SARS-CoV-2 Infection of Rhesus Macaques Treated Early with Human COVID-19 Convalescent Plasma

Abstract

Human clinical studies investigating use of convalescent plasma (CP) for treatment of coronavirus disease 2019 (COVID-19) have produced conflicting results. Outcomes in these studies may vary at least partly due to different timing of CP administration relative to symptom onset. The mechanisms of action of CP include neutralizing antibodies but may extend beyond virus neutralization to include normalization of blood clotting and dampening of inflammation. Unresolved questions include the minimum therapeutic titer in the CP units or CP recipient as well as the optimal timing of administration. Here, we show that treatment of macaques with CP within 24 h of infection does not reduce viral shedding in nasal or lung secretions compared to controls and does not detectably improve any clinical endpoint. We also demonstrate that CP administration does not impact viral sequence diversity in vivo, although the selection of a viral sequence variant in both macaques receiving normal human plasma was suggestive of immune pressure. Our results suggest that CP, administered to medium titers, has limited efficacy, even when given very early after infection. Our findings also contribute information important for the continued development of the nonhuman primate model of COVID-19. These results should inform interpretation of clinical studies of CP in addition to providing insights useful for developing other passive immunotherapies and vaccine strategies. IMPORTANCE Antiviral treatment options for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain very limited. One treatment that was explored beginning early in the pandemic (and that is likely to be tested early in future pandemics) is plasma collected from people who have recovered from coronavirus disease 2019 (COVID-19), known as convalescent plasma (CP). We tested if CP reduces viral shedding or disease in a nonhuman primate model. Our results demonstrate that administration of CP 1 day after SARS-CoV-2 infection had no significant impact on viral loads, clinical disease, or sequence diversity, although treatment with normal human plasma resulted in selection of a specific viral variant. Our results demonstrate that passive immunization with CP, even during early infection, provided no significant benefit in a nonhuman primate model of SARS-CoV-2 infection.

Keywords: COVID-19; SARS-CoV-2; animal models of infectious diseases; convalescent plasma; microbial pathogenesis; nonhuman primate; passive immunization; virology.

FIG 1

Impact of CP treatment on viral RNA and clinical outcomes. (A) Study design. (B, C) Quantitative PCR analysis of total vRNA, full-length vRNA, and subgenomic RNA (sgRNA) in nasal lavages (B) and tracheal aspirates (C) from individual animals over time. Graphs show mean of four independent qPCRs for each RNA target. (D) Median sgRNA levels in nasal lavages and tracheal aspirates of each experimental group over time. The dashed line indicates limit of detection. (E) Levels of infectious virus in tracheal aspirates as determined by plaque assay. Right graph shows the correlation between full-length vRNA (gRNA) detected by qPCR and viral plaque counts in these samples. (Spearman correlation r = 0.86, P ≤ 0.001). (F) Clinical outcomes, including total clinical scores, blood oxygen content (% oxygen saturation [SpO₂]), and total radiographic (Rad.) scores. Nx indicates those samples collected at the time of necropsy. Horizontal lines on scatterplots show medians; n = 2 (CP treatment), n = 2 (NP treatment), and n = 4 (not treated [NT]).

FIG 2

Antibody levels to SARS-CoV-2 in CP and plasma of animals. (A) Binding antibody levels to the viral nucleocapsid, spike, and RBD antigens in the CP pool and NP pool used for transfusion, a rhesus macaque anti-SARS-CoV-2 antibody positive plasma pool positive control, and an NHP monoclonal antibody to SARS-CoV-2 spike (NHP spike MAb). (B) Total S1 antibody levels in the plasma of each animal over the course of the study using an antigen sandwich assay. (C) Antibody levels to nucleocapsid, spike, and RBD in the plasma of each animal over the course of the study using a multiplex assay. (D) Pseudovirus 50% neutralization titer (50% NT) levels in the plasma of each animal over the course of the study.

FIG 3

CD8 T-cell responses to SARS-CoV-2. (A) SARS-CoV-2-specific CD8 T cell responses in blood collected at necropsy. (B) Relationship between full-length vRNA (gRNA) copies (cps) in nasal lavages and nucleocapsid (NC)-specific CD8 T-cell responses at necropsy. (C) Relationship between antigen-specific antibody responses and corresponding CD8 T-cell responses assessed at necropsy. Results of a Spearman correlation are shown; IFN-γ, interferon-γ.

FIG 4

Impact of CP treatment on viral replication and evolution. (A) Comparison of allele frequencies determined using amplicon (ARTIC) and metagenomic sequencing (mNGS) methods. The square of the Pearson correlation coefficient values are shown. (B) Impact of treatment on viral sequence nucleotide diversity in nasal lavages and tracheal aspirates was assessed by ARTIC for comparison with the virus stock used for inoculation. A box plot of pairwise nucleotide diversity shows the median and the 1st and 3rd quartiles. (C) Impact of treatment on viral allele frequencies was assessed in comparison to the virus stock. Asterisks indicate expansion of a H655Y substitution in the viral spike protein. (D) Heat map showing antibody cross-reactivity to other related viruses in the human plasma pools and in plasma samples from study animals treated with NP and CP.