTRA™-ID30,34. Virus challenge occurred 4 weeks after the final vaccination timepoint. NHPs were administered a target dose of 1000 PFU (plaque forming units) of LASV Josiah suspended in 1 mL of sterile saline via intramuscular (IM) injection to the upper arm. Blood samples were collected prior to virus challenge on Day 0 and on Days 3, 6, 10, 14, 21, 28, and 38 (end of study) post-challenge. Blood samples were used for blood chemistry and blood cell population assessments. Serum was isolated and used for virus titration by plaque assay and plaque reduction neutralization tests (PRNTs). Post-challenge clinical scoring was performed daily by the same operator, blinded to the study groups. Clinical scoring for euthanasia determinations at RML was based upon appearance, recumbency, respiration, and appetite. Predetermined euthanasia criteria were established based on ACUC-approved scoring criteria. Animals were euthanized when predefined endpoint criteria were met, or at the end of the study. To monitor NHP for hearing loss, Brainstem Auditory Evoked Response (BAERCOM®) analysis was performed on each animal under anesthesia at the start of the study to obtain baseline measurements and prior to euthanasia to assess outcome.
One vs two-dose study (Arm 2)
The one vs two-dose study was conducted at USAMRIID in Frederick, MD. Eighteen cynomolgus macaques of Vietnamese and Cambodian origins (7 male and 11 female, ranging between 2.1 and 8.8 kg) were divided in three groups of six NHPs/group. One group was left unimmunized and the other two groups received one or two doses of INO-4500 at 2 mg per dose intradermally, followed by ID-EP. Both groups were immunized at week 0; the second dose for the two-dose group was administered four weeks after the first dose. Blood samples were collected at multiple time-points following immunization after which NHPs were challenged with a target of 1000 PFU of LASV Josiah via IM injection at week 8. Blood samples were collected just prior to virus challenge on Day 0 and on Days 3, 6, 10, 14, 21, and 28 post-challenge, as well as at the end of study on Day 30 post-challenge. Blood samples were used for blood chemistry and blood cell population assessments. Serum samples were separated and used for virus titration by plaque assay and PRNTs. Post-challenge clinical scoring was performed daily by members of the study team who were blinded to the study groups. Clinical scoring for euthanasia determinations at USAMRIID were based upon recumbency. Predetermined clinical scoring and euthanasia criteria were established based on USAMRIID’s IACUC-approved protocol. Animals were monitored for body weight and temperature changes and evaluated for clinical signs of disease. Animals were humanely euthanized when moribund according to predefined criteria in the IACUC protocol, or at the end of the study. NHPs were also monitored for hearing loss using BAERCOM® at the start and at the end of the study to assess outcome.
Durability study (Arm 3)
The Durability study was conducted at RML, in Hamilton MT. A total of eighteen cynomolgus macaques of Chinese origin (50% male and female, ranging between 2.3 and 5.2 kg) were randomly assigned (stratified by weight and sex) to two groups consisting of six NHPs per group. An age/weight-matched control group consisting of six NHPs was populated prior to challenge. The control group was unvaccinated and the other two groups received two doses of 2 mg or 1 mg of INO-4500 + IP-EP four weeks apart. All NHPs were challenged with a target IM dose of 1000 PFU LASV (Josiah) 59 weeks after the final vaccination was administered. Blood samples were collected as described for Arms 1 and 2. Post-challenge assessments were performed as described for Arm 1 study. All animals were euthanized at the end of the study on Day 35 post-challenge. Tissue samples were collected for histopathology after euthanasia.
Heterologous immune response study in NHP (supplemental study)
NHPs were housed at BIOQUAL facility following the approved ACUP protocol. Five cynomolgus NHPs (2 male and 3 female, ranging from 3.2 to 9.5 kg) received 2 mg of INO-4500 with ID-EP at weeks 0, 4, 8, and 37 (booster dose). Serum and PBMCs were collected at selected time-points for assessment of cross-reactive antibody and T cell responses.
PBMC isolation
Blood was collected in K2 EDTA tubes and shipped overnight to Inovio for peripheral blood mononuclear cell (PBMC) isolation. Whole blood was diluted 1:1 with an equal volume of PBS supplemented with 2% FBS. PBMCs were isolated using a 50 mL SepMate tube (Stemcell Technologies) pre-filled with 15 mL of 90% Ficoll (GE Healthcare) and 10% PBS. Red blood cells present in the PBMC suspension were lysed with 5 mL ACK buffer for 5 min before the addition of 1x PBS to stop the reaction. Cells were then centrifuged at 1500 rpm for 5 min and resuspended in 10 mL of RPMI supplemented with 10% FBS and 10% PenStrep (R10 medium) for ELISpot assay.
IFNγ Enzyme-linked Immuno Spot (ELISpot) assay
To assess the cellular IFNγ responses to vaccinations, monkey interferon (IFN)-γ ELISpot assays were performed using a Mabtech IFNγ ELISpotPRO (ALP) kit (Mabtech, Sweden) following manufacturer’s protocol. Briefly, 96-well ELISpot plates were blocked with R10 medium overnight at 4 °C. The following day, 200,000 freshly isolated PBMCs from NHPs were added to each well and incubated at 37 °C in 5 % CO2 in the presence of media with DMSO (negative control), PMA/ Ionomycin (positive control for monkey, 5000 cells/well plated), or media with peptide pools consisting of 15-mers overlapping by nine amino acids and spanning the length of LASV lineages I, II, III, or IV GPC protein (GenScript, custom made). After 18-20 h, plates were washed and developed. Antigen-specific responses were determined by subtracting the number of spots in the negative control wells from the wells containing peptide pools.
Enzyme-linked Immunosorbent Assay (ELISA)
To determine antigen-specific antibody responses in sera, 96-well ELISA plates were coated with 1 µg/ml recombinant LASV lineage IV GP prefusion protein (Zalgen Labs) or 1 µg/ml recombinant LASV lineages I, II and III GP prefusion protein (kindly donated from Dr. Erica Sapphire, La Jolla Institute for Immunology) in 1x PBS overnight at 4 °C. Plates were then washed with 1x PBS + 0.05% Tween 20 and blocked with 3% BSA PBS-Tween 20 for 2 h at 37 °C. After blocking, plates were washed and serum was serially diluted in 1% BSA PBS-Tween 20 and incubated for 2 h at 37 °C. Plates were washed and incubated with 1:10,000 dilution of secondary anti-human IgG HRP (BD Bioscience cat #555788) for 1 h. Plates were washed and SureBlue TMB peroxidase substrate (ref# 95059-286, KPL) was added to the plates for 10 min, after which the reaction was stopped with TMB stop solution (ref# 5150-0020, KPL). The absorbance signal was acquired at 450 nm using an automated plate reader (Biotek).
LASV pseudovirus neutralization assay
Pseudovirus production: HIV-based LASV IV pseudovirus carrying a luciferase reporter gene was generated by co-transfecting 293T cells with pLASV-GPC IV and envelope-defective HIV-1 pNL4-3.Luc.R-E plasmids (NIH AIDS reagent program) using Lipofectamine 3000 (Life Technologies). Pseudovirus-containing culture supernatant was harvested 72 h post-transfection. Supernatants were filtered using a 0.22 μm filter (Sigma-Millipore), aliquoted, and stored at −80 °C until further use. For pseudovirus neutralization assays, NHP sera samples were heat inactivated for 1 h at 56 °C and serial dilutions of heat-inactivated sera were mixed with equal volumes of LASV pseudovirus and incubated 1 h at 37 °C. Pseudovirus-sera mixture was added to 96-well plates with 293 T cells. After a 72 h incubation, cells were lysed, and luciferase signal acquired by luminescence using an automated plate reader.
