key: cord-0946246-eid0qrew
authors: Peled, Yael; Afek, Arnon; Kreiss, Yitshak; Rahav, Galia; Nemet, Ital; Kliker, Limor; Indenbaum, Victoria; Ram, Eilon; Lavee, Jacob; Segev, Amit; Matezki, Shlomi; Sternik, Leonid; Raanani, Ehud; Lustig, Yaniv; Patel, Jignesh K.; Mandelboim, Michal
title: Kinetics of cellular and humoral responses to third BNT162B2 COVID-19 vaccine over six months in heart transplant recipients – implications for the omicron variant
date: 2022-05-24
journal: J Heart Lung Transplant
DOI: 10.1016/j.healun.2022.05.014
sha: b195fd9e38eb219bfe6899b07989119b567ff755
doc_id: 946246
cord_uid: eid0qrew

BACKGROUND: The durability of the immune response following the 3-dose BNT162b2 vaccination is unknown. The complexity of the situation is enhanced by the threat that highly transmissible variants may further accelerate the decline in the protection afforded by mRNA vaccines. METHODS: 103 3-dose-vaccinated heart transplant recipients were longitudinally assessed for the kinetics of variant-specific neutralization (Cohort 1, n=60) and SARS-CoV-2-specific-T-cell response (Cohort 2, n=54) over 6 months. Neutralization and T-cell responses were compared between paired samples at 2 time points, using the Kruskal-Wallis test followed by Dunn's multiple comparison test for continuous variables and McNemar's test for dichotomous variables. The Bonferroni method of P values adjustment for multiple comparison was applied. RESULTS: The third dose induced high neutralization of the wild-type virus and delta variant [geometric mean titer (GMT), 137.2 (95%CI, 84.8-221.9) and 80.6, (95%CI, 49.3-132.0), respectively], and to a lesser degree of the omicron variant [GMT, 10.3 (95%CI, 5.9-17.9)]. At 6 months, serum neutralizing activity declined but was still high for the wild-type virus and for the delta variant [GMTs 38.1 (95% CI, 21.2-69.4), P=0.011; and 28.9 (95% CI, 16.6-52.3), P=0.022, respectively], but not for the omicron variant [GMT 5.9 (95% CI, 3.4-9.8), P=0.463]. The percentages of neutralizing sera against the wild-type virus, delta and omicron variants increased from 70%, 65%, and 38%, before the third dose, to 93% (P<0.001), 88% (P<0.001), and 48% (P=0.021) at 3 weeks after, respectively; and remained high through the 6 months for the wild-type (80%, P=0.06) and delta (77%, P=0.102). The third dose induced the development of a sustained SARS-CoV-2-specific-T-cell population, which persisted through 6 months. CONCLUSIONS: The third BNT162b2 dose elicited a durable SARS-CoV-2-specific T-cell response and induced effective and durable neutralization of the wild-type virus and the delta variant, and to a lesser degree of the omicron variant.

In July 2021, Israel took the pioneering decision to administer a third dose of the BNT162b2 vaccine. The first population to receive the third dose consisted of immunocompromised patients, including heart transplant (HT) recipients, who had demonstrated an inadequate immune response to the two-dose BNT162b2 vaccine regimen and remained at high risk of developing severe complications from COVID-19 infection. 1 Our group showed that the third dose of the BNT162b2 vaccine, given 6 months after the second dose, induced enhanced humoral and cellular immune responses in HT recipients with a good safety profile. 2 This positive immune response laid the ground for the administration of a third dose for the general population, driven by observations of waning immunity in the 6 months following the second dose and a lowered protection afforded by the vaccine. 3, 4 The third dose of the BNT162b2 vaccine did indeed reduce the rates of confirmed infection and severe COVID-19 illness in the general population. 4 However, the durability of the immune response following the three-dose vaccination regimen still remains unclear, and the complexity of the situation is enhanced by the ever-present threat that highly transmissible variants may further accelerate the decline in the protection afforded by mRNA vaccines. 5, 6 These concerns take on added urgency when governments are faced with making decisions in light of the emergence of variants of concern (VOCs), particularly the current rapid emergence of the SARS-CoV-2 omicron (B.1.1.529) variant responsible for the dramatic rise in cases of COVID-19 cases worldwide.

