key: cord-0722022-xblkbrsq
authors: Pillai, Shiv
title: Sub-optimal Humoral immunity in SARS CoV-2 infection and viral variant generation
date: 2021-11-03
journal: Clin Lab Med
DOI: 10.1016/j.cll.2021.10.001
sha: 8faf312c31d032536bdd090c8b77005583563b6c
doc_id: 722022
cord_uid: xblkbrsq

This review describes the underlying basis for the sup-optimal humoral immune response in COVID-19 including the absence of evidence for affinity maturation in the vast majority of patients and the absence of germinal centers even in severe disease. Sub-optimal humoral and cellular immunity provide the optimal conditions for the generation and selection of viral variants.

disease progression and disease sequelae. The emergence of B cells with a switched-memory phenotype is not in itself a marker for durability, (the length of time for which there is effective protection from re-infection by circulating antibodies). Memory B cells disappeared during a six-year follow up period in patients with SARS (5) . In COVID-19, natural immunity (immunity generated by infection) has been documented to decline and does not effectively generate protection or herd immunity (6) (7) (8) (9) (10) . We believe that sub-optimal adaptive immunity against SARS-CoV-2 as documented by us in terms of the cells involved in humoral immunity (11) , but which also likely applies to cellular immunity, facilitates viral persistence and variant generation.

In this review we will first discuss the durability of natural immunity in SARS, MERS and COVID-19. We will then discuss the biology of germinal centers and the loss of germinal centers in severe COVID-19 and focus on the loss of Bcl-6+ T follicular helper cells in this disease. We will subsequently discuss the loss of germinal centers in other severe infections in animal models and examine what that might suggest in a mechanistic sense. Finally, we will consider the potential pathological consequences of the extrafollicular B cell response in COVID-19.

Slifka, Antia, Ahmed and colleagues have highlighted the dichotomy between the robust, virtually life-long, humoral immunity seen with natural infections with viruses such as measles, mumps and rubella or vaccination with live-attenuated viral vaccines (with calculated antibody half-lives ranging roughly from 50 to 100 years) in comparison to the steadily declining humoral immunity seen in the majority of infections with SARS and MERS and after immunization with killed parenteral vaccines such as the one used in influenza (12) (13) (14) (15) . In one of the deepest follow-J o u r n a l P r e -p r o o f up studies in SARS, a disease in which the virus was rapidly eliminated, and no vaccine was administered subsequently, patients were followed for up to six years after infection (5) . Virusspecific IgG antibodies steadily waned and had completely disappeared in 21 of 23 patients; specific memory B cells had also become undetectable in all 23 by the end of the study, though about half preserved memory T cells.

In COVID-19, vaccine availability may make conducting a similar study difficult, but all indications are that a trajectory for humoral immunity that is similar to that observed in SARS is likely. Both antigen-specific switched memory B cells and plasma cells can therefore either be relatively short-lived, in the order of 2-4 years, after an infection like SARS, or very long-lived after infection with measles or mumps or after yellow fever vaccination. Just as we recognize Most of our understanding of the pathogenesis of severe COVID-19 has been derived from generally non-quantitative studies of the blood, with few systematic studies of adaptive J o u r n a l P r e -p r o o f immune cells at the sites of infection and in draining lymph nodes. While the virus attenuates type I interferon production by infected cells (16, 17) and the anti-viral state is further compromised in some susceptible individuals by mutations or by pre-existing antibodies to type I interferons (18, 19) , most of the initial tissue damage in the lungs is likely generated by excessive unregulated inflammation.

One of the most remarkable phenomena in adaptive immunity that has been of great interest to immunologists as well as molecular biologists is the germinal center response. This response is the key to protective responses against most pathogens and is central to the success of vaccination.

Over a century ago, pathologists had recognized the presence of proliferating cells in organized collections in lymph nodes. (reviewed in 20) It had originally been assumed, incorrectly, that these were the sites at which lymphocytes were generated, hence they were called "germinal centers". Little was known then however as to what lymphocytes actually did.

The function of lymphocytes in adaptive immunity would only be established in 1957 by Gowans (21) . It soon became clear that there were two types of lymphocytes. B lymphocytes are generated in the bursa of Fabricius in birds (or the bone marrow in other vertebrates), while T lymphocytes are generated in the thymus (22, 23) . In the early 1960s it was eventually recognized that germinal centers were not the sites at which lymphocytes are made. Over the next decade it became apparent that germinal centers are induced structures that emerge after immunization, but their precise functional role remained mysterious for a while.

Although the increase in the affinity of antibodies after repeated immunization, a phenomenon called affinity maturation, had been described well before the function of lymphocytes was appreciated, nothing was known about how this phenomenon occurred until the early 1980s (reviewed in 24, 25) . The theoretical possibility that antibody diversity might be caused by a process of somatic mutation had been entertained even in the 1960s by Burnet and others (26) . In the early 1970s the sequencing of antibodies using Edman degradation started to reveal the theoretical possibilities of somatic mutation in antibody diversification, but whether this phenomenon occurred during lymphocyte development or after an immune response was 

We have demonstrated the loss of germinal centers in thoracic lymph nodes in severe COVID-19, as shown in figure 2 (11) . A pathological description consistent with our data has also been reported, (30) . Thoracic lymph nodes, like Peyer's patches and mesenteric lymph nodes, constitutively contain germinal centers; age-matched elderly individuals who died of non-COVID causes in the same time window (and were autopsied in a similar accelerated manner to those who succumbed to COVID-19) had robust germinal centers presumably induced by protein antigens from microbes or allergens constitutively present in the respiratory tract (11) . The Our previous studies also showed that SARS-CoV-2-specific switched memory B cells were identifiable in the blood of patients with severe COVID-19 (11) . These data raised typically extending for decades (13) .

