Episode 315 Correlates of protection

Sun, 02/12/2023 - 20:07

Episode 315: Overview of the importance of various antibody and T cell function in SARS-CoV-2 natural infection and vaccination


Dear colleagues,


Most attention has gone to neutralizing antibodies as correlates of protection.  This episode will show that both non-neutralizing antibody functions and T cells have also important roles, although the evidence is less strong, because these aspects have been less systematically studied.


The episode is meant to be very didactic. Please, if you don’t understand my text, read the original papers/reviews




I hope you enjoy  ….  




Schematic representation of the integrated adaptive and innate immune responses that result in the elimination of the viral infection. (Manish Dhawan Vaccines 2023 -Ep 315-1)





The virus enters individuals following contact with the upper respiratory tract. Antigen-presenting cells (APCs), including dendritic cells and alveolar macrophages, endocytose and destroy the SARS-CoV-2 virus via a procedure termed antigen processing. Antigen segments are subsequently expressed on the cell membrane by MHC proteins, allowing T cells to recognize them. Following the interaction with T cells like CD4+ and CD8+, different types of responses occur.

  • When CD4+ cells interact with the presented antigen on the MHC class II molecule, the activation of B-cells occurs. This will lead to the clonal expansion of CD4+ cells and B cells. After eliciting the B cells with the help of CD4+ cells, secretion of the antibodies occurs, which clears the viral infection.
  • Upon interaction with the CD8+ T cell with peptides presenting on MHC class I, clonal expansion of T cells occurs, which leads to the apoptosis of the infected cells through various mechanisms and leads to the release of other pro-inflammatory mediators.



The association of the T cells with the excessive release of cytokines, disease severity, and

ARDS (acute respiratory distress syndrome). (Manish Dhawan Vaccines 2023 – ep 315-1)




During a mild infection or low viral load, the T cell n umber remains normal, and possibly normal functioning of the Th2 type cells will not lead to excessive inflammation and normal clonal expansion of T cells.


However, under the severe form of the infection, monocyte accumulation and excessive antigen presentation lead to an increase in pro-inflammatory cytokine levels concurrently, causing T cell depletion and exhaustion.

Importantly excessive activation of Th1 and Th17 type T cells lead to increased levels of cytokines, importantly IL-6.


Severe infection causes lymphopenia by directly affecting lymphatic organs such as the spleen and lymph nodes.


The further figure represents various pathways for the excessive release of cytokines.

  • Firstly, CD4+ T cells may be stimulated quickly into Th1 cells that secrete GM-CSF, generating CD14+CD16+ monocytes with high IL-6 levels.
  • Secondly, an increase in the CD14+ IL-1+ monocyte subpopulation stimulates the production of IL-1.
  • Additionally, Th17 cells generate IL-17, which recruits more monocytes, macrophages, and neutrophils while also stimulating other cytokine cascades, such as IL-1 and IL-6.

This can lead to tissue damage and excessive accumulation of fluid in the lung, which in turn leads to multiple organ damage.


Immunological differences between mild self-limiting and severe life-threatening COVID (Areez Shafqat Front Immunol Nov 2022)



Protective immunity to SARS-CoV-2 infection is characterized by

  • early and robust type I and III IFN.
  • corresponds to the early recruitment and expansion of T-cell responses, which correlates with SARS-CoV-2 viral clearance and asymptomatic or mild COVID-19.
  • also features early plasmablast expansion and intact B-cell germinal center responses.


Immunopathogenesis is characterized by

  • delayed T-cell activation,
  • a consequent delay in viral clearance
  • and overactivation of innate immunity.

→ Innate immune cells then overproduce pro-inflammatory cytokines that spillover into the systemic circulation, causing a cytokine storm. Concomitantly, exaggerated neutrophil responses, platelet activation, and endothelial inflammation (i.e., endotheliosis) precipitate a hypercoagulable state manifesting clinically as the thrombotic microangiopathy that affects critically ill COVID-19 patients.



Layered defenses against SARS-CoV-2, or the “Swiss cheese” model of immunity. (David Goldblatt Imm Rev Sept 2022 Ep 315-3)




Conceptually, layered defenses are like a “Swiss cheese model”: even though each layer is imperfect, all together they make it highly unlikely that the pathogen breaches all of the layers of defense.


Key:  nAbs = neutramizing antibodies; non-nAbs = non-neutralizing antibodies; mem = memory  




The underlying mechanism of vaccine antigen delivery forms and adjuvants shape the type of

cellular immune response.   (Zhonjie Sun Vaccines July 2022 Ep 315-4 )





Generally, the antigens of the conventional vaccine platform are degraded in lysosomes and then subjected to MHC II to be presented to CD4+ T cells after being phagocytized by APCs (antigen-presenting cells).

  • The antigens of nucleic acid vaccines that are expressed and degraded in the cytoplasm of APCs interact with MHC I molecules to be presented to CD8+ T cells, while the antigens expressed in other non-APCs is similar to the conventional vaccine platform.
  • The adjuvant that stimulates the TLR1/-2 on the cell membrane promotes APCs secreting IL-4 and IL-10 and help Th2 differentiation. TLR3, -7/-8, and -9 elevate the expression of IL-12 and IFN a/b and induce Th1 differentiation through different transcription factors upon activation.



2.1. Lymphoid and circulating TFH (T follicular Helper)  responses in COVID-19



SARS-CoV-2 antigen in the lymph nodes results in activation of antigen-specific B cells and TFH cells. Their interaction leads to the initiation of the germinal center reaction. This results in the development of memory B cells with increased somatic hypermutation (SHM) and increased affinity, as well as long-lived plasma cells that traffic to the bone marrow and provide a long-term source of neutralizing antibodies. A population of short-lived antibody-secreting cells (ASCs) appears in the circulation and provides a raid source of neutralizing antibodies. Concurrently, a population of activated (CD38+, PD-1+, ICOS+) cTFH cells appears in the circulation. This population contains antigen-specific cTFH cells (not depicted). Although memory B cells and ASCs are primarily located in lymphoid tissues, they are typically measured in blood samples, where they correlate with activated cTFH cells. Activated cTFH cells also correlate with the development of neutralizing antibodies. These cTFH cells are a potential biomarker of TFH activity in lymphoid tissues but it remains to be determined if this population of cTFH cells are predictive of long-term neutralizing antibodies, or of the development of long-lived plasma cells and the prolonged evolution of the MBC pool. T


2.2. Importance of both Spike-binding and virus-neutralizing antibodies is well established in vaccine trials

with binding antibody titers giving the highest statistical correlation, across variants (David Goldblatt Imm Rev Sept 2022 Ep 315-3)








2.3. Importance of non-neutralizing functions  (David Goldblatt Imm Rev Sept 2022 Ep 315-3)



Antibody-mediated effector functions via the Fc part

  • ADCC = antibody-dependent cellular cytotoxicity by NK cells,
  • ADCP = antibody-dependent phagocytosis by neutrophils and macrophages
  • CDC = complement-dependent cytolysis.



Relevance of antibody effector functions throughout the SARS-CoV-2 viral life cycle. (David Goldblatt Imm Rev Sept 2022 Ep 315-3)





The cartoon depicts the interactions of the virus with the host cell, and the moments when the spike antigen may be visible to circulating antibodies.

  • As the virus roles across the cell surface and may be targetable by many effector mechanisms including those driven by phagocytic cells and natural killer (NK) cells (left).
  • However, once binding to ACE2 has occurred, the virus is rapidly endocytosed, leaving limited to no spike on the surface of cells.
  • New viruses assemble and release from the Golgi, leaving little to no spike on the surface at the time of egress (right).  Thus, functional spike-specific antibodies likely confer the bulk of their protective functions via the recognition and elimination of free particles prior to infection or soon after egress, providing a critical bottleneck for the virus


Non-survivors showed attenuated IgG responses, accompanied by compromised Fc effector activity  (Tomer Zohar Cell Dec 2020 Ep 315-5)




Analyses of the functional humoral trajectories associated with the resolution of SARS-CoV-2 infection find that despite equivalent IgM and IgA immunity to the virus across all levels of disease severity, survival and recovery are linked to early class switching to IgG and the ability to leverage Fcγ receptors targeting the spike protein.


Subtle differences in FcR functions between Moderna (mRNA-1273) and Pfizer (BNT 162b2): associate with consistently slightly higher vaccine protection by Moderna  

  • Higher concentrations of receptor binding domain (RBD)– and N-terminal domain–specific IgA after Moderna.
  • Antibodies eliciting neutrophil phagocytosis (ADNP) and natural killer cell activation were also increased in Moderna



Fc functional assays [antibody-dependent complement deposition (ADCD), antibody-dependent cellular phagocytosis (ADCP), antibody-dependent neutrophil phagocytosis (ADNP), and antibody-dependent NK cell activation (ADNKA; CD107a surface expression and MIP-1α or IFN-γ production)]


Importance of non-neutralizing functions to recognize VOC (after previous infection with another VOV or vaccination with WT  (David Goldblatt Imm Rev Sept 2022 Ep 315-3)



Non-neutralizing Fc-functional antibodies (yellow) may bind to the entire surface of the spike.

Yet, with the incorporation of mutations, in Variations of Concern, changes that may impede neutralizing antibody binding may disrupt a few, but only a fraction of non-neutralizing antibodies.


PAR 3 CD4 and CD8 T CELL FUNCTIONS and IMPORTANCE in COVID disease and vaccination


3.1. Overview of important T cell functions (David Goldblatt Imm Rev Sept 2022 Ep 315-3)



T foll Helper



CD4 T Helper cells restricted by MHC class II (HLA DR/DQ/DS): mainly active as Th1 and T follicular cells

CD8 T cells restricted by MHC class I (HLA-A/B/C): mainly active as cytolytic cells



CD4 T cells  are important in anti-viral defense in three ways:

  • to induce Ab production via the T follicular Helper (TfH) phenotype with crucial role for IL-21
  • direct antiviral effect via the Th1 phenotype (IFN-gamma)
  • Cytolytic activity against MHC class II infected cells (e.g. macrophages)

CD8 T cells are mainly active as cytolytic T cells (CTL): killing via perforin-granzyme or Fas pathways


3.2. Repertoire of antigenic SARS-CoV-2 epitopes

  1. Total possible CD4+ and CD8+ T epitope repertoire of SARS-CoV-2 (Xiaoxiao Jin Virus Res 2023 Ep 315-6)



Most CD8 T cell epitopes are located in the “non-structural proteins” (encoding mainly the replication complex, including polymerase, proteases and co-factors), follow ed by Spike and other structural proteins (Envelop, Membrane and Nucleoprotein)

Most CD4+ T cell epitopes are located in the Spike protein, followed by N and non-structural proteins.


  1. HLA restriction (Xiaoxiao Jin Virus Res 2023 Ep 315-6)


Not surprisingly, higher numbers of epitopes have been found to bind to the more common HLA types, but this may become more balanced when the less common will be more investigated.


  1. T cell epitope repertoire after SARS-CoV-2 infection or vaccination in each individual. (Griffoni Curr Res Immunol 2022 Ep 315-7)


Overall, T cell responses recognize up to 10 different SARS-CoV-2 proteins representing 80% or more of the overall response, with the spike (S) being the most immunodominant, followed by Nucleocapsid (N), Membrane (M), and other Non-structural proteins (Nsps). (as shown by  Xiaoxiao Jin see above)


At the epitope level recognition by each individual is restricted by his/her HLA type and TCR (= T cell receptor repertoire.

Hence, only 30-40 epitopes are recognized by either CD4+ or CD8+ T cells after natural infection considering all SARS-CoV-2 proteins recognized. A median of 10–11 epitopes are recognized specifically for S protein in natural infection similarly to what is observed after vaccination. Obviously vaccination only elicits T cells against Spike, while responses after infection are much broader, explaining why post-infection immunity offers a betters protection (at the risk of primo-infection)


3.3. How to measure T cell functions? (Megan Schwarz Curr Res Immunol June 2022 Ep 315-8)

Two approaches to quantifying cellular immunity.


  1. TCR repertoire analysis involves quantification of the number of T cells that recognize specific epitopes.
  • MHC tetramer assays use fluorescently labeled, synthesized MHC-peptide complexes to bind antigen-specific TCRs. The bound TCRs can then be quantified and characterized by flow cytometry due to the fluorescent label.
  • TCR sequencing allows for the profiling of an individual’s entire T cell repertoire.
  1. Functional analyses require the activation of antigen-specific T cells (via peptides or proteins) and downstream quantification (either of activated T cells or byproducts of activation (e.g. cytokines)).

Comparison of various cellular immunity assays.


Each assay is compared in terms of their required starting material, turnaround time, effort needed in terms time and skill-level or personnel involved, and finally cost. A. Next Generation Sequencing; B. ELISpot; C. ELISA/ELLA; D. Flow Cytometry; E. qPCR-based assays (i.e. qTACT/dqTACT).


Functional assays based on PBMC


After blood is drawn, PBMCs (peripheral blood mononuclear cells) are isolated. Once isolated, PBMCs are stimulated with a peptide pool overnight. Activated T cells can be directly quantified by ELISpot/Fluorospot and ELISA/ELLA assays, ICS (intracellular cytokine staining), and surface staining (e.g. AIM = activation-induced markers). If cells are stimulated for longer periods of time (4–7) days and stained with a cell trace dye, a proliferation assay can be performed.


Functional assays based on whole blood


Freshly drawn blood is stimulated with a peptide pool overnight. During this time, peptides are processed and presented to T cells by APCs. Antigen-specific T cells are activated and produce cytokines/chemokines (e.g. IFN-γ), which, in turn, stimulates other cells in the blood (i.e. monocytes) that upregulate CXCL10 mRNA expression. ELISA/ELLA assays quantify extracellular cytokines in the supernatant of the blood. Flow cytometry can be used to quantify upregulated T cell surface receptors (e.g. AIM) as well as intracellular cytokines (ICS). Whole blood total RNA can be extracted and used for TACTseq or qTACT. Alternatively, RNA extraction can be avoided and the whole blood used directly in the dqTACT assay.



3.4. Importance of T cells to protect against severe infection:

In 6 studies that measured SARS-CoV-2-specific CD4 T cells and CD8 T cells: early and/or larger T cell responses were associated with faster viral clearance and/or better clinical outcomes,

Example 1: Takya Sekine Cell Oct 2020 Ep 315-9: Robust T Cell Immunity in Convalescent Individuals

with Asymptomatic or Mild COVID-19


  • Acute-phase SARS-CoV-2-specific T cells display an activated cytotoxic phenotype
  • Convalescent patients generate broad balanced “polyfunctiinal” T cell responses

SARS-CoV-2 elicits broadly directed and functionally replete memory T cells that may protect against recurrent episodes of severe COVID-19.


Example 2: Anthony Tan Early induction of functional SARS-CoV-2-specific T cells Cell Reports  Feb 2021 (Ep 315-10)


  • Rapid induction and quantity of humoral responses associate with an increase in disease severity,
  • Early induction of interferon (IFN)-g-secreting SARS-CoV-2-specific T cells is present in patients with mild disease and accelerated viral clearance.


3.5. Importance of T cells  in vaccine-mediated protection (David Goldblatt Imm Rev Sept 2022 Ep 315-3)

No T cell correlates-of-protection COVID-19 vaccine studies have been done in humans !

