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COVID-19 and X-linked agammaglobulinemia (XLA) – insights from a monogenic antibody deficiency

Purpose of review The clinical outcomes from COVID-19 in monogenic causes of predominant antibody deficiency have pivotal implications for our understanding of the antiviral contribution of humoral immunity. This review summarizes the lessons learned from COVID-19 infection in X-linked agammaglobulinemia (XLA) due to genetic defects in Bruton's tyrosine kinase (BTK). Recent findings Key molecular pathways underlying the development of severe COVID-19 are emerging, highlighting the possible contribution of BTK to hyperinflammation. SARS-CoV-2 specific T-cell responses and complement activation appear insufficient to achieve viral clearance in some B-cell deficient individuals. Whilst appearing efficacious in this group, use of convalescent plasma has been recently associated with the evolution of viral escape variants. Early data suggests individuals with XLA can mount a viral-specific T-cell vaccine response, however, the clinical significance of this is still emerging. Summary In contrast to reports made early in the pandemic, we show XLA patients remain susceptible to severe disease. Persistent infection was common and is likely to carry a significant symptom burden and risk of novel variant evolution. COVID-19 infection in this vulnerable, antibody deficient group due to genetic, therapeutic or disease causes may require prompt and specific intervention for both patient and societal benefit.


INTRODUCTION
Since its emergence in 2019, the novel pandemic coronavirus (SARS-CoV-2) causing COVID-19 has placed enormous pressure on health-care systems worldwide. Individuals with primary immunodeficiency (PID) appear at particularly elevated risk of mortality [1 && ,2 && ]. The diagnosis of PID encompasses a heterogeneous patient group, however, with over 430 distinct monogenic disorders recognised to date, accompanied by a spectrum of immunological and clinical phenotypes [3]. Analysis of young individuals hospitalised with COVID-19 has revealed inbornand acquired-dysregulation of the innate type I interferon signalling pathway is highly enriched within those with risk of severe disease [4 && ,5 && ].
However less is known about the impact of monogenic defects affecting other immune compartments.
X-linked agammaglobulinemia (XLA) is a congenital antibody deficiency caused by mutations in Bruton's Tyrosine Kinase (BTK). Treatment consists of lifelong immunoglobulin replacement therapy (IgRT). It should be noted this treatment does not replace IgA or IgM, nor can current therapies compensate for the other lost roles of BTK outside of B-cell development. These limitations in therapy are being increasingly recognised of late, particularly in relation to ongoing risk of recurrent respiratory tract infections and the frequent development of end organ damage such as bronchiectasis.
Initial experience during the pandemic suggested that individuals with congenital forms of agammaglobulinaemia appeared to experience only asymptomatic or milder disease, relative to those with common variable immunodeficiency (CVID), but considered only 2 congenital agammaglobulinemia cases and 7 patients overall [6 & ]. We and others have recently described the heavy burden associated with persistent and symptomatic respiratory and gastro-intestinal tract infections in individuals with both XLA and CVID [7 & ,8 & ,9,10], suggesting a reevaluation of this assessment as more reports became available. Here, we conduct a rapid review of the peer-reviewed and preprint literature for the outcomes from COVID-19 in cases of congenital agammaglobulinaemia in relation to disease severity and persistence, and consider biological and epidemiological factors which may contribute to these findings. PubMed, MedRxiv, Medline and EMBASE were interrogated for articles published until June 30 th 2021 with the search terms; 'COVID-19', 'SARS-CoV-2', 'XLA', 'Agammaglobulinaemia', 'BTK' and 'Bruton'. Finally, given continued occurrence of new viral variants with enhanced transmissibility, we assess the evidence for T-cell responses to vaccination in this patient group. Together, the findings emphasise the unique balance between pathogen clearance and immunopathology with specific immunological defects, and highlight a need for greater focus on individualised approaches to protect and treat immunocompromised patients.

