Spike-Directed Vaccination Elicits Robust Spike- Speci c T cell Responses Including to Mutant Strains

Maja Stanojevic Children's National Hospital Ashley Geiger Children's National Hospital Brita Ostermeier Children's National Hospital Danielle Sohai Children's National Hospital Christopher Lazarski Children's National Hospital Haili Lang Children's National Hospital Mariah Jensen-Wachspress Children's National Hospital Kathleen Webber Children's National Hospital Peter Burbelo National Institute of Dental and Craniofacial Research Jeffrey Cohen National Institute of Allergy and Infectious Disease Michael Keller Children's National Hospital Catherine Bollard Children's National Hospital Conrad Russell Cruz (  CCruz@childrensnational.org ) Children's National Hospital


SARS-CoV
The mutations within the Spike protein are within the receptor binding domain, which influences virus attachment and entry into cellswith some studies suggesting increased binding affinity, and consequently, increased infectivity and transmission. 4 Recently, different studies have suggested that there is diminished neutralization of the two strains even after vaccination: 5 sera from vaccinated individuals had neutralization titers several logs lower than those of the reference strain. 6,7 This raises appreciable concerns regarding the efficacy of current vaccine strategies to control the pandemic.
T cell responses are a critical complementary immune response to antibody responses. Indeed, in individuals with combined variable immune deficiency, rates of COVID-19 hospitalizations and mortality parallel those of the general population, suggesting that in these B cell deficient individuals, T cell response plays a key immunologic role for viral control. 8 Previous studies have already determined the ability of T cells derived from convalescent individuals to recognize SARS-CoV-2, 9 and presence of virus-specific T cells correlates with protection from severe COVID-19and with disease severity and recovery. 10 However, while antibody responses have been well characterized in individuals who have received most of the approved vaccines, 6,7,[11][12][13] there is currently a paucity of data available on the breadth and cross-reactivity of the T cell responses to mutant viral strains post vaccination. A study by Tarke et al 14  Amplification of specific responses by culturing PBMC in cytokines and antigen for 10 days can be used to enhance sensitivity of determining immune responses. 15 Hence, we evaluated SARS-CoV-2 specific T cell responses in the peripheral blood immediately after blood collection (D0) and after a 10-day expansion step to amplify the response (Supplemental Figure 1). With this approach, we demonstrate reliable amplification post vaccination, similar to what we have observed in antigen-exposed, convalescent donors. 15 However, even after this expansion step, no detectable responses were observed in our pre-vaccine donor cohort ( Figure 1B,   Supplemental Figure 2). In contrast, after two doses of vaccine, increased T cell responses to Spike were observed in all donors ( Figure 1B, Supplemental Figure 2). Of note, Spike responses were reliably detectable after the second vaccination, in contrast to antibody responses which were detected after the first dose ( Figure 1A). 16 Because current circulating SARS-CoV-2 variants threaten to prolong the pandemic, and observations of decreased neutralization activity from antibodies have been made, 6 We confirmed the potential cross-reactivity of the post vaccine T cell responses following observations that T cell products stimulated with the reference Spike (Supplemental Table 6) were also able to recognize mutated peptides in the B.  Table 7 Table 2).
To determine whether CD4+ T cells or CD8+ T cells contribute to the reactivity to Spike, we performed intracellular cytokine stains, and showed that both CD4 and CD8 compartments show specificity against reference Spike protein following vaccination (Figure 2D, 2E, Supplemental This data supports the hypothesis that COVID-19 vaccination elicits immunological responses that are cross-reactive with novel strains, and may be protective through a combination of humoral and adaptive T cell immunity. This data also suggests that immunity may be incomplete after the first vaccine dose. Cellular immune responses peaked after the second Spike mRNA vaccine dose, emphasizing the importance of completing the validated 2-dose vaccine strategy advocated by the manufacturers to elicit both humoral and cell-mediated immunity to SARS-CoV-2 in vivo.
The presence of CD8+ T cell responses in donors post vaccination is therefore encouraging since cytotoxic T cells directly lyse their targets, and we posit that their presence may offset the unwanted excessive cytokine secretion mediated by unregulated T cell activity.
In conclusion, we contend that ongoing surveillance of both antibody and T cell responses to emerging variants is an important measure to evaluate the immune protection afforded by current mRNA vaccines. T cell response data can potentially serve as indicators for future vaccination strategies including the need for booster vaccines, as well as designs for more global, universal vaccines.

