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Presently, the world needs safe and effective vaccines to overcome the COVID-19 pandemic. Our work has focused on formulating two types of mRNA vaccines that differ in capacity to copy themselves inside the cell. These are non-amplifying mRNA (NRM) and self-amplifying mRNA (SAM) vaccines. Both the vaccine candidates encode an engineered viral replicon which can provoke an immune response. Hence we predicted and screened twelve epitopes from the spike glycoprotein of SARS-CoV-2. We used five CTL, four HTL, and three B-cell-activating epitopes to formulate each mRNA vaccine. Molecular docking revealed that these epitopes could combine with HLA molecules that are important for boosting immunogenicity. The B-cell epitopes were adjoined with GPGPG linkers, while CTL and HTL epitopes were linked with KK linkers. The entire protein chain was reverse translated to develop a specific NRM-based vaccine. We incorporate gene encoding replicase in the upstream region of CDS encoding antigen to design the SAM vaccine. Subsequently, signal sequences were added to human mRNA to formulate vaccines. Both vaccine formulations translated to produce the epitopes in host cells, initiate a protective immune cascade, and generate immunogenic memory, which can counter future SARS-CoV-2 viral exposures before the onset of infection. Supplementary Information The online version contains supplementary material available at 10.1007/s12033-021-00432-6.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a life-threatening complication described after administration of recombinant adenoviral vector encoding the spike protein antigen of Severe Acute Respiratory Syndrome CoronaVirus-2. The syndrome is characterized by platelet consumption and thrombosis. High levels of antibodies to platelet factor 4 (PF4)–polyanion complexes were identified in many patients with VITT by enzyme-linked immunosorbent assay (ELISA). A 64-year-old woman presented with thrombocytopenia, right renal vein thrombosis with renal infarction, right intra-right atrium and intra-right ventricle thrombosis and pulmonary embolism after ChAdOx1-S vaccine administration. ELISA for antibodies to PF4–polyanion complexes tested positive, while functional tests were not. Thrombocytopenia was refractory to intravenous immunoglobulins and corticosteroids. Eltrombopag was introduced and platelet gradually rose to normal values. VITT is a novel complication yet to be understood. The clinical case we reported highlights the difficulties in the management of this disorder and discloses a new potential therapy in refractory conditions.

With recent advances, chimeric antigen receptor (CAR) immunotherapy has become a promising modality for patients with refractory cancer diseases. The successful results of CAR T cell therapy in relapsed and refractory B-cell malignancies shifted the paradigm of cancer immunotherapy by awakening the scientific, clinical, and commercial interest in translating this technology for the treatment of solid cancers. This review elaborates on fundamental principles of CAR T cell therapy (development of CAR construct, challenges of CAR T cell therapy) and its application on solid tumors as well as CAR T cell therapy potential in the field of neuro-oncology. Glioblastoma (GBM) is identified as one of the most challenging solid tumors with a permissive immunological milieu and dismal prognosis. Standard multimodal treatment using maximal safe resection, radiochemotherapy, and maintenance chemotherapy extends the overall survival beyond a year. Recurrence is, however, inevitable. GBM holds several unique features including its vast intratumoral heterogeneity, immunosuppressive environment, and a partially permissive anatomic blood–brain barrier, which offers a unique opportunity to investigate new treatment approaches. Tremendous efforts have been made in recent years to investigate novel CAR targets and target combinations with standard modalities for solid tumors and GBM to improve treatment efficacy. In this review, we outline the history of CAR immunotherapy development, relevant CAR target antigens validated with CAR T cells as well as preclinical approaches in combination with adjunct approaches via checkpoint inhibition, bispecific antibodies, and second-line systemic therapies that enhance anticancer efficacy of the CAR-based cancer immunotherapy.

Coronavirus disease of 2019 (COVID-19) pandemic, taking place globally, occurs as a result of the SARS-CoV-2 viral infection which has caused death of innumerable numbers of people and is responsible for a massive drop in the global economy. Millions of people are infected, and the death rate is also quite high in different countries. So, there is an urgent requirement of the invention of some effective and efficient drugs that can be effective against this deadly viral infection. The invention of new drugs and vaccine has become a matter of utmost importance to stop the mayhem of coronavirus pandemic. In the middle of such a deadly pandemic, the necessity of development of a vaccine is of high importance in this context. Among all the popular methods of vaccine development, the mRNA vaccines turned out to be the one of the most versatile vaccine with quick responses. However, in this review, we have explained all the possible types of vaccines available including DNA vaccines, RNA vaccines, and live and attenuated vaccines. Their effectiveness, importance, and application of the vaccines against the SARS-CoV-2 virus have been discussed. Research is also being conducted in the field of gene silencing, and one of the best possible ways to combat the virus at the molecular level is by applying RNAi technology. The modified siRNA molecules can be used to silence the gene expression of the virus. A summarization of the virus's behavior, characteristics, and the methods by which RNAi technology can be administered to control the virus is depicted in this study.

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Paratuberculosis, a contagious, untreatable, and chronic granulomatous enteritis that results in diarrhea, emaciation, and death in farmed ruminants (i.e., cattle, sheep, and goats). In this study, the Ag85B antigen from MAP was expressed in transgenic alfalfa as an attractive vaccine candidate. Agrobacterium-mediated transformation allowed the rescue of 56 putative transformed plants and transgenesis was confirmed in 19 lines by detection of the Ag85B gene (MAP1609c) by PCR. Line number 20 showed the highest Ag85B expression [840 ng Ag85B per gram of dry weight leaf tissue, 0.062% Total Soluble Protein (TSP)]. Antigenicity of the plant-made Ag85B was evidenced by its reactivity with a panel of sera from naturally MAP-infected animals, whereas immunogenicity was assessed in mice immunized by either oral or subcutaneous routes. The plant-made Ag85B antigen elicited humoral responses by the oral route when co-administered with cholera toxin as adjuvant; significant levels of anti-85B antibodies were induced in serum (IgG) and feces (IgA). Long-lasting immunity was evidenced at day 180 days post-first oral immunization. The obtained alfalfa lines expressing Ag85B constitute the first model of a plant-based vaccine targeting MAP. The initial immunogenicity assessment conducted in this study opens the path for a detailed characterization of the properties of this vaccine candidate. Supplementary Information The online version contains supplementary material available at 10.1007/s12033-021-00307-w.

Zika virus (ZIKV) is a positive-stranded RNA virus within the Flaviviridae family. After decades of circulation in Asia, ZIKV was introduced to Brazil in 2014–2015, associated with a rise in congenital malformations. Unlike the genetically related dengue virus (DENV), ZIKV constitutes only one serotype. Although assumed that ZIKV infection may engender lifelong immunity, the long-term kinetics of ZIKV antibody responses are unclear. We assessed long-term kinetics of ZIKV NS1-IgG response in 144 individuals from 3 different subpopulations: HIV patients, tuberculosis patients and healthy individuals first tested in 2016 and retested 1.5–2 years after the 2015–2016 ZIKV epidemic in Salvador de Bahia, Brazil, using a widely distributed NS1-based commercial ELISA. The seropositivity in 2016 reached 59.0% (85/144, 95% confidence interval (CI) 50.7–66.7%), and decreased to 38.6% (56/144, CI 31.3–47.0%) 1.5–2 years later. In addition, the median ZIKV NS1-ELISA reactivity for individuals that remained positive in both timepoints significantly decreased from a ratio of 4.4 (95% CI 3.8–5.0) to 1.6 (95% CI 1.6–1.9) over the 2-year interval (Z: − 6.1; p

Animal bioprosthetic heart valves (BHV) are used to replace defective valves in patients with valvular heart disease. Especially young BHV recipients may experience a structural valve deterioration caused by an immune reaction in which α-Gal and Neu5Gc are potential target antigens. The expression of these and other carbohydrate antigens in animal tissues used for production of BHV was explored. Protein lysates of porcine aortic and pulmonary valves, and porcine, bovine and equine pericardia were analyzed by Western blotting using anti-carbohydrate antibodies and lectins. N-glycans were released by PNGase F digestion and O-glycans by β-elimination. Released oligosaccharides were analyzed by liquid chromatography – tandem mass spectrometry. In total, 102 N-glycans and 40 O-glycans were identified in animal heart tissue lysates. The N- and O-glycan patterns were different between species. α-Gal and Neu5Gc were identified on both N- and O-linked glycans, N,N´-diacetyllactosamine (LacdiNAc) on N-glycans only and sulfated O-glycans. The relative amounts of α-Gal-containing N-glycans were higher in bovine compared to equine and porcine pericardia. In contrast to the restricted number of proteins carrying α-Gal and LacdiNAc, the distribution of proteins carrying Neu5Gc-determinants varied between species and between different tissues of the same species. Porcine pericardium carried the highest level of Neu5Gc-sialylated O-glycans, and bovine pericardium the highest level of Neu5Gc-sialylated N-glycans. The identified N- and O-linked glycans, some of which may be immunogenic and remain in BHVs manufactured for clinical use, could direct future genetic engineering to prevent glycan expression rendering the donor tissues less immunogenic in humans.

Immunohistochemistry and all techniques that use antibodies and fluorescence are widespread, essential and irreplaceable tools used in both research laboratory settings and diagnostic pathology laboratories. The field was born approximately 80 years ago, with the idea that antibodies could be tagged with fluorescent substances and used to detect antigens in cells and microorganisms, and has vertiginously evolved since; these advances have come in all aspects of the methodology, tissue fixation, generation of antibodies, monoclonal antibodies, signal amplification, antigen retrieval, signal amplification, microscopy and have become increasingly sophisticated, from in situ hybridization, in situ proximity ligation assay, flow cytometry, comet assay, to multiplexing and green fluorescent protein reconstitution, yielding Nobel Prizes along the way and generating invaluable scientific and diagnostic advances as well as timeless beautiful images.

Flow cytometry is a widely used diagnostic tool in many laboratories, which generates information that is essential for the diagnosis and classification of different hematolymphoid neoplasms (Reichard KK, KS, Flow cytometry in the assessment of hematologic disorders. In: Orazi A, Foucar K, Knowles DM et al (eds) Neoplastic hematopathology. Lippincott, Williams and Wilkins, Baltimore, MD, pp 119-145, 2013). Flow cytometry allows us to identify individual cells within heterogeneous populations. It is also useful for the quantification of cells, such as CD4 counts in HIV patients and CD34 stem cells on bone marrow and peripheral blood specimens. Lastly, it can also be used to describe the pattern of antigen expression on cells known as immunophenotyped (Craig FE, Foon KA, Blood 111(8):3941-3967, 2008).

Ticks are obligate hematophagous ectoparasites that infect domestic animals, humans, and wildlife. Ticks can transmit a wide range of pathogens (viruses, rickettsia, bacteria, parasites, etc.), and some of those are of zoonotic importance. Tick-borne diseases have a negative economic impact in several tropical and subtropical countries. With climate change, tick distribution and tick-associated pathogens have increased. Currently, tick control procedures have more environmental drawbacks and there are pitfalls in vaccination process. Since vaccinations have helped to prevent several diseases and infections, several vaccination trials are ongoing to control ticks and tick-borne pathogens. However, autoimmune reactions to vaccinations are reported as an adverse reaction since vaccines were used to protect against disease in humans and animals. The antibodies against the vaccine antigen might harm similar antigen in the host. Therefore, in this chapter, we attempt to shed light on the importance of raising awareness of possible adverse events associated with vaccinations and the methods that should be used to address this problem. In silico and lab work should be performed ahead of the vaccination process to evaluate the vaccine candidates and avoid the vaccination opposing consequences.

Vaccines are the most effective and economic way of combating poultry viruses. However, the use of traditional live-attenuated poultry vaccines has problems such as antigenic differences with the currently circulating strains of viruses and the risk of reversion to virulence. In veterinary medicine, reverse genetics is applied to solve these problems by developing genotype-matched vaccines, better attenuated and effective live vaccines, broad-spectrum vaccine vectors, bivalent vaccines, and genetically tagged recombinant vaccines that facilitate the serological differentiation of vaccinated animals from infected animals. In this chapter, we discuss reverse genetics as a tool for the development of recombinant vaccines against economically devastating poultry viruses.

Antibodies produced by plasma cells are a major arm of adaptive immunity. Germinal center reactions that include germinal center B cells and follicular T cells are fundamental players for antibody production, particularly antigen specific antibodies. Here we describe multiple methods that we and others have developed to analyze the production of antigen-specific antibodies in mice, with protocols for assessing antibody affinity and antibody isotype. The detection of antigen-specific IgE in serum using a traditional enzyme-linked immunosorbent assay (ELISA) method is often problematic due to much higher amounts of IgG. Thus we provide a specialized protocol for the detection of antigen-specific IgE in serum using ELISA.

Every T cell clone has its unique T cell receptor that results from somatic recombination of V(D)J genes in developing T cells. This process leads to a highly diverse TCR repertoire of naive T cells, which is selected, upon antigenic recognition, to form the repertoires of effector and memory T cells. The advent of next-generation sequencing (NGS) technology allows for the high-throughput analysis of the TCR repertoires in the different T cell populations. TFH cells, since their initial discovery in human tonsils and in mouse lymphoid organs, have become the subject of intense investigations due to their essential role in regulating B cell responses and the process of antibody affinity maturation. Circulating follicular helper T cells (cTFH) are considered a helper T cell linage in the blood that to some extent relates with bona fide TFH cells in the germinal centers of secondary lymphoid organs. Due to the limited access to the secondary lymphoid organs, cTFH have become a more accessible immunological readout. The assessment of the TCR repertoires of TFH and of cTFH cells is of both fundamental and clinical importance being instrumental to define the linage relationship of cTFH with other T cell subsets and to monitor response to infections or vaccination or disease states. In this chapter, we will provide detailed methods for isolation of antigen-specific cTFH cells in vitro and subsequent protocols for the high-throughput TCR sequencing, followed by repertoire data analysis.

Germinal centers (GCs) are microanatomical structures in secondary lymphoid organs where B cells undergo affinity maturation for antigen during the course of an immune response. This process is driven by a subset of T cells termed T follicular helper cells (Tfh) that through a multistep process gain access to the GC niche within the B cell follicle. This protocol details how to study Tfh behavior in vivo, on a single cell level, using two-photon intravital microscopy of the murine popliteal lymph node.

T Follicular helper (Tfh) cells stimulate, whereas T follicular regulatory (Tfr) cells inhibit, effector B cell responses. Although new tools have been developed to assess the functional roles of Tfh and Tfr cells in vivo, methods to assess mechanisms have been limited. One such limitation has been the ability of in vitro functional assays to recapitulate robust germinal center-like responses. Although previous in vitro Tfh-mediated and Tfr-suppressed assays to assess antibody regulation have been developed, these classically have relied on polyclonal stimulation. To understand Tfh and Tfr cell functionality, more robust assays that utilize specific antigen are needed. Here we describe an in vitro approach for sensitively and quantitatively assessing the capacity of Tfh and Tfr cells to regulate B cell responses in an antigen-driven system. These assays allow the study of Tfh and Tfr cells in specific disease contexts, such as IgG production after vaccination or IgE responses during allergic airway disease.

Humoral adaptive immune responses trigger the establishment of plasma B cells secreting antibodies of various isotypes that bind antigen specifically and with high affinity. Moreover, memory B cells will be generated. To accomplish this, B cells need assistance from a special subset of CD4 T cells, the so called follicular T cells that differentiate from naive T cells in the course of the immune response. Therefore, the study of follicular T cells is of primordial interest when investigating the molecular and cellular determinants of adaptive immune responses. This is done by direct analysis of the cells isolated from mice following an immunological challenge but in many instances such analyses must involve follow-up studies in cell culture requiring living cells. Especially, in vitro experimentation necessitates isolation and sorting of follicular T cells. However, follicular T cells are generally difficult to handle because they are prone to apoptosis and cell death. This is particularly evident when dealing with follicular T cells residing in the gut since we observed that isolation and processing from murine gut notoriously results in very high loss rates when compared for example to cells obtained from immunized peripheral lymph nodes. To bypass these limitations, we developed a protocol that allows for efficient isolation of intact follicular T cells. The protocol introduced here illustrates isolation and handling of follicular T cells using murine Peyer's Patches as an example because they constantly harbor significant amounts of these cells.

Nonclassical T cells are a heterogeneous group of T lymphocytes that are activated during the early stages of infection and act as a bridge between the innate and adaptive immune system. Among them, Natural Killer T (NKT) cells have been extensively studied in the last two decades due to their unique ability to recognize foreign/self-lipid antigens in the context of CD1d, a nonclassical major histocompatibility complex molecule. In this chapter, we describe our protocols to track murine NKT cells in lymph nodes by flow cytometry and confocal microscopy.

There is no vaccine available to prevent Neisseria gonorrhoeae infection, however there is currently a high level of interest in developing gonococcal vaccines due to the increasing number of cases and continuing emergence of antimicrobial resistance worldwide. A key aspect of vaccine development is the investigation of the functional immune response raised to the vaccine targets under investigation. Here, we describe two assays used to assess the functional immune response raised against gonococcal vaccine targets: the serum bactericidal assay (SBA) and the opsonophagocytic assay (OPA).

Vaccines induce a highly complex immune reaction in secondary lymphoid organs to generate immunological memory against an antigen or antigens of interest. Measurement of post immunization immune responses generated by specialized lymphocyte subsets requires time-dependent sampling, usually of the blood. Several T and B cell subsets are involved in the reaction, including CD4 and CD8 T cells, T follicular helper cells (Tfh), and germinal center B cells alongside their circulating (c) counterparts; cTfh and antibody secreting cells. Multicolor flow cytometry of peripheral blood mononuclear cells (PBMC) coupled with high-dimensional analysis offers an opportunity to study these cells in detail. Here we demonstrate a method by which such data can be generated and analysed using software that renders multidimensional data on a two dimensional map to identify rare vaccine-induced T and B cell subsets.

High-density protein microarray is an established technology for characterizing host antibody profiles against entire pathogen proteomes. As one of the highest throughput technologies for antigen discovery, proteome microarrays are a translational research tool for identification of vaccine candidates and biomarkers of susceptibility or protection from microbial challenge. The application has been expanded in recent years due to increased availability of bacterial genomic sequences for a broader range of species and strain diversity. Panproteome microarrays now allow for fine characterization of antibody specificity and cross-reactivity that may be relevant to vaccine design and biomarker discovery, as well as a fuller understanding of factors underlying themes of bacterial evolution and host-pathogen interactions. In this chapter, we present a workflow for design of panproteome microarrays and demonstrate statistical analysis of panproteomic human antibody responses to bacterial vaccination and challenge. Focus is particularly drawn to the bioinformatics and statistical tools and providing nontrivial, real examples that may help foster hypotheses and rational design of panproteomic studies.

Antigen selection is a critical step in subunit vaccine design, especially if the goal is to identify antigens that can be bound by neutralizing antibodies to prevent invasion of cells by intracellular bacteria. Here, we describe a method involving two dimensional gel electrophoresis (2-DE) coupled with western blotting (WB) and mass spectrometry (MS) to identify bacterial proteins that: (1) interact with the host target cell proteins, and (2) are targeted by antibodies from sera from infected animals. Subsequent steps would be performed to validate that the bacteria are targeted by neutralizing antibodies to prevent invasion of the eukaryotic cells.

Immunoprecipitation is an affinity purification technique that exploits the highly specific interactions formed between antibodies and their cognate antigens to purify molecules of interest from complex biological solutions. The generation of an effective humoral response provides protection against a wide range of gram-positive pathogens, and thus immunoprecipitation using antibodies purified from immune humans or animals provides a simple but effective means of isolating prospective vaccine antigens from fractionated bacterial cells for downstream identification. The commercial availability of antibody preparations from donated human plasma, containing antibodies against many common gram-positive pathogens, allows the protocol to be performed in the absence of bespoke vaccination experiments. Thus, immunoprecipitation has the potential to reduce the number of animals used in vaccine studies by allowing an initial screen for promising antigens to be conducted in vitro.

The bacterial cell surface (surfaceome) is the first site encountered by immune cells and is thus an important site for immune recognition. As such, the characterization of bacterial surface proteins can lead to the discovery of novel antigens for potential vaccine development. In this chapter, we describe a rapid 5-min surface shaving proteomics protocol where live bacterial cells are incubated with trypsin and surface peptides are "shaved" off. The shaved peptides are subsequently identified with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Several checkpoints, including colony forming unit (CFU) counts, flow cytometry, and a false positive unshaved control, are introduced to ensure cell viability/membrane integrity are maintained and that proteins identified are true surface proteins. The protein topology of shaved peptides can be bioinformatically confirmed for surface location. Surface shaving facilitates identification of surface proteins expressed under different conditions, by different strains as well as highly abundant essential and immunogenic bacterial surface antigens for potential vaccine development.

Outer membrane vesicles (OMV) represent a promising platform for the development of vaccines against bacterial pathogens. More recently, bacteria have been genetically modified to increase OMV yield and modulate the design of resulting particles, also named generalized modules for membrane antigens (GMMA). OMV/GMMA resemble the bacterial surface of the pathogen, where key antigens to elicit a protective immune response are and contain pathogen-associated molecular patterns (e.g., lipopolysaccharides, lipoproteins) conferring self-adjuvanticity. On the other hand, OMV/GMMA are quite complex molecules and a comprehensive panel of analytical methods is needed to ensure quality, consistency of manufacture and to follow their stability over time. Here, we describe several procedures that can be used for OMV/GMMA characterization as particles and for analysis of key antigens displayed on their surface.

Vaccines are regarded as the most cost-effective countermeasure against infectious diseases. One challenge often affecting vaccine development is antigenic diversity or pathogen heterogeneity. Different strains produce immunologically heterogeneous virulence factors, therefore an effective vaccine needs to induce broad-spectrum host immunity to provide cross-protection. Recent advances in genomics and proteomics, particularly computational biology and structural biology, establishes structural vaccinology and highlights the feasibility of developing effective and precision vaccines. Here, we introduce the epitope- and structure-based vaccinology platform multiepitope-fusion-antigen (MEFA), and provide instructions to generate polyvalent MEFA immunogens for vaccine development. Conceptually, MEFA combines epitope vaccinology and structural vaccinology to enable a protein immunogen to present heterogeneous antigenic domains (epitopes) and to induce broadly protective immunity against different virulence factors, strains or diseases. Methodologically, the MEFA platform first identifies a safe, structurally stable and strongly immunogenic backbone protein and immunodominant (ideally neutralizing or protective) epitopes from heterogeneous strains or virulence factors of interest. Then, assisted with protein modeling and molecule dynamic simulation, MEFA integrates heterogeneous epitopes into a backbone protein via epitope substitution for a polyvalent MEFA protein and mimics epitope native antigenicity. Finally, the MEFA protein is examined for broad immunogenicity in animal immunization, and assessed for potential application for multivalent vaccine development in preclinical studies.

Metal ion transporters in the outer membrane of gram-negative bacteria that are responsible for acquiring iron and zinc are attractive vaccine targets due to their essential function. The core function is mediated by an integral outer membrane TonB-dependent transporter (TBDT) that mediates the transport of the metal ion across the outer membrane. Some TBDTs also have a surface lipoprotein (SLP) that assists in the efficient capture of the metal ion-containing host protein from which the metal ion is extracted. The challenges in producing the integral outer membrane protein for a commercial subunit vaccine prompted us to develop a hybrid antigen strategy in which surface loops of the TBDT are displayed on the lipoprotein, which can readily be produced as a soluble protein. The focus of this chapter will be on the methods for production of hybrid antigens and evaluating the immune response they elicit.

Reverse vaccinology (RV) was first introduced by Rappuoli for the development of an effective vaccine against serogroup B Neisseria meningitidis (MenB). With the advances in next generation sequencing technologies, the amount of genomic data has risen exponentially. Since then, the RV approach has widely been used to discover potential vaccine protein targets by screening whole genome sequences of pathogens using a combination of sophisticated computational algorithms and bioinformatic tools. In contrast to conventional vaccine development strategies, RV offers a novel method to facilitate rapid vaccine design and reduces reliance on the traditional, relatively tedious, and labor-intensive approach based on Pasteur"s principles of isolating, inactivating, and injecting the causative agent of an infectious disease. Advances in biocomputational techniques have remarkably increased the significance for the rapid identification of the proteins that are secreted or expressed on the surface of pathogens. Immunogenic proteins which are able to induce the immune response in the hosts can be predicted based on the immune epitopes present within the protein sequence. To date, RV has successfully been applied to develop vaccines against a variety of infectious pathogens. In this chapter, we apply a pipeline of bioinformatic programs for identification of Shigella flexneri potential vaccine candidates as an illustration immunoinformatic tools available for RV.

Western blot processing is a well-established procedure that includes protein extraction from tissues and cells, gel electrophoresis separation, transfer to a membrane, and immunodetection with specific antibodies. Here, we show that optimization of washing helps to maximize the specific interactions of antigens and antibodies. Performing all washing steps at 4 °C ensures a maximal signal to noise ratio and reduces nonspecific signals.

A reliable and specific diagnosis is imperative in viral diagnosis, both for clinical management and surveillance, and to ensure that early treatment and control measures are carried out. The number of days of illness is important to choose the most appropriate method to be used and for the correct interpretation of the results obtained. Specific IgM is elicited after that period, indicating an active infection and usually lasts up to 3 months. However, in DENV secondary infections, IgM levels may be significantly lower or undetectable. After 10-12 days, a lifetime specific IgG is produced. Routinely, the laboratory diagnosis of DENV infections can be performed by viral isolation and/or detection of viral nucleic acid, serological assays for the detection of specific antibodies (IgM/IgG), antigen (NS1) and the detection of viral antigens in tissues, which are suitable during certain phases of the disease. For serological diagnosis, serum, plasma, or cerebrospinal fluid (CSF) samples may be investigated. If the test is carried out a few days after collection, the specimens can be stored at 4 °C, since the immunoglobulins are stable in serum or plasma. If the storage period is extended, the material must be kept at -20 °C or -70 °C. In serology, several methods can be used to detect specific viral antigens and/or antibodies, produced by the host in response to DENV infection. Routinely, serological tests include the hemagglutination inhibition (HI) assay, the plaque reduction neutralizing test (PRNT), the gold standard assay for dengue immune response characterization, and ELISAs to detect IgM (MAC-ELISA) and IgG (IgG-ELISA).

Despite many advances on the understanding of dengue pathogenesis in the last decades, some questions remained to be clarified. The virulence of the pathogen and the host immune response are the main factors involved in pathogenesis of dengue infection. In addition, skin dendritic cells (DCs) are one of the primary targets for dengue virus infection. After infection, DCs process and present antigens to T cells and also secrete cytokines that shape the immune response. Although relevant for the development of antiviral immune response, an imbalance in the cytokine production by immune cells could lead to cytokine storm observed in severe dengue fever cases. Therefore, this chapter will describe the protocols for the in vitro differentiation of human monocytes into human monocyte-derived dendritic cells (mdDCs), followed by dengue virus infection, as well as the cytokine quantification produced by mdDCs using a cytometric bead array method.

Immunohistochemistry (IHC) is a combination of immunological, biochemical, and pathological methods to visualize the presence and distribution of specific epitopes in tissue sections. Selected antigens are stained by differently labeled antibodies binding to their target antigens in situ. Here we describe sample preparation and sample staining in order to diagnose and analyze tissue samples infected with M. ulcerans from human as well as animal source.