key: cord-0954243-cn26081v
authors: Chen, Ji‐Ming
title: SARS‐CoV‐2 replicating in nonprimate mammalian cells probably have critical advantages for COVID‐19 vaccines due to anti‐Gal antibodies: A minireview and proposals
date: 2020-08-02
journal: J Med Virol
DOI: 10.1002/jmv.26312
sha: cc66b7c562ae41490d8e744efc00a5b70aba69ab
doc_id: 954243
cord_uid: cn26081v

Glycoproteins of enveloped viruses replicating in nonprimate mammalian cells carry α‐1,3‐galactose (α‐Gal) glycans, and can bind to anti‐Gal antibodies which are abundant in humans. The antibodies have protected humans and their ancestors for millions of years, because they inhibit replication of many kinds of microbes carrying αGal glycans and aid complements and macrophages to destroy them. Therefore, severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) replicating in nonprimate mammalian cells (eg, PK‐15 cells) carry αGal glycans and could be employed as a live vaccine for corona virus 2019 (COVID‐19). The live vaccine safety could be further enhanced through intramuscular inoculation to bypass the fragile lungs, like the live unattenuated adenovirus vaccine safely used in US recruits for decades. Moreover, the immune complexes of SARS‐CoV‐2 and anti‐Gal antibodies could enhance the efficacy of COVID‐19 vaccines, live or inactivated, carrying α‐Gal glycans. Experiments are imperatively desired to examine these novel vaccine strategies which probably have the critical advantages for defeating the pandemic of COVID‐19 and preventing other viral infectious diseases.

Corona virus 2019 (COVID-19) caused by the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in December 2019 in Wuhan, China, and has resulted in an ongoing pandemic. As of 13 July 2020, over 12.9 million cases have been reported across 188 countries and territories, resulting in over 560 000 deaths. 1,2 So far, particularly effective antiviral agents against COVID- 19 have not been identified. 3 It is thus imperatively desired to develop safe and effective vaccines for the disease, and accordingly, various vaccines for COVID- 19 have been under development with unprecedented rapidity. [4] [5] [6] [7] [8] [9] These vaccines can be largely divided into whole-virus vaccines and subunit vaccines. [4] [5] [6] [7] [8] [9] Whole-virus vaccines include live vaccines and inactivated vaccines, and subunit vaccines are those based on proteins, DNA, messenger RNA, or viral vectors.

Through decades of competition, live vaccines, inactivated vaccines, and subunit vaccines are employed for prevention of 13, 6, and 4 human viral infectious diseases, respectively (Table 1) . 10 Therefore, it is of great significance to develop whole-virus vaccines, particularly live vaccines which are usually more efficacious and less costly than other vaccines, for COVID-19. 11 Currently, several inactivated vaccines for COVID-19 have entered clinical trials, 8, 9 but progress of live vaccine development for COVID-19 has yet to be reported.

The inactivated vaccines for COVID-19 currently under development are produced using Vero cells. 8, 9 It has been found that SARS-CoV-2 replicates in PK-15 cells as efficiently as in Vero cells, and can reach to the titer of 10 11 PFU/mL. 12 As elucidated below, SARS-CoV-2 replicating in nonprimate mammalian cells, including porcine PK-15 cells, probably has critical advantages for inactivated and live vaccines for COVID-19, due to interaction of the glycans of α-1,3-galactose (Galα1-3Galβ1-4GlcNAc-R, αGal) and anti-Gal antibodies.

Glycans at the N-linked glycosylation sites on enveloped viruses including coronaviruses are synthesized by the relevant cellular enzymes in host cells. [13] [14] [15] [16] [17] [18] Accordingly, enveloped viruses, such as influenza virus, vesicular stomatitis virus (VSV), eastern-equineencephalitis virus (EEEV), replicating in cells of nonprimate mammals, lemurs, or New-World monkeys, including murine 3T3 fibroblasts, canine MDCK, and porcine PK-15, carry Gal-Gs, because these cells express active α-1,3-galactosyltransferase (α-1,3-GT). [13] [14] [15] [16] [17] [18] These cells also decorate their surface glycoprotein and glycolipids with many Gal-Gs synthesized within the cells. In contrast, enveloped viruses replicating in cells of humans, apes, Old-World monkeys, including human Hela cells and African green monkey Vero cells, lack Gal-Gs, because nucleotide mutations inactivated the enzyme α1,3GT in these cells. [13] [14] [15] [16] [17] [18] The surfaces of these cells also lack Gal-Gs. Many bacteria (eg, Serratia marcescens) and protozoa (eg, Trypanosoma cruzi) synthesize and present Gal-Gs on the glycoproteins and glycolipids. 13, 18, 19 3 | ANTI-GAL ANTIBODIES (ANTI-GAL-ABS) Nonprimate mammals, lemurs, or New-World monkeys do not produce anti-Gal-Abs due to immune tolerance to self-antigens. 14 In contrast, humans, apes, and Old-World monkeys produce anti-Gal-Abs throughout life because of frequent antigenic stimulation by many gastrointestinal bacteria carrying Gal-Gs. 19 Anti-Gal-Abs are abundant in humans, constituting~1% of serum IgG, IgM, and IgA immunoglobulins. 18, 20 Anti-Gal-Abs also exist as IgA and IgG in milk, colostrum, saliva, and bile. 20 Beyond anti-Gal-Abs specifically binding to Gal-Gs, there are other natural anti-glycan antibodies in human serum which bind to over 100 types of glycans, including anti-A and anti-B antibodies produced against blood-groups A and B glycans in the ABO system. 21 The natural anti-glycan antibodies of anti-A and anti-B are produced due to stimulation of gastrointestinal bacteria carrying blood-groups A or B antigen. 13, 22 It remains unknown whether anti-A and/or anti-B antibodies are responsible for the observations that blood group O humans are probably less susceptible to COVID-19 and group A humans are probably the most susceptible to COVID-19. [23] [24] [25] Anti-Gal-Abs induce immune rejection of xenografts (eg, porcine organs) carrying Gal-Gs transplanted into humans. 13, 26 However, their major contribution to humans is to help the immune system to eliminate many kinds of zoonotic viruses carrying Gal-Gs synthesized by the source animals, and fight bacteria and protozoa carrying Gal-Gs. [13] [14] [15] [16] [17] [18] In terms of evolution and immunology, anti-Gal-Abs have contributed greatly to protection of humans and their ancestors from infectious diseases for millions of years. 13, 27 The protection of anti-Gal-Abs against viruses is based on the following three mechanisms:

First, anti-Gal-Abs can prevent the virus from attaching to the viral receptors, and thus block the entry and replication of the virus. 13, 23, 28 This is because when anti-Gal-Abs bind to Gal-Gs on the viral surface glycoproteins, the binding can present a spatial obstacle to prevent the virus from attaching to the viral receptors on host cells. In-vitro incubation of EEEV carrying Gal-Gs with purified human anti-Gal-Abs blocked the replication of ∼50% of the virions in Vero cells, whereas such inhibition was not detected with EEEV lacking Gal-Gs. 13, 17 It is noteworthy that anti-Gal-Abs may be unable to neutralize or block all virions carrying Gal-Gs. 13, 17 Second, the immune complexes of anti-Gal-Abs and Gal-Gs on a virus can target the virus to the complement system and activate the system to destroy the virus. 13, [29] [30] [31] [32] [33] Through this complementmediated mechanism, anti-Gal-Abs have been found to be able to facilitate destruction of Gal-Gs presenting lymphocytic choriomeningitis virus, measles virus, Newcastle disease virus, porcine endogenous retrovirus, pseudorabies virus, Sindbis virus, Type C retrovirus, vaccinia virus, and VSV. 13, [29] [30] [31] [32] [33] An in-vitro study showed that anti-Gal-Abs enhanced complement-mediated killing of measles virus by over three folds compared with killing of the virus lacking Gal-Gs. 31 Another in-vitro study showed that anti-Gal-Abs could enhance complement-mediated killing of VSV over 10-fold compared with killing of the virus lacking Gal-Gs, and over 99.99% VSV virions could be destroyed through this approach. 16 Third, anti-Gal-Abs can target a Gal-Gs-carrying virus to macrophages and other antigen presenting cells (APCs), through the interaction of their Fc tails and the Fc receptors on APCs, and aid these immune cells to destroy the virus and present relevant antigenic epitopes to T and B cells. 13, [34] [35] [36] [37] This process is termed immune opsonization which reduces the virus pathogenesis and induces higher protective immunity as compared with the virus alone (ie, lacking a-gal epitopes). Mice in which the α1,3GT gene was knockout (GT-KO mice) and which produce anti-Gal-Abs have been used for 35 For the same reason, immunogenicity of recombinant HIV viral glycoprotein gp120 and bovine serum albumin carrying α-Gal-Gs increased by over 4 to 30 folds in GT-KO mice compared with these glycoproteins lacking Gal-Gs. 36, 37 Although anti-Gal-Abs are abundant in human bodies, their activity varies among individuals. 13 should not be used for those not in good health or with immunodeficiency. 46 Those people with immunodeficiency could be inoculated with the inactivated vaccines carrying Gal-Gs to help them to establish specific immunity against COVID-19.

Animal experiments and clinical trials are imperatively desired to confirm the potential critical advantages elucidated above for defeating COVID-19.

First, it should be confirmed through experiments that SARS-CoV-2 replicating in PK-15 cells stably carry Gal-Gs and can thus bind to anti-Gal-Abs in humans. In this respect isolectin 1-B4 from

Griffonia simplicifolia (GS-1-B4) can be used as an immunodiagnostic reagent because of its high specificity for the glycan. 49 To confirm that SARS-CoV-2 replicating in PK-15 cells can induce higher immunity, two inactivated vaccines can be prepared in the same way except that the cells for culturing SARS-CoV-2 are different. Then, two randomized groups of rhesus macaques are inoculated with these two inactivated vaccines, and the antibodies against SARS-CoV-2 are qualitatively evaluated using mini-neutralization assay. 5, 6 To confirm the potential that SARS-CoV-2 replicating in non- Notably, all relevant data should be collected from the above experiments and clinical trials to identify or exclude the possibility that the anti-Gal-Abs could weaken the immune response to the vaccine, and that anti-Gal-Abs have some potential limitations and disadvantages. 

The author thanks Meng Yang and Jiwang Chen for their helpful assistance, and thanks deeply an anonymous scientist for his very precious direction. This work was conducted at Qingdao Agricultural University and Qingdao Six-Eight Nearby Sci-Tech Company.

World Health Organization. Coronavirus disease (COVID-2019) situation reports

Preventive and treatment strategies of COVID-19: from community to clinical trials

The early landscape of COVID-19 vaccine development in the UK and rest of the world

Progress and prospects on vaccine development against SARS-CoV-2. Vaccines (Basel)

SARS-CoV-2 mRNA vaccine development enabled by prototype pathogen preparedness

Safety, tolerability, and immunogenicity of a recombinant adenovirus type-5 vectored COVID-19 vaccine: a doseescalation, open-label, non-randomised, first-in-human trial

Development of an inactivated vaccine candidate for SARS-CoV-2

Development of an inactivated vaccine candidate, BBIBP-CORV, with potent protection against SARS-CoV-2

Potential of live pathogen vaccines for defeating the COVID-19 pandemic: history and mechanism

Comparative tropism, replication kinetics, and cell damage profiling of SARS-CoV-2 and SARS-CoV with implications for clinical manifestations, transmissibility, and laboratory studies of COVID-19: an observational study

Human natural antibodies to mammalian carbohydrate antigens as unsung heroes protecting against past, present, and future viral infections