Donors
Blood was obtained from seronegative donors with no history of SARS-CoV2 infection after

Antibody Testing
Antibodies were determined as previously described. 18 Plasma was obtained from samples up to 24 hours following blood draw by centrifugation at 1000 G for 15 minutes. Samples were incubated with spike and nucleocapsid proteins fused to Gaussia and Renilla luciferase enzymes.

Luciferase activity was measured in light units with a Berthold 165 LB 960 Centro Microplate
Luminometer.

Expansion of SARS-CoV-2 T Cells
In Figure 1, peripheral blood mononuclear cells (PBMCs) were pulsed with an overlapping peptide mix (as described in Keller et al 15 ) of viral structural proteins: Nucleocapsid, Spike, Envelope and Membrane. In Figure 2, PBMCs were separately pulsed with a mix of (1) overlapping peptides spanning the reference Spike (Supplemental Table 6 and supplemented with GlutaMax (Gibco, Grand Island, NY), as previously described. 15 Cells were fed regularly and split when confluent. Cells were harvested on day ten or eleven and evaluated for antigen specificity by ELISpot.

Intracellular Cytokine Stain
Ex vivo expanded T cells were frozen after 10 days culture, and thawed the day before intracellular cytokine staining in the presence of 40 U/mL IL2. Intracellular cytokine stain was performed as previously described, with some modifications. 15 Briefly, T cells were stimulated with either actin (negative control), Spike peptides, or PHA (positive control) at a concentration of FastImmune), and incubated at 37°C 5% CO2. After 6 hours, T cells were then washed in 2% FBS phosphate-buffered saline and surface stained with fluorochrome-conjugated antibodies to CD3, CD4, and CD8. Cells were then fixed, permeabilized using BD Cytofix/Cytoperm solution, and stained with anti-IFN-γ and anti-TNF-α. T cells were fixed until acquired the following day and then were analyzed on a BD Cytoflex, with data analysis performed using FlowJo (FlowJo LLC, Ashland, OR).

Epitope Analysis
Predicted HLA Class I were derived from artificial neural network prediction by NetMHCpan 4.1. 19 Sequences were inputted with no predetermined peptide length selection, alleles of the recruited donors (where no specific type was available, the HLA supertype representative was used), and with a threshold of 0.5% rank for strong binders. Predicted HLA Class II epitopes were derived from the Immune Epitope Database and Analysis Resource. 20 Sequences were inputted as peptides (listed in Supplemental Table 7) with the IEDB recommended 2.22 prediction method, selecting for the full HLA reference set with select alpha and beta chains analyzed separately where applicable. Percentile ranks <2 were considered strong binders, and percentile ranks between 2-10 were considered weak binders. nonstructural antigens Spike, membrane, nucleocapsid, and envelope to zoom in on specific T cells, measured immediately before vaccination (blue circles), three weeks after the first vaccine and before administration of the second vaccine (red circles), and three to four weeks after administration of the second vaccine (green circles).  Table 7). All T cells were expanded for 10-11 days from peripheral blood mononuclear cells against reference Spike peptides. Figure 1 Antibodies and T cells from seronegative donors demonstrate enhanced reactivity to Spike post vaccination. A. Antibody to nucleocapsid and to Spike, measured in six donors immediately before vaccination (blue circles), three weeks after the rst vaccine and before administration of the second vaccine (red circles), and three to four weeks after administration of the second vaccine (green circles). B. T cell responses as measured by IFN-γ ELISpot after 10-11 day expansion of peripheral blood mononuclear cells with COVID-19 nonstructural antigens Spike, membrane, nucleocapsid, and envelope to zoom in on speci c T cells, measured immediately before vaccination (blue circles), three weeks after the rst vaccine and before administration of the second vaccine (red circles), and three to four weeks after administration of the second vaccine (green circles).