Human antiviral B cell responses: Emerging lessons from hepatitis B and COVID‐19

Abstract Humoral immunity is a critical component of the coordinated response required to resolve viral infections and mediate protection following pathogen clearance or vaccination. A better understanding of factors shaping the memory B cell response will allow tailored development of efficient preventative vaccines against emerging acute viral infections, therapeutic vaccines, and immunotherapies for chronic viral infections. Here, we use recent data obtained by profiling antigen‐specific B cell responses in hepatitis B as a framework to explore lessons that can be learnt from different viral infections about the diverse influences on humoral immunity. Hepatitis B provides a paradigm where successful B cell responses in resolved or vaccinated individuals can be contrasted to the failed response in chronic infection, while also exemplifying the degree to which B cell responses within infected individuals can differ to two antigens from the same virus. Drawing on studies in other human and murine infections, including emerging data from COVID‐19, we consider the influence of antigen quantity and structure on the quality of the B cell response, the role of differential CD4 help, the importance of germinal center vs extrafollicular responses and the emerging concept that responses residing in non‐lymphoid organs can participate in B cell memory.


| INTRODUC TI ON
While the GC has remained the focus of B cell research, it has long been appreciated that antibody responses can also develop outside of the B cell follicle in the absence of notable GCs. Extrafollicular differentiation of naive B cells into short-lived antibody secreting cells has been shown to mediate early antiviral immune protection in mice, 1 with extrafollicular B cells also able to undergo affinity maturation and generate both memory and long-lived plasma cells independently of T cell help. [2][3][4][5] These responses provide malleable first line defense against replicating pathogens, yet may also contribute to autoantibody production and immunopathology.
Although the roles of B cells in viral infections are diverse and wide-ranging, including immunoregulatory cytokine production and antigen presentation, this review will concentrate on findings from new studies in hepatitis B, SARS-CoV-2 infection and other human and murine infections to consider emerging concepts regarding the factors that govern memory B cell differentiation and their impact on humoral immunity. Due to the role of virus surface proteins in facilitating virus entrance, antibodies targeting HBsAg have strong neutralizing activity, 7,8 interfering with the attachment of the "a"-determinant region of the virus to heparan sulfate proteoglycans on hepatocytes, or blocking binding of the pre-S1 domain of HBsAg to the host cellular receptor, sodium taurocholate co-transporting polypeptide (NTCP). Therefore, a lack of neutralizing anti-HBs, in combination with weak and exhausted HBV-specific CD8 T cells, is thought to contribute to viral persistence in chronically infected patients 9,10 and remains an elusive goal of therapies aiming to achieve a functional cure. Clinically, CHB can be further divided into discrete phases of disease classified on the basis of ongoing viremia, liver inflammation, and HBsAg load, facilitating detailed analysis as to how fluctuations in these disease parameters can influence immunity. Thus, divergent humoral immune responses can be studied not only between HBV-vaccinated, resolved, or chronically infected individuals but also within patients with CHB, comparing responses to HBsAg and HBcAg and different phases of disease.

| US ING HBV A S A MODEL
It is increasingly recognized that chronic viral infections, such Understanding these factors may reveal key insights into how B cell immunity is established following viral infection and how these pathways may be disrupted in chronic infection.
One key piece of evidence may reside in the observation that B cell depletion by anti-CD20 or anti-CD52 antibody therapies (eg, the use of Rituximab in the management of lymphoma) significantly increases the risk of HBV-reactivation in HBV-resolved patients and of viremic flares in chronically infected patients with low viral loads (HBsAg + inactive carriers). [11][12][13] Long-lived plasma cells lack expression of CD20 and so should be preserved following Rituximab treatment; as a key cell population responsible for providing long-term antibody production, these cells might be expected to maintain serum antibody responses in the absence of memory B cells. 14 The observation that Rituximab can lead to loss of humoral immunity in these settings therefore suggests that long-lived plasma cells may not be reliably