Man, apes, and Old-World monkeys differ from other mammals in the expression of α-Galactosyl glycans on nucleated cells

Enhancement of antigen presentation of influenza virus hemagglutinin by the natural anti-Gal antibody

Xenoantigen-dependent complement-mediated neutralization of LCMV glycoprotein pseudotyped VSV in human serum

Differential host dependent expression of α-galactosyl glycans on viral glycoproteins: A study of Eastern equine encephalitis virus as a model

Gene sequences suggest inactivation of α1-3 Galactosyltransferase in catarrhines after the divergence of apes from monkeys

Distribution of bacterial α1,3-galactosyltransferase genes in the human gut microbiome

Anti-alpha-galactosyl immunoglobulin A (IgA), IgG, and IgM in human secretions

Microbial glycan microarrays define key features of host-microbial interactions

Biochemistry and genetics of the ABO, Lewis, and P blood group systems

Harnessing the natural antiglycan immune response to limit the transmission of enveloped viruses such as SARS-CoV-2

Relationship between the ABO blood group and the COVID-19 susceptibility

ABO phenotype and death in critically ill patients with COVID-19

Removal of anti-porcine natural antibodies from human and nonhuman primate plasma in vitro and in-vivo by a Gal α 1-3Gal β 1-4 β Glc-X immuneaffinity column

Catastrophic-selection" interplay between enveloped virus epidemics, mutated genes of enzymes synthesizing carbohydrate antigens, and natural anti-carbohydrate antibodies

Coronaviruses' sugar shields as vaccine candidates

Sensitization of cells and retroviruses to human serum by.α1-3 Galactosyltransferase

Evaluation of the Galα1-3Gal epitope as a host modification factor eliciting natural humoral immunity to enveloped viruses

Expression of ABO or related antigenic carbohydrates on viral envelopes leads to neutralization in the presence of serum containing specific natural antibodies and complement

A novel mechanism of retrovirus inactivation in human serum mediated by anti-α-Galactosyl natural antibody

The neutralization of pseudorabies virus by anti-α-Galactocyl natural antibody in normal serum

From therapeutic antibodies to immune complex vaccines

Increased immunogenicity of influenza virus vaccine by anti-Gal mediated targeting to antigen presenting cells

Increased immunogenicity of HIV-1 p24 and gp120 following immunization with gp120/p24 fusion protein vaccine expressing α-Gal epitopes

The influence of natural antibody specificity on antigen immunogenicity

Quantitation and characterization of anti-Galα1-3Gal antibodies in sera of 200 healthy persons

Interaction of baboon anti-α-galactosyl antibody with pig tissues

Boosting anti-alpha-Gal immune response to control COVID-19

Lytic anti-αgalactosyl antibodies from patients with chronic Chagas' disease recognize novel O-linked oligosaccharides on mucin-like glycosylphosphatidylinositol-anchored glycoproteins of Trypanosoma cruzi

Antibody-dependent SARS coronavirus infection is mediated by antibodies against spike proteins

COVID-19: immunopathology and its implications for therapy

Immunization with types 4 and 7 adenovirus by selective infection of the intestinal tract

Safety evaluation of adenovirus type 4 and type 7 vaccine live, oral in military recruits

Immunization against viral diseases

Live-attenuated H1N1 influenza vaccine candidate displays potent efficacy in mice and ferrets

Potential for elimination of SAR-CoV-2 through vaccination as inspired by elimination of multiple influenza viruses through natural pandemics or mass vaccination

M109919200 How to cite this article: Chen J-M. SARS-CoV-2 replicating in nonprimate mammalian cells probably have critical advantages for COVID-19 vaccines due to anti-Gal antibodies: A minireview and proposals

The authors declare that there are no conflict of interests.

Ji-Ming Chen completed this work with the aid of Meng Yang, Jiwang Chen, and an anonymous scientist.

The article does not contain the participation of animals and humans other than the authors.

http://orcid.org/0000-0002-0404-0830