Unravelling humoral immunity in SARS-CoV-2: Insights from infection and vaccination

2.1 Extra-follicular and germinal center responses

The SARS-CoV-2 genome encodes spike (S), nucleocapsid (N), membrane, and envelope structural proteins, with 12 open reading frames (ORFs) coding for 27 proteins [11]. The genomic organization includes 5 ′ - leader sequence- ORF1/ab- S- ORF3a- E- MORF6a- ORF7a- ORF7b- ORF8- N- ORF10-3 ′ from left to right [11]. The S protein is essential in viral infection and pathogenesis [12]. It consists of subunits S1 and S2, with S1 containing the N-terminal domain (NTD) and the receptor-binding domain (RBD), while S2 comprises heptad repeat 1 (HR1) and HR2 [13]. The RBD initiates the binding process by interacting with its receptor, angiotensin-converting enzyme 2 (ACE-2). This interaction initiates conformational changes in the S protein, which in turn leads to membrane fusion mediated through HR1 and HR2. Ultimately, this process results in the entry of the virus into target cells [13]. After encountering a viral pathogen or receiving a vaccine, the primary phase in the initiation of humoral immunity involves the triggering of a temporary, extrafollicular response by antigen-specific plasmablasts. This response leads to the production of antibodies with low affinity targeting the pathogen [14] (Fig. 1) [14, 15].

OPEN IN VIEWER

OPEN IN VIEWER

B cells undergo a development process that includes stages such as pro-B cells, pre-B cells, and immature B cells before maturing into functional B cells [17]. This development occurs initially in the foetal liver before birth and continues in the bone marrow afterward. Within the maturing of B cells, distinct subpopulations, including B-1, B-2, and regulatory B cells, play crucial roles in antiviral B cell responses. The B-1 cell subset, originating from the foetal liver, can be further categorized into B-1a and B-1b subpopulations [17]. Initially, in COVID-19 patients, naive B cells – characterized as CD19 ⁺ IgD ⁺ CD27^– CD38 ^{+ / –}, and CD24 ^{+ / –} (Fig. 2) – recognize the antigen through their BCR receptor [14]. Subsequently, the receptor CD40 binds to CD40L (CD154) [18] (Fig. 2), which is located on the Th lymphocytes (LTH) membrane, activating the LTH cells via the CK-Rc pathway to produce interleukin 4, which activates unswitched B cell proliferation recognized as CD21^lowCD27 ⁺ CD38 ⁺ CD71 ⁺ (Fig. 2) in convalescent and severe infected COVID-19 patients [8, 18]. B-2 cells, which originate from the bone marrow, encompass both follicular B cells and marginal zone B cells. Typically, marginal zone B cells and B-1 cells contribute to T-independent responses, leading to the generation of short-term immunity. Conversely, follicular B cells play a role in T-dependent responses, providing enduring protection against reinfection by the same pathogen [19].

To further explore the B cell phenotypes linked to extrafollicular B cell proliferations in patients infected with COVID-19, a study employed the use of CD11c ⁺ for identifying “activated naive” B cells, which undergo differentiation into first double-negative-type-1 (DN1) and then double-negative-type-2 (DN2) B cells characterized by CD27 ^- CD21 ^- and IgD ^-  [14]. DN1 (CD21 ⁺ CD11c ^- ) B cells exhibited comparable levels between healthy subjects and individuals with mild/moderate COVID-19. However, there was a significant reduction observed in severe and critical cases [20]. In contrast, another study confirmed that individuals with severe COVID-19 exhibited elevated frequencies of DN2 B cells when contrasted with those experiencing mild illness. Moreover, there were higher frequencies of plasmablasts in patients with severe symptoms [14]. Thus, the majority of peripheral blood ASCs had low SHM frequencies in class-switched cells [21]. At this point, B cells differentiate into effector cells producing IgM as the first line of humoral defence as shown in Fig. 1 [22]. In fact, in PCR-positive patients, the IgM detection was reported as reduced to 68.8 percent by 40 days post-onset of disease or from the day of PCR positivity [23]. In the case of reinfection, these cells migrate to the dark area of the germinal center, which emerges in several copies within secondary lymphoid organs upon exposure to antigen [24]. In this area, B cells undergo class hypermutation, and thus affinity maturation [22]. The basis for affinity maturation is the SHM of the immunoglobulin germline genes, which is carried out by a protein known as activation-induced cytosine deaminase (AID) [25]. An increased proliferation of B cells is established and migration to the light area of the GC or through activation by Th cells undergoes a class switch of immunoglobulins [22]. This differentiation is mediated by follicular Th cells (LTHF) which activate a panel of mutated B cells that are selected, based on their affinity, to proliferate and differentiate into antibody-secreting PCs and MBC cells. Throughout an infection, Abs improve their affinity for an antigen (binding strength) and change class or isotype (Immunoglobulin IgM, IgG, IgA, IgE), to better bind and neutralize pathogens [26]. Regarding activation of broad-spectrum neutralizing antibody responses in mildly infected patients, they were principally generated against RBD and N protein in four of the five spike epitopes (249-261, 597-606, 805-816, 1256-1265), and three of the nine nucleoprotein epitopes (164-216, 232-269, 361-390) [27]. In addition, epitopes were also predicted for Orf3a, Orf3b, Orf7a, and Orf8 SARS-CoV-2 proteins [27]. Furthermore, IgG antibodies also have other important roles, particularly, Fc-mediated effector activities such as cell activations and ADCC. IgM immunoglobulins, which are the most antibody generated between B cell activation and class switching, are often the first to be expressed together with IgD during naive B cell development. IgM represents approximately 10% of all antibodies in the serum [28] (Fig. 1). IgM antibodies exhibit a comparatively low affinity as compared to IgG antibodies that go through affinity maturation by somatic mutations, which results in high affinity for the target antigen, they first show later in the immune response (Fig. 1) [28].