We showed that in patients with severe COVID-19 lymph node architecture was well preserved, there were well-defined follicles and T cell zones and that that most CD4+ T cell subsets and regulatory T cells were well preserved (11) . There was however a striking loss of T follicular helper cells, especially Bcl-6+ T follicular helper cells. This loss of Bcl-6+ T follicular helper cells would suffice to explain the loss of germinal center B cells (11) .

We showed that there were high levels of TNF- expression in the thoracic lymph nodes in severe COVID-19 (11) and we postulated that, as had been more mechanistically examined in murine models of severe intracellular infections discussed briefly in the next section, the high levels seen of TNF- in lymph nodes might account for the loss of germinal centers in severe COVID-19.

There are a few murine models of intracellular infections in which germinal centers are lost, and in some a block in TFH cell differentiation has also been observed (35) (36) (37) . In a murine malaria model, the loss of TFH cells and germinal centers was observed and this was reversed by blockade of TNF-or IFN-. Genetic deletion of T-bet also prevented the loss of germinal centers. The TFH cell precursors did not express high levels of PD-1 and CXCR5 but expressed genes such as T-bet and CXCR3, characteristic of TH1 cells (35) .

In a study of a murine rickettsial infection caused by Ehrlichia muris, the loss of germinal centers was reversed by TNF- blockade as well as by the use of mice that have an engineered deletion of TNF-. In another study involving Salmonella infection, IL-12 was shown to be responsible for the block in TFH cell differentiation and the loss of germinal centers that was seen (37) . Given the known functional and sequential links between IL-12, TH1 cells and the J o u r n a l P r e -p r o o f downstream production of TNF-, we suspect that signaling through TNFRII on CD4+ T cells might cause a block in TFH cell differentiation in these models and in severe viral infections.

There is a slightly artificial murine viral infection model in which germinal centers are lost (32) . When regular inbred mice are first immunized with a specific LCMV peptide that activates CD4+ T cells and then later infected with LCMV clone 13, they develop a severe viral infection that results in lymphopenia, the loss of germinal centers in lymph nodes and an eventually lethal severe viral infection that involves the lungs and other organs. This disease resembles severe COVID-19 in many ways. Very high levels of IL-12 were observed in these mice. Overall a number of severe intracellular infections result in the loss of germinal centers, and this may involve in some poorly defined way, the sequential induction of high levels of IL-12, IFN- and TNF-.

Some activated B cell subsets appear to be key drivers of inflammatory and fibrotic diseases, many of which respond therapeutically to B cell depletion. Our previous studies revealed the presence of subsets of antigen-specific disease-related IgD -CD27double negative (DN) B cells that accumulate in the blood of COVID-19 patients including those with severe disease (11) and similar overall B cell populations have been observed by others (38) . We have demonstrated that DN B cells in COVID-19 include SARS-CoV-2 specific cells, but there is no definitive evidence as yet that these cells actually produce the large number of autoantibodies now described in COVID-19 patients (39, 40) . The presence of these cells B cell in the blood correlates with immune dysregulation and a break in B and T cell tolerance in COVID-19. It has however never been established whether DN B cells actually accumulate in the lesions of J o u r n a l P r e -p r o o f inflammatory or fibrotic diseases, even in diseases that respond to B cell depletion, or if they interact with CD4+ T cells in end-organs. Whether or not specific DN B cell subsets may be more relevant in a tissue context on inflammatory and fibrotic diseases has also not been investigated.

While the contribution, if any, of B cells to the progression or sequelae of COVID-19 is unclear, the global lack of B cells, however, appears to correlate with less severe COVID-19, and this infection has been reported to be less likely to be lethal in patients with X-linked agammaglobulinemia (41) (42) (43) . This could be due to the paucity of B cells in these patients rather or possibly defective BTK signaling in myeloid and lymphoid cells. BTK inhibition appears to reduce hospitalization rates and disease progression in COVID-19 patients (44) though the results of randomized clinical trials are awaited, Anti-CD20 mediated B cell depletion has been seen to be clinically useful in ameliorating severe progressive interstitial lung disease (45, 46) and also in reversing progression in severe combined immunodeficiency with associated granulomatous-lymphocytic interstitial lung disease (47). While there is no consensus view about B cell depletion in COVID-19 (in patients receiving anti-CD20 for other diagnoses), it has frequently been suggested to be beneficial. Since no clear-cut negative outcomes have been observed with anti-CD20, trials using this therapeutic in various clinical contexts were allowed to resume in Europe in early 2021.

Mild COVID-19 generates very poor antibody responses likely because the virus is contained by type I interferons generated by plasmacytoid dendritic cells, is rapidly cleared, and antigen does not accumulate in draining lymph nodes. Affinity maturation is rare with moderate 

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