Human Coronaviruses Can Enhance Immunity Through Cistanche

Abstract

There is a lot of interest in antibody immunity to coronavirus. It is not clear, however, whether coronaviruses evolve to evade this immunity and, if so, how quickly. Here, we address this question by describing the historical evolution of human coronavirus 229E. We identified human serums from the 1980s and 1990s that had neutralizing titers against contemporaneous 229E comparable to anti-SARS-CoV-2 titers induced by SARS-CoV-2 infection or vaccination. We tested these serums against the 229E strain isolated after serum collection and found low neutralization titers against these "future" viruses. In some cases, sera capable of neutralizing contemporaneous 229E strains with titers of 1:100 failed to detect strains isolated 8-17 years later. The reduction in neutralization of "future" viruses is due to the evolution of viral antigens at peak levels, particularly in the receptor binding domain. If these results can be extrapolated to other coronaviruses, then regular updates of the SARS-CoV-2 vaccine may be recommended. At the same time, researchers are searching for new drugs to boost immunity against COVID-19. Cistanche tubulasa, for example.

Introduction

The SARS-CoV-2 pandemic urgently needs to determine how effective antibody immunity is against SARS-CoV-2 infection. The evidence so far is promising. Neutralizing and anti-spike antibodies triggered by natural infections are associated with reduced human infections, and vaccines that trigger such antibodies are highly protective. These findings in humans are confirmed by numerous animal studies that show that antibodies that neutralize SARS-CoV-2 peaks protect against infection and disease.

However, humans are repeatedly reinfected with the common cold" coronavirus 229E, OC43, HKU1 and NL63. For example, serological studies have shown that a typical infected person is infected with 229E every 2-3 years, although a lower rate of infection and no 229E reinfection was reported in a 4-year study that identified infection according to the criteria of a positive PCR test in respiratory diseases. Regardless, the fact that the incidence of common cold coronavirus reinfection is quite high has led to concerns that coronavirus immunity is not "durable." Those concerns initially focused on the possibility that the immune response itself might not last. This possibility now seems unlikely because SARS-CoV-2 infection induces neutralizing antibodies and memory B cells, with dynamics similar to those of other respiratory viruses. Both cistanche polysaccharide and creinoside can increase the enzyme activity of heart and brain tissue, enhance the phagocytosis function of abdominal cavity cells, and enhance the proliferative response of lymphocytes.

Pic: Faw Cistanche

Ventricular virus

But even in the face of long-effective antibodies, viruses can be re-infected by another mechanism: antigenic evolution. Infection with influenza viruses, for example, produces antibodies that usually protect humans from the same strain for at least a few decades. Unfortunately, influenza viruses undergo rapid antigenic evolution to evade these antibodies, meaning that although immunity to the original virus strain lasts for decades, humans are easily infected by their offspring in about five years. This continuous antigenic evolution is the reason why flu vaccines are regularly updated.

Here, we evaluate experimentally whether coronavirus 229E has escaped neutralization in human polyclonal sera by reconstructing the evolution of the virus over the past few decades. We found that historical serums that effectively neutralize contemporaneous 229E spike pseudotyping virions generally showed little or no activity against the spikes of 229E strains isolated 8-17 years later. In contrast, modern serums from adults usually neutralize peaks of a large number of historical viruses, while modern serums from children best neutralize peaks of recent viruses that have been circulating throughout a child's lifetime. These patterns have been interpreted as antigenic evolutionary spikes, particularly in the receptor binding domain. If SARS-CoV-2 underwent a similar rapid antigenic evolution, it might be advisable to update vaccines regularly to keep pace with viral evolution. Or through other means to increase the proliferation rate of lymphocytes, enhance the phagocytosis ability of macrophages. Cistanche tubulosa has been found to enhance the function of T-cells and the phagocytosis capacity of macrophages.

