Title : ABO isoagglutinins inhibit coronavirus infection?
COVID-19, caused by SARS-CoV-2, requires the coronavirus Spike (S)-protein, host receptor ACE2 and the protein TMPRSS2 for infection. Emerging progeny viruses use host plasma membrane, which may or may not contain ABH(O) antigens, as part of their virus envelope. Multiple studies have reported that blood group O is protective against severe COVID-19 disease, while blood group A patients have an increased susceptibility to COVID-19. This suggests that anti-A antibodies (isoagglutinins) from blood group O patients could provide natural protection against COVID-19. SARS-CoV-2 work requires a containment level 3 (CL3) environment. However, the structural and functional properties of the S-protein are mutual between SARS-CoV-2 and the endemic strain, NL63 requiring only CL2 containment. Thus, NL63, acting as a surrogate for SARS-CoV-2, may be useful for investigating the role of ABO isoagglutinins in coronavirus infection. Selecting cell lines that express ABH antigens would be ideal for investigating a role for viruses that carry A/B glycans in their envelope and if they can be inhibited by corresponding ABO isoagglutinins.
Methodology: Mammalian cell lines LLC-MK2 (monkey), HT-29 (human), and Vero (monkey) were checked for ACE2 expression via Western Blot and stained with anti-A/B antibodies to determine ABO expression via immunofluorescence. LLC-MK2 were used to propagate NL63 and optimize RT-qPCR for detecting early infection (subgenomic RNA), cytopathic effects (CPE), or virus production. HEK293T/17 cells were transfected with CoV2-Spike pseudotyped-luciferase-reporter lentivirus and glycosyltransferases that synthesize ABH antigens, to produce CoV2-S lentivirus that expresses ABO antigens on its envelope and used to infect Vero cells. Cells that expressed low levels or no ACE2/TMPRSS2 were transfected with ACE2/TMPRSS2 plasmids to increase permissiveness to coronavirus infection. Monoclonal anti-A/B antibodies were used to determine if ABO isoagglutinins could inhibit infection of CoV2-S lentivirus (Vero) and SARS-CoV-2 (HT29).
Results: LLC-MK2 were B positive, while HT-29 were A positive. 293 cells transfected with ABO transferases expressed ABH antigens on their plasma membrane. LLC-MK2 cells were found to be permissive to NL63 infection, showing CPE, early detection of subgenomic RNA, and productive infection, and were used to propagate NL63 in vitro. CoV2-S pseudotyped lentivirus was found to infect all cells.
ACE2/TMPRSS2-transduced HT-29 were observed to be permissive to SARS-CoV-2 infection but not NL63 infection. VeroTMP+/CSTL- (TMPRSS2-transduced + Cathepsin-deletion) had a significantly greater CoV2-S lentivirus infection rate than wildtype Vero. A-expressing CoV2-S lentivirus infection in VeroTMP+/CSTL- was completely inhibited by monoclonal anti-A, whereas the antibody showed no inhibitory effects on wildtype and O-expressing virus.
Conclusions/Future Directions: Cells that are permissive to either coronaviruses NL-63 (MK2), SARS-CoV-2 (HT-29ACE2+TMP+), or CoV2-S pseudotyped lentivirus (VeroTMP+/CTSL-) have been identified and ABO confirmed on plasma membrane. ACE2/TMPRSS2-transduction of cell lines were successful in increasing their permissiveness to coronavirus infection. Proof-of-concept has been shown with monoclonal anti-A inhibition of only A-expressing CoV2-S lentivirus. Currently, we are testing both anti-A and anti-B inhibition of A- and B-expressing CoV2-S lentivirus in VeroTMP+/CTSL-, and of SARS-CoV-2 produced in A-expressing HT-29. We are also transfecting MK2 cells with ABO glycosyltransferases to examine if monoclonal anti-A/B can inhibit A- or B-expressing NL63 infection.
- Audience will gain a better understanding of ABO biochemistry and their role in coronavirus infections.
- Helps to explain the pathophysiology of COVID-19 and may support more aggressive treatment in patients who are blood group A that become infected with SARS-CoV-2.
- Yes, if my study confirms the role of ABO in coronavirus disease, this research could be expanded to study their effects in other types of viral diseases, particularly enveloped virus.
- If ABO is confirmed to have a protective role in COVID-19 disease, IVIg transfusions which contain high levels of IgG anti-A/B would be justified to be used as a treatment to reduce disease severity.
- My study may provide a surrogate virus to use instead of SARS-CoV-2 which is safer for researchers to handle and that can be used in a containment level 2 environment.
- May provide rationale as to why some people that are in close contact acquire the disease while others do not. It could be based on blood group.