Search Results (106)

Toxoplasma gondii is an obligate intracellular protozoan parasite that causes toxoplasmosis, posing a significant threat to human health and livestock production worldwide. Although monovalent DNA or mRNA vaccines often confer only partial protection, whether these platforms can be effectively integrated into a heterologous prime–boost regimen against T. gondii remains to be fully elucidated. Here, we constructed GRA35-encoding DNA and mRNA vaccines and evaluated their immunogenicity and protective efficacy, administered either alone or in heterologous prime–boost combinations, in C57BL/6 and BALB/c mice. Both vaccines induced strong antigen-specific immune responses, with the heterologous prime–boost regimen eliciting the strongest effects and conferring the most robust and consistent protection across both mouse strains. Immunization triggered a predominantly Th1-skewed response characterized by significantly elevated IFN-γ production, accompanied by balanced antigen-specific IgG responses. Moreover, vaccinated mice developed rapid and potent cytotoxic T lymphocyte (CTL) responses. Following challenge with the RH and PRU strains, vaccinated mice exhibited prolonged survival and significantly reduced brain cyst burdens following PRU challenge compared with control groups. Collectively, these findings indicate that GRA35-based nucleic acid vaccines, particularly when administered in a heterologous prime–boost regimen, elicit multifaceted protective immune responses and represent promising vaccine candidates against T. gondii infection. Full article

Background/Objectives: Pathogenic mammarenaviruses cause severe hemorrhagic and neurologic disease in humans. Machupo virus (MACV), a New World (NW) mammarenavirus, causes Bolivian hemorrhagic fever in humans, and there are no approved vaccines. Methods: Here, we describe and compare the immunogenicity of three vaccines expressing the MACV glycoprotein complex (GPC) in C57BL/6 mice: a recombinant vesicular stomatitis virus (rVSV) and two different lipid nanoparticle (LNP)-encapsulated nucleoside-modified mRNA (mRNA-LNP) vaccines. The first mRNA-LNP vaccine, designated MACV mRNA, expresses the full-length MACV GPC. The second mRNA-LNP vaccine, called MACV VLP mRNA, encodes MACV GPC with appended sequences that induce the budding of virus-like particles (VLPs) with MACV GPC on the surface. This is the first description of any mRNA-LNP vaccine for MACV and the first comparison of mRNA and rVSVs as vaccine candidates for MACV. Results: We find that two doses of either MACV mRNA or MACV VLP mRNA are required for the induction of robust humoral and cellular immune responses including total MACV GPC IgG, neutralizing antibodies, cross-reactive antibodies that bind the related Junín virus GPC, and MACV-specific T-cell responses. To further investigate vaccination strategies for MACV, we also evaluated a heterologous prime-boost regimen involving the MACV mRNA vaccine coupled with the rVSV-based MACV vaccine. We find that the highest levels of MACV GPC-specific IgG and neutralizing titers were achieved when heterologous mRNA and rVSV prime-boost regimens were employed. Conclusions: These results elucidate differences in the immune response to different vaccine platforms for MACV and can inform future vaccine development for NW arenaviruses. Full article

Background: House dust mites (HDM) are one of the significant indoor allergen sources which cause IgE-mediated responses in most of the allergic individuals. HDMs are found in human habitats worldwide and Der p 2 is one of the major clinically relevant HDM allergens involved in triggering allergic diseases. The recombinant production of Der p 2 in plant systems provides a cost-effective and viable platform for developing diagnostic kits and allergen-specific immunotherapy. Methods: The D. pteronyssinus Der p 2 allergen was transiently expressed in Nicotiana benthamiana and its immunogenicity was evaluated in mice. The Der p 2 coding sequence was cloned into a geminiviral plant expression vector and introduced into N. benthamiana leaves via Agrobacterium tumefaciens-mediated infiltration. Recombinant Der p 2 proteins were purified from the crude extracts and confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot. The immunogenicity of the plant-produced Der p 2 proteins was further evaluated by immunizing mice following a prime–boost immunization regimen, and Der p 2-specific antibody responses were assessed by ELISA. Results: Recombinant Der p 2 was successfully expressed and purified from N. benthamiana, and immunized mice developed high levels of Der p 2-specific IgG antibodies, with antibody titers increased after booster immunization. Conclusions: The results demonstrate that the transient expression of Der p 2 in plants is a feasible and effective strategy for producing immunologically active recombinant allergen proteins for diagnostic and potential clinical applications. Full article