T cell activation assay
T-cell activation assay was performed as previously described with modifications35. Briefly, PBMCs from study Arm 3 were thawed (Day 0 pre-immunization, Day 42, 270 and 379 pre-challenge) and resuspended in complete RPMI (cRPMI) medium supplemented with 10% FBS, 1% Penn/Strep, 1% L-glutamine, 1% HEPES, 1% Non-essential amino acids, 1% sodium pyruvate, 0.1% DNAse I, 0.1% 2-mercapto-ethanol, and allowed to rest overnight at 37 °C and 5% CO2. PBMCs were washed with cRPMI and resuspended in medium containing overlapping peptides spanning the Josiah GPC for 24 h. Cells were washed with cRPMI, and supplemented with 10 U/mL IL-2 and 10 ng/mL of IL-7 every 2 days for 6 days. IL-2 and IL-7 were removed, PBMCs washed with cRPMI, and allowed to rest overnight at 37 °C and 5% CO2. The next day cells were incubated with peptide megapools or DMSO control for 20 h. PBMCs were stained for 1 h at 4 °C with LIVE/DEAD stain (ThermoFisher), anti-CD3 (2 µL/well, clone SP34-2, APC-Cy7), anti-CD4 (4 µL/well, clone L200, PerpCp-Cy5.5), anti-CD8 (2 µL/well, clone RPA-T8, BV650), anti-CD69 (3 µL/well, clone FN50, APC), anti-CD134 (2 µL/well, clone L106, BV786), anti-CD137 (2 µL/well, clone 4-1BB, BV421), anti-CD25 (4 µL/well, clone BC96, AF488) and anti-CD154 (2 µL/well, clone 24-31, BV605) antibodies. Staining was removed and cells were fixed with 1% paraformaldehyde until acquisition. PBMCs were acquired by flow cytometry using LSRFortessa cytometer (BD Biosciences). Data were analyzed with FlowJo V10 Software.
Virus challenge
USAMRIID studies were conducted under an Institutional Animal Care and Use Committee (IACUC) approved protocol in compliance with the Animal Welfare Act, PHS Policy, and other Federal statutes and regulations associated with animal experiments and procedures. The IACUC of Rocky Mountain Laboratories, National Institutes of Health (NIH) approved all animal experiments. The facilities where the research was conducted are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) and adhere to principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council, 201136. Rhesus macaques were single-housed in adjacent primate cages to allow social interactions. Animals were subject to a fixed 12 h/12 h light-dark cycle. Commercial monkey chow was provided twice daily and water was available ad libitum. The diet was supplemented with treats, vegetables, or fruit at least once a day. Environmental enrichment included a variety of human interactions, manipulanda, commercial toys, videos, and music. Animals were monitored at least twice daily throughout the experiment.
Viremia analysis
Serum samples collected prior to virus challenge on Day 0 and the post-challenge schedule described above were assayed for the presence of infectious virus via a standard plaque assay with modifications37. Briefly, Vero-76 cells plated on six-well plates were incubated with gentle rotation at 37 °C, 5% CO2 with ten-fold serial dilutions of serum for 1 h. After incubation, 0.8% molecular grade agarose in EBME (basal medium Eagle with Earle’s salts) with 10% FBS and 20 μg/ml gentamicin was applied to each well and allowed to solidify. Plates were incubated at 37 °C for 4 days, then stained with a neutral red overlay (Invitrogen, Carlsbad, CA). Plates were incubated overnight at 37 °C in the staining overlay, and plaques were counted and recorded. Lower limit of detection for the LASV plaque assay has been empirically determined to be 10 PFU.
Plaque-reduction neutralization test (PRNT)
LASV neutralizing responses were assessed in serum samples collected pre- and post-exposure by a modified PRNT assay38. Briefly, two-fold dilutions of sera (in 100 µl volumes) were incubated for 1 h at 37 °C with LASV diluted to approximately 100 PFU per serum dilution. After incubation, each serum dilution/virus mixture was then added to Vero-76 cells at ~90% confluency in six-well tissue culture plates. The downstream procedure was performed as described above for the standard plaque assay. Plaques were counted and compared to control wells containing cells infected with 100 PFU LASV pre-incubated with a LASV naïve NHP serum. Neutralizing antibody titers yielding a 50% reduction (PRNT50) in plaques were determined.
Cells lines
Vero-76 CRL-1587 was obtained from ATCC (Manassas VA) and tested free of mycoplasma. HEK293T (Lenti-X™ 293T cat# 632180) was obtained from Takara Bio (San Jose, CA) and validated free of mycoplasma by the manufacturer.
Blood chemistry and hematology analysis
Serum samples collected from NHPs were analyzed for glucose, blood urea nitrogen, creatinine, uric acid, calcium, albumin, total protein, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin, gamma-glutamyl transferase, and amylase. 100 μl of serum was added to a General Chemistry 13-panel rotor and evaluated in a Piccolo point-of-care blood chemistry analyzer (Abaxis). For studies conducted at RML, hematology analysis was performed using an IDEXX Instrument, using a volume of 500 µl of EDTA-treated whole blood. For studies conducted at USAMRIID, hematology analysis was performed using an DxH 520 instrument (Beckman Coulter) using a volume of 100 µl of EDTA-treated whole blood.
Cytokine/chemokine analysis
Serum collected at Days 0, 3, 9-11, 14, and 35 were used to assess fold change in cytokine/chemokine levels post-challenge using a 23-plex magnet immunology multiplex panel kit specific for nonhuman primates (EMD Millipore). This kit allows for simultaneous measurements of G-CSF, GM-CSF, IFN-γ, IL-1ra, IL-1β, IL-2,IL-4, IL-5, IL-6, IL-8, IL-10, IL-12/23 (p40), IL-13, IL-15, IL-17, IL-18, MCP-1, MIP-1α, MIP-1β, sCD40L, TGF-α, TNF-α, and VEGF. To conduct this assay, 25 µl of undiluted serum from each NHP at the time-points listed above was added to the 96-well plate according to manufacturer’s instructions. Once all samples, controls, and standards were added, 25 µl of magnetic bead conjugates were added to the wells, and plates were incubated for 2 h at room temperature. Plates were washed with a magnetic plate washer, then 25 µl of detection antibodies were added to each well, followed by a 1 h incubation with agitation at RT. A final incubation of 30 min after addition of 25 µl of Streptavidin-Phycoerythrin was performed, and plates were washed. 150 µl sheath fluid was added to each well and mixed prior to plate reading. After 10 min, plates were loaded onto the Magpix instrument (Luminex). Fluorescence intensity values were recorded for each multiplexed cytokine/chemokine per well and exported to an Excel file. Values of all 23 cytokines/chemokines per time-point for each NHP were obtained and analyzed.
Tissue pathology
Tissue samples collected at necropsy were inactivated with 10% buffered formalin for 30 days prior to removal from the containment laboratory. Once inactivated, tissues were processed and embedded in paraffin. The paraffin-embedded tissues were sectioned to 5 μm thick and placed on glass slides. The histology slides were deparaffined, stained with hematoxylin and eosin (H&E), cover slipped, and labeled in accordance with USAMRIID SOPs.
