By Author
  By Title
  By Keywords

September 2022, Volume 72, Issue 9

Research Article

Impact of Age, Gender, post infection and post vaccination status on antibody response in COVID 19 patients

Sidra Sadiq  ( Department of Chemical Pathology and Endocrinology, Rehman Medical Institute, Peshawar, Pakistan; )
Maria Khan  ( Department of Microbiology, Rehman Medical Institute, Peshawar, Pakistan; )
Tayyaba Sadiq  ( Department of Chemical Pathology and Endocrinology, Rehman Medical Institute, Peshawar, Pakistan; )
Sheryar Orakzai  ( Rehman College of Allied Health Sciences, Peshawar, Pakistan. )
Mirza Muhammad Dawood  ( Department of Chemical Pathology and Endocrinology, Rehman Medical Institute, Peshawar, Pakistan; )
Najeeb Ul Haq  ( Rehman College of Allied Health Sciences, Peshawar, Pakistan. )

Abstract

Objective: To evaluate severe acute respiratory syndrome coronavirus-2 spike protein antibodies against coronavirus disease-2019 in post-infection and post-vaccinated individuals.

 

Method: The cross-sectional study was conducted from June, 1 to July 31, 2021 at the Rehman Medical Institute, Peshawar, Pakistan, and comprised subjects of either gender in whom immunogenicity was checked 35 days post-vaccination and 90 days post-infection. Correlation with age and gender was checked. Specimens were collected and investigated for severe acute respiratory syndrome coronavirus-2 spike protein antibodies by consuming electro-chemiluminescence immunoassay. Data was analysed using SPSS 23.

 

Results: Of the total 256 patients enrolled, 70(27.34%) were included; 49(69%) males and 21(29.6%) females. The overall mean age was 44±7.75 years. Among 30(42.8%) patients with positive polymerase chain reaction test, the mean time between the positive test and antibody screening was 90±30 days. Among the 40(57.2%) vaccinated individuals, the time between vaccination and antibody screening was 35±9.74 days. Overall, 68(97%) patients revealed robust positive findings to severe acute respiratory syndrome coronavirus-2 spike proteins antibodies >50IU/mL. Male subjects had significantly higher immunogenic response compared to females (p=0.001), and immunogenicity decreased with advancing age (p<0.001). Also, post-vaccinated patients’ antibody response was significant compared to post-infection patients’ response (p=0.001).

 

Conclusion: Majority of the patients had significantly higher antibody titers against severe acute respiratory syndrome coronavirus-2 post-infection and post-vaccination. Males and younger individuals developed a significant humoral immunity compared to females and the elderly.

 

Keywords: Antibodies, COVID-19, SARS-CoV-2, Vaccination. (JPMA 72: 1805; 2022) 

 

DOI: https://doi.org/10.47391/JPMA.5309

 

Introduction

 

The coronavirus diseased-2019 (COVID-19), a novel positive-strand ribonucleic acid (RNA) coronavirus of the Coronaviridae family was declared a pandemic by the World Health Organisation (WHO) on March 11, 2020.1 It continued to spread despite repeated lockdowns and long-term control measures in most countries. The envelope (E), membrane (M), nucleocapsid (N) and spike (S) proteins in the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genome are structural proteins. S1 and S2 are the two subunits of the S protein.2

The S1 subunit contains the receptor binding domain (RBD), which has a high affinity for the angiotensin converting enzyme 2 (ACE2) receptor on the cell surface membrane. The interaction of SARS-CoV-2 RBD with ACE2 viral receptor on the host cell is what causes the infection.2,3

The humoral immune response to infection or vaccination has two basic outcomes: antibody production by antibody secreting cells (ASCs), which can provide rapid serological immunity, and the development of long-lived memory B cells, which can mount recall responses.4,5 Memory B cells drive the recall response by producing new antibodies by establishing new ASCs or re-entering germinal centres for subsequent rounds of somatic hyper-mutation if circulating antibodies fail to protect against a future exposure.6,7 COVID-19-infected persons are expected to have antibodies and memory B cells for at least 6-8 months.8-12 In SARS-CoV-2-exposed persons, no memory responses or effective vaccine dose protocols have been investigated.13

Control techniques, such as mask-wearing, physical separation, and the use of contact tracing, were beneficial to decrease the virus spread, but no significant advantages were observed, and it was realised that vaccinations were the only realistic way out of the pandemic.14 Most COVID-19 vaccination approaches to date have aimed at generating neutralising antibodies against S protein, hence preventing SARS-CoV-2 infection in its early stages. Several vaccine candidates have been demonstrated to be safe and effective in clinical studies15 and vaccination in mass numbers (when used in combination with other existing control measures) is recognised as one of the most important aspects of pandemic management. Although the results of clinical trials are encouraging, real-world evidence on vaccines is still lacking.

The current study was planned to evaluate SARS-CoV-2 S antibody levels among post-infected and post-vaccinated individuals in a tertiary care setting.

 

Materials and Methods

 

The cross-sectional study was conducted from June, 1 to July 31, 2021, at the Rehman Medical Institute, Peshawar, Pakistan. After approval from the institutional ethics approval board, the sample size was calculated using the WHO calculator16 on the basis of literature.17 The sample was raised using purposive sampling technique. Those included were individuals of either gender aged 18-80 years, who were selected retrospectively, while those with negative polymerase chain reaction (PCR) test and those not vaccinated were excluded. Also excluded were patients receiving immunosuppressive therapy or suffering from an immunosuppression-related disease, having no history of infection or vaccination, those aged <18 years and pregnant women.

Post-vaccinated patients had received two doses of either Sinopharm or Sinovac 21 days apart as validated by the vaccination certificate. Both the vaccines contained inactivated virus. The post-infection status was validated by PCR report. Post-infection patients were divided into three groups on the basis of mild, severe or carrier state, based on the Chinese Centre for Disease Control fuidelines.18 Samples for immunoglobulin-G (IgG) quantitative antibodies were obtained by trained personnel on day 90 and day 35 in post-infection and post-vaccinated individuals, respectively. Written informed consent was obtained from all the subjects..

Standard protocols were used to collect a nasopharyngeal swab, and the presence of SARS-CoV-2 was identified by reverse transcriptase PCR (RT-PCR) (Light Mix Modular SARS-CoV-2 COVID-19 RdRP, Roche, Switzerland) testing. According to manufacturer’s recommendations, a cycle threshold (CT) result of 15-30 was considered positive.

An electro-chemiluminescence immunoassay (ECLIA) (Abbot Architect, United States) based on double-antigen sandwich assay principle was tested (Abbot Advice Dx SARS-CoV-2 IgG II assay, US) for quantitatively determining antibody levels to the SARS-CoV-2 S protein. Blood samples were collected using acid citrate dextrose, sodium citrate, potassium ethylenediaminetetraacetic acid (EDTA), tri-potassium EDTA or lithium heparin tubes. This competitive serological assay simultaneously determined an individual’s seropositivity against the SARS-CoV-2 S protein and estimated the neutralising capacity of anti-S antibodies to block interaction with the human ACE2 required for viral entry. Natively-folded viral S protein RBD-containing antigens via avidin-biotin interactions were noted. Sera were then supplemented with soluble ACE2-fragment crystallizable (Fc) fusion to compete for RBD-binding serum antibodies, and antibody binding was quantified. Comparison of signal from untreated serum and ACE2-Fc-treated serum revealed the presence of antibodies that compete with ACE2 for RBD-binding, as evidenced by loss of signal with ACE2-Fc treatment. The manufacturers recommended antibodies cutoff level of 50.0AU/mL, above which the value was considered positive. The diagnostic measuring interval of 22.0 to 25000.0 AU/mL was found to be the linearity.

Data was analysed using SPSS23. Age, gender, time duration of sample collection were all subjected to descriptive statistics. Pearson correlation was used to establish the correlation of age, gender, post-infection and post-vaccination status with antibody levels. P<0.05 was taken as statistically significant.

 

Results

 

Of the total 256 patients enrolled, 70(27.34%) were included; 49(69%) males and 21(29.6%) females. The overall mean age was 44±7.75 years. Among 30(42.8%) patients with positive PCR test, the mean time between positive the test and antibody screening was 90±30 days. Among the 40(57.2%) vaccinated individuals, the time between vaccination and antibody screening was 35±9.74 days (Table 1).

 

 

Overall, 68(97%) patients revealed robust positive findings to SARS-CoV-2 S protein antibodies >50IU/mL. Male subjects had significantly higher immunogenic response compared to females (p=0.001), and immunogenicity decreased with advancing age (p<0.001). Also, post-vaccinated patients’ antibody response was significant compared to post-infection patients’ response (p=0.001) (Table2).

 

 

Among the 30(42.8%) post-infection patients 12(40%) had severe symptoms (Table 3), and among those with severe symptoms, the most common was decrease in oxygen saturation 5(41.6 %) (Table 4) None of the vaccinated individuals had previously encountered Covid-19 infection.

 

 

 

 

Discussion

 

In the current study, SARS CoV-2 post-infection or post-vaccination individuals were selected for investigation. A substantial portion of the enrolled patients in study presented with remarkably higher anti-SARS-CoV-2 antibody levels of >50 AU/mL, and the results are comparable with other clinical trials.17,19

People who had been previously exposed to COVID-19 had significant humoral immunogenic response compared to non-infected individuals which was consistent with  earlier findings.20

Only a few studies have assessed gender differences in COVID-19 incidence and disease progression and an independent review of the responsible factors is still absent. Based on differences in innate and adaptive immunity, steroid hormone synthesis by the gonads, and sex chromosomal variables, it is interesting to note that males showed a greater immunological response than females in an Italian study.21 In viral infections, sex hormones are known to regulate innate immune responses. Oestrogens influence receptor responsiveness and the creation of pro-inflammatory cytokines, which can be life threatening if released in excess. Oestrogens that bind to the oestrogen receptor alpha (ERa) or beta receptor (ERb) can thereby influence the immune system. All immune cells express ERa, which is essential in their maturation and regulation. It is also immunologically protective, since it is involved in the generation of interferon (IFN) type I and the activation of natural killer (NK) cells. ERb is engaged in pro-inflammatory events and has the opposite effects. The decrease of ERa in elderly women is linked with immunosuppression, indicating that oestrogen can protect against COVID-19.22

It has been reported that antibody levels decline with advancing age.19,23 In one study on 34 individuals in the USA, peak antibody titers occurred between 30 and 152 days post-infection.24 Similar findings have been reported from Israel.19

Protective correlations have already been established for a range of viral diseases. These associations are typically established on a certain titer of antibody obtained from vaccination or spontaneous infection, which considerably diminishes the chance of (re)infection, like for example, the Hem agglutination inhibition level for influenza virus, where a 1:40 level minimises the possibility of transmission by 50%.25 It is uncertain whether human infection with SARS-CoV-2 prevents against reinfection26 and if so, for how long. We know that neutralising antibodies are produced by common human coronaviruses and that these antibodies can last for decades, preventing against reinfection or attenuating symptoms if reinfection occurs.27

It is perceived that infection with SARS-CoV-2 protects non-human primate models from reinfection for at least some duration.28 Although there is no definitive proof that these antibody responses guard against reinfection, that is extremely probable to reduce the likelihood of reinfection and, in the instance of breakthrough infection, may attenuate symptoms.

When immune reactions to vaccine were studied among 100 participants in Pakistan, considerably robust immune response was noted after a single vaccine shot17 and similar results were stated by another study.17,29 The majority of the patients developed humoral response 35 days post-administration of the second dose of  COVID-19 vaccine.19

The current study had the limitation of a small sample size. Establishing immunogenicity in post-infection and post-vaccination individuals, as well as the impact of age and gender on humoral immune responses, requires further research.

 

Conclusions

 

Maximum number of patients developed immunity post-infection and post-vaccination. Male and younger individuals were more efficient in developing humoral immune responses than female and older patients.

 

Acknowledgments: We are grateful to laboratory staff Afzal, Danish, Waqas, Israr and Shakeel for their technical assistance.

 

Disclaimer: None.

 

Conflict of interest: None.

 

Source of Funding:  None.

 

References

 

1.       Emmert EA. Biosafety guidelines for handling microorganisms in the teaching laboratory: development and rationale. J Microbiol Biol Educ 2013;14:78-83. doi: 10.1128/jmbe.v14i1.531.

2.       Chen X, Pan Z, Yue S, Yu F, Zhang J, Yang Y, et al. Disease severity dictates SARS-CoV-2-specific neutralizing antibody responses in COVID-19. Signal Transduct Target Ther 2020;5:180. doi: 10.1038/s41392-020-00301-9..

3.       Legros V, Denolly S, Vogrig M, Boson B, Siret E, Rigaill J, et al. A longitudinal study of SARS-CoV-2-infected patients reveals a high correlation between neutralizing antibodies and COVID-19 severity. Cell Mol Immunol 2021;18:318-27. doi: 10.1038/s41423-020-00588-2..

4.       Kurosaki T, Kometani K, Ise W. Memory B cells. Nat Rev Immunol 2015;15:149-59. doi: 10.1038/nri3802.

5.       Akkaya M, Kwak K, Pierce SK. B cell memory: building two walls of protection against pathogens. Nat Rev Immunol 2020;20:229-38. doi: 10.1038/s41577-019-0244-2.

6.       Mesin L, Schiepers A, Ersching J, Barbulescu A, Cavazzoni CB, Angelini A, et al. Restricted Clonality and Limited Germinal Center Reentry Characterize Memory B Cell Reactivation by Boosting. Cell 2020;180:92-106.e11. doi: 10.1016/j.cell.2019.11.032.

7.       Goel RR, Apostolidis SA, Painter MM, Mathew D, Pattekar A, Kuthuru O, et al. Distinct antibody and memory B cell responses in SARS-CoV-2 naïve and recovered individuals following mRNA vaccination. Sci Immunol 2021;6:eabi6950. doi: 10.1126/sciimmunol.abi6950.

8.       Seow J, Graham C, Merrick B, Acors S, Pickering S, Steel KJA, et al. Longitudinal evaluation and decline of antibody responses in SARS-CoV-2 infection. Nat Microbiol 2020;5:1598-607. doi: 10.1038/ s41564-020-00813-8.

9.       Yao XY, Liu W, Li ZY, Xiong HL, Su YY, Li TD, et al. Neutralizing and binding antibody kinetics of COVID-19 patients during hospital and convalescent phases. MedRxiv 2020. doi: 10.1101/2020.07.18. 20156810. [preprints]

10.     Isho B, Abe KT, Zuo M, Jamal AJ, Rathod B, Wang JH, et al. Persistence of serum and saliva antibody responses to SARS-CoV-2 spike antigens in COVID-19 patients. Sci Immunol 2020;5:eabe5511. doi: 10.1126/sciimmunol.abe5511..

11.     Beaudoin-Bussières G, Laumaea A, Anand SP, Prévost J, Gasser R, Goyette G, et al. Decline of Humoral Responses against SARS-CoV-2 Spike in Convalescent Individuals. mBio 2020;11:e02590-20. doi: 10.1128/mBio.02590-20.

12.     Wu J, Liang B, Chen C, Wang H, Fang Y, Shen S, et al. SARS-CoV-2 infection induces sustained humoral immune responses in convalescent patients following symptomatic COVID-19. Nat Commun 2021;12:1813. doi: 10.1038/s41467-021-22034-1.

13.     Saadat S, Rikhtegaran Tehrani Z, Logue J, Newman M, Frieman MB, Harris AD, et al. Binding and Neutralization Antibody Titers After a Single Vaccine Dose in Health Care Workers Previously Infected With SARS-CoV-2. JAMA 2021;325:1467-9. doi: 10.1001/jama.2021.3341.

14.     ElBagoury M, Tolba MM, Nasser HA, Jabbar A, Elagouz AM, Aktham Y, et al. The find of COVID-19 vaccine: Challenges and opportunities. J Infect Public Health 2021;14:389-416. doi: 10.1016/j.jiph. 2020.12.025.

15.     Polack FP, Thomas SJ, Kitchin N, Absalon J, Gurtman A, Lockhart S, et al. Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine. N Engl J Med 2020;383:2603-15. doi: 10.1056/NEJMoa2034577.

16.     World Health Organization. Sample size calculator for evaluation of COVID-19 vaccine effectiveness. [Online] 2021 [Cited 2021 March 17]. Available from URL: https://apps.who.int/iris/handle/10665/340303

17.     Saeed U, Uppal SR, Piracha ZZ, Khan AA, Rasheed A, Waheed A, et al. Evaluation of SARS-CoV-2 spike antibody levels among Sputnik V first dose vaccinated people in Pakistan: formulation of national anti-COVID-19 mass vaccination strategy. Res Sq 2021. DOI: 10.21203/rs.3.rs-480406/v1. [preprints]

18.     Shi H, Han X, Jiang N, Cao Y, Alwalid O, Gu J, et al. Radiological findings from 81 patients with COVID-19 pneumonia in Wuhan, China: a descriptive study. Lancet Infect Dis 2020;20:425-34. doi: 10.1016/S1473-3099(20)30086-4.

19.     Abu Jabal K, Ben-Amram H, Beiruti K, Batheesh Y, Sussan C, Zarka S, et al. Impact of age, ethnicity, sex and prior infection status on immunogenicity following a single dose of the BNT162b2 mRNA COVID-19 vaccine: real-world evidence from healthcare workers, Israel, December 2020 to January 2021. Euro Surveill 2021;26:2100096. doi: 10.2807/1560-7917.ES.2021.26.6.2100096.

20.     Rodda LB, Netland J, Shehata L, Pruner KB, Morawski PA, Thouvenel CD, et al. Functional SARS-CoV-2-Specific Immune Memory Persists after Mild COVID-19. Cell 2021;184:169-83.e17. doi: 10.1016/ j.cell.2020.11.029..

21.     Conti P, Younes A. Coronavirus COV-19/SARS-CoV-2 affects women less than men: clinical response to viral infection. J Biol Regul Homeost Agents 2020;34:339-43. doi: 10.23812/Editorial-Conti-3..

22.     Angele MK, Pratschke S, Hubbard WJ, Chaudry IH. Gender differences in sepsis: cardiovascular and immunological aspects. Virulence 2014;5:12-9. doi: 10.4161/viru.26982..

23.     Pellini R, Venuti A, Pimpinelli F, Abril E, Blandino G, Campo F, et al. Obesity may hamper SARS-CoV-2 vaccine immunogenicity. MedRxiv 2021. doi: 10.1101/2021.02.24.21251664. [preprints]

24.     Crawford KHD, Dingens AS, Eguia R, Wolf CR, Wilcox N, Logue JK, et al. Dynamics of Neutralizing Antibody Titers in the Months After Severe Acute Respiratory Syndrome Coronavirus 2 Infection. J Infect Dis 2021;223:197-205. doi: 10.1093/infdis/jiaa618.

25.     Krammer F, Weir JP, Engelhardt O, Katz JM, Cox RJ. Meeting report and review: Immunological assays and correlates of protection for next-generation influenza vaccines. Influenza Other Respir Viruses 2020;14:237-43. doi: 10.1111/irv.12706.

26.     Hall VJ, Foulkes S, Charlett A, Atti A, Monk EJM, Simmons R, et al. Do antibody positive healthcare workers have lower SARS-CoV-2 infection rates than antibody negative healthcare workers? Large multi-centre prospective cohort study (the SIREN study), England June to November 2020. MedRxiv 2021. doi: 10.1101/2021. 01.13.21249642 [preprints]

27.     Huang AT, Garcia-Carreras B, Hitchings MDT, Yang B, Katzelnick LC, Rattigan SM, et al. A systematic review of antibody mediated immunity to coronaviruses: kinetics, correlates of protection, and association with severity. Nat Commun 2020;11:4704. doi: 10.1038/s41467-020-18450-4.

28.     Deng W, Bao L, Liu J, Xiao C, Liu J, Xue J, et al. Primary exposure to SARS-CoV-2 protects against reinfection in rhesus macaques. Science 2020;369:818-23. doi: 10.1126/science.abc5343.

29.     Poland GA, Ovsyannikova IG, Kennedy RB. SARS-CoV-2 immunity: review and applications to phase 3 vaccine candidates. Lancet 2020;396:1595-606. doi: 10.1016/S0140-6736(20)32137-1.

Journal of the Pakistan Medical Association has agreed to receive and publish manuscripts in accordance with the principles of the following committees: