All of us are now either looking forward to receiving our Covid-19 Vaccine, or have been fortunate enough to have recently been given our first or second dose. Upon departure from the vaccine clinic, you were most likely reminded to maintain social distancing and facemask wearing for the foreseeable future. But why do we still need to do these things even after we are vaccinated? Well, there are several reasons: firstly, it takes several days to weeks for the immune system to build a robust response to the  Vaccine, and during this time, the individual has limited or no protection from infection; secondly, the Vaccine works to a different degree for every one of us, and so our level of protection, and the duration of that protection is not well understood right now. To complicate things more, it is still unclear if vaccination prevents an individual from spreading the virus, even if they do not display any symptoms. With so many unknowns, it makes sense that we should continue to take these safety precautions for the foreseeable future.

What Is Vaccine Efficacy

COVID-19 Vaccine efficacy can be very complicated to understand. Given that both the Moderna and Pfizer vaccines claim to have “95% efficacy”, we should start by clarifying how they determined this number and precisely what 95% efficacy really means.

Let’s take a deeper dive into Pfizer’s clinical trial for their Vaccine that enrolled 46,661 people- half were given the Vaccine and half were given the placebo. 

Within the placebo group, of 21,830, 162 individuals became infected with Covid-19 throughout the study compared with only eight individuals who had received the Vaccine. We can calculate the infection risk; for the placebo group, this is (162/21830) = 0.74%, compared with (8/21830) = 0.04% in the group given the Vaccine.

Both of these percentages (0.74% in the placebo group and 0.04% in the vaccine group) look small, and they are both less than 1%. However, if we now reflect those numbers in terms of the USA population, the difference is more apparent: that 0.74% from the placebo group would equate to ~2.5 million Covid-19 infections, compared with only 131,000 in the group given the Vaccine.

So, where does this 95% efficacy figure come from? Well basically it's determined by taking the infection risk of the placebo minus the infection risk of the vaccine group, divided by the infection risk of the vaccine group:

The difference in infection risks between the control placebo and the vaccine group = 0.7%

0.74%- 0.04% = 0.7%

(0.7%/0.74%) = 94.59% efficacy rate.

(i.e., a 95% reduction in new cases of Covid-19 in the vaccine group compared with the placebo group).

To be clear, it is essential to note that this does not mean that 95% of the vaccine group is protected and 5% are not; it means that within the vaccinated population, there is a 95% decrease in infection rates.

But the ideal conditions within a clinical trial, particularly such a high-profile clinical trial as this Covid-19 vaccine trial, do not necessarily reflect what is happening in the real world. The term Vaccine Effectiveness is often used to reflect how well a vaccine performs outside of clinical trials, where underlying health conditions, medications, diet, microbiome composition and the overall wellness of an individual can have profound impact on the efficacy of the Vaccine for them.

How will experts evaluate the effectiveness of Covid-19 vaccines in the real world? 

There are several approaches being taken by epidemiologists:

1. Case-control studies were carried out in clinics and hospitals to assess patients who come in and are diagnosed with Covid-19 and those who do not test positive for the virus. The patient's background with respect to vaccination status will be recorded and the data tracked over time. 
2. Cohort studies are performed similar to that within the clinical trials- two groups a vaccinated cohort and a non-vaccinated cohort. These studies are performed both retrospectively and prospectively, often coupled with medical records.
3. Screening Studies compare infection rates within a small population to the general population e.g. a student population at a college, compared with the general population in the surrounding county. 
4. Ecologic Analysis Studies assess groups of people, such as those in different geographical locations or different months of the year- and compare the number of people vaccinated with the number who are diagnosed with Covid-19. This can be complicated to collate overtime and frequently mathematical models are employed (Shim and Galvani, 2012). 

Importantly, all of these studies will need to be completed across different real-world demographics in terms of age-, racial-, ethnic-, socioeconomic- groups with various underlying medical conditions, and such studies can take years to complete (D. S. Fedson, 1998). 