Immunohistochemistry
Inactivated tissue samples for studies conducted at RML were shipped to USAMRIID for analysis. Immunohistochemistry (IHC) was performed in accordance with USAMRIID SOPs using the Dako Envision system (Dako Agilent Pathology Solutions, Carpinteria, CA, USA). Briefly, after deparaffinization, peroxidase blocking, and antigen retrieval, sections were covered with mouse anti-Lassa virus monoclonal antibody (clone 52-2074-7 A, USAMRIID) diluted 1:8000 and incubated for 45 min at RT. After incubation, samples were washed, and the peroxidase-labeled secondary antibody added for 35 min. Slides were washed and the chromogenic substrate 3,3′ Diaminobenzidine (DAB) solution (Dako Agilent Pathology Solutions) added and incubated for 80 min, after which substrate-chromogen solution was removed and samples were counterstained with hematoxylin. After last wash, the sections were dehydrated, cleared with xyless, and cover slipped. Slides were evaluated using a Nikon Eclipse 600 light microscope.
BAERCOM® analysis
Hearing screenings for the DR and One vs Two studies were performed using a BAERCOM® device (UFI, Inc.) according to manufacturer’s instructions. Briefly, needle probes were placed sub-dermally into the skin above each ear around the temporal bones. An additional probe was placed sub-dermally on the crown of the head. The probes were plugged into the BAERCOM device which was set to medium resolution recording. An earplug was placed into one ear at a time and audiometric readings were collected at 0 dB to obtain a baseline measurement, followed by 75 dB for each ear. The BAERCOM® software recorded waveforms for each reading.
Statistics and reproducibility
GraphPad Prism 7.02 was used to analyze and plot the data. Where appropriate, the statistical difference between immunization groups at each time point was assessed using parametric one-way ANOVA t-test or nonparametric Kruskal-Wallis Mann-Whitney test, and p < 0.05 was defined as significant. Two-way ANOVA was used to compare differences between time-points and treatment groups. Six NHPs were used per treatment group. Technical duplicates were used to run experimental assays. Predicted probability of survival plots were generated using SAS software. Survival was analyzed using logistic regression statistical model. P-value from this model was used to determine whether there was strong association between peak antibodies and overall survival.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Results
Immunogenicity and protective efficacy of a dose-range of INO-4500
We have previously shown that INO-4500 (pLASV-GPC), when administered via intradermal (ID) delivery with electroporation (EP) using a 2 mg dose of vaccine, induces antigen-specific immune responses and 100% protective efficacy against lethal LASV challenge in NHPs30,34. To evaluate whether lower doses of INO-4500 also induce LASV-specific immune response and protective efficacy from lethal LASV challenge, we designed a Dose-Ranging (DR) NHP study (Arm 1). NHPs were divided into four groups (n = 6/group) that received 0.1 mg, 1 mg, or 2 mg dose of INO-4500 at weeks 0 and 4. The fourth group was left unvaccinated. At week 8, animals were challenged intramuscularly with 1000 PFU of LASV Josiah (Fig. 1a).
Fig. 1 [Images not available. See PDF.]
Pre-challenge immunogenicity and post-challenge outcomes in the Dose-ranging study Arm 1.
a NHPs were divided into four groups of 6 animals/group. Animals received 2, 1 or 0.1 mg of INO-4500 (with intradermal (ID) EP; groups 1, 2 and 3, respectively), and a fourth group served as unvaccinated control. NHPs were dosed at weeks 0 and 4, after which animals were challenged with LASV Josiah at week 8. Serum and PBMCs were collected to assess the presence of LASV-specific antibody and T cell responses before and after immunizations. b LASV-specific antibody responses were assessed at week 6 by IgG Binding ELISA to lineage IV Prefusion GPC. IgG titers were calculated based on the highest serum dilution with OD450 equal to or higher than 0.150 times 2.5 standard deviations of the experimental duplicates. Error bars represent the geometric mean (GMT) ± 95% confidence interval (CI). c IFNγ T cell responses at week 6 for each group was measured in PBMCs stimulated with lineage IV Josiah GPC peptide megapools by IFNγ ELISpot assay. Data were calculated after DMSO subtraction from peptide-stimulated wells. Error bars represent the mean ± standard error of the mean (SEM). b-c Each datapoint represents the mean of technical duplicates for each NHP. d Kaplan-Meier survival curves for vaccinated and control animals after challenge with 1000 PFU LASV Josiah at week 8. e Quantification of LASV Josiah in PFU per mL of serum by group. Serum samples were collected every 3–5 days to monitor viremia in vaccinated and control NHPs. Surviving NHPs were monitored until Day 38 post-challenge. Shaded areas indicate viral levels above 4 log10 PFU/mL, considered to be peak at critical phase. Animals noted with a black X indicate vaccinated NHPs that were euthanized during critical phase. f LASV neutralization in serum as measured by plaque reduction neutralization test (PRNT).
We assessed INO-4500 elicited LASV-specific antibodies in the serum collected at week 6 (after two immunizations). INO-4500 vaccination induced LASV-specific antibodies in all animals that received 2 mg of INO-4500. Groups that received 1 and 0.1 mg had a seroconversion rate of 83% (5/6 NHP) after the second immunization (Fig. 1b). A dose of 2 mg of INO-4500 elicited higher levels of LASV-specific antibodies, compared to NHPs receiving 1 or 0.1 mg doses. None of the unvaccinated animals had detectable LASV antibodies. Of note, we previously assessed the cross-reactive antibody responses of 2 mg of INO-4500 in a separate NHP study and observed INO-4500 induction of cross-reactive binding IgG antibodies directed against lineages I, II and III GPCs (Supplementary Fig. 1a).
We next assessed the levels of LASV-specific T cells elicited by INO-4500. Results demonstrated that vaccination induced LASV-specific T cell responses in a dose-dependent manner (week 6 mean [min-max] for 2 mg:171 [3.3-552]; 1 mg: 56 [1.7-173]; 0.1 mg: 20 [0-67] SFU per 106 cells) (Fig. 1c). Overall, immunization with 2 mg of INO-4500 confers higher levels of LASV-specific cellular responses compared to lower dose groups. Additionally, INO-4500 cross-cellular immune responses to lineages I, II and III were previously assessed in a separate NHP study (Supplementary Fig. 1b). INO-4500 induced levels of cellular responses in PBMCs stimulated with overlapping GPC peptides to all lineages tested, although cellular responses to lineage I were significantly lower compared to the levels observed for lineage IV (Supplementary Fig. 1b).
To assess the protective efficacy of INO-4500, NHPs were challenged 4 weeks after second vaccine dose with LASV Josiah. All NHPs in the 2 mg group maintained baseline body weights and temperatures and survived to the study endpoint (Fig. 1d, Supplementary Figs. 2–4). Several NHPs in the lower dose groups experienced breakthrough disease and were euthanized (2/6 and 3/6 in the 1 and 0.1 mg groups, respectively) (Fig. 1d). All surviving NHPs that did not meet euthanasia criteria, regardless of disease severity, recovered by Day 19 post-challenge. These NHPs did not develop signs of persistent illness and remained healthy until the end of study (Supplementary Fig. 2). After challenge, individual body weights remained stable, with only the 1 mg group trending toward weight loss. All surviving NHPs returned to baseline body weight by Day 20 (Supplementary Fig. 3). Body temperature for all groups increased from baseline and remained elevated until approximately Day 14 post-challenge (Supplementary Fig. 4).
