RESUMO
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.
Assuntos
Inteligência Artificial , Leucócitos Mononucleares , ELISPOT/métodos , Algoritmos , Linfócitos B , AntígenosRESUMO
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.
Assuntos
Imunidade Humoral , Células B de Memória , Linfócitos B , Antígenos , Anticorpos Neutralizantes , Anticorpos AntiviraisRESUMO
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.
Assuntos
Linfócitos B , Leucócitos Mononucleares , Leucócitos Mononucleares/metabolismo , ELISPOT , Antígenos , Células Produtoras de Anticorpos , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismoRESUMO
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.
Assuntos
Linfócitos B , Leucócitos Mononucleares , Leucócitos Mononucleares/química , Antígenos , Células Produtoras de Anticorpos , Imunoglobulina G , ImunoglobulinasRESUMO
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.
Assuntos
COVID-19 , Infecções por Vírus Epstein-Barr , Influenza Humana , Humanos , SARS-CoV-2 , Herpesvirus Humano 4 , Anticorpos Antivirais , Células B de Memória , Estações do AnoRESUMO
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.
Assuntos
Anticorpos Antivirais/imunologia , Testes de Inibição da Hemaglutinação/métodos , Influenza Humana/diagnóstico , Animais , Antígenos Virais/genética , Antígenos Virais/imunologia , Reações Cruzadas , Modelos Animais de Doenças , Epitopos , Furões/imunologia , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/virologia , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/genética , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/imunologia , VacinaçãoRESUMO
Assessment of humoral immunity to SARS-CoV-2 and other infectious agents is typically restricted to detecting antigen-specific antibodies in the serum. Rarely does immune monitoring entail assessment of the memory B-cell compartment itself, although it is these cells that engage in secondary antibody responses capable of mediating immune protection when pre-existing antibodies fail to prevent re-infection. There are few techniques that are capable of detecting rare antigen-specific B cells while also providing information regarding their relative abundance, class/subclass usage and functional affinity. In theory, the ELISPOT/FluoroSpot (collectively ImmunoSpot) assay platform is ideally suited for antigen-specific B-cell assessments since it provides this information at single-cell resolution for individual antibody-secreting cells (ASC). Here, we tested the hypothesis that antigen-coating efficiency could be universally improved across a diverse set of viral antigens if the standard direct (non-specific, low affinity) antigen absorption to the membrane was substituted by high-affinity capture. Specifically, we report an enhancement in assay sensitivity and a reduction in required protein concentrations through the capture of recombinant proteins via their encoded hexahistidine (6XHis) affinity tag. Affinity tag antigen coating enabled detection of SARS-CoV-2 Spike receptor binding domain (RBD)-reactive ASC, and also significantly improved assay performance using additional control antigens. Collectively, establishment of a universal antigen-coating approach streamlines characterization of the memory B-cell compartment after SARS-CoV-2 infection or COVID-19 vaccinations, and facilitates high-throughput immune-monitoring efforts of large donor cohorts in general.
Assuntos
Antígenos Virais/análise , Linfócitos B/imunologia , ELISPOT/métodos , Memória Imunológica , SARS-CoV-2/imunologia , Proteínas Virais/imunologia , Animais , COVID-19 , Histidina , Humanos , Camundongos , Oligopeptídeos , SARS-CoV-2/metabolismoRESUMO
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.
Assuntos
Envelhecimento/imunologia , Anticorpos Antivirais/imunologia , Linfócitos B/imunologia , Memória Imunológica , Vacinas contra Influenza/administração & dosagem , Vacinação , Adolescente , Adulto , Idoso , Feminino , Humanos , Vacinas contra Influenza/imunologia , MasculinoRESUMO
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.
Assuntos
COVID-19/imunologia , COVID-19/virologia , Interações Hospedeiro-Patógeno/imunologia , SARS-CoV-2/imunologia , Linfócitos T/imunologia , Antígenos Virais/imunologia , Biomarcadores , COVID-19/metabolismo , Reações Cruzadas/imunologia , Citocinas/metabolismo , Epitopos de Linfócito T/imunologia , Humanos , Epitopos Imunodominantes/imunologia , Memória Imunológica , Peptídeos/imunologia , Ligação Proteica , Glicoproteína da Espícula de Coronavírus/imunologia , Linfócitos T/metabolismoRESUMO
Recent advances in high-throughput single cell sequencing have opened up new avenues into the investigation of B cell receptor (BCR) repertoires. In this study, PBMCs were collected from 17 human participants vaccinated with the split-inactivated influenza virus vaccine during the 2016-2017 influenza season. A combination of Immune Repertoire Capture (IRCTM) technology and IgG sequencing was performed on ~7,800 plasmablast (PB) cells and preferential IgG heavy-light chain pairings were investigated. In some participants, a single expanded clonotype accounted for ~22% of their PB BCR repertoire. Approximately 60% (10/17) of participants experienced convergent evolution, possessing public PBs that were elicited independently in multiple participants. Binding profiles of one private and three public PBs confirmed they were all subtype-specific, cross-reactive hemagglutinin (HA) head-directed antibodies. Collectively, this high-resolution antibody repertoire analysis demonstrated the impact evolution can have on BCRs in response to influenza virus vaccination, which can guide future universal influenza prophylactic approaches.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Adolescente , Adulto , Idoso , Reações Cruzadas/imunologia , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Hemaglutininas/imunologia , Humanos , Imunoglobulina G/imunologia , Influenza Humana/virologia , Masculino , Pessoa de Meia-Idade , Vacinação/métodos , Vacinas de Produtos Inativados/imunologia , Adulto JovemRESUMO
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.
RESUMO
Influenza viruses infect millions of people each year, resulting in significant morbidity and mortality in the human population. Therefore, generation of a universal influenza virus vaccine is an urgent need and would greatly benefit public health. Recombinant protein technology is an established vaccine platform and has resulted in several commercially available vaccines. Herein, we describe the approach for developing stable transfected human cell lines for the expression of recombinant influenza virus hemagglutinin (HA) and recombinant influenza virus neuraminidase (NA) proteins for the purpose of in vitro and in vivo vaccine development. HA and NA are the main surface glycoproteins on influenza virions and the major antibody targets. The benefits for using recombinant proteins for in vitro and in vivo assays include the ease of use, high level of purity and the ability to scale-up production. This work provides guidelines on how to produce and purify recombinant proteins produced in mammalian cell lines through either transient transfection or generation of stable cell lines from plasmid creation through the isolation step via Immobilized Metal Affinity Chromatography (IMAC). Collectively, the establishment of this pipeline has facilitated large-scale production of recombinant HA and NA proteins to high purity and with consistent yields, including glycosylation patterns that are very similar to proteins produced in a human host.
RESUMO
Detection of antigen-specific memory B cells for immune monitoring requires their activation, and is commonly accomplished through stimulation with the TLR7/8 agonist R848 and IL-2. To this end, we evaluated whether addition of IL-21 would further enhance this TLR-driven stimulation approach; which it did not. More importantly, as most antigen-specific B cell responses are T cell-driven, we sought to devise a polyclonal B cell stimulation protocol that closely mimics T cell help. Herein, we report that the combination of agonistic anti-CD40, IL-4 and IL-21 affords polyclonal B cell stimulation that was comparable to R848 and IL-2 for detection of influenza-specific memory B cells. An additional advantage of anti-CD40, IL-4 and IL-21 stimulation is the selective activation of IgM+ memory B cells, as well as the elicitation of IgE+ ASC, which the former fails to do. Thereby, we introduce a protocol that mimics physiological B cell activation through helper T cells, including induction of all Ig classes, for immune monitoring of antigen-specific B cell memory.
Assuntos
Linfócitos B/imunologia , Memória Imunológica/imunologia , Interleucina-4/metabolismo , Interleucinas/metabolismo , HumanosRESUMO
Influenza is a highly contagious viral pathogen with more than 200,000 cases reported in the United States during the 2017-2018 season. Annual vaccination is recommended by the World Health Organization, with the goal to reduce influenza severity and transmission. Currently available vaccines are about 60% effective, and vaccine effectiveness varies from season to season, as well as between different influenza subtypes within a single season. Immunological imprinting from early-life influenza infection can prominently shape the immune response to subsequent infections. Here, the impact of preexisting B cell memory in the response to quadrivalent influenza vaccine was assessed using blood samples collected from healthy subjects (18-85 years old) prior to and 21-28 days following influenza vaccination. Influenza vaccination increased both HA-specific antibodies and memory B cell frequency. Despite no apparent differences in antigenicity between vaccine components, most individuals were biased toward one of the vaccine strains. Specifically, responses to H3N2 were reduced in magnitude relative to the other vaccine components. Overall, this study unveils a potentially new mechanism underlying differential vaccine effectiveness against distinct influenza subtypes.