VIRAL INFECTIONS IN X-LINKED AGAMMAGLOBULINEMIA
Whilst the predisposition to bacterial infection is well recognised in XLA there is in addition both consistent historical evidence of viral susceptibility before and after the advent of IgRT able to maintain stable levels of IgG well into the normal range. This includes enteroviral infection leading to chronic and potentially fatal enteroviral infections (chronic meningoencephalitis in agammaglobulinaemia), from Echovirus type 11 and Coxsackievirus B5 [11,12]. The exact mechanism underlying this susceptibility in XLA is unknown, although it has been suggested the loss of BTK in TLR signalling may play a role [13]. Severe herpesvirus infections are described prior to optimal IgRT and vaccine-associated paralytic poliomyelitis is reported [14]. Live vaccinations are therefore contraindicated in XLA. Respiratory viruses occur more frequently in antibody deficiency and are prolonged with rhinoviral persistence described for 4 months in XLA [10]. Chronic Type 2 norovirus induced gut failure necessitating parenteral nutrition and ultimately HSCT to cure the infection and the antibody deficiency has been recently reported [8 & ]. Taken together the findings suggest an important role for antibody in viral defence with the caveat regarding the other potential functions of BTK.

A ROLE FOR THERAPEUTIC ANTIBODIES AND BRUTON'S TYROSINE KINASE INHIBITION IN COVID-19 INFECTION?
Antibody responses are active in the early immune response to COVID-19, as demonstrated by detectable SARS-CoV-2 specific antibody responses within the first days to weeks of infection [15]. Neutralising antibodies to viral spike protein are an important correlate of protective immunity following vaccination [16]. Studies of antibody therapy in COVID-19 support the importance of early intervention for convalescent plasma [17 & ], and whilst initial results from the 5795 patients allocated to convalescent plasma within the RECOVERY trial suggested this to be ineffective, subsequent reanalysis strongly suggests benefit in sero-negative individuals [18]. In addition, 2 monoclonal antibodies targeting the receptor-binding domain of the SARS-CoV-2 spike protein reduce viral load [19], and offer clear benefit in reducing 28-day inpatient mortality amongst hospitalised sero-negative adults with COVID-19 [20 benefit only when given early and also benefit in seronegative over seropositive individuals [18].
B-cell aplasia and pan-hypogammaglobulinaemia are hallmarks of X-linked and autosomal recessive antibody deficiency [3] which mean that these patients will neither mount an antibody response to COVID-19 and nor are they likely to have antiinterferon antibodies -in contrast to autoimmune polyglandular syndrome type 1 (APS-1), a rare autoimmune disease that results from autosomal recessive mutations of the autoimmune regulatory (AIRE) gene. Instances of severe COVID-19 in individuals with APS-1 coinciding with preexisting neutralising antibodies type I interferons stimulated the search and discovery of similar auto-antibodies in up to 10% of individuals with severe COVID-19 pneumonia [5 && ]. As noted in Fig. 1, BTK has wide ranging roles beyond B-cell development, and is expressed across haematopoietic lineages apart from T-cells, where it participates in Toll-like receptor (TLR) and NLRP3 inflammasome signalling (including thromboinflammation in platelets) ( Fig. 1) [21 -23]. It has been suggested that BTK deficiency may protect from the cytokine storm seen in severe disease based on the role of BTK in macrophage activation and IL-6 production [6 & , [24][25][26], although murine and human studies have shown conflicting results [27]. Recent studies of blood monocytes from general adult patients with severe COVID-19 showed consistent increases in BTK activation and production of interleukin-6 [28 && ]. Blockade of IL-6 using Tocilizumab has been shown also to improve survival in severe disease [29], and BTK inhibitors (such as ibrutinib and acalabrutinib) are now being studied for the treatment of severe COVID-19 following promise in case series [28 It is therefore plausible that agammaglobulinaemia or genetic BTK deficiency may confer protection from severe COVID-19.

OUTCOMES FROM COVID-19 IN X-LINKED AGAMMAGLOBULINEMIA PATIENTS
The clinical outcomes 28 XLA patients with SARS-CoV-2 infection identified from peer-reviewed and preprint literature are summarised in Table 1. Age was reported in 23/28 individuals, with a median age at presentation of COVID-19 infection of 30.5 years (range: 5-55 years). Clinical presentations were available in 24 patients, with asymptomatic presentations uncommon (2/24, 8%). Fever was present in 83%, cough in 50%, and shortness of breath in 38%; a background of bronchiectasis There is increasing evidence for involvement of BTK in roles beyond B cell signalling in phagocytosis, the NLRP3 inflammasome (including thromboinflammation in platelets, [23]), microbe recognition via TLRs, nucleic acid recognition, maturation, differentiation signals and chemotaxis [53]. BTK, Bruton's tyrosine kinase; XLA, X-linked agammaglobulinemia. where CPT was successful in achieving viral clearance after two independent courses of remdesivir had failed. Interestingly, remdesivir achieved symptomatic response and suppression of viral load, but its cessation was followed by recrudescence of symptoms and rise in viral load. Genomic sequencing indicated persistent infection rather than re-infection. Detailed longitudinal investigation of this patient confirmed the absence of viral-specific antibodies produced by the patient and within replacement immunoglobulin therapy. Additionally, complement activation similar to critically ill COVID-19 admissions was demonstrated [34 && ,35]. Polyfunctional CD8 T-cell responses were also observed, comparable to those seen in healthcare workers, and these increased in magnitude during the course of COVID-19 infection [34 && ]. This case illustrates the power of a personalised experimental approach, and suggests that SARS-CoV-2 specific antibodies provide a nonredundant contribution to reduction in viral load and eventual viral clearance. The patient continued regular IgRT throughout their admission, suggesting that clearance was not due to correction of defects of dendritic cell maturation arising in the absence of IgRT [34 && ]. Overall, the small sample size in the XLA patient group reported to date, possible reporting bias, and high mortality in this age-range leaves us underpowered to address the hypothesis that individuals with XLA have a different inpatient mortality rate to the general population (Table 2). Mortality appears comparable to modelling of inpatient mortality for COVID-19 in the UK population of a similar age-band [36]. Time until viral clearance was reported in 8/23 patients (35%) (Fig. 2). Mean duration was 38.3 days (95% confidence interval: 20.3-56.2 days) and ranged from '6 to 14' days (handled as Table 2. Age stratified mortality for individuals with Primary Immunodeficiency, X-linked Agammaglobulinemia, and the general UK population ], symptomatic and inflammatory marker improvement occurred in all but 1 within 48 h, followed by falling viral load and reduction in oxygen requirements [38 & ,39]. Together, this provides evidence for viral persistence of SARS-CoV-2 in patients with XLA and iatrogenic B-cell depletion and suggests a role for antibodies in reduction in viral load and contribution to viral clearance. The United States Food and Drug Administration reissued emergency use authorisation for high-titre COVID-19 CPT in hospitalized patients with impaired humoral immunity [39] in March 2021, however, a potential concern in immunosuppressed individuals was raised by Kemp et al. [40 && ]. Performing phylogenetic analysis on ultra-deep whole-genome sequential sequencing on samples from a chronically infected immunosuppressed individual with SARS-CoV-2, they showed CPT was accompanied by viral evolution and reduced sensitivity to neutralizing antibodies [40 && ]. Recent analysis of a second immunocompromised adult with hypogammaglobulinaemia and SARS-CoV-2 infection for over 290 days has subsequently demonstrated similar viral escape mutations can also arise independently of CPT [41 && ]. In this instance, following failure of remdesivir, treatment with the combination of casirivimab and imdevimab at day 265 achieved progressive resolution of all symptoms over the next 8 weeks, and clearance by RT-PCR was achieved by day 311 [41 && ].

