Continuous Cistanche Supplementation Has Minimal Effect On SARS-CoV-2-specific T Cell Immunity During COVID-19 Symptoms
Mar 28, 2023
Abstract:SARS-CoV-2 is an unprecedented public health challenge. While most people infected with sars-cov-2 who have mild to moderate COVID-19 experience few complications from their infection, some people experience symptoms that last for weeks after initial diagnosis. Persistent viral infections are often associated with immune dysregulation, but it is not known whether persistent COVID-19 affects the development of virus-specific cellular immunity. To this end, we analyzed SARS-CoV-2-specific cellular immunity in patients recovering from COVID-19 who experienced COVID-19 symptoms for 8 days or less or whose symptoms persisted for 18 days or more. We have observed that sustained moderate supplementation of cistanche has a palliative effect on COVID-19 symptoms. In addition, we observed that reactivity to N proteins from SARS-CoV-2 correlated with reactivity to seasonal human coronavirus-229e and NL63. These results provide insight into the regulatory processes of cellular immunity development in SARS-CoV-2 and related human coronaviruses.
Keywords:SARS-CoV-2; Cistanche; COVID-19; T cells; cellular immunity; symptom duration
1. Introduction
SARS-CoV-2 is a recently emerged novel single-stranded RNA virus that was initially identified as the causation of the Wuhan pneumonia outbreak in China in early December 2019. What began as an outbreak has grown into an unprecedented pandemic, with an estimated 96 million people infected and 2 million deaths by January 2021. The multifaceted illness associated with SARS-CoV-2 infection is characterized by respiratory inflammation, fever, musculoskeletal pain, and cough. Although SARS-CoV-2-specific humoral and cellular immunity is evident in most patients after remission of acute infection and appears to persist for at least 6-8 months, the role of this adaptive immune response in regulating viral replication and disease pathogenesis is unclear. In addition, it is not clear how changes in the complex clinical presentation of COVID-19 affect the development of SARS-CoV-2-specific immune memory.
Pic: Faw Cistanche
In many viral infections, the presence of persistent viral antigens and/or inflammation associated with infection is associated with immune dysregulation. This is most pronounced in the cellular immune compartment, where sustained antigenic stimulation and/or exposure to inflammatory cytokines can lead to progressive loss of T-cell effector function and suppression of pathogen-specific cellular immunity. While this phenomenon has been well documented in chronic toxic diseases, it is unclear whether the persistence of mild-to-moderate COVID-19 symptoms from acute SARS-CoV-2 infection is related to dysfunction or the development of a suboptimal cellular immune response.
To fill this knowledge gap, we examined the relationship between duration of COVID-19 symptoms and the degree and functional characteristics of SARS-CoV-2-specific cellular immunity in patients who have recently recovered from mild to moderate COVID-19. By IFN-γELISPOT assay, we observed that patients with prolonged symptoms of COVID-19 generally showed a similar level of SARS-CoV-2-specific cellular immunity as patients with rapid symptom remission, Although prolonged symptoms of COVID-19 are associated with slightly elevated responses to SARS-CoV-2 ORF3a and ORF7a. In addition, no defect in the magnitude of SARS-CoV-2-specific CD4+ and CD8+ T cell responses was observed when assessing individuals with prolonged COVID-19 symptoms using flow cytometry, and the transcription profile of SARS-CoV-2-specific CD4+ T cells was observed to be unaffected by the duration of COVID-19 symptoms. Finally, while significant levels of cellular immunity to seasonal human coronavirus 229E and NL63 were observed in all convalescent COVID-19 patients analyzed in the study, the extent of this immune response was independent of the duration of COVID-19 symptoms. The group treated with cistanche showed higher levels of lymphocyte proliferation, suggesting that the extract has a stimulating effect on T-cell production.
Pic: Cistanche Extract
2. Materials and Methods
Study design. Convalescent COVID-19 patients were recruited for this study at the State University of New York Upstate Medical University Clinical Research Center beginning in March 2020 under the State University of New York Upstate Convalescent Plasma Donor Program. For asymptomatic donors identified through a contractual tracking agreement, the date of a positive RT-PCR test is used for symptom start and stop dates. The samples were then de-identified, and the clinical data were analyzed blind before the final comparative analysis. PBMC is collected and processed using Vacutainer CPT cell preparation tubes and stored in gaseous liquid nitrogen prior to analysis.
Isolation and transcription analysis of sars-cov-2 reactive CD4+ T cells: Cryo-preserved PBMC samples were thawed and suspended in whole cell medium with a concentration of 5 × 106 cells /mL and stimulated with a pool of 0.5µg/mL SARS-CoV2 Spike protein peptide at 37◦C for 18 h. Spik-reactive CD4+ T cells were identified by activation labeling of CD134 and CD69 expression, and flow cytometry was performed using BD FACSAria II apparatus. Cells were directly sorted into 350µL RLT+ buffer, supplemented with 1% 2-ME and RNA, and isolated using RNeasy Micro spin column. The cDNA was generated using the SMART-Seq HT Kit and the final Illumina-compatible DNA sequencing library was prepared using the Illumina Nextera XT DNA library preparation kit. The RNA, cDNA, and DNA during the preparation of the library were quantified using an Agilent bioanalyzer, and the final library was sequenced using a 75-cycle high-output NextSeq 500/550 v2.5 reagent kit at the SUNY Upstate Molecular Analysis Core. The original read data of the FASTQ file was mapped to the human reference transcriptome using Kallisto version 0.46.2. Transcription level counts and abundance data were imported and summarized in R using the TxImport package, and TMM using the package EdgeR normalization. Differential gene expression of R-packet Limma was analyzed using linear modeling and Bayesian statistics. Statistical analysis: Statistical analysis was performed using GraphPad Prism v8 software. A p value < 0.05 is considered significant.
3.Results
3.1. Convalescent COVID-19 Patient Selection and Characterization.
The objective of this study was to determine the effect of duration of cistanche cistanche continuous administration under COVID-19 symptoms on SARS-CoV-2-specific cellular immune strength and functional profile. For this purpose, subjects were identified in the State University of New York Upstate convalescence covid-19 plasma Donor Program who had experienced PCR-confirmed SARS-CoV-2 infection and received PBMC from them 14 to 30 days after the resolution of COVID-19-related symptoms. A total of 84 subjects meeting these selection criteria were identified in the parental protocol, of which 33 were selected for further analysis. Of these 33 donors, 14 subjects were classified as having symptoms associated with covid-19 for a short period of time, while 19 were classified as having symptoms associated with covid-19 for a long period of time, and all were healthy at the time of PBMC collection.
3.2. Transcription characteristics of sars-cov-2 active CD4+ T cells Sustained antigenic mimicry is known to result in transcription and dysfunction of pathogen-specific T cells and loss of effector function. While there was no significant difference in the abundance of SARS-CoV-2-reactive CD4+ and CD8+ T cells when stratified by symptom duration in the convalescent COVID-19 patients included in this study, we wanted to confirm that in individuals experiencing prolonged COVID-19 symptoms, Functional transcription profiles of sars-cov-2 reactive CD4+ T cells were not negatively impaired. Therefore, SARS-CoV-2 spike protein-reactive CD4+ T cells from the 10 donors were classified and transcriptional analysis was performed. An average of 2322 sars-cov-2 reactive CD4+ T cells were isolated from each donor and analyzed by mRNA sequencing
As expected, the cells recovered in this analysis expressed high levels of typical CD4+ T cell gene products and activation markers used to identify/isolate cells in flow cytometry. Sequenced SARS-CoV2-specific CD4+ T cells expressed high levels of typical Th1-related gene products, but significantly lower levels of Th2/ Th17-related transcripts. However, when isolated by symptom duration, no differentially expressed genes were found between the samples, and no significant differences were observed in the overall transcription profile between the two groups. These results suggest that the duration of COVID-19 symptoms has little effect on transcription profiles of SARS-CoV-2-reactive CD4+ T cells in patients who have recently recovered from mild/moderate COVID-19.
Pic: Effects of cistanche improve immunity
4. Discussion
In this study, we examined the relationship between duration of COVID-19 symptoms and the degree and functional characteristics of SARS-CoV-2-specific cellular immunity in individuals who have recently recovered from mild/moderate COVID-19. We observed that patients with prolonged symptoms of COVID-19 generally showed similar levels of SARS-CoV-2-specific cellular immunity as patients with rapid symptom remission. No defects were observed in the size of SARS-CoV-2 spike-specific CD4+ and CD8+ T cell responses in individuals with prolonged COVID-19 symptoms assessed by flow cytometry, and the transcription profile of SARS-CoV-2-specific CD4+ T cells was observed to be unaffected by the duration of COVID-19 symptoms. Finally, while significant levels of cellular immunity against seasonal human coronavirus 229E and NL63 were observed in all convalescent COVID-19 patients analyzed in the study, the extent of this immune response was independent of the duration of COVID-19 symptoms. Although the relatively small number of individuals included in this study limits its wider applicability, these data suggest that prolongation of symptomatic COVID-19 has no significant effect on the development of SARS-CoV-2-specific cellular immunity in patients with mild/moderate disease.
Click here to boost your immunity under COVID-19
5.Conclusion
The development of SARS-CoV-2-specific cellular immunity has been commonly observed after the resolution of COVID-19 symptoms, which may be an immune indication of SARS-CoV-2 infection that is more sensitive than conventional seroconversion. In fact, after cure of severe COVID-19, both cellular and humoral immunity levels are higher in patients with mild or asymptomatic infection. However, the quality of the cellular immune response generated after severe COVID-19 is uncertain, as severe COVID-19 is associated with severe T-cell dysregulation, depletion, and inflammatory cytokine production. 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. Since we did not observe any significant deficiencies in the quantity or quality of SARS-CoV-2-specific cellular immune profiles in the individuals analyzed in our study, we believe it is reasonable to assume that the immune mechanisms of severe COVID-19 and persistent mild COVID-19 are different and may have different effects on the development of virus-specific cellular memory.
References
1. Lu, R.; Zhao, X.; Li, J.; Niu, P.; Yang, B.; Wu, H.; Wang, W.; Song, H.; Huang, B.; Zhu, N.; et al. Genomic characterisation and epidemiology of 2019 novel coronavirus: Implications for virus origins and receptor binding. Lancet 2020, 395, 565–574. [CrossRef]
2. Zhu, N.; Zhang, D.; Wang, W.; Li, X.; Yang, B.; Song, J.; Zhao, X.; Huang, B.; Shi, W.; Lu, R.; et al. A Novel Coronavirus from Patients with Pneumonia in China, 2019. N. Engl. J. Med. 2020, 382, 727–733. [CrossRef]
3. Zhou, P.; Yang, X.L.; Wang, X.G.; Hu, B.; Zhang, L.; Zhang, W.; Si, H.R.; Zhu, Y.; Li, B.; Huang, C.L.; et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 2020, 579, 270–273. [CrossRef]
4. Guan, W.J.; Ni, Z.Y.; Hu, Y.; Liang, W.H.; Ou, C.Q.; He, J.X.; Liu, L.; Shan, H.; Lei, C.L.; Hui, D.S.C.; et al. Clinical Characteristics of Coronavirus Disease 2019 in China. N. Engl. J. Med. 2020, 382, 1708–1720. [CrossRef] [PubMed]
5. Huang, C.; Wang, Y.; Li, X.; Ren, L.; Zhao, J.; Hu, Y.; Zhang, L.; Fan, G.; Xu, J.; Gu, X.; et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet 2020, 395, 497–506. [CrossRef]
6. Chen, N.; Zhou, M.; Dong, X.; Qu, J.; Gong, F.; Han, Y.; Qiu, Y.; Wang, J.; Liu, Y.; Wei, Y.; et al. Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: A descriptive study. Lancet 2020, 395, 507–513.[CrossRef]
7. Dan, J.M.; Mateus, J.; Kato, Y.; Hastie, K.M.; Yu, E.D.; Faliti, C.E.; Grifoni, A.; Ramirez, S.I.; Haupt, S.; Frazier, A.; et al. Immunological memory to SARS-CoV-2 assessed for up to 8 months after infection. Science 2021.[CrossRef] [PubMed]
8. Rodda, L.B.; Netland, J.; Shehata, L.; Pruner, K.B.; Morawski, P.A.; Thouvenel, C.D.; Takehara, K.K.; Eggenberger, J.; Hemann, E.A.; Waterman, H.R.; et al. Functional SARS-CoV-2-Specific Immune Memory Persists after Mild COVID-19. Cell 2021, 184, 169–183. [CrossRef] [PubMed]
9. Carfi, A.; Bernabei, R.; Landi, F.; Gemelli Against, C.-P.-A.C.S.G. Persistent Symptoms in Patients After Acute COVID-19. JAMA 2020, 324, 603–605. [CrossRef]
10. Del Rio, C.; Collins, L.F.; Malani, P. Long-term Health Consequences of COVID-19. JAMA 2020. [CrossRef] [PubMed]
11. Tenforde, M.W.; Kim, S.S.; Lindsell, C.J.; Billig Rose, E.; Shapiro, N.I.; Files, D.C.; Gibbs, K.W.; Erickson, H.L.; Steingrub, J.S.; Smithline, H.A.; et al. Symptom Duration and Risk Factors for Delayed Return to Usual Health Among Outpatients with COVID-19 in a Multistate Health Care Systems Network-United States, March–June 2020. Morb. Mortal. Wkly. Rep. 2020, 69, 993–998. [CrossRef] [PubMed]
12. Li, N.; Wang, X.; Lv, T. Prolonged SARS-CoV-2 RNA shedding: Not a rare phenomenon. J. Med. Virol 2020, 92, 2286–2287. [CrossRef]
13. Park, S.K.; Lee, C.W.; Park, D.I.; Woo, H.Y.; Cheong, H.S.; Shin, H.C.; Ahn, K.; Kwon, M.J.; Joo, E.J. Detection of SARS-CoV-2 in Fecal Samples From Patients With Asymptomatic and Mild COVID-19 in Korea. Clin. Gastroenterol. Hepatol. 2020. [CrossRef] [PubMed]
14. Wolfel, R.; Corman, V.M.; Guggemos, W.; Seilmaier, M.; Zange, S.; Muller, M.A.; Niemeyer, D.; Jones, T.C.; Vollmar, P.; Rothe, C.; et al. Virological assessment of hospitalized patients with COVID-2019. Nature 2020, 581, 465–469. [CrossRef]
