Search Results (26)

The measurement of serum antibodies that specifically recognize self-antigens is a critical diagnostic in autoimmunity. A limitation of such an approach is sensitivity to detect the antibody, particularly when abundant self-antigens in the body may bind and sequester circulating specific antibodies. The presence of specific memory B cells (B_mem) may provide a more sensitive and robust indicator of an autoimmune response, as is suggested for certain anti-viral responses. B cell enzyme-linked ImmunoSpot (ELISPOT) is capable of detecting antigen-specific B_mem cells in blood at the single cell level, following stimulation of peripheral blood mononuclear cells (PBMCs) to expand and differentiate the B_mem cells into functional antibody-secreting cells (ASCs). While this assay has been widely utilized in infectious diseases and vaccination, detection is more difficult for autoantigens due to self-tolerance and specific tissue compartmentalization of immune responses, making autoantigen-specific B cells rare in the circulation. The cycles of re-activation of B_mem cells to become ASCs, that may reflect disease flare-ups in autoimmunity, are not well defined. For several autoimmune diseases (ADs), the targeting of B cells via depleting monoclonal antibodies has proven to be an effective treatment, where B_mem cells are likely being targeted. The measurement of autoantigen-reactive B_mem cells may aid in diagnosis and staging of clinical severity, or be a metric for efficacious treatments, thus providing an additional informative biomarker of ADs. How B cell ELISPOT has been utilized to characterize B_mem cells in human ADs is described here, including the advantages and disadvantages of the assay. Full article

Background: Tuberculosis (TB) is a respiratory infectious disease, and the current TB vaccine has low local lung protection. We aim to optimize immune pathways to improve the immunogenicity of vaccines. Methods: In the immunogenicity study, 50 BALB/c mice were randomly divided into the following: (1) phosphate buffered saline (PBS)+intramuscular injection combined with electroporation (EP) group (100 μL), (2) pVAX1+EP group (50 μg/100 μL), (3) ag85ab+EP group (50 μg/100 μL), (4) pVAX1+pulmonary delivery (PD) group (50 μg/50 μL), and (5) ag85ab+PD group (50 μg/50 μL). Immunization was given once every 2 weeks for a total of three times. The number of IFN-γ-secreting lung and spleen lymphocytes was determined by enzyme-linked immunospot assay (ELISPOT). The levels of Th1, Th2, and Th17 cytokines in the culture supernatants of lung and spleen lymphocytes were detected with the Luminex method. The proportion of FoxP3 regulatory T cells in splenocytes was determined by flow cytometry. The levels of IgG-, IgG1-, and IgG2a-specific antibodies in plasma and IgA antibody in bronchoalveolar lavage fluid (BALF) were determined by enzyme-linked immunosorbent assay (ELISA). Results: The PD and EP routes of Mycobacterium tuberculosis (M. tb) ag85ab DNA vaccine can effectively induce the responses of IFN-γ-secreting lung and spleen lymphocytes, and induce dominant Th1 and Th17 cell immune responses. The PD route can induce earlier, greater numbers and stronger responses of pulmonary effector T cells, with higher levels of the specific antibody IgA detected in BALF. High levels of the specific antibodies IgG, IgG1, and IgG2α were detected in the plasma of mice immunized by the EP route. Conclusions: The PD route of DNA vaccines can more effectively stimulate the body to produce strong cellular and mucosal immunity than the EP route, especially local cellular immunity in the lungs, which can provide early protection for the lungs. It can significantly improve the immunogenicity of the ag85ab DNA vaccine, suggesting a feasible and effective approach to DNA immunization. Full article

We previously reported on HIV vaccines that elicited autologous Tier 2 neutralizing antibodies (nAbs) in rabbits. In the current study, we sought to establish a proof of concept that HIV vaccines using identical designs elicit Tier 2 nAbs in arhesus macaque (RM) model. DNA and MVA vaccines expressing SIV Gag and HIV-1 Env antigens were constructed, and in vitro expression was confirmed. A soluble envelope protein (gp140 Env) was expressed from a stable HEK293 cell line and purified using lectin affinity and size exclusion chromatography. The expression and secretion of SIV Gag and HIV-1 Env by the DNA and MVA vaccines was verified in vitro. Five RMs were inoculated with two DNA, followed by two MVA, and finally with two gp140 Env vaccines at weeks 0, 4, 8, 12, 20 and 28. Vaccine-induced T cell immunity was measured by IFN-γ ELISpot while nAbs were evaluated against MW965 (Tier 1A), 6644 (Tier 1B), autologous ZM109.5A and a closely-related ZM109.B4 (Tier 2) pseudovirions. Vaccinated RMs were challenged intrarectally with simian-human immunodeficiency virus (SHIV), four weeks after the final vaccination, as was an unvaccinated control group (n = 4). Following vaccination, all the animals developed moderate IFN-γ ELISpot responses after the DNA vaccinations which were boosted by the MVA vaccine. After the gp140 Env boost, all animals developed nAbs with peak median titres at 762 (MW965) and 263 (ZM109.5A). The vaccinated animals became infected after a similar number of challenges to the unvaccinated controls, and the resultant number of viral copies in the blood and the lymphoid tissues were similar. However, the duration of detectable viraemia in the vaccinated animals (median: 2 weeks) was shorter than the controls (median: 8.5 weeks). These data show that the vaccines elicited robust cellular and functional humoral immune responses that resulted in a quicker control of viraemia. Full article

Determining an individual’s humoral immune reactivity to a pathogen, autoantigen, or environmental agent is traditionally accomplished through the assessment of specific antibody levels in blood. However, in many instances, titers of specific antibodies decline over time and thus do not faithfully reveal prior antigen exposure or establishment of immunological memory. To estimate an individual’s humoral immune competence, it is therefore necessary to assess functional B cell memory. Here, we describe novel B cell ELISPOT and FluoroSpot assays (collectively referred to as ImmunoSpot) that can be rapidly developed and validated to characterize the memory B cell (B_mem) repertoire specific for any desired antigen ex vivo and at single-cell resolution. Moreover, multiplexed variants of the B cell FluoroSpot assay enable high-throughput testing of antigen-specific B cells secreting distinct antibody classes and/or IgG subclasses, with minimal cell material requirements. B cell ImmunoSpot assays also enable measurement of affinity distributions within the antigen-specific B_mem compartment and permit cross-reactivity measurements that can provide insights into B_mem established against future pathogen variants. Collectively, the ImmunoSpot^® system presented here is highly reproducible, and can be readily validated for regulated tests. The newly gained ability to monitor the antigen-specific B_mem compartment should catalyze a more comprehensive understanding of humoral immunity in health and disease. Full article

Background/Objectives: Understanding the behavior of B cells during infection and vaccination is important for determining protective humoral immunity. We evaluated the profile of humoral immunity and B cell pool in individuals who were acutely infected with SARS-CoV-2, recovered from COVID-19, or received two doses of the AZD1222 vaccine. Methods: Peripheral blood mononuclear cells (PBMCs) from these individuals were subjected to in vitro stimulation to promote the differentiation of B cells into antibody-secreting cells (ASCs), and the ELISpot evaluated the abundance of pan and SARS-CoV-2 Spike S1-reactive IgG+ ASC. Stimulated PBMCs were characterized using flow cytometry. Culture supernatants were assessed for soluble B-cell-activating factors. The IgA and IgG for the S1 were evaluated through ELISA. Results: The recovered individuals displayed a robust S1 ASC compared to acute and vaccinated individuals. Although the frequency of total B cells or B cell subsets did not vary among the groups, plasmablast cells were increased in naïve and double-negative B cells in the acute, recovered, and vaccinated individuals. Similar IgA and IgG production appeared to be present in the acute and recovered individuals. During vaccination, more IgG is produced than IgA. In acute patients, BAFF levels were positively correlated with total B cells and IgG+ plasmablast cells but negatively correlated with IgA+ plasmablast cells. Conclusions: Vaccination and natural infection with COVID-19 induce a differential profile and functionality of B cells. We suggest that new vaccines against COVID-19 incorporate molecular adjuvants that regulate B lymphocyte functionality and consider the beneficial aspects of the IgA response in addition to IgG. Full article

Backgrounds: A contemporary public health challenge is the increase in the prevalence rates of herpes zoster (HZ) worldwide. Methods: In this work, the gE gene structure was analyzed using bioinformatics techniques, and three plasmids of varying lengths, tgE537, tgE200, and tgE350, were expressed in Chinese hamster ovary (CHO) cells. These proteins were used to immunize BALB/c mice with Al/CpG adjuvant; ELISPOT and FCM were used to evaluate cellular immunity; and ELISA, VZV microneutralization, and FAMA assays were performed to detect antibody titers. Results: Target protein concentrations of 1.8 mg/mL for tgE537, 0.15 mg/mL for tgE200 and 0.65 mg/mL for tgE350 were effectively produced. The ability of the three protein segments to stimulate CD4⁺ and CD8⁺ T cells, as well as to cause lymphocytes to secrete IFN-γ and IL-4, did not significantly differ from one another. Both tgE537 and tgE350 were capable of generating VZV-specific antibodies and neutralizing antibodies, while tgE350 had the highest neutralizing antibody titer (4388). There was no equivalent humoral immune response induced by tgE200. Conclusions: The results of this investigation provide the groundwork for the creation of HZ recombinant vaccines using truncated proteins as antigens. Full article

Background/Objectives: The body’s immune response to infections and vaccination leads to the formation of memory B cells (MBCs), which protect against future infections. MBCs circulate in the blood, and the strength of the MBC response is measured with different tests. In this study, tests to measure the MBC response were compared. Methods: An MBC enzyme-linked immunospot assay (MBC-ELISpot), which counts the antibody-releasing cells (MASCs) that arise from memory B cells in vitro, and two versions of an MBC enzyme-linked immunosorbent assay (MBC-ELISA), which measures the concentration of antibodies released by the MASCs, were compared. The lower measurement limit of MBC-ELISpot and ELISA was determined, and it was investigated how the measurement results of individual samples and in a sample of test persons correlate. Results: Both methods had similar lower limits of detection, and the antibody concentration correlated strongly with the number of MASCs in individual samples. The antibody concentrations measured on a sample correlated with Pearson correlation coefficients of R = 0.83–0.87 with the number of MASCs, and the proportion of antigen-specific antibodies in total IgG correlated with R = 0.74–0.82 with the proportion of antigen-specific MASCs in all antibody-secreting cells. Conclusions: Since the measurement sensitivity of MBC-ELISA and MBC-ELISpot is similar and the measurement results correlate strongly in a random sample, the tests are interchangeable. The MBC-ELISA has an advantage over the ELISpot in that the antibody measurements can be divided up over time, repeated, and extended. This creates new possibilities for measuring the MBC response. Full article

An intranasal COVID-19 vaccine, DelNS1-based RBD vaccines composed of H1N1 subtype (DelNS1-nCoV-RBD LAIV) was developed to evaluate the safety and immunogenicity in healthy adults. We conducted a phase 1 randomized, double-blinded, placebo-controlled study on healthy participants, age 18–55 and COVID-19 vaccines naïve, between March and September 2021. Participants were enrolled and randomly assigned (2:2:1) into the low and high dose DelNS1-nCoV-RBD LAIV manufactured in chicken embryonated eggs or placebo groups. The low and high-dose vaccine were composed of 1 × 10⁷ EID₅₀/ dose and 1 × 10^7.7 EID₅₀/ dose in 0.2 mL respectively. The placebo vaccine was composed of inert excipients/dose in 0.2 mL. Recruited participants were administered the vaccine intranasally on day 0 and day 28. The primary end-point was the safety of the vaccine. The secondary endpoints included cellular, humoral, and mucosal immune responses post-vaccination at pre-specified time-points. The cellular response was measured by the T-cell ELISpot assay. The humoral response was measured by the serum anti-RBD IgG and live-virus neutralizing antibody against SARS-CoV-2. The saliva total Ig antibody responses in mucosal secretion against SARS-CoV-2 RBD was also assessed. Twenty-nine healthy Chinese participants were vaccinated (low-dose: 11; high-dose: 12 and placebo: 6). The median age was 26 years. Twenty participants (69%) were male. No participant was discontinued due to an adverse event or COVID-19 infection during the clinical trial. There was no significant difference in the incidence of adverse events (p = 0.620). For the T-cell response elicited after full vaccination, the positive PBMC in the high-dose group increased to 12.5 SFU/10⁶ PMBC (day 42) from 0 (baseline), while it increased to 5 SFU/10⁶ PBMC (day 42) from 2.5 SFU/10⁶ PBMC (baseline) in the placebo group. The high-dose group showed a slightly higher level of mucosal Ig than the control group after receiving two doses of the vaccine (day 31, 0.24 vs. 0.21, p = 0.046; day 56 0.31 vs. 0.15, p = 0.45). There was no difference in the T-cell and saliva Ig response between the low-dose and placebo groups. The serum anti-RBD IgG and live virus neutralizing antibody against SARS-CoV-2 were undetectable in all samples. The high-dose intranasal DelNS1-nCoV-RBD LAIV is safe with moderate mucosal immunogenicity. A phase-2 booster trial with a two-dose regimen of the high-dose intranasal DelNS1-nCoV-RBD LAIV is warranted. Full article

Anti-dsDNA autoantibodies quantification and complement levels are widely used to monitor disease activity in systemic lupus erythematosus (SLE). However, better biomarkers are still needed. We hypothesised whether the dsDNA antibody-secreting B-cells could be a complementary biomarker in disease activity and prognosis of SLE patients. Fifty-two SLE patients were enrolled and followed for up to 12 months. Additionally, 39 controls were included. An activity cut-off (comparing active and non-active patients according to clinical SLEDAI-2K) was established for SLE-ELISpot, chemiluminescence and Crithidia luciliae indirect immunofluorescence tests (≥11.24, ≥374.1 and ≥1, respectively). Assays performances together with complement status were compared regarding major organ involvement at the inclusion and flare-up risk prediction after follow-up. SLE-ELISpot showed the best performance in identifying active patients. High SLE-ELISpot results were associated with haematological involvement and, after follow-up, with an increased hazard ratio for disease flare-up (3.4) and especially renal flare (6.5). Additionally, the combination of hypocomplementemia and high SLE-ELISpot results increased those risks up to 5.2 and 32.9, respectively. SLE-ELISpot offers complementary information to anti-dsDNA autoantibodies to evaluate the risk of a flare-up in the following year. In some cases, adding SLE-ELISpot to the current follow-up protocol for SLE patients can improve clinicians’ personalised care decisions. Full article

Vaccines are a promising therapeutic alternative to monoclonal antibodies against HER-2⁺ breast cancer. We present the preclinical activity of an ES2B-C001, a VLP-based vaccine being developed for human breast cancer therapy. FVB mice challenged with HER-2⁺ mammary carcinoma cells QD developed progressive tumors, whereas all mice vaccinated with ES2B-C001+Montanide ISA 51, and 70% of mice vaccinated without adjuvant, remained tumor-free. ES2B-C001 completely inhibited lung metastases in mice challenged intravenously. HER-2 transgenic Delta16 mice developed mammary carcinomas by 4–8 months of age; two administrations of ES2B-C001+Montanide prevented tumor onset for >1 year. Young Delta16 mice challenged intravenously with QD cells developed a mean of 68 lung nodules in 13 weeks, whereas all mice vaccinated with ES2B-C001+Montanide, and 73% of mice vaccinated without adjuvant, remained metastasis-free. ES2B-C001 in adjuvant elicited strong anti-HER-2 antibody responses comprising all Ig isotypes; titers ranging from 1–10 mg/mL persisted for many months. Antibodies inhibited the 3D growth of human HER-2⁺ trastuzumab-sensitive and -resistant breast cancer cells. Vaccination did not induce cytokine storms; however, it increased the ELISpot frequency of IFN-γ secreting HER-2-specific splenocytes. ES2B-C001 is a promising candidate vaccine for the therapy of tumors expressing HER-2. Preclinical results warrant further development towards human clinical studies. Full article

Previous reports demonstrated that FLU-v, a peptide-based broad-spectrum influenza vaccine candidate, induced antibody and cellular immune responses in humans. Here, we evaluate cellular effector functions and cross-reactivity. PBMC sampled pre- (day 0) and post-vaccination (days 42 and 180) from vaccine (n = 58) and placebo (n = 27) recipients were tested in vitro for responses to FLU-v and inactivated influenza strains (A/H3N2, A/H1N1, A/H5N1, A/H7N9, B/Yamagata) using IFN-γ and granzyme B ELISpot. FLU-v induced a significant increase in the number of IFN-γ- and granzyme-B-secreting cells responding to the vaccine antigens from pre-vaccination (medians: 5 SFU/10⁶ cells for both markers) to day 42 (125 and 40 SFU/10⁶ cells, p < 0.0001 for both) and day 180 (75 and 20 SFU/10⁶ cells, p < 0.0001 and p = 0.0047). The fold increase from pre-vaccination to day 42 for IFN-γ-, granzyme-B-, and double-positive-secreting cells responding to FLU-v was significantly elevated compared to placebo (medians: 16.3-fold vs. 1.0-fold, p < 0.0001; 3.5-fold vs. 1.0-fold, p < 0.0001; 3.0-fold vs. 1.0-fold, p = 0.0012, respectively). Stimulation of PBMC with inactivated influenza strains showed significantly higher fold increases from pre-vaccination to day 42 in the vaccine group compared to placebo for IFN-γ-secreting cells reacting to H1N1 (medians: 2.3-fold vs. 0.8-fold, p = 0.0083), H3N2 (1.7-fold vs. 0.8-fold, p = 0.0178), and H5N1 (1.7-fold vs. 1.0-fold, p = 0.0441); for granzyme B secreting cells reacting to H1N1 (3.5-fold vs. 1.0-fold, p = 0.0075); and for double positive cells reacting to H1N1 (2.9-fold vs. 1.0-fold, p = 0.0219), H3N2 (1.7-fold vs. 0.9-fold, p = 0.0136), and the B strain (2.0-fold vs. 0.8-fold, p = 0.0227). The correlation observed between number of cells secreting IFN-γ or granzyme B in response to FLU-v and to the influenza strains supported vaccine-induced cross-reactivity. In conclusion, adjuvanted FLU-v vaccination induced cross-reactive cellular responses with cytotoxic capacity, further supporting the development of FLU-v as a broad-spectrum influenza vaccine. Full article

Since BNT162b2 was approved to prevent COVID-19 in children, we aim to compare the safety and immunogenicity of the BNT162b2 vaccine in liver-transplanted (LT) and healthy adolescents. LT and healthy adolescents received two doses of 30 µg of BNT162b2. All were evaluated for total COVID-19 antibodies directed against the receptor-binding domain (RBD) and interferon-γ using the ELISpot at all time points; anti-nucleocapsid immunoglobulin was evaluated at week 8 and the surrogate virus-neutralizing antibody (sVN) to Omicron at day 0 and week 8. Adverse effects were recorded during days 0–7. In total, 16 LT and 27 healthy adolescents were enrolled (aged 14.78 ± 1.70 years). After completion, all LT and healthy adolescents were positive for anti-RBD immunoglobulin, with geometric mean titers of 1511.37 (95% CI 720.22–3171.59) and 6311.90 (95% CI 4955.46–8039.64)) U/mL (p < 0.001). All tested negative for anti-nucleocapsid immunoglobulin, indicating no COVID-19 infection after vaccination. However, the sVNs to Omicron were positive in only nine (33.33%) healthy adolescents and none of the LT adolescents. Interferon-γ-secreting cells were lower in LT adolescents than healthy adolescents. The LT adolescents had a lower immunogenic response to BNT162b2 than the healthy adolescents. Administrating two doses of BNT162b2 was safe, but was less effective against the Omicron variant. Full article

The long-term effect of protection by two doses of SARS-CoV-2 vaccination in patients receiving chronic intermittent hemodialysis (CIHD) is an urging question. We investigated the humoral and cellular immune response of 42 CIHD patients who had received two doses of SARS-CoV-2 vaccine, and again after a booster vaccine with mRNA-1273 six months later. We measured antibody levels and SARS-CoV-2-specific surrogate neutralizing antibodies (SNA). Functional T cell immune response to vaccination was assessed by quantifying interferon-γ (IFN-γ) and IL-2 secreting T cells specific for SARS-CoV-2 using an ELISpot assay. Our data reveal a moderate immune response after the second dose of vaccination, with significantly decreasing SARS-CoV-2-specific antibody levels and less than half of the study group showed neutralizing antibodies six months afterwards. Booster vaccines increased the humoral response dramatically and led to a response rate of 89.2% for antibody levels and a response rate of 94.6% for SNA. Measurement in a no response/low response (NR/LR) subgroup of our cohort, which differed from the whole group in age and rate of immunosuppressive drugs, indicated failure of a corresponding T cell response after the booster vaccine. We strongly argue in favor of a regular testing of surrogate neutralizing antibodies and consecutive booster vaccinations for CIHD patients to provide a stronger and persistent immunity. Full article

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. Full article

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. Full article