key: cord-1048899-9splodhi
authors: Dailey, J.; Kozhaya, L.; Dogan, M.; Hopkins, D.; Lapin, B.; Herbst, K.; Brimacombe, M.; Grandonico, K.; Karabacak, F.; Schreiber, J.; Liang, B. T.-L.; Salazar, J.; Unutmaz, D.; Hyams, J.
title: Antibody Responses to SARS-CoV-2 after Infection or Vaccination in Children and Young Adults with Inflammatory Bowel Disease
date: 2021-06-15
journal: medRxiv : the preprint server for health sciences
DOI: 10.1101/2021.06.12.21258810
sha: d3a53d5c21a06808aa91d42fb84bd48f1ef54488
doc_id: 1048899
cord_uid: 9splodhi

Abstract Background: Characterization of neutralization antibodies to SARS-CoV-2 infection or vaccination in children and young adults with inflammatory bowel disease (IBD) receiving biologic therapies is crucial. Methods: We performed a prospective longitudinal cohort study evaluating SARS-CoV-2 Spike protein receptor binding domain (S-RBD) IgG positivity along with consistent clinical symptoms in patients with IBD receiving infliximab or vedolizumab. Serum was also obtained following immunization with approved vaccines. IgG antibody to the spike protein binding domain of SARS-CoV-2 was assayed with a fluorescent bead-based immunoassay that takes advantage of the high dynamic range of fluorescent molecules using flow cytometry. A sensitive and high-throughput neutralization assay that incorporates SARS-CoV-2 Spike protein onto a lentivirus and measures pseudoviral entry into ACE2 expressing HEK-293 cells was used. Results: 436 patients were enrolled (mean age 17 years, range 2-26 years, 58% male, 71% Crohn disease, 29% ulcerative colitis, IBD-unspecified). 44 (10%) of enrolled subjects had SARS-CoV-2 S-RBD IgG antibodies. Compared to non-IBD adults (ambulatory) and hospitalized pediatric patients with PCR documented SARS-CoV-2 infection, S-RBD IgG antibody levels were significantly lower in the IBD cohort and by 6 months post infection most patients lacked neutralizing antibody. Following vaccination (n=33) patients had a 15-fold higher S-RBD antibody response in comparison to natural infection, and all developed neutralizing antibodies to both wild type and variant SARS-CoV-2. Conclusions and Relevance: The lower and less durable SARS-CoV-2 S-RBD IgG response to natural infection in IBD patients receiving biologics puts them at risk of reinfection. The robust response to immunization is likely protective.

Individuals with inflammatory bowel disease (IBD) potentially have higher risk of symptomatic or severe SARS-CoV-2 disease because of the immunosuppressive therapies they receive. However, results from large multicenter international studies performed in adults and children with IBD to date show similar severity of illness and risk of hospitalization or death compared with age-matched individuals without IBD (1) (2) (3) . Nonetheless, corticosteroid therapy, thiopurine therapy, or combination anti-TNF/thiopurine at the time of infection have been associated with more severe disease (1, 4) .

SARS-CoV-2 elicits a robust humoral and cellular immune response (5) (6) (7) (8) (9) (10) (11) (12) . A strong antibody response generated by effector B cells is critical for the eventual clearance of viruses, and the development of potent neutralizing antibodies is a major part of the memory response that prevents reinfection. Indeed, SARS-CoV-2 elicits virus-specific IgM, IgG and IgA, and neutralizing IgG antibodies (nAbs) following 7-14 days post-infection (9, 10) . IgG levels to SARS-CoV-2 nucleocapsid (N) and spike proteins increase gradually following infection.

Antibodies that bind to the receptor binding domain (RBD) of spike protein may neutralize viral entry into cells and play an important role in the protective immune response to SARS-CoV-2 (11) . A recent study from the United Kingdom of 7000 adult patients receiving either the biological therapies infliximab (anti-TNFα) or vedolizumab (anti-α4β7 integrin) (13) revealed in fewer infliximab-treated than vedolizumab-treated patients (48% (39/81) vs 83% (30/36), p=0.00044) and the magnitude of anti-SARS-CoV-2 reactivity was lower.

In the late winter/spring 2020 Connecticut (U.S.A) was a "hot spot" for SARS-CoV-2 infection presenting a unique opportunity to perform longitudinal surveillance on a geographically defined population of children and young adults with IBD receiving biologics in our ambulatory infusion center. Here we report IgG S-RBD antibody response to SARS-CoV-2 infection and its durability over time, assess the neutralization capability of serum from our patient cohort to native and variant virus, and the relationship of this humoral response to clinical expression of COVID-19. The recent availability of SARS-CoV2 vaccine for young individuals further allowed us to assess SARS-CoV2 S-RBD IgG responses following vaccination.

Between May 2020 and April 2021, we performed a single-center prospective longitudinal study evaluating seropositivity to SARS-CoV2 spike protein binding domain in an ambulatory infusion center in Farmington, CT. Patient eligibility included: (1) diagnosis of IBD, (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

To measure SARS-CoV-2 S-RBD IgG antibodies we used a fluorescent bead-based immunoassay developed as previously described (15). We screened patient serum samples for SARS-CoV-2 wild type (WT) S-RBD or K417N, E484K, N501 mutant (mt S-RBD) Spike protein receptor binding domain specific IgG antibodies. Both WT S-RBD and mt S-RBD proteins were Biotinylated and purchased from Acro Biosystems. Tenfold serially diluted serum samples were assayed then analyzed by flow cytometry using iQue Screener Plus (IntelliCyt, MI)(15). Flow cytometry data were analyzed using FlowJo (BD biosciences). Titration curves for each sample were used to normalize the area under the curve (AUC) values to quantitate the antibody levels. Statistical analyses were performed using GraphPad Prism 8.0 software (GraphPad Software).

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(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

To determine patient characteristics and the humoral immune response to SARS-CoV-2, correlations between antibody AUC levels, 50% neutralization titer (NT50) values and demographics of the study subjects were analyzed. All immunological data were entered into a common Excel-based database. These data were then merged with cohort information drawn from the Redcap database, using study identification numbers. The database was updated monthly as the study proceeded and the monthly updates integrated to provide a longitudinal database for analysis. To compare differences across groups, chi-square and Fisher exact tests were used to compare count or categorical data. T-tests and ANOVA were used to compare mean differences and exact p values are reported. In small sample settings, non-parametric ranksum tests were used to compare continuous measures. Multiple comparison corrections for All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 15, 2021. ; https://doi.org/10.1101/2021.06.12.21258810 doi: medRxiv preprint pairwise comparisons were used as appropriate. All statistical analyses were performed using GraphPad Prism V8 software. Numbers of repeats for each experiment and sample sizes were described in the associated figure legends.

Of 472 patients with IBD treated in our infusion center, 436 (92%) were enrolled (mean Table 1 . The group with anti-SARS-CoV-2 positivity was significantly older than the group without S-RBD IgG antibodies (p=0.02). Twenty-four of the 44 reported a positive PCR test, 22 had a household member who tested positive, and 25 reported a household member who experienced symptoms consistent with infection. Duration between most recent positive PCR test and serum sampling was 3.9 weeks (median 2.6, range 1.0-12.6) in seropositive IBD subjects who had documented PCR. For 3/9 asymptomatic patients where PCR+ date was known the average duration between PCR+ and positive serum was 5 weeks. For the other 6 the time of infection was unknown but less than 12 weeks. All adult control patients were PCR+ (mean age 41 years) with a mean duration between +PCR and serum for study of approximately 6 weeks.

All non-IBD pediatric controls (mean age 13 years) were PCR+ and had serum drawn during their initial hospital admission for moderate to severe COVID-19.

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(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

We first compared anti-S-RBD IgG responses in our IBD study population to non-IBD adults and pediatric patients with COVID-19. Serum anti-S-RBD IgG was significantly lower than either comparator group, most notably (9.9x difference) between IBD patients and non-IBD adults ( Figure 1a ). There was no significant difference between the biologic treatment groups.

We next assessed the neutralizing activity of the serum from each of the 3 study groups using a pseudovirus test to block cell entry, as described (15). While the neutralization titer (NT50) between adult and pediatric non-IBD groups were similar, both were significantly higher compared to the IBD group (Figure 1b) . No neutralizing activity was observed in 5/44 of the IBD group as well as 1/23 of the adult non-IBD subjects and 1/11 of the pediatric non-IBD All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 15, 2021. ; https://doi.org/10.1101/2021.06.12.21258810 doi: medRxiv preprint subjects. We did not find any significant differences between the biologic treatment groups.

There was also significant correlation between NT50 and S-RBD IgG levels (Supplemental (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 15, 2021. ; https://doi.org/10.1101/2021.06.12.21258810 doi: medRxiv preprint weeks. S-RBD IgG responses were significantly higher (~15x) following vaccination in comparison to natural infection in the IBD subgroup (Figure 3a) . Moreover, all IBD subjects had higher neutralizing IgG post vaccination compared to natural infection (~10x), including patients receiving both infliximab monotherapy and infliximab along with methotrexate ( Figure 3b) . 

Our data have significant and immediate implications for patients with IBD receiving biologic therapy and potentially for others with chronic inflammatory disease receiving similar therapies. The decreased S-RBD IgG response compared to adult non-IBD controls and the relatively rapid disappearance of neutralizing antibodies post-infection implies increased risk for All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 15, 2021. ; https://doi.org/10.1101/2021.06.12.21258810 doi: medRxiv preprint reinfection. However, the strong response to vaccination in these subjects with the generation of S-RBD neutralizing antibody of higher titer than natural infection, underscores the critical importance of immunization in this patient group.

During the period we performed our antibody screens SARS-CoV-2 infection in the United States (16) was around 10% underscoring that our study population, which also had around 10% evidence of SARS-CoV-2 infection, was not at increased risk of infection. The clinical expression of disease was mild as has been observed (1, 2). Yet, our data differ from the study in the United Kingdom (13) where less than half of PCR+ patients with IBD receiving infliximab were found to have antibodies to SARS-CoV-2. In our sampling, we found 44/436 (about 10%) to have S-RBD antibodies, including 24 IBD patients who had tested PCR+ and 23/24 had S-RBD IgG antibodies and had detectable neutralizing antibody. Our overall prevalence of antibody positivity to SARS-CoV-2 was about twice that noted in the United Kingdom study, likely reflecting the epidemiology of infection in Connecticut compared to the United Kingdom.

Data are now emerging on the response to immunization with mRNA and other vaccines.

A recent study demonstrated that infliximab was associated with attenuated immunogenicity to a single dose of the BNT162b2 and ChAdOx1 nCoV-19 SARS-CoV-2 vaccines (17). However, vaccination after SARS-CoV-2 infection, or a second dose of vaccine, led to seroconversion in most patients. Our data, acquired after 2 doses of mRNA vaccine showed robust seroconversion despite infliximab therapy and all patients had neutralizing antibody. We did not sample patients after a single dose. Our observations strongly support the recent vaccination endorsement by the International Organization for the Study of IBD (18) and lessen theoretical concerns about diminished response to immunization for influenza (19, 20) and hepatitis B (21) as has been All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 15, 2021. ; https://doi.org/10.1101/2021.06.12.21258810 doi: medRxiv preprint reported in patients on anti-TNF therapy. Indeed, a recent systematic review and meta-analysis suggested that immunosuppressive therapy does not significantly reduce immunogenicity of vaccinations in children with IBD (22) .

The potential effect of anti-metabolites on response to immunization remains unclear and a recent report in solid organ transplant patients suggested a lessened response to mRNA vaccination (23) . In adult patients with rheumatoid arthritis a temporary discontinuation of methotrexate for 2 weeks prior to influenza vaccination improved immunogenicity (24).

Methotrexate is often used either alone or in combination with anti-TNF agents in the treatment of IBD. In our cohort, the number of patients on combination infliximab and methotrexate who had also been vaccinated was too small to compare, albeit all had similar S-RBD IgG levels.

Future studies assessing larger numbers of IBD patients on combination infliximab and antimetabolites would be useful to determine if the addition of anti-metabolite therapies lessen the antibody responses to either natural infection or immunization. Finally, 4 of our immunized patients were documented to have had SARS-CoV-2 infection prior to immunization and all received 2 doses of a mRNA vaccine. It has been suggested that only one dose may be necessary in healthy subjects with prior infection (25) but we did not obtain samples after one dose of vaccine in our patient cohort, thus the impact of the second dose remains to be determined.

Our study has several significant strengths. We performed longitudinal surveillance on a defined population allowing us to identify both symptomatic and asymptomatic infection. In contrast to previous studies, which just measured antibody levels, we determined neutralization activity of the anti-S-RBD IgG to both wild-type and variant virus. Thus, we were able to determine in an assay assumed to be a surrogate for protection from clinical infection, that most of our patients generated titers of neutralizing antibody post-infection that rapidly declined over All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 15, 2021. ; https://doi.org/10.1101/2021.06.12.21258810 doi: medRxiv preprint a 6-month period. There were also several limitations. We did not have control data from IBD patients not receiving biologics, particularly those on methotrexate or thiopurine monotherapy.

We also do not yet have longitudinal data on the durability of antibody response following vaccination. Finally, we did not test antibody neutralization ability from our patients against emerging SARS CoV-2 variants which may reveal lower titers and a more rapid decline in protective levels. However, given almost perfect correlation of S-RBD IgG levels with the NT50 (Supplemental Figure 1b) and a 34.3x lower antibody response to mutant S-RBD in natural infection compared to vaccine (Figure 4c ), we anticipate neutralization activity to decline as we observed for the S-RBD antibody level.

In conclusion, the humoral response to SARS-CoV-2 infection in children and young adults was less than in adult and pediatric non-IBD controls and neutralizing antibody was absent in most infected IBD patients by 6 months. The robust response to immunization was reassuring and supports the utility of vaccinating these patients. Further data will be required to understand the durability of the response to the vaccine, whether previous infection will enhance the antibody response to the vaccine in this patient group lessening the need for a second dose, and whether chronic anti-metabolite administration will have a mitigating influence over time. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (n= 21 for baseline and +2 months, n=20 for +4 months, n=19 for +6 months) b. S-RBD IgG All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Dotted lines indicate the neutralization threshold which was NT50 of 5. Two-tailed Mann-Whitney U test was used to determine the statistical significance. All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. with lentiviruses pseudotyped with wild-type and N501Y-mutated SARS-CoV-2 spike proteins as described in the Methods. Blue, orange, and red dots indicate the subjects with Vedolizumab monotherapy, Infliximab monotherapy, and Infliximab + Methotrexate co-therapy, respectively.

Green dots represent the COVID-19 seropositive subjects among the vaccinated group. Circles

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The authors thank the patients and families cared for at the Connecticut