Detection of antibodies against SARS-CoV-2 both in plasma pools for fractionation and in commercial intravenous immunoglobulins produced from plasma collected in Italy during the pandemic

Abstract

Background

We investigated the presence of anti-SARS-CoV-2 antibodies in Italian plasma pools and intravenous immunoglobulins sent to our Institute (Italian National Institute of Health - Istituto Superiore di Sanità) in the context of the Official Control Authority Batch Release. The plasma pools were made up from donations collected in several different Italian regions from May 2017 to October 2020, i.e. in the pre-pandemic and pandemic periods.

Materials and methods

All plasma pools were initially tested for the qualitative detection of anti-SARS-CoV-2 antibodies against the nucleocapsid protein using the Roche Elecsys^® Anti-SARS-CoV-2 test kit. Plasma pools positive for these antibodies were further tested using the Roche Elecsys^® Anti-SARS-CoV-2 S test kit for the quantitative detection of antibodies against SARS-CoV-2 spike receptor binding domain. All plasma pools showing reactivity to these antibodies were tested undiluted for the presence of SARS-CoV-2 RNA using the Grifols Procleix SARS-CoV-2 transcription-mediated amplification assay. Intravenous immunoglobulins were tested using both test kits to determine the presence of anti-SARS-CoV-2 antibodies.

Results

All plasma pools made up from donations collected in the pre-pandemic period were negative for anti-SARS-CoV-2 antibodies against the nucleocapsid protein. Of the plasma pools made up from donations collected from December 2018 to March 2020, only 1 pool out of 68 (1.4%), that was made up from donations from the Lombardy region, was reactive for these antibodies. Interestingly, 105 out of 174 (60.3%) of the plasma pools made up from donations collected from November 2018 to October 2020 showed the presence of these antibodies. All plasma pools positive for these antibodies were tested for antibodies against SARS-CoV-2 spike receptor binding domain and were confirmed positive.

Discussion

None of these plasma pools tested were reactive for SARS-CoV-2 RNA. In the case of intravenous immunoglobulins, 20 out of 25 (80%) batches showed the presence of both anti-SARS-CoV-2 antibodies, reflecting the concentration in the plasma pools used for their production.

INTRODUCTION

The first cases of severe acute respiratory syndrome (SARS) “viral pneumonia” were reported by the Chinese authorities in Wuhan, the capital of the Hubei province in China, in early December 2019¹. In January 2020, it was determined that the outbreak was caused by a novel coronavirus^2,3. On 11 February 2020, the International Committee on Taxonomy of Viruses announced that the new betacoronavirus, closely related to SARS-CoV, was to be referred to as “SARS-CoV-2”. The same day, the World Health Organization (WHO) announced that the disease caused by the novel coronavirus would be named COVID-19. On 11 March 2020, the WHO declared the COVID-19 outbreak a global pandemic. Since then, SARS-CoV-2 has continued to spread across the world and, as of 16 February 2021, there have been 109 million confirmed infections and 2.4 million deaths reported⁴.

In Italy, the first confirmed cases were reported on 21 February 2020 in the towns of Codogno, in the region of Lombardy, and Vo’ Euganeo, in the Veneto region⁵. Over the following weeks, the pandemic spread quickly across the country, especially in Northern Italy, and COVID-19 diagnosed cases increased exponentially, reaching a daily peak of 6,557 newly confirmed cases on 21 March 2020. Two months later, COVID-19 cases reported in Italy dropped to approximately 600 per day, indicating a decline in the first pandemic wave (March–May 2020). The second wave started in October 2020 after a low incidence transition phase (June–September 2020) and continued until January 2021⁶.

SARS-CoV-2 is primarily transmitted by air and so far there has been no evidence of transmission through transfusion of blood or blood components. As with previous MERS-CoV or SARS-CoV outbreaks, blood RNA levels were detected during secondary complications of COVID-19 and the blood viral load was directly related to clinical disease progression⁷. Since the onset of the COVID-19 pandemic, donations of blood and blood components have continued regularly at the blood establishments (BE) and at the blood collection units (BCU) of blood donor associations and federations, in compliance with national public health prevention measures and national legislative selection criteria to ensure the quality and safety of donations. Furthermore, donors’ medical histories have been subjected to a more thorough investigation to identify at-risk donors. Plasma collected through apheresis (source plasma) or recovered from whole blood donations (recovered plasma) has been sent to manufacturers for the production of plasma-derived medicinal products (PDMPs). Convalescent plasma (CP) has been collected at the BE and BCU from donors who had been infected with SARS-CoV-2 but who showed complete resolution of symptoms before donating and had measurable titres of neutralising antibodies; this CP has been destined to the clinical treatment of COVID-19.

This study investigates the presence of antibodies against SARS-CoV-2 in plasma pools for fractionation in Italy in a defined time frame covering the pre-pandemic and pandemic periods. Since the plasma pools studied were obtained from thousands of donations collected in various Italian Regions from healthy donors, this investigation can help trace the appearance of anti-SARS-CoV-2 antibodies over time in the asymptomatic population, even on a regional scale, allowing trends to be defined, with respect also to cases detected in the Italian population as a whole. In this study, we have also investigated the presence of antibodies against SARS-CoV-2 in intravenous immunoglobulins (IVIG) that are PDMPs obtained through industrial-scale processing of pooled plasma made up from thousands of donations. They contain a broad range of antibody specificities and are thus polyvalent immunoglobulins. The major component of IVIG is the serum IgG fraction consisting mainly of IgG1 and IgG2 subclasses⁸. Traces of IgA and IgM can also be found. We hereby show that commercial IVIG produced from Italian plasma contain detectable antibodies against SARS-CoV-2, with a concentration that is influenced by the different circulation patterns of the virus in the Italian Regions.

MATERIALS AND METHODS

Plasma pools for fractionation

Plasma pools made up from blood and plasma donations collected in 11 Italian Regions, internally organised in consortia, were produced by Kedrion Biopharma (Castelvecchio Pascoli, Barga, Italy). An aliquot of a cryosupernatant sample from each plasma pool to be subjected to ethanol fractionation was collected and tested for virological markers by the manufacturer in compliance with the relevant European Pharmacopoeia monographs. Aliquots were also analysed by the Italian Official Medicine Control Laboratory, the Istituto Superiore di Sanità, in the context of the Official Control Authority Batch Release. The aliquots were stored at −20°C before testing.

Plasma pools were produced between January 2019 and November 2020, with dates of donation collection covering years 2017–2020. These plasma pools can be grouped as follows:

• Period 1, or pre-pandemic, for plasma collected from May 2017 to December 2019;

• Period 2, for plasma collected from December 2018 to March 2020;

• Period 3, for plasma collected from November 2018 to October 2020.

The overlap in the three groups is due to the fact that plasma pools consist of thousands of donations collected in a wide timeframe that can date back to three years from their date of production.

IVIG

A total of 25 batches of 5% IVIG fractionated from plasma collected in Periods 1, 2 and 3 were analysed. The IVIG batches were manufactured from source and recovered plasma.

WHO International Standard for anti-SARS-CoV-2 antibody

The first WHO International Standard (IS) for anti-SARS-CoV-2 immunoglobulin (NIBSC code: 20/136) is a freeze-dried preparation equivalent to 0.25 mL of pooled plasma obtained from 11 individuals who had recovered from SARS-CoV-2 infection. A content of 250 IU/vial, or binding antigen units (BAU), was assigned after a WHO collaborative study⁹.

Serological tests

All plasma pools were initially tested for the qualitative detection of anti-SARS-CoV-2 antibodies using the Elecsys^® Anti-SARS-CoV-2 test kit, an electrochemiluminescence immunoassay by Roche Diagnostics (Basel, Switzerland), on the cobas e411 instrument. The assay is a double-antigen sandwich using recombinant nucleocapsid protein (NP) for the detection of total antibodies (IgA, IgM, and IgG) against SARS-CoV-2 (anti-SARS-CoV-2 NP antibodies). Results are reported as numeric values in the form of a cutoff index (COI; signal sample/cutoff) as well as in the form of a qualitative “non-reactive” (COI <1.0; negative) or “reactive” (COI ≥1.0; positive) result.

Plasma pools positive for anti-SARS-CoV-2 NP antibodies were further tested using the Elecsys^® Anti-SARS-CoV-2 S test kit, recently released by Roche Diagnostics for the quantitative detection of antibodies against SARS-CoV-2 spike receptor binding domain (anti-SARS-CoV-2 S-RBD antibodies). The total antibody content in the sample is expressed as U/mL, traceable to the Roche Diagnostics internal standard for anti-SARS-CoV-2 S. This standard consists of an equimolar mixture of two monoclonal antibodies that bind Spike-1 RBD at two different epitopes; 1 nM of these antibodies correspond to 20 U/mL of the Elecsys^® Anti-SARS-CoV-2 S assay. The cutoff is 0.8 U/mL and the linear range is up to 250 U/mL. Samples with a concentration >250 U/mL can be diluted 1:10 in specimen diluent. Both test kits were used to determine the presence of anti-SARS-CoV-2 antibodies in IVIG.

Validation of Elecsys^® Anti-SARS-CoV-2 for use in plasma pools

The specificity and reliability of the qualitative test kit on plasma pools were evaluated by testing 361 plasma pools made up from donations collected in Period 1; of these plasma pools, 107 originated from Italian plasma and 254 from plasma collected abroad. All plasma pools tested negative for anti-SARS-CoV-2 NP antibodies with a COI mean value of 0.102 (min 0.086; max 0.179).

Adoption of the IU for Elecsys^® Anti-SARS-CoV-2 S

The quantitative test kit was evaluated against the WHO IS in order to convert U/mL into IU/mL. To do this, two independent dilutions obtained from two vials of IS at a final concentration of 100, 50, 25 and 12.5 IU/mL of neutralising antibodies were tested in quadruplicate for a total of eight determinations for each point of the IS. Good linearity was obtained and a mean conversion factor was estimated to be 0.92 in the range 12–100 IU/mL (see Online Supplementary Content, Table SI and Table SII).

Nucleic acid amplification testing

All plasma pools positive for anti-SARS-CoV-2 S-RBD antibodies were tested for the presence of SARS-CoV-2 RNA using the Grifols Procleix^® SARS-CoV-2 transcription-mediated amplification (TMA) assay on the Procleix Panther system (Grifols International SA, Barcelona, Spain). The test has a 95% limit of detection (LOD) of 16.5 copies/mL (95% CI: 12.8–23.6 copies/mL) by probit analysis. Testing was performed according to the manufacturer’s instructions and all plasma pools were tested without dilution.

Statistical analysis

Statistical analysis was performed using one-way ANOVA in order to compare the log¹⁰ COI values obtained from plasma pools in the three collection periods that are reported in Figure 1. Similarly, the log¹⁰ COI values as well as the log¹⁰ IU/mL values obtained across four Italian areas (see Figure 2) were compared. Since the underlying data distributions of both COI and IU/mL values are Log-Normal, the values were log-transformed in order to obtain Normal distributions of data, which is, furthermore, a pre-requisite to perform a parametric statistical method such as ANOVA. In case of significant ANOVA, the pairwise comparisons were carried out using post-hoc tests for multiple comparisons in order to test the significance of the mean difference between two groups (periods or areas). With this aim in view, the False Discovery Rate correction for multiplicity was applied.

Figure 1.

Log₁₀ cutoff index (COI) values obtained from plasma pools tested for the presence of anti-SARS-CoV-2 NP antibodies in the three collection periods were statistically compared using one-way ANOVA

Results show a more than highly significant difference for Period 3 with respect to the other two periods while there was no significant difference between Periods 2 and 1.

Figure 2. Distribution of the log10 COI values (A) and of the log10 IU/mL values (B) observed in various Italian Regions in Period 3.

Area 1= Lombardy, Piedmont, Sardinia; Area 2= Emilia Romagna; Area 3 = Tuscany; Area 4 = Latium, Apulia, Calabria, Campania, Molise, Sicily Central and Southern Italian Regions. The log₁₀ COI values (2A) and the log₁₀ IU/mL values (2B) observed in four Italian areas in period 3 were statistically compared using One-way ANOVA. The results show no significant difference for areas 3 and 4, neither in the case of the log₁₀ COI values nor in the case of the log₁₀ IU/mL values, while all the other comparisons show a more than highly significant difference between the areas.

RESULTS

Anti-SARS-CoV-2 antibodies in Italian plasma pools

A total of 349 Italian plasma pools were tested for the presence of anti-SARS-CoV-2 NP antibodies. As expected, 107 plasma pools made up from donations collected in Period 1 (May 2017–December 2019) were negative for anti-SARS-CoV-2 NP antibodies: mean COI value 0.10 (min 0.087; max 0.13).

With respect to the 68 plasma pools made up from donations collected in Period 2 (December 2018–March 2020), only one (1.4%), made up from donations from the Lombardy Region, was reactive: COI value 2.01.

Regarding plasma pools made up from donations collected in Period 3 (November 2018-October 2020), 105 out of 174 (60.3%) were reactive for anti-SARS-CoV-2 NP antibodies: mean COI value 4.49 (min 0.099; max 50.69).

All plasma pools positive for anti-SARS-CoV-2 NP antibodies were tested for anti-SARS-CoV-2 S-RBD antibodies and were confirmed positive. Quantitative results are expressed in IU/mL; a mean value 6.86 IU/mL was observed (min 0.04; max 34.15).

Figure 1 reports the distribution of the log¹⁰ COI values obtained from plasma pools tested for the presence of anti-SARS-CoV-2 NP antibodies in the three collection periods. Considering only Period 3, a different distribution of the reactivity (expressed as COI for anti-SARS-CoV-2 NP antibodies and as IU/mL for anti-SARS-CoV-2 S-RBD antibodies) can be observed among the Italian Regions (see Figure 2A and B, respectively) grouped into four areas corresponding to the different consortia (Area 1: Lombardy, Piedmont and Sardinia; Area 2: Emilia Romagna; Area 3: Tuscany; Area 4: Apulia, Calabria, Campania, Lazio, Molise and Sicily). In fact, when the log¹⁰ COI values (Figure 2A) and the log¹⁰ IU/mL values (Figure 2B) observed in Period 3 were statistically compared using one-way ANOVA, the results showed no significant difference for Areas 3 and 4, either in the case of the log¹⁰ COI values or in the case of the log¹⁰ IU/mL values, while all the other comparisons showed a more than highly significant difference between the areas.

In addition, 17 plasma pools (made up from plasma collected in Period 3), that were negative when tested using the qualitative test kit (COI <1.0 but >0.46), were weakly reactive using the quantitative test kit, showing values ranging from 1 to 2 IU/mL indicating a low presence of anti-SARS-CoV-2 S-RBD antibodies (see Online Supplementary Content, Table SII).

SARS-CoV-2 RNA

All plasma pools showing reactivity for anti-SARS-CoV-2 S-RBD antibodies were tested undiluted for the presence of the viral genome by TMA assay. None of the plasma pools tested reactive for SARS-CoV-2 RNA.

Anti-SARS-CoV-2 antibodies in IVIG produced from Italian plasma pools

All IVIG were tested with both Elecsys^® test kits in order to detect the presence of anti-SARS-CoV-2 NP antibodies and anti-SARS-CoV-2 S-RBD antibodies (see Table I).

Table I.

Results obtained from IVIG batches tested using both Elecsys^® test kits for the presence of anti-SARS-CoV-2 nucleocapsid protein (NP) antibodies and anti-SARS-CoV-2-spike receptor binding domain (RBD) antibodies

IVIG	Region	N. of donations	N. of plasma pools collected in Period 1	N. of plasma pools collected in Period 2	N. of plasma pools collected in Period 3 (n. of positive plasma pools)	COI for anti- SARS-CoV-2 NP antibodies	IU/mL for anti-SARS-CoV-2S-RBD antibodies
1	Lombardy, Piedmont	103,774	1	15	0	0.196 (non-reactive)	<0.4 (non-reactive)
2	Lombardy, Piedmont, Sardinia	103,579	16	0	0	0.190 (non-reactive)	<0.4 (non-reactive)
3	Lombardy, Piedmont	82,778	0	9	3 (2)	31.0	28.0
4	Lombardy, Piedmont	62,202	0	3	7 (6)	66.49	66.6
5	Lombardy, Piedmont	41,628	0	0	6 (6)	95.64	95.7
6	Lombardy, Piedmont	84,318	0	3	11 (10)	83.42	98.4
7	Lombardy, Piedmont	83,525	0	0	14 (14)	106.0	109.6
8	Lombardy, Piedmont	69,233	0	0	10 (10)	106.6	112.8
9	Lombardy, Piedmont, Sardinia	86,509	1	0	13 (11)	115.8	105.7
10	Lombardy, Piedmont, Sardinia	54,453	1	0	7 (5)	68.05	68.4
11	Lombardy, Piedmont, Sardinia	68,867	0	10	0	0.195 (non-reactive)	<0.4 (non-reactive)
12	Lombardy, Piedmont, Sardinia	44,376	0	6	0	0.186 (non-reactive)	<0.4 (non-reactive)
13	Emilia Romagna	45,515	0	0	8 (8)	36.89	42.0
14	Emilia Romagna	42,295	0	0	8 7)	29.2	25.8
15	Tuscany	83,148	0	0	14 (4)	4.14	5.3
16	Tuscany	40,572	0	0	6 (3)	5.83	6.9
17	Tuscany	47,761	0	0	8 (6)	10.3	16.8
18	Apulia, Calabria, Campania, Lazio, Molise	104,180	0	1	14 (5)	2.47	3.5
19	Apulia, Calabria, Molise, Lazio, Sicily	77,532	0	0	12 (9)	4.18	6.3
20	Apulia, Calabria, Molise, Sicily	83,820	0	3	9 (0)	0.396 (non-reactive)	<0.4 (non-reactive)
21	Apulia, Calabria, Molise, Sicily	43,747	0	0	7 (7)	4.09	5.3
22	Apulia, Campania, Calabria, Lazio, Molise, Sicily	68,051	0	1	10 (6)	2.30	4.1
23	Apulia, Calabria, Sicily	85,524	0	0	12 (3)	2.44	4.1
24	Apulia, Calabria, Sicily	55,148	0	0	8 (2)	1.49	2.7
25	Apulia, Calabria, Lazio, Molise, Sicily	96,295	0	2	12 (2)	1.65	2.2

N/n: number; COI: cutoff index.

Five IVIG batches (coded 1, 2, 11, 12 and 20) tested negative for both SARS-CoV-2 antibodies. The absence of SARS-CoV-2 antibodies in these batches of finished product reflect the negativity of the plasma pools used for their production.

The remaining 20 batches showed the presence of anti-SARS-CoV-2 NP antibodies and anti-SARS-CoV-2 S-RBD antibodies. In particular, the anti-SARS-CoV-2 S-RBD antibody content ranged from approximately 2 IU/mL to 112 IU/mL. The different concentration of antibodies in the batches of finished product reflect the content of the plasma pools used for their production.

Statistical analysis

Results are shown in Figure 1 and 2 where p≥0.01 and <0.05 was considered statistically significant, p≥0.001 and <0.01 was considered highly significant, p<0.001 was considered more than highly significant. Data were processed using R software, version 4.0.2 (R Foundation, Vienna, Austria).

DISCUSSION

Plasma pools, obtained from donations collected between May 2017 and October 2020 in 11 Italian Regions and produced by Kedrion Biopharma between January 2019 and November 2020, were grouped into three collection periods: Period 1 or pre-pandemic period, from May 2017 to December 2019; Period 2, a pandemic period that includes donations up to March 2020; and Period 3, a pandemic period that includes donations up to October 2020.

A first screening of 349 plasma pools demonstrated the presence of anti-SARS-CoV-2 NP antibodies in 1 plasma pool (1.4%) made up from plasma collected in the Lombardy region in Period 2 and on 60.3% of the plasma pools made up from plasma collected in Period 3. All plasma pools positive for anti-SARS-CoV-2 NP antibodies were shown to be reactive also for anti-SARS-CoV-2 S-RBD antibodies. Our findings reflect the epidemiology of the Italian population as no reactivity was observed in the plasma pools made up from plasma collected in the pre-pandemic period (Period 1). In addition, our results show that in the period corresponding to the first pandemic wave (Period 3) there is a clear prevalence of positivity in Areas 1 and 2 that include Lombardy, Piedmont, Emilia Romagna and Sardinia, with respect to the other Italian Regions. It is noteworthy that even if Sardinia is included in Area 1 as part of the corresponding consortium, the circulation of the virus on this island was very low during the first pandemic wave and not comparable to that observed in the other Regions in Areas 1 and 2.

