Methods:
After institutional review board (IRB) approval, a prospective study
analyzing immune response to COVID-19 vaccination in pediatric cancer
patients was undertaken. Eligibility criteria included cancer patients
aged 0-25 years of age actively receiving therapy, including
chemotherapy, immunotherapy, and targeted therapy. We excluded patients
who were completely vaccinated prior to their cancer diagnosis. Patients
may have received any of the vaccines approved for emergency use by the
U.S. Food and Drug Administration. This includes BNT162b2, developed by
Pfizer and BioNTech, mRNA-1273, developed by Moderna. For the remainder
of this paper, these vaccines will be referred to as Pfizer and Moderna.
Blood samples were collected pre-vaccination, as well as 2, 4-6, and
8-12 weeks after completion of COVID vaccination. Completion was defined
as completion of the two dose series. As a control group, we enrolled
patients in our clinic aged 0-25 years who were not receiving cancer
directed therapy, followed for a benign hematology issue, or were more
than 6 months after completion of therapy for their cancer diagnosis.
These participants had one sample drawn between 2 weeks and 6 months of
completion of vaccination.
Demographic data was collected from the medical record, including age,
gender, ethnicity, cancer diagnosis, personal history of COVID, current
therapy, type of vaccine received, and dates of vaccination. Therapy
details included if it was chemotherapy, immunotherapy, targeted
therapy, or combination therapy. We also collected if therapy was
intravenous, oral, or both. If available, we also collected complete
blood count (CBC) prior to vaccination and CBCs within 4 days of all
sample time points.
Once blood samples were obtained in clinic, they were transported to the
lab and processed within two hours. In order to maximize T cell yield
for analysis in patients who may have low lymphocyte counts due to
therapy, participants blood samples were collected in Vacutainer® CPT™
Cell Preparation Tube (BD) and processed according to the manufacturer’s
instructions. The cells were then re-suspended in 1ml of freezing medium
(RPMI containing 40% FBS and 12.5% DMSO) and stored in liquid
nitrogen.
To measure immunogenicity, we performed two assays. First, we measured
neutralizing IgG antibodies, which has been shown to be highly
predictive of immune protection from symptomatic SARS-CoV2
infection.10 This was done using the
SARS-CoV2-Surrogate Virus Neutralization Test Kit (GenScript) according
to the manufacturer’s instructions. All the samples were run in
duplicates. The quantitative detection range of this assay is between 47
to 185U/ml. Titers ≤60.8 U/ml (lower 10% of the quantifiable detection
range) was taken as inadequate B-cell response. Titers ≥60.8U/ml was
taken as adequate B-cell response. The second assay measures interferon
gamma (IFNγ) secretion, which is a marker of T cell response to viral
antigens and another established method of measuring cellular immune
response after vaccination.11 This is done using an
enzyme-linked immunosorbent spot (ELISPOT) kit for IFNγ (Mabtech).
Briefly, frozen peripheral blood mononuclear cells were thawed and then
seeded onto anti-human IFNγ monoclonal antibody pre-coated plates at a
concentration of 250,000 cells/well. These cells were then stimulated
with SARS –CoV-2 spike peptide pools at a concentration of 2µg/ml by
incubating for 36 hours in 5% CO incubator and sent to Mabtech, Inc.
Ohio, USA, for quantitation. Results were reported as Spot forming units
per million cells (SFU/106cells). SFU values ≤ Mean-
standard deviation (i.e. ≤ 659 SFU/106 cells) of the
control sample values were defined as an inadequate T-cell response.
When analyzing what clinical features may impact vaccine response, we
utilized B cell response by neutralizing IgG antibodies to define our
adequate and inadequate response groups. This was because this assay
allowed for replicates to strengthen the validity of our results as
compared to the T cell assay, which could be only done once. Statistical
analysis was done using Graphpad Prism 8.0. When appropriate, unpaired t
tests were obtained to evaluate for statistical significance, which was
defined as p value <0.05.