Search Results (103)

Hepatitis E virus (HEV) causes significant morbidity and mortality globally, particularly affecting vulnerable populations such as pregnant women. HEV239 (Hecolin^®), a recombinant vaccine containing the immunodominant protruding (E2) domain of the HEV capsid protein, has demonstrated effectiveness, yet detailed human cellular immune responses remain understudied. This study characterized humoral and cellular immune responses following vaccination with HEV239 or natural HEV infection in healthy Bangladeshi women aged 16–39 years. Using dual IFNγ and IL-4 ELISpot assays, we found robust, predominantly Th1-mediated cellular responses at 30 days after the third vaccine dose, comparable to responses during acute infection. Longitudinal antibody assessments confirmed sustained antibody production, primarily against the E2 domain of genotypes 1 and 3, persisting up to two years post-vaccination. Despite limitations related to sample size and assay sensitivity, our findings underscore the immunogenic potential of HEV239 and support a broader use in HEV-endemic regions. Full article

Background: The prevalence of pulmonary nontuberculous mycobacteria (NTM) is increasing in Europe and North America. Most pulmonary NTM cases are caused by Mycobacterium avium complex (MAC). The treatment of pulmonary MAC is suboptimal with failure rates ranging from 30% to 40% and there is a need to develop new vaccines. Methods: We tested the ability of two whole-cell vaccines, DAR-901 (heat-killed M. obuense) and BCG (live-attenuated M. bovis), to induce MAC cross-reactive immunity by first immunizing BALB/c mice and then performing IFN-γ ELISPOT assays after overnight stimulation of splenocytes with live MAC. To study the ability of these vaccines to protect against MAC infection, BALB/c mice were vaccinated with DAR-901 (intradermal) or BCG (subcutaneous or intranasal) and challenged with aerosolized MAC 4 weeks later. A group of mice vaccinated with BCG were also treated with clarithromycin via gavage. Lung colony-forming units (CFU) in immunized mice and unvaccinated controls were quantified 4 weeks after infection. Histopathology was used to quantify lung inflammation and flow cytometry was used to study lung immunity in BCG-vaccinated and unvaccinated mice following MAC infection. To increase the safety profile of mucosal BCG vaccination, we studied BCG with a “kill switch” (tetR BCG) in scnn1b-transgenic mice (i.e., mice prone to cystic fibrosis-type lung diseases). Results: Our results showed that (i) DAR-901 induced cross-reactive immunity to MAC to a similar level as BCG, (ii) DAR-901 and BCG protected against aerosol MAC challenge, (iii) mucosal BCG vaccination, compared to systemic BCG and DAR-901 vaccinations, provided the best protection against MAC challenge, (iv) BCG vaccination did not interfere with anti-MAC activities of clarithromycin, (v) BCG-vaccinated mice had increased inflammation and increased frequencies of activated CD4 and CD8 T cells following MAC infection, and (vi) doxycycline treatment of tetR BCG-vaccinated mice decreased lung BCG CFU without affecting MAC immunity. Conclusions: Both DAR-901 and BCG vaccinations induce MAC cross-reactive immunity and protect against aerosolized MAC challenges. Mucosal BCG vaccination provides the best protection and TetR BCG could enhance the safety of mucosal BCG vaccination. Full article

Background: Breast cancer stem cells (CSCs), particularly those enriched in triple-negative breast cancer (TNBC), are key contributors to tumor recurrence, metastasis, and resistance to therapy. CSCs often undergo epithelial-to-mesenchymal transformation (EMT), enhancing their invasiveness. Immune-based strategies that selectively target CSC/EMT antigens offer a promising therapeutic approach. Methods: Twelve candidate CSC/EMT-associated proteins were identified through a systematic literature review. Human serum samples were assessed for antigen-specific IgG using ELISA. Th1/Th2 cytokine profiles, in response to predicted MHC II epitopes, were measured by ELISPOT in PBMCs. Epitope immunogenicity and tumor inhibition were evaluated in murine models, using either TNBC or luminal B syngeneic breast cancer cell lines. Results: Six of the candidate proteins (SOX2, YB1, FOXQ1, MDM2, CDH3, CD105) elicited antigen-specific IgG in human serum. Th1-selective epitopes, defined by high Th1/Th2 ratios, were identified for five of these proteins. Immunization of mice with peptide pools derived from CD105, CDH3, MDM2, SOX2, and YB1 induced significant antigen-specific IFN-γ responses. Tumor growth was significantly inhibited in the vaccinated mice across both the TNBC and luminal B breast cancer models, with mean tumor volume reductions ranging from 61% to 70%. Conclusions: CSC/EMT-associated antigens are immunogenic in humans and can be targeted using Th1-selective epitope-based vaccines. Immunization with these epitopes effectively inhibits tumor growth in multiple murine models of breast cancer. These findings support further clinical evaluation of CSC/EMT-targeted vaccines, especially for high-risk or advanced-stage breast cancer patients. Full article

