Search Results (116)

Background/Objectives: The continued evolution and cross-species transmission of clade 2.3.4.4b H5Nx highly pathogenic avian influenza (HPAI) viruses underscores the need for broadly protective vaccines in swine, a key intermediary host. This study aimed to evaluate systemic and mucosal immune responses elicited by adenoviral-vectored (Ad) vaccines encoding a centralized consensus hemagglutinin antigen (H5CC) in mice and swine. Methods: We constructed H5CC-based vaccines that were delivered using replication-defective (Ad5 and Ad6) and replication-competent (Ad28 and Ad48) human adenoviral vectors. Using a serotype-switched prime-boost strategy, vaccines were delivered intramuscularly (IM) or intranasally (IN) in mice and swine. We determined humoral, mucosal, and cell-mediated immune responses by hemagglutination inhibition (HI), microneutralization assay (MNA), ELISA, and IFN-γ ELISpot. Protective efficacy was evaluated by lethal H5N1 challenge in mice. Results: All vaccine strategies and routes induced significant levels of anti-H5 immunity. Ad5/Ad6 IM immunization elicited strong systemic IgG and MNA titers and robust T cell responses. IN delivery with Ad5/Ad6 induced superior mucosal IgA levels in lungs and nasal secretion. In swine, Ad5/Ad6 IM conferred the highest MNA titer and T cell responses, while the IN route enhanced mucosal IgA. The Ad28/Ad48 vaccines induced immunity in a similar pattern as compared to the Ad5/Ad6 strategy, but to a slightly lesser degree, in general. The commercial H1/H3 swine influenza vaccine failed to elicit cross-protective immunity. All H5CC vaccinated mice survived lethal H5N1 challenge without weight loss. Conclusions: Adenoviral-vectored H5CC vaccines elicit broad, cross-clade immunity with route-dependent immune profiles. IM vaccination is optimal for systemic and cellular responses, while IN delivery enhances mucosal immunity. These findings support the advancement of adenoviral platforms for influenza control in swine and pandemic preparedness. Full article

Immunotherapy, particularly approaches that combine tumor-specific vaccines with immune checkpoint modulation, represents a promising strategy for overcoming tumor immune evasion. While most mRNA-based cancer vaccines focus solely on antigen delivery, there is a need for platforms that simultaneously enhance antigen presentation and modulate the tumor microenvironment to increase therapeutic efficacy. This study presents a novel dual-nanolipid exosome (NLE) platform that simultaneously delivers MUC1 mRNA and CTLA-4-targeted siRNA in a single system. These endogenous lipid-based nanoparticles are structurally designed to mimic exosomes and are modified with mannose to enable selective targeting to dendritic cells (DCs) via mannose receptors. The platform was evaluated both in vitro and in vivo in terms of mRNA encapsulation efficiency, nanoparticle stability, and uptake by DCs. The co-delivery platform significantly enhanced antitumor immune responses compared to monotherapies. Flow cytometry revealed a notable increase in tumor-infiltrating CD8⁺ T cells (p < 0.01), and ELISPOT assays showed elevated IFN-γ production upon MUC1-specific stimulation. In vivo CTL assays demonstrated enhanced MUC1-specific cytotoxicity. Combined therapy resulted in immune response enhancement compared to vaccine or CTLA-4 siRNA alone. The NLE platform exhibited favorable biodistribution and low systemic toxicity. By combining targeted delivery of dendritic cells, immune checkpoint gene silencing, and efficient antigen expression in a biomimetic nanoparticle system, this study represents a significant advance over current immunotherapy strategies. The NLE platform shows strong potential as a modular and safe approach for RNA-based cancer immunotherapy. Full article

Background: Characterization of cellular responses to vaccinations in immunocompromised patients remains an evolving area of research. This particularly applies for pneumococcal vaccination in diseases such as psoriasis and in the setting of immunosuppressive therapy. Methods: This prospective study included 42 patients with moderate-to-severe psoriasis. Following German guidelines at the time, patients underwent a sequential vaccination protocol against Streptococcus pneumoniae, consisting of Prevenar 13 (PCV13) and Pneumovax 23 (PPSV23). Over a 7-month period, we analyzed T-cell responses to common serotypes of Streptococcus pneumoniae using an interferon-γ ELISpot assay. For comparison, we performed an ELISA to measure pneumococcus-specific antibody production. Results: Patients undergoing anti-TNF-α blocker therapy, monoclonal antibody therapy (specifically anti-IL-12/23, IL-23, and IL-17), and methotrexate therapy showed significantly different responses to the pneumococcal serotype PS14 at onset (p = 0.02). T-cell responses ranged from strong (PS9N, PS14, PS25F) and intermediate (PS2) to weak (PS6A and PS11A). We did not observe a significant correlation of IgG antibodies with the magnitude of cellular immune responses. Conclusions: Immunosuppressive therapy alters vaccination-induced cellular immunity in psoriasis patients. Further research is needed to clarify the mechanisms involved. Full article

