In vivo monoclonal antibody efficacy against SARS-CoV-2 variant strains

Abstract

Rapidly emerging SARS-CoV-2 variants jeopardize antibody-based countermeasures. Although cell culture experiments have demonstrated a loss of potency of several anti-spike neutralizing antibodies against variant strains of SARS-CoV-2^1-3, the in vivo importance of these results remains uncertain. Here we report the in vitro and in vivo activity of a panel of monoclonal antibodies (mAbs), which correspond to many in advanced clinical development by Vir Biotechnology, AbbVie, AstraZeneca, Regeneron and Lilly, against SARS-CoV-2 variant viruses. Although some individual mAbs showed reduced or abrogated neutralizing activity in cell culture against B.1.351, B.1.1.28, B.1.617.1 and B.1.526 viruses with mutations at residue E484 of the spike protein, low prophylactic doses of mAb combinations protected against infection by many variants in K18-hACE2 transgenic mice, 129S2 immunocompetent mice and hamsters, without the emergence of resistance. Exceptions were LY-CoV555 monotherapy and LY-CoV555 and LY-CoV016 combination therapy, both of which lost all protective activity, and the combination of AbbVie 2B04 and 47D11, which showed a partial loss of activity. When administered after infection, higher doses of several mAb cocktails protected in vivo against viruses with a B.1.351 spike gene. Therefore, many-but not all-of the antibody products with Emergency Use Authorization should retain substantial efficacy against the prevailing variant strains of SARS-CoV-2.

Extended Data Figure 1.. Neutralization curves with mAbs and variant SARS-CoV-2 strains.

Anti-SARS-CoV-2 human mAbs were tested for inhibition of infection of the indicated viruses on Vero-TMPRSS2 cells using a focus reduction neutralization test. One representative experiment of two performed in duplicate is shown.

Extended Data Figure 2.. SARS-CoV-2 variant infection after antibody prophylaxis of K18-hACE2 mice.

(a-l) 8-10-week-old female and male K18-hACE2 transgenic mice received 40 μg of the indicated mAb treatment by intraperitoneal injection one day before intranasal challenge with 10³ FFU of the indicated SARS-CoV-2 strain. At 6 dpi, viral RNA levels in the heart (a-d), spleen (e-h), and brain (i-l) were measured (line indicates median; in order left to right n = 9, 5, 5, 6, 6, 6 (a); n = 12, 6, 6, 6, 5, 6 (b, c, f, g, j, k); n = 10, 6, 6, 6, 6, 5 (d, h); n = 6, 5, 5, 3, 3, 3 (e); n = 9, 4, 4, 6, 6, 6 (i); n = 11, 6, 6, 6, 6, 5 (l) mice per group, two experiments; one-way ANOVA with Dunnett’s test with comparison to control mAb: ns, not significant, **** P < 0.0001; in order left to right ** P = 0.0066, 0.0032, 0.0080, 0.0016, 0.0052 (a); *** P = 0.0002, ** P = 0.0017, 0.0052 (b); *** P = 0.0004, 0.0004, * P = 0.0140, ** P = 0.0043 (c); ** P = 0.0080, 0.0080, * P = 0.0226 (d); *** P = 0.0001, 0.0001, 0.0008, 0.0008, 0.0008 (e); *** P = 0.0008 (h); ** P = 0.0014, 0.0014, 0.0029, 0.0032 (j); *** P = 0.0002, 0.0002, ** P = 0.0061, 0.0028 (k); * P = 0.0371 (l)). Dotted line indicates the limit of detection of the assay.

Extended Data Figure 3.. Reduced infectious virus in the lungs of antibody-treated mice.

(a-b) 8-10-week-old female and male K18-hACE2 transgenic mice received 40 μg (~2 mg/kg) of the indicated mAb treatment by intraperitoneal injection one day before intranasal inoculation with 10³ FFU of the indicated SARS-CoV-2 strain. Tissues were collected at 6 dpi. (c-f) 6-7-week-old female and male immunocompetent 129S2 mice received 40 μg (~ 2 mg/kg) of the indicated mAb treatment by intraperitoneal injection one day before intranasal inoculation with 10³ FFU of WA1/2020 N501Y/D614G, Wash-B.1.351, or Wash-B.1.1.28 and 10⁵ FFU of B.1.1.7. Tissues were collected at 3 dpi. (g-h) 8-10-week-old female and male K18-hACE2 transgenic mice were administered 10³ FFU of the indicated SARS-CoV-2 strain by intranasal inoculation. One day later, animals received 200 μg (~10 mg/kg) of the indicated mAb treatment by intraperitoneal injection. Tissues were collected at 6 dpi. For all panels, infectious virus in lung homogenates was determined by plaque assay using Vero-TMPRSS2-hACE2 cells (line indicates median; in order left to right n = 5, 5, 5, 6, 6, 6 (a); n = 6, 6, 6, 6, 6, 6 (b-h) mice per group, one-way ANOVA with Dunnett’s test with comparison to control mAb: ns, not significant, **** P < 0.0001; ** P = 0.0012, *** P = 0.0003 (c); ** P = 0.0048, *** P = 0.0005 (e); ** P = 0.0031, 0.0019, 0.0020 (f left to right)). Dotted lines indicate the limit of detection of the assay.

Extended Data Figure 4.. Antibody neutralization and protection against a natural B.1.351 isolate.

(a) Selected anti-SARS-CoV-2 mAbs (one from each cocktail) were tested for neutralization of infection by WA1/2020 D614G, Wash-B.1.351 or B.1.351 on Vero-TMPRSS2 cells using an FRNT. One representative experiment of two performed in duplicate is shown. (b-e) 8-10-week-old female K18-hACE2 transgenic mice received 40 μg (~2 mg/kg) of control mAb or COV2-2130/COV2-2196 by intraperitoneal injection one day before intranasal inoculation with 10³ FFU of B.1.351. (b) Weight change following infection (mean ± SEM; n = 6 mice per group, two experiments; one-way ANOVA with Dunnett’s test of area under the curve: ns, not significant, **** P < 0.0001). Viral RNA levels in the lung (c), nasal wash (d), and brain (e) (line indicates median; n = 6 mice per group, two experiments; Mann Whitney test: ** P = 0.002 (c-e)). Dotted line indicates the limit of detection of the assay.

Extended Data Figure 5.. Antibody-mediated protection against SARS-CoV-2-induced inflammation.

8-10-week-old female and male K18-hACE2 transgenic mice received 40 μg (~2 mg/kg) of the indicated mAb treatment by intraperitoneal injection one day before intranasal inoculation with 10³ FFU of the indicated SARS-CoV-2 strain. Heat map of cytokine and chemokine protein expression levels in lung homogenates collected at 6 dpi from the indicated groups. Data are presented as log₂ fold-change over naïve animals. Blue, reduction; red, increase.

Extended Data Figure 6.. Cytokine and chemokine induction following SARS-CoV-2 infection.

Individual plots for cytokine and chemokine protein levels in the lungs of antibody treated K18-hACE2 transgenic mice at 6 dpi after infection with the indicated SARS-CoV-2 strain (line indicates mean; n = 3 naïve, n = 5 for all other groups of B.1.1.7 and N501Y infected mice; n = 3 naïve, 10 control, 5 for all other groups of Wash-B.1.351 and Wash-B.1.1.28 infected mice; one-way ANOVA with Dunnett’s test with comparison to control mAb: ns, not significant, **** P < 0.0001; all other P values are listed in Supplementary Table S5). Select cytokines and chemokines were used to generate the heat-map in Extended Data Figure 5.

Extended Data Figure 7.. Low-dose antibody prophylaxis against SARS-CoV-2 variants in K18-hACE2 mice.

(a-p) 8-10-week-old female and male K18-hACE2 transgenic mice received 4 μg (~0.2 mg/kg) of the indicated mAb treatment by intraperitoneal injection one day before intranasal inoculation with 10³ FFU of the indicated SARS-CoV-2 strain. Tissues were collected at 6 dpi. (a-d) Weight change following infection with SARS-CoV-2 (mean ± SEM; in order left to right n = 11, 6, 6, 7, 7 (a); n = 10, 6, 6, 5, 7 (b); n = 10, 6, 6, 7, 7 (c-d) mice per group, two experiments; one-way ANOVA with Dunnett’s test of area under the curve with comparison to control mAb: **** P < 0.0001). Viral RNA levels in the lung (e-h), nasal wash (i-l), or brain (m-p) were measured (line indicates median; in order from left to right n = 11, 6, 6, 6, 7 (e); n = 10, 6, 6, 5, 7 (f, n); n = 10, 6, 6, 7, 7 (g, h, i, k, m, and o); n = 8, 6, 6, 5, 6 (j); n = 10, 6, 6, 6, 7 (l, p) mice per group, two experiments; one-way ANOVA with Dunnett’s test with comparison to isotype control mAb: ns, not significant, **** P < 0.0001); * P = 0.034 (e); * P = 0.0422, *** P = 0.0004 (g); ** P = 0.0080 (h); * P = 0.0209 (left), 0.0365 (right) (j); * P = 0.0124 (left), 0.0497 (right), *** P = 0.0001 (k); ** P = 0.0069 (l); ** P = 0.0087 (left), 0.0061 (right), * P = 0.0264 (n); * P = 0.0378 (left), 0.0446 (right), *** P = 0.0004 (o); ** P = 0.0045 (left), 0.0035 (right), *** P = 0.0002, * P = 0.0107 (p)). Dotted line indicates the limit of detection of the assay.

Extended Data Figure 8.. In vivo correlation of antibody-mediated protection against SARS-CoV-2.

For each panel, the fold-change in the EC₅₀ values of the indicated mAb or mAb cocktails between the N501Y/D614G strain and one or more variants of concern (B.1.1.7, Wash-B.1.351, or Wash-B.1.1.28) were plotted on the x-axis. Next, the fold-change in lung viral RNA titer corresponding to the indicated treatment group (top labels) between the N501Y/D614G strain and one or more variants of concern (B.1.1.7, Wash-B.1.351, or Wash-B.1.1.28) were plotted on the y-axis. Best-fit lines were calculated using a simple linear regression. Two-tailed Pearson correlation was used to calculate R² and p values indicated within each panel.

Extended Data Figure 9.. Antibody-mediated protection against SARS-CoV-2 variants in 129S2 mice.

(a-h) 6-7-week-old female and male immunocompetent 129S2 mice received 40 μg (~ 2 mg/kg) of the indicated mAb treatment by intraperitoneal injection one day before intranasal inoculation with 10³ FFU of WA1/2020 N501Y/D614G, Wash-B.1.351, or Wash-B.1.1.28 or 10⁵ FFU of B.1.1.7. Tissues were collected at 3 dpi. Viral RNA levels in the lung (a-d) or nasal washes (e-h) were determined (line indicates median; in order left to right n = 12, 8, 6, 5 (a); n = 12, 7, 9, 10 (b); n = 11, 9, 8, 10 (c); n = 14, 9, 6, 5 (d); n = 14, 5, 9, 9 (e); n = 11, 7, 9, 10 (f); n = 10, 9, 8, 10 (g); n = 15, 9, 7, 10 (h) mice per group, pooled from two to three experiments; one-way ANOVA with Dunnett’s test with comparison to control mAb: ns, not significant, *** P < 0.001; *** P = 0.0009, * P = 0.0176, ** P = 0.0077 (c); ** P = 0.0042, *** P = 0.0001 (left), 0.0010 (right) (d); * P = 0.0467 (left), 0.0188 (right) (e); ** P = 0.0059, *** P = 0.0002 (left), 0.0004 (right) (f); * P = 0.0184 (g), * P = 0.0129, ** P = 0.0090 (h)). Dotted line indicates the limit of detection of the assay.

Extended Data Figure 10.. COV2-2130/COV2-2196 antibody cocktail protects hamsters against historical and variant SARS-CoV-2 strains.

Six-week-old male Syrian golden hamsters received a single 800 μg (~10 mg/kg) (a-h) or 320 μg (~4 mg/kg) dose (i-p) of COV2-2130/COV2196 mAb cocktail or control mAb by intraperitoneal injection one day before intranasal inoculation with 5 x 10⁵ FFU of WA1/2020 D614G or Wash-B.1.351 viruses. Nasal swabs and lung tissues were collected at 3 and 4 dpi, respectively. (a, i) Weight change following infection with SARS-CoV-2 (line indicates mean; n = 5 hamsters per group, one experiment). Infectious virus in the lung (b, j) or viral RNA levels in the lung (c, k) and nasal swabs (d, l) were determined (line indicates median; n = 5 animals per group, one experiment). Dotted line indicates the limit of detection of the assay. (e-h, m-p) Cytokine and inflammatory gene expression in lung homogenates collected at 6 dpi from indicated groups (line indicates mean; n= 5 hamsters per group). Values were calculated using the ΔΔC_t method compared to a naïve control group. Because data were obtained from a single experiment (even with multiple animals), statistical analysis was not performed.

Extended Data Figure 11.. Antibody protection against SARS-CoV-2 induced lung pathology.

8-10-week-old female K18-hACE2 transgenic mice received 200 μg (~10 mg/kg) of the indicated mAb treatment or isotype control by intraperitoneal injection one day after intranasal inoculation with 10³ FFU of Wash-B.1.351 SARS-CoV-2. At 6 dpi, animals were sacrificed and lungs fixed for sectioning prior to staining with hematoxylin and eosin. A lung section from a naïve, uninfected animal is shown (top panels) as a reference control. Images show low (left panels for each treatment) and high (right panels for each treatment; boxed region from left) resolution. Scale bars for all images, 100 μm. Representative images from n = 3 mice per group.

Extended Data Figure 12.. Post-exposure antibody therapy against SARS-CoV-2 variants in K18-hACE2 mice.

(a-b) 8-10-week-old female and male K18-hACE2 transgenic mice were administered 10³ FFU of the indicated SARS-CoV-2 strain by intranasal inoculation. One day later, mice received 200 μg (~10 mg/kg) of the indicated mAb treatment by intraperitoneal injection. Tissues were collected at 6 dpi. Viral RNA levels in the brain (a, b) (line indicates median; in order left to right n = 9, 6, 6, 6, 6, 6, 6 (a); n = 7, 6, 6, 6, 6, 5, 7 (b) mice per group, two experiments; one-way ANOVA with Dunnett’s test with comparison to control mAb: ns, not significant, **** P < 0.0001). Dotted line indicates the limit of detection of the assay.

Extended Data Figure 13.. Neutralization curves with mAb LY-CoV016 as monotherapy and combination with variant SARS-CoV-2 strains.

Anti-SARS-CoV-2 human mAb LY-CoV016 and combination LY-CoV555/LY-CoV016 (parental mAbs of bamlanivimab and etesevimab, respectively) were tested for inhibition of infection of the indicated SARS-CoV-2 viruses using a FRNT. EC₅₀ values (ng/ml) (a) and one representative experiment of two performed in duplicate is shown (b).

Figure 1.. Neutralization of SARS-CoV-2 variant strains by clinically relevant mAbs.

(a-b) SARS-CoV-2 variant substitutions mapped onto the structure of the spike protein. Schematic layout of the spike protein monomer is depicted at the top. Structure of spike monomer (PDB: 7C2L with RBD from PDB: 6W41) is depicted as a cartoon, with NTD, RBD, RBM, and S2 colored orange, green, magenta, and light blue, respectively. Substitutions for each variant are shown as spheres and colored according to the legend. Substitutions shown in black are shared between multiple variants. Purple triangle, pink square, purple pentagon, and black hexagon represent approximate locations of L5, S13, D253 and P681 respectively, which were not modeled in the original structures. (a) Structural figure generated using UCSF ChimeraX. (b) Viruses used with indicated colored mutations in the spike protein. (c) Summary of EC₅₀ values (ng/mL) of neutralization of SARS-CoV-2 viruses performed in Vero-TMPRSS2 cells. Blue shading of cells indicates a partial (EC₅₀ > 1,000 ng/mL) or complete (EC₅₀ > 10,000 ng/mL) loss of neutralizing activity. (d) Neutralization curves comparing the sensitivity of SARS-CoV-2 strains to the indicated individual or combinations of mAbs. Data are representative of two to five experiments, each performed in technical duplicate.

Figure 2.. Antibody prophylaxis against SARS-CoV-2 variants in K18-hACE2 mice.

(a-l) 8-10-week-old female and male K18-hACE2 transgenic mice received 40 μg (~2 mg/kg) of the indicated mAb treatment by intraperitoneal injection one day before intranasal inoculation with 10³ FFU of the indicated SARS-CoV-2 strain. Tissues were collected at 6 dpi. (a, d, g, j) Weight change following infection with SARS-CoV-2 (mean ± SEM; n = 6-12 mice per group, two experiments; one-way ANOVA with Dunnett’s test of area under the curve: ns, not significant, **** P < 0.0001). Viral RNA levels in the lung (b, e, h, k) and nasal washes (c, f, i, l) were measured (line indicates median; in order left to right n = 9, 6, 7, 6, 6, 6 (a); n = 11, 5, 5, 6, 6, 6 (b); n = 9, 5, 5, 6, 6, 6 (c); n = 12, 6, 6, 6, 5, 6 (d-f); n = 12, 6, 6, 6, 6, 6 (g-h, and j-k); n = 12, 6, 6, 6, 6, 5 (i); n = 10, 6, 6, 6, 6, 5 (l) mice per group, two experiments; one-way ANOVA with Dunnett’s test with comparison to control mAb: ns, not significant, **** P < 0.0001; * P = 0.026 (c); ** P = 0.0016, *** P = 0.0002 (l)). Dotted line indicates the limit of detection of the assay.

Figure 3.. Post-exposure antibody therapy against SARS-CoV-2 variants in K18-hACE2 mice.

(a-f) 8-10-week-old female and male K18-hACE2 transgenic mice were administered 10³ FFU of the indicated SARS-CoV-2 strain by intranasal inoculation. One day later, mice received 200 μg (~10 mg/kg) of the indicated mAb treatment by intraperitoneal injection. Tissues were collected at 6 dpi. (a, d) Weight change following infection with SARS-CoV-2 (mean ± SEM; in order left to right n = 15, 6, 6, 6, 6, 7, 7 (a, d) mice per group, two experiments; one-way ANOVA with Dunnett’s test of area under the curve: ns, not significant, **** P < 0.0001). Viral RNA levels in the lung (b, e) and nasal wash (c, f) (line indicates median; in order left to right n = 7, 6, 6, 6, 6, 7, 7 (b); n = 9, 6, 6, 6, 6, 7, 7 (c, e, and f) mice per group, two experiments; one-way ANOVA with Dunnett’s test with comparison to control mAb: ns, not significant, **** P < 0.0001; ** P = 0.0014 (left), 0.0088 (right), *** P = 0.0007 (left), 0.0003 (right) (b); ** P = 0.0026 (left), 0.0041 (middle), 0.0049 (right) (c); ** P = 0.0049, *** P = 0.0004 (e); ** P = 0.0094, *** P = 0.0005, * P = 0.0442 (f). Dotted line indicates the limit of detection of the assay.