We next assessed blood cell populations and blood chemistry parameters following LASV exposure. White blood cells (lymphocytes, neutrophils, and monocytes) increased in all vaccinated NHPs post-challenge, remaining elevated in surviving NHPs throughout the study (Supplementary Fig. 5). In particular, increases in lymphocytes were higher in both the 1 and 2 mg groups, remaining elevated until the study endpoint. Platelets initially dropped in vaccinated NHPs, but all surviving animals began to recover platelets to baseline levels by approximately Day 14 post-challenge (Supplementary Fig. 5). Most blood chemistry values remained stable in vaccinated NHPs post-challenge (Supplementary Fig. 6). Levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), creatinine (CRE), and gamma glutamyl transferase (GGT), indicating liver and kidney damage became elevated in control, but not in vaccinated NHPs. Levels of serum calcium (CA), albumin (ALB), and total protein (TP) decreased in all NHPs initially, but surviving vaccinated NHPs returned to baseline levels around Day 14 (Supplementary Fig. 6).
To assess virus levels after LASV challenge, serum viremia was monitored by plaque assay. On Day 6, all NHPs, regardless of group, demonstrated low-level viremia (Fig. 1e). By Day 9, 2/6 (2 mg) and 1/4 (1 mg) NHPs immunized with INO-4500 had cleared virus in serum. Vaccinated and control animals that reached peak viremia levels above 4 Log10 PFU/mL were unable to control the infection and succumbed to disease (Fig. 1e). All surviving vaccinated NHPs showed undetectable viremia by Day 21 (Fig. 1e). Viremia levels in all control animals and in 4/5 vaccinated non-survivors continued to increase until euthanasia.
We next assessed the presence of neutralizing antibodies (nAb) before and after LASV challenge. nAb titers were low or undetectable in all NHPs prior to challenge on Day 0 (Fig. 1f). nAbs increased rapidly in most vaccinated NHPs and were sustained in all vaccinated NHPs that survived LASV challenge. No neutralizing activity above background was detected in the control group. We also assessed the presence of nAbs at week 6 pre-challenge using a pseudovirus-based assay. None of the vaccinated or control animals had detectable nAbs, except for one NHP in the 2 mg group (DR-3) (Supplementary Fig. 7). Importantly, vaccinated NHPs that did not survive LASV challenge had no detectable nAb, suggesting that nAb may play an important role at controlling viremia and preventing death.
Evaluation of persistent disease and sensorineural hearing loss after challenge
Hearing loss is a commonly sequela observed in Lassa Fever survivors. BAERCOM® has been a useful tool to assess hearing loss in NHPs following LASV exposure39. It allows for a qualitative assessment of a positive or negative hearing response to a click sound generated by the device at varying decibel (dB) levels. To assess the ability of INO-4500 to prevent development of hearing loss after LASV exposure, BAERCOM® measurements were obtained before and after challenge (Supplementary Fig. 8). Readings were made at 0 and 75 dB for the left and right ears. All NHPs exhibited waveforms consistent with a normal hearing response at the baseline measurement. The acute phase analysis did not reveal any hearing loss in NHPs that met euthanasia criteria, nor in any NHP surviving to Day 14 post-challenge. All surviving NHPs maintained normal hearing response waveforms at study end. Hearing assessments were not performed in unvaccinated NHPs since there were no survivors, though we have previously reported hearing loss in those unvaccinated NHPs that survived LASV challenge39.
We performed histological analysis to assess signs of persistent disease. Pathologic findings in survivors were minimal and followed a clear dose response pattern consisting mainly of minimal lymphoid hyperplasia typically seen after viral antigen stimulation that increased with lower vaccine doses (data not shown). In contrast, non-survivors developed lesions consistent with Lassa fever in NHP (Supplementary Fig. 9). Lesions identified in the control group and non-surviving vaccinated NHP were similar in severity and scope, and consisted of typical LASV lesions that have been previously described30,39,40.
Single dose of INO-4500 confers protective efficacy and immune responses against LASV lineage IV Josiah in NHPs
In the previous NHP study Arm 1, data indicated that two doses INO-4500 at 2 mg each conferred complete protection from lethal LASV challenge in vaccinated NHPs (Fig. 1d). To further explore vaccination regimen impact, we designed a NHP study Arm 2 to evaluate whether a single 2 mg dose of INO-4500 is sufficient to induce LASV-specific immune responses and protective efficacy against viral challenge (One vs Two study). NHPs were divided into three groups (n = 6/group) that received two or a single dose of 2 mg of INO-4500 at weeks 0 and 4. The third group remained unvaccinated (Fig. 2a). NHPs were monitored for the presence of LASV-specific binding IgG antibodies and T cells after first and second doses at week 4 and 6, respectively. All animals were intramuscularly challenged with a lethal dose of LASV Josiah (target of 1000 PFU) at week 8.
Fig. 2 [Images not available. See PDF.]
Pre-challenge immunogenicity and post-challenge outcomes in One vs Two dose NHP study Arm 2.
a NHPs were divided into four groups of 6 animals/group. Animals received one or two 2 mg doses of INO-4500 with ID-EP at week 0 or weeks 0 and 4, respectively. The third group served as control. All animals were challenged at week 8 with a target of 1000 PFU LASV Josiah. Serum and PBMCs were collected to assess the presence of LASV-specific antibody and T cell responses before and after immunizations. b LASV-specific antibody response was assessed by IgG Binding ELISA to LASV lineage IV Prefusion GPC at weeks 4 and 6. IgG titers were calculated based on the highest serum dilution with OD450 equal to or higher than 0.250 + 2.5 standard deviations of the experimental duplicates. Error bars represent the GMT ± 95% CI. c IFNγ responses at week 4 and 6 for each group were measured in PBMCs stimulated with lineage IV Josiah GPC peptide megapools by IFNγ ELISpot assay. Data were calculated after DMSO subtraction from peptide-stimulated wells. Error bars represent the mean ± SEM. b, c Each datapoint represents the mean of technical duplicates for each NHP. d Kaplan-Meier survival curves for vaccinated and control animals after challenge with 1000 PFU LASV Josiah at week 8. e Quantification of LASV in PFU per mL of serum by group. Serum samples were collected every 3–5 days to monitor viremia in vaccinated and control NHPs. In NHPs that succumbed to disease, viremia was monitored until the time of death or euthanasia. Surviving NHPs were monitored until Day 30 post-challenge. Shaded areas indicate peak viremia levels above 4 log10 PFU/mL at critical phase. Animals noted with a black X indicate vaccinated NHPs that succumbed to disease. f LASV neutralization in serum as measured by plaque reduction neutralization test (PRNT).
We assessed the levels of LASV-specific IgG antibodies after dosing with INO-4500. All animals in the two-dose group seroconverted after the second immunization (Fig. 2b), a finding consistent with the corresponding group in Arm 1 (Fig. 1b). All NHPs that received a single dose of INO-4500 seroconverted 4 weeks after immunization. Except for one NHP in the one-dose group (1v2-7), LASV-specific antibodies in this group remained detectable and stable for up to week 6 post-immunization (Fig. 2b). The magnitude of antibody response following immunizations was dose dependent. No nAb was detected at weeks 4 and 6 following initial dosing using a pseudovirus neutralization assay (Supplementary Fig. 10).