VACCINE RESPONSES TO SARS-CoV-2 IN X-LINKED AGAMMAGLOBULINEMIA
We identified 2 reports assessing vaccine response following 2 doses of Pfizer-BioNTech in 10 individuals with genetically confirmed XLA [42 && ,43 & ]. As expected, these individuals mounted no detectable serological response, however, robust production of IFNy was evident following stimulation with spike peptide using an ex vivo ELISpot approach in 9/10 individuals (and IL-2 production in 5/5). Stimulation with peptides representing SARS-CoV-2 membrane protein induced no cytokine responses, consistent with vaccine-induced cellular immunity over postinfection responses [42 && ]. The relevance of such T-cell assessments, as a reflection of nature and durability of cellular immunity and risk of future severe COVID-19, remain uncertain. The presence of polyfunctional CD8þ T-cells during prolonged infection, described by Buckland et al.    Fig. 3. The efficacy of alternative vaccination strategies, such as subunit or adenoviral vectors has yet to be examined. However, previous assessment of a trivalent inactivated influenza vaccine in 12 XLA patients showed induction of influenza virus-specific CD4 and CD8 T-cell responses comparable to healthy controls when assessed up to 6 months postvaccination [44].

CONCLUSION
In this review we draw together the international experience of COVID-19 in XLA, representing the largest description of outcomes for this rare patient group to date, highlighting the vulnerability of such individuals to chronic SARS-CoV-2 infection. In contrast to reports made early in the pandemic, we show these patients remain susceptible to severe disease despite any immunomodulation mediated by their underlying selective immunodeficiency. Although low, the crude case fatality rate of 4% associated with acute COVID-19 in XLA should be viewed in the context of the young age of the cohort described, likelihood of reporting bias, and the high level of background lung disease (i.e. bronchiectasis). Protracted SARS-CoV-2 infection appeared common, with fever and respiratory symptoms in over 80% of cases at presentation, often accompanied by systemic inflammation and respiratory impairment. This is closely analogous to the marked symptom burden and impaired quality of life associated with persistent viral infections of the respiratory tract, gastro-intestinal, and central nervous system reported in this patient group [7 11,12,14]. The psychosocial impact of protracted COVID-19 infection on these individuals and their families is likely multiplied given requirements to self-isolate accompanied by experiencing progressive debilitation due to declining respiratory function [41 && ]. We also highlight the far-reaching societal and clinical implications of chronic viral infection within primary and the expanding field of secondary predominant antibody deficiency disorders [45]. Whilst viral reversion of attenuated vaccine strains during chronic infection in XLA can result in vaccine-associated paralytic poliomyelitis for the individual [14], evolution of novel highly transmissible SARS-CoV-2 variants in hypogammaglobulinemic individuals poses a continued threat to vaccine-induced herd immunity [40 && ,41 && ]. The significance of increasing titres of anti-SARS-CoV-2 antibody within pooled purified IgRT products over time warrants further investigation, as they may complement T-cell induced vaccine responses. Caution is necessary, however, given immune selection pressure from CPT or future IgRT may accelerate generation of escape variants [40 && ]. This suggests virological surveillance with immunological risk stratification should go hand in hand in order to recognise infection in this subset of vulnerable individuals and support a rapid individualised response.
The paradigm that XLA is simply an antibody deficiency is slowly being challenged. In particular, susceptibility to both severe and persistence viral infections is also being recognised. Beyond the lack of ongoing of IgA/IgM in IgRT, the wider roles of BTK that cannot be compensated for in XLA are likely to play a major role in the control of viral infections.

Acknowledgements
None. Conflicts of interest S.J. has participated in advisory boards, trials, projects, and has been a speaker with Baxalta, CSL Behring, Shire, Thermofisher, Swedish Orphan Biovitrum, Biotest, Binding Site, Grifols, BPL, Octapharma, LFB, GSK, Weatherden, Zarodex, Sanofi, and UCB Pharma. All authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Author contributions: S.J. and A.G. conceived the study. All authors contributed to the literature review. M.J.P. and B.M.J.S. wrote the first draft.  6.