Pigeon Newcastle disease poses a persistent threat to the global pigeon industry, underscoring the need for effective vaccination strategies. While both inactivated and DNA vaccines offer distinct advantages, the immunogenicity of a combined heterologous regimen remains underexplored. This study evaluated and compared three immunization strategies in pigeons: a DNA vaccine encoding the NDV F protein fused with chicken IL-18, an inactivated vaccine from a local virulent strain, and a DNA prime-inactivated boost regimen. The preparation workflows for both vaccine platforms are described in detail to provide methodological context for the immunological comparison. Critically, the prime–boost regimen elicited significantly higher hemagglutination inhibition (HI) antibody titers than either vaccine administered alone, demonstrating a clear synergistic effect. These findings highlight the complementary roles of the two platforms and provide a strong immunological rationale for further evaluation of this sequential strategy. Future studies incorporating viral challenge experiments and long-term immune monitoring are needed to determine whether the enhanced HI antibody response translates into protective efficacy under field conditions. Full article

Background/Objectives: Porcine epidemic diarrhea (PED) is a highly contagious enteric disease caused by porcine epidemic diarrhea virus (PEDV) and is associated with severe clinical signs and high mortality in neonatal piglets. Vaccination is an important strategy for PED control through the induction of humoral immunity. This study aimed to compare immune responses induced by inactivated and live-attenuated PEDV vaccines and to evaluate a heterologous prime-boost vaccination strategy in PEDV-naïve replacement gilts. Methods: Twenty-four PEDV-naïve replacement gilts were randomly assigned to four groups: unvaccinated control, inactivated vaccine administered twice (K/K), live-attenuated vaccine administered twice (L/L), and live-attenuated priming followed by an inactivated booster (L/K). Pigs received two intramuscular vaccinations at 16 weeks of age and two weeks later. Serum samples collected up to 42 days post-vaccination were analyzed for PEDV-specific IgG and IgA antibodies by ELISA, and serum-neutralizing antibody titers were determined using a serum neutralization test. Results: The L/K regimen induced the highest PEDV-specific IgG responses, with peak levels at day 28 post-vaccination that were significantly higher than those in the K/K and control groups. Serum-neutralizing antibody titers were significantly higher in the L/K and L/L groups than in the K/K and control groups. Serum IgA responses were low and transient across all vaccination groups. Conclusions: A heterologous prime-boost vaccination strategy using a live-attenuated PEDV vaccine followed by an inactivated booster induces strong systemic humoral immune responses in replacement gilts and represents a promising approach for PEDV vaccination programs. Full article

Background/Objectives: SARS-CoV-2, the causative agent of COVID-19, has been responsible for more than seven million deaths worldwide since its emergence. The Bacillus Calmette–Guérin (BCG) vaccine, used for over 100 years to prevent tuberculosis, induces a Th1-prominent immune response that is important for protection against Mycobacterium tuberculosis, other mycobacteria, and intracellular pathogens. BCG has also been shown to induce innate immune memory and heterologous protection against non-related infections. Additionally, BCG has been used as a vector to express heterologous proteins, showing protective effects against various diseases, particularly respiratory viral infections, including SARS-CoV-2. In this report, we constructed two recombinant BCG strains as potential vaccine candidates based on the receptor-binding domain (RBD) of the Spike antigen: one expressing only the RBD protein (rBCG-RBD) and another expressing the RBD protein in fusion with the LTB (Escherichia coli Labile Toxin subunit B) adjuvant (rBCG-LTB-RBD). Methods: We evaluated the induction of SARS-CoV-2-specific humoral and cellular immune responses using these vaccine candidates in a prime–boost strategy with a booster dose using the rRBD protein (produced in cell culture) and the Alum adjuvant. Antisera were evaluated for neutralization of the Wuhan and Omicron SARS-CoV-2 pseudotyped virus. Results: Either immunization scheme (rBCG-RBD/rRBD or rBCG-LTB-RBD/rRBD) induced high IgG antibody titers, with antibody neutralization against a Wuhan SARS-CoV-2 pseudotyped virus after 10 weeks. The antibody levels induced by rBCG-RBD/rRBD were maintained for up to 9 months. Interestingly, only the sera from mice receiving the prime–boost with rBCG-LTB-RBD/rRBD showed cross-reactive neutralization against the Omicron SARS-CoV-2 pseudotyped virus. Immunization with rBCG-RBD or rBCG-LTB-RBD and a rRBD booster dose promoted the induction of specific CD4+ and CD8+ T cells producing Th1/Th2 cytokines (IL-4, TNF-α and IFN-γ). Conclusions: Our study highlights the potential of the prime–boost immunization strategy using rBCG-RBD/rRBD to induce long-term immunity and rBCG-LTB-RBD/rRBD to induce cross-protection against different variants, both of which could serve as promising vaccine candidates. Full article

Background: Developing an effective vaccine is crucial for the prevention and control of AIDS. Viral vector-based vaccines, particularly those utilizing homologous or heterologous prime-boost strategies, represent an important direction in current HIV vaccine research. Methods: In this study, replication-defective chimeric adenovirus Ad5F35 and modified vaccinia virus Ankara (rMVA) vector vaccines expressing the HIV-1 AEgp145 were successfully constructed, designated as Ad5F35-AEgp145 and rMVA-AEgp145, respectively. Sixty BALB/c mice were randomly divided into three groups: Ad5F35 alone, rMVA prime/Ad5F35 boost, and PBS control. The mice were immunized intramuscularly at weeks 0 and 3, and humoral and cellular immune responses were assessed at 4, 8, 12, and 16 weeks after the initial immunization. Results: The homologous Ad5F35 and heterologous rMVA/Ad5F35 vaccination regimens elicited comparable levels of HIV Env-specific cellular immune responses, peaking at 2100 ± 222 SFCs/million splenocytes and 2200 ± 619 SFCs/million splenocytes, respectively (p > 0.05). Compared to the heterologous regimen, the homologous Ad5F35 regimen induced significantly higher levels of gp120-binding antibodies at weeks 4 and 8 post-initial immunization, with geometric mean titers of 1:25,600 ± 7011 versus 1:1280 ± 150.7 and 1:10,240 ± 4048 versus 1:2560 ± 391.9, respectively. Furthermore, neutralizing activity at week 8 was significantly higher in the homologous group, with a 50% neutralization titers of 1:45 compared to 1:12 in the heterologous group (p < 0.01). Conclusion: This study demonstrates that the Ad5F35-AEgp145 vaccine, whether administered alone or in combination with rMVA-AEgp145, effectively induces strong and comparable cellular immune responses targeting HIV-1 Env in mice. While both regimens are effective, homologous immunization elicits moderately higher levels of antibody responses. These findings provide an important foundation for the further investigation of vector-based HIV vaccine formulations. Full article

Background: Emerging immune-evasive viral variants threaten the efficacy of current vaccines, underscoring the need for strategies that elicit broad and durable protection. Heterologous prime–boost regimens combining distinct vaccine platforms can enhance humoral and cellular immunity through complementary mechanisms. Methods: Using an intramuscular immunization scheme aligned with clinical vaccination practice, CD-1 mice received homologous or heterologous prime–boost regimens combining a replication-deficient Orf virus (Parapoxvirus orf, ORFV)-based spike vaccine (ORFV-S) with the licensed adjuvanted recombinant protein vaccine VidPrevtyn Beta. Spike-specific humoral and cellular immune responses were assessed. Results: ORFV-S alone induced potent and broad spike-specific IgG responses and achieved the strongest ACE2-binding inhibition across variants of concern. ORFV-S priming followed by VidPrevtyn Beta boosting markedly enhanced the magnitude and cross-variant breadth of antibody responses compared with homologous protein vaccination. Both homologous ORFV-S and heterologous regimens incorporating ORFV-S elicited strong CD4⁺ and CD8⁺ T-cell responses, whereas VidPrevtyn Beta alone induced only modest T-cell activity, demonstrating that ORFV-S effectively complements protein-based vaccines. Conclusions: The ORFV-S vector represents a potent vaccine platform capable of inducing broad humoral and cellular immunity. Its use in heterologous prime–boost combinations enhances both antibody magnitude and breadth beyond homologous protein vaccination, supporting its application in vaccination strategies against evolving viral pathogens. Full article

Background/Objectives: Tuberculosis (TB) remains the leading cause of death from a single infectious agent worldwide. In line with the World Health Organization’s (WHO) goal to end TB by 2035, the rapid development and clinical implementation of new, effective vaccines is urgently needed. To support global TB control efforts, we developed a novel candidate subunit multistage vaccine. Methods: This vaccine incorporates multiple Mycobacterium tuberculosis antigens expressed during both dormant and active stages of infection, fused into a single recombinant protein (ESAT6-CFP10-Ag85A-Rv2660c-Rv1813c). The antigen was encapsulated in biodegradable poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles along with the pattern recognition receptor (PRR) agonists monophosphoryl lipid A (MPLA) and muramyl dipeptide (MDP), which function as adjuvants. Results: Using a mixed intramuscular/nasal prime-boost regimen, the vaccine elicited a mixed Th1/Th17 cell-mediated immune response, as well as a robust humoral response characterized by sustained systemic IgG (lasting at least one year) and prominent local secretory IgA. The vaccine demonstrated protective efficacy as a prophylactic booster following BCG priming in both murine and guinea pig models and was also effective in a therapeutic setting in a murine infection model. Conclusions: The results of this study provide empirical evidence that multistage tuberculosis vaccines represent a promising strategy for achieving global TB control. Full article