Protective humoral immune memory has two major cellular components, circulating memory B cells that act as immune sentinels by carrying out vital immune surveillance tasks and long-living, bone marrow-resident plasma cells with high affinity for secreting antibodies [29]. After SARS-CoV-2 infection, detectable classical CD24 ⁺ class-switched memory B cells, activated CD24 ^- negative and natural unswitched CD27 ⁺ IgD ⁺ IgM ⁺ subsets [30] (Fig. 2E, G, H) appear two weeks after the onset of symptoms in convalescent patients. Notably, while extrafollicular antibody-secreting cells, such as short-lived plasmablasts and plasma cells, are initially present, their numbers decline after six months [14]. A study revealed using the scRNA-seq dataset that CD19 ⁺ IgD ^- B cells could be divided into five major clusters (MBC, Activated, PC, Naive/Transi, antibody-secreting plasmablasts (PB)) according to their gene expression profile [8]. Unexpectedly, frequencies of MBCs continue to increase throughout 3 to 6 months following infection [8]. This increase is shown in MBCs that are spike ^- , RBD ^- , and nucleocapsid-specific. About 4 months after infection, SARS-CoV-2-specific MBC frequencies stabilize and are sustained for at least 15 months [26]. The increased frequency of MBCs binding to the spike and RBD is linked to significant SHM over 6 months. In addition, a reduction in the quantity of switched memory B cells has been identified in COVID-19 patients, and this decrease is independently correlated with the mortality rate among this patient cohort [31]. MBCs circulate throughout the body, distinguishing them from Plasma cells (PCs). Upon reinfection, MBCs can rapidly respond by generating antibody-secreting plasmablasts (PBs) or initiating secondary germinal centers (GCs) [24]. This phenomenon may manifest in situations where the organism is deficient in a requisite quantity of Abs to obstruct the onset of a novel infection. Alternatively, it may transpire in instances where the infection is induced by a variant of a pathogenic agent that eludes recognition by the extant array of highly specialized antibodies [32]. MBCs exhibit a broader spectrum of affinity and reactivity compared to antibodies secreted over the long term. This includes their initial responsiveness and the ability to recognize variations [33].

OPEN IN VIEWER

MBCs possess the capability to promptly initiate the secondary GC reaction, leading to the generation of PBs (Figs 2, 3). These PBs, in turn, secrete antibodies (Abs) that contribute to antigen capture and provide feedback to augment the secondary immune responses [34]. Novel GCs foster the development of MBCs and Plasma cells (PCs) characterized by heightened specificity and affinity towards the emerging challenge. These GCs play a pivotal role in enhancing affinity for the mutated pathogen. The immune response is initiated by MBCs, distinguished by elevated affinity and prevalence compared to their counterparts in the initial response. Consequently, the attainment of protective Abs levels, of the appropriate class, transpires considerably more expeditiously than in situations where long-lived PCs have dwindled to levels insufficient for achieving neutralizing immunity [34]. Populations of MBCs that lack both CD80 ^- and PD ^- L2 ^- and are generated through a pathway independent of GCs, can undergo differentiation into antibody-secreting cells. These cells possess the capacity to re-enter the germinal center for activation and further development into ASCs that produce high-affinity NAbs [35] (Fig. 3).

The class switching phenomenon is upregulated and downregulated necessary through several cytokines such as IL-5, IL-10, IFN-y, and TGF-B [22]. Some of the memory B cells that preferentially express the T-box transcription factor (T-bet) and Eomesodermin as well as surface molecules like CD80, CD180, Transmembrane Activator, and CAML Interactor (TACI), are inherently programmed to differentiate quickly into long-lived, high-affinity plasmablasts that secrete antibodies. Studies have shown immune modifications in the biomarkers of memory B cells. Specifically, SARS-CoV-2-spike-specific memory B cells in patients with non-severe COVID-19 exhibit elevated levels of T-bet and Fc-receptor-like 5 (FcRL5) compared to individuals with severe infection [36]. Somatic mutations in the VH genes of memory B cells are correlated with a sustained antibody response and a swift recovery from SARS-CoV-2 infection [22]. For long-term protection, a study on 25 patients grouped as mild, moderate, or severe reported from assessing RBD or N-specific MBCs that all groups had increased MBCs from the beginning to 150 days after infection [37]. Moreover, an analysis of a cohort comprising 188 patients revealed that circulating MBCs with specificity for the Receptor-Binding Domain (RBD), Nucleocapsid (N), and Spike (S) proteins persisted for a duration exceeding six months, extending up to eight months Post-Symptom Onset (PSO). Additionally, among longitudinally tracked individuals twenty-nine of 36 had higher frequencies of RBD-specific memory B cells extending from one month to six months PSO [38]. Another long-term investigation that looked at MBC levels in moderate instances discovered that three months following COVID-19 infection, IgG ⁺ MBCs continued to grow [39]. Furthermore, an alternate investigation indicated that functional Immunoglobulin G (IgG) MBCs persist in infected individuals for a duration spanning 5 to 8 months. However, it is noteworthy that the serum levels of SARS-CoV-2-specific IgG inevitably diminish over time [40]. This suggests that assessing MBC levels may be more sensitive in predicting prolonged protection and identifying previous infections. In conclusion, these discoveries underscore the crucial role of humoral responses orchestrated by B cells, leading to the formation of both short- and long-term MBCs and antibodies, as a fundamental defence mechanism against reinfection [41].

