Early post-infection treatment of SARS-CoV-2 infected macaques with human convalescent plasma with high neutralizing activity reduces lung inflammation

Abstract

Early in the SARS-CoV-2 pandemic, there was a high level of optimism based on observational studies and small controlled trials that treating hospitalized patients with convalescent plasma from COVID-19 survivors (CCP) would be an important immunotherapy. However, as more data from controlled trials became available, the results became disappointing, with at best moderate evidence of efficacy when CCP with high titers of neutralizing antibodies was used early in infection. To better understand the potential therapeutic efficacy of CCP, and to further validate SARS-CoV-2 infection of macaques as a reliable animal model for testing such strategies, we inoculated 12 adult rhesus macaques with SARS-CoV-2 by intratracheal and intranasal routes. One day later, 8 animals were infused with pooled human CCP with a high titer of neutralizing antibodies (RVPN NT ₅₀ value of 3,003), while 4 control animals received normal human plasma. Animals were monitored for 7 days. Animals treated with CCP had detectable levels of antiviral antibodies after infusion. In comparison to the control animals, they had similar levels of virus replication in the upper and lower respiratory tract, but had significantly reduced interstitial pneumonia, as measured by comprehensive lung histology. By highlighting strengths and weaknesses, data of this study can help to further optimize nonhuman primate models to provide proof-of-concept of intervention strategies, and guide the future use of convalescent plasma against SARS-CoV-2 and potentially other newly emerging respiratory viruses.

Author summary

The results of treating SARS-CoV-2 infected hospitalized patients with COVID-19 convalescent plasma (CCP), collected from survivors of natural infection, have been disappointing. The available data from various studies indicate at best moderate clinical benefits only when CCP with high titer of neutralizing antibodies was infused early in infection. The macaque model of SARS-CoV-2 infection can be useful to gain further insights in the value of CCP therapy. In this study, animals were infected with SARS-CoV-2 and the next day, were infused with pooled human convalescent plasma, selected to have a very high titer of neutralizing antibodies. While administration of CCP did not result in a detectable reduction in virus replication in the respiratory tract, it significantly reduced lung inflammation. These data, combined with the results of monoclonal antibody studies, emphasize the need to use products with high titers of neutralizing antibodies, and guide the future development of CCP-based therapies.

SciScore for 10.1101/2021.09.01.458520: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Ethics	IACUC: Ethics Statement: The study was approved by the Institutional Animal Care and Use Committee of the University of California, Davis (study protocol 21735). Euthanasia Agents: Euthanasia and evaluation of pathology: All animals were euthanized at day 7 after infection with an overdose of pentobarbital and subjected to a full necropsy under BSL-3 conditions.
Sex as a biological variable	Each of the 2 study groups had equal sex distribution (half males, half females) and similar age and weight (table S4).
Randomization	Using a 1.5 mm spaced grid, 25 randomly selected x40 fields from each slide were evaluated for the severity of the interstitial inflammation (mostly mononuclear cells sometimes with neutrophils).
Blinding	Clinical observations and sample collections: Daily cage-side clinical monitoring was performed by a veterinarian who was blinded to the group assignments, and included recording of responsiveness, discharge, respiratory rate and character, evidence of coughing/sneezing, appetite, stool quality.
Power Analysis	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
The VITROS® Anti-SARS-CoV-2 Total Test (which measures total antibodies to the SARS-CoV-2 virus (IgG, IgM, IgA and other isotypes) was performed following the manufacturer’s instructions [46].	Anti-SARS-CoV-2 suggested: None SARS-CoV-2 virus ( IgG suggested: None IgG suggested: None IgM suggested: None IgA suggested: None
The S1 antigens coated on the assay wells bind S1 antibodies from human serum which, in turn, bind to a secondary HRP-labeled S1 antigen in the conjugate reagent forming a sandwich.	S1 suggested: None
Fresh whole blood (150µl) from each study animal was stained with a panel of fluorophore conjugated monoclonal antibodies against the following surface antigens: CD3, CD4, CD8, CD11c, CD14, CD16, CD20, CD28, CD66, CD95, CD123, HLA-DR, and PD1 for 30 minutes at room temperature.	CD3 suggested: (GeneTex Cat# GTX13464, RRID:AB_369206) CD4 suggested: (Nanostring Cat# 121300104, RRID:AB_2893077) CD8 suggested: (BD Biosciences Cat# 557939, RRID:AB_2802162) CD11c suggested: (Nanostring Cat# 121300104, RRID:AB_2893077) CD14 suggested: (Creative Diagnostics Cat# CABT-45572MH, RRID:AB_2528792) CD16 suggested: (BD Biosciences Cat# 557939, RRID:AB_2802162) CD20 suggested: None CD28 suggested: None CD66 suggested: None CD95 suggested: None CD123 suggested: None HLA-DR suggested: None PD1 suggested: None
Experimental Models: Cell Lines
Sentences	Resources
The resulting mix was incubated and then added to 96-well plates containing ACE2 and TMPRSS2 expressing HEK293T cells.	HEK293T suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
In addition, the donor plasma pools and samples from the treated animals were also assessed for antibody responses to SARS-CoV-2, SARS-CoV, middle east respiratory syndrome coronavirus (MERS-CoV), various seasonal human coronaviruses (HCoV-229E, HCoV-NL63, HCoV-HKU1, and HCoV-OC43), influenza, and several other common-cold viruses by using a coronavirus antigen microarray (CoVAM) described earlier [18, 47].	HCoV-NL63 suggested: RRID:CVCL_RW88)
Software and Algorithms
Sentences	Resources
GraphPad Software, San Diego, CA).	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)
Serum biochemistry: Biochemistry analysis on serum samples was performed using Piccolo® BioChemistry Plus disks, that were run on the Piccolo® Xpress Chemistry Analyzer (Abbott), according to the manufacturer’s instructions.	Piccolo® BioChemistry suggested: None Abbott suggested: (Abbott, RRID:SCR_010477)
Compensation, gating, and analysis were performed using FlowJo (Versions 9 and 10).	FlowJo suggested: (FlowJo, RRID:SCR_008520)
Statistical analyses: Statistical analyses were performed using Prism version 9 (GraphPad), with selection of the test as outlined in the results.	Prism suggested: (PRISM, RRID:SCR_005375)

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

The current study provides insights on the efficacy and limitations of CCP therapy against SARS-CoV-2 replication and COVID-19 disease, as well as the opportunities and challenges associated with the use of a nonhuman primate model in testing passive immunotherapy strategies. We demonstrate that administration of a pooled human CCP with high titer of neutralizing and spike-binding antibodies, administered one day after virus inoculation, conferred therapeutic benefits to SARS-CoV-2 infected macaques in terms of reduced interstitial pneumonia, despite no detectable effect on reducing virus replication. The lack of a detectable effect of the CCP on RNA levels in mucosal samples is likely multifactorial, with insufficient antiviral activity as the primary explanation, but influenced by additional experimental factors. Although we used a CCP with high neutralizing activity in vitro, the antibodies became diluted so much upon transfusion that by the time they reached mucosal sites, their concentration was probably too low to have a drastic impact on reducing virus replication in vivo. In this context, it is important to note that we inoculated animals with a very high dose of SARS-CoV-2, to induce rapid wide-spread infection of upper and respiratory tract, with peak virus replication occurring within the first 1-2 days. Having high levels of viral replication at the time of CCP administration sets a high bar to detect efficacy, especially as it takes time for passively infused ant...

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

Results from rtransparent:

Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

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Early post-infection treatment of SARS-CoV-2 infected macaques with human convalescent plasma with high neutralizing activity reduces lung inflammation

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