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Hepatitis B virus (HBV) infection is responsible for one of the most prevalent infectious disease: approximately one third of the world’s population has been exposed to the virus, and 350 million people are chronically infected. HBV infection is responsible for over 1 million deaths per year and currently represents 5–10% of cases of liver transplantation. Despite the availability of an effective vaccine, HBV is still a problem of global public health importance in European Community, because of the profound dynamicity of viral epidemiology, due to migratory effluxes from and to the areas of high endemicity. The current treatment strategies of chronic infection remain unsatisfactory, leading just to a partial, temporary control of viral replication that needs uninterrupted drug’s administration. Thus, increasing scientific attention is concentrated on the possibility to potentiate the natural and most effective defense against the virus, the antiviral immune response, eventually reversing its abnormalities that have been associated to the status of chronic infection. In order to achieve a deeper understanding of mechanisms orchestrating anti-HBV immune response, the project aimed to extend the study of immune response to the areas that have attracted less scientific attention so far, and in particular:
1) CD4 T helper cells analysis in both self-limited (resolution after acute phase) and chronic infection with different virological profiles, by the identification and characterization of antigen-specific T cells in the memory repertoire of patients.
The project has taken advantage from the high through-put interrogation technique of memory T cells, developed by the Host institution. This method relies on the separation of cells into distinct functional categories and then in several replicate cultures, each containing a limited number of cells, that once activated by polyclonal stimuli, generate a pool of amplified T cells blasts. The purpose of this method is to enrich the cells of a given specificity by clustering them in the subset(s) where they are most prominently present, and to make easier their detection and enumeration. This approach was thought particularly suitable for HBV infection, given the limited number of proteins encoded by viral genome, and the need to expand in vitro the cells to detect a response as general interrogation approach. Briefly, the distinct T helper subsets have been separated from total memory pool (by excluding the CD4+CD45RA+CCR7+ naïve cells), according to the surface expression of chemokine receptors CXCR3 and CCR6, in the different combinations, in order to evaluate the response in the distinct T helper subsets, defined by phenotypic expression of these different homing receptors. The antigen specificity was assessed by measurement of proliferative response to HBV antigens (recombinant proteins or synthetic overlapping peptides): core antigen, envelope antigen (or HBsAg) and polymerase. As far as self-limited patients are concerned, despite the homogeneity of clinical conditions (time point of test in the early follow-up after acute phase), the frequencies of specific CD4+ T cells are variable between different donors, ranging from 0 to 500 cells per million cells. Memory HBV-specific T cells are prominently found in the CCR6– subset and, within this subset, more frequently detected in CXCR3– compartment, enriched in Th2 cells, than in CXCR3+ subset, enriched in Th1 cells. The frequency of HBV-positive response in the CCR6+ subsets is almost negligible. The responses to the three antigens under evaluation were equally distributed in the two CCR6– subsets, without any compartmentalization or preferential clustering of antigen-specific response in a given subset. Despite the high sensitivity of the experimental approach, the HBV+ CD4+ cells were very low, or even completely undetectable in chronic patients. Furthermore, according to previous data, although core and polymerase antigens are the most preferentially targeted viral proteins by CD4+ response, the only specificity able to persist in the limited CD4+ response of chronic infection is the one against core antigen. The data show that peripheral CD4+ HBV specific can be still detected after virological resolution of the disease, while they are missing during chronic infection. Interestingly, to underline the centrality of the role of CD4+ response in the rare cases of control after chronic phase, the only chronic patient with a frequency of response comparable to that of self-limited ones was a patient experiencing rapid decline of HBsAg levels at the time of test, and who would reach complete viral control (HBsAg loss) some months later.

2) Dissection of anti-HBV B memory response against structural antigens (core antigen and HBsAg), to investigate its potential role in the different degrees of viral control: indeed, anti-HBV humoral response data arise from diagnostic serology, but no information at cellular levels was available. This is particularly important for anti-HBsAg response, as this antigen is typically produced in large excess both in acute and chronic infections, potentially masking the antibody production, such as the timing and the extent of anti-HBsAg antibodies production cannot be easily addressed by limiting the analysis on the antibodies level measurement.
To address this issue, the project used the highly sophisticated methodologies developed at the Host Institution, based on high throughput cell culture systems, allowing the interrogation of immune repertoires with high efficiency and the isolation of monoclonal antibodies of a given specificity.
This analysis has been performed on patients with self-limited infection, in the follow-up of acute phase and in patients with chronic infection, with low and high antigenemia.
The results demonstrated an opposite behavior of two structural antigens in eliciting humoral response. The frequency of IgG+ memory B cells reactive against core antigen was pretty high in almost all donors, ranging from 0,1 to 8,8% IgG+ memory B cells, irrespective of clinical status.
Unexpectedly, the anti-HBsAg response resulted to be completely absent in all patients, also in the follow-up of acute phase, after achieving HBsAg clearance. To try to understand the meaning of this finding, the analysis was moved to later time points (patients after one and three years acute infection), to take into account the usual delayed development of neutralizing antibodies, typical of non-cytopathic viruses, such as HBV, but same results were found. The reasons of this finding have not yet been completely elucidated, they deserve further investigations (compartmentalization of anti-HBsAg B cells in bone marrow or in the liver? Selective exhaustion of deletion of this specificity?), considering the important implication of the features and kinetics of neutralizing response in clinical context.

3) Finally, in order to better explore also “indirect roles” of antibodies in HBV control, and the interplay between B cells and CD4+ T cells, the antigen presentation to Th cells was evaluated, starting from the availability of specific B cell clones, obtained by the isolation procedures described above. These B cells, carrying BCR specific for HBV antigens, were compared in their ability to present the antigen to CD4+ blasts to the CD14+ monocytes, and also to B cells of unrelated specificities. The results pointed out that specific B cell clones were consistently superior than the monocytes in eliciting the CD4+ response, both in terms of proliferation and cytokine production. This finding held true for both core antigen (in the natural infection) and also for HBsAg (in case of vaccination, given the above mentioned data, that B cells specific for HBsAg were not isolated from any donors). Interestingly, monocytes have been proven unable to present HBV antigens in the context of in vitro expanded cells (Th blasts and Th cell clones), while the ability of monocytes to present antigen is maintained in case of primary lymphocytes (Figure 1 and Figure 2)

Altogether, the interrogation of multiple repertoires performed during the project reveals some unusual behaviors of response against HBV, that need further investigations: the absence or unresponsiveness of memory B cells specific for HBsAg; the almost complete lack of CD4 arm in chronic infection; the defect of antigen presentation to activated memory specific cells; the importance of the role of antigen presentation by B cells. The results suggest the complex, tightly coordinated interplay between B cells and CD4+ T cells. These data are limited to the peripheral compartment, as the study of other sites probably more relevant for the dynamics of infection (liver, lymphnodes) is difficult to evaluate for their inaccessibility and limited number of cells obtained; but the consistency of immunogenicity for core and HBsAg, at both B and Th cell levels, needs to be deeply evaluated, to better define the abnormalities to be reverted for improving the control of HBV infection.