A Competitive Hemagglutination Inhibition Assay for Dissecting Functional Antibody Activity against Influenza Virus.

Influenza remains one of the most contagious infectious diseases. Approximately, 25 to 50 million people suffer from influenza-like illness in the United States annually, leading to almost 1 million hospitalizations. Globally, the World Health Organization (WHO) estimates 250,000 to 500,000 mortalities associated with secondary respiratory complications due to influenza virus infection every year. Currently, seasonal vaccination represents the best countermeasure to prevent influenza virus spread and transmission in the general population. However, presently licensed influenza vaccines are about 60% effective on average, and their effectiveness varies from season to season and among age groups, as well as between different influenza subtypes within a single season. The hemagglutination inhibition (HAI) assay represents the gold standard method for measuring the functional antibody response elicited following standard-of-care vaccination, along with evaluating the efficacy of under-development influenza vaccines in both animal models and clinical trial settings. However, using the classical HAI approach, it is not possible to dissect the complexities of variable epitope recognition within a polyclonal antibody response. In this paper, we describe a straightforward competitive HAI-based method using a combination of influenza virus and recombinant hemagglutinin (HA) proteins to dissect the HAI functional activity of HA-specific antibody populations in a single assay format. IMPORTANCE The hemagglutination inhibition (HAI) assay is a well-established and reproducible method that quantifies functional antibody activity against influenza viruses and, in particular, the capability of an antibody formulation to inhibit the binding of hemagglutinin (HA) to sialic acid. However, the HAI assay does not provide full insights on the breadth and epitope recognition of the antibody formulation, especially in the context of polyclonal sera, where multiple antibody specificities contribute to the overall observed functional activity. In this report we introduce the use of Y98F point-mutated recombinant HA (HAΔSA) proteins, which lack sialic acid binding activity, in the context of the HAI assay as a means to absorb out certain HA-directed (i.e., strain-specific or cross-reactive) antibody populations. This modification to the classical HAI assay, referred to as the competitive HAI assay, represents a new tool to dissect the magnitude and breadth of polyclonal antibodies elicited through vaccination or natural infection.

A Computationally Optimized Broadly Reactive Antigen Subtype-Specific Influenza Vaccine Strategy Elicits Unique Potent Broadly Neutralizing Antibodies against Hemagglutinin.

Computationally optimized broadly reactive Ags (COBRA) targeting H1 elicit a broad cross-reactive and cross-neutralizing Ab response against multiple H1N1 viral strains. To assess B cell breadth, Mus musculus (BALB/c) Ab-secreting cells elicited by a candidate COBRA hemagglutinin (HA) (termed P1) were compared with Ab-secreting cells elicited by historical H1N1 vaccine strains. In addition, to evaluate the Ab response elicited by P1 HA at increased resolution, a panel of P1 HA-specific B cell hybridomas was generated following immunization of mice with COBRA P1 and the corresponding purified mAbs were characterized for Ag specificity and neutralization activity. Both head- and stem-directed mAbs were elicited by the P1 HA Ag, with some mAbs endowed with Ab-dependent cell-mediated cytotoxicity activity. P1 HA-elicited mAbs exhibited a wide breadth of HA recognition, ranging from narrowly reactive to broadly reactive mAbs. Interestingly, we identified a P1 HA-elicited mAb (1F8) exhibiting broad hemagglutination inhibition activity against both seasonal and pandemic H1N1 influenza strains. Furthermore, mAb 1F8 recognized an overlapping, but distinct, epitope compared with other narrowly hemagglutination inhibition-positive mAbs elicited by the P1 or wild-type HA Ags. Finally, P1 HA-elicited mAbs were encoded by distinct H chain variable and L chain variable gene segment rearrangements and possessed unique CDR3 sequences. Collectively, the functional characterization of P1 HA-elicited mAbs sheds further insights into the underlying mechanism(s) of expanded Ab breadth elicited by a COBRA HA-based immunogen and advances efforts toward design and implementation of a more broadly protective influenza vaccine.

Infection of Ferrets with Influenza Virus Elicits a Light Chain-Biased Antibody Response against Hemagglutinin.