We measured the levels of IFNγ-producing T cells in isolated PBMCs stimulated with LASV lineage IV peptide megapools. Cellular responses peaked 2 weeks after the second immunization (week 6) in the two-dose group (mean [min-max]: 354 [17-1397] SFU per 106 cells), and at week 4 for the one-dose group (mean [min-max]: 115.8 [11.7-348] SFU per 106 cells) (Fig. 2c). T cell data suggest that immunization with two doses of 2 mg of INO-4500 generates higher levels of LASV-specific cellular responses compared to a single dose.
To evaluate whether a single 2 mg dose of INO-4500 confers protection against lethal LASV exposure, NHPs were challenged with LASV Josiah at week 8 post-immunization. Consistent with the findings from study Arm 1 (Fig. 1d), all NHPs in the two-dose group were protected from LASV challenge (Fig. 2d). In the one-dose group, 67% of NHPs (4/6 animals) survived challenge. All unvaccinated animals met euthanasia criteria by Day 21 post-challenge (Fig. 2a). In the one-dose group, 2/6 NHPs developed severe disease and loss of body weight and required euthanasia (Fig. 2d, Supplementary Fig. 11–13). The four NHPs that survived to the study endpoint developed only mild, transient disease signs that resolved by Day 16 post-challenge (Supplementary Fig. 11). Surviving NHPs in the one-dose group displayed mild weight loss that was regained by end of study (Supplementary Fig. 12). Body temperatures increased in most NHPs starting at Day 3 and continued to increase until approximately Day 10 post-challenge (Supplementary Fig. 13). Hematology results demonstrated a decrease in platelets in all three groups initially, with surviving vaccinated NHPs returning to baseline by the end of the study (Supplementary Fig. 14j). Blood chemistry levels for analytes indicative of liver and kidney damage remained stable for all surviving vaccinated NHPs, compared to the control group, whose levels of ALT, AST, ALP, GGT, TBIL, BUN, and CRE were increased in NHPs requiring euthanasia (Supplementary Fig. 15). Levels of calcium, albumin, and TP decreased for all groups initially but stabilized in vaccinated groups after Day 10 post-challenge.
To assess post-challenge viral burden, NHPs were monitored for the presence of infectious LASV in the serum by plaque assay. Viremia was not detected until Day 6 post-challenge (Fig. 2e). Two doses of INO-4500 prevented viremia in 5/6 NHPs. In the one-dose group, all NHPs were viremic or transiently viremic, with all surviving NHPs (4/6) clearing virus by Day 21 post-challenge. Viremia levels in vaccinated surviving NHPs were lower compared to the levels observed in the control group (Fig. 2e).
We next assessed the presence of nAbs before and after LASV exposure. Neutralizing antibody titers were low to undetectable for all NHPs prior to LASV challenge (Fig. 2f). After challenge, neutralizing responses rapidly increased and peaked on Day 14 for both vaccinated groups, with neutralizing titers ≥64 detected for 4/6 and 5/6 NHPs receiving one or two doses of INO-4500, respectively. Of note, the single NHP (1v2-2) that experienced low-level viremia until the study endpoint had a delayed response to detectable neutralizing antibody titers. This NHP was negative for a neutralizing titer response until the study end (Fig. 2f). No neutralizing activity above background was detected in the control NHPs, nor for the two non-surviving NHPs in the one-dose group (1v2-7 and 1v2-12). Interestingly, the same two non-surviving NHPs in the one-dose group had viremia titers above 104 PFU/mL, and low to undetectable binding antibodies and T cells at weeks 4 and 6 following INO-4500 vaccination. Overall, pre-challenge and post-challenge immune response profiles suggest a potential recall of memory immune cells and their reactivation upon antigen re-exposure following LASV challenge.
Single dose of INO-4500 protected NHPs from persistent disease and hearing loss
To investigate whether INO-4500 had an impact on hearing loss, BAERCOM® analysis was performed on surviving NHPs at the end of the study. A hearing response to click stimulus was observed in all NHPs tested. All surviving NHPs had normal behavioral hearing response throughout the study (Supplementary Fig. 16). Pathology findings were similar to what was observed for the DR study in that all surviving NHPs were free of LASV-associated gross lesions at necropsy, including the inner ear. Except for one NHP that experienced extended low-level viremia in the two-dose vaccination group (1v2-2), all other vaccinated NHPs experienced only mild lesions consistent with a normal response to recent infection. The viremic NHP in the two-dose group cleared the virus by study end and was not exhibiting clinical signs of disease; however, this animal had mild residual inflammatory lesions in the brain, lymphoid, lung, and genitourinary systems consistent with recent systemic viral infection (Suppl. Fig. 17). The noted lesions are thought to be resolving acute lesions in an animal that experienced an extended disease course. Of note, this NHP did not show any lesions in the inner ear that would be consistent with sensorineural hearing loss as we have described previously, nor did this NHP exhibit any behaviors consistent with hearing loss at the end of the study. Histologic lesions identified in the two non-survivors in the one-dose group were consistent with severe LASV infection and were indistinguishable from the six NHPs in the control group that succumbed to disease in the acute phase (Suppl. Fig. 17).
INO-4500 induces durable antigen-reactive T cells and protection against LASV challenge in NHPs one year after primary immunization series
To determine the durability of INO-4500-mediated protection we assessed the outcome of LASV Josiah challenge 1 year after vaccination in a study Arm 3. NHPs were separated into two vaccinated (2 doses of 2 mg and 1 mg of INO-4500) and one control groups (Fig. 3a).
Fig. 3 [Images not available. See PDF.]
Durability of immune responses after INO-4500 immunization and survival percentages following LASV challenge in Durability NHP study Arm 3.
a Overview of study Arm 3. NHPs were immunized with INO-4500 + ID-EP at weeks 0 and 4. NHPs were challenged with LASV at week 59. b LASV-specific IgG responses by IgG Binding ELISA to LASV Clade IV Prefusion GPC for 2 mg and 1 mg groups. LASV IgG antibody response was assessed at baseline, after two immunizations at week 6, and prior to LASV challenge at week 54. Error bars represent the GMT ± 95% CI. c Individual IFNγ cellular responses after INO-4500 immunization for NHPs from 2 mg and 1 mg groups. PBMCs were isolated at baseline, at week 6 and prior to LASV challenge at week 54. PBMCs were stimulated with Clade IV Josiah GPC peptide megapools. Data were calculated after DMSO subtraction from peptide-stimulated wells. Error bars represent the mean ± SEM. b-c Each datapoint represents the mean of technical duplicates for each NHP. Quantification of d LASV-specific CD4+ and e CD8 + T cells upon stimulation with peptide megapools spanning the Josiah lineage IV GPC. Cells were stained with antibodies against markers that are upregulated upon T cell activation, including CD69, CD134, CD137, CD154, and CD25. Frequency of LASV + T cells were calculated based on any T cell expressing at least two AIM markers. Percentage of positive CD4+ and CD8 + T cells were calculated after DMSO subtraction from peptide-stimulated samples. Bars represent the mean ± SD. Each symbol represents an individual NHP test sample. *p ≤ 0.05, Two-way ANOVA multiple comparison. f Kaplan-Meier survival curves following LASV challenge with Josiah strain one year post-initial immunization.
We assessed the levels of LASV-specific IgG antibodies in the serum before and after vaccination, and just prior to LASV challenge. All vaccinated animals seroconverted after two doses with INO-4500 at week 6 (Fig. 3b). At week 54 prior to challenge, antibody levels remained detectable in 50% of NHPs for vaccinated groups. NHPs were monitored for the presence of LASV-specific T cells. All NHPs in the 1 mg dose group, except for one NHP (D-8), had measurable levels of LASV-specific T cells after two vaccinations by IFNγ ELISpot. All NHPs in the 2 mg group demonstrated levels of T cells after vaccination (Fig. 3c). The levels of T cells one year after initial immunization were low or undetectable for all vaccinated NHPs, regardless of dose. Since IFNγ ELISpot assay can underestimate the frequencies of antigen-specific T cells41, we carried out a T cell activation assay to characterize the short- and long-term cellular immune responses induced from INO-4500 vaccination. The frequency of LASV-specific CD4 and CD8 T cells were assessed at week 0 (pre-immunization) and at weeks 6, 39, and 54 prior to LASV challenge. CD4 and CD8 T cells in PBMCs were analyzed by quantifying the frequency of cells expressing markers of T cell activation, including CD69, CD134, CD137, CD25, and CD154 after stimulation with overlapping peptides spanning the Josiah GPC. Cells expressing at least two markers were considered LASV-specific (Supplementary Fig. 18a, b, d, e). LASV-specific CD4 and CD8 T cell responses peaked at week 6, after which a reduction in activated T cells was observed at week 39 but remained stable until week 54 (Fig. 3d, e). The majority of vaccinated NHPs presented with LASV-specific T cells prior to challenge (Supplementary Fig. 18c, d). CD4 T cell responses in the 1 mg group were slightly increased compared to the 2 mg group (Fig. 3d). In contrast, overall CD8 T cell responses were slightly elevated for the 2 mg group, compared to the 1 mg group (Fig. 3e).
At week 59 animals were challenged with LASV Josiah. Despite the lower frequency of LASV-specific antibodies and T cells, all vaccinated animals survived lethal LASV challenge one year after immunization (Fig. 3f). NHPs in the control group experienced severe disease complications and succumbed to disease (Fig. 3f and Supplementary Fig. 19c). NHPs were monitored for the development of clinical signs of disease. In both vaccinated groups, NHPs lacked disease signs, except for one NHP (D-6) in the 2 mg group that showed transient, mild signs on Day 8 post-challenge (Supplementary Fig. 19a). Accordingly, weights and temperatures of vaccinated NHPs were stable after challenge through the end of study (Supplementary Figs. 20 and 21). No unexpected changes in hematology parameters were noted in the vaccinated NHPs post-challenge compared to control NHPs (Supplementary Fig. 22). Similarly, blood chemistry parameters for vaccinated NHPs remained within normal range compared to control NHPs that experienced increases in analytes indicative of liver and kidney dysfunction (Supplementary Fig. 23).
To further investigate the disease pathology in vaccinated survivors and control NHPs, we assessed viral replication, presence of nAbs and tissue histopathology post-LASV exposure. Three NHPs in each of the two vaccinated groups lacked detectable virus in the serum as measured by plaque assay throughout the study (Fig. 4a). The remaining NHPs experienced transient, low-level viremia that became undetectable by Day 10 (4/6 that were viremic) or Day 14 (2/6 that were viremic) post-challenge (Fig. 4a). Despite a lower dose, viremia in NHPs in the 1 mg vaccinated group was comparable with the levels experienced by the 2 mg group. NHPs in the control group demonstrated an increase in viremia throughout the study until euthanasia criteria were met. Vaccinated NHPs also demonstrated rapidly increasing nAb titers post-challenge compared to the unvaccinated control group. Neutralizing titers were detectable above baseline in vaccinated NHPs on Day 3 post-challenge (6/6 NHP in the 2 mg group and 4/6 NHP in the 1 mg group) (Fig. 4b). Neutralizing titers continued to rise in vaccinated NHPs, reaching levels higher than any previously conducted study for this vaccine candidate. Neutralizing titers remained elevated and dose- dependent for vaccinated NHPs through the end of study.
Fig. 4 [Images not available. See PDF.]
Post-challenge outcomes and cytokine/chemokine changes post-challenge induced from INO-4500 vaccination in NHPs from Durability Study.
a Quantification of LASV Josiah replication in PFU per ml of serum for each individual group. Serum samples were collected every 3-5 days to monitor viremia in vaccinated and control NHPs. In NHPs that succumbed to disease, viremia was monitored until their time of death or euthanasia. Surviving NHPs were monitored until Day 35 post-challenge. b LASV neutralization in the serum by plaque reduction and neutralization test (PRNT). c Levels of cytokines and chemokines post-challenge are expressed as fold changes in median fluorescence intensity (MFI) values compared to Day 0 values. Cytokines were placed into the following basic functional groupings: Adaptive Immunity (IL-2, IL-4, IL-5, IL-15, IL-13, G-CSF, GM-CSF; Pro-Inflammatory (IL-1β, IL-1RA, IL-6, IL-8, IL-17°, IL-18, IFNγ, MCP-1, MIP-1°, MIP-1β, TNFα, sCD40L, VEGF; and Anti-Inflammatory (IL-10, IL-12P40, TGF-α).
Neutralizing responses in the control group were transiently low or undetectable after LASV-challenge. Vaccinated NHPs were clinically well and were clear of most pathologic lesions at necropsy (Supplementary Fig. 24). The lesions identified in the pathology report were mild and included lesions typically seen in NHP recovering from viral infection. The main finding in these animals was lymphoid hyperplasia, an expected and common finding consistent with recent infection (Supplementary Fig. 24).
INO-4500 induces a controlled release of soluble mediators after LASV infection
We proceeded to investigate the inflammatory profile induced by LASV challenge. Changes in soluble mediators involved in LASV infection were assessed and levels in serum post-challenge quantified as median fluorescence intensity (MFI) fold change over pre-challenge baseline. Vaccinated NHPs experienced a modest increase in IFNγ (3.5–3.6-fold) post-exposure. IFNγ response was lower (2.5-fold) in control NHPs at Day 3, increasing to nearly 16-fold during the acute phase when these NHPs experienced severe disease signs and required euthanasia (Fig. 4c). Moreover, a reduction in IL-8 was observed in control NHPs over baseline at Days 9-11 compared to the vaccinated groups. Low levels of IL-8 have been linked to fatal outcomes in Lassa fever patients42,43. In contrast, levels of the pro-inflammatory cytokine IL-6 are increased to >10-fold over baseline in control NHPs during acute phase. IL-6 has been shown to induce the production of hepatic inflammatory markers during acute phase of infection. Indeed, control NHPs experienced acute liver damage as evidenced by elevated levels of ALT and AST (Supplementary Fig. 22a, b) but increased only slightly above baseline in vaccinated NHPs. This observation is consistent with previous observations that elevated IL-6 is associated with a fatal LASV outcome44. Differences observed in levels of IL-1RA between vaccinated and control groups was striking, with the control NHPs experiencing over 100-fold increase by Day 9-11 (Fig. 4c). INO-4500-vaccinated NHPs experienced modest vaccine dose-dependent increases early in the infection (Day 3), continuing to increase at Day 9-11, returning to baseline levels by Day 14. Increases in IL-1RA for all groups indicate the activation of an acute response to infection. Finally, we observed a decrease in sCD-40L in control NHPs compared to vaccinated NHPs. sCD-40L is produced by activated T cells and platelets and can bind to CD40 on B cells, leading to the proliferation and differentiation of B cells. Indeed, we observed an upregulation of CD-40L (CD154) on the surface of CD4 T cells co-expressing CD134 in INO-4500-vaccinated NHPs (Supplementary Fig. 18e). A reduction in sCD-40L levels in control NHPs indicates that there are fewer activated T cells present to release sCD-40L, thus preventing antigen-specific B cell production. These results indicate that control NHPs experienced an acute and fatal disease characterized by an uncontrolled immune response as evidenced by the elevation of pro-inflammatory cytokines.