So, what can you do for yourself to determine how well the Vaccine is working for you? 

Well, there are several ways to examine and measure the strength/robustness of your immune response following vaccination. 

B-Cell (Antibody) Responses 

Typically, one of the most widely used means of immune response evaluation is the quantification of antibody levels that have been generated by B-cells in response to the Vaccine. These serology tests require a blood sample, and using ELISA or bead-capture methods, can determine the relative levels of antibodies that target specific SARS-CoV-2 antigens. The higher the antibody levels to a viral antigen target, the stronger the immunological response. One caveat to this is that not all antibodies are equal, and the detection of neutralizing antibodies (nAbs) is needed to confirm protection from the virus. It has been demonstrated that in the case of SARS-CoV-2, nAbs are able to neutralize the virus by blocking the RBD regions binding to the cell surface ACE2 receptor. Tracking of these antibody levels or titers, particularly those associated with binding to the Receptor Binding Domain (RBD) region of the SARS-CoV-2 spike protein, provides a relatively simple method of vaccine effectiveness overtime.  

However, there have been concerns expressed over the use of antibody titers as performance indicators for coronaviruses such as SARS-CoV-2, for which the antibody response is often short-lived. In fact, during infection with either SARS or MERS viruses, high antibody levels are associated with worsening disease symptoms, clinical lung damage, and poorer patient outcome. This is potentially exacerbated through antibody-dependent enhancement that results in an increase in the uptake of the virus into cells. This effect has been well described for diseases such as Dengue and Zika. Interestingly, it has been demonstrated that the T-cell responses to Dengue and Zika are key to maintaining high levels of antibodies while preventing or counteracting antibody-dependent enhancement. In the case of SARS-CoV-2 infection, there is coordination of activities performed by both B-cells and T-cells to control and eliminate the infection and to build the immunological memory to manage re-infection (Fig. 1). So, should we also be looking at T-cell responses to assess vaccine effectiveness against SARS-CoV-2?

T-Cell Responses 

The importance of T-cell responses for vaccine effectiveness were manifested by the yellow fever vaccine, first developed 83 years ago. This vaccine stimulates a long-lasting and highly protective T-cell response. In the case of natural SARS-CoV-2 infection, strong T-cell responses are  correlated with less severe disease, with the T-cells rapidly clearing the virus, and enhancing an appropriate antibody response. Low CD8+ T-cells (<165/µL) correlates with a higher predictor of death or poor outcome. Experts in coronavirus vaccines agree that vaccine effectiveness will rely on both a strong antibody response and an optimal memory CD8+ T-cell response. 

Natural infection with SARS-CoV-2 induces a broad spectrum of CD8+ and CD4+ T-cell responses, not just to the spike protein (S) but also with the Membrane (M) and Nucleocapsid (N) proteins (Hellerstein, 2021). However, with most vaccines focusing on the Spike protein as the immunizing agent, researchers will need to understand the relationship between the breath, duration and overall robustness of T-cell responses and the resulting protective immunity generated by these vaccines versus natural infection.

Fig. 1. (adapted from Cox, R.J & Brokstad, K. A. 2021. Nature Reviewed Immunology 20 581-582). The action of T-cells and B-cells in immunity to SARS-CoV-2. A. the onset of infection with the SARS-CoV-2 virus results in the activation of innate immunity and dendritic cells which then drive the induction of the virus-specific T cell and B cell responses. The development of effective vaccines will require us to understand more about the memory response to Covid-19. B. A predictive time-course of adaptive immunity to SARS-CoV-2. 

CTL- cytotoxic T-lymphocyte, T_FH -follicular helper cell; T_H -T helper cell; T_reg- regulatory T cell

The durability of virus-specific T-cells targeting SARS-CoV-2 is a feature of both natural infection and effective vaccination. So how do we measure an effective T-cell response? 