Nevertheless we know that T cell responses vary depending on the type of COVID-19 vaccine:

  • In response to the adenoviral COVID-19 vaccines (AstraZeneca ChAdOx1 or J&J Ad26.COV2.S), TH1 cell, TFH cell, and CD8 T cell responses are detected in many individuals, and T cell memory does develop.
  • The protein vaccine NVX-CoV2373 elicits substantial TH1 and TFH cells responses, as well as detectable CD8 cell responses in a minority of individuals (unpublished data)
  • The inactivated vaccines (CoronaVac) generates relatively weak CD4 T cell responses with a mixture of TH1 and TH2 cells, and no CD8 T cells
  • mRNA: with both Pfizer and Moderna there are a number of animal studies, showing CD4 T and CD8 T cell responses that are correlated with protection, even if antibodies are low.  The epidemiological evidence in people, who are still protected against severe disease, while antibodies are low and ineffective against newer VOC, is also an argument for T cell mediated protection. 


3.6. T cells have less problems with variants than neutralizing antibodies

1) Stable Spike specific T cell immune responses to Omicron. (Jinyan Liu Nature March 2022 Ep 315-11)







T cell responses at months 1 and 8 following final vaccination with Ad26.COV2.S (n = 20) or BNT162b2 (n = 27). a, b, Pooled peptide spike-specific IFNγ CD8+ T cell responses (a) and CD4+ T cell responses (b) by intracellular cytokine staining assays. Responses were measured against the SARS-CoV-2 WA1/2020, B.1.617.2 (Delta) and

B.1.1.529 (Omicron) variants. Responses in five unvaccinated, uninfected individuals are also shown.


2) Divergent SARS-CoV-2 Omicron–reactive T and B cell responses in COVID-19 vaccine recipients

(GeurtsvanKessel Sci Immunol March 2022 Ep 315-12)


This study shows that vaccinated individuals retain T cell immunity to the SARS-CoV-2 Omicron variant, potentially

balancing the lack of neutralizing antibodies in preventing or limiting severe COVID-19. Booster vaccinations are

needed to further restore Omicron cross-neutralization by antibodies.


Evaluation of antibody and T cell responses to Spike early and late after basic vaccination


Ab (ELISA) and T cells (IFN-g ELISPOT) to wild type



Ab and T cell responses after Adeno (ChAdOx = Astra-Zeneca or Ad26.COV2.S = Janssen) are lower but more stable over 6 months as compared to mRNA (Moderna or Pfizer)  


T cell responses towards variants after basic vaccination = stable after 6 months

AIM = activation-induced markers Ox40 CD69 CD137 (= CD40-ligand)

Note: Some individuals have no response to for CD4 or CD8 T cells


Variant-specific immune responses early after booster vaccination.




Note: CD4 T cell response in all boosted individuals, but CD8 T cell response is lacking in some.



  1. Most VOC mutation occur in sites which are not T cell epitopes  (Marina Brand Immunogenetics Jan 2023 Ep 315-13)






Although neutralizing Ab were a good correlate of protection in vaccine trials, it is evident that other arms of the immune system are involved in protection against severe disease, both during infection and after vaccination, These include:

  • Non-neutralizing Ab functions, which engage Natural Killer cells, phagocytes, complement to clear virally infected cells
  • CD4 T cells are not only important to sustain and mature neut and non-neut antibodies, but have also direct antiviral functions: they focus on Spike
  • CD8 T cells can kill virally infected cells efficiently, but they focus more on non-structural proteins, which may explain why they are not induced in every person by vaccinating against Spike.


While neut Ab (induced by either infection or repeated vaccination) lose potency against omicron, T cell responses are better preserved. Maybe they do explain in part why the still widespread epidemic has become less severe


These potential “complementary” correlates of protection have not been thoroughly investigated, partly because it is not immediately evident which of the many possible functional or molecular tests will be most useful.  


Food for research !!!


Best wishes,