The domestic ferret (Mustela putorius furo) is a commonly used animal model for the study of influenza virus infection and vaccination. Recently, our group has developed murine mAbs with specificity for the κ (Igκ) and λ (Igλ) L chains of ferret Ig. These mAbs were used to quantify the abundance of Igκ and Igλ in serum and to evaluate L chain usage of the Ab response against the hemagglutinin (HA) protein elicited by influenza infection. After influenza A infection of immunologically naive ferrets with various H1N1 or H3N2 strains, the acute Ab response against HA exhibited an inherent bias toward λ L chain usage. In contrast, secondary infection of H1N1 preimmune ferrets with an antigenically distinct H1N1 virus elicited a recall response against the original HA that was no longer biased toward Igλ and possessed differential specificity. Moreover, sequential infection of ferrets with H1N1 influenza viruses elicited an Igκ-biased Ab response directed against the HA globular head and stem regions. Furthermore, sequential infection of ferrets with viral vectors expressing chimeric HA, aimed at boosting Ab reactivity against the HA stem region, also elicited an Igκ-biased response. Collectively, these findings suggest that ferret B cells expressing an Igκ or Igλ BCR possess differential specificities, and highlight the utility of our recently developed mAbs for studying the immune response to influenza virus infection and vaccination in the ferret model.

Elicitation of Protective Antibodies against a Broad Panel of H1N1 Viruses in Ferrets Preimmune to Historical H1N1 Influenza Viruses.

Most preclinical animal studies test influenza vaccines in immunologically naive animal models, even though the results of vaccination may not accurately reflect the effectiveness of vaccine candidates in humans that have preexisting immunity to influenza. In this study, novel, broadly reactive influenza vaccine candidates were assessed in preimmune ferrets. These animals were infected with different H1N1 isolates before being vaccinated or infected with another influenza virus. Previously, our group has described the design and characterization of computationally optimized broadly reactive hemagglutinin (HA) antigens (COBRA) for H1N1 isolates. Vaccinating ferrets with virus-like particle (VLP) vaccines expressing COBRA HA proteins elicited antibodies with hemagglutination inhibition (HAI) activity against more H1N1 viruses in the panel than VLP vaccines expressing wild-type HA proteins. Specifically, ferrets infected with the 1986 virus and vaccinated with a single dose of the COBRA HA VLP vaccines elicited antibodies with HAI activity against 11 to 14 of the 15 H1N1 viruses isolated between 1934 and 2013. A subset of ferrets was infected with influenza viruses expressing the COBRA HA antigens. These COBRA preimmune ferrets had superior breadth of HAI activity after vaccination with COBRA HA VLP vaccines than COBRA preimmune ferrets vaccinated with VLP vaccines expressing wild-type HA proteins. Overall, priming naive ferrets with COBRA HA based viruses or using COBRA HA based vaccines to boost preexisting antibodies induced by wild-type H1N1 viruses, COBRA HA antigens elicited sera with the broadest HAI reactivity against multiple antigenic H1N1 viral variants. This is the first report demonstrating the effectiveness of a broadly reactive or universal influenza vaccine in a preimmune ferret model.IMPORTANCE Currently, many groups are testing influenza vaccine candidates to meet the challenge of developing a vaccine that elicits broadly reactive and long-lasting protective immune responses. The goal of these vaccines is to stimulate immune responses that react against most, if not all, circulating influenza strains, over a long period of time in all populations of people. Commonly, these experimental vaccines are tested in naive animal models that do not have anti-influenza immune responses; however, humans have preexisting immunity to influenza viral antigens, particularly antibodies to the HA and NA glycoproteins. Therefore, this study investigated how preexisting antibodies to historical influenza viruses influenced HAI-specific antibodies and protective efficacy using a broadly protective vaccine candidate.

Towards a universal influenza vaccine: different approaches for one goal.

Influenza virus infection is an ongoing health and economic burden causing epidemics with pandemic potential, affecting 5-30% of the global population annually, and is responsible for millions of hospitalizations and thousands of deaths each year. Annual influenza vaccination is the primary prophylactic countermeasure aimed at limiting influenza burden. However, the effectiveness of current influenza vaccines are limited because they only confer protective immunity when there is antigenic similarity between the selected vaccine strains and circulating influenza isolates. The major targets of the antibody response against influenza virus are the surface glycoprotein antigens hemagglutinin (HA) and neuraminidase (NA). Hypervariability of the amino acid sequences encoding HA and NA is largely responsible for epidemic and pandemic influenza outbreaks, and are the consequence of antigenic drift or shift, respectively. For this reason, if an antigenic mismatch exists between the current vaccine and circulating influenza isolates, vaccinated people may not be afforded complete protection. There is currently an unmet need to develop an effective "broadly-reactive" or "universal" influenza vaccine capable of conferring protection against both seasonal and newly emerging pre-pandemic strains. A number of novel influenza vaccine approaches are currently under evaluation. One approach is the elicitation of an immune response against the "Achille's heel" of the virus, i.e. conserved viral proteins or protein regions shared amongst seasonal and pre-pandemic strains. Alternatively, other approaches aim toward eliciting a broader immune response capable of conferring protection against the diversity of currently circulating seasonal influenza strains.In this review, the most promising under-development universal vaccine approaches are discussed with an emphasis on those targeting the HA glycoprotein. In particular, their strengths and potential short-comings are discussed. Ultimately, the upcoming clinical evaluation of these universal vaccine approaches will be fundamental to determine their effectiveness against preventing influenza virus infection and/or reducing transmission and disease severity.

γδ T Cells Shape Preimmune Peripheral B Cell Populations.

We previously reported that selective ablation of certain γδ T cell subsets, rather than removal of all γδ T cells, strongly affects serum Ab levels in nonimmunized mice. This type of manipulation also changed T cells, including residual γδ T cells, revealing some interdependence of γδ T cell populations. For example, in mice lacking Vγ4(+) and Vγ6(+) γδ T cells (B6.TCR-Vγ4(-/-)/6(-/-)), we observed expanded Vγ1(+) cells, which changed in composition and activation and produced more IL-4 upon stimulation in vitro, increased IL-4 production by αß T cells as well as spontaneous germinal center formation in the spleen, and elevated serum Ig and autoantibodies. We therefore examined B cell populations in this and other γδ-deficient mouse strains. Whereas immature bone marrow B cells remained largely unchanged, peripheral B cells underwent several changes. Specifically, transitional and mature B cells in the spleen of B6.TCR-Vγ4(-/-)/6(-/-) mice and other peripheral B cell populations were diminished, most of all splenic marginal zone (MZ) B cells. However, relative frequencies and absolute numbers of Ab-producing cells, as well as serum levels of Abs, IL-4, and BAFF, were increased. Cell transfers confirmed that these changes are directly dependent on the altered γδ T cells in this strain and on their enhanced potential of producing IL-4. Further evidence suggests the possibility of direct interactions between γδ T cells and B cells in the splenic MZ. Taken together, these data demonstrate the capability of γδ T cells of modulating size and productivity of preimmune peripheral B cell populations.

γδ T cells affect IL-4 production and B-cell tolerance.

γδ T cells can influence specific antibody responses. Here, we report that mice deficient in individual γδ T-cell subsets have altered levels of serum antibodies, including all major subclasses, sometimes regardless of the presence of αß T cells. One strain with a partial γδ deficiency that increases IgE antibodies also displayed increases in IL-4-producing T cells (both residual γδ T cells and αß T cells) and in systemic IL-4 levels. Its B cells expressed IL-4-regulated inhibitory receptors (CD5, CD22, and CD32) at diminished levels, whereas IL-4-inducible IL-4 receptor α and MHCII were increased. They also showed signs of activation and spontaneously formed germinal centers. These mice displayed IgE-dependent features found in hyper-IgE syndrome and developed antichromatin, antinuclear, and anticytoplasmic autoantibodies. In contrast, mice deficient in all γδ T cells had nearly unchanged Ig levels and did not develop autoantibodies. Removing IL-4 abrogated the increases in IgE, antichromatin antibodies, and autoantibodies in the partially γδ-deficient mice. Our data suggest that γδ T cells, controlled by their own cross-talk, affect IL-4 production, B-cell activation, and B-cell tolerance.

Sequential Infection in Ferrets with Antigenically Distinct Seasonal H1N1 Influenza Viruses Boosts Hemagglutinin Stalk-Specific Antibodies.

UNLABELLED: Broadly reactive antibodies targeting the conserved hemagglutinin (HA) stalk region are elicited following sequential infection or vaccination with influenza viruses belonging to divergent subtypes and/or expressing antigenically distinct HA globular head domains. Here, we demonstrate, through the use of novel chimeric HA proteins and competitive binding assays, that sequential infection of ferrets with antigenically distinct seasonal H1N1 (sH1N1) influenza virus isolates induced an HA stalk-specific antibody response. Additionally, stalk-specific antibody titers were boosted following sequential infection with antigenically distinct sH1N1 isolates in spite of preexisting, cross-reactive, HA-specific antibody titers. Despite a decline in stalk-specific serum antibody titers, sequential sH1N1 influenza virus-infected ferrets were protected from challenge with a novel H1N1 influenza virus (A/California/07/2009), and these ferrets poorly transmitted the virus to naive contacts. Collectively, these findings indicate that HA stalk-specific antibodies are commonly elicited in ferrets following sequential infection with antigenically distinct sH1N1 influenza virus isolates lacking HA receptor-binding site cross-reactivity and can protect ferrets against a pathogenic novel H1N1 virus. IMPORTANCE: The influenza virus hemagglutinin (HA) is a major target of the humoral immune response following infection and/or seasonal vaccination. While antibodies targeting the receptor-binding pocket of HA possess strong neutralization capacities, these antibodies are largely strain specific and do not confer protection against antigenic drift variant or novel HA subtype-expressing viruses. In contrast, antibodies targeting the conserved stalk region of HA exhibit broader reactivity among viruses within and among influenza virus subtypes. Here, we show that sequential infection of ferrets with antigenically distinct seasonal H1N1 influenza viruses boosts the antibody responses directed at the HA stalk region. Moreover, ferrets possessing HA stalk-specific antibody were protected against novel H1N1 virus infection and did not transmit the virus to naive contacts.

Enhanced Influenza Virus-Like Particle Vaccination with a Structurally Optimized RIG-I Agonist as Adjuvant.

UNLABELLED: The molecular interaction between viral RNA and the cytosolic sensor RIG-I represents the initial trigger in the development of an effective immune response against infection with RNA viruses, resulting in innate immune activation and subsequent induction of adaptive responses. In the present study, the adjuvant properties of a sequence-optimized 5'-triphosphate-containing RNA (5'pppRNA) RIG-I agonist (termed M8) were examined in combination with influenza virus-like particles (VLP) (M8-VLP) expressing H5N1 influenza virus hemagglutinin (HA) and neuraminidase (NA) as immunogens. In combination with VLP, M8 increased the antibody response to VLP immunization, provided VLP antigen sparing, and protected mice from a lethal challenge with H5N1 influenza virus. M8-VLP immunization also led to long-term protective responses against influenza virus infection in mice. M8 adjuvantation of VLP increased endpoint and antibody titers and inhibited influenza virus replication in lungs compared with approved or experimental adjuvants alum, AddaVax, and poly(I·C). Uniquely, immunization with M8-VLP stimulated a TH1-biased CD4 T cell response, as determined by increased TH1 cytokine levels in CD4 T cells and increased IgG2 levels in sera. Collectively, these data demonstrate that a sequence-optimized, RIG-I-specific agonist is a potent adjuvant that can be utilized to increase the efficacy of influenza VLP vaccination and dramatically improve humoral and cellular mediated protective responses against influenza virus challenge. IMPORTANCE: The development of novel adjuvants to increase vaccine immunogenicity is an important goal that seeks to improve vaccine efficacy and ultimately prevent infections that endanger human health. This proof-of-principle study investigated the adjuvant properties of a sequence-optimized 5'pppRNA agonist (M8) with enhanced capacity to stimulate antiviral and inflammatory gene networks using influenza virus-like particles (VLP) expressing HA and NA as immunogens. Vaccination with VLP in combination with M8 increased anti-influenza virus antibody titers and protected animals from lethal influenza virus challenge, highlighting the potential clinical use of M8 as an adjuvant in vaccine development. Altogether, the results describe a novel immunostimulatory agonist targeted to the cytosolic RIG-I sensor as an attractive vaccine adjuvant candidate that can be used to increase vaccine efficacy, a pressing issue in children and the elderly population.

Functionally responsive self-reactive B cells of low affinity express reduced levels of surface IgM.

Somatic gene rearrangement generates a diverse repertoire of B cells, many which have receptors possessing a range of affinities for self-Ag. Newly generated B cells express high and relatively uniform amounts of surface IgM (sIgM), while follicular (FO) B cells express sIgM at widely varying levels. It is plausible, therefore, that downmodulation of sIgM serves as a mechanism to maintain weakly self-reactive B cells in a responsive state by decreasing their avidity for self-Ag. We tested this hypothesis by performing comparative functional tests with FO IgM(hi) and IgM(lo) B cells from the unrestricted repertoire of WT C57BL/6 mice. We found that FO IgM(lo) B cells mobilized Ca(2+) equivalently to IgM(hi) B cells when the same number of sIgM molecules was engaged. In agreement, FO IgM(lo) B cells were functionally competent to produce an antibody response following adoptive transfer. The FO IgM(lo) cell population had elevated levels of Nur77 transcript, and was enriched with nuclear-reactive specificities. Hybridoma sampling revealed that these B-cell receptors were of low affinity. Collectively, these results suggest that sIgM downmodulation by low-affinity, self-reactive B cells preserves their immunocompetence and circumvents classical peripheral tolerance mechanisms that would otherwise reduce diversity within the B cell compartment.

Antigen-specific suppression of humoral immunity by anergic Ars/A1 B cells.

Autoreactive anergic B lymphocytes are considered to be dangerous because of their potential for activation and recruitment into autoimmune responses. However, they persist for days and constitute ∼5% of the B cell pool. We assessed their functional potential in the Ars/A1 transgene model, where anergic B cells express a dual-reactive Ag receptor that binds, in addition to a self-Ag, the hapten p-azophenylarsonate (Ars). When Ars/A1 B cells were transferred into adoptive recipients that were immunized with foreign proteins covalently conjugated with Ars, endogenous IgG immune responses to both were selectively and severely diminished, and the development of T helper cells was impaired. Approximately 95% inhibition of the anti-Ars response was attained with ∼4000 transferred Ars/A1 B cells through redundant mechanisms, one of which depended on their expression of MHC class II but not upon secretion of IL-10 or IgM. This Ag-specific suppressive activity implicates the autoreactive anergic B cell as an enforcer of immunological tolerance to self-Ags.

Artificial Intelligence-Based Counting Algorithm Enables Accurate and Detailed Analysis of the Broad Spectrum of Spot Morphologies Observed in Antigen-Specific B-Cell ELISPOT and FluoroSpot Assays.

Antigen-specific B-cell ELISPOT and multicolor FluoroSpot assays, in which the membrane-bound antigen itself serves as the capture reagent for the antibodies that B cells secrete, inherently result in a broad range of spot sizes and intensities. The diversity of secretory footprint morphologies reflects the polyclonal nature of the antigen-specific B cell repertoire, with individual antibody-secreting B cells in the test sample differing in their affinity for the antigen, fine epitope specificity, and activation/secretion kinetics. To account for these heterogeneous spot morphologies, and to eliminate the need for setting up subjective counting parameters well-by-well, CTL introduces here its cutting-edge deep learning-based IntelliCount™ algorithm within the ImmunoSpot® Studio Software Suite, which integrates CTL's proprietary deep neural network. Here, we report detailed analyses of spots with a broad range of morphologies that were challenging to analyze using standard parameter-based counting approaches. IntelliCount™, especially in conjunction with high dynamic range (HDR) imaging, permits the extraction of accurate, high-content information of such spots, as required for assessing the affinity distribution of an antigen-specific memory B-cell repertoire ex vivo. IntelliCount™ also extends the range in which the number of antibody-secreting B cells plated and spots detected follow a linear function; that is, in which the frequencies of antigen-specific B cells can be accurately established. Introducing high-content analysis of secretory footprints in B-cell ELISPOT/FluoroSpot assays, therefore, fundamentally enhances the depth in which an antigen-specific B-cell repertoire can be studied using freshly isolated or cryopreserved primary cell material, such as peripheral blood mononuclear cells.

Monitoring Memory B Cells by Next-Generation ImmunoSpot® Provides Insights into Humoral Immunity that Measurements of Circulating Antibodies Do Not Reveal.

Memory B cells (Bmem) provide the second wall of adaptive humoral host defense upon specific antigen rechallenge when the first wall, consisting of preformed antibodies originating from a preceding antibody response, fails. This is the case, as recently experienced with SARS-CoV-2 infections and previously with seasonal influenza, when levels of neutralizing antibodies decline or when variant viruses arise that evade such. While in these instances, reinfection can occur, in both scenarios, the rapid engagement of preexisting Bmem into the recall response can still confer immune protection. Bmem are known to play a critical role in host defense, yet their assessment has not become part of the standard immune monitoring repertoire. Here we describe a new generation of B cell ELISPOT/FluoroSpot (collectively ImmunoSpot®) approaches suited to dissect, at single-cell resolution, the Bmem repertoire ex vivo, revealing its immunoglobulin class/subclass utilization, and its affinity distribution for the original, and for variant viruses/antigens. Because such comprehensive B cell ImmunoSpot® tests can be performed with minimal cell material, are scalable, and robust, they promise to be well-suited for routine immune monitoring.

Assessing the Affinity Spectrum of the Antigen-Specific B Cell Repertoire via ImmunoSpot®.

The affinity distribution of the antigen-specific memory B cell (Bmem) repertoire in the body is a critical variable that defines an individual's ability to rapidly generate high-affinity protective antibody specificities. Detailed measurement of antibody affinity so far has largely been confined to studies of monoclonal antibodies (mAbs) and are laborious since each individual mAb needs to be evaluated in isolation. Here, we introduce two variants of the B cell ImmunoSpot® assay that are suitable for simultaneously assessing the affinity distribution of hundreds of individual B cells within a test sample at single-cell resolution using relatively little labor and with high-throughput capacity. First, we experimentally validated that both ImmunoSpot® assay variants are suitable for establishing functional affinity hierarchies using B cell hybridoma lines as model antibody-secreting cells (ASC), each producing mAb with known affinity for a defined antigen. We then leveraged both ImmunoSpot® variants for characterizing the affinity distribution of SARS-CoV-2 Spike-specific ASC in PBMC following COVID-19 mRNA vaccination. Such ImmunoSpot® assays promise to offer tremendous value for future B cell immune monitoring efforts, owing to their ease of implementation, applicability to essentially any antigenic system, economy of PBMC utilization, high-throughput capacity, and suitability for regulated testing.

Four-Color ImmunoSpot® Assays Requiring Only 1-3 mL of Blood Permit Precise Frequency Measurements of Antigen-Specific B Cells-Secreting Immunoglobulins of All Four Classes and Subclasses.

The B lymphocyte response can encompass four immunoglobulin (Ig) classes and four IgG subclasses, each contributing fundamentally different effector functions. Production of the appropriate Ig class/subclass is critical for both successful host defense and avoidance of immunopathology. The assessment of an antigen-specific B cell response, including its magnitude and Ig class/subclass composition, is most often confined to the antibodies present in serum and other biological fluids and neglects monitoring of the memory B cell (Bmem) compartment capable of mounting a faster and more efficient antibody response following antigen reencounter. Here, we describe how the frequency and Ig class and IgG subclass use of an antigen-specific Bmem repertoire can be determined with relatively little labor and cost, requiring only 8 × 105 freshly isolated peripheral blood mononuclear cells (PBMC), or if additional cryopreservation and polyclonal stimulation is necessary, 3 × 106 PBMC per antigen. To experimentally validate such cell saving assays, we have documented that frequency measurements of antibody-secreting cells (ASC) yield results indistinguishable from those of enzymatic (ELISPOT) or fluorescent (FluoroSpot) versions of the ImmunoSpot® assay, including when the latter are detected in alternative fluorescent channels. Moreover, we have shown that frequency calculations that are based on linear regression analysis of serial PBMC dilutions using a single well per dilution step are as accurate as those performed using replicate wells. Collectively, our data highlight the capacity of multiplexed B cell FluoroSpot assays in conjunction with serial dilutions to significantly reduce the PBMC requirement for detailed assessment of antigen-specific B cells. The protocols presented here allow GLP-compliant high-throughput measurements which should help to introduce high-dimensional Bmem characterization into the standard immune monitoring repertoire.

Antibody Levels Poorly Reflect on the Frequency of Memory B Cells Generated following SARS-CoV-2, Seasonal Influenza, or EBV Infection.

The scope of immune monitoring is to define the existence, magnitude, and quality of immune mechanisms operational in a host. In clinical trials and praxis, the assessment of humoral immunity is commonly confined to measurements of serum antibody reactivity without accounting for the memory B cell potential. Relying on fundamentally different mechanisms, however, passive immunity conveyed by pre-existing antibodies needs to be distinguished from active B cell memory. Here, we tested whether, in healthy human individuals, the antibody titers to SARS-CoV-2, seasonal influenza, or Epstein-Barr virus antigens correlated with the frequency of recirculating memory B cells reactive with the respective antigens. Weak correlations were found. The data suggest that the assessment of humoral immunity by measurement of antibody levels does not reflect on memory B cell frequencies and thus an individual's potential to engage in an anamnestic antibody response against the same or an antigenically related virus. Direct monitoring of the antigen-reactive memory B cell compartment is both required and feasible towards that goal.