Well, it can be as simple as looking for the secretion of IFN-gamma in vitro by T-cells exposed to their target SARS-CoV-2 antigen, to more complex epigenetic and metabolic analysis of CD8+ T-cells following programming to target SARS-CoV-2 antigens.

There is undoubtedly a need for tests that can be used to correlate vaccine-induced T-cell immunity (summarized in Fig. 2). Currently used platforms include T-cell proliferation assays, cytokine profiling and ELISPOT assays, the quantification of antigen-specific T-cells, cytotoxic T lymphocyte and T-cell immune-phenotyping assays, along with Phosphoflow analysis of T-cell signaling in vitro. Typically, PBMCs are the primary source of cells for these types of assays; however, this might not ideally reflect activities within the major lymphoid compartments or disease affected tissue. Despite these limitations, these assays can help in the assessment of vaccine candidates. 

T-cell Proliferation Assays: The proliferation of PBMCs after stimulation with antigen/peptide has been used as an integrated measure of cellular immunity for many years, through the incorporation of ³H-thymidine or stable dyes such as carboxyfluorescein succinimidyl ester (CFSE) and the subsequent semi-quantitative loss of signal through cell division. More recently, intracellular staining for Ki67 nuclear antigen has been used as a marker for recently or actively dividing cells as a marker for PBMC proliferation and T-cell responses after human vaccination (Stubbe, et. al. 2006). 

Cytokine-based T-cell Assays: The secretion of IFN-gamma, TNF-a, and IL-2 are common markers for bulk T_h1 response of T-cells, either by ELISA or Cytometric Bead Array. ELISPOT and intracellular cytokine staining offer the advantage of measuring cytokine production on a per-cell basis, even at lower frequencies such as those generated by antigen-specific T-cell responses. 

Peptide tetramers are synthetic structures made from 4 or more identical HLA structures coupled together and then loaded with antigenic peptides and are designed to bind to T-cell receptors of antigen-specific cells with very high avidity and enable the detection and enumeration of viral antigen-specific CD4+ and CD8+ T-cells within a sample. However, this requires knowledge of the sample HLA type, and is typically limited to use in clinical trials during control study conditions. 

Cytotoxic T Lymphocyte (CTLs) Response Assays: CTLs are produced from precursor T cells after stimulation with antigen-peptide presented on Antigen Presenting Cells (APC) in the presence of costimulatory signals and CD4 T_helper cells. CTLs kill their target cells using the release of lytic granules containing perforin and granzymes, or by activating target cell death receptors such as FAS (Lowin et. al. 1994). CTL activity can be measured using a ⁵¹Cr release assay, granzyme ELISPOT, PI or 7-AAD staining and CD107a surface expression. 

T-cell Immuno-phenotyping: The frequency of memory T-cells following antigen exposure (natural or through vaccination) has been shown to correlate with a protective immune response and vaccine efficacy. Coupling flow cytometry immunophenotyping with tetramer staining or intracellular cytokine staining (ICS) of IFN-gamma has been useful in assessing memory T-cells, and the subcategories central memory (T_CM) and effector memory (T_EM). Cellular proliferation determination with CFSE or Ki67 staining, can be used to assess the magnitude of the T-cell response to vaccination. Additional surface marker staining can help to assess CTL activity (CD107a) and identify T_h1 cells expressing CD154 that support the humoral response and activate DCs.

PhosphoFlow: This flow cytometry technique is used to assess phosphorylation events regulating cell signaling, and T-cell responsiveness to cytokine activation. Pathways that can be monitored using this technique include ERK, ZAP70, NFκB and STAT5, and infer memory potential of subsets based on these phosphorylation profiles (Puronen et. al. 2012).

Fig. 2. Assays available for measuring the T-cell response to vaccines. The success of a T-cell vaccine requires the induction of specific T-cell memory against the cognate Vaccine antigen. A variety of assays are currently available for evaluating the function, immune phenotype, and frequency of T-cell responses including: tetramers to quantify total memory T cells, proliferation assays to measure T-cell activation, cytokine-based assays to measure functionality and the profile of the vaccine response, and immunophenotyping via flow cytometry-based methods to integrate many of the aforementioned assays. Finally, T-cell effector function assays are critical to show that vaccine-induced T cells are able to kill antigen-expressing target cells. BrdU: 5-bromo-2′-deoxyuridine; CBA: Cytometric bead array; CFSE: Carboxyfluoresceinsuccinimidyl ester; CyTOF: Cytometry by time-of-flight; ELISPOT: Enzyme-linked immunosorbent spot; ICS: Intracellular cytokine staining.

What can we learn from T-cell responses to natural Covid-19 infection that might help produce more effective vaccines to SARS-CoV-2?

Functional profiling of T-cells from convalescent patients reveals that their long-lived T-cells display certain specific characteristics, in particular the expression of surface markers associated with naïve and effector cells, coupled with a lack of Granzyme B expression (a characteristic of naïve T-cell functionality). Upon exposure with their target antigen in vitro, these T-cells will proliferate, secrete effector cytokines, and establish gene expression patterns that are distinct from other T-cells. Typically, these T-cells are sustained in the circulation for many months after infection.

Examination of the antigen reactivity of the T-cells from natural infection has revealed that recovered patients with mild disease display prominent profiles of CD8+ T-cells targeting M- and N- proteins compared with the S- protein. Patients with severe symptoms were shown to display high serum cytokine levels and express PD-1 on their T-cells, consistent with exhaustion of their T-cells, which typically showed bias towards the viral Spike protein. These studies indicate that an adaptive CD8+ T-cell response with broad T-cell specificity confers the strongest protection and that this could become important for future rational design of Covid-19 vaccines. 

Real Hope from Vaccines 

In the case of SARS-CoV-1, recovered patients had reactive T-cells for over 17 years post-infection, suggesting that long-term immunological memory to beta-coronaviruses is achievable. In one study, these T-cells were shown to cross react with SARS-CoV-2, even though the patients were not exposed to the new virus and indicated that even previous exposure with common beta-coronaviruses that cause common colds, provided some protection against Covid-19 (Ng. et. al. 2016). Studies like these provide hope that the emergence of variants during this pandemic will not diminish the promise of the new vaccines. 

FlowMetric is developing novel flow cytometry panels and methods to assess the immune responses to vaccine candidates within clinical trials. From this our clients gain insights into key biomarkers for vaccine performance in the real world, and even provide personal profiling of vaccine efficacy for individuals as we continue towards reestablishing a more normal life again.

Dr. Julie Bick is a medicinal biochemist who has spent close to 7 years with FlowMetric Life Sciences. After receiving her doctorate in Biochemistry at Southampton University in the UK, she began her career as Associate Professor at Rutgers University, NJ, before moving to the west coast to perform biomedical research with Syngenta and Novartis at the Torrey Mesa Research Institute in San Diego. Dr. Bick specializes in biomedical engineering of cells and proteins in order to provide innovative therapeutic and diagnostic solutions. She brings to FlowMetric a clinical expertise across a wide range of therapeutic areas from autoimmunity to oncology and chronic inflammatory conditions, acquired over 25 years of research experience in academic, biotechnology and pharmaceutical laboratories. In leading FlowMetric Life Sciences’ innovation initiatives, Dr. Bick has been collaborating with BurstIQ to implement Block Chain solutions into the company’s Contract Research Organization division, with a focus on enhanced big data analytics and process control solutions in the regulated clinical environment. Dr. Bick is committed to working with local Community Colleges to support STEM programs for the next generation of scientists.

Reference 

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Hellerstein, M. (2021) What are the roles of antibodies versus a durable, high quality T-cell response in protective immunity against SARS-CoV-2? Vaccine: X. 6, 100076. https://doi.org/10.1016/j.jvacx.2020.100076

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