Background: The recent global outbreak with clade IIb and the concurrent emergence of clade I mpox virus in Africa show that mpox is a challenging problem. MVA-BN induces low-level mpox-neutralizing antibody responses that wane rapidly. This study was conducted to compare the mpox immunity induced by a replication-competent smallpox vaccine and non-replicating MVA-BN. Methods: Stored sera (n = 302) and PBMCs (n = 244) collected pre-vaccination and at five post-vaccination time points in MVA-BN and six post-vaccination time points in Dryvax clinical trials were used. Antibody titers that neutralized at least 50% of mpox in cell culture were determined by the focus reduction neutralization test (FRNT) 50, and the mpox-specific T cell responses were measured using an IFN-γ ELISPOT assay. Results: The peak geometric fold rise (95% CI) (i.e., the maximum GMFR across all study visits) in the mpox FRNT50 for subcutaneous (SC) MVA-BN, intradermal (ID) MVA-BN, and Dryvax was 22.1 (8.3, 59.1), 18.5 (8.0, 43.1), and 245.8 (100.4, 601.6), respectively. The GMFR at day 180 post-vaccination for MVA-BN (SC), MVA-BN (ID), and Dryvax was 2.4, 2.7, and 64, respectively. The mean (95% CI) peak number of mpox-specific IFN-γ-producing SFCs was 127 (43.1, 238.3), 87.3 (46, 137), and 61.2 (44.3, 77.7) for MVA-BN (SC), MVA-BN (ID), and Dryvax, respectively. On day 180, the mean SFCs in the three groups decreased to 10.8 (−34.4, 3.8), 3.3 (−6.2, 18.6), and 2.2 (−9, 12.5), respectively. Conclusions: The peak mpox-neutralizing antibody titer was >10-fold lower in MVA-BN recipients compared to those who received a replication-competent smallpox vaccine, and the level at day 180 was >20 times lower in MVA-BN recipients. MVA-BN induced similar or higher T cell responses. Full article

Background/Objectives: Chlamydia trachomatis (Ct) is the leading bacterial cause of sexually transmitted infection globally. If undiagnosed or left untreated, these infections can lead to serious complications such as infertility, ectopic pregnancies, and chronic pelvic pain. Despite the high prevalence and potential for serious health complications, no vaccine has been licensed. Pigs offer a valuable biomedical model for chlamydia research: they have an overall high degree of similarity to humans and serve as natural hosts for Chlamydia suis (Cs), a close relative of Ct. Thus, in this study, the pig model was used to evaluate a vaccine candidate against Ct. Methods: The vaccine candidate consists of chlamydial-protease-like activity factor (CPAF) protein adjuvanted with STING (Stimulator of Interferon Genes) pathway agonist cyclic-di-AMP (c-di-AMP). Pigs received two doses intramuscularly followed by two intranasal doses. Each week, the systemic T cell response was assessed via IFN-γ and IL-17 ELISpots, as well as multi-parameter flow cytometry on 0, 14, and 28 days post vaccination (dpv). The humoral immune response was analyzed by measuring CPAF-specific antibody levels and avidity via ELISAs. Results: Vaccination with c-di-AMP adjuvanted CPAF triggered low-level systemic IFN-γ and multifunctional IFN-γ⁺TNF-α⁺ CD4 T cell responses. Despite the rather low systemic effector cytokine production, robust anti-CPAF IgG responses were detected in serum, vaginal swab eluates, and oviduct flushes. Genital Ct challenge 42 dpv resulted in only transient infection, precluding a confident assessment of vaccine efficacy of the tested CPAF/c-di-AMP vaccine candidate. However, after challenge, vaccinated pigs exhibited boosted systemic anti-CPAF IFN-γ and mucosal IgG responses compared to unvaccinated pigs. Conclusions: Thus, while vaccine efficacy remains elusive, the CPAF/c-di-AMP vaccine candidate was immunogenic: it elicited a low-level systemic cell-mediated response and robust humoral immune responses. Future studies will incorporate a STING agonist directly conjugated to CPAF as well as addition of other Th1-inducing adjuvants to enhance cellular immunity. Full article

Background: Hepatitis B virus (HBV) is a major cause of morbidity and mortality globally, and chronic infections are associated with cirrhosis and hepatocellular carcinoma. Issues with conventional treatments and vaccines mean there is a need for new therapeutic vaccines, which must elicit a strong and sustainable immune response. Here, we evaluated the immunogenicity of dual-antigen vaccines containing hybrid surface (hy-LHBs) and core (HBc) antigens, combined with a carboxyl-vinyl polymer (CVP) as a mucoadhesive excipient, following intranasal administration in mice. Methods: Mice were intranasally administered a mixed vaccine (10 µg of hy-LHBs and 2.5 or 10 µg of HBc) with or without a CVP excipient, and they were assessed for their immune response (levels of IgGs or IgA antibodies in an ELISA, IFN-γ level in splenocytes in an ELISpot assay, and cytokine/chemokine levels in a BioPlex assay). A protein stability assay was also conducted for vaccine formulations with and without excipients. Results: Significantly enhanced IgG production was noted targeting hy-LHBs and (less markedly) HBc at 10 µg/antigen, but only a non-significant elevation was noted with the vaccine containing 2.5 µg HBc. The BioPlex assay showed a significant increase in IL-2 (#00-07, 0B), IL-12(p40)(#00), eotaxin (#00), MIP1α (#00, #00-07, 0B), and MCP-1 (#00-07, 0B) in mice that received treatment compared to those of untreated mice. The endpoint titers of IgG1 and IgG2a were measured, which were higher with CVP excipients than without. From the IgG2a/IgG1 ratio, a higher IgG1 response was induced by CVPs to hy-LHBs and a higher IgG2a response was induced to HBc. Th2-dominant phenotype to hy-LHBs was induced with CVP#00 in an ELISpot assay. The highest anti-hy-LHBs antibody titer was noted with the conventional CVP#00 excipient. Consistent with these results, a higher amount of neutralizing antibodies of HBV was induced with CVP#00 treatment and followed by #00-03 and #14-00. Conclusions: We consider that the addition of CVP excipients to vaccine formulation enhances immunogenicity and HBV antigen stability for intranasal vaccines. This effect was seen for both humoral and cell-mediated immune responses, indicating the potential of CVPs as excipients in intranasal HBV vaccines. Full article

Background: COVID-19, caused by SARS-CoV-2, poses a significant threat to human health. Vaccines designed for T-cell epitopes play an important role in eliminating the virus. However, T cell epitope screening often requires the use of a large number of peripheral blood mononuclear cells (PBMCs) from infected or convalescent patients, and if MHC humanized mice can be used for epitope screening, they will not have to wait for enough PBMCs to be available to screen for epitopes, thus buying time for epitope confirmation and vaccine design. Methods: In this study, we used SARS-CoV-2 BA.5 to infect HLA-A11/DR1, C57BL/6, hACE2 mice, and detected body weight changes, viral load, and pathological changes after infection. Fourteen days after the HLA-A11/DR1 and C57BL/6 mice were immunized against inactivated viruses, IgG antibodies were detected in mouse serum using ELISA, and IFN-γ produced by peptide stimulation of splenocytes was detected by ELISpot. Results: There is no obvious pathogenic phenotype of SARS-CoV-2 infection in HLA-A11/DR1 mice. Specific IgG antibodies were detected in serum after immunization of inactivated virus in both HLA-A11/DR1 and C57BL/6 mice, but specific IFN-γ was detected in splenocytes of HLA-A11/DR1 mice. Conclusions: Although HLA-A11/DR1 mice are unable to replicate the virus effectively in vivo, they are able to generate cellular immune responses after immunization inactivated viruses. Therefore, it can be used as a tool to substitute for human PBMCs in epitope screening, thus shortening the timeliness of T cell epitope screening and obtaining the immunogenicity information of new epitopes in a timely manner. Full article

Background/Objectives: Developing durable cellular immunity remains a critical challenge for HIV vaccine development. Methods: We evaluated a sequential and repeated heterologous prime–boost vaccination regimen using four distinct vector-based vaccines (DNA, rAd5, rSeV, and rMVA) expressing HIV-1 gag in rhesus macaques over a decade-long observation period. Results: Compared to the two-vector and control groups, the four-vector regimen elicited potent gag-specific cellular immune responses, as evidenced by IFN-γ ELISPOT assays showing sustained responses exceeding 500 SFCs/10⁶ PBMCs for up to 52 or 69 weeks post-vaccination. Intracellular cytokine staining revealed multifunctional CD4+ and CD8+ T-cell responses, while humoral immunity against Ad5 vectors remained manageable despite repeated administrations. Conclusions: These findings demonstrate that sequential and repeated heterologous vaccination effectively induces and maintains durable cellular immunity, providing a strategic framework for HIV vaccine design. 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

Background: Available assays to measure pox virus neutralizing antibody titers are laborious and take up to 5 days. In addition, assays to measure T cell responses require the use of specific antigens, which may not be the same for all pox viruses. This study reports the development of robust assays for the measurement of mpox-specific neutralizing antibodies and IFN-γ-producing T-cell responses. Methods: Fourteen samples from 7 volunteers who received Modified Vaccinia Ankara-Bavarian Nordic (MVA-BN) were used. The focused reduction neutralization test (FRNT) was performed using the mpox-specific A29 monoclonal antibody. Optimization and further development of FRNT were conducted using the plaque reduction neutralization test (PRNT) as the gold standard. The mpox-specific IFN-γ ELISPOT assay was optimized using different mpox antigen preparations. Results with pre-vaccination samples were compared with post-vaccination samples using the Wilcoxon matched-pairs test. Results: Pre-vaccination and post-vaccination sera (n = 7) had FRNT50 (i.e., titers that inhibited at least 50% of the virus) of 109.1 ± 161.8 and 303.7 ± 402.8 (mean ± SD), respectively. Regression analysis of fold changes in FRNT50 and PRNT50 showed that the two assays closely agree (n = 25 tests on paired samples, R² of 0.787). Using UV-inactivated mpox as an antigen, the number of IFN-γ spot-forming T cells (SFC) in pre-vaccination samples (16.13 ± 15.86, mean ± SD) was significantly lower than SFC in post-vaccination samples (172.9 ± 313.3, mean ± SD) with p = 0.0078. Conclusions: Our newly developed microneutralization test has a good correlation with PRNT. UV-inactivated mpox is an appropriate antigen for the ELISPOT assay that measures mpox cross-reactive T cells. These assays will be useful in future mpox vaccine studies. Full article

Background/Objectives: New SARS-CoV-2 variants are continuously emerging, making it essential to assess the efficacy of vaccine-induced immune protection. Limited information is available regarding T cell responses to BA.2.86 and JN.1 variants, particularly in elderly individuals. Methods: We evaluated T cell and total IgG responses against the receptor-binding domain (RBD) of the ancestral SARS-CoV-2 strain, as well as BA.2.86 and JN.1 omicron subvariants, in two groups of subjects. One group consisted of SARS-CoV-2-exposed elderly individuals who were fully vaccinated with the BNT162B2 mRNA vaccine, with a booster dose of the updated 2023–2024 COVID-19 vaccine (XBB.1.5) at least 15 days after receiving a booster dose of the updated 2023–2024 COVID-19 vaccine. The second group consisted of healthcare workers who were unexposed to SARS-CoV-2 one month after the booster dose of the first-generation BNT162b2 mRNA vaccine. T cell activation-induced markers (AIM) and IFN-γ secretion were evaluated by flow cytometry and ELISpot assays, respectively. Results: Elderly subjects showed reduced IgG levels against JN.1 compared with the ancestral strain. BA.2.86 stimulation resulted in lower IFN-γ levels in the elderly versus the COVID-19-naïve group. AIM analysis showed that among T cells, CD4+ were the most responsive, with a reduced proportion of JN.1-reactive CD4+ T cells compared with the ancestral strain in the SARS-CoV-2-unexposed group. Despite receiving the updated booster, the elderly group showed reduced CD4+ T cell reactivity to BA.2.86. Conclusions: The XBB.1.5-containing vaccine induced lower CD4+ T cell responses against BA.2.86 in the elderly. CD4+ T cells from BNT16b2-vaccinated, COVID-19-naïve subjects recognized ancestral and BA.2.86 RBD strains while showing reduced responses to JN.1. These results emphasize the need for tailored vaccine strategies for emerging variants, particularly in vulnerable populations. Full article

Background/Objectives: The receptor binding motif (RBM) of the SARS-CoV-2 spike protein is critical for viral entry into host cells. Development of a vaccine targeting this region is a promising strategy for COVID-19 prevention. To enhance the immunogenicity of SARS-CoV-2 vaccines, we developed an adenoviral vector expressing the RBM from the SARS-CoV-2 spike protein that fused to the human Fc (hFc) domain. Methods: The recombinant RBM_hFc fusion protein was successfully cloned into the pacAd5CMV-N-pA (pAd5) vector and expressed in HEK293 cells as a ~40 kDa protein. A recombinant adenovirus encoding RBM_hFc was subsequently generated and confirmed by cytopathic effect assay. Results: Western blot analysis verified the expression of RBM_hFc in the adenovirus (AdV). ELISA assays, validated for IgG detection, demonstrated a twofold increase in IgG antibody levels (M–1.090 at 450 nm; SD—±0.326; and 95% CI—0.250 [0.839 to 1.340]) in sera from BALB/c mice immunized with Ad/RBM_hFc, compared to the negative control group. Result suggests a robust humoral immune response induced by the Ad/RBM_hFc vaccine. Moreover, ELISpot assays demonstrated a tenfold increase in IFN-γ -producing cells (M—440 spot-forming cells; SD—±124.976; and 95% CI—75.522 [364.478 to 515.522]) in mice immunized with AdV/RBM_hFc compared to the negative control group. Result proved that AdV/RBM_hFc-stimulated a robust cellular immune response in animal model. Conclusions: Our findings indicate that the RBM_hFc fusion protein enhances both humoral and cellular immune responses. These results suggest the potential of adenoviral vectors carrying RBM_hFc as vaccine candidates. However, comprehensive evaluation of the protective efficacy of these adenoviral vectors will necessitate rigorous experimental studies. Full article

In Europe and North America, the prevalence of pulmonary nontuberculous mycobacteria (NTM) is increasing. Most pulmonary NTM infections are caused by the Mycobacterium avium complex (MAC). Sadly, the treatment of pulmonary MAC is suboptimal with failure rates ranging from 37% to 58%. Therefore, there is a need to develop new therapeutics. Developing new immunotherapies and studying their interaction with standard or new drugs requires reliable assays. Four different assays including CFSE-based flow cytometry, in vitro protection assays, IFN-γ ELISPOT, and murine infection models were optimized using a reference strain of MAC (ATCC 700898) to help with the development of immunotherapies for MAC. Expansion of proliferating and IFN-γ producing human T cells is optimal after 7 days of stimulation with MAC at a multiplicity of infection (MOI) of 0.1, achieving a stimulation index of 26.5 ± 11.6 (mean ± SE). The in vitro protection assay for MAC works best by co-culturing T cells expanded for 7 days with MAC (MOI 1)-infected autologous macrophages. Aerosol MAC infection of mice allows measurement of the effects of the BCG vaccine and clarithromycin. IFN-γ ELISPOT assays with live MAC (MOI 3) stimulation of splenocytes from mice immunized with BCG help identify differences between unimmunized mice and mice immunized with BCG. In conclusion, multiple assays are available for use to identify MAC-specific effector T cells, which will help in the development of new therapeutics or vaccines against pulmonary MAC. Full article

COVID-19 vaccine evaluations are mainly focused on antibody analyses, but there is growing interest in measuring the cellular immune responses from the researchers evaluating these vaccines. The cellular responses to several COVID-19 vaccines have been studied using the enzyme-linked immunospot (ELISPOT) assay for IFN-γ. However, the ELISPOT assay is no longer used only for research purpose and so the performance of this assay must be validated. Since the bioanalytical validation of ELISPOT-IFN-γ is essential for evaluating the method’s effectiveness and establishing confidence in a vaccine’s immunogenicity, the present work validates the ELISPOT-IFN-γ assay’s performance in determining the frequency of IFN-γ-producing cells after stimulation with the SARS-CoV-2 spike protein. The validation was performed in peripheral blood mononuclear cells from volunteers immunized with anti-COVID-19 vaccines. According to the findings, the LOD was 17 SFU and the LLOQ was 22 SFU, which makes the method highly sensitive and suitable for evaluating low levels of cellular responses. The procedure’s accuracy is confirmed by the correlation coefficients for the spike protein and anti-CD3⁺, being 0.98 and 0.95, respectively. The repeatability and intermediate precision tests were confirmed to be reliable by obtaining a coefficient of variation of ≤25%. The results obtained in this validation enable the assay to be employed for studying antigen-specific cells and evaluating cellular responses to vaccines. 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