Predominantly defective CD8+ T cell immunity to SARS-CoV-2 mRNA vaccination in lung transplant recipients.

BACKGROUND: Although mRNA vaccines have overall efficacy preventing morbidity/mortality from SARS-CoV-2 infection, immunocompromised persons remain at risk. Antibodies mostly prevent early symptomatic infection, but cellular immunity, particularly the virus-specific CD8+ T cell response, is protective against disease. Defects in T cell responses to vaccination have not been well characterized in immunocompromised hosts; persons with lung transplantation are particularly vulnerable to vaccine failure with severe illness. METHODS: Comparison groups included persons with lung transplantation and no history of COVID-19 (21 and 19 persons after initial mRNA vaccination and a third booster vaccination respectively), 8 lung transplantation participants recovered from COVID-19, and 22 non-immunocompromised healthy control individuals after initial mRNA vaccination (without history of COVID-19). Anti-spike T cell responses were assayed by stimulating peripheral blood mononuclear cells (PBMCs) with pooled small overlapping peptides spanning the SARS-CoV-2 spike protein, followed by intracellular cytokine staining (ICS) and flow cytometry for release of cytokines in response to stimulation, including negative controls (no peptide stimulation) and positive controls (phorbol myristate acetate [PMA] and ionomycin stimulation). To evaluate for low frequency memory responses, PBMCs were cultured in the presence of the mRNA-1273 vaccine for 14 days before this evaluation. RESULTS: Ionophore stimulation of PBMCs revealed a less inflammatory milieu in terms of interleukin (IL)-2, IL-4, and IL-10 profiling in lung transplantation individuals, reflecting the effect of immunosuppressive treatments. Similar to what we previously reported in healthy vaccinees, spike-specific responses in lung transplantation recipients were undetectable (< 0.01%) when tested 2 weeks after vaccination or later, but were detectable after in vitro culture of PBMCs with mRNA-1273 vaccine to enrich memory T cell responses. This was also seen in COVID-19-recovered lung transplantation recipients. Comparison of their enriched memory responses to controls revealed relatively similar CD4+ T cell memory, but markedly reduced CD8+ T cell memory both after primary vaccination or a booster dose. These responses were not correlated to age or time after transplantation. The vaccine-induced CD4+ and CD8+ responses correlated well in the healthy control group, but poorly in the transplantation groups. CONCLUSIONS: These results reveal a specific defect in CD8+ T cells, which have key roles both in transplanted organ rejection but also antiviral effector responses. Overcoming this defect will require strategies to enhance vaccine immunogenicity in immunocompromised persons.

Persistent memory despite rapid contraction of circulating T Cell responses to SARS-CoV-2 mRNA vaccination

Introduction While antibodies raised by SARS-CoV-2 mRNA vaccines have had compromised efficacy to prevent breakthrough infections due to both limited durability and spike sequence variation, the vaccines have remained highly protective against severe illness. This protection is mediated through cellular immunity, particularly CD8+ T cells, and lasts at least a few months. Although several studies have documented rapidly waning levels of vaccine-elicited antibodies, the kinetics of T cell responses have not been well defined. Methods Interferon (IFN)-γ enzyme-linked immunosorbent spot (ELISpot) assay and intracellular cytokine staining (ICS) were utilized to assess cellular immune responses (in isolated CD8+ T cells or whole peripheral blood mononuclear cells, PBMCs) to pooled peptides spanning spike. ELISA was performed to quantitate serum antibodies against the spike receptor binding domain (RBD). Results In two persons receiving primary vaccination, tightly serially evaluated frequencies of anti-spike CD8+ T cells using ELISpot assays revealed strikingly short-lived responses, peaking after about 10 days and becoming undetectable by about 20 days after each dose. This pattern was also observed in cross-sectional analyses of persons after the first and second doses during primary vaccination with mRNA vaccines. In contrast, cross-sectional analysis of COVID-19-recovered persons using the same assay showed persisting responses in most persons through 45 days after symptom onset. Cross-sectional analysis using IFN-γ ICS of PBMCs from persons 13 to 235 days after mRNA vaccination also demonstrated undetectable CD8+ T cells against spike soon after vaccination, and extended the observation to include CD4+ T cells. However, ICS analyses of the same PBMCs after culturing with the mRNA-1273 vaccine in vitro showed CD4+ and CD8+ T cell responses that were readily detectable in most persons out to 235 days after vaccination. Discussion Overall, we find that detection of spike-targeted responses from mRNA vaccines using typical IFN-γ assays is remarkably transient, which may be a function of the mRNA vaccine platform and an intrinsic property of the spike protein as an immune target. However, robust memory, as demonstrated by capacity for rapid expansion of T cells responding to spike, is maintained at least several months after vaccination. This is consistent with the clinical observation of vaccine protection from severe illness lasting months. The level of such memory responsiveness required for clinical protection remains to be defined.

Persistent memory despite rapid contraction of circulating T Cell responses to SARS-CoV-2 mRNA vaccination.

