La ciencia es diversión

DETECCIÓN DE LA NUEVA ENFERMEDAD POR CORONAVIRUS 2019 (COVID-19) MEDIANTE ENSAYOS DE SERODIAGNÓSTICO RÁPIDO

Detección de la nueva enfermedad por coronavirus 2019 (COVID-19) mediante ensayos de serodiagnóstico rápido

On 31 December 2019, WHO was informed of a cluster of cases of pneumonia of unknown cause detected in Wuhan City, Hubei Province of China. The coronavirus disease (COVID-2019) was identified as the causative virus by Chinese authorities on 7 January. As part of WHO’s response to the outbreak, the R&D has been activated to accelerate diagnostics, vaccines and therapeutics for this novel coronavirus.

Coronavirus 2020
Figure 1. Coronavirus, 2020. Illustration by David S. Goodsell, RCSB Protein Data Bank doi: 10.2210/rcsb_pdb/goodsell-gallery-019

Regarding diagnostics, the new coronavirus COVID-19 (SARSCoV2 or SARSCoV(2019)) requires a rapid point of care diagnostics for use at the community level in order to accelerate the therapeutics and vaccines. Let´s have a look at the possible antigens which can be used in these rapid serodiagnosis test:


NUCLEOCAPSID

The nucleocapsid phosphoprotein (NP) is a highly phosphorylated protein which not only is responsable for construction of the ribonucleoprotein complex by interacting with the viral genome and regulating the synthesis of viral RNA and protein, but also serves as a potent immunogen that induces humoral and cellular immunity. The nucleoprotein is one of the major antigens of the SARS-CoV and there are abundant antigenic sites predicted in this protein. Compared with other viral structural proteins, the low variation rate of the N protein with regards to its size suggests its importance to the survival of the virus.

SARS-CoV N-terminal
Figure 2. Structure of the N-terminal RNA binding domain of the SARS-CoV, nucleocapsid protein (PDB code: 1SSK)

If we perform an alignment of the NP amino acid sequences of both related coronaviruses, the high identity between both of them, predict a clear cross-reactivity in a serological test:

ALIGNMENT OF SARSCoV (2003) and SARSCoV2 (2019)/COVID-19

COVID-19 MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHG
SARSCoV(2003) MSDNGPQNQRSAPRITFGGPSDSSDNSKNGERNGARPKQRRPQGLPNNTASWFTALTQHG
**********.************:...:****.***.***********************
COVID-19 KEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAG
SARSCoV(2003) KENLTFPRGQGVPINTNSSKDDQIGYYRRATRRIRGGDGKMKELSPRWYFYYLGTGPEAG
**:*.************** **********************:*****************
COVID-19 LPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGS
SARSCoV(2003) LPYGANKEGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGS
*******:****************************************************
COVID-19 QASSRSSSRSRNSSRNSTPGSSRGTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQ
SARSCoV(2003) QASSRSSSRSRNSSRNSTPGSSRGTSPARMAGNGGDTALALLLLDRLNQLENKVSGKGQQ
************************************:**************.*:******
COVID-19 QQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKH
SARSCoV(2003) QQGQTVTKKSAAEASKKPRQKRTATKQYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKH
************************** *********************************
COVID-19 WPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAY
SARSCoV(2003) WPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYHGAIKLDDKDPQFKDNVILLNKHIDAY
********************************* **********:***:***********
COVID-19 KTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAADLDDFSKQLQQSMSSADSTQA
SARSCoV(2003) KTFPPTEPKKDKKKKADELQALPQRQKKQQTVTLLPAADLDEFSKQLQQSMSGTDSTQA
****************** **********************:**********.:*****

This protein is highly similar to both, SarsCoV-1 (2003) and SarsCoV-2 (COVID-19), therefore, with the NP protein from SarsCoV (2003) in your serodiagnosis assay, your test will be able to detect the current coronavirus infections (COVID-19) as they share an identity of 94% and similarity of 97% in their amino acid sequence.

At the moment, at Rekom Biotech, we have developed three antigens from the nucleocapsid of the SARSCoV(2003): NP-NTD (N-terminal domain, RAG0082); NP-MID (middle domain, RAG0081) and NP-CTD (C-terminal domain, RAG0080). The three of them contain together all the antigenic determinants described by Wang et al., The structure analysis and antigenicity study of the N protein of SARS-CoV, 2003, Genomics Proteomics Bioinformatic, 1 (2): 145-54.

We are not going to develop the COVID-19 NP protein as it is so identical to SARSCoV(2003) NP that the obtained result in a serodiagnosis test would be exactly the same: detection.


SPIKE PROTEIN

The spike (S) protein is a surface trimer glycoprotein located on the envelope of the virions. It is cleaved into S1 and S2 subunits. The S1 subunit is responsible for host-receptor binding while the S2 subunit contains the membrane-fusion machinery. There are two domains in coronavirus S1: N-terminal domain (S1-NTD) and C-terminal domain (S1-CTD). One or both of these S1 domains potentially bind receptors and function as the receptor-binding domain (RBD).

SARS-CoV Spike
Figure 3. Structure of the trimer of the spike protein (PDB code: 6CRV). In green is shown the S1 domain and in yellow the S2 domain.

If we perform an alignment of the S glycoprotein amino acid sequences of both related coronaviruses, maybe it is possible to find out special regions with less identity which can theoretically avoid this cross-reactivity in a serological test:

ALIGNMENT OF SARSCoV (2003) and SARSCoV2 (2019)/COVID-19

COVID-19 -MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFF
SARSCoV(2003) MLFFLFLQFALVNSQCVNLTGRTPLNPNYTNSSQRGVYYPDTIYRSDTLVLSQGYFLPFY
:*.::: :.**.******* ** * * **** *******.::**..* :*. ****:
COVID-19 SNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLI
SARSCoV(2003) SNVSWYYSLTTN-NAATKRTDNPILDFKDGIYFAATEHSNIIRGWIFGTTLDNTSQSLLI
***:*:::: .. . .*** ***:* *:**:***:**:**************..:*****
COVID-19 VNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDL
SARSCoV(2003) VNNATNVIIKVCNFDFCYDPYLSGYYHNN-KTWSIREFAVYSSYANCTFEYVSKSFMLNI
*******:****:*:** **:*. ***:* *:* ** **** ********:.*::::
COVID-19 EGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQ
SARSCoV(2003) SGNGGLFNTLREFVFRNVDGHFKIYSKFTPVNLNRGLPTGLSVLQPLVELPVSINITKFR
.*: * *:.******:*:**:******.**:** *.** *:*.*:***:**:.****:*:
COVID-19 TLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSET
SARSCoV(2003) TLLTIHRGDPMP---NNGWTAFSAAYFVGYLKPRTFMLKYNENGTITDAVDCALDPLSET
***::**. * ..**** :***:****:****:***********************
COVID-19 KCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRIS
SARSCoV(2003) KCTLKSLTVQKGIYQTSNFRVQPTQSVVRFPNITNVCPFHKVFNATRFPSVYAWERTKIS
******:**:**************:*:********:*** :*******.*****:*.:**
COVID-19 NCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIA
SARSCoV(2003) DCIADYTVFYNSTSFSTFKCYGVSPSKLIDLCFTSVYADTFLIRFSEVRQVAPGQTGVIA
:*:***:*:***:************:** *****.****:*:** .****:****** **
COVID-19 DYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTP
SARSCoV(2003) DYNYKLPDDFTGCVIAWNTAKQDVGN-----YFYRSHRSTKLKPFERDLSSDENGVR---
******************: : * *:** .*.::*******:*:: .
COVID-19 CNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCV
SARSCoV(2003) -----------TLSTYDFNPNVPLEYQATRVVVLSFELLNAPATVCGPKLSTQLVKNQCV
.*.:*.*:*. : **. **********:********* **:****:**
COVID-19 NFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVIT
SARSCoV(2003) NFNFNGLKGTGVLTDSSKRFQSFQQFGKDASDFIDSVRDPQTLEILDITPCSFGGVSVIT
*******.******:*.*:* .*****:* :* *:************************
COVID-19 PGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNS
SARSCoV(2003) PGTNTSLEVAVLYQDVNCTDVPTTIHADQLTPAWRIYATGTNVFQTQAGCLIGAEHVNAS
****** :***********:**.:********:**:*:**:*****:*********** *
COVID-19 YECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFT
SARSCoV(2003) YECDIPIGAGICASYHTAS----ILRSTSQKAIVAYTMSLGAENSIAYANNSIAIPTNFS
***************:* : **.:.::*:***********:**:**********:
COVID-19 ISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQ
SARSCoV(2003) ISVTTEVMPVSMAKTSVDCTMYICGDSIECSNLLLQYGSFCTQLNRALSGIAIEQDKNTQ
******::****:************** ********************:***:*******
COVID-19 EVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGD
SARSCoV(2003) EVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGD
************************************************************
COVID-19 CLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFA
SARSCoV(2003) CLGGISARDLICAQKFNGLTVLPPLLTDEMIAAYTAALISGTATAGWTFGAGAALQIPFA
***.*:************************** **:**::** *:***************
COVID-19 MQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQAL
SARSCoV(2003) MQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQESLTSTASALGKLQDVVNQNAQAL
************************************:**:********************
COVID-19 NTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIR
SARSCoV(2003) NTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIR
************************************************************
COVID-19 ASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAP
SARSCoV(2003) ASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYIPSQEKNFTTAP
************************************************:*:*********
COVID-19 AICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYD
SARSCoV(2003) AICHEGKAHFPREGVFVSNGTHWFVTQRNFYEPKIITTDNTFVSGNCDVVIGIINNTVYD
****:****************************:*******************:******
COVID-19 PLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLID
SARSCoV(2003) PLQPELDSFKEELDKYFKNHTSPDIDLGDISGINASVVNIQKEIDRLNEVARNLNESLID
************************:**************************:********
COVID-19 LQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDED
SARSCoV(2003) LQELGKYEQYIKWPWYVWLGFIAGLIAIVMVTILLCCMTSCCSCLKGCCSCGSCCKFDED
****************:****************:**************************
COVID-19 DSEPVLKGVKLHYT
SARSCoV(2003) DSEPVLKGVKLHYT
**************

With ectodomain S1 (red) of the spike glycoprotein, it is possible maybe to discriminate better COVID-19 from SARSCoV(2003) as this protein is less similar than nucleocapsid, with a 70% identities and 81% similarities. If your test wants to discriminate, the best option is select S1 region of the spike protein. In this domain, we can find the N-terminal domain of S1 ectodomain (S1 NTD) and the C-terminal domain of the S1 ectodomain (S1 CTD). On regards these two regions, S1-NTD has an identity of 66% and 79% similarities, meanwhile S1-CTD has 79% identities and 89% similarities.

Regarding the S protein, at Rekom Biotech, we are working on improving the development of the COVID-19 spike protein (S1-CTD) and also a chimera, to increase the number of epitopes in the recombinant antigen.


CONCLUSSION

In summary, both coronaviruses are extremely similar but maybe, during this outbreak, the greatest urgency is to be able to detect in a reliable and viable manner the presence of antibodies against coronavirus in the blood of potentially infected people, and I presume that the therapeutics and vaccine will be equally effective in both cases (if the patient is infected by SARSCoV1 or SARSCoV2). Therefore, in my point of view, it is feasible, as an emergency, to use the NP SARSCoV (2003) as antigen to detect the new COVID-19 due to its high identity and also taking in mind that the main aim is detection and not discrimination at the moment.

Written by Ana Camacho.

CATEGORÍA: NOTAS CIENTÍFICAS