Third doses of some vaccines have been shown to increase neutralization efficiency against variants. For example, in non-immunocompromised individuals, at 5 months following the second BNT162b2 dose, only a low neutralization efficiency against the wild-type virus was observed. The third dose of the BNT162b2 vaccine induced effective neutralization of the wild-type virus and the omicron variant at 1 month after administration, but the durability of this effect remains unknown. 2 

The cohort comprised 103 adult (≥18 years) stable HT patients vaccinated with 3 doses of the Pfizer 

To calibrate and determine the 50% tissue culture infectious dose (TCID50) for each variant, Vero-E6 cells at a concentration of 20*10 3 /well were seeded in 3 sterile 96-well plates with 10% FCS MEM-Eagle medium and stored at 37°C for 24 hours. Tenfold serial dilutions of each variant were prepared using 2% FCS MEM-Eagle medium and incubated for 5 days with the Vero-E6 cells. Following gentian violet staining, the TCID50 of each variant was calculated using the Spearman-Karber method.

Vero-E6 cells were seeded at 20*10 3 cells/well in sterile 96-wells plates with 10% FCS MEM-Eagle medium and stored at 37°C for 24 hours. For the wild-type, delta, or omicron isolates, 100 TCID50 were incubated with inactivated serum diluted 1:8 to 1:16384 in 96-well plates for 60 min at 33°C. Virus-serum mixtures were added to the Vero E-6 cells and incubated for five days at 33°C, after which gentian violet (1%) was used to stain and fix the cell culture layer. The neutralizing dilution of each serum sample was determined by identifying the well with the highest serum dilution without an observable cytopathic effect. A dilution equal to 1:10 or above was considered neutralizing.

Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using UNI-SEP+(Novamed). Plasma was collected and spun at 1000 × g for 20 min to remove platelets before collection of PBMCs. Following one wash with phosphate-buffered saline and one wash with 4Cell®

Nutri-T Medium (Sartorius), cells were resuspended in 4Cell Nutri-T-Medium and counted using a Countess II Cell counter (Invitrogen).

Fresh PBMCs were used in the ELISpot assay, performed with the ELISpot IFN-γ kit [Autoimmun Diagnostika GmbH] according to the manufacturer's instructions. Briefly, fresh PBMCs were added to duplicate wells at 2 × 10 5 cells in 50 μL per well and stimulated with 50 μL of SARS-CoV-2 peptide pools (S-complete, Miltenyi Biotech) (2 μg/mL per peptide). 4Cell Nutri-T Medium was used as the negative control, and phytohemagglutinin (PHA), as the positive control. After 16-20 hours at 37 °C, 5% CO 2 , 95% humidity, cells were removed, and secreted IFN-γ was detected by adding an alkaline-phosphataseconjugated secondary antibody for 2 hours. The plates were developed using the BCIP/NBT substrate.

ELISpot plates were scanned on an AID ELISpot Reader. The unspecific background [mean spot forming units (SFU) from negative control wells] was subtracted from the experimental readings.

Continuous variables were tested for distribution by using the Shapiro-Wilk test, and results are presented as means ± standard deviation if normally distributed, and as median (interquartile range) if non-normally distributed. T-cell response and neutralizing activity were compared between paired samples at two time points (T0 versus T1, T1 versus T2, T0 and diabetes mellitus (45%) being the most common. Immunosuppression with a calcineurin inhibitor and mycophenolate was the most frequently followed protocol (72%); 16 (27%) patients were treated with everolimus and a low dose of a calcineurin inhibitor, and 17 (27%) had been weaned off chronic steroids (Table 1) The percentages of sera demonstrating neutralizing activity (i.e., above the threshold) against the wild-type virus, delta and omicron variants increased from 70%, 65%, and 38%, before the third dose (T0), to 93% (P<0.001), 88% (P<0.001), and 48% (P=0.021) at 3 weeks after the third dose (T1), respectively; and remained high through the 6 months for the wild-type (80%; P=0.060, T2 vs. T1) and delta (77%; P=0.102, T2 vs. T1) ( Figure 2B ). The percentage of neutralizing sera against the omicron variant remained low (39%;P=0.618, T2 vs. T1).

Of a total 103 BNT162b2-3-dose-vaccinated patients, whole blood samples were obtained for 54 patients [Cohort 2; mean age 56.3 (±15.0) years, and 39 (72%) male; Figure 1 and supplementary Table 1 ]. The lymphocyte count on the day of third vaccine dose was 1.5 K/μL (1.1-2.2), with a neutrophil/lymphocyte ratio of 3.2 (2.3-4.5). There was a significant increase in IFN-γ spot numbers from the time of the third dose to 3 weeks after the dose (P=0.007). The IFN-γ spot response was maintained through the 6 months after the third dose (P=1.0; T2 vs. T1, Figure 3A) . Notably, over 70% of individuals in the cohort showed an inducible SARS-CoV-2-specific T-cell response at 3 weeks after the third dose (T1), and this proportion was maintained during the 6 months after the booster dose (P=0.951) ( Figure 3B ). In 12 of the patients, the SARS-CoV-2-specific T-cell response was measured in two time periods, namely, 3 weeks (T1) and 6 months (T2) following the third dose ( Figure 4) . Comparison of paired samples from the same patient at 3 weeks (T1) and 5-6 months after the third dose (T2) showed no significant difference in IFNγ spot numbers (Figure 4) , supporting the durability of the third dose-induced T-cell response.

Importantly, an inducible SARS-CoV-2-specific T-cell response but negative neutralization was observed for 3 patients. No correlation was found between the SARS-CoV-2-specific T-cell response and neutralization.

Several important findings emerged from this prospective longitudinal study in severely immunocompromised but third-dose vaccinated individuals. First, the third BNT162b2 dose induced high neutralization of the wild-type virus and the delta variant and to a lesser degree of the omicron variant.

Second, at 6 months following the third dose, serum neutralizing activity elicited by the third BNT162b2 vaccination was still evident for the wild-type virus and for the delta variant, albeit to a lesser degree, but not for the omicron variant. Third, the third dose induced the development of a sustained SARS-CoV-2specific T-cell population. Fourth, cellular responses were evident in the absence of measurable neutralizing antibodies, suggesting a cellular benefit, even when there did not appear to be an antibody response.

The BNT162b2 vaccine was previously reported to have >90% efficacy in the general population against the ancestor Wuhan virus, 9, 10 but not for HT recipients. Detectable antibodies against the receptor-binding domain were demonstrated in only 10-57% and a cellular response in 10-70% of HT recipients at different time points following 2 doses of mRNA vaccines. 1, [11] [12] [13] With the emergence of new VOCs, significant concerns continue to be raised regarding the effectiveness of the vaccines against these novel variants. We have, uniquely, longitudinally assessed the three-dose BNT162b2-induced neutralization response to the wild-type virus and to two variants responsible for COVID-19 surges by using microneutralization assays involving cell cultures infected with live viruses (wild type and variants). In contrast to previous studies reporting the waning of vaccine (two doses)-induced or disease-induced neutralization responses, we provide longitudinal data on the ability of the three-dose vaccine regimen to induce initial variant-specific neutralizing responses at different time points following 2 and 3 vaccine doses.

We demonstrate that the third dose elicited high neutralization of the wild-type virus and delta variant and to a lesser degree of the omicron variant. The clinical significance of this observation has yet to be determined, but it raises concerns regarding efficacy of the BNT162b2 vaccine in the immunocompromised population against this now dominant variant. Whether a fourth dose will induce higher neutralization response against the omicron variant in this population is yet to be determined.

Notably, for the general population, a third dose induced an increase in neutralization of the wild-type virus (GMT, 891.4) and, to a lesser degree, of the omicron variant (GMT, 108) at 1 month after the third dose, 7 with the latter titer being close to that observed for the wild-type in our HT population 3 weeks after the third dose (GMT, 137). The low neutralization for the HT patients might be partially explained by the low inducible SARS-CoV-2-specific T-cell response observed in our HT population after the second dose. The high inducible SARS-CoV-2-specific T-cell response observed in our study following the third dose might indicate that a fourth dose would induce more effective neutralization against the omicron variant in severely immunocompromised populations.

The balance between the adaptive immune responses, comprising both humoral and SARS-CoV-2specific T-cell responses, that work together is an important factor in the development of protective immunity against viral infections. 14 Activated CD4+ T-cells are important for B-cell activation and the generation of neutralizing antibodies to maintain a durable antibody response. 14 Whether SARS-CoV-2-specific T-cells, in the absence of an effective neutralization response, induce immunity/protection is unknown, but numerous studies support the correlation between the induction of neutralization and immunity. The utility of vaccine-induced neutralizing activity as a predictive metric of protection has been demonstrated in the preclinical and clinical studies of SARS-CoV-2 vaccines designed to elicit robust T-cell responses based on the induction of neutralizing antibodies. [15] [16] [17] We should, however, keep in mind that the interplay and balance between the humoral and cellular immune responses against SARS-CoV-2 is complex and much is still to be learnt, particularly as recent data suggest that exposure to SARS-CoV-2 can induce virus-specific T-cell responses without seroconversion. 18

The strength of our study lies in several directions: 1) The research is timely, deals with urgent public health concerns, is relevant to the medical community due to the exponential rise in omicron variant 19, 20 Thus, despite the ability of emerging variants to alter T-cell specificities, these variants do not escape the entire repertoire of spike-specific T cells. Indeed, it has been demonstrated that, for most vaccinated individuals, VOCs are recognized by the spike-specific T cells induced by mRNA vaccines. 21, 22 Here, we present novel data indicating a third dose-induced durable SARS-CoV-2 specific T-cell response for up to 6 months.