Introduction: While antibodies raised by SARS-CoV-2 mRNA vaccines have had compromised efficacy to prevent breakthrough infections due to both limited durability and spike sequence variation, the vaccines have remained highly protective against severe illness. This protection is mediated through cellular immunity, particularly CD8+ T cells, and lasts at least a few months. Although several studies have documented rapidly waning levels of vaccine-elicited antibodies, the kinetics of T cell responses have not been well defined. Methods: Interferon (IFN)-γ enzyme-linked immunosorbent spot (ELISpot) assay and intracellular cytokine staining (ICS) were utilized to assess cellular immune responses (in isolated CD8+ T cells or whole peripheral blood mononuclear cells, PBMCs) to pooled peptides spanning spike. ELISA was performed to quantitate serum antibodies against the spike receptor binding domain (RBD). Results: In two persons receiving primary vaccination, tightly serially evaluated frequencies of anti-spike CD8+ T cells using ELISpot assays revealed strikingly short-lived responses, peaking after about 10 days and becoming undetectable by about 20 days after each dose. This pattern was also observed in cross-sectional analyses of persons after the first and second doses during primary vaccination with mRNA vaccines. In contrast, cross-sectional analysis of COVID-19-recovered persons using the same assay showed persisting responses in most persons through 45 days after symptom onset. Cross-sectional analysis using IFN-γ ICS of PBMCs from persons 13 to 235 days after mRNA vaccination also demonstrated undetectable CD8+ T cells against spike soon after vaccination, and extended the observation to include CD4+ T cells. However, ICS analyses of the same PBMCs after culturing with the mRNA-1273 vaccine in vitro showed CD4+ and CD8+ T cell responses that were readily detectable in most persons out to 235 days after vaccination. Discussion: Overall, we find that detection of spike-targeted responses from mRNA vaccines using typical IFN-γ assays is remarkably transient, which may be a function of the mRNA vaccine platform and an intrinsic property of the spike protein as an immune target. However, robust memory, as demonstrated by capacity for rapid expansion of T cells responding to spike, is maintained at least several months after vaccination. This is consistent with the clinical observation of vaccine protection from severe illness lasting months. The level of such memory responsiveness required for clinical protection remains to be defined.

Dominant CD8+ T Cell Nucleocapsid Targeting in SARS-CoV-2 Infection and Broad Spike Targeting From Vaccination.

CD8+ T cells have key protective roles in many viral infections. While an overall Th1-biased cellular immune response against SARS-CoV-2 has been demonstrated, most reports of anti-SARS-CoV-2 cellular immunity have evaluated bulk T cells using pools of predicted epitopes, without clear delineation of the CD8+ subset and its magnitude and targeting. In recently infected persons (mean 29.8 days after COVID-19 symptom onset), we confirm a Th1 bias (and a novel IL-4-producing population of unclear significance) by flow cytometry, which does not correlate to antibody responses against the receptor binding domain. Evaluating isolated CD8+ T cells in more detail by IFN-γ ELISpot assays, responses against spike, nucleocapsid, matrix, and envelope proteins average 396, 901, 296, and 0 spot-forming cells (SFC) per million, targeting 1.4, 1.5, 0.59, and 0.0 epitope regions respectively. Nucleocapsid targeting is dominant in terms of magnitude, breadth, and density of targeting. The magnitude of responses drops rapidly post-infection; nucleocapsid targeting is most sustained, and vaccination selectively boosts spike targeting. In SARS-CoV-2-naïve persons, evaluation of the anti-spike CD8+ T cell response soon after vaccination (mean 11.3 days) yields anti-spike CD8+ T cell responses averaging 2,463 SFC/million against 4.2 epitope regions, and targeting mirrors that seen in infected persons. These findings provide greater clarity on CD8+ T cell anti-SARS-CoV-2 targeting, breadth, and persistence, suggesting that nucleocapsid inclusion in vaccines could broaden coverage and durability.

Primary, Recall, and Decay Kinetics of SARS-CoV-2 Vaccine Antibody Responses.

Studies of two SARS-CoV-2 mRNA vaccines suggested that they yield ∼95% protection from symptomatic infection at least short-term, but important clinical questions remain. It is unclear how vaccine-induced antibody levels quantitatively compare to the wide spectrum induced by natural SARS-CoV-2 infection. Vaccine response kinetics and magnitudes in persons with prior COVID-19 compared to virus-naïve persons are not well-defined. The relative stability of vaccine-induced versus infection-induced antibody levels is unclear. We addressed these issues with longitudinal assessments of vaccinees with and without prior SARS-CoV-2 infection using quantitative enzyme-linked immunosorbent assay (ELISA) of anti-RBD antibodies. SARS-CoV-2-naïve individuals achieved levels similar to mild natural infection after the first vaccination; a second dose generated levels approaching severe natural infection. In persons with prior COVID-19, one dose boosted levels to the high end of severe natural infection even in those who never had robust responses from infection, increasing no further after the second dose. Antiviral neutralizing assessments using a spike-pseudovirus assay revealed that virus-naïve vaccinees did not develop physiologic neutralizing potency until the second dose, while previously infected persons exhibited maximal neutralization after one dose. Finally, antibodies from vaccination waned similarly to natural infection, resulting in an average of ∼90% loss within 90 days. In summary, our findings suggest that two doses are important for quantity and quality of humoral immunity in SARS-CoV-2-naïve persons, while a single dose has maximal effects in those with past infection. Antibodies from vaccination wane with kinetics very similar to that seen after mild natural infection; booster vaccinations will likely be required.