Assuntos
Linfócitos B/imunologia , Vacinas contra Influenza , Influenza Humana/prevenção & controle , Vacinação , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antivirais/sangue , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Imunidade , Imunoglobulina G/sangue , Memória Imunológica , Vírus da Influenza A Subtipo H3N2/imunologia , Influenza Humana/imunologia , Masculino , Pessoa de Meia-Idade , Estados Unidos , Adulto JovemRESUMO
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.
Assuntos
Anticorpos Monoclonais/metabolismo , Anticorpos Antivirais/metabolismo , Anticorpos Amplamente Neutralizantes/metabolismo , Vírus da Influenza A Subtipo H1N1/fisiologia , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Infecções por Orthomyxoviridae/imunologia , Animais , Anticorpos Monoclonais/química , Anticorpos Antivirais/química , Anticorpos Amplamente Neutralizantes/química , Biologia Computacional , Cães , Mapeamento de Epitopos , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/metabolismo , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB CAssuntos
Controle de Doenças Transmissíveis , Doenças Transmissíveis/imunologia , Vacinas/imunologia , Animais , Controle de Doenças Transmissíveis/métodos , Doenças Transmissíveis/epidemiologia , Doenças Transmissíveis/etiologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Celular , Imunidade HumoralRESUMO
T cell immunity is traditionally assessed through functional recall assays, which detect the consequences of the T cells' antigen encounter, or via fluorescently labeled multimers that selectively bind peptide-specific T cell receptors. Using either approach, if the wrong antigen or peptide of a complex antigenic system, such as a virus, is used for immune monitoring, either false negative data will be obtained, or the magnitude of the antigen-specific T cell compartment will go largely underestimated. In this work, we show how selection of the "right" antigen or antigenic peptides is critical for successful T cell immune monitoring against human cytomegalovirus (HCMV). Specifically, we demonstrate that individual HCMV antigens, along with previously reported epitopes, frequently failed to detect CD8+ T cell immunity in test subjects. Through systematic assessment of T cell reactivity against individual nonamer peptides derived from the HCMVpp65 protein, our data clearly establish that (i) systematic testing against all potential epitopes encoded by the genome of the antigen of interest is required to reliably detect CD8+ T cell immunity, and (ii) genome-wide, large scale systematic testing of peptides has become feasible through high-throughput ELISPOT-based "brute force" epitope mapping.
Assuntos
Antígenos Virais/imunologia , Linfócitos T CD8-Positivos/imunologia , Citomegalovirus/imunologia , Mapeamento de Epitopos , Epitopos de Linfócito T/imunologia , Ensaios de Triagem em Larga Escala , Peptídeos/imunologia , Adolescente , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
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.
Assuntos
Vírus da Influenza A/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Vigilância da PopulaçãoRESUMO
Influenza viruses represent a threat to the world population. The currently available standard of care influenza vaccines are offered for each influenza season to prevent infection and spread of influenza viruses. Current vaccine formulations rely on using wild-type Ags, including the hemagglutinin (HA) and neuraminidase (NA) proteins as the primary immune targets of the vaccine. However, vaccine effectiveness varies from season to season, ranging from 10 to 75% depending on season and on age group studied. To improve rates of vaccine effectiveness, a new generation of computationally optimized broadly reactive Ags (COBRA)-based vaccines have been developed as a next-generation influenza vaccine. In this report, mice were intranasally, i.p., or i.m. primed with reassortant influenza viruses expressing different H1N1 COBRA HA proteins. These mice were subsequently boosted i.p. or i.m. with the same viruses. Sera collected from mice that were intranasally infected and i.p. boosted with COBRA-based viruses had broad anti-HA IgG binding, hemagglutination inhibition, and neutralizing activity against a panel of seasonal and pandemic H1N1 viruses. Mice immunized with viruses expressing a seasonal or pandemic H1N1 HA protein had antisera that recognized fewer viruses in the panel. Overall, COBRA-based HA proteins displayed on the surface of a virus elicited a breadth of Abs that recognized and neutralized historical H1N1 strains as well as more contemporary H1N1 viruses.