Deconvoluting the T Cell Response to SARS-CoV-2: Specificity Versus Chance and Cognate Cross-Reactivity.

SARS-CoV-2 infection takes a mild or clinically inapparent course in the majority of humans who contract this virus. After such individuals have cleared the virus, only the detection of SARS-CoV-2-specific immunological memory can reveal the exposure, and hopefully the establishment of immune protection. With most viral infections, the presence of specific serum antibodies has provided a reliable biomarker for the exposure to the virus of interest. SARS-CoV-2 infection, however, does not reliably induce a durable antibody response, especially in sub-clinically infected individuals. Consequently, it is plausible for a recently infected individual to yield a false negative result within only a few months after exposure. Immunodiagnostic attention has therefore shifted to studies of specific T cell memory to SARS-CoV-2. Most reports published so far agree that a T cell response is engaged during SARS-CoV-2 infection, but they also state that in 20-81% of SARS-CoV-2-unexposed individuals, T cells respond to SARS-CoV-2 antigens (mega peptide pools), allegedly due to T cell cross-reactivity with Common Cold coronaviruses (CCC), or other antigens. Here we show that, by introducing irrelevant mega peptide pools as negative controls to account for chance cross-reactivity, and by establishing the antigen dose-response characteristic of the T cells, one can clearly discern between cognate T cell memory induced by SARS-CoV-2 infection vs. cross-reactive T cell responses in individuals who have not been infected with SARS-CoV-2.

Impaired memory B-cell recall responses in the elderly following recurrent influenza vaccination.

Influenza is a highly contagious viral respiratory disease that affects million of people worldwide each year. Annual vaccination is recommended by the World Health Organization with the goal of reducing influenza severity and limiting transmission through elicitation of antibodies targeting the hemagglutinin (HA) glycoprotein. The antibody response elicited by current seasonal influenza virus vaccines is predominantly strain-specific, but pre-existing influenza virus immunity can greatly impact the serological antibody response to vaccination. However, it remains unclear how B cell memory is shaped by recurrent annual vaccination over the course of multiple seasons, especially in high-risk elderly populations. Here, we systematically profiled the B cell response in young adult (18-34 year old) and elderly (65+ year old) vaccine recipients that received annual split inactivated influenza virus vaccination for 3 consecutive seasons. Specifically, the antibody serological and memory B-cell compartments were profiled for reactivity against current and historical influenza A virus strains. Moreover, multiparametric analysis and antibody landscape profiling revealed a transient increase in strain-specific antibodies in the elderly, but with an impaired recall response of pre-existing memory B-cells, plasmablast (PB) differentiation and long-lasting serological changes. This study thoroughly profiles and compares the immune response to recurrent influenza virus vaccination in young and elderly participants unveiling the pitfalls of current influenza virus vaccines in high-risk populations.

Functional T Cell Reactivity to Melanocyte Antigens Is Lost during the Progression of Malignant Melanoma, but Is Restored by Immunization.

Healthy human subjects develop spontaneous CD8+ T cell responses to melanoma associated antigens (MA) expressed by normal melanocytes, such as Tyrosinase, MAGE-A3, Melan/Mart-1, gp100, and NY-ESO-1. This natural autoimmunity directed against melanocytes might confer protection against the development of malignant melanoma (MM), where MA are present as overexpressed tumor-associated antigens. Consistent with this notion we report here that functional T cell reactivity to MA was found to be significantly diminished to MAGE-A3, Melan-A/Mart-1, and gp100 in untreated MM patients. Three lines of evidence suggest that the MA-reactive T cells present in healthy subjects undergo exhaustion once MM establishes itself. First, only the MA-specific T cell reactivity was affected in the MM patients; that to third party recall antigens was not. Second, in these patients, the residual MA-specific T cells, unlike third party antigen reactive T cells, were functionally impaired, showing a diminished per cell IFN-γ productivity. Third, we show that immunization with MA restored natural CD8+ T cell autoimmunity to MA in 85% of the MM patients. The role of natural T cell autoimmunity to tumor-associated MA is discussed based on discrete levels of T cell activation thresholds.