key: cord-0960121-xy013ecy
authors: Focosi, Daniele; Maggi, Fabrizio; Mazzetti, Paola; Pistello, Mauro
title: Viral infection neutralization tests: A focus on severe acute respiratory syndrome‐coronavirus‐2 with implications for convalescent plasma therapy
date: 2020-09-21
journal: Rev Med Virol
DOI: 10.1002/rmv.2170
sha: bfd3efbf85def6e4d6660c343bf9dd592ed83712
doc_id: 960121
cord_uid: xy013ecy

Viral neutralization tests (VNTs) have long been considered old‐fashioned tricks in the armamentarium of fundamental virology, with laboratory implementation for a limited array of viruses only. Nevertheless, they represent the most reliable surrogate of potency for passive immunotherapies, such as monoclonal or polyclonal antibody therapy. The recent interest around therapy with convalescent plasma or monoclonal antibodies for the Covid‐19 pandemic has paralleled the revival of VNTs. We review here the available methods by dissecting variations for each fundamental component of the VNT (i.e., virus type and dose, replication‐competent cell line, serum, and detection system).

2 (ACE2) and bind only to up RBDs, 2 ACE2-blocking hNAbs that bind both up and down RBDs and can contact adjacent RBDs, 3 hNAbs that bind outside the ACE2 site and recognize up and down RBDs. 9 S variants that resist commonly elicited nAb are now present at low frequencies in circulating SARS-CoV-2 populations. 10 While vaccines are still under development and reinfection from next pandemic waves is still under observation, CP donor selection represents the most urgent challenge.

To date more than 90,000 patients across the world have already been treated with CP, mostly in non-randomized studies (65,000 in the United States only thanks to expanded access programs approved by FDA 11 ). The efficacy of CP therapy is believed to rely on nAb content. 3 Convalescent patients should hence be screened for the presence of nAb levels, and donations collected only from convalescent individuals with high nAb titres. In the setting of Covid-19, several regulatory authorities recommend threshold values, but none of them specifies nAb assay details that could alter test output: FDA says that 'when a measurement of nAb titres is available, we recommend nAb titres of at least 1:160. A titre of 1:80 may be considered acceptable if an alternative matched unit is not available. When the measurement of nAb titres is not available, consider storing a retention sample from the CP donation for determining antibody titres at a later date. 12 The ECDC basically endorse FDA recommendations, suggesting that immunocompromised recipients are transfused with CP units having a titre ≥1:320. 13 As previously said, those thresholds have poor meaning unless details are disclosed, making trial results poorly comparable.

Neutralizing antibody assessment has historically been performed using time-consuming and hazardous methods that required high technical skills. While high throughput platform surrogate tests having substantially shorter turnaround time and good correlations with nAb are heavily under study, old-fashioned methods remain the gold standard for exact nAb quantification. The Covid-19 pandemic has had the side benefit of expediting research on nAb testing upgrades. This manuscript reviews the principle behind classical nAb testing by dissecting each key component (as depicted in Figure 1 ), and the developments that have been released in the last years. [VSV] pseudotype assays, respectively; see paragraph below) in high throughput situations, as great care is necessary when using 293T-derived cells whose adhesive properties during washing steps are suboptimal. 20

Intact virions or several different surrogates can be used to represent the viral challenge, as detailed below. facilities. For SARS-CoV-2, the expression of full-length S protein was enhanced over 10-fold by deleting the last C-terminal 18 18 or 19 19, 26 amino acids of the cytoplasmic tail, or by codon optimization. 17 Such methodology has previously been used to produce pseudotyped viruses for SARS-CoV-1 27 and MERS-CoV. 28 Modification of a single amino acid in the Furin cleavage site of S (R682Q) enhanced infectious particle production another 10-fold. With all enhancing elements combined, the titre of pseudotyped particles reached almost 10 6 infectious particles/ ml. 19 Nevertheless, lower Spike densities in pseudotypes viruses could affect the avidity of bivalent antibodies, particularly those that are unable to engage two S-protein monomers within a single trimer and whose potency is dependent on engaging 2 adjacent trimers.