Post-challenge serum viremia and neutralizing antibody levels and associated outcome after LASV challenge
To expand our knowledge of the parameters associated with survival in the LASV challenge model, we plotted viremia and neutralizing antibody levels associated with various vaccination regimens against the survival outcome. We used peak serum viremia and PRNT50 titers obtained post-challenge from Arms 1, 2, and 3 studies. We compared how these two parameters are associated with a 70% survival rate. Using serum viremia values from Arm 1, a 70% probability of survival is achieved when NHPs present a titer below 3.6 log10 PFU/mL at any time-point post-challenge, regardless of vaccine dose (Fig. 5a). Similar viremia titers were observed in Arm 3, where a value below 3.5 log10 PFU/mL predicts 70% survival (Fig. 5b). In NHPs from Arm 2, a titer of 4.5 log10 PFU/mL or below predicts 70% survival (Fig. 5c). The higher viremia value observed in study Arm 2 may be due to the elevated viremia observed in the control group as compared to Arms 1 and 3 (Figs. 1e, 2e and 4a). Taken together, these data suggest that the lower the viremia, the higher the chance of survival following LASV challenge. In fact, all NHPs in the Dose-ranging and Durability studies that presented peak viremia above 4 Log10 PFU/mL, regardless of vaccine regimen, succumbed to LASV disease (Figs. 1e and 4a).
Fig. 5 [Images not available. See PDF.]
Survival probabilities based on serum viremia and neutralizing antibodies post-LASV challenge.
a–c Analysis of 70% probability of survival calculated based on serum viremia titers as log10 PFU/mL for (a) Dose-ranging (Arm 1) (b) One vs Two dose (Arm 2) and (c) Durability (Arm 3) studies. d–f Analysis of 70% probability of survival calculated based on PRNT50 titers for (d) Dose-ranging (e) One vs two dose and (f) Durability studies. Predicted probability plots were generated using SAS software. Survival was analyzed using a logistic regression statistical model. Blue lines indicate predictive survival probability at a given measurement. The value beside circular symbols indicates the number of NHPs with corresponding assay readout.
We evaluated the relation of nAbs after LASV exposure with survival. Peak PRNT50 values were plotted against survival status for all NHPs across the three studies (Fig. 5d–f). All three studies followed a similar pattern where minimal PRNT50 values ranging from 17.6 and 23.2 predict 70% survival from LASV challenge. INO-4500 vaccinated NHPs demonstrated increased levels of nAbs post-challenge; this was shown to be associated with survival. Interestingly, prior to challenge, vaccinated NHPs had no detectable nAbs, suggesting potential memory B cell recall after LASV exposure, although memory B cells were not assessed. In contrast, all NHPs that succumbed to LASV disease, regardless of vaccine status, were unable to generate LASV-specific nAbs above the minimum protective threshold. The generation of nAbs against LASV may be an important component involved in disease protection and prevention from death in this model.
Discussion
We have previously demonstrated that two doses of INO-4500 conferred complete protection from Lassa fever in NHPs29,34. Here, we expanded our knowledge to investigate the protective efficacy and immunogenicity of INO-4500 at low doses, and as a single shot, in addition to assessing durability of immune response. INO-4500 conferred dose-dependent, durable protection from Lassa fever in NHPs, eliciting antibodies and T-cell responses. INO-4500 also induces cross-reactive humoral and cellular responses against heterologous LASV lineages, including I, II, and III. Despite LASV-specific antibody levels waning, INO-4500 protected NHPs from Lassa fever one year after initial immunization. The presence of long-term LASV-specific CD4 and CD8 T cells suggests a potential recall of immune memory cells after LASV exposure that may be critical to prevent the acute onset of symptoms and ultimately to protect NHPs from succumbing to disease.
The lowest (1 mg) vaccine dose used to assess protective durability induced 100% protection in NHPs one year after vaccination, but only partial protection (67%) 4 weeks after vaccination. We hypothesized this phenomenon may be due to the development of memory immunity. LASV-specific T cell responses quantified one year after immunization indicated the presence of a population of LASV-specific CD4 T cells that were recalled upon LASV peptide exposure. This suggests a potential T-cell mediated activation of B cells to rapidly engage the production of neutralizing antibodies observed after challenge. Moreover, assessment of cytokine/chemokine release post-challenge indicate that a programmed response occurred in vaccinated NHPs, regardless of vaccine dose, compared to the uncontrolled release of pro-inflammatory mediators observed in control NHPs.
We demonstrated that INO-4500 induces high levels of LASV-specific T cells after a single or two immunizations. T cell-mediated immunity is critical in the prevention of severe disease, especially in the context of viral infections45–47. In severe cases of LASV infection, poor LASV-specific effector T cell responses were observed45. Moreover, studies in humans and NHP have shown that early T cell activation and strong T cell responses are associated with disease recovery44,48. In guinea pigs, immunization with a Lassa mRNA vaccine induced antigen-specific T cells and conferred protection against LASV infection49.
Antibodies against LASV have also been shown to play a critical role in viral clearance and survival. Recently, identification of neutralizing antibodies and their use in therapeutic studies has opened opportunities for the development of monoclonal antibody therapy and vaccines50,51. Cynomolgus macaques treated with LASV-GPC neutralizing monoclonal antibodies after challenge with lethal LASV showed 100% protection and survival with low to undetectable viremia52. Here we showed that INO-4500 vaccination can rapidly induce production of neutralizing antibodies after LASV exposure in NHPs, and that presence of neutralizing antibodies is associated with survival from LASV challenge.
A LASV vaccine candidate in order to be successful, must not only protect against severe disease and death, but also prevent the debilitating persistent disease state, including sensorineural hearing loss, that has been well described in Lassa fever survivors8. In the present study, INO-4500 prevented the development of hearing loss post-challenge in all vaccinated NHPs, and demonstrated the absence of pathologic lesions that have been previously associated with hearing loss in NHPs that survive beyond the critical phase of disease39.
Our study has some limitations. We were unable to measure LASV T cells post-challenge. Thus, we measured the durability of LASV-specific T cells prior to LASV challenge. Despite low to undetectable IFNγ + T cells one year after vaccination by IFNγ ELISpot, LASV-specific T cells were detected in a flow cytometry-based T cell activation assay. The latter is a more sensitive assay compared to ELISpot, as it measures the frequency of peptide-mediated stimulation of markers of T cell activation expressed on the surface of activated T cells, compared to single quantification of IFNγ + T cells41,53. This could also explain the lower frequency of IFNγ + T cells assessed at the peak of T cell response at week 6 post-vaccination. The small sample size of each NHP study also limited our understanding of the role of T cells and IgG antibodies elicited after vaccination with survival from the LASV challenge. Moreover, we observed an unusually high viremia in NHPs from study Arm 1 compared to Arm 2 and to what we reported previously30. It is possible that the observed difference is because study Arm 1 was conducted at a different institute (RML) and is a result of Institute-to-Institute variability in available resources.
Taken together, the studies presented here indicate that vaccination with INO-4500 induces long-term protective efficacy in NHPs, and that protection is associated with a synergy of antibodies and T cells against Lassa virus.
Acknowledgements
The authors are grateful for the assistance of members of the Veterinary Medicine Branch at Rocky Mountain Laboratories, Laboratory of Virology for assistance during the challenge phase of the dose-response and durability studies, and staff within the Veterinary Medicine and Pathology Divisions at USAMRIID for assistance during the challenge phase of the One vs Two Dose study. The excellent work of the technicians in the Veterinary Branch at RML and the Pathology Division at USAMRIID for tissue preparation is highly appreciated. All NHP studies described in this work were funded by the Coalition for Epidemic Preparedness Innovations (CEPI), through a grant awarded to Inovio Pharmaceuticals Inc. RML-conducted components were supported by the Intramural Research Program of NIAID, NIH. Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the U.S. Army. Figures depicting each NHP study design were produced using BioRender (Ontario, Canada) and can be accessed using the URLs provided: https://BioRender.com/b78x022https://BioRender.com/g13k072 and https://BioRender.com/n80i107.
Author contributions
Conceptualization, V.M.A., K.C., J.J., S.J.R., K.E.B., C.S., and L.M.H. Methodology, V.M.A., K.C., J.J. Investigation, V.M.A., K.C., K.R., J.J., A.P., Resources, K.C., K.R., E.W., N.J., G.L., J.S., S.V., J.W., C.S., P.F., F.F., P.H., J.L., K.W., H.F. Writing-original draft preparation, V.M.A., K.C., and P.F. Writing-review and editing V.M.A., K.C., K.R., J.J., J.B., T.R.F.S., N.J. and L.M.H., Project administration, K.C., K.R., H.F., S.J.R., K.E.B., C.S., L.M.H., and T.R.F.S.
Peer review
Peer review information
Communications Medicine thanks Peter Okokhere and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Data availability
The datasets used and analyzed in main Figs. 1, 2, 3 and 4 of this article are available in Supplementary Data 1, under separate tabs for ELISA, ELISpot, viremia, PRNT, survival, T cell activation and cytokine release assays. Additional histopathology images and all other datasets are available from the corresponding author upon reasonable request.
Competing interests
V.M.A., T.R.F.S., and L.M.H. are employees of Inovio Pharmaceuticals and as such receive salary and benefits, including ownership of stock and stock options from the company. J.J., J.B., A.P., S.R., and K.E.B. are former Inovio employees. All other authors declare no competing interest.
Supplementary information
The online version contains supplementary material available at https://doi.org/10.1038/s43856-024-00684-8.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Abstract
Background
We have previously developed a DNA-based vaccine, INO-4500, encoding the Lassa lineage IV glycoprotein precursor. INO-4500, when delivered with electroporation, elicited humoral and cellular responses, and conferred 100% protection in cynomolgus non-human primates. Here, we expanded the characterization of INO-4500 assessing immunogenicity and protective efficacy of lower doses and single immunization, and the durability of immune responses.
Methods
The study was divided into three arms evaluating INO-4500 vaccination: Arm 1 – Dosing regimen; Arm 2 – Single immunization; and Arm 3—Durability of immune responses and protective efficacy. Humoral and T cell responses were assessed by IgG binding ELISA, IFNγ ELISpot and flow cytometry-based T cell activation assays. NHPs were challenged with a lethal dose of Lassa lineage IV 8 weeks (Arms 1 and 2) or one year (Arm 3) after immunization. NHPs were assigned clinical scores and monitored for survival. Viremia, virus neutralization and release of soluble mediators were assessed post-challenge, as well as disease pathology following NHPs death or euthanasia.
Results
INO-4500 induces dose-dependent immune responses and protective efficacy. Animals receiving two doses of 2 mg of INO-4500 show complete short- and long-term LASV protection. NHPs receiving 1 mg of INO-4500 are protected from LASV challenge one year after vaccination but are only partially protected 8 weeks post-vaccination. LASV-specific memory T cells are present in vaccinated NHPs one year after vaccination. INO-4500 vaccination prevents NHPs from developing severe disease.
Conclusions
These studies demonstrate that INO-4500 can provide short- and long-term protection in NHPs from lethal LASV challenge.
Plain language summary
Lassa Fever is an infection that leads to excessive bleeding and often, death. We previously developed a DNA vaccine, named INO-4500, that protected monkeys from becoming infected with Lassa. In the present study, we tested whether different vaccine schedules, including lower doses and single dose, also confer disease protection in monkeys. In addition, we evaluated the long-term protection of INO-4500 and confirmed that INO-4500 protects monkeys from infection with Lassa one year after vaccination. These findings confirm that our vaccine prevents infection with Lassa and provides long-term protection from Lassa Fever in this animal model of the disease. This work supports further investigation being undertaken into whether the vaccine can prevent Lassa fever in humans.
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Details
; Cashman, Kathleen 2 ; Rosenke, Kyle 3 ; Wilkinson, Eric 2 ; Josleyn, Nicole 2 ; Lynn, Ginger 2 ; Steffens, Jesse 2 ; Vantongeren, Sean 2 ; Wells, Jay 2 ; Schmaljohn, Connie 4 ; Facemire, Paul 5 ; Jiang, Jingjing 1 ; Boyer, Jean 1 ; Patel, Aditya 1 ; Feldmann, Friederike 6
; Hanley, Patrick 6
; Lovaglio, Jamie 6
; White, Kimberly 3 ; Feldmann, Heinz 3
; Ramos, Stephanie 1 ; Broderick, Kate E. 1 ; Humeau, Laurent M. 1 ; Smith, Trevor R. F. 1
1 Inovio Pharmaceuticals Inc., Plymouth Meeting, USA (GRID:grid.421774.3) (ISNI:0000 0004 0417 098X)
2 United States Army Medical Research Institute of Infectious Diseases (USAMRIID), Virology Division, Fort Detrick, USA (GRID:grid.416900.a) (ISNI:0000 0001 0666 4455)
3 Rocky Mountain Laboratories (RML), Laboratory of Virology (LV), Division of Intramural Research (DIR), National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Hamilton, USA (GRID:grid.419681.3) (ISNI:0000 0001 2164 9667)
4 United States Army Medical Research Institute of Infectious Diseases, Office of the Chief Scientists, Headquarters, Fort Detrick, USA (GRID:grid.416900.a) (ISNI:0000 0001 0666 4455); Integrated Research Facility (IRF), National Institutes of Health (NIH), National Institute of Allergy and Infectious Diseases (NIAID), Frederick, USA (GRID:grid.94365.3d) (ISNI:0000 0001 2297 5165)
5 United States Army Medical Research Institute of Infectious Diseases (USAMRIID), Pathology Division, Fort Detrick, USA (GRID:grid.416900.a) (ISNI:0000 0001 0666 4455)
6 Rocky Mountain Laboratories (RML), Rocky Mountain Laboratory Veterinary Branch (RMVB), Division of Intramural Research, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Hamilton, USA (GRID:grid.419681.3) (ISNI:0000 0001 2164 9667)