Absence of pathogenic viruses in COVID‐19 convalescent plasma

Abstract

Background

It is important to maintain the safety of blood products by avoiding the transfusion of units with known and novel viral pathogens. It is unknown whether COVID‐19 convalescent plasma (CCP) may contain pathogenic viruses (either newly acquired or reactivated) that are not routinely screened for by blood centers.

Methods

The DNA virome was characterized in potential CCP donors (n = 30) using viral genome specific PCR primers to identify DNA plasma virome members of the Herpesviridae [Epstein Barr Virus (EBV), cytomegalovirus (CMV), human herpesvirus 6A/B, human herpesvirus 7] and Anelloviridae [Torque teno viruses (TTV), Torque teno mini viruses (TTMV), and Torque teno midi viruses (TTMDV)] families. In addition, the RNA plasma virome was characterized using unbiased metagenomic sequencing. Sequencing was done on a HiSeq2500 using high output mode with a read length of 2X100 bp. The sequencing reads were taxonomically classified using Kraken2. CMV and EBV seroprevalence were evaluated using a chemiluminescent immunoassay.

Results

TTV and TTMDV were detected in 12 (40%) and 4 (13%) of the 30 study participants, respectively; TTMDV was always associated with infection with TTV. We did not observe TTMV DNAemia. Despite CMV and EBV seroprevalences of 33.3% and 93.3%, respectively, we did not detect Herpesviridae DNA among the study participants. Metagenomic sequencing did not reveal any human RNA viruses in CCP, including no evidence of circulating SARS‐CoV‐2.

Discussion

There was no evidence of pathogenic viruses, whether newly acquired or reactivated, in CCP despite the presence of non‐pathogenic Anelloviridae. These results confirm the growing safety data supporting CCP.

1. INTRODUCTION

The novel pathogen SARS‐CoV‐2 has infected over 500 million people globally, causing the clinical disease called COVID‐19 and resulting in over 6 million deaths. COVID‐19 convalescent plasma (CCP) became one of the most common therapies early in the pandemic and numerous randomized trials were conducted. ¹ , ² , ³ , ⁴ , ⁵ , ⁶ , ⁷ , ⁸ The AABB reviewed more than 30 randomized trials and published clinical practice guidelines that recommend CCP for outpatients at high risk of disease progression and hospitalized patients who are immunosuppressed or do not have SARS‐CoV‐2 antibodies detected at admission. ⁹ Similar to standard plasma, CCP is screened for human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), and other transfusion transmitted infections. However, emerging viruses or reactivated viruses found in blood could be present in CCP, a blood product obtained from individuals recovering from SARS‐CoV‐2.

Healthy humans have a plasma virome that can undergo changes in composition in altered immunological states or physiology. ¹⁰ Individuals with resolved COVID‐19 have different cytokine and chemokine profiles ¹¹ ; IL‐6 is higher among hospitalized CCP donors when compared to non‐hospitalized donors. Studies have also revealed elevated IL‐6 concentrations to be associated with SARS‐CoV‐2 serum RNAemia. ¹² These changes in cytokine distribution can also lead to herpesvirus reactivation. ¹³ Similarly, while DNA metagenomic sequencing has revealed Anelloviridae families in blood in the absence of disease, members of the family have been suggested as indicators of immune health in transplant settings. ¹⁴ There are no data on the plasma virome composition of CCP, which could have clear implications for transfusion. Hence, we conducted an intensive study of CCP using metagenomics and targeted PCR to characterize the plasma virome.

2. STUDY DESIGN AND METHODS

2.1. Study participants

This cross‐sectional study evaluated potential CCP donors (n = 30) collected between April and May 2020 during the alpha variant phase prior to vaccination who had a confirmed positive nasopharyngeal SARS‐CoV‐2 PCR test, as previously described. ¹⁵ , ¹⁶ Among the first 126 CCP donors at a single center, samples were grouped according to anti‐SARS‐CoV‐2 IgG antibody titers and 10 samples were randomly selected from each tertile. All potential donors met standard criteria for blood donation (at least 18 years of age, had never been diagnosed with HIV or HBV, were not pregnant, etc.). The selected samples were collected a median of 43 days (IQR, 37.5–48) after the initial positive PCR test. These samples were also negative for standard transfusion transmitted infections (TTIs) screened by the blood collection centers. Plasma was separated from whole blood within 12 h and stored in aliquots at −80°C.

This study was approved by the Johns Hopkins University School of Medicine Institutional Review Board. All samples were de‐identified prior to laboratory testing, and all participants provided informed consent.

2.2. Serology testing

The plasma samples were evaluated for antibodies to human cytomegalovirus (CMV) and Epstein–Barr virus (EBV) viral capsid antigen p18 synthetic peptide using the DiaSorin chemiluminescent immunoassay on the LIASON Analyzer (Stillwater, MN) in a CLIA‐certified laboratory.

2.3. Sample preparation for next generation sequencing (NGS) and PCR

1. Nucleic Acid Extraction: Extraction was performed as previously described. ¹⁷ Pre‐extraction steps done on plasma to minimize non‐viral sequences included spinning the samples at 1600 g for 15 min at 4°C followed by filtration using 0.2 μM syringe filters (Whatman, Amersham Place, UK). This was followed by separate extraction of DNA and RNA from 200 uL of plasma using ZR‐Duet DNA/RNA miniprep kit (Zymo Research, USA).

2. Next Generation Sequencing (NGS) for RNA viruses: The NGS library constructions were performed using Ovation® SoLo® Single Cell RNA‐Seq system. Libraries were assessed prior to sequencing using a bioanalyzer to visualize fragment size distribution and a Kappa PCR library kit (Roche, IN) to assess sample concentration. All samples were pooled in a single lane prior to sequencing on a HISeq2500 (Illumina). Sequencing of pooled samples was done in technical replicates (two lanes) using high output mode with a read length of 2X100 bp and 500 million reads/lane.

3. Targeted PCR for DNA viruses: We used a targeted PCR approach for DNA viruses, given past reports that the DNA virome is largely composed of members of the Herpesviridae and Anelloviridae families. ¹⁸ Plasma Herpesviridae testing was done to identify HHV4 (EBV), HHV5 (CMV), HHV6A/B, and HHV7. Similarly, specific Anelloviridae PCRs were used to detect torque teno viruses (TTV), torque teno midi viruses (TTMDV), and torque teno mini viruses (TTMV). ¹⁹ , ²⁰

Herpesviridae testing for study participants was done using previously published primers on 3 μl of extracted DNA. ²¹ Prior to testing, the lower limit of detection (LOD) of each of the Herpesviridae PCRs was determined using custom‐designed gBlock (IDT, USA) and, when available, AcroMetrix panels (Thermo Fisher, USA). Briefly, a 273 bp double‐stranded gBlock gene fragment from IDT spanning the primer binding regions for each of the included Betaherpesvirinae (HHV5[CMV], HHV6A/B, and HHV7) were designed. Following determination of the number of molecules in the stock, half log₁₀ serial dilutions spanning concentrations from 9 log₁₀ to 3.5 log₁₀ copies/mL were tested in duplicates using virus specific primers. The dilutions were made in herpesvirus negative plasma to also assess possible PCR inhibition. We further verified CMV qPCR LOD using an AcroMetrix CMVtc panel. The AcroMetrix EBV plasma panel was used to determine the LOD of EBV qPCR. The concentration for the EBV plasma panel ranged from 6 log₁₀ to 2 log₁₀ IU/ml. The LOD was determined as the lowest concentration at which both duplicates were detected by PCR.

For amplification of members of the Anelloviridae family, rolling circle amplification using TempliPhi amplification kit (Millipore Sigma, USA) was done to enrich for circular templates prior to PCR using specific primers. ²⁰

Limits of detection: Based on the commercially available AcroMetrix standards, the LOD for EBV and CMV were 1000 and 3000 IU/mL, respectively. Using gblocks, the LOD of HHV6A/B, and HHV7 assays were observed to be 3200 copies/mL.

Analysis of NGS data: Kraken2 was used for metagenomics read classification with a custom database containing 10,109 viral sequences and human genome sequences built from NCBI RefSeq. ²² Specificity and sensitivity of our approach were assessed by testing different in silico datasets obtained from Sequence Read Archive ²³ against the custom database. The in‐silico datasets contained human sequences, SARS‐CoV‐2 sequences, and cell line ‐omics data. For the human sequences and SARS‐CoV‐2 sequences dataset, the base calling error rates ranged from 1% to 25%. The cell line omics dataset contained whole genome sequences of Genome in a Bottle (n = 5), ²⁴ whole exome sequences of DSMZ cell lines (n = 10), and RNA seq data from melanoma cells (n = 5). We observed in a database containing 100% human samples and a base calling error rate of 10% that the false positivity rate (assignment of human reads to a virus) was ~0.0001%. The inclusion of these cell line omics datasets confirmed the accuracy of the approach as many of the cell lines were positive for EBV, and a follow‐up search in the literature revealed that they were indeed EBV‐transformed cell lines (e.g., the Genome in a Bottle cell lines). Based on the results, Kraken2 performance was optimized for sensitivity and specificity using a score threshold of 0.05. For the study samples, quality trimming of the study paired‐end read fragments was done using FASTP. ²⁵

3. RESULTS

Among 30 potential CCP donors, the median age of these participants was 40 (IQR: 32–55) years, with the majority of the participants being male (60.0%) (Table 1). Twenty‐four participants identified themselves as White, three as Asians, one as Hispanic, and two as mixed/unknown. CCP samples were collected a median of 43 days (IQR:38–48) after the initial positive SARS‐CoV‐2 PCR test. Two of the participants had been hospitalized due to COVID‐19. The seroprevalences of CMV and EBV were 33.3% and 93.3%, respectively.

TABLE 1.

Observed virome composition of the study participants

Subject ID	Age (years)	Gender	Race/ethnicity	Days since positive SARS‐CoV‐2 PCR swab	CMV seroprevalence	EBV seroprevalence	Virus(es) detected
Vir‐1	68	Female	Mixed/other/unknown	30	+	+	TTV
Vir‐2	28	Male	Asian	37	−	+	ND
Vir‐3	32	Male	Asian	41	−	+	TTV
Vir‐4	40	Male	White	37	+	+	ND
Vir‐5	64	Male	White	40	−	+	TTV, TTMDV
Vir‐6	61	Female	White	27	−	+	ND
Vir‐7	50	Female	White	33	−	+	ND
Vir‐8	38	Male	White	33	+	+	ND
Vir‐9	19	Male	White	43	−	+	TTV
Vir‐10	28	Male	White	50	−	−	ND
Vir‐11	40	Male	White	52	−	+	TTV
Vir‐12	27	Female	White	39	−	+	TTV
Vir‐13	63	Male	White	47	−	+	ND
Vir‐14	61	Female	White	42	+	+	ND
Vir‐15	43	Female	White	50	+	+	ND
Vir‐16	53	Male	Asian	48	−	−	ND
Vir‐17	43	Male	White	41	−	+	ND
Vir‐18	48	Male	White	35	+	+	ND
Vir‐19	19	Female	White	44	−	+	ND
Vir‐20	37	Male	White	48	+	+	TTV
Vir‐21	28	Female	White	53	+	+	TTV, TTMDV
Vir‐22	29	Male	Mixed/other/unknown	43	+	+	ND
Vir‐23	37	Male	White	39	+	+	TTV, TTMDV
Vir‐24	77	Female	White	51	−	+	ND
Vir‐25	56	Male	White	37	−	+	TTV
Vir‐26	31	Female	White	47	−	+	TTV, TTMDV
Vir‐27	50	Male	White	47	−	+	ND
Vir‐28	32	Male	White	57	−	+	ND
Vir‐29	40	Female	White	62	−	+	ND
Vir‐30	62	Female	Hispanic	42	−	+	TTV

Abbreviations: −, negative; +, positive; CMV, cytomegalovirus; EBV, epstein barr virus; ND, none detected; TTMDV, torque teno midi virus; TTV, torque teno virus.

To assess the possibility of transmitted pathogenic viruses that are not routinely tested by blood centers, DNA and RNA were extracted from plasma samples. To address RNA viruses, we used an unbiased metagenomic sequencing approach on RNA extractions from plasma, effectively characterizing the entire plasma RNA virome. We additionally addressed the possibility of prolonged SARS‐CoV‐2 shedding by populating the reference database with SARS‐CoV‐2 sequences. ²⁶ Metagenomic sequencing did not reveal any human RNA viruses in CCP, including no evidence of SARS‐CoV‐2 RNAemia.

We used a targeted PCR strategy to address the majority of bloodborne DNA viruses that are not routinely tested for by blood collection centers. We interrogated each CCP sample for several members of the Herpesviridae family, testing for EBV, CMV, HHV6A/B, and HHV7 using distinct PCR assays. None of the individual Herpesviridae PCRs were positive, indicating that neither new acquisition nor reactivation of these viruses was relevant in the cohort (Table 1). This finding is notable since SARS‐CoV‐2 infection has been associated with reactivation of members of the Herpesviridae family in the course of COVID‐19 and its convalescence. ¹³

We also performed targeted PCR for members of the Anelloviridae family since they are reportedly abundant in plasma and can be underestimated by NGS methods given their circular genomes. ¹⁷ We found Anelloviridae viremia in 40% (n = 12) of the study participants. All 12 participants with Anelloviridae infection had TTV plasma DNAemia. In addition, among these 12 participants, four were co‐infected with TTMDV. We found no TTMV DNAemia.

4. DISCUSSION

CCP has been used to treat COVID‐19 and has been demonstrated to be effective when high‐titer units are provided early in infection, especially among the immunocompromised. ⁹ Although safety measures during clinical trials and observational studies are comprehensive, ⁶ , ²⁷ they do not fully address asymptomatic transmission of plasma virome. By intensively studying plasma from a cohort of potential CCP donors, we demonstrate that this risk is negligible. We address the thoroughness of our approach in two ways, noting our limits of detection and the ability to detect non‐pathogenic bloodborne viruses, Anelloviridae, in 40% of participants.

Healthy humans have a plasma virome that can undergo changes in composition in altered immunological states or physiology. In blood, DNA metagenomic sequencing has revealed viremia with members of the Herpesviridae family in the absence of disease, including HHV‐7 in 20% of individuals and EBV in 14% of individuals. ¹⁸ Metagenomic approaches done on pre‐ and post‐transfusion recipients have also revealed the presence of novel RNA viral pathogens and non‐pathogens. ²⁸ In the study of over 8000 people, Anelloviridae were identified in 9% of the study participants when using a metagenomic approach, while a PCR based approach done on over 1000 participants identified a prevalence of 65%. ¹⁸ , ²⁹ Though no disease has been associated with this viral family, recent studies among solid organ transplants have indicated that they can serve as surrogate markers of transplant outcomes with lower levels of plasma associated with graft rejection compared to people with higher levels. ¹⁴ It is unclear what underlies the inverse association between Anelloviridae prevalence and transplant outcomes, nor whether these are transmitted with the transplant. Reports of transmission in the setting of blood transfusion have been reported for Anelloviridae. ³⁰ However, transmission of Anelloviridae was not evaluated in this study.

The strengths of this study include the multi‐pronged rigorous testing and the well‐characterized cohort. However, there are limitations associated with this study. One limitation includes the lack of samples from the same participants prior to their infection with SARS‐CoV‐2. However, the existing data from asymptomatic individuals allowed for cross‐sectional comparison between groups. The number of participants in this study was limited compared to standard donor TTI studies, but the techniques were much more intensive to detect a broad range of potential viruses. In addition, sampling after SARS‐CoV‐2 positivity occurred in a relatively narrow interval and prior to the FDA mandating high‐titer CCP. The lack of detection of RNA viruses in human plasma could be attributed to the lower sensitivity of metagenomic approaches compared to more targeted approaches like PCR. We have observed metagenomic approaches to be sensitive for detection of viral loads >10,000 copies/ml. ¹⁷ So, although the techniques used in this study could have missed low levels of RNAemia, it is unlikely to have missed acute RNA viral infections. Importantly, we did not detect SARS‐CoV‐2 RNAemia in any of the participants, supporting the potential absence of a long‐term SARS‐CoV‐2 reservoir in convalescing patients.

In summary, we found no evidence of pathogenic RNA or DNA viruses in CCP despite the abundance of Anelloviridae DNA that we found in blood. This study adds further strength to the safety of CCP as a valuable therapeutic for persons at high risk for progression to severe disease for whom few other options exist.

FUNDING INFORMATION

This work was supported in part by the National Institutes of Health (R01AI120938, R01AI120938S1, R01AI128779, and R01DK131926 to Aaron A. R. Tobian; R01AI05273, R01HL059842, and R01AI152078 to Arturo Casadevall; R01AI138810 for Ashwin Balagopal; K23HL151826 to Evan M. Bloch and R01DA01380 and R21DA053145 for Abraham J. Kandathil), National Institute of Allergy and Infectious Diseases Division of Intramural Research (Andrew D. Redd, Thomas C. Quinn), Bloomberg Philanthropies (Arturo Casadevall), and Department of Defense (W911QY2090012 to David Sullivan). The funders had no role in study design, data analysis, decision to publish, or preparation of the manuscript.

CONFLICT OF INTEREST

Evan M. Bloch reports personal fees and non‐financial support from Terumo BCT, personal fees and non‐financial support from Grifols Diagnostics Solutions, outside of the submitted work; Evan M. Bloch is a member of the United States Food and Drug Administration (FDA) Blood Products Advisory Committee. Any views or opinions that are expressed in this manuscript are that of the author's, based on his own scientific expertise and professional judgment; they do not necessarily represent the views of either the Blood Products Advisory Committee or the formal position of FDA, and also do not bind or otherwise obligate or commit either Advisory Committee or the Agency to the views expressed. All other authors declare no conflicts of interest.

Kandathil AJ, Benner SE, Bloch EM, Shrestha R, Ajayi O, Zhu X, et al. Absence of pathogenic viruses in COVID‐19 convalescent plasma. Transfusion. 2023;63(1):23–29. 10.1111/trf.17168