Assembly of the VSV occurs at the plasma membrane and involves budding of virions from the cell surface. During budding, VSV acquires an envelope consisting of a lipid bilayer derived from the plasma membrane and spike proteins consisting of trimers of the VSV-glycoprotein (VSV-G). When the VSV-G is absent and the glycoprotein from a heterologous virus is complacently expressed in cells infected with recombinant vesicular stomatitis virus with protein G deletion (rVSV-dG), the glycoprotein of the heterologous virus could be assembled into the VSV membrane (30) . Recently, VSVdGluc bearing S chimeras has been used to study the cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses. 29 A PsVNA assay for SARS-CoV-2, which consists of pseudotyped VSV bearing the full-length Sprotein of SARS-CoV-2 and Huh7 cell, has been successfully tested. 30 Interestingly, the absence of proof-reading activity in VSV-L polymerase has been exploited to generate virus stocks with greater diversity (especially in S protein) than authentic SARS-CoV-2. 10 The assay provides results in 12-16 h.

Because single-cycle, replication-defective pseudotypes viruses do not allow for any viral spread and this could impact the sensitivity of the VNT, replication-competent VSV/SARS-CoV-2 chimeric viruses have been generated for usage in multicycle replication-based assays. 20

Lentiviral pseudotype bearing the truncated spike protein of SARS-CoV-2 was also constructed and used to study the virus entry and its immune cross-reactivity with SARS-CoV-1. 20, 31, 32 In one study, Moloney murine leukemia virus (MMLV) was able to pseudotype the VSV-G glycoprotein efficiently but was unable to pseudotype the SARS-CoV-2 Spike protein. 16 However, in a different study, MMLV was able to pseudotype the SARS-CoV-2 Spike protein. 24 These differences could be due to the constructs used to express the Spike protein with the former study expressing a full-length Spike protein, whilst the latter study expressed a Spike protein with a deletion in the C-terminal 19 amino acid (aa) that could aid better expression as alluded to earlier. 33 In contrast, pLV pseudotyped both glycoproteins efficiently; however, much higher titres of pLV-G particles were produced. Among all the tested mammalian cells, HEK 293Ts expressing human ACE2 (hACE2) were most efficiently transduced using the pLV-S system. 16 The neutralizing activity of both CP and human monoclonal antibodies measured using each virus correlated quantitatively with nAb measured using an authentic SARS-CoV-2 neutralization assay. The assay provides results in approximatively 48 h. As previously stated, when the firefly luciferase reporter gene is inserted in the viral construct, cells are lysed and assayed for luciferase expression. 17, 22 The evaluability of the PRNT technique may be further improved by overlaying the cells with cellulose or by using specific antibodies to detect remaining viral antigens in the cells. 46 70 This test is performed on the same platform and in parallel with an ELISA for the detection of antibodies against the RBD. 71, 72 An entirely different approach is based on the antibody detection by agglutination PCR (ADAP) methodology. A cell-free neutralization polymerase chain reaction (PCR) assay using SARS-CoV-2 S protein and human ACE2 receptor-DNA conjugates has been developed to quantify nAbs. Briefly, the neutralizing antibodies in the specimen will engage with S1-DNA conjugate in step 1 to decrease S1-DNA binding with ACE2-DNA in step 2. Even this assay can be run in BSL2 and provide results in 2À 3 h. 73 

While the overall antibody responses for other beta coronaviruses typically declines after 6-12 months, 76 SARS-CoV-specific nAb usually persist for 2 years. 77 In most of Covid-19 inpatients, nAb reached a plateau 2 weeks post-symptom onset and then declined, reaching a low or undetectable level ≥40 days post-symptom onset. 21 In less severe cases, nAb in serum reached a peak about 4 weeks after disease onset but dropped to a lower level about 6 weeks later. 78 

Anti-S1 RBD Anti-S1

Anti-S1/S2 trimers Anti-N symptoms onset but RBD-specific IgM decreased much more abruptly. Similarly, a significant decrease in the capacity of CP to neutralize pseudo particles bearing SARS-CoV-2 S wild-type or its D614G variant has been reported. 82 The magnitude of the nAb response is correlated with disease severity, but this does not affect the kinetics of the nAb response.

Whilst some individuals with high peak ID 50 (>10,000) maintained titres >1000 at >60 days after onset of symptoms, some with lower peak ID 50 had titres approaching baseline within a 94 days followup. 83 four-fold from one to four months post-symptom onset. 88 The decline in anti-RBD antibodies was not related to the number of donations but strongly correlated with the number of days after symptoms onset (r ¼ 0.821). 89 The rapid decline in nAb may be attributed to the rapid decay of IgM in the acute phase. However, the relative contribution of IgG to nAb increased and that of IgM further decreased after 6 weeks after symptom onset. 12 

This manuscript contains no original data.

https://orcid.org/0000-0001-8811-195X

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