Background/Objectives: Effective prophylaxis for Mycobacterium tuberculosis (Mtb) requires greater understanding of immune correlates of protection. With renewed interest in BCG as an Mtb vaccine, particularly via the intravenous (IV) route, our objective was to characterize both innate and adaptive immune correlates of vaccine-induced pulmonary immunity as potential biomarkers for protective efficacy in a murine model of Mtb infection. Methods: Mice were given BCG via different routes and some boosted with recombinant virus constructs encoding Mtb Ag85B. Responding innate lymphoid cell (ILC) populations, T cells and B cells were analyzed by fluorescence activated cell sorting (FACS) for surface markers and by intracellular cytokine staining or antibody ELISPOT. Some immunized mice were challenged with aerosolized Mtb and monitored for bacterial growth in the lungs and spleen. Results: BCG given IV, but not intranasally or subcutaneously, resulted in marked increases in IFNγ expression at 72 h by pulmonary CD49⁺ NK cells, CD69⁺ ILC1, and two ILC3 populations, NCR-ILC3 and LTi cells, the latter also producing IL-22. Pulmonary ILC2 populations in these mice had significantly increased IL-13 expression at 24 h compared to the other routes. Interestingly, high levels of NK cells and ILC1 expressing IFNγ and/or TNFα were sustained at 8 wk, with sustained expression of IL-17A by pulmonary NCR-ILC3 and pronounced tissue-resident and effector memory CD4⁺ and CD8⁺ T cell responses. Intranasal boosting with Ad-Ag85B enhanced these T cell responses and generated Mtb-specific pulmonary IgA and IgG B cells, correlating with significantly reduced bacterial loads following Mtb challenge. Conclusions: BCG given IV primed for both early and persistent pulmonary ILC1/ILC3 responses of a predominantly Th1/Th17-type profile along with local Mtb-specific memory T cell and B cell populations, correlating with enhanced protective efficacy. These are worthy of further study as compartmentalized biomarkers for effective vaccine-induced local immunity against Mtb. Full article

West Nile virus (WNV) is a mosquito-borne neurotropic virus that causes neurologic disease in both humans and horses. Yet the long-term cellular immune response following natural infection in horses remains poorly understood. This study aims to evaluate the WNV-specific T-cell response in horses recovered from West Nile neuroinvasive disease (WNND). Twelve client-owned horses (4 Hungarian sport horses, 2 Lippizaners, 1 KWPN, 1 Shagya Arabian, 1 Friesian, 1 Gidran, 1 Andalusian, and 1 draft cross horse) with confirmed clinical WNV infection were enrolled, and peripheral blood mononuclear cells (PBMCs) were collected approximately 290 days post infection. An equine interferon-gamma (IFNγ) Enzyme-Linked Immunospot (ELISpot) assay was performed using a WNV capsid peptide pool as an antigen to assess virus-specific cellular immunity. Results: Ten of twelve horses (83%) exhibited a significant IFNγ response. Statistical analyses revealed no association between ELISpot responses and clinical severity, age, sex, breed, or neutralizing antibody titers. These results demonstrate that naturally infected horses are capable of mounting robust WNV-specific T-cell responses independent of humoral immunity. The findings support a potentially important role for cellular immune memory in long-term protection against WNV reinfection and suggest that the capsid peptide-based ELISpot assay may serve as a useful diagnostic or research tool for the evaluation of orthoflavivirus immunity in equines. Full article

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: 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

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: Mucin-1 (MUC1) is a glycoprotein that is hypoglycosylated and overexpressed in most adenocarcinomas, making it a promising target for cancer vaccines. Our group previously demonstrated that C3 (OPSS)-liposomes enhance antigen uptake by antigen-presenting cells (APCs) via the complement C3 pathway and, when combined with toll-like receptor (TLR) agonists, reduce tumor growth in murine cancer models. Methods: In the present study, we evaluate the immunogenicity of MUC1 peptide vaccines encapsulated in C3-liposomes, with and without TLR agonists, using MUC1-tolerant transgenic mice challenged with Lewis lung carcinoma (LLC.MUC1) cells. To assess vaccine effectiveness, tumor volumes were measured, and flow cytometry and ELISA and ELISPOT assays were used to assess the immune response. Results: Both male and female C57BL/6 transgenic mice vaccinated with MUC1 C3-liposomes developed significantly smaller tumors than those vaccinated with free MUC1 peptide or PBS. Notably, a sex-dependent response emerged in mice vaccinated with MUC1 C3-liposomes with TLR agonists (TLR4, TLR7/8, and TLR9); male mice exhibited greater tumor suppression than females. Flow cytometry analysis revealed that female mice had significantly higher levels of CD11b⁺, LY6C⁺, and LY6G⁺ MDSC cells, suggesting a potential mechanism for the sex difference. Additionally, MUC1 C3-liposome vaccination elicited robust adaptive immune responses, including significantly higher levels of IFN-γ-producing T cells and MUC1-specific IgG antibodies compared to non-encapsulated MUC1 or TLR adjuvant-only formulations. Conclusions: These findings underscore the potential of C3-liposome-based antigen vaccines to enhance anti-tumor immunity and highlight the impact of sex differences in vaccine efficacy. 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