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. 2021 Oct 26;12(5):e0239521.
doi: 10.1128/mBio.02395-21. Epub 2021 Sep 14.

Neutralizing Monoclonal Antibodies That Target the Spike Receptor Binding Domain Confer Fc Receptor-Independent Protection against SARS-CoV-2 Infection in Syrian Hamsters

Wen Su #  1 , Sin Fun Sia #  1 , Aaron J Schmitz #  2 , Traci L Bricker #  3 , Tyler N Starr  4 , Allison J Greaney  4 , Jackson S Turner  2 , Bassem M Mohammed  2 , Zhuoming Liu  5 , Ka Tim Choy  1 , Tamarand L Darling  3 , Astha Joshi  3 , Ka Man Cheng  1 , Alvina Y L Wong  1 , Houda H Harastani  3 , John M Nicholls  6 , Sean P J Whelan  5 , Jesse D Bloom  4   7 , Hui-Ling Yen  1 , Ali H Ellebedy  2 , Adrianus C M Boon  2   3   5
Affiliations

Neutralizing Monoclonal Antibodies That Target the Spike Receptor Binding Domain Confer Fc Receptor-Independent Protection against SARS-CoV-2 Infection in Syrian Hamsters

Wen Su et al. mBio. .

Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein is the main target for neutralizing antibodies. These antibodies can be elicited through immunization or passively transferred as therapeutics in the form of convalescent-phase sera or monoclonal antibodies (MAbs). Potently neutralizing antibodies are expected to confer protection; however, it is unclear whether weakly neutralizing antibodies contribute to protection. Also, their mechanism of action in vivo is incompletely understood. Here, we demonstrate that 2B04, an antibody with an ultrapotent neutralizing activity (50% inhibitory concentration [IC50] of 0.04 μg/ml), protects hamsters against SARS-CoV-2 in a prophylactic and therapeutic infection model. Protection is associated with reduced weight loss and viral loads in nasal turbinates and lungs after challenge. MAb 2B04 also blocked aerosol transmission of the virus to naive contacts. We next examined three additional MAbs (2C02, 2C03, and 2E06), recognizing distinct epitopes within the receptor binding domain of spike protein that possess either minimal (2C02 and 2E06, IC50 > 20 μg/ml) or weak (2C03, IC50 of 5 μg/ml) virus neutralization capacity in vitro. Only 2C03 protected Syrian hamsters from weight loss and reduced lung viral load after SARS-CoV-2 infection. Finally, we demonstrated that Fc-Fc receptor interactions were not required for protection when 2B04 and 2C03 were administered prophylactically. These findings inform the mechanism of protection and support the rational development of antibody-mediated protection against SARS-CoV-2 infections. IMPORTANCE The ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by SARS-CoV-2, has resulted in the loss of millions of lives. Safe and effective vaccines are considered the ultimate remedy for the global social and economic disruption caused by the pandemic. However, a thorough understanding of the immune correlates of protection against this virus is lacking. Here, we characterized four different monoclonal antibodies and evaluated their ability to prevent or treat SARS-CoV-2 infection in Syrian hamsters. These antibodies varied in their ability to neutralize the virus in vitro. Prophylactic administration of potent and weakly neutralizing antibodies protected against SARS-CoV-2 infection, and this effect was Fc receptor independent. The potent neutralizing antibody also had therapeutic efficacy and eliminated onward aerosol transmission. In contrast, minimally neutralizing antibodies provided no protection against infection with SARS-CoV-2 in Syrian hamsters. Combined, these studies highlight the significance of weakly neutralizing antibodies in the protection against SARS-CoV-2 infection and associated disease.

Keywords: COVID-19; SARS-CoV-2; Syrian hamster; monoclonal antibodies; transmission.

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Figures

FIG 1
FIG 1
A single amino acid substitution abrogates MAb 2B04 binding to SARS-CoV-2 RBD. (a and b) ELISA binding to recombinant RBD (a) and binding affinity measured by biolayer interferometry (b) of RBD-specific MAbs. OD490 nm, optical density at 490 nm. (c) Percent relative infection by chimeric vesicular stomatitis virus harboring the SARS-CoV-2 S protein as a function of MAb concentration in a GFP reduction neutralization test. (d) Mutational antigenic profiling of RBD-specific MAbs. The sum of the escape fractions of all amino acid substitutions at each site in the RBD for each Mab is shown. Sites of strong binding escape for any of the four antibodies are indicated by pink bars along the x axis and shown in greater detail in panel e. (e) Logo plots showing the effects of each amino acid substitution on antibody binding, with taller letters indicating more binding escape, and letters colored according to how deleterious mutations are for ACE2 binding (20). Interactive visualizations of escape data can be found at https://jbloomlab.github.io/SARS-CoV-2-RBD_MAP_Ellebedy_Abs/. (f) Antibody escape mapped to the RBD surface (PDB accession no. 6M0J [37]) for 2B04, 2C02, 2C03, and 2E06 MAbs. The ACE2 contact surface is outlined in black. Sites are colored according to the maximum effect of a substitution at a given site, from white (no effect on antibody binding) to red (mutation with the strongest effect on antibody binding). (g) ELISA to measure binding of MAbs to wild-type (black) and E484A mutant (red) RBD. Values are means plus standard errors of the means (SEM) (error bars).
FIG 2
FIG 2
Neutralizing MAb 2B04 protects Syrian hamsters against SARS-CoV-2. (a) Experimental scheme for prophylactic or therapeutic treatment of Syrian hamsters with chimeric human MAb. Hamsters received 1 mg of hu-Ig 2B04 or isotype control (IgG) 24 h prior to (prophylactic) or 16 h after (therapeutic) IN challenge with 105 TCID50 of SARS-CoV-2. D-1, day −1; NT, nasal turbinate. (b and c) Infectious virus titer (left) and viral RNA level (right) detected in nasal turbinates (b) and lungs (c). Statistical significance by two-way ANOVA followed by Tukey’s multiple-comparison test is indicated by asterisks as follows: *, P < 0.05; **, P < 0.01. (d) Infectious virus titer (bars) and viral RNA level (circles) detected in nasal washes on the indicated day postinoculation (dpi). Paired measurements share colors. (e) Percent weight change of hamsters receiving isotype control (left), hu-Ig 2B04 prophylactically (center), or therapeutically (right). Statistical significance by ANOVA with Holm-Sidak correction for multiple comparisons is indicated by asterisks as follows: *, P < 0.05, **, P < 0.01. Lines indicate means ± standard deviations (SD). The dotted lines in panels b to d show the limit of detection of the assay, and each symbol represents the value for one hamster. The dotted line in panel e represents no weight loss, and each symbol represents the value for one hamster.
FIG 3
FIG 3
Chimeric hamster neutralizing MAb protects Syrian hamsters against SARS-CoV-2. (a) Detection of RBD-specific chimeric human (black) or hamster (orange) MAbs with anti-hamster (α-hamster) or anti-human secondary (2°) antibodies, respectively. (b) Experimental scheme for SARS-CoV-2 challenge of Syrian hamsters receiving 1 mg of chimeric hamster 2B04 (ham-Ig) or isotype control 24 h prior to IN challenge with 105 TCID50 of SARS-CoV-2. (c) Serum virus neutralizing titer (PRNT90) 1 day after IP administration of 1 mg of chimeric hamster 2B04 or isotype control (IgG). (d and e) Infectious virus titer (left) and viral RNA copy numbers (right) detected in nasal turbinate (d) and lungs (e) 2 and 5 dpi. Lines indicate mean ± SD. The dotted lines in panels c to e show the limit of detection of the assay, and each symbol represents the value for one hamster. Statistical significance by two-way ANOVA followed by Tukey’s multiple-comparison test is indicated by asterisks as follows: *, P < 0.05, **, P < 0.01, ***, P < 0.001, ****, P < 0.0001. (f and g) Hematoxylin and eosin staining (left panels) and immunohistochemistry for SARS-CoV-2 N protein (brown color, middle and right panels) in nasal turbinates (f) and lungs (g) of SARS-CoV-2-challenged hamsters 2 dpi. Scale bars = 500 μm. Higher-magnification images (right panels) of the boxed areas in the immunochemistry sections (middle panels) are shown for lungs and nasal turbinates. (h) Weight changes of hamsters receiving isotype (IgG, black circles) or 2B04 (blue circles) after SARS-CoV-2 challenge. Statistical significance by two-way ANOVA with Holm-Sidak correction for multiple comparisons is indicated by asterisks as follows: **, P < 0.01; ****, P < 0.0001. The dotted line in panel h represents no weight loss and each symbol represents one hamster.
FIG 4
FIG 4
Chimeric hamster neutralizing MAb protect against SARS-CoV-2 transmission. (a) Experimental scheme for SARS-CoV-2 challenge and aerosol transmission in Syrian hamsters receiving 1 mg of ham-Ig 2B04. (b) Infectious virus titer (bars) and viral RNA copy numbers (circles) detected in nasal washes of inoculated donor animals (left) and the aerosol-contact animals (right). (c) Weight changes of donor (left) and aerosol-contact (right) hamsters. The dotted line in panel b shows the limit of detection of the assay, and each symbol represents the value for one hamster. The dotted line in panel c represents no weight loss, and each symbol represents the value for one hamster.
FIG 5
FIG 5
Neutralizing MAbs protect Syrian hamsters from SARS-CoV-2 infection in a Fc-independent manner. (a) ELISA binding of wild-type (circles) and LALA mutant (triangles) chimeric hamster MAbs to human (top panels) and mouse (bottom panels) FcγR1. (b) Experimental scheme for SARS-CoV-2 challenge of hamsters receiving 1 mg of wild-type or LALA mutant ham-Ig 2B04, 2C02, 2C03, or 2E06 24 h prior to intranasal challenge with 2.5 × 105 PFU of SARS-CoV-2. (c) Infectious virus titer in the lungs of isotype or wild-type and LALA mutant ham-Ig 2B04-, 2C02-, 2C03-, or 2E06-treated hamsters. Statistical significance by Kruskal-Wallis test with a multiple comparison correction between isotype and wild- type or LALA 2B04-, 2C02-, 2C03-, and 2E06-treated groups is indicated by asterisks as follows: *, P < 0.05; ****, P < 0.0001. Lines represent geometric mean ± geometric SD, and the dotted line is the limit of detection for the assay. Each symbol represents the value for one hamster. (d) Viral RNA copy numbers in lungs of isotype or wild-type and LALA mutant ham-Ig 2B04-, 2C02-, 2C03-, or 2E06-treated hamsters. Statistical significance by one-way ANOVA and a multiple comparison correction between isotype and wild type or LALA 2B04-, 2C02-, 2C03-, and 2E06-treated groups is indicated by asterisks as follows: *, P < 0.05, ****, P < 0.0001. Lines represent the geometric mean, and the dotted line is the limit of detection for the assay. Each symbol represents the value for one hamster. (e) Weight changes of hamsters receiving the indicated wild-type (WT) (circles) or LALA mutant (triangles) MAb 3 dpi. Statistical significance by one-way ANOVA with a multiple comparisons between isotype and wild-type or LALA 2B04-, 2C02-, 2C03-, and 2E06-treated groups is indicated by asterisks as follows: *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. The dotted line represents no weight loss. Each symbol represents the value for one hamster.
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