PEMERIKSAAN

PASCA VAKSINASI COVID-19

Prof. Dr. Aryati, dr., MS, SpPK(K)

Ketua Umum Pengurus Pusat
Perhimpunan Dokter Spesialis Patologi Klinik (PDS PatKLIn)
Sabtu, 20 Maret 2021
3
25 Februari 2021

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Mahmood et al. 6 August 2020. Vaccines 2020, 8, 443; doi:10.3390/vaccines8030443
 VAKSIN YANG AKAN MASUK KE INDONESIA

https://indonesia.go.id/narasi/indonesia-dalam-angka/sosial/vaksinasi-mandiri-masih-
https://sehatnegeriku.kemkes.go.id
menunggu-hari
Ophinni et al, 2020. Acta Med Indones - Indones J Intern Med • Vol 52 • Number 4 • October 2020
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APA YANG DIHARAPKAN DARI VAKSINASI?

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Potential Endpoints of An Efficacious COVID-19 Vaccine (Hodgson et al, 2020)

Penilaian efikasi vaksin dengan

membandingkan clinical endpoint antara
kelompok yang divaksin dan tidak divaksin
adalah metode yang paling efektif

Penilaian yang mengandalkan paparan alami

SARS-CoV-2 rentan dipengaruhi banyak faktor

Hodgson et al, 27 October 2020 https://doi.org/10.1016/S1473-3099(20)30773-8 9

A variety of approaches have been used to identifiy, confirm
and evaluate immunological markers as indicators of vaccine-
induced protection
Trials and Other Experimental Designs

• Randomized Controlled Trials with Clinical Endpoints

• Immunogenicity Studies
• Passive Immunization Studies
• Challenge Studies

Observational Studies

• Cohort Studies
• Natural history studies
• Maternal-newborn studies
• Case-control studies
• Ecological studies
WHO, 2013. Correlates of Vaccine-Induced Protection: Methods and Implications
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 Phase 3 efficacy trial:
• Very costly and time
consuming
• Involve more than
• Immune marker (either humoral/cellular thousands individuals in
response) could reliably predict risk of SARS-CoV-2
protection against disease exposure2
• Can be used as surrogate endpoint to
assess vaccine efficacy2
1 WHO, 2013. Correlates of Vaccine-Induced Protection: Methods and Implications
2Jin et al,2021 Signal Transduction and Targeted Therapy (2021) 6:48 11
ANTIBODI KUANTITATIF SEBAGAI PENANDA
PROTEKSI?

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STRUKTUR SARS-COV-2

Mahmood et al. 6 August 2020. Vaccines 2020, 8, 443; doi:10.3390/vaccines8030443


• Spike proteins mediate the contact with the host cell by
binding to ACE2 receptor, a process necessary for the virus
entry
• S protein contains 2 subunits: S1N-terminal domain,
responsible for receptor binding, and S2 C-terminal domain,
responsible for the fusion.
• S2 subunit is the most conserved one
• While the S1 subunit differs even within species of the same
coronaviruses. The S1 contains two sub-domains (N-
terminal and C-terminal), which are both show receptor-
binding functions
Viruses 2019, 11, 59; doi:10.3390/v11010059
The Nucleocapsid protein regulates the viral
genome transcription, replication and packaging,
and it is essential for viability. The N protein is of
potential interest for vaccine development as it is
highly immunogenic and its amino acid sequence is
highly conserved
Forcelloni et al, 2020. https://doi.org/10.1101/2020.05.14.095133 14
• Life cycle of highly pathogenic human CoVs. 15
Jiang et al, 2020. https://doi.org/10.1016/j.it.2020.03.007
Schematic representation of convalescent plasma components
and its mechanisms of action
Rojas et al, 2020 https://doi.org/10.1016/j.autrev.2020.102554
Galipeau et al, 2020. Front. Immunol., 18 December 2020 | https://doi.org/10.3389/fimmu.2020.610688
 Pan-SARS-CoV-2 S1-RBD Ig in levels at the first follow-up was a
median of 66 U/L (IQR, 25–174).