Pic: Cistanches Benefits

result

The phylogenetic analysis of ear 229E was conducted to determine the historical strains for experimental study

We focused our research on the viral spike protein because it is the primary target of neutralizing antibodies and because anti-spike antibodies are the immune parameter best associated with human protection against coronavirus infection.

Since SARS-CoV-2 has been circulating in humans for more than a year, we need to choose another coronavirus with a broader evolutionary history. Of the four common cold coronaviruses endemic to humans, two A coronaviruses 229E and NL63 are similar to SARS-CoV-2 in that they bind protein receptors via receptor-binding domains in spikes. In contrast, two beta corona-viruses OC43 and HKU1 bind glycan receptors through the n-terminal domain of the spike. Antibodies that block receptor binding dominate the immune neutralization activity induced by SARS-CoV-2 infection, so we conclude that even though SARS-CoV-2 is a type B coronavirus, its antigenicity evolution is more likely to be predicted by two human A coronaviruses that also use its RBD-binding protein receptor. Of the two viruses, we chose 229E because it was first identified in humans 50 years ago, while NL63 was only discovered in 2003.

We extrapolated the phylogenetic tree of 229E spines from direct or underpassed human isolates, excluding older strains that had been extensively passed in the laboratory. This tree has several important features. First, it acts like a clock, with sequence divergence proportional to the date of virus isolation. Second, the tree is "ladder shaped" with a short branch on the trunk. The ladder shape of 229E's phylogeny has previously been noted, a characteristic of viruses such as influenza, where immune pressures drive population turnover by selecting for antigenic variants. Third, sequence grouping by date rather than by quarantine country. Phylogeny organized by date rather than geographic location indicates rapid global spread, another characteristic of human influenza viruses. Finally, although there is some intra-ear recombination, it is between closely related strains and does not affect the broader topology of the tree. The MDA content in the heart, liver and brain tissue of D-galactose induced subacute aging model mice was significantly decreased by litenosin, the telomerase activity in the heart and brain tissue was significantly increased, the phagocytosis function of peritoneal macrophages was enhanced, and the lymphocyte proliferation response and IL-2 content in peripheral blood were significantly increased. It showed that litenoside can enhance immune function.

Pic: Effects of cistanche improve immunity

Discussion

We have experimentally demonstrated that the spikes of human coronaviruses have sufficient antigenic evolution rate to escape neutralization of many polyclonal human serums within one to two decades. The finding suggests that one reason humans are repeatedly reinfected with seasonal coronaviruses may be that the evolution of viral spikes erodes immunity triggered by previous infections.

How fast does the antigen of 229E evolve compared to the influenza virus? Notably, we did not find studies that measured the rate at which influenza evolution eroded neutralization in human serum. However, many studies have tested influenza antigen evolution using hemagglutination inhibition in the sera of ferrets infected with a single virus strain. The rate at which 229E is neutralized by human serum is several times slower than the rate at which A/H3N2 influenza viruses are neutralized by ferret serum, but comparable to the rate at which influenza b viruses escape. However, ferret sera infected with a single strain of influenza virus tend to recognize fewer strains than human sera infected multiple times with multiple strains. Thus, ferret serum HAI may overestimate the rate of erosion of influenza evolution on actual human serum neutralization. In this regard, further work is needed to make a positive comparison of the antigenic evolution of these viruses.

The big question is what our work means for the evolution of possible antigens for SARS-CoV-2. While it is impossible to know whether SARS-CoV-2 will evolve like 229E, it is certain that mutations affecting serum neutralization in polyclonal humans are already present in the SARS-CoV-2 lineage, although a large proportion of the population is still naive and therefore likely to have little immune stress to the virus. But according to our observations, even if human coronaviruses evolve to evade neutralization immunity, two facts offer hope. First, the level of immunity required to prevent severe COVID-19 may be lower, possibly because a slower course of disease has more time to mount an immune response than a "faster" virus such as influenza. An optimistic explanation is that the disease may usually be mild, even if the viral antigens evolve to allow reinfection. Second, many of the leading SARS-CoV-2 vaccines use new technologies, such as MRNa-based delivery, that can easily update the vaccine if antigen evolution occurs. Therefore, we suggest that the evolution of SARS-CoV-2 should be monitored for antigenic mutations that might suggest regular vaccine updates.

Click here to boost your COVID-19 immunity

Neutralization assays

293T cells used in neutralization experiments were transfected with APN and TMPRSS2 and seeded according to the virus titer described above. Neutralization experiments were performed 20-24 hours after cells were seeded into 96-well plates. First, the heat-inactivated serum samples were diluted 1:10 in D10 growth medium, then consecutively 3 times in the 96-well "setting" plate of the tc coating, eventually diluting each sample a total of 7 times. Each serum sample was diluted twice. Each 229E spike-pseudotyped lentivirus was then diluted to achieve a luciferase reading of about 200,000 RLUs per well. Equal amounts of virus were then added to each well of virus and serum "culture" plates, which were incubated at 37˚C and 5% CO2 for 1 hour, after which 100 uL of each virus and serum mixture was transferred to 293T-APN-TMPRSS2 cell plates seeded the day before. Culture dishes were incubated at 37˚C and 5% CO2 for about 50-52 h, and the luciferase signal was read as described above for viral titer determination.

Each neutralization plate contains a list of positive control Wells consisting of cells and viruses, cultured in D10 medium but free of serum, and negative controls consisting of viruses but free of cells (we also confirmed that similar results were obtained using cell-only negative controls). After subtracting the background readings for the negative control group, the infectivity score was calculated at each serum dilution as the signal score for the positive control group (averaged across the two positive control holes in each row). We then use neutral curves to fit 2-parameter Hill curves with baselines fixed at 1 and 0. Note that the serum concentrations reported in these curves are the concentrations of the virus pre-incubated with the serum for 1 hour. All tests were repeated, but some serovirus pairs had additional readings as we re-ran selected serovirus pairs to confirm that the results remained consistent across trial days. In all cases, daily consistency was good and reported values were average ic50 for all measurements. Desert Cistanche polysaccharide can reduce MDA content in brain, heart and liver of immunocompromised animal models, increase telomerase activity in heart and brain tissue, phagocytosis function of peritoneal macrophages, proliferation response of lymphocytes, IL-2 content in spleen T lymphocytes and calcium ion concentration in thymus cells. These indicators indicate that cistanche polysaccharide can enhance immune function.

References

1. Addetia A, Crawford KHD, Dingens A, Zhu H, Roychoudhury P, Huang M-L, et al. Neutralizing Antibodies Correlate with Protection from SARS-CoV-2 in Humans during a Fishery Vessel Outbreak with aHigh Attack Rate. J Clin Microbiol. 2020; 58. https://doi.org/10.1128/JCM.02107-20 PMID: 32826322

2. Lumley SF, O’Donnell D, Stoesser NE, Matthews PC, Howarth A, Hatch SB, et al. Antibody Status andIncidence of SARS-CoV-2 Infection in Health Care Workers. N Engl J Med. 2021; 384: 533–540. https://doi.org/10.1056/NEJMoa2034545 PMID: 33369366

3. Polack FP, Thomas SJ, Kitchin N, Absalon J, Gurtman A, Lockhart S, et al. Safety and Efficacy of theBNT162b2 mRNA Covid-19 Vaccine. N Engl J Med. 2020;0: null. https://doi.org/10.1056/NEJMoa2034577 PMID: 33301246

4. Alsoussi WB, Turner JS, Case JB, Zhao H, Schmitz AJ, Zhou JQ, et al. A Potently Neutralizing AntibodyProtects Mice against SARS-CoV-2 Infection. J Immunol. 2020; 205: 915–922. https://doi.org/10.4049/jimmunol.2000583 PMID: 32591393

5. McMahan K, Yu J, Mercado NB, Loos C, Tostanoski LH, Chandrashekar A, et al. Correlates of protection against SARS-CoV-2 in rhesus macaques. Nature. 2020; 1–8. https://doi.org/10.1038/s41586-020-03041-6 PMID: 33276369

6. Walls AC, Park Y-J, Tortorici MA, Wall A, McGuire AT, Veesler D. Structure, Function, and Antigenicityof the SARS-CoV-2 Spike Glycoprotein. Cell. 2020; 181: 281–292.e6. https://doi.org/10.1016/j.cell.2020.02.058 PMID: 32155444

7. Zost SJ, Gilchuk P, Case JB, Binshtein E, Chen RE, Nkolola JP, et al. Potently neutralizing and protective human antibodies against SARS-CoV-2. Nature. 2020; 584: 443–449. https://doi.org/10.1038/s41586-020-2548-6 PMID: 32668443

8. Edridge AWD, Kaczorowska J, Hoste ACR, Bakker M, Klein M, Loens K, et al. Seasonal coronavirusprotective immunity is short-lasting. Nat Med. 2020; 26: 1691–1693. https://doi.org/10.1038/s41591-020-1083-1 PMID: 32929268

9. Hendley JO, Fishburne HB, Jack M. Gwaltney J. Coronavirus Infections in Working Adults. Am RevRespir Dis. 1972; 105: 805–811. https://doi.org/10.1164/arrd.1972.105.5.805 PMID: 5020629

10. Schmidt OW, Allan ID, Cooney MK, Foy HM, Fox JP. Rises in titers of antibody to human corona virusesOC43 and 229E in Seattle families during 1975–1979. Am J Epidemiol. 1986; 123: 862–868. https://doi.org/10.1093/oxfordjournals.aje.a114315 PMID: 3008551

11. Aldridge RW, Lewer D, Beale S, Johnson AM, Zambon M, Hayward AC, et al. Seasonality and immunityto laboratory-confirmed seasonal coronaviruses (HCoV-NL63, HCoV-OC43, and HCoV-229E): resultsfrom the Flu Watch cohort study. Wellcome Open Res. 2020; 5: 52. https://doi.org/10.12688/wellcomeopenres.15812.2 PMID: 33447664

12. Ibarrondo FJ, Fulcher JA, Goodman-Meza D, Elliott J, Hofmann C, Hausner MA, et al. Rapid Decay ofAnti–SARS-CoV-2 Antibodies in Persons with Mild Covid-19. N Engl J Med. 2020; 383: 1085–1087.https://doi.org/10.1056/NEJMc2025179 PMID: 32706954

13. Crawford KHD, Dingens AS, Eguia R, Wolf CR, Wilcox N, Logue JK, et al. Dynamics of neutralizing antibody titers in the months after SARS-CoV-2 infection. J Infect Dis. 2020; 223: 197–205. https://doi.org/10.1093/infdis/jiaa618 PMID: 33000143

14. Gaebler C, Wang Z, Lorenzi JCC, Muecksch F, Finkin S, Tokuyama M, et al. Evolution of AntibodyImmunity to SARS-CoV-2. Nature. 2021. https://doi.org/10.1038/s41586-021-03207-w PMID:33461210

15. Rodda LB, Netland J, Shehata L, Pruner KB, Morawski PA, Thouvenel CD, et al. Functional SARSCoV-2-specific immune memory persists after mild COVID-19. Cell. 2020 [cited 8 Dec 2020]. https://doi.org/10.1016/j.cell.2020.11.029 PMID: 33296701

16. Wajnberg A, Amanat F, Firpo A, Altman DR, Bailey MJ, Mansour M, et al. Robust neutralizing antibodies to SARS-CoV-2 infection persist for months. Science. 2020; 370: 1227–1230. https://doi.org/10.1126/science.abd7728 PMID: 33115920

17. Couch RB, Kasel JA. Immunity to influenza in man. Annu Rev Microbiol. 1983; 37: 529–549. https://doi.org/10.1146/annurev.mi.37.100183.002525 PMID: 6357060