Correlates of protection against SARS-CoV-2 in rhesus macaques

Abstract

Recent studies have reported the protective efficacy of both natural¹ and vaccine-induced^2-7 immunity against challenge with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in rhesus macaques. However, the importance of humoral and cellular immunity for protection against infection with SARS-CoV-2 remains to be determined. Here we show that the adoptive transfer of purified IgG from convalescent rhesus macaques (Macaca mulatta) protects naive recipient macaques against challenge with SARS-CoV-2 in a dose-dependent fashion. Depletion of CD8⁺ T cells in convalescent macaques partially abrogated the protective efficacy of natural immunity against rechallenge with SARS-CoV-2, which suggests a role for cellular immunity in the context of waning or subprotective antibody titres. These data demonstrate that relatively low antibody titres are sufficient for protection against SARS-CoV-2 in rhesus macaques, and that cellular immune responses may contribute to protection if antibody responses are suboptimal. We also show that higher antibody titres are required for treatment of SARS-CoV-2 infection in macaques. These findings have implications for the development of SARS-CoV-2 vaccines and immune-based therapeutic agents.

Extended Data Figure 1.. Pseudovirus NAb titers in plasma and purified IgG of donor macaques.

9 rhesus macaques were infected and re-challenged with SARS-CoV-2. IgG was purified from plasma and formulated at 10 mg/ml.

Extended Data Figure 2.. Antibody titers following prophylactic adoptive transfer of IgG prior to SARS-CoV-2 challenge.

(a) Log₁₀ pseudovirus neutralizing antibody (NAb) titers, (b) Log₁₀ S-specific ELISA titers, and (c) Log₁₀ RBD-specific ELISA titers in rhesus macaques following intravenous infusion of 250 mg/kg, 25 mg/kg, 2.5 mg/kg of purified SARS-CoV-2 IgG or sham IgG on day −3. On day 0, all animals were challenged with 10⁵ TCID50 SARS-CoV-2. Days following challenge is shown on the x-axis. Red lines reflect median responses. The number of animals is denoted in each panel and the median line overlaps with data lines.

Extended Data Figure 3.. Systems serology following adoptive transfer of IgG in rhesus macaques.

(a) S-specific IgG subclass, FcR, and antibody-dependent cellular phagocytosis (ADCP), antibody-dependent neutrophil phagocytosis (ADNP), antibody-dependent complement deposition (ADCD) and antibody-dependent NK cell activation (ADNKA) responses. (b) Radar plot showing the mean percentile of the antibody levels across passive transfer groups. The size and color intensity of the wedges indicate the mean percentile of the feature for the corresponding group (antibody subclass, red; FcR binding, blue; effector function, green). (c) Principal component analysis (PCA) plot showing the multivariate antibody profiles across groups. Each dot represents one animal (n=12 independent animals) and the color of the dot indicates the group. Red lines reflect median responses.

Extended Data Figure 4.. Systems serology immune correlates.

(a) The heat map shows the Spearman correlations between antibody features and peak sgRNA in BAL and NS (*q-value < 0.05, **q-value < 0.01 with q-value obtained by Benjamini-Hochberg correction for multiple testing). (b) Relations between the top four antibody features (n=12 independent animals) that are highest correlated to peak viral loads in BAL (upper row) and NS (bottom row). Red lines indicate fitted exponential curves. (c) The radar plots depict the mean percentile of each antibody feature for protected and non-protected animals. The size and color intensity of the wedges indicate the mean percentile of the feature for the corresponding group (antibody subclass, red; FcR binding, blue; effector function, green).

Extended Data Figure 5.. Decline of NAb titers in convalescent rhesus macaques following SARS-CoV-2 infection.

Log₁₀ pseudovirus NAb titers in the 9 re-challenged macaques used as donors in the adoptive transfer study and in the 10 macaques used in the CD8 depletion study at week 4 and week 7 following SARS-CoV-2 infection. Red lines reflect median values. P values reflect two-sided Mann-Whitney tests.

Extended Data Figure 6.. CD8+ cells following CD8 depletion and SARS-CoV-2 re-challenge.

Rhesus macaques were infected with SARS-CoV-2 and received 50 mg/kg anti-CD8α mAb, anti-CD8β mAb, or sham mAb at week 7, reflecting day −3 relative to re-challenge. On day 0, all animals were re-challenged with 10⁵ TCID50 SARS-CoV-2. Total CD8+ cells, CD8+CD3+ cells, and CD8+CD3- cells per ul peripheral blood are shown. Days following challenge is shown on the x-axis. Red lines reflect median values.

Extended Data Figure 7.. CD8+ T cell responses following CD8 depletion and SARS-CoV-2 re-challenge.

(a) IFN-γ S-specific CD8+ T cell responses assessed by intracellular cytokine staining in non-depleted (Sham) and CD8 depleted (anti-CD8α) rhesus macaques prior to and following SARS-CoV-2 re-challenge. Days following re-challenge is shown on the x-axis. Red lines reflect median values. P values reflect two-sided Mann-Whitney tests. (b) IFN-γ, TNF-α, and IL-2 S-specific CD8+ T cell responses in non-depleted (Sham) animals prior to re-challenge (n=5 independent animals). Red lines reflect median responses.

Extended Data Figure 8.. CD4+ T cell responses following CD8 depletion and SARS-CoV-2 re-challenge.

IFN-γ S-specific CD4+ T cell responses assessed by intracellular cytokine staining in non-depleted (Sham) and CD8 depleted (anti-CD8α) rhesus macaques prior to and following SARS-CoV-2 re-challenge. Days following re-challenge is shown on the x-axis. Red lines reflect median values.

Extended Data Figure 9.. NAb responses following CD8 depletion and SARS-CoV-2 re-challenge.

Log₁₀ pseudovirus NAb titers in non-depleted (Sham) and CD8 depleted (anti-CD8α) rhesus macaques prior to and following SARS-CoV-2 re-challenge. Days following re-challenge is shown on the x-axis.

Figure 1.. Viral loads following prophylactic adoptive transfer of IgG prior to SARS-CoV-2 challenge.

Rhesus macaques were challenged by the intranasal and intratracheal routes with 10⁵ TCID50 SARS-CoV-2. (a) Log₁₀ sgRNA copies/ml (limit of quantification 50 copies/ml) were assessed in bronchoalveolar lavage (BAL) following challenge. (b) Log₁₀ sgRNA copies/swab (limit of quantification 50 copies/swab) were assessed in nasal swabs (NS) following challenge. Days following challenge is shown on the x-axis. Red lines reflect median values. The number of animals is denoted in each panel and the median line overlaps with data lines.

Figure 2.. Regression analysis of antibody threshold for protection.

Differences in (a) pseudovirus NAb titers, (b) RBD ELISA titer, and (c) S (FLED) ELISA titer between protected and not protected animals. Animals were considered to be protected if they had no detectable virus in bronchoalveolar lavage (BAL, n = 5 protected animals, left), nasal swab (NS, n = 5 protected animals, middle) or in both (n = 4 protected animals, right). The threshold titers are indicated as horizontal lines and were obtained by logistic regression for scenarios where the groups were not completely separable using the corresponding titer (95% predicted probability of protection, solid lines), or represent the mean between the highest and lowest titers of not protected and protected animals, respectively (dotted lines). In the box plots, whiskers indicate range, and boxes indicates 25^th to 75^th percentile. Relationship between (d) pseudovirus NAb titers, (e) RBD ELISA titer, and (f) S (FLED) ELISA titer and peak viral loads in BAL (left) and NS (right). Red lines indicate fitted exponential curves.

Figure 3.. Viral loads following therapeutic adoptive transfer of IgG after SARS-CoV-2 challenge.

Rhesus macaques were challenged by the intranasal and intratracheal routes with 10⁵ TCID50 SARS-CoV-2 on day 0 and then received intravenous infusion of 250 mg/kg, 25 mg/kg, or 0 mg/kg purified SARS-CoV-2 IgG on day 1. Log₁₀ sgRNA copies/ml (limit of quantification 50 copies/ml) were assessed in bronchoalveolar lavage (BAL) and log₁₀ sgRNA copies/swab (limit of quantification 50 copies/swab) were assessed in nasal swabs (NS) following challenge. Days following challenge is shown on the x-axis. Red lines reflect median values. Arrows represent the day of IgG infusion. The number of animals is denoted in each panel and the median line overlaps with data lines.

Figure 4.. Viral loads following CD8 depletion and SARS-CoV-2 re-challenge.

Rhesus macaques were infected with SARS-CoV-2 and received 50 mg/kg anti-CD8α mAb, anti-CD8β mAb, or sham mAb at week 7, reflecting day −3 relative to re-challenge. On day 0, all animals were re-challenged with 10⁵ TCID50 SARS-CoV-2. (a) Log₁₀ sgRNA copies/ml (limit of quantification 50 copies/ml) were assessed in bronchoalveolar lavage (BAL) following re-challenge. (b) Log₁₀ sgRNA copies/swab (limit of quantification 50 copies/swab) were assessed in nasal swabs (NS) following re-challenge. Days following challenge is shown on the x-axis. (c) Comparison of peak log sgRNA in NS following re-challenge in sham and anti-CD8 groups. Peak log sgRNA in NS from naïve animals following primary challenge from the adoptive transfer study are shown for comparison. Red lines reflect median values. The number of animals is denoted in each panel (Sham n=5, Anti-CD8 n=8, Naïve n=3 independent animals). P values reflects two-sided Mann-Whitney tests.