The ongoing global monkeypox outbreak since 2022 has highlighted the urgent need for vaccine development. Current vaccination strategies rely on cross-protective immunity provided by orthopoxvirus-based live-attenuated vaccines. However, these vaccines not only exhibit suboptimal efficacy against monkeypox virus (MPXV) but also raise safety concerns, particularly given the significant global overlap between MPXV infections and HIV. Owing to their superior safety profile and accessibility, recombinant subunit vaccines represent a highly promising platform for monkeypox vaccine development. In this study, we developed a subunit vaccine comprising A29L, B6R, and M1R antigens formulated with a proprietary nanoemulsion adjuvant and evaluated its immunogenicity and protective efficacy. In mice immunized with a prime-boost regimen of the three individual antigens combined with the nanoemulsion adjuvant, comparable serum IgG levels against each antigen were elicited. Both A29 and M1 formulations induced serum antibodies with potent neutralizing activity against MPXV and Vaccinia virus Western Reserve strain (VACV-WR). Notably, M1 antiserum exhibited stronger neutralization than A29 antiserum, whereas B6R immune serum showed no significant neutralizing activity. Splenocytes from B6R-immunized mice mounted a robust IFN-γ response, which was markedly lower in those immunized with A29 or M1. All three monovalent vaccines conferred complete survival following an intranasal lethal MPXV challenge, with M1 providing the strongest protection. In a lethal VACV-WR challenge model, only M1 immunization conferred significant protection. Histopathological analysis of lung tissues on day 5 post-infection revealed more pronounced inflammatory features in B6R-immunized mice compared to the nanoemulsion adjuvant control group. Furthermore, the nanoemulsion-adjuvanted bivalent A29L + B6R formulation induced significantly higher IgG and neutralizing antibody titers and demonstrated superior protective efficacy compared to the aluminum hydroxide-adjuvanted formulation. This comparative preclinical evaluation provides important evidence to support the development of a safe and effective subunit vaccine against monkeypox. Full article

Background/Objectives: In previous studies, we demonstrated that the HIV-1 Env-Gag VLP mRNA vaccine elicited superior cellular immune responses. In this study, we further evaluated the immunogenicity of the Env-Gag VLP mRNA and adenovirus vector vaccines when administered individually or in combination in mice. Methods: BALB/c mice were divided into four groups and immunized twice at a 3-week interval. The three groups received either the Env-Gag VLP mRNA vaccine, the adenovirus vector vaccines expressing env and gag genes, or PBS as a control. The fourth group received a prime-boost regimen, primed with the Env-Gag mRNA vaccine and boosted with the adenovirus vector vaccines. The HIV-1 specific cellular and humoral immune responses were measured 1, 2, 4 and 8 weeks after the last immunization. Results/Conclusions: The results showed that the mRNA vaccines prime-adenovirus vector vaccines boost elicited higher cellular immune responses than those induced by homologous regimens at multiple time points, especially 8 weeks after the last immunization. Although the level of gp120 binding antibody in the combined immunization group is significantly lower than that of in the VLP mRNA vaccine group, a more balanced Th1/Th2 responses were induced in the combined immunization group, and significantly higher and longer-lasting neutralizing antibody levels were detected in this group making it a very promising HIV vaccine strategy. Full article

Vaccines have been pivotal in reducing the incidence and severity of infectious diseases, improving population health, and lowering mortality rates globally. While substantial progress has been made in optimizing vaccine formulations, adjuvants, and schedules, comparatively less attention has been given to how the site of vaccination may influence immunologic outcomes. This review examines the impact of the administration of prime and booster vaccine doses in the same (ipsilateral) versus the opposite arms (contralateral) on vaccine immunogenicity. We review animal model and human studies evaluating the impact of ipsilateral versus contralateral COVID-19 and non-COVID-19 vaccine boosting on immunologic outcomes with a focus on the germinal center response, antibody production, and T cell activation. While some studies suggest that ipsilateral administration may enhance the quality of germinal center B cell responses and antibody magnitude, data across different studies have been inconsistent. Relatively few studies have compared ipsilateral versus contralateral boosting, and differences in study design and outcomes have limited the ability to draw conclusions as to whether one is superior to the other. This review highlights a noteworthy and underexplored area in vaccinology and the need for future research to clarify whether ipsilateral/contralateral boosting strategies matter. To answer this question, high-quality, randomized controlled trials evaluating different types of vaccines that consider immunologic mechanisms, capture key time points and appropriate specimens, and evaluate early and long-term immunogenicity endpoints are required. Full article

Background/Objectives: Flaviviruses, including West Nile virus (WNV), pose global health challenges due to their worldwide distribution, pathogenicity, and lack of effective treatments or vaccines. Today, WNV is considered the most important causative agent of viral encephalitis worldwide. This study investigated the different forms of the main WNV antigen—the preM/E protein—in the context of its immunogenic and protective properties. Methods: The recombinant adenovirus type 2 (rAd2) vectors expressing different forms of the WNV preM/E genes were obtained using standard molecular biology techniques. Immunogenicity in mice was assessed by enzyme-linked immunosorbent assay (ELISA) and virus neutralization assay. Immunological efficacy was evaluated in a mouse viral challenge model. Results: The rAd2 vector expressing the West Nile virus preM/E gene with mutations in the fusion loop exhibited robust immunogenicity when administered intramuscularly either once or in a homologous prime-boost regimen. This antigen form, as part of an adenoviral vector, protected mice from death in viral challenge experiments, providing 100% survival following WNV challenge. Conclusions: We believe that a vaccination strategy involving a recombinant adenoviral vector based on human adenovirus type 2 and the WNV antigen represented by the preM/E gene with mutations in the fusion loop may be a promising approach for combating West Nile virus infection. Full article

Background/Objectives: Varicellovirus bovinealpha1 and Varicellovirus bovinealpha5 (BoAHV-1 and BoAHV-5), respectively, are widely distributed pathogens that cause distinct clinical conditions in cattle including infectious bovine rhinotracheitis, infectious pustular vulvovaginitis/balanoposthitis, and meningoencephalitis. Due to the establishment of viral latency, controlling these infections is challenging, and vaccination remains the most effective strategy. In this study, vaccine candidates targeting both BoAHV-1 and BoAHV-5 were developed. Methods: A synthetic gene encoding immunodominant epitopes from the gB and gD proteins and tegument phosphoprotein of BoAHV-1 and BoAHV-5 was designed to produce a multi-epitope recombinant antigen, expressed both in a prokaryotic system (RecBoAHV) and by a modified vaccinia Ankara (MVA-BoAHV) viral vector. The binding affinity of MHC-I to bovine leukocyte antigens (BoLA) was predicted using the NetMHCpan tool (version 4.1). The immunogenicity of the vaccine candidates was evaluated in rabbit and mouse models, using prime-boost immunization protocols. Sera from bovines naturally infected with BoAHV-1 and/or BoAHV-5 were used to evaluate the chimeric protein antigenicity. Immune responses were assessed by indirect ELISA and Western blot. Results: The recombinant multi-epitope protein was effectively recognized by IgG and IgM antibodies in sera from cattle naturally infected with BoAHV-1 or BoAHV-5, confirming the antigenic specificity. Both RecBoAHV and MVA-RecBoAHV induced strong and specific humoral immune responses in rabbits following a homologous prime-boost regimen. In mice, both homologous and heterologous prime-boost protocols revealed robust immunogenicity, particularly after the second booster dose. Conclusions: These findings highlight the immunogenic potential of the RecBoAHV multi-epitope vaccine candidates for controlling BoAHV-1 and BoAHV-5 infections. Further characterization of these vaccine formulations is currently underway in bovine, the target specie. Full article

Background: In response to the emergence of immune-evasive variants of SARS-CoV-2, this study explores a novel heterologous vaccination strategy using a microparticulate formulation approach that is delivered via oral dissolving film (ODF) formulations into the buccal cavity. Heterologous administration has the potential to generate cross-reactive antibodies, which can be especially beneficial against viruses with ever-mutating variants. Moreover, the microparticulate oral dissolving film-based vaccine approach is a non-invasive vaccine delivery platform. Methods: The vaccine design incorporated whole inactivated Delta and Omicron variants of the virus, administered at prime and booster doses, respectively, effectively encapsulated in a Poly(lactic-co-glