Catch-up antibody responses and hybrid immunity in mRNA vaccinated patients at risk of severe COVID-19

Abstract Background The immunogenicity of repeated vaccination and hybrid immunity in vulnerable patients remains unclear. Methods We studied the impact of iterative Covid-19 mRNA vaccination and hybrid immunity on antibody levels in immunosuppressed subjects. Patients with liver cirrhosis (n = 38), survivors of allogeneic haematopoietic stem cell transplantation (allo-HSCT) (n = 36) and patients with autoimmune liver disease (n = 14) along with healthy controls (n = 20) were monitored for SARS-CoV-2-S1 IgG after their 1st–3rd vaccine doses, 31 of whom became infected with the Omicron variant after the 2nd dose. Ten uninfected allo-HSCT recipients received an additional 4th vaccine dose. Results Unexpectedly, immunosuppressed patients achieved antibody levels in parity with controls after the 3rd vaccine dose. In all study cohorts, hybrid immunity (effect of vaccination and natural infection) resulted in approximately 10-fold higher antibody levels than vaccine-induced immunity alone. Conclusions Three doses of the Covid-19 mRNA vaccine entailed high antibody concentrations even in immunocompromised individuals, and hybrid-immunity resulted further augmented levels than vaccination alone. Clinical trial registration: EudraCT 2021-000349-42


Introduction
Irrespective of the underlying aetiology, end-stage liver disease is characterised by compromised immune function. Cirrhosis-associated immune dysfunction (CAID) is thought to occur as a result of hepatic reticuloendothelial cell injury, reduced production of vital innate immunity proteins by the liver [1] as well as systemic inflammation [2], and subsequently leads to an increased susceptibility to severe and life-threatening infections. Although CAID is primarily associated with impaired innate immune responses [3][4][5], recent studies indicate that certain subsets of T cells in patients with cirrhosis exhibit markers of exhaustion, such as TIM-3, CTLA-4, and PD-1, implying that T cell deficiency may also contribute to the observed vulnerability to infections [6,7]. Thus, not surprisingly, individuals with cirrhosis who contract SARS-CoV-2 are at heightened risk of decompensation, severe morbidity, and death [8][9][10].
Likewise, recipients of allogeneic haematopoietic stem cell transplantation (allo-HSCT) are severely immunocompromised, especially the first year following transplantation. Innate immunity generally recovers rapidly, although some functions may be impaired for prolonged periods of time. The adaptive immune system recovers laggardly during the first 1-2 years. However, a successful immune reconstitution can be hampered by the presence of graft versus host disease (GvHD) and its treatment as well as other post-transplant interventions (e.g. renewed chemotherapy or rituximab treatment), higher age and infectious complications [11,12]. As a successful response to vaccination necessitates a partial reconstitution of B-and T-cell immunity, allo-HSCT recipients often experience prolonged suboptimal responsiveness [13]. Several studies reported COVID-19-associated mortality exceeding 20% in this population during the first pandemic wave [14,15], but over time mortality rates have declined to 1-8% in fully vaccinated recipients [16,17].
Two mRNA vaccine doses entail abated immunity in patients with liver cirrhosis [18] especially in the presence of more advanced disease [19] as well as among survivors of allo-HSCT [20], though a third dose improve responses among both patient cohorts [21,22]. Recently, additional vaccine doses are recommended to enhance the protection of susceptible populations as vaccineinduced immune responses substantially wane over time [23].
Although Omicron is reportedly more transmissible and prone to vaccine breakthroughs than prior variants, it also appears less virulent [24]. Recent observations suggest that protection against reinfection with SARS-CoV-2 Delta (B.1.617.2) is higher among individuals with hybrid immunity, referring to the combined effect of vaccination and natural infection, as compared with vaccineor infection-induced immunity alone [25].
This study aimed at clarifying the immunogenicity of repeated vaccination and hybrid immunity in three cohorts of vulnerable patients. We thus monitored patients with the liver disease where patients received a 3rd dose of mRNA COVID-19 vaccine along with a group of allo-HSCT recipients immunised with up to 4 doses.

Study design and endpoints
The DurIRVac study (EudraCT 2021-000349-42) was conducted at the Sahlgrenska University Hospital between March 2021 and July 2022. The researchers in this study had no influence regarding which mRNA COVID-19 vaccine (mRNA-1273 Moderna Spikevax V R or BNT162b2 Pfizer-BioBTech Comirnaty V R ) participants received nor the number or time-point of vaccinations, as doses were administered in accordance with regional prioritisation. Research participants gave written informed consent before enrolment. The study was approved by the Swedish Ethical Review Authority (permit nos. 2020-03276, 2021-00374 and 2021-00539) and by the Swedish Medical Products Agency (no. 5.1-2021-11118) and is registered at the European Union Drug Regulating Authorities Clinical Trials Database (EudraCT no. 2021-000349-42). The original study primary endpoints were to quantify anti-RBD Spike lgG quantitative serology levels as well as concentrations achieved using a novel rapid spike cytokine release assay [26] immediately before and 4 weeks after administered COVID-19 vaccine doses for enrolled research participants, and thereafter strive for sampling every 3 months for 2 years. The sampling schedule was later reduced from the original study protocol for logistical reasons. Additionally, when the origin study protocol was written in 2021, available data on immune responses to COVID-19 vaccination was limited, making it challenging to provide a statistical basis for calculating the study population in our application to the Swedish Ethical Review Authority and Swedish Medical Products Agency. The original study protocol states that for these reasons the immune responses of approximately 100 healthy research participants, 100 patients with cirrhosis, 100 patients with impaired renal function, 100 transplant recipients and 100 patients with impaired immune systems for other reasons were intended to be evaluated. Subsequently, these enrolment goals could not be achieved, and instead all potential research participants in each respective cohort that could be reached and were willing to participate were enrolled in the study (CONSORT diagrams in Supplementary Figures S1-S3).

Study population
Liver disease cohorts Thirty-eight patients with cirrhosis (baseline characteristics in Supplementary Table S1, CONSORT diagram in Supplementary Figure S1) and 14 non-cirrhotic patients, 5 of whom were receiving immunosuppressive medication, with autoimmune liver diseases (ALD) were enrolled (baseline characteristics in Supplementary Table  S2, CONSORT diagram in Supplementary Figure S1). The liver fibrosis stage was confirmed using acoustic radiation force impulse (ARFI) by Acuson S2000 (Siemens Medical Solutions, Erlangen, Germany).

Healthy controls
Twenty healthy volunteers (baseline characteristics in Supplementary Table S4, CONSORT diagram in Supplementary Figure S3) were recruited.
Analysis of IgG against the RBD within S1 of SARS-CoV-2 (anti-RBD-S1 IgG) Chemiluminescent microparticle immunoassays were performed on serum using the automated Alinity system for quantitative measurement of anti-RBD-S1 IgG (SARS-CoV-2 IgG II Quant, Abbott, Abbott Park, Illinois, USA) with levels reported in the WHO international standard Binding Antibody Units (BAU)/mL.

Documentation of COVID-19
All participants were COVID-19-naïve prior to the 2nd vaccine dose as per reverse transcription polymerase chain reaction (RT-PCR), COVID antigen test, analyzes for antibodies against the nucleocapsid protein of SARS-CoV-2 (SARS-CoV-2 IgG, Abbott, Abbott Park, Illinois, USA), review of medical records, or evaluation of participant self-reporting questionnaire. All documented COVID-19 infections had detectable SARS-CoV-2 RNA, reactive COVID antigen test, or presence of antibodies against the nucleocapsid in a sample obtained after the 2nd vaccine dose. The Omicron variants (B.1.1.529) BA.1 and BA.2 dominated the local circulation during this study.

Adverse events related to mRNA COVID-19 vaccination
Participants completed a standardised questionnaire and adverse events were categorised per CTCAE (Common Terminology Criteria for Adverse Events).

Statistical analysis
Continuous variables are described in the text as median and range and as logarithmic values with mean and 95% confidence interval in the figures. Mann-Whitney U test, paired t-test, and t-test with Welch's correction on logarithmic values were applied to calculate differences in serologic response and time to sampling between groups. Data analyses were performed using SPSS for MacOS and GraphPad Prism 8 for macOS. Statistical significance was set to p < .05. All indicated p-values are two-sided.

Antibody response
After the 3rd dose of mRNA COVID-19 vaccine, all but 2 patients with cirrhosis (36/38, 95%) achieved anti-RBD IgG levels exceeding the proposed protective level of 141 BAU/ml [27] (Figure 1(A)); please note that this putative preventative threshold likely varies across SARS-CoV-2 variants though antibody titres reportedly are associated with both risk of breakthrough infection as well as disease severity [28]. One cirrhotic patient continued to have undetectable antibodies despite receiving a 3rd vaccine dose. Anti-RBD IgG levels were significantly higher after the 3rd vaccine dose among uninfected participants (i.e. vaccine-induced immunity, median 563 vs. 944 BAU/ml, after 2nd and 3rd dose respectively, p ¼ .005, paired t-test on logarithmic values). Ten of 38 patients with cirrhosis were infected with SARS-CoV-2 following the 2nd dose. Hybrid Figure 1. Antibody response to COVID-19 mRNA vaccination with and without COVID-19 during the study period. Scatter plots with mean and 95% confidence interval demonstrating logarithmic IgG antibody levels in serum against the receptor-binding domain (RBD) within spike-1 (S1) among patients with liver disease with (A) or without cirrhosis (B; autoimmune hepatitis) as well as allogeneic haematopoietic stem-cell transplantation recipients (C) and healthy controls (D). Patients are stratified as to whether they acquired a SARS-CoV-2 Omicron subvariant infection following the second vaccine dose (i.e. developed hybrid immunity (HI), open circles) or not (i.e. only vaccine-induced immunity (VII), grey filled circles for those receiving 3 and black filled circles for those receiving 4 vaccine doses). The dotted line represents the limit of detection of the assay (i.e. 14 BAU/mL). Statistics were calculated by unpaired t-test with welch's correction ( Ã p < .05, ÃÃÃ p < .001, ÃÃÃÃ p < .0001, ns ¼ not significant) and paired t-test on logarithmic values ( # p < .05, ## p < .01, ### p < .001, ns ¼ not significant). immunity, i.e. the combined effect of vaccination and naturally acquired COVID-19, in cirrhotic patients, was associated with strikingly higher antibody levels (11,275 vs. 944 BAU/ml, p < .0001, unpaired t-test with Welch's correction on log values) as compared with antibody levels achieved through 3 doses of vaccination alone. The time elapsed between vaccination to a blood draw after the 3rd dose of the vaccine was significantly longer than after the 2nd dose (118 vs. 90 days, p < .001, Mann-Whitney U-test). Eight patients (8/38, 21%) achieved anti-RBD IgG levels that were lower than those achieved after the 2nd vaccine dose. However, the time from vaccination to sampling was protracted in 7 of these 8 patients, allowing for the possible waning of antibody levels.
After the 2nd mRNA COVID-19 vaccine dose, 3/14 (20%) of non-cirrhotic ALD patients had antibody concentrations <141 BAU/mL, but following the 3rd dose, all achieved levels of anti-RBD IgG exceeding 141 BAU/ml (Figure 1(B)). A non-significant trend towards higher concentrations after the 3rd vaccine dose was noted among 9 uninfected ALD participants (i.e. vaccineinduced immunity, median 709 vs. 1497 BAU/ml after 2nd and 3rd dose respectively, p ¼ .09, paired t-test on logarithmic values). However, hybrid immunity in ALD was associated with significantly higher antibody levels (median 5307 vs. 200 and 1497 BAU/ml, p ¼ .01 and .008, unpaired t-test with Welch's and paired t-test on logarithmic values) as compared with levels achieved following the 2nd and 3rd vaccine doses without infection. The time elapsed between vaccination and sampling after the 3rd vaccine dose was significantly longer than after the 2nd dose (92 vs. 68 d, p ¼ .01, Mann-Whitney U-test).
Antibody responses were also compared between vaccine-induced immunity achieved in COVID-19 naïve allo-HSCT patients following the 3rd and 4th doses of mRNA vaccine as well as hybrid immunity after documented Omicron infection (Figure 1(C)). After the 3rd dose, all patients achieved levels exceeding 141 BAU/ml. A cohort of slightly older patients had received a 4th dose following which 9 of 10 (90%) achieved antibody levels exceeding 141 BAU/ml. Hybrid immunity (n ¼ 8) was significantly associated with higher antibody levels (median 11,401 vs. 1,426 and 6471 BAU/ml, p < .001 and <.05, t-test with Welch's correction on logarithmic values) as compared with levels achieved following 3 (n ¼ 18) or 4 (n ¼ 10) vaccine doses without infection.
For comparison the anti-RBD IgG concentrations achieved among healthy controls following their 2nd and 3rd mRNA COVID-19 vaccine doses as well as hybrid immunity after documented Omicron infection were analysed (Figure 1(D)). Controls with previous COVID-19 infection, i.e. hybrid immunity, had significantly higher levels of anti-RBD IgG (median 6566 vs. 1458 BAU/ml, p ¼ .01, t-test with Welch's correction on logarithmic values) after the third dose of vaccine compared to COVID-19 naïve participants.

Severity of breakthrough COVID-19
There were 22 reported mild SARS-CoV-2 Omicron subvariant infections among the patient cohorts and 8 among healthy controls during the study period. Additionally, a 54-year-old male allo-HSCT recipient developed a pulmonary embolism but promptly recovered after initiation of direct oral anticoagulants.

Tolerability and safety of mRNA COVID-19 vaccines
For the patient cohorts with liver disease the most common adverse events were reaction at the injection site (64%) and fatigue (22%). Similarly, for the allo-HSCT recipients, the most frequent adverse events were local injection site reactions (24% and 40% after the 3rd and 4th dose respectively) as well as fatigue or fever (11% and 0% after the 3rd and 4th dose respectively). One patient reported worsening of GvHD following both the 3rd and 4th doses but required no change in immunosuppressive treatment. The frequency or severity of adverse events from vaccination were lower among patients than in healthy controls, and no serious adverse events were recorded.

Discussion
The main findings in this study were that a first or second booster immunisation after the initial 2-dose mRNA COVID-19 vaccine regimen resulted in high antibody levels in most, albeit not all, at-risk patients enrolled, and that hybrid immunity elicited significantly higher antibody concentrations than vaccine-induced immunity alone in all 3 patient cohorts as well as in healthy controls. Additionally, despite sluggish responses to the first 2 doses of COVID-19 mRNA vaccination, especially among cirrhotic patients, the median concentrations achieved in the 3 patient cohorts after their 3rd dose were in parity with those observed in healthy controls indicating an encouraging catch-up effect. Indeed, among allo-HSCT recipients Omicron infection together with 3 doses led to significantly higher levels than those achieved following both a 3rd and 4th mRNA vaccine dose without prior COVID-19.
Whether or not the observed higher humoral responses induced by hybrid immunity also translate into increased durability remains to be seen though preliminary reports are propitious as is the suggested protective roll against reinfection [25]. A recent auspicious report indicates that an early transient presence of multipolar, antigen-specific T helper (T H ) cells after verified COVID-19 is followed by a durable T H 1-like reactivity against nucleocapsid reflecting long-lasting T cell memory [29]. However, though both vaccine-induced and hybrid immunity from early Omicron variants protect against severe COVID-19, newer publications indicate that this protection is diminished against more recently emerged Omicron sublineages, e.g. XBB [30,31], suggesting the continued need for updated booster vaccine doses.
Despite a 3rd or 4th vaccine dose 2 of 28 patients with cirrhosis and 1 of 28 allo-HSCT recipient did not achieve antibody levels exceeding the putative protective level 141 BAU/mL [27]. Whether or not this finding warrants immune surveillance of these at-risk populations, however, is debateable, but our findings are in line with a similar study highlighting the benefits of a 3rd mRNA vaccine dose in both patients with cirrhosis [22] and allo-HSCT recipients [28,32,33]. Recent publications suggested health benefits from a 4th mRNA COVID-19 vaccine dose [34][35][36], but to our knowledge there currently are no reports beyond 4 doses.
This study has strengths and limitations. Strengths include a unique documentation of the impact of hybrid immunity on antibody levels, in both at-risk populations and healthy controls, following first exposure to the SARS-CoV-2 Omicron variant after having received two doses of mRNA COVID vaccine. Over time, this will be crescively difficult to demonstrate because of increasing limitations regarding access to diagnostic testing as well as the accrued risk of reiterated SARS-COV-2 infections. Limitations in this study include a relatively small sample size and logistical difficulties regarding adherence to the original study protocol with regards to analyses other than antibody concentrations.
In conclusion, our findings imply that protective humoral immunity is achievable in vulnerable populations by reiterated COVID-19 vaccination, and especially by hybrid immunity.

Disclosure statement
No potential conflict of interest was reported by the author(s).