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Literature reports the association between aging and decline in the immune system function. The elderly have a higher risk of developing infectious diseases and are often less responsive to vaccines that are effective in the young. The case fatality rate of invasive meningococcal disease is higher in the elderly; therefore, vaccination for this population should be evaluated. Although new vaccines have been developed against Neisseria meningitidis, there is still a need to evaluate a vaccine for those older than 60 years, as the currently licensed vaccines are not indicated for this population. [ABSTRACT FROM AUTHOR]

Highlights • Individual variability in humoral response was evaluated after immunization with OMVs from N. meningitidis strain B:4:P1.9. • Cross-reactivity with meningococcal clinical strains prevalent in Brazil in 2014–2016 were analyzed by Dot-blot ELISA. • Animals presented different humoral responses after immunization. • Antibodies elicited by vaccination cross-reacted with strains B:4,7:P1.19,15, B:19:nt, W:2a:P1.5,2, Y:22:nt and Y:4:nt. [ABSTRACT FROM AUTHOR]

Neisseria meningitidis bacterium is a Gram-negative diplococcus. Among their serogroups, the B is one of the main causes of invasive meningococcal disease. Newborns and children are particularly susceptible to this infection because of their immune systems that are still maturing and relatively inexperienced. Thus, further studies on the use of maternal immunization for protection against this disease are needed. The purpose of this study was to evaluate the potential immunogenic antigens from the outer membrane of N. meningitidis serogroup B in outbred mice and the influence of maternal immunization in the offspring, and analyze the adjuvant effect of bilayer fragments of dioctadecyldimethylamonium bromide (DODAB-BF) and hydroxide aluminium (alum) in enhancing antibodies production and transference to offspring. IgG and IgG1, IgG2a and IgG2b subclasses of antibodies in serum from immunized mice and controls were quantified and compared. Immunization by subcutaneous and intramuscular routes exhibited evidence of IgG, and both adjuvants promoted the production of IgG1 and IgG2b that were transferred to the offspring. These antibodies also showed specificity with the outer membrane vesicles from homologous strain and were capable to cross react with different strains. The use of DODAB-BF seems to enhance immune response on mothers and offspring and may have immunological advantages. [ABSTRACT FROM AUTHOR]

Neisseria lactamica, a commensal bacterium that is non-pathogenic to humans and is usually found in the upper respiratory tract of children, is closely related to the pathogenic species Neisseria meningitidis. Colonization by Neisseria lactamica can be responsible for the development of natural immunity to meningococcal infection in childhood, when rates of meningococcal carriers are low. These features suggest that N. lactamica components can be key elements in the production of a new vaccine for N. meningitidis. The production of monoclonal antibodies for N. lactamica is an important tool in the selection of new antigens for the preparation of a vaccine for N. meningitidis B. [ABSTRACT FROM AUTHOR]

Vaccines are the most effective tool to control infectious diseases, which provoke significant morbidity and mortality rates. Most vaccines are administered through the parenteral route and can elicit a robust systemic humoral response, but they induce a weak T‐cell‐mediated immunity and are poor inducers of mucosal protection. Considering that most pathogens enter the body through mucosal surfaces, a vaccine that elicits protection in the first site of contact between the host and the pathogen is promising. However, despite the advantages of mucosal vaccines as good options to confer protection on the mucosal surface, only a few mucosal vaccines are currently approved. In this review, we discuss the impact of vaccine administration in different mucosal surfaces; how appropriate adjuvants enhance the induction of protective mucosal immunity and other factors that can influence the mucosal immune response to vaccines. [ABSTRACT FROM AUTHOR]

The Enzyme-Linked Immunosorbent Assay is a versatile technique, which can be used for several applications. It has enormously contributed to the study of infectious diseases. This review highlights how this methodology supported the science conducted in COVID-19 pandemics, allowing scientists to better understand the immune response against SARS-CoV-2. ELISA can be modified to assess the functionality of antibodies, as avidity and neutralization, respectively by the standardization of avidity-ELISA and surrogate-neutralization methods. Cellular immunity can also be studied using this assay. Products secreted by cells, like proteins and cytokines, can be studied by ELISA or its derivative Enzyme-linked immunospot (ELISpot) assay. ELISA and ELISA-based methods aided the area of immunology against infectious diseases and is still relevant, for example, as a promising approach to study the differences between natural and vaccine-induced immune responses against SARS-CoV-2. [ABSTRACT FROM AUTHOR]

Adjuvants are important components of vaccines, increasing immunogenicity and modulating the immune response. SARS-CoV-2 vaccines are still being developed in order to improve worldwide access to immunization. Specific populations should be addressed in these investigations, such as pregnant women—to protect both mothers and neonates. In this study, female adult mice were immunized with Receptor-binding domain (RBD) from SARS-CoV-2 adjuvanted by a mixture of DDA and Saponin and put to mating to verify the maternal transference of IgG. For comparison, other group received RBD adjuvanted by OMVs from Neisseria meningitidis and Alum. The adjuvants enhanced IgG production and neutralization. DDA/Sap contributed to increase IgG1, IgG2a, IgG2b, and IgG3 isotypes. Total IgG avidity was considered high, as well as IgG1, IgG2a, and IgG2b avidity. IgG antibodies were effectively transferred to the offspring, predominantly IgG2a, IgG2b, and IgG3. The passive transferred immunoglobulin maintained the neutralizing ability, although it lost avidity. ELISA data was confirmed in Dot-ELISA and immunoblotting assays. DDA and Saponin seem a promising adjuvant mixture to enhance the humoral response of SARS-CoV-2 antigens. Further studies considering the effects of maternal immunization in the protection of offspring are needed, regardless the platform used in COVID-19 vaccines. [ABSTRACT FROM AUTHOR]

Highlights • We evaluated the OMVs of N. meningitidis B complexed with dioctadecyldimethylammonium bromide (DODAB-BF) and aluminum hydroxide. • We used prime-boost immunization for the first time, comparing the evaluation of intranasal and subcutaneous routes in neonatal mice. • The immunization routes used produced IgG, IgG1, and IgG2a antibodies. • The ELISpot assay was used to determine the cytokines INF-γ- and IL-4. Abstract Background The Neisseria meningitidis bacterium is a Gram-negative diplococcus that can be classified into different serogroups according to the capsular structure. Six of them (A, B, C, W, X, Y) are responsible for causing Invasive Meningococcal Disease (IMD). The strategies for the development of a vaccine for serogroup B have been directed to the use of outer membrane vesicles (OMVs). The aim of this study was to evaluate the immunogenicity of antigenic determinants from OMVs of N. meningitidis B complexed with two different adjuvants: DODAB-BF and aluminum hydroxide (alum), comparing the evaluation of intranasal and subcutaneous route of immunization. Methods We used prime-boost immunization for the first time in outbred neonatal mice evaluating the cellular and humoral immune response. Results Immunoblot, ELISA DOT-ELISA and ELISpot were used universal methods of antibody detection, in order to detect the humoral and cellular immune response in male and female mice. Immunoblot analyzes the specificity of antibodies with the homologous N. meningitidis strain. ELISA served to quantify and compare the titers of antibodies in the serum of mice immunized with DODAB-BF + OMVs and alum + OMVs for IgG, IgG1, and IgG2a. Intranasal immunization produced a mixed response in the T helper cells Th1 and Th2, while subcutaneous immunization exhibited a Th1 profile. The DOT-ELISA identified cross-reactivity with DODAB-BF to different serogroups of N. meningitidis (B, C, W, and Y) that was not observed with alum. ELISpot analyzed IFN-γ- and IL-4 and the results showed the response directly to Th1 and Th2 profile. Conclusion Our findings indicate that DODAB-BF can be an alternative adjuvant for mucosal cell activation with OMVs of N. meningitidis B and that DODAB-BF was similar to aluminum hydroxide as an adjuvant for subcutaneous immunization. [ABSTRACT FROM AUTHOR]

The article highlights a study on the preparation of monoclonal antibodies (Mab) against Neisseria (N.) meningitidis B. The native outer membrane complex (NOMC) of N. meningitidis B was used in a mice for immunization. The antibodies produced were FL24(PL)Br, a new serotype 24 class 3 antigen of nontypeable serogroup B strain and FL14(PL)Br, which was specific for the serotype 14 and reacted with the S3446 reference strain of N. meningitidis.

The article provides information on the production of the monoclonal antibody (MAb) 1D41D8 that fights 35 kilodaltons lipoprotein of Mycoplasma penetrans. Lipid-associated membrane proteins (LAMPs) were used as antigen for immunization of a mice. It cites the selection of hybridoma cell lines through the enzyme-linked immunosorbent assay (ELISA). It indicates that such monoclonal antibody recognized 35 kilodalton LAMPs.

Immunization is the key to prevent invasive meningococcal disease (IMD), caused by Neisseria meningitidis. Outer membrane vesicles (OMVs) can be used as meningococcal antigens. Isogenic mice A/Sn (H2a) were immunized with low antigenic doses of OMVs of an N. meningitidis C:2a:P1.5 strain, via intranasal/intramuscular route, adjuvanted by cholera toxin subunit B (CTB) or via intramuscular route only, adjuvanted by aluminium hydroxide (AH). Mice were followed until old age and humoral and cellular responses were assessed by ELISA, Immunoblotting, Dot-blot, Serum-bactericidal assay, Immunohistochemistry and ELISpot. OMV+CTB and OMV+AH groups presented statistically higher antibodies titers, which persisted until middle and old ages. IgG isotypes point to a Th2 type of response. Avidity indexes were considered high, regardless of adjuvant use, but only groups immunized with OMVs and adjuvants (OMV+CTB and OMV+AH) presented bactericidal activity. The antibodies recognized antigens of molecular weights attributed to porin and cross-reactivity proteins. Although the spleen of old mice did not present differences in immunohistochemistry marking of CD68+, CD4+, CD79+ and CD25+ cells, splenocytes of immune groups secreted IL-4 and IL-17 when stimulated with OMVs and meningococcal C polysaccharide. We concluded that both adjuvants, CTB and AH, improved the immunogenicity of low doses of OMVs and contributed to a persistent immune response. Even though AH is well established in the vaccinology area, CTB seems to be a promising adjuvant candidate for meningococcal vaccines: it is suitable for mucosal delivery and supports a Th2 type of response. Therefore, OMVs are still a relevant vaccine platform. [ABSTRACT FROM AUTHOR]

Highlights: [•] We establish a new experimental model to study T. crassiceps infection in mice. [•] This model was compared to the previously established intraperitoneal infection model. [•] The air pouch provides a convenient cavity that allows studying T. crassiceps parasite. [Copyright &y& Elsevier]

SARS-CoV-2 is considered a global emergency, resulting in an exacerbated crisis in the health public in the world. Although there are advances in vaccine development, it is still limited for many countries. On the other hand, an immunological response that mediates protective immunity or indicates that predict disease outcome in SARS-CoV-2 infection remains undefined. This work aimed to assess the antibody levels, avidity, and subclasses of IgG to RBD protein, in symptomatic patients with severe and mild forms of COVID-19 in Brazil using an adapted in-house RBD-IgG ELISA. The RBD IgG-ELISA showed 100% of specificity and 94.3% of sensibility on detecting antibodies in the sera of hospitalized patients. Patients who presented severe COVID-19 had higher anti-RBD IgG levels compared to patients with mild disease. Additionally, most patients analyzed displayed low antibody avidity, with 64.4% of the samples of patients who recovered from the disease and 84.6% of those who died in this avidity range. Our data also reveals an increase of IgG1 and IgG3 levels since the 8th day after symptoms onset, while IgG4 levels maintained less detectable during the study period. Surprisingly, patients who died during 8–14 and 15–21 days also showed higher anti-RBD IgG4 levels in comparison with the recovered (P < 0.05), suggesting that some life-threatening patients can elicit IgG4 to RBD antibody response in the first weeks of symptoms onset. Our findings constitute the effort to clarify IgG antibodies' kinetics, avidity, and subclasses against SARS-CoV-2 RBD in symptomatic patients with COVID-19 in Brazil, highlighting the importance of IgG antibody avidity in association with IgG4 detection as tool laboratory in the follow-up of hospitalized patients with more significant potential for life-threatening. [ABSTRACT FROM AUTHOR]

Purpose: In the present study, meningococcal serogroup B outer membrane vesicles (OMVs) were associated with bilayer fragments of a cationic lipid, dioctadecyldimethylammonium (DDA-BF), used as adjuvant, in an antigenic preparation tested in adult female outbred mice. This adjuvant was compared to the traditional adjuvant aluminum hydroxide. Materials and Methods: The potential in generating humoral response was evaluated by enzyme-linked immunosorbent assay (ELISA). Individual serum was collected and immunoglobulin G (IgG), IgG1, IgG2a, and IgG2b were quantified. Analyses were carried out 15 and 60 days after immunization. Antibodies avidity index were also analyzed by ELISA. Immunoblot and dot-ELISA were carried out to evaluate specific reaction for homologous strains and crossreactive antigens present in other meningococcal strains isolated in 2011-2012 year, in Brazil. Delayed type hypersensitivity was used as indicative of cellular immunity and compared between two experimental groups, 24 hours after homologous strain challenge. Results: The OMVs of Neisseria meningitidis, and N. lactamica (related species) were characterized by electrophoretic separation of proteins in 13% polyacrylamide gel. The strains presented antigens in the range of 8 to 130 kDa, showing a heterogeneous protein migration pattern. In the group immunized with OMVs/DDA-BF, we found no significant production of total IgG 15 days after the first immunization. On the other hand, 60 days after first immunization both adjuvants act benefiting total IgG production similarly. The antibodies of the IgG isotype produced by animals immunized after one or two doses after first immunization, showed intermediate and high avidity, independent on the adjuvant used. In both experimental groups the swelling of the footpads was significantly higher than those of the controls, suggesting that only one dose was enough to stimulate the generation of cellular immunity. Conclusion: The use of this cationic adjuvant for N. meningitidis OMVs preparation revealed good potential for future new antigen preparation for N. meningitidis vaccine. [ABSTRACT FROM AUTHOR]

Outer membrane vesicles (OMVs) of Neisseria meningitidis contain important antigens to trigger an immune response against meningococci and have been studied as vaccines compounds. The immune response to a vaccine may be affected by its constitution and route of administration. Therefore, Swiss mice were immunized by different routes with OMVs of N. meningitidis B with dimethyl dioctadecyl ammonium bromide in bilayer fragments (DDA-BF) or aluminum hydroxide (AH) as adjuvants. The adjuvants and different routes were compared regarding the immune responses by ELISA, western blot, delayed type hypersensitivity (DTH) and histopathologic analysis. The antigenic preparation generated humoral and cellular immune responses. In quantitative analyzes, in general, AH was superior to DDA-BF. However, analysis such as IgG avidity index, bactericidal activity and immunoblot, revealed no important differences regarding the adjuvant or route of immunization. Regarding the parameters tested, it was not possible to define a superiority between the adjuvants and routes of immunization proposed by this study. [ABSTRACT FROM AUTHOR]

The elderly are more likely to die when infected with Neisseria meningitidis. Aging is associated with immune system dysfunctions that impair responses to vaccines and infections. Therefore, immunization of middle-aged individuals could be beneficial. This study aims to evaluate the immunogenicity of N. meningitidis B outer membrane vesicles (OMVs) complexed to two different adjuvants. Middle-aged BALB/c and A/Sn mice were immunized and subsequent immune response was assessed by ELISA, immunoblotting and ELISpot. IgG levels were similar between the animals immunized with OMVs complexed to adjuvants. A total of 235 days after the last immunization only A/Sn mice presented higher IgG levels than those observed in the baseline, especially the group immunized with OMVs and aluminum hydroxide. The predominant IgG subclasses were IgG2a and IgG2b. Immunization with the three-dose regimen generated IgG antibodies that recognized a variety of antigens present in the homologous and heterologous meningococcal OMVs evaluated. There was an increase in the frequency of antigen-specific IFN-γ secreting splenocytes, after in vitro stimulation, in mice immunized with OMVs and adjuvants compared to the control group, almost 1 year after the last immunization. Both adjuvants showed similar performance. Immunization of middle-aged mice has generated a robust immune response and it appears to be advantageous. [ABSTRACT FROM AUTHOR]

The avidity index (AI) measures the binding strength between the antibody and the antigen, reflecting the affinity maturation. It can be measured by a modified ELISA, adding a chaotropic agent to disrupt the antigen x antibody interaction. However, details of the protocols used affect the final results. We compared the AI of mice sera after a three-dose immunization with meningococcal antigens using different adjuvants. The AI was assessed using potassium thiocyanate (KSCN) and urea as chaotropic agents, incubated at 4 °C, room temperature (RT) and 37 °C. KSCN presented statistically different results when the incubation was set at 4 °C vs RT and 4 °C vs 37 °C, thus, the mean AI obtained were lower. For Urea, 4 °C vs 37 °C presented relevant differences. Using whole-cells suspensions or OMVs as coating antigen provided similar results in some protocols. Thus, the affinity maturation was assessed after each immunization dose and adjuvant use (aluminium hydroxide and dimethyldioctadecylammonium bromide) supported affinity maturation. It is important to study the AI as a functional parameter of humoral response, and both KSCN and Urea are suitable chaotropic agents, however, the protocols should be standardized considering the nature of the antigen, the chaotropic activity and overall laboratory conditions. Adjuvants are important tools to improve antibody avidity following immunization. • Incubation of KSCN or Urea at 4 °C or 37 °C • Correlation of OMVs and WCs were better using KSCN 4 °C/RT or Urea 37 °C. • Affinity maturation curves were similar with different protocols. • Adjuvants improved avidity after immunization in middle-aged mice. [ABSTRACT FROM AUTHOR]

Introduction: In Brazil, various isolates of rabies virus (RABV) show antigenic profiles distinct from those established by the reduced panel of eight monoclonal antibodies (MAbs) determined by the Centers for Disease Control and Prevention (CDC), utilized for the antigenic characterization of RABV in the Americas. The objective of this study was to produce MAbs from RABV isolates from insectivorous bats with an antigenic profile incompatible with the pre-established one. Methodology: An isolate of RABV from the species Eptesicus furinalis that showed an antigenic profile incompatible with the panel utilized was selected. Hybridomas were produced utilizing the popliteal lymph nodes of mice immunized with ribonucleoproteins purified from the isolate. Results: Two MAbs-producing clones were obtained, BR/IP1-3A7 and BR/IP2-4E10. Fifty-seven isolates of RABV from different species of animals and different regions of Brazil were analyzed utilizing the MAbs obtained. In the analysis of 23 RABV isolates from nonhematophagous bats, the MAbs cross-reacted with ten isolates, of which four were of the species Nyctinomops laticaudatus, one of the species Eptesicus furinalis, and five of the genus Artibeus. Of the nine isolates of non-hematophagous isolates that displayed an incompatible profile analyzed, characteristic of insectivorous bats, BR/IP1-3A7 reacted with five (55.55%) and BR/IP2-4E10 with four (44.44%). Conclusions: The MAbs obtained were able to recognize epitopes common between the three genera, Artibeus, Eptesicus, and Nyctinomops, thereby allowing the antigenic characterization of RABV isolates in Brazil. [ABSTRACT FROM AUTHOR]

Abstract: The aim of the present study was to investigate the immune response to native outer membrane vesicles (NOMVs) of Neisseria lactamica with and without Bordetella pertussis (BP) as adjuvant in intranasal (i.n./i.m) immunization. N. lactamica NOMVs delivered intranasally (i.n) to BALB/c mice in a final volume of 5μl that was gradually introduced with a micropipette, Animals received 1, 2, 3, or 4 doses of antigens at 3, 7, 9 and 12 days after birth. On the 35th day, the animals were immunized intramuscularly (i.m.) with (NOMV) of N. lactamica. The prime-booster strategy using NOMV of N. lactamica with BP as adjuvant in the primer (i.n.) and booster (i.m.) is an effective immunization protocol for inducing humoral immune responses producing IgG antibodies of intermediate to high avidity. [Copyright &y& Elsevier]

Background: A simple filter paper method was developed for, the transport and storage of monoclonal antibodies (Mabs) at room temperature or -20°C after spotting on filter paper, for subsequent sero-typing of outer membrane antigens of N.meningitidis by dot-blot ELISA. Methods: Monoclonal antibodies (Mabs) were spotted within a 0.5-1 cm diameter area of Whatman grade 903 paper, which were stored individually at room temperature or at -20°C. These MAbs were stored and analyzed after periods of one week, 4 weeks, 12 months, or 13 years in the case of frozen Mab aliquots, or after 4 weeks at -20°C or at room temperature (RT) in the case of Mabs dried on filter paper strips. Assays were performed in parallel using dot-blot ELISA. In addition to the MAbs specific for serotyping class 1, 2 or 3, we used a larger number of Mabs for polysaccharides, lipooligosaccharides (LOS), class 5 and cross-reactive antigens for native outer membrane of N.meningitidis. The Mabs dried on filter paper were eluted with phosphate-buffered saline (PBS) containing 0.2% gelatin. Results: Mabs of the isotypes IgG and IgM dried on filter papers were not affected by duration of storage. The detection by serotyping Mabs was generally consistent for dried filter paper MAb samples stored frozen for over 1 year at -20°C, and although decreased reactive antibody titers were found after storage, this did not interfere with the specificity of the Mabs used after 13 years as dry spots on filter paper. Conclusion: The use of filter paper is an inexpensive and convenient method for collecting, storing, and transporting Mab samples for serotyping studies. In addition, the samples occupy little space and can be readily transported without freezing. The efficiency of using immunoglobulin G (IgG) or M (IgM) eluted was found to be consistent with measurement of IgG or IgM titers in most corresponding, ascites Mabs stored frozen for over 1 year. The application of meningococcal typing methods and designations depend on the question being asked. [ABSTRACT FROM AUTHOR]

A rapid and efficient method for preparing monoclonal antibody (MAb) serotypes using Neisseria meningitidis outer membrane were used in BALB/c mouse footpads for the immunization. The popliteal lymph nodes were isolated 19 days later for MAb-producing hybridomas, from which the MAbs against the 37 kDa protein were screened. Variations in class 2/3 (PorB) proteins form the basis for meningococcal serotyping. This is the first report on the preparation of MAbs against N. meningitidis that is specific to PorB protein using popliteal lymph nodes. The new monoclonal antibodies were specific for PorB outer membrane protein FL24(PL)Br, a new serotype 24 class 3 antigens of non-typeable (NT:NST) serogroup B strain, and FL14(PL)Br specific for the serotype 14, and reacted with the S3446 reference strain analyzed. A total of 12% of the case isolates reacted with one or more of the monoclonal antibodies. The high-affinity MAbs produced by hybridoma methodology provide a basis for further research on the pathogenesis and early diagnosis of meningococcus. [ABSTRACT FROM AUTHOR]

To develop a rapid and efficient method for preparing monoclonal antibodies (MAb) against 35 kDa lipoprotein of Mycoplasma penetrans, BALB/c mice were injected into the footpads for immunization, and the popliteal lymph nodes were isolated 19 days later for MAb-producing hybridomas, from which the mAbs against the 35 kDa lipoprotein were screened. The identification of the mAb against the 35 kDa lipoprotein was performed using indirect enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Using popliteal lymph node procedures, we generated several positive clones, one of which we characterized by ELISA and immunoblot. The clone 1D41B8 was identified as the immunoglobulin G1 (IgG1) isotype, kappa chain with affinity constants (Ka) of 2.95 x 109M-1. The MAbs did not cross-react with a number of control bacteria, which included Mycoplasma fermentans, Mycoplasma hominis, and Mycoplasma genitalium. This is the first report on the preparation of mAbs against M. penetrans that is specific to 35 kDa lipoprotein using popliteal lymph nodes. The high-specificity and high-affinity MAbs produced by two methodologies used of hybridomas provide a basis for further research on the pathogenesis and early diagnosis of M. penetrans. This simple approach may become a method of choice for the generation and production of MAbs in a short period of time. [ABSTRACT FROM AUTHOR]