It is noteworthy that all donations used to produce the plasma pools were obtained from healthy donors. In fact, absence of fever, cough or other respiratory symptoms related to SARS-CoV-2 infection was a prerequisite for donor eligibility, while a previous history of COVID-19 in the donor was generally followed by the segregation of his/her plasma which was then directed to use as CP.

It appears that, in the context of SARS-CoV-2 infections, the actual role of IVIG is not to boost the immune system but to modulate the systemic inflammatory syndrome by involving elevated levels of circulating cytokines and immune-cell hyperactivation, as observed in some COVID-19 patients. This overwhelming response, which is described as a “cytokine storm”, becomes the major cause of lung injury¹⁰. The clinical use of anti-SARS-CoV-2 specific immunoglobulins is currently being investigated and it is not yet possible to draw any conclusions as to their mechanisms of action or therapeutic indications. While waiting for further confirmation of the clinical use of these products, it is to be hoped that, once collected, CP will be stored for the preparation of IVIG in order to obtain products with a high anti-SARS-CoV-2 antibody titre. The integration of a specific programme of plasma collection, finalised to the production of hyperimmune anti-SARS-CoV-2 IVIG, into the European project Support-e is also advisable. In fact, another important point to be considered is that the number of donors carrying anti-SARS-CoV-2 antibodies will progressively increase over time, making hyperimmune plasma available worldwide.

The considerable content of anti-SARS-CoV-2 antibodies in the plasma pools made up from plasma collected during the first pandemic wave points to the presence of a high percentage of asymptomatic subjects that were not identified by the national surveillance system as the preventive measures in place did not foresee the use of screening tests. The COVID-19 cases that were detected at the beginning of the pandemic were typically symptomatic subjects who needed therapy or hospitalisation. Our study, therefore, highlights the fact that the percentage of anti-SARS-CoV-2 positive subjects had been greatly underestimated at the beginning of the pandemic. Only after the subsequent introduction of screening tests across the population, by means of rapid molecular or antigenic tests, was it possible to detect also subjects that, though asymptomatic, were capable of transmitting the SARS-CoV-2 infection. It should be noted, however, that despite post-donation information notifications concerning blood donor SARS-CoV-2 positivities collected by the National Blood Centre, the national epidemiological surveillance system did not report cases of SARS-CoV-2 transmission through blood transfusions. This is in line with previous observations of other respiratory viruses and during outbreaks of SARS-CoV and MERS, i.e. that there is no evidence of SARS-CoV-2 transmission through blood transfusions¹¹.

Twenty-five IVIG batches produced from plasma pools composed of plasma collected in the three periods were also screened for the presence of anti-SARS-CoV-2 NP antibodies and anti-SARS-CoV-2 S-RBD antibodies. Twenty of them (80%) were found positive with a different content of antibodies that reflects the concentration in the plasma pools used for their production.

Finally, all plasma pools that we found positive for anti-SARS-CoV-2 NP antibodies and anti-SARS-CoV-2 S-RBD antibodies showed the absence of SARS-CoV-2 RNA.

CONCLUSIONS

In the present study, we report the presence of anti-SARS-CoV-2 antibodies in plasma pools for fractionation collected in Italy in the pandemic period. None of the plasma pools tested were reactive for SARS-CoV-2 RNA. This further supports the safety of commercial IVIG obtained by blood donations during the COVID-19 pandemic, also considering the implementation of inactivation/reduction steps in the manufacturing process. Anti-SARS-CoV-2 antibodies were detected in 80% of IVIG batches, reflecting the concentration in the plasma pools used for their production. These findings can contribute to a better understanding of the SARS-CoV-2 pandemic in Italy during the first wave.

Further studies aimed at confirming the presence of anti-SARS-CoV-2 neutralising antibodies in the investigated IVIG should be carried out to verify the actual neutralising capacity of their anti-SARS-CoV-2 S-RBD antibodies, as recently reported in IVIG produced from US plasma collected during the 2020 pandemic¹².