Interestingly, the antibody levels significantly increased to the

second follow-up (median 139 days) to a median of 109 U/L
(IQR, 46–227) (p < 0.001).
Anti-RBD S1 antibody = Elecsys® Anti-SARS-CoV-2
S antibody assay (pan-SARS-CoV-2 S1-RBD Ig) Schaffner et al, J Clin Med 2020 Dec 9;9(12):3989. doi: 10.3390/jcm9123989
 Titer antibodi anti-RBD tinggi
berkorelasi dengan netralisasi,
antibody-mediated complement
deposition (ADCD), and antibody-
dependent neutrophil phagocytosis
(ADNP)
Respon imun humoral terbatas tampak
pada individu dengan titer anti-RBD
rendah
0,1 ug/mL 0,1 ug/mL 0,25 ug/mL

IgG RBD = in-house ELISA

Positive cutoff = mean OD - converted μg/ml values

of the negative control wells on the respective
plate + five times the standard deviation of the
concentration from negative plasma samples

Bartsch et al, 15 February 2021

https://doi.org/10.1038/s41467-021-21336-8
 In order to mount a robust immune
response against an invading pathogen,
both adaptive and innate arms of the
immune system work in conjunction.

Though antibodies are traditionally

considered as necessary molecules of
immune defense, their generation relies
on effective cross-talk with the T-cells.

MARKER OF PROTECTION?

Jain et al, 3 November 2020. Vaccines 2020, 8(4), 649; https://doi.org/10.3390/vaccines8040649

Zhou & Zhao, 2020; 16(10): 1718-1723. doi: 10.7150/ijbs.45123

The titers of NAb correlated with the

spike-binding antibodies targeting S1,
RBD, and S2 regions (Wu et al, 2020)
21
Speiser & Bachmann. 22 July 2020. Vaccines 2020, 8, 404; doi:10.3390/vaccines8030404
Terdapat ikatan spesifik dose-
dependent antara hACE2
dengan RBD atau S1, namun
tidak terhadap nucleocapsid Berbagai tes antibodi terhadap Spike atau S-RBD berkorelasi
positif dengan Neutralization Test (r 0,237 – 0,447), kecuali anti-
RBD lebih baik dibandingkan NP (r = 0,094)2
S1 dalam mendeteksi Nab
(pada sVNT)1 Neutralizing Ab = HIV-1 pseudotyped virus neutralizing assay

Anti-RBD/S1/N = indirect ELISA

sVNT = Genscript
1Tan et al. 23 July 2020 Nature Biotechnology volume 38, pages1073–1078(2020)
Neutralizing Ab = cVNT & pVNT
2Luchsinger et al, 2020. Journal of Clinical Microbiology doi: 10.1128/JCM.02005-20
 SUMMARY OF THE KEY COMPONENTS OF A VIRAL NEUTRALIZATION ASSAY

Plaque Reduction Neutralization Test

(PRNT) → gold standard neutralizing
antibody

Prosedur rumit, butuh kultur sel

Membutuhkan BSL-2 atau BSL-3 Focosi et al. 12 August 2020 Rev Med Virol. 2020;e2170. https://doi.org/10.1002/rmv.2170
 Surrogate Virus Neutralization Test → deteksi Nab
dengan meniru interaksi virus-host pada plate ELISA

Anti-SARS-CoV-2 neutralizing antibodies block the SARS- Anti-SARS-CoV-2 neutralizing antibodies block HRP-
CoV-2 spike protein from binding to hACE2 receptor conjugated RBD protein from binding to the hACE2
proteins on the host cell surface protein pre-coated on an ELISA plate
Tan et al. 23 July 2020 Nature Biotechnology volume 38, pages1073–1078(2020)
 Terdapat korelasi antara sVNT (Genscript
Pass) dengan PRNT-50 (r = 0,73) serta
dengan PRNT-90 (r = 0,65)

Kelemahan sVNT:
• Deteksi antibodi non netralisasi
• Crossreactivity dengan SARS-CoV-1 dan
syphilis

Valcourt et al. 24 December 2020. https://doi.org/10.1016/j.diagmicrobio.2020.115294

All binding assays except the DiaSorin S1/2 IgG showed a
quadratic relationship with the sVNT. However, for the DiaSorin
S1/2, the quadratic curve approached a straight line, indicating
a mostly linear relationship between this binding assay and the
sVNT within the observed range. Perkmann et al, 08 March, 2021 . medRxiv (preprint) doi: https://doi.org/10.1101/2021.03.05.21252977
Kelompok sera pasien konvalesen COVID-19 dengan
kadar IgM dan IgG rendah (ELISA) masih menunjukkan
inhibisi 70-90% pada sVNT (Genscript) Tan et al, 23 July 2020. Nat Biotechnol 38, 1073–1078 (2020).
https://doi.org/10.1038/s41587-020-0631-z

IgG anti-RBD berkorelasi dengan
antibodi netralisasi
IgG-RBD = ELISA
Ab neutralisasi = neutralization assay
Peterhoff et al. Infection volume 49, pages75–82(2021)
Anti-RBD IgG dan anti-ECD (anti-spike ectodomain)
berkorelasi dengan microneutralization assays (0.67 versus
0.62)
Terdapat 80% of donor memiliki titer virus neutralization
≥1:160 pada plasma konvelesen ketika titer serum anti-RBD
atau anti-EDC titers ≥ 1:1350
IgG-RBD/EDC = ELISA
Ab neutralisasi = microneutralization assay
Salazar et al, 2020 bioRxiv. Preprint. 2020 Jun 9. doi: 10.1101/2020.06.08.138990
RESPON IMUN PASCA VAKSINASI
 GMT 121 GMT 247 GMT 74 GMT 215

Total IgG anti (whole virus) SARS-CoV-2 = ELISA

Effect of an Inactivated Vaccine Against SARS-CoV-2 on Safety Neutralizing Ab = PRNT50
and Immunogenicity Outcomes (Sinopharm) GMT = Geometric Mean Titer
Xia et al. 13 August 2020 JAMA. doi:10.1001/jama.2020.15543
 Immune responses induced by the days 0 and 28 vaccination The level of neutralising antibodies (of vaccination) was
schedule were larger than those induced by the days 0 and 14 lower than those of convalescent patients who previously
vaccination schedule, regardless of the dose. had COVID-19

IgG anti-RBD = in-house ELISA Zhang et al. 17 November 2020. https://doi.org/10.1016/ S1473-
Neutralizing Ab = micro cytopathogenic effect assay (pseudovirus) 3099(20)308434 (CoronaVac Phase 1/2 Clinical Trial)
Studi pada 69 partisipan 21 hari pasca vaksinasi Pfizer
dosis pertama

In conclusion, the results of the investigated test

systems correlate well but are not necessarily
interchangeable
Perkmann et al, 08 March, 2021 . medRxiv (preprint) doi: https://doi.org/10.1101/2021.03.05.21252977
 GMT 235,228 GMT 151,761 GMT 157,946

GMT 182 GMT 167 GMT 109

At the 100-μg dose, mRNA-1273 produced high levels of binding and GMT: Geometric Mean Titer
neutralizing antibodies that declined slightly over time, as expected, but they
remained elevated in all participants 3 months after the booster vaccination
(the injections were received 28 days apart) Felson & Smolen, 7 January 2021 DOI: 10.1056/NEJMc2032195
 GMT 775 GMT 685 GMT 552

GMT 430 GMT 269 GMT 165

At day 119, the binding and neutralizing GMTs exceeded the median
GMTs in a panel of 41 controls who were convalescing from Covid-19,
with a median of 34 days since diagnosis (range, 23 to 54)
Felson & Smolen, 7 January 2021 DOI: 10.1056/NEJMc2032195
Exponential Increase in Neutralizing and Spike Specific Antibodies
Following Vaccination of COVID-19 Convalescent Plasma Donors
Spike IgG level

14 days post
2nd dose
4137 AU/mL

Male 60s

1452 AU/mL

Female
60s
3410 AU/mL

Male 50s
Spike IgG = Diasorin (positive >15 AU/mL)
Total Ig Nucleocapsid = Roche (positive COI>1,0)
Rata-rata kadar IgG-Spike pasca vaksinasi
pada 10 subjek dengan riwayat COVID-19
4166 AU/mL (rentang 1235 – 7854)
Vickers et al, 05 Fen 2021. https://doi.org/10.1101/2021.02.02.21250836
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Studi terhadap 126 subjek terkonfirmasi COVID-19

Terdapat satu subjek konvalesen dengan

antibodi negatif (<3,8 AU/mL) pasca
terkonfirmasi COVID-19 (waktu pemeriksaan
tidak diketahui) → meningkat menjadi <1000
AU/mL pasca vaksinasi

Subjek tersebut tidak menghasilkan respon

antibodi terhadap infeksi COVID-19 dan
berespon terhadap vaksinasi seperti pada
subjek yang tidak terinfeksi COVID-19

Terdapat 21% pasien dengan antibodi negatif (<15 AU/mL) PERLU PERIKSA ANTIBODI PRA-VAKSINASI PADA
PENYINTAS COVID

Vickers et al, 05 Fen 2021. https://doi.org/10.1101/2021.02.02.21250836

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BAGAIMANA MENENTUKAN KADAR
SEROPROTEKTIF?

VE = Vaccine efficacy

WHO, 2013. Correlates of Vaccine-Induced Protection: Methods and Implications

Jin et al, 2 Feb 2021 Signal Transduction and Targeted Therapy (2021) 6:48
37
Other Issues Related to Immunological Markers
for Protection
Endpoint Definition
• Protection against clinical
endpoints may require not just
different quantities of a specific
immune marker but involve
different markers
Antigen Factors
• Immunity observed from natural
exposure can be qualitatively
and/or quantitatively different
from those vaccinated
• Composition of the vaccine
Exposure intensity /
challenge dose
• The level of protection provided
by a specific Ab titre in one
population may be higher than in
different population setting
Immunological Factors
• Type of antibody (qualitative
and quantitative aspects,
include avidity and titre of
protection)
• Kinetics of immune response
WHO, 2013. Correlates of Vaccine-Induced Protection: Methods and Implications
Host Factors
• Age
• Socioeconomic status
• Environmental factors
Measurement Error and Noise
• Lack of standardization, laboratory
variability, high intra-individual
variability
• The need for valid and reliable
assays is thus emphasized by
regulatory agencies
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Angka yang tertera pada tabel
merupakan batas cut-off “high titer”
donor plasma konvalesen yang
direkomendasikan
• Plasma konvalesen akan terdilusi
ketika masuk ke tubuh pasien
• Batas cut-off tidak dapat
dikatakan berkorelasi dengan
kadar seroprotektif pasca
vaksinasi
Jin et al, 2 Feb 2021 Signal Transduction and Targeted Therapy (2021) 6:48
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FAKTOR YANG MEMPENGARUHI RESPON IMUN PASCA VAKSINASI

Zimmerman & Curtis. 13 March 2019. Clin Microbiol Rev. 2019 Apr; 32(2): e00084-18.
BAGAIMANA REKOMENDASI PEMERIKSAAN
ANTIBODI KUANTITATIF PASCA VAKSINASI?

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Anti SARS-CoV-2 antibodies titer (preferably quantitative, anti-spike/RBD) shall be assesed before
vaccination, so that SARS-CoV-2 vaccine administration can be prioritized to sero-negative individuals

IgG anti-spike/RBD shall be monitored for up to 6-8 months, preferably starting 1-2 weeks after the last dose of
vaccine, using always the same assay. This would timely identify lack of seropositivity or seronegativization

Antibodies assesment and monitoring, before and after vaccination, are necessary in patients with
immunodeficiencies and cancer

The performance characteristics (both analytical and diagnostic) of each specific test shall then be
validated

When feasible and clinically advisable, assesment of cellular immunity may also be an option

Lippi et al, 2021. Clin Chem Lab Med 2021; aop. https://doi.org/10.1515/cclm-2021-0038
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 5 March, 2021

• Antibody testing is not currently recommended to assess for immunity to SARS-CoV-2 following
COVID-19 vaccination because the clinical utility of post-vaccination testing has not been
established.
• Antibody tests currently authorized under an EUA have variable sensitivity, specificity, as well
as positive and negative predictive values, and are not authorized for the assessment of
immune response in vaccinated people
• The serologic correlates of protection have not been established, and antibody testing does
not evaluate the cellular immune response, which may also play a role in vaccine-mediated
protection.
• Finally, antibody testing against nucleocapsid will not detect immune responses resulting from
vaccination, but patients may not always know what type of antibody test was used.
• If antibody testing was performed following vaccination, additional doses of the same or
different COVID-19 vaccines are not recommended based on antibody test results at this time.
• If antibody testing was done after the first dose of an mRNA vaccine, the vaccination series
should be completed regardless of the antibody test result.
Pembentukan antibodi pasca vaksinasi tidak mengurangi kewajiban protokol kesehatan 5 M
→ belum diketahui pasti efektivitas vaksin dalam mencegah penularan
TAKE HOME MESSAGES
1. Pasca vaksinasi respon imun pasti terjadi.
2. Pembentukan antibodi bergantung pada banyak faktor (faktor vaksin,
faktor host, dan faktor assay).
3. Belum terdapat standarisasi internasional antibodi netralisasi yang sesuai
dengan berbagai tes antibodi kuantitatif yang beredar. Gold standard
adalah PRNT.
4. Pemeriksaan antibodi kuantitatif RBD-S untuk monitoring mandiri respon
imun vaksinasi harus menggunakan alat dan reagen yang sama.
5. Target utama vaksinasi saat ini adalah untuk mencapai meningkatkan
cakupan untuk mencapai herd immunity → belum ada rekomendasi
penambahan/perubahan jenis vaksin/dosis berdasarkan hasil antibodi
6. Penentuan cut-off kadar antibodi seroprotektif masih membutuhkan
penelitian
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TERIMA
KASIH