Research ArticleEMERGING INFECTIONS

A lipid-encapsulated mRNA encoding a potently neutralizing human monoclonal antibody protects against chikungunya infection

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Science Immunology  17 May 2019:
Vol. 4, Issue 35, eaaw6647
DOI: 10.1126/sciimmunol.aaw6647
  • Fig. 1 Prophylactic efficacy of CHKV-24 IgG protein.

    (A) Concentration of human IgG in AG129 mouse serum after CHKV-24 IgG protein infusion. Total human IgG levels were measured 24 hours after infusion of purified human mAb IgG protein for CHIKV-24 (orange) or an irrelevant control mAb to influenza (flu; blue). Animals receiving 10 mg/kg (200 μg), 2 mg/kg (40 μg), or 0.4 mg/kg (8 μg) of recombinant CHKV-24 IgG protein had mean systemic IgG concentrations of CHKV-24 of 78, 10, or 3 μg/ml, respectively. The serum concentration of the influenza control antibody was similar. Five animals per group were tested. The mean values are indicated, and error bars show the SD. (B) Survival of AG129 mice treated with mAb CHKV-24 IgG protein and challenged with CHIKV. Mice were treated with a single intravenous injection of 10 mg/kg (left), 2 mg/kg (middle), or 0.4 mg/kg (right) of mAb CHKV-24 (red) or an irrelevant human IgG to influenza A virus (flu; blue) 24 hours before virus challenge with 102.5 TCID50 of CHIKV-LR06. The group shown in gray contained animals that were neither treated nor challenged. Challenged animals were anesthetized with isoflurane before subcutaneous injection in the footpad and hock of the right leg with a total volume of 0.1 ml of the diluted virus (0.05 ml each site). Kaplan-Meier survival plot is shown. Survival data were analyzed using the Wilcoxon log-rank survival analysis. **P < 0.01, as compared with control. The number of animals in each group was 10. Animals receiving 2 or 10 mg/kg of CHKV-24 were completely protected (100% survival) from lethal challenge. Animals receiving 0.4 mg/kg of chikungunya IgG were partially protected (50% survival). All animals receiving the flu IgG at 10, 2, or 0.4 mg/kg succumbed (0% survival) to infection by day 5.

  • Fig. 2 Prophylactic efficacy of CHKV-24 mRNA.

    (A) Expression of human mAb IgG in serum after intravenous infusion of mRNA. Expression levels of human IgG in the serum of AG129 mice after infusion of mRNA encoding CHKV-24 or a control (influenza; flu) mAb mRNA. The CHKV-24 mRNA was administered to mice at a dose of 0.5 mg/kg (orange), 0.1 mg/kg (blue), or 0.02 mg/kg (purple) or the influenza mRNA at a dose of 0.5 mg/kg (cyan), by intravenous tail vein injection. Animals were bled at 24 hours after infusion to measure systemic levels of IgG. Each group had five animals. An additional group of 10 animals was infused with these mRNAs and doses at the same time and challenged with virus 24 hours after infusion [results shown in (B) and (C)]. The mean values are indicated, and error bars show the SD. (B) Protection against lethal CHIKV infection mediated by human mAb expressed from mRNA. CHKV-24 mRNA was administered to mice as a prophylaxis at 0.5 mg/kg (orange), 0.1 mg/kg (blue), or 0.02 mg/kg (purple) by intravenous tail vein injection. An irrelevant IgG mRNA was used at 0.5 mg/kg as a control (cyan). Each group of animals was challenged 24 hours after infusion with CHIKV strain LR06 and monitored for mortality. The number of animals in each group was 10. **P < 0.01, which indicates that the survival differed significantly from that of the group treated with 0.5 mg/kg of the irrelevant IgG (Wilcoxon log-rank survival test). (C) Titer of CHIKV in AG129 mice treated with mRNA encoding mAb CHKV-24 IgG or an mRNA encoding an irrelevant control mAb. Serum samples obtained 2 days after virus challenge were assayed on Vero cell monolayer cultures to determine virus titer (log10TCID50/ml). The limit of detection (LOD) was 1.7. The mean values are indicated, and error bars show the SD. Comparisons were made by Kruskal-Wallis test with Dunn’s posttest. ***P < 0.0003, as compared with control IgG. The number of animals in each group was five.

  • Fig. 3 Therapeutic administration of CHKV-24 mRNA reduces clinical disease and viral titer in WT mice.

    C57BL/6 mice received human IgG mRNA (10 mg/kg) by intravenous injection 4 hours after inoculation with CHIKV-LR06. (A) Foot swelling was monitored by digital calipers [n =15 per group, two experiments, two-way analysis of variance (ANOVA) with Sidak’s posttest]. Line indicates significance between the groups at each time point. Error bars indicate SEM. (B) Serum was collected at 2 dpi or (C) ipsilateral (i.) and contralateral (c.) ankles were harvested on 7 dpi, and viral RNA was quantified by qRT-PCR (serum: n = 15 per group, two experiments; ankles: n = 10 per group, two experiments, Mann-Whitney test for each tissue). Bars indicate median values. Dotted lines indicate the limit of detection. (D) Ipsilateral feet were collected at 7 dpi, fixed in PFA, decalcified, paraffin-embedded, sectioned, and stained with H&E. Images show low magnification (scale bar, 100 μm) with a high-magnification inset (scale bar, 10 μm). Top and bottom panels are representative images of the joint space and midfoot, respectively (n = 5 per group, two experiments). Arrows indicate cellular infiltrate in joint space.

  • Fig. 4 Pharmacodynamics of CHK-24 mRNA in cynomolgus monkeys.

    Data show the total human IgG1 concentrations from two NHP studies in which animals were treated with 0.5 mg/kg of CHKV-24 mRNA by intravenous infusion. CHKV-24 mRNA was delivered over 60 min, in a volume of 5 ml/kg and dose concentration of 0.02 mg/ml. Four or six animals were tested in each group for study 1 or 2, respectively. The mean values are indicated, and error bars show the SD. Maximum concentration of 35.9 and 10.1 μg/ml was observed at 24 hours after infusion for study 1 (red curve) and study 2 (blue curve), respectively.

  • Fig. 5 Functional concentrations of mRNA-expressed CHKV-24 IgG in NHP serum.

    Macaques were infused with 0.5 mg/kg of mRNA encoding CHKV-24, and 24 hours later, serum samples were obtained and tested for the presence of CHIKV-specific binding or neutralizing antibodies. Antibody function was assessed by a focus reduction neutralization test (FRNT50; left) and by ELISA (right); a standard curve for concentration versus activity in each assay was generated using dilution curves of purified CHKV-24 at defined concentrations. A group of six animals was tested, and the in vitro experiments were performed twice. The mean values are indicated, and error bars show the SD.

  • Fig. 6 Concentrations of mRNA-expressed CHKV-24 IgG in NHP serum after repeat mRNA dosing.

    In a nonclinical GLP repeat-dose study using groups of four animals per treatment, macaques were infused with mRNA encoding CHKV-24 on days 0 and 7, and serum samples were collected for human IgG1 quantification at 6, 24, 48, 72, or 120 hours after the start of infusion of doses 1 and 6, 12, 24, 48, 72, 120, 168, 216, 288, 360, 432, 528, 720, 1080, or 2160 hours after the start of infusion of dose 2. A dose response was observed after each administration of mRNA encoding CHKV-24. Antibody concentrations after day 8 were calculated only for the highest dose level (3 mg/kg). At 24 hours after dosing with 3.0 mg/kg mRNA, the maximum CHKV-24 IgG serum concentration was 16.2 or 28.8 μg/ml for dose 1 or dose 2, respectively. The mean values are indicated, and error bars show the SD.

  • Table 1 Neutralizing activity of CHKV-specific human mAbs.

    IC50 FRNT, fifty percent maximal inhibitory concentration of antibody in a focus reduction neutralization test. > symbol indicates neutralization was not detected when tested at concentrations up to 10 µg/ml.

    MAb clone
    (CHKV-)
    IC50 FRNT (ng/ml)
    against CHIKV
    244
    3511
    2717
    824
    1225
    4837
    2986
    3281
    53319
    31684
    502266
    1>
    4>
    9>
    13>
    19>
    22>
    23>
  • Table 2 Human IgG pharmacokinetic parameters of CHKV-24 in macaques after delivery of antibody-encoding mRNA.

    The CHKV-24 NHP serum samples from study 2 were analyzed using the Human Therapeutic IgG1 ELISA Kit, with serum dilutions ranging from 1:100 to 1:1000. Parameters were calculated using Phoenix pharmacokinetic software (Certara, USA). A standard curve of absorbance at 450 nm versus log (concentration) was fit with a 4-parameter logistic equation for IgG1 quantification.

    Tmax (hours)Cmax (μg/ml)AUC0–720 hour (hour*μg/ml)t1/2 (hours)
    MeanSDCV%MeanSDCV%MeanSDCV%MeanSDCV%
    240010.15.36533720195052.456165.811.7

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