| ANTIG EN -DRIVEN DE TERMINATION OF MEMORY B CELL PROG R AMS
What can be learned from the dichotomous response to the surface and core antigens in hepatitis B about the role of antigen load or specific antigen structure in regulating humoral responses? In line with their lack of detectable antibodies against HBsAg, patients with CHB are shown to have reduced numbers of functional HBsAg-specific responses by ELISpot compared to those with acute-resolving infection. [17][18][19][20][21] However, application of fluorescently labeled antigen-bait systems was able to reveal for the first time that HBsAg-specific B cells in fact persist at equivalent levels in patients with chronic or acute-resolving infection and HBV-vaccinated controls but are functionally defective, failing to produce detectable anti-HBs upon stimulation in vitro. 22,23 Comparatively, HBcAg-specific B cells are present at much higher frequencies than HBsAg-specific B cells, 24 with their number associated with elevated liver inflammation and ongoing viral replication. 25 In stark contrast to HBsAg-specific B cells, HBcAg-specific B cells maintain an ability to secrete anti-HBc antibodies upon IL-2, IL-21 and CD40 stimulation. 24 These data therefore suggest that the absence of anti-HBs in patient sera is a result of defective HBsAg-specific B cells rather than a complete loss of this B cells with HBsAg-specificity. HBsAg-specific B cells, HBcAg-specific B cells were phenotypically dominated by IgG-switched CD27 + CD21 + classical memory B cells (cMBCs), 24 which persist long-term independently of antigen 30,31 and retain the capacity to proliferate and differentiate upon secondary exposure faster than naive B cells. 32,33 Hence, the comparative dominance of atMBCs and cMBCs in HBsAg-and HBcAg-specific responses respectively may explain the difference in the formation of productive and long-lived humoral responses.
Antigen-experienced atMBCs have been described in many different infection settings and are thought to represent a distinct memory B cell population that diverges from cMBCs. 34 Their presence in these contexts may give some clues as to factors that perturb memory B cell responses in chronic infections (summarized in Figure 1).
Accumulating evidence suggests that atMBCs arise following repeated antigen challenge in an inflammatory setting inferring that these cells are perpetuated by persistent antigenic stimulation 35 ; in support, the frequency of atMBCs observed in HIV infection falls in line with treatment-induced reductions in viral load [36][37][38] and in those that spontaneously resolve infection. 39,40 Thus, the accumulation of these cells in CHB appears to be, at least in part, driven by chronic exposure to HBV-viral proteins, as evidenced by the higher frequency of atMBCs in patients with ongoing infection compared with vaccinated or resolved controls. In addition to secreting full replicating virions, HBV-infected hepatocytes also release vast quantities of subviral particles-or "empty" viruses comprised of HBsAg in various sizes. These subviral particles flood the system, outnumbering infectious virus by 1000-to 100 000-fold, 41  Humoral immunity may also be regulated via properties intrinsic to the antigen in question. A key differential between HBcAg-and HBsAg-specific responses is the ability of HBcAg to promote B cell differentiation and isotype switching independently of CD4 T cell help. 59 HBcAg is distinct from HBsAg in its capacity to self-assemble into highly immunogenic virus-like particles that can bind and activate naive B cells in a T cell-independent fashion. 60   helper cells in vivo. 61

| CD 4 T CELL DE TERMINATI ON OF B CELL FATE
Since GC formation and function are critically dependent on T FH cell function, defective humoral immunity is closely associated with im-

ACK N OWLED G EM ENTS
ARB and MKM are funded by a WT Investigator award (101849/Z/13/A) and a NIHR EME grant.

MKM has received collaborative research funding from Gilead
Sciences, F. Hoffmann-La Roche and Immunocore and has served as a consultant or on advisory boards for Gilead Sciences, F.
Hoffmann-La Roche, Immunocore and GSK. MKM and ARB have filed IP on a method to enhance HBV-specific immune responses.