Immune Maturation Effects on Viral Neutralization and Avidity of Hyperimmunized Equine Anti-SARS-CoV-2 Sera

Abstract

Mass-vaccination against COVID-19 is still a distant goal for most low-to-middle income countries. The experience gained through decades producing polyclonal immunotherapeutics (such as antivenoms) in many of those countries is being redirected to develop similar products able to neutralize SARS-CoV-2 infection. In this study we analyzed the biological activity (viral neutralization or NtAb) and immunochemical properties of hyperimmune horses' sera (HHS) obtained during initial immunization (I) and posterior re-immunization (R) cycles using the RBD domain of the SARS-CoV-2 spike protein as antigen. HHS at the end of the R cycle showed higher NtAb titers when compared to those after the I cycle (35,585 vs. 7000 mean NtAb, respectively). Moreover, this increase paralleled an increase in avidity (95.2% to 65.2% mean avidity units, respectively). The results presented herein are relevant for manufacturers of these therapeutic tools against COVID-19.

Keywords: COVID-19; anti-SARS-CoV-2; hyperimmune equine; hyperimmunization; immune maturation; therapeutics.

Figure 1

rRBD-specific IgG immune response kinetics in horses’ sera. Blood was extracted from the jugular vein of horses at regular intervals during each immunization cycle. RBD-specific IgG response in sera obtained from these blood samples was estimated by the indirect ELISA technique described in Section 2. Figure 1 displays the level of rRBD-specific IgG response detected (OD_{450 nm}) for each sera sample at the different times of extraction (Time). Results of sera obtained during the initial immunization cycle (white circles) and during the following reimmunization cycle (black circles) are represented together with their estimated group means (black lines).

Figure 2

(a) NtAb contrast in HHS at final blood collection date during I and R cycles. HHS were collected from horses 7 days after receiving their last immunization dose. These HHS are the starting material for EpAbs production. Their viral infection neutralization titer (NtAb) was estimated as described in Section 2 and grouped according to their corresponding immunization cycles (as stated in x-axis). Paired dot and lines are represented for each horse to aid visual inspection and analysis of both individual and group difference. Statistical inference analysis between groups is included in the text. (b) Correlation analysis between indirect ELISA and viral neutralization results. Indirect ELISA results (OD_{450 nm}) on HHS of horses from the I (white circles) and R (black circles) were plotted against log transformed viral neutralization antibodies titers (Log(NtAb)). Spearman’s rank correlation coefficient is depicted in the chart together with the p value associated with rejecting the null hypothesis (that OD_{450 nm} and Log(NtAb) are not correlated) whether it is true. A linear regression line is shown in blue to aid visual inspection of the data.

Figure 3

(a) Affinity maturation between hyperimmunization cycles. rRBD-specific avidity testing for IgG antibodies in HHS samples collected in each cycle at the moment of final blood extraction was analyzed as described in Section 2. Individual avidity scores (%) are grouped according to their respective immunization cycle (as stated in x-axis). Paired dot and lines are represented for everyone to aid visual inspection and analysis of both individual and group differences. Statistical inference analysis between groups is included in the text. (b) Correlation analysis affinity maturation and viral neutralization. Avidity on HHS of horses from the I (white circles) and R (black circles) were plotted against log transformed viral neutralization antibodies titers (Log(NtAb)). Spearman’s rank correlation coefficient is depicted in the chart together with the p value associated with rejecting the null hypothesis (that Avidity and Log(NtAb) are not correlated) whether it is true. A linear regression line is shown in blue to aid visual inspection of the data.