The limitations of the study include the relatively small number of patients (although this is the leading and largest cohort for long-term boosting data). In addition, only peripheral circulatory virusspecific T cells were analyzed (i.e., no information on other localized SARS-CoV-2-specific T cells, e.g., bone marrow), and the IFN-γ ELISpot assay can detect only peripheral T cells secreting the Th1 cytokine IFN-γ. The values for the SARS-CoV-2-specific T cell response that confer protection are unknown. 23 We did not longitudinally routinely perform polymerase-chain-reaction testing for SARS-CoV-2, which could have resulted in underdiagnosis of SARS-CoV-2 infection. Also, the study was not designed to establish predictors of vaccine-induced neutralization, since its aim was to assess the long-term kinetics of vaccine-induced neutralizing antibody. Finally, clinical correlation of these data will be needed.

The third BNT162b2 dose elicited a durable SARS-CoV-2-specific T-cell response and induced high and durable neutralization of the wild-type virus and the delta variant, and to a lesser degree of the omicron variant, providing an encouraging indication of vaccine neutralization against virulent variants. Any hint of vaccine immunological efficacy in HT patients may be magnified several fold in the non-suppressed vaccinated population. Our findings may inform vaccination strategies to control the future trajectory of the COVID-19 pandemic, particularly the need for scheduling booster doses into immunization protocols. 

BNT162b2 vaccination in heart transplant recipients: Clinical experience and antibody response

Third dose of the BNT162b2 vaccine in heart transplant recipients: Immunogenicity and clinical experience

Waning immune humoral response to BNT162b2 Covid-19 vaccine over 6 months

Protection of BNT162b2 vaccine booster against Covid-19 in Israel

Effectiveness of COVID-19 vaccines in preventing SARS-CoV-2 infection among frontline workers before and during B.1.617.2 (Delta) variant predominance-Eight

Effectiveness of Pfizer-BioNTech and Moderna vaccines in preventing SARS-CoV-2 infection among nursing home residents before and during widespread circulation of the SARS-CoV-2 B.1.617.2 (delta) variant-National Healthcare Safety Network

Third BNT162b2 vaccination neutralization of SARS-CoV-2 omicron infection. Accepted for publication

Safety and efficacy of the BNT162b2 mRNA Covid-19 vaccine

Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: An interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK

Antibody response to 2-dose SARSCoV-2 mRNA vaccine series in solid organ transplant recipients

Cellular and humoral immune response after mRNA-1273 SARS-CoV-2 vaccine in liver and heart transplant recipients

Poor humoral and T-cell response to two-dose SARS-CoV-2 messenger RNA vaccine BNT162b2 in cardiothoracic transplant recipients

Decline in neutralising antibody responses, but sustained T-cell immunity, in COVID-19 patients at 7 months post-infection

Safety and immunogenicity of the ChAdOx1 nCoV-19 vaccine against SARS-CoV-2: a preliminary report of a phase 1/2, single-blind, randomised controlled trial

Evaluation of the mRNA-1273 vaccine against SARS-CoV-2 in nonhuman primates

Phase 1-2 trial of a SARS-CoV-2 recombinant spike protein nanoparticle vaccine

Intrafamilial exposure to SARS-CoV-2 associated with cellular immune response without seroconversion

Impact of SARSCoV-2 variants on the total CD4 + and CD8+ T cell reactivity in infected and vaccinated individuals

SARS-CoV-2 mRNA vaccines induce broad CD4+ T cell responses that recognize SARS-CoV-2 variants and HCoVNL63

Prior SARS-CoV-2 infection rescues B and T cell responses to variants after first vaccine dose

Minimal cross-over between mutations associated with Omicron variant of SARS-CoV-2 and CD8+ T cell epitopes identified in COVID-19 convalescent individuals

SARS-CoV-2-specific T cells in infection and vaccination

Figure 1: Recruitment of participants, testing, and follow-up

The study population comprised adult HT patients vaccinated with 3 doses of the Pfizer BNT162b2

COVID-19 vaccine. Patients were longitudinally assessed for the kinetics of variant-specific neutralization (Cohort 1) and for SARS-Co-V-2-specific T-cell response (Cohort 2) over six months

Spike-specific T cell responses induced by the third booster dose of the BNT162b2 vaccine

The authors gratefully acknowledge the invaluable contribution of Ms. Hana Algazi-Patal, the coordinator 

None of the authors has a financial relationship with a commercial entity that has an interest in the subject of the presented manuscript or other conflicts of interest to disclose.