Neutralizing antibodies (NAbs) play a crucial role in eradicating the virus and providing protection against SARS-CoV-2 [32]. They exert their effects through various mechanisms, including preventing the attachment of virions to receptors, hindering virus uncoating in endosomes, impeding virus uptake into host cells, or inducing the aggregation of virus particles [32]. In the context of COVID-19, natural infection typically results in the production of varying titers of NAbs in most individuals between days 14 and 20 post-infection [42]. Several studies have shown detectable SARS-CoV-2 antibody responses in most patients up to 13 months after infection, raising optimism for potential longer-lasting immunity [43]. However, the levels of neutralizing antibodies start to decline around 6 to 8 months post-infection. It is estimated that within this timeframe from the onset of symptoms, approximately 24% of convalescent donors experience a loss of their NAbs [44]. Patients who recovered from severe illness tend to exhibit higher levels of NAbs compared to those with mild or asymptomatic infections [42]. This difference could be attributed to prolonged stimulation of the B-cell receptor or elevated levels of interferon type I (IFN-I) generated during severe illness. IFN-I, in turn, stimulates dendritic cell activation, facilitating the presentation of antigens to immature CD4 ⁺ and CD8 ⁺ T lymphocytes [32]. A study observed a gradual decline in SARS-CoV-2 neutralizing antibodies in individuals who were outpatient or asymptomatic, occurring approximately five months after the initial infection [45]. Additionally, researchers reported changes in the rate of NAbs against the S protein of SARS-CoV-2 over time, exhibiting a decline. In contrast, antibodies against non-neutralizing viral targets, such as N and ORF8, increased [46].

Other researchers have proposed that pre-existing memory responses resulting from a natural infection can be mobilized to promptly generate neutralizing antibodies upon re-exposure to SARS-CoV-2. These scientists have identified Immunoglobulin G (IgG) memory B cells specific to the S glycoprotein and Receptor-Binding Domain (RBD) in the blood of individuals who have experienced COVID-19 [39]. IgM is the first antibody to be released against SARS-CoV-2 during the acute phase of infection, as depicted in Fig. 1, and it takes about 5 days. Finally, the most often utilized antibody in diagnostic testing and vaccination immunity detection is IgG, which is secreted from the RNA peak during prolonged COVID-19 phases. IgA is secreted mostly in the mucus routes during the phases of recovery or covalence instances [28]. Among those infected with SARS-CoV-2, RBD-specific IgG1 and IgG3 were among the detectable antibody subtypes. However, IgG2 and IgG4 were hardly ever seen [28]. 94% of infected individuals showed detectable levels of IgM against the RBD of the SARS-CoV-2 S protein and 88% against the N protein by day 14 after the onset of symptoms, suggesting that the majority of patients had seroconverted [28]. In a different context, numerous studies have examined the diminishing levels of antibodies following infection and found that infections caused by various coronaviruses resulted in comparable rates of antibody reduction. The decline in antibody levels after infection with endemic coronaviruses HCoV-OC43 (lasting 109–164 days) and HCoV-229E (lasting 109–144 days) was like that observed with SARS-CoV-2 (with a half-life to baseline ranging from 148 to 185 days). Notably, estimates of the half-life to baseline after HCoV-NL63 infection indicated a more prolonged decline, spanning from 207 to 386 days. The estimates for SARS-CoV also suggested an extended half-life to baseline, with the duration of this extension varying across anti-N IgG datasets [47].

3.1 Memory B cells after vaccination

In response to COVID-19 vaccination, MBCs are produced. After two doses of RNA vaccines or SARS-CoV-2 infection, similar rates of RBD-binding IgG ⁺ MBCs are produced. SHM levels are also significant and comparable between individuals who have received 2 doses of RNA vaccines and those who have experienced SARS-CoV-2 infection at the 5-month mark [55]. As a result, 2-doses of RNA vaccinations produce a significant number of affinity-developed MBCs. The affinity maturation following a typical 2-dose RNA vaccination regimen, however, is qualitatively worse than that following SARS-CoV-2 infection. Months after infection, significant gains in NAbs breadth were seen, but not after RNA vaccination (for instance, 69% of NAbs from previously infected participants had enhanced potency, whereas just 19% of NAbs from 2-dose RNA vaccines did) [56]. The brief interval between dose 1 and dose 2 of the RNA vaccines may be responsible for these qualitative changes. The priming period can be important for the quality of a B cell response (49). The NAb titers and NAb breadth are significantly increased when the dosing interval between RNA vaccine vaccinations is increased from 3 to 10 weeks [57], most likely due to an effect on affinity maturation [57]. Between 3 and 6 months after vaccination with mRNA vaccines, an adenoviral vector vaccine, or a recombinant protein vaccine, spike and RBD IgG ⁺ MBCs frequencies rise [56].

The vaccination elicited a strong response from SARS-CoV-2-specific germinal center (GC) B cells and T follicular helper (TFH) cells. The administration of a second dose of the vaccine resulted in a notable increase in the percentage of GC B cells [22]. The response in the germinal center (GC) involved attracting recently activated naive B cells that specifically recognize epitopes located within the spike protein of SARS-CoV-2. Additionally, pre-existing memory B cell clones specific to seasonal coronaviruses contributed to this response. Remarkably, SARS-CoV-2-specific GC B cells persisted in lymph nodes for at least 15 weeks post-vaccination, maintaining levels close to the peak frequency. This observation suggests that these cells are likely undergoing affinity maturation [57]. Memory B cells recognizing variations of SARS-CoV-2 demonstrate higher levels of somatic hypermutation compared to cells targeting only the wild-type virus. This indicates the role of the germinal center in the development of broadly protective immunity, as the increased somatic hypermutation suggests an adaptive response that enhances the ability to recognize diverse variants of the virus [58]. Using Flow cytometry analysis it was observed that 10 out of the 15 individuals investigated exhibited a lasting SARS-CoV-2-specific germinal center (GC) B cell response (characterized by CD19 ⁺ CD4 ^- IgD^low CD20 ⁺ 866 CD38^int CXCR5^high CD71 ⁺ ) [59], as shown in Fig. 2. Conversely, in the remaining five individuals, no spike protein-binding GC B cell clones were detected using ELISpot (Enzyme-Linked Immunospot) [59]. This discrepancy suggests that the duration of the SARS-CoV-2-specific GC response induced by vaccination varies among individuals [22]. It’s crucial to highlight that vaccination has proven effective in generating Memory B Cells (MBCs) and Neutralizing Antibodies (NAbs) against SARS-CoV-2 variants. A single dose of mRNA vaccine is successful in eliciting robust antibody responses, marked by increased antibody titers specific to the Alpha (B.1.1.7) and Beta (B.1.351) variants, especially in individuals with a history of prior infection. However, to achieve sufficient neutralizing antibodies against the Spike protein of the Alpha, Beta, and Delta (B.1.617) variants, two doses are required [60]. This dual-dose approach ensures a more comprehensive and sustained immune response, enhancing the effectiveness of the vaccine in protecting a range of SARS-CoV-2 variants.

3.2 NAbs induced by vaccination

Most studies have consistently observed that a significant proportion of individuals undergo seroconversion and generate neutralizing antibodies (NAbs) a few days after receiving SARS-CoV-2 vaccination. However, it is commonly noted that these antibody levels tend to diminish over time [38]. Nevertheless, memory B cells can generate additional antibodies upon re-exposure to the virus [60]. A team of researchers conducted a 7-month follow-up on six patients who received the same vaccine (BNT162b2). Overall, the titers of anti-SARS-CoV-2 spike RBD IgG and neutralizing antibodies showed a gradual decline over the observed period [61]. The modelling of the decay of neutralization titers post-immunization reveals a significant decrease in protection against SARS-CoV-2 infection as neutralization levels decline. This implies a potential need for booster vaccines within a year to sustain sufficient protection [62]. Nevertheless, the emergence of SARS-CoV-2 variants featuring mutations that allow them to evade antibodies poses a challenge to the NAbs induced by vaccination. There is evidence indicating a correlation between the titers of NAbs and effectiveness against certain viral strains [63]. Indeed, in response to heightened transmissibility and/or pathogenicity, the WHO has classified certain SARS-CoV-2 variants as Variants of Concern (VOC). These include alpha (B.1.1.7), beta (B.1.351), gamma (B.1.1.248), delta (B.1.617.2), and omicron (B.1.1.529) Additionally, there are Variants of Interest (VOI) such as epsilon (B.1.427/B.1.429) and iota (B.1.5 (C.37) [64]. In the given study [65] involving twenty individuals infected with the Omicron variant and fully vaccinated, the NAbs titers of sera from the Wild-Type (WT) cohort against four VOCs – Alpha, Beta, Delta, and Omicron – were found to be lower compared to NAb titers against WT itself. The observed decreases were 1-, 8-, 3-, and 13-fold, respectively. In the Delta cohort, in comparison to the NAb titer against Delta, NAb titers of sera against WT, Alpha, Beta, and Omicron decreased by 1.5-, 1.4-, 4.9-, and 6.0-fold, respectively. For the Omicron cohort, relative to the NAb titer against Omicron, NAb titers of sera against WT, Alpha, Beta, and Delta decreased by 1.0-, 0.9-, 2.1-, and 1.2-folds, respectively. Based on the aforementioned factors and additional studies, it can be said that the current vaccines continue to offer clinical protection against most dangerous variants by lessening the severity of COVID-19 disease; however, the decline in neutralization potency is still an issue that needs more research.

3.3 Specific B cells and Nbs elicited by different vaccine platforms

Following a typical two-dose immunization schedule, COVID-19 vaccines licensed by the WHO under the Emergency Use Listing (EUL) have demonstrated efficacy and effectiveness in avoiding infection and illness development [66]. However, waning of the immune response against SARSCoV-2 has been reported [67]. A study conducted in Morocco reported IgG levels in 82 health workers who received the second dose of ChAdOx1-S/nCoV-19 by AstraZeneca at least five months before the enrolment date. In this research, two distinct tests were employed: Euroimmun ELISA and the Abbott Architect^TM SARS-CoV-2 IgG assay. The study observed that 65.85% of the cohort tested positive for IgG using ELISA Euroimmun. Additionally, the Abbott Architect^TM SARS-CoV-2 IgG assay indicated a percentage of 52.11% for IgG positivity within the same cohort [68]. Furthermore using the Activation-Induced Marker (AIM), it was reported in a noCOVID-19 group, an activation of memory B cells (CD19 ⁺ CD27 ⁺ CD38 ⁺ ) after the first and second doses of the COVID-19 vaccine [69]. A study examined the immune memory of mRNA-1273, BNT162b2, and Ad26.COV2.S, and NVX-CoV2373 vaccines [70] using IgG ELISA assay against RBD and N. They detected NAbs titers using SARS-CoV-2 pseudovirus assay. For mRNA-1273, after 1^st dose immunization, 100% of vaccinees had detectable spike IgG and RBD IgG titers. Additionally, 86% of vaccinees had detectable neutralization antibody titers after the 1^st dose. 100% of vaccinees for BNT162b2 had measurable RBD and spike IgG titers following the first dose of immunization. Following the first dose, 76% of vaccinated individuals exhibited detectable neutralizing antibodies, a number marginally lower than the 86% with mRNA-1273. Following the second immunization with BNT162b2, neutralization antibody titers increased 20-fold, and spike and RBD IgG were enhanced 9–16 times [70]. For the second dose of mRNA-1273 antibody levels of both spike and RBD IgG were boosted 9-fold and neutralizing antibody titers were boosted 25-fold (GMT 1,399). 86% of recipients of the Ad26.COV2. S 1-dose vaccination exhibited detectable Spike IgG and 79% showed RBD IgG at T2 (15 ± 4 days). At T2, measurable neutralizing antibodies were present in 64% of vaccinees, which was somewhat less than the 86% with mRNA-1273 and the 76% with BNT162b2. For NVX-CoV2373, antibody titers were available for 3.5 and 6 months. Spike and RBD IgG titers were substantial at 3.5 months post-vaccination and were marginally decreased at T5 (185 ± 8 days). Neutralization antibody titers were comparable at both time points [70]. A follow-through case study of one patient (70 years old) from 2021 to 2023 who had been vaccinated six times; three doses of CoronaVac, one dose of AZD1222, half-dose BNT162b2(PF), and one dose of Covovax^TM (CO), and had a breakthrough infection with the Omicron sublineage BA.5, 126 days after receiving Covovax^TM as the sixth dose [71]. The total IgG RBD exhibited a remarkable 25-fold surge rising from 5,691 U/mL before the breakthrough infection to 146,528 U/mL by day 29 after the antigen test kit. Moreover, NAbs titers were assessed using the focus reduction neutralization test (FRNT50) against live viruses, including Omicron BA.5, BA.2.75, BQ.1.1, and XBB.1.5. The results indicated detectable levels of NAbs following the administration of the fourth half-dose Pfizer (PF) and the sixth Covovax (CO) booster vaccination [71].

To point out the immunogenicity of the third “booster” dose regarding the broad-spectrum antibody responses and infection protection, several studies with cohorts of no history of COVID-19 reported a significant increase in the median neutralizing Ab anti-spike concentration post-third dose compared to the median neutralizing Ab anti-spike concentration pre-third dose. In a systematic review encompassing 30 studies, the focus was on third-dose responses, specifically examining IgG levels against COVID-19 antigens before and after receiving the third booster dose [72]. In the first study, IgG levels were reported to be less than 50 AU/ml after 1 month of vaccination, compared to 586 AU/ml in responders (1 month). Another study indicated 284 AU/ml versus 7554 AU/ml ( ⩾ 21 days) using the Roche Elecsys Assay for both measurements. Additionally, a separate study observed 0 (-) IgG versus 298 AU/ml (1 month) IgG. Furthermore, two cohorts showed IgG levels of 1056 UI/ml and 17.8 U/mL ( ⩾ 21 days), in contrast to 6464 UI/ml and 1180 U/ml in two other cohorts ( ⩾ 21 days), all measured against the RBD antigen (Table 1) [72].

4.1 Hybrid immunity

Hybrid immunity which is conferred to the combination of previous infection and vaccination offers greater broad-spectrum protection against COVID-19 [73], and larger amounts of neutralizing antibodies are produced [74], which increases protection against infection compared to immunity brought on by infection or vaccination alone [75]. A study on individuals who had PCR-confirmed infections by SARS-CoV-2 or who had received at least two doses of the BNT162b2 vaccine at least 7 days before the end of the study period, confirmed that after a few months, hybrid immune individuals exhibited superior protection against reinfection compared to uninfected individuals who had previously received two doses of the vaccine (the two-dose cohort) [73]. These findings were confirmed from databases after the selection of reinfection cases in a great number of individuals [73]. Other cellular and serological studies confirmed this fact when working on convalescent and vaccinated people with the BNT162b2 mRNA vaccine. They noted that more than 400 cells neutralized the original SARS-CoV-2 virus that was first discovered in Wuhan, China, and over 3,000 cells developed monoclonal antibodies against the spike protein, 70% of these neutralizing antibodies escaped other variants [76]. Moreover, vaccinated participants who were seropositive showed a 2.46-fold increase in S-protein-specific CD19 ⁺ CD27 ⁺ IgD ^- IgM ^- memory B cells compared with participants who were seronegative and an overall 10% higher count of CD19 ⁺ CD27 ⁺ IgD ^- IgM ^- memory B cells [76]. In addition, a single sorting assay of the prefusion S protein trimer-specific (S protein ⁺ ) and class-switched memory B cells (CD19 ⁺ CD27 ⁺ IgD ^- IgM ^- ) followed by ELISA assay for NAbs, noted that the fraction of S-protein-specific B cells producing NAbs were 7.5% for participants who were seronegative and 14.8% for seropositive participants [76]. However, it was shown that the rates of SARS-CoV-2 infections were similar in different profiles noting the group of individuals who had a previous infection and no vaccination, the group of individuals who had an infection and were then vaccinated with a single dose after at least 3 months and the group of individuals who were vaccinated with two doses and then got infected [77]. In a nationwide study in Sweden, that included over 4 million individuals; it was concluded that one-dose hybrid immunity was associated with a 58% lower risk of reinfection (aHR 0 ⋅ 42 [95% CI 0 ⋅ 38–0 ⋅ 47]) than natural immunity, and two-dose hybrid immunity was associated with a 66% lower risk of reinfection (aHR 0 ⋅ 34 [95% CI 0 ⋅ 31–0 ⋅ 39]) than natural immunity [78]. All this data supports the fact that vaccination seemed to further decrease the risk of severe cases and hospitalizations. As a general conclusion, instead of relying solely on vaccination, it is well noted that either a prior infection or vaccination is proof of immunity.

4.2 Cross-reactive immunity

A major unresolved question is whether prior immunity to endemic, human common cold coronaviruses (H-CoVs) impacts susceptibility to SARS-CoV-2 infection or immunity following infection and vaccination. Four Human Coronaviruses (H-CoVs) are prevalent worldwide and have been endemic in humans for decades and typically induce mild upper respiratory disease and account for 30% of “common colds” [79]. In contrast to 229E and NL63, which are alphacoronaviruses, HKU1 and OC43 are betacoronaviruses, as is SARS-CoV-2. The endemic H-CoVs share 30% homology within the spike proteins, despite the virus’s dramatically different ability to cause severe disease [80]. Several studies demonstrated that multiple epitopes of H-CoVs in vitro are recognized by sera with cross-reactive antibodies [81]. Not only do these antibodies cross-react with those of SARS-COV-2 and its antigens, but studies have shown that they increase following SARS-CoV-2 infection which may be due to activation of pre-existing memory B cells that were generated after previous infection with H-CoVs [82]. A study on humoral response against six selected epitopes of HCoV-NL63 (NL63-RBM1, NL63-RBM2_1, NL63-RBM2_2, NL63-RBM3, NL63-SPIKE_541–554, and NL63-DISC-like) and two epitopes of SARS-CoV-2 (COV2-SPIKE_421–434 and COV2-SPIKE ) 742 ⁢ – ⁢ 759 evaluated IgG responses using indirect ELISA in the plasma of pre-pandemic, mid-pandemic, and SARS-CoV-2 positive groups, has found that 63.1% of the pre-pandemic population (101 patients out of 160) and 48.9% in mid-pandemic population (69 patients out of 141) were seropositive [83]. Furthermore, a study on pre-pandemic, high-prevalence symptomatic donors and high-prevalence asymptomatic donors noted that there is a potential cross-reactivity of SARS-CoV-2 IgG antibodies with all four spike proteins corresponding to SARS-CoV, MERS-CoV, HCoV-OC43, and HCoV-HKU1 using recombinant soluble spike proteins of each virus in Expi293 expression system [80]. Controversially, a study has pointed out that in mice exposed to H-CoVs spike proteins have the potential to inhibit the generation of neutralizing antibodies specific for the RBD of SARS-CoV-2 [82] indicating that prior exposure to coronaviruses with sufficient homology can block the immune response to a novel coronavirus [82].

4.3 B cell immunodeficiency and COVID-19

A study of clinical cases with different immunodeficiencies reported that in contrast to patients with dysfunctional B cells caused by common variable immunodeficiency (CVID), those with agammaglobulinemia tended towards a milder form of COVID-19, a shorter duration of the disease, and a lack of necessity for treatment with IL-6-blocking medications [7]. Given that B cells are a significant source of IL-6, the absence of IL-6 derived from B cells has been hypothesized to prevent systemic autoimmunity and inflammatory responses [84]. A study reported that in patients undergoing iatrogenic B-cell depletion, especially with agents targeting CD20, the analysis revealed an increased risk of severe COVID-19 and death, irrespective of the underlying disease states. Among individuals with humoral inborn errors of immunity and COVID-19, their synthesis suggested that those with dysregulated humoral immunity, primarily common variable immunodeficiency (CVID), might face a higher susceptibility to severe COVID-19 compared to individuals with humoral immunodeficiency states due to X-linked agammaglobulinemia and other miscellaneous forms of humoral immunodeficiency. However, there were insufficient data available to assess the risk of COVID-19 infection in both patient populations [85]. A case report investigated a patient undergoing chemotherapy for malignant B cell lymphoma, revealing an active virus that persisted for over three months. Notably, there was an evolution of the viral strain observed during this period in the same individual [86].

4.4 The mechanism of breakthrough infection

Emerging variants, like the Delta variant, can evade the detection by diagnostic tests, show reduced sensitivity to antiviral drugs, monoclonal antibodies, and convalescent plasma, and can reinfect previously healthy and immunized people [87]. SARS-CoV-2 variants increase transmissibility, morbidity, and mortality. Since the SARS-CoV-2 genome is vulnerable to a variety of mutations that cause antigenic drift and enable immunological identification. During the pandemic, the Delta variant has been found to have the following mutations on the RBD of the spike protein: T19R, G142D, Δ 156–157, R158G, L452R, T478K, D614G, P681R, E484Q/K, and D950N [54]. The mutation L452R may promote the RBD and ACE2 receptor’s interaction, which would facilitate the interconnection of the membranes between the host cell and the virus, allowing more viral RNA to enter the cell [54]. A study of 6076 participants with no documented SARS-CoV2 infection reported a dose-dependent association between the level of total anti-spike IgG and the risk of breakthrough infection with the Delta variant. The study found that individuals in the highest quintile of total anti-spike IgG ( $>$ 3.02 log10 BAU) had a 71% lower rate of breakthrough infection with the Delta variant (incidence rate ratio [IRR] 0.29, 95% confidence interval [CI]: 0.15–0.56), while those in the lowest quintile ( ⩽ 1.77 log10 BAU) had a 57% lower rate (IRR 0.43, 95% confidence interval [IRR 0.25–0.75) [88]. In another study of a cohort of 12248 healthcare professionals in June 2021, a group of researchers found a 2.57% incidence of breakthrough infections. From this cohort, 58.5% had received at least one dose of the ChAdOx1-S/nCoV-19 vaccine (AZD1222) [87]. A third scientific team evaluated healthcare professionals who had received two doses of the BNT162b2 vaccination. Among them, 0.4% were infected post-vaccination with only mild or asymptomatic cases [55]. The rise in the reinfection rate further demonstrates that the virus breached the barrier of defence offered by the current vaccines. Due to the weakening of neutralization and the failure of monoclonal antibodies, the vaccination efficiency of current vaccines drastically decreased after the appearance of the Delta variation compared to the original strain and prior VOCs [55]. An in vitro investigation revealed that the Delta variant had an 8-fold lower sensitivity to vaccine-elicited antibodies and was less responsive to neutralizing antibodies in sera from recovered people [89]. The data of a group of researchers suggested that the Delta variant escaped natural infection-mediated neutralization with an average of 3.2-fold reduction in live virus neutralization assays. The neutralization of Beta was similarly reduced in an analysis employing serum from vaccine recipients, with an average reduction of 7.1-fold for a non-replicating vector platform and 4.1-fold for an mRNA platform [42]. It is reasonable to assume that the Delta version can resist immunological reactions, particularly humoral immunity. However, because natural infections or vaccinations generate memory immunity and can offer long-lasting protection mediated by memory cells, severe instances are rarely discovered from breakthrough infections [90]. For the omicron variant, there was no association between protection from the infection unlike the Delta variant, suggesting that a quantitative level of anti-spike IgG has limited impact on the risk of breakthrough infection. However, one case was observed of severe COVID-19 disease among the Delta variant cases, and none among participants infected with the Omicron variant, suggesting a dissociation between anti-Spike antibody levels and the risk of progression to severe Omicron infection [88].

5.1 Convalescent plasma therapy

In cases where a new pathogen can stimulate the production of neutralizing antibodies and evoke an immune response, the passive transfusion of convalescent blood products, particularly convalescent plasma, has demonstrated effectiveness as a feasible therapeutic approach [91]. Convalescent blood products can be produced by collecting whole blood or apheresis plasma from a donor who has recovered from the illness [91]. Donor selection is generally based on neutralizing antibody titer, as assessed with a plaque reduction neutralization test (PRNT) [91]. Nevertheless, research findings have suggested that ELISA ratios/indices exhibit strong correlations with PRNT titers. For instance, the Euroimmun ELISA IgG score, featuring a signal/cutoff reactivity index of 9.1, accurately identified 60% of samples with PRNT titers of approximately 1:100, achieving a specificity of 100% [92]. The Convalescent plasma (CP) transfusion has been used for all stages of infections [91]. In an early case series from China, five patients requiring mechanical ventilation, with four of them having no pre-existing medical conditions, received convalescent plasma transfusions. The CP used had an ELISA IgG titer of about 1:1,000 and a PRNT titer of approximately 40. These transfusions took place between days 10 to 22 after admission [93]. Following the initiation of medication, three patients were successfully taken off mechanical ventilation within two weeks, and four patients recovered from acute respiratory distress syndrome (ARDS). The remaining patients maintained a stable condition [93]. In another Chinese study (ChiCTR2000030046) involving 10 severe cases, the administration of a single dose of 200 ml of convalescent plasma with a neutralizing antibody titer of 1:640 led to an undetectable viral load in 7 patients. These individuals also showed radiological and clinical improvement [94]. In Wuhan, a separate cohort of six cases with COVID-19 pneumonia demonstrated that administering a single 200-ml dose of convalescent plasma at a late stage led to viral clearance in two patients and radiological resolution in five patients. The titers of anti-S antibodies were assessed exclusively by chemiluminescent immunoassay (CLIA) [95]. In addition, reported successful treatment of 2 out of 3 patients with different doses of CP [96]. Moreover, a team also documented a single patient’s recovery from mechanical breathing following the titration of CP antibodies using an anti-N protein ELISA [97]. In a retrospective observational study, six late-stage critically ill patients treated with convalescent plasma, titrated using gold immunochromatography, exhibited no mortality improvement, despite achieving viral clearance. This contrasts with the outcomes observed in thirteen untreated controls [98]. There was also one documented case of recovery in a centenarian patient who got two CP units (S-RBD-specific IgG titer of 1:640) [99]. Moreover, a single-arm phase II trial (NCT04321421) conducted in Lombardy, they treated 49 patients with moderate to severe illness using up to 3 units of pathogen-reduced-treated convalescent plasma (250 to 300 ml/48 [100]. 96% of the patients had neutralizing antibody titers of 1:160 [100]. Crucially, rather than the customary 100 tissue culture infective doses (TCID50), the viral inoculum contained 50% TCID50. The seven-day death rate was 6% as opposed to 16% in the previous group [100].

5.2 B-1 cells

The distinct subgroup of B lymphocytes known as B-1a cells in contrast to conventional B-2 cells, is essential to the innate immune response against bacterial, viral, and acute inflammatory illnesses [101]. These cells function early in the immune system’s response [101]. These cells secrete polyreactive natural immunoglobulin M (IgM), which neutralizes and detect microorganisms indiscriminately [101, 102]. Therefore, B1a cells in both mice and humans are functionally characterized and quantified based on their ability to express detectable levels of IL-10 following ex vivo stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin [101]. The cell surface phenotype of murine B-1 cells is CD45R(B220)^low, surface IgM (sIgM)^high, sIgD^low, CD23^{low / -} , CD19^high and CD43 ⁺ and can be either CD5 ⁺ (B-1a) or CD5 ^- (B-1b) [103]. Human B-1 cells were defined as CD20 ⁺ CD27 ⁺ CD43 ⁺ with little to no surface CD69 and CD70, which are both markedly upregulated after activation of CD20 ⁺ CD27 ^- CD43 ^- (naive) and CD20 ⁺ CD27 ⁺ CD43 ^- (memory) B cells [104]. Indeed, these cells also produce spontaneously Immunomodulatory molecules like the granulocyte-monocyte colony-stimulating factor (GM-CSF), an immune-boosting substance, and the anti-inflammatory cytokines interleukin IL-35 [101]. Through this particularity, B-1a can neutralize a broad range of pathogens and play a protective anti-inflammatory role in controlling hyperinflammation [105]. Consequently, it makes sense to assume that B-1a cells that produce IL-10 will shield COVID-19 patients from developing acute respiratory distress syndrome (ARDS) since it may inhibit the generation of reactive oxygen species (ROS), interfere with macrophage activation, and stop neutrophil extracellular traps (NETs) from forming [35]. B-1a cells demonstrate proficiency as antigen-presenting cells, establishing effective communication with T cells via constitutively expressed co-stimulatory molecules CD80/CD86. Additionally, B-1a cells play a role in promoting T cell growth and contributing to the differentiation of CD4 T cells into pro-inflammatory Th17 cells [106]. Hence, the immunomodulatory effects of B-1a cells suggest a potential benefit for the treatment of COVID-19.

5.3 B regulatory cells (Bregs) modulation

In general, the function of regulatory B (Breg) cells in viral infections can be linked to their suppression of other suppressor cells like regulatory T cells and their ability to block effector cells of the adaptive immune system like CD4 ⁺ and CD8 ⁺ T cells [107]. Bregs phenotypes identified in humans include CD19 ⁺ CD24 ⁺ CD38 ⁺ Bregs [107, 108, 109], CD19 ⁺ CD24 ⁺ CD27 ⁺ Bregs [110], CD19 ⁺ CD5 ⁺ CD1d ⁺ Bregs [111] and CD19 ⁺ TIM-1 ⁺ Bregs. The primary function of these cells has been discovered to be the production of inhibitory cytokines [107]. While studies have investigated the involvement of Breg cells in various viral infections [107] none specifically address their role in COVID-19 infection. IL-10 is so crucial to Breg cells’ inhibitory actions during viral infections that it might be viewed as the cytokine’s executive branch. Numerous studies have demonstrated how Breg cells can regulate Treg cells to suppress immunological responses. Since IL-10 is essential for the generation of Treg cells, blocking it will halt the process of turning CD4 ⁺ CD25 ^- T cells into Treg cells [112]. A study reported after stimulation of PBMCs for IL-10 induction, using multiparametric flow cytometry to determine B10 frequencies in severe and critical COVID-19 patients [113]. The study first segregated total CD19 ⁺ B cells; then CD27 ⁺ CD24^hi cells were gated to identify IL-10-producers as B10 cells. There was a notable decrease in the frequencies of regulatory B10 cells in severe and critical COVID-19 patients when compared to healthy controls [113]. The waning of the B10 subpopulation in hospitalized COVID-19 patients implies the potential association or contribution of the loss of these cells to the inflammatory pathogenesis of severe disease.

5.4 Neutralizing monoclonal antibodies (nMAb)

The administration of neutralizing monoclonal antibodies (nMAbs) targeting the SARS-CoV-2 spike protein has demonstrated effectiveness in preventing hospitalization and mortality. This evidence is derived from randomized clinical trials involving individuals who are not hospitalized and have not received vaccination, particularly those at a heightened risk of experiencing adverse outcomes [114]. To date, the US Food and Drug Administration (FDA) has granted emergency use authorization (EUA) to several neutralizing monoclonal antibody (nMAb) therapies In a comprehensive systematic review, a collective cohort of 25,241 patients underwent scrutiny, having received one of four distinct treatments: bamlanivimab, bamlanivimab-etesevimab, casirivimabimdevimab, and sotrovimab. The primary objective of this investigation was to evaluate the safety of neutralizing monoclonal antibodies (nMAbs) employed in the treatment of COVID-19 and to discern any potential associations with adverse outcomes. This extensive analysis aimed to provide insights into the overall safety profile of these therapeutic interventions and their impact on patients grappling with the virus [115]. Indeed, the use of nMAbs demonstrated an association with decreased probabilities in various clinical outcomes, including reduced odds of emergency department (ED) visits within a 14-day timeframe (odds ratio [OR], 0.76; 95% CI, 0.68–0.85), diminished likelihood of hospitalization within the same period (OR, 0.52; 95% CI, 0.45–0.59), and lower mortality rates within 30 days (OR, 0.14; 95% CI, 0.10–0.20). Furthermore, the application of nMAbs was correlated with a lowered risk of hospitalization in individuals who had not received vaccination (OR, 0.51; 95% CI, 0.44–0.59). Conversely, in immunocompromised patients, there was an observed heightened risk of hospitalization and mortality, with an OR of 0.31 (95% CI, 0.24–0.41) for hospitalization within 14 days and an OR of 0.13 (95% CI, 0.06–0.27) for death within 30 days [115]. REGN-COV2 is a combination of two potent neutralizing monoclonal antibodies (nMabs) named casirivimab and imdevimab. These antibodies are of the IgG1 class and have unmodified Fc regions [114]. During the examination of a placebo-controlled trial (NCT04425629) in the early stages (Phase I/II), a noteworthy finding emerged. Patients who underwent treatment with casirivimab and imdevimab exhibited a significantly lower occurrence of COVID-19-related hospitalizations or visits to the emergency department compared to those who received a placebo (2% versus 4%). This observation underscores the potential efficacy of this combination in preventing severe outcomes associated with COVID-19. In a distinct context, when bamlanivimab was employed in isolation as a monotherapy, it demonstrated substantial potency. This neutralizing monoclonal antibody, belonging to the IgG1 class and featuring an unaltered Fc region, specifically targets the S protein. Importantly, bamlanivimab was derived from the convalescent plasma of individuals who had recovered from COVID-19. This underscores its origin from the immune response of those who successfully battled the virus, adding a layer of natural immunity to its therapeutic profile [116].