Antibody Responses to SARS-CoV-2 Antigens in Humans and Animals

Abstract

To optimize the public health response to coronavirus disease 2019 (COVID-19), we must first understand the antibody response to individual proteins on the severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) and the antibody's cross reactivity to other coronaviruses. Using a panel of 37 convalescent COVID-19 human serum samples, we showed that the magnitude and specificity of responses varied across individuals, independent of their reactivity to seasonal human coronaviruses (HCoVs). These data suggest that COVID-19 vaccines will elicit primary humoral immune responses in naïve individuals and variable responses in those previously exposed to SARS-CoV-2. Unlike the limited cross-coronavirus reactivities in humans, serum samples from 96 dogs and 10 cats showed SARS-CoV-2 protein-specific responses focused on non-S1 proteins. The correlation of this response with those to other coronaviruses suggests that the antibodies are cross-reactive and generated to endemic viruses within these hosts, which must be considered in seroepidemiologic studies. We conclude that substantial variation in antibody generation against coronavirus proteins will influence interpretations of serologic data in the clinical and veterinary settings.

Keywords: COVID-19; SARS-CoV-2; antibody; respiratory viruses; vaccine.

Figure 1

Antibody responses in patients with COVID-19. (A) Levels of serum IgG, IgA, and IgM antibodies against S, S1, RBD, S2, N, E, and NS3 of SARS-CoV-2 were measured by ELISA. Blue (IgG), green (IgA), and purple (IgM) lines represent the antibody titers of individual COVID-19 positive sera, pink lines represent negative control sera. (B) Each dot represents an individual antibody titer, and groups are divided by age and sex. (C) Correlation of anti-N responses with other antiviral protein responses in patients with COVID-19. Correlations of the values were assessed by linear regression. (D) Correlations between neutralization titer (x-axis) and each antibody response to the viral protein of SARS-CoV-2 (y-axis) are shown. Each dot indicates the neutralization titer of serum antibodies of patients with COVID-19, as determined by calculating the highest dilution of serum that prevents infection of 50% of inoculations and antiviral protein titers (IgG) of human sera, as determined by ELISA.

Figure 2

Preexisting and cross-reactive antibodies to human respiratory viruses in sera of patients with COVID-19. (A) Respiratory virus-specific antibody profiling. Heatmap indicating IgG antibody responses in 46 sera samples (37 COVID-19 samples and 9 control samples) measured by protein microarray. The microarray slides were probed with human sera and labeled with secondary antibodies to human IgG conjugated to a quantum dot fluorophore. The slides were imaged using the GenePix 4000B Microarray Scanner to measure background-subtracted median spot fluorescence. Mean fluorescence intensity (MFI) of the 4 replicates for each antigen was used for analysis. Each column represents a paired sample, and each row, a respiratory virus protein. Box graph indicates the sum of antibody responses of all sera samples for each respiratory virus protein. (B) Correlation between protein microarray and ELISA-based readouts. Each dot indicates the antibody response of serum from a single patient with COVID-19 against S, S1, RBD, S2, and N of SARS-CoV-2, as measured by fluorescence intensity (y-axis) and absorbance at 450 nm, as determined by ELISA (x-axis). (C) Relations of antibody response between specific antibody levels against SARS-CoV-2 and those against endemic HCoVs NL63, 229E, HKU1, and OC43 in patients with COVID-19.

Figure 2

Preexisting and cross-reactive antibodies to human respiratory viruses in sera of patients with COVID-19. (A) Respiratory virus-specific antibody profiling. Heatmap indicating IgG antibody responses in 46 sera samples (37 COVID-19 samples and 9 control samples) measured by protein microarray. The microarray slides were probed with human sera and labeled with secondary antibodies to human IgG conjugated to a quantum dot fluorophore. The slides were imaged using the GenePix 4000B Microarray Scanner to measure background-subtracted median spot fluorescence. Mean fluorescence intensity (MFI) of the 4 replicates for each antigen was used for analysis. Each column represents a paired sample, and each row, a respiratory virus protein. Box graph indicates the sum of antibody responses of all sera samples for each respiratory virus protein. (B) Correlation between protein microarray and ELISA-based readouts. Each dot indicates the antibody response of serum from a single patient with COVID-19 against S, S1, RBD, S2, and N of SARS-CoV-2, as measured by fluorescence intensity (y-axis) and absorbance at 450 nm, as determined by ELISA (x-axis). (C) Relations of antibody response between specific antibody levels against SARS-CoV-2 and those against endemic HCoVs NL63, 229E, HKU1, and OC43 in patients with COVID-19.

Figure 3

Immune responses of mice to SARS-CoV-2. (A) Viral protein-specific IgG antibody responses of mice immunized with S, S1, RBD, S2, N, E, or NS3 of SARS-CoV-2. Viral protein-specific IgG responses of murine sera and cross-reactivities to the viral proteins of SARS-CoV, CCoV 1-71, HCoV NL63, and influenza virus were measured by ELISA. Blue bars represent homologous antibody responses, and bars with slash lines display cross-reactive antibody responses. Red lines are the cut-offs calculated as 3 times the mean absorbance value of the preimmune sera. (B) Antibody response was measured in mice immunized with S, N, E, M, or 3CLpro of SARS-CoV. Purple bars show homologous antibody responses, and bars with slash lines represent cross-reactive antibody responses. (C) S-, S1-, RBD-, S2-, N-, E-, and NS3-specific IgG PCs in spinal bone marrow (left) and ASCs in cervical lymph nodes (right) were quantified ex vivo in ELISpot assays. Bone marrow and lymph node cells were obtained from the mice immunized with respective viral protein of SARS-CoV-2. (D) The presence of neutralizing antibodies in mice immunized with S, S1, RBD, S2, N, E, or NS3 of SARS-CoV-2; inactivated whole virus of SARS-CoV-2; and S, N, E, M, or 3CLpro of SARS-CoV was measured by in vitro neutralization assays using a pseudotyped virus. The transduction efficacy was determined by measuring the luciferase activity. Nonlinear regression analysis was used to obtain the IC₅₀ in the neutralization assays.

Figure 3

Immune responses of mice to SARS-CoV-2. (A) Viral protein-specific IgG antibody responses of mice immunized with S, S1, RBD, S2, N, E, or NS3 of SARS-CoV-2. Viral protein-specific IgG responses of murine sera and cross-reactivities to the viral proteins of SARS-CoV, CCoV 1-71, HCoV NL63, and influenza virus were measured by ELISA. Blue bars represent homologous antibody responses, and bars with slash lines display cross-reactive antibody responses. Red lines are the cut-offs calculated as 3 times the mean absorbance value of the preimmune sera. (B) Antibody response was measured in mice immunized with S, N, E, M, or 3CLpro of SARS-CoV. Purple bars show homologous antibody responses, and bars with slash lines represent cross-reactive antibody responses. (C) S-, S1-, RBD-, S2-, N-, E-, and NS3-specific IgG PCs in spinal bone marrow (left) and ASCs in cervical lymph nodes (right) were quantified ex vivo in ELISpot assays. Bone marrow and lymph node cells were obtained from the mice immunized with respective viral protein of SARS-CoV-2. (D) The presence of neutralizing antibodies in mice immunized with S, S1, RBD, S2, N, E, or NS3 of SARS-CoV-2; inactivated whole virus of SARS-CoV-2; and S, N, E, M, or 3CLpro of SARS-CoV was measured by in vitro neutralization assays using a pseudotyped virus. The transduction efficacy was determined by measuring the luciferase activity. Nonlinear regression analysis was used to obtain the IC₅₀ in the neutralization assays.

Figure 4

Antibody responses in dogs and cats. (A) Serum antibody responses of dogs to S, S1, RBD, S2, N, E, and NS3 viral proteins of SARS-CoV-2, and inactivated viruses, CCoV 1-71, HCoV NL63, and H1N1 influenza virus were measured by IgG ELISA. The OD values were normalized by subtracting the OD values generated by sera from SPF dogs. (B) In canine serum samples, IgG levels against CCoV/HCoV and those against S, S1, RBD, S2, N, E, and NS3 of SARS-CoV-2, as detected by ELISA, were used in correlation analyses. (C) Serum IgG antibody responses of cats to SARS-CoV-2 S, S1, RBD, S2, N, E, and NS3, and and inactivated viruses, CCoV 1-71, HCoV NL63, and H1N1 influenza virus were measured by ELISA. The crude OD values generated by ELISA were normalized by subtracting the OD values generated by sera from SPF cats. (D) ELISA was also used to determine IgG antibody levels against N of SARS-CoV-2, CCoV 1-71, and HCoV NL63 in the sera of cats for correlation analyses.

Figure 4

Antibody responses in dogs and cats. (A) Serum antibody responses of dogs to S, S1, RBD, S2, N, E, and NS3 viral proteins of SARS-CoV-2, and inactivated viruses, CCoV 1-71, HCoV NL63, and H1N1 influenza virus were measured by IgG ELISA. The OD values were normalized by subtracting the OD values generated by sera from SPF dogs. (B) In canine serum samples, IgG levels against CCoV/HCoV and those against S, S1, RBD, S2, N, E, and NS3 of SARS-CoV-2, as detected by ELISA, were used in correlation analyses. (C) Serum IgG antibody responses of cats to SARS-CoV-2 S, S1, RBD, S2, N, E, and NS3, and and inactivated viruses, CCoV 1-71, HCoV NL63, and H1N1 influenza virus were measured by ELISA. The crude OD values generated by ELISA were normalized by subtracting the OD values generated by sera from SPF cats. (D) ELISA was also used to determine IgG antibody levels against N of SARS-CoV-2, CCoV 1-71, and HCoV NL63 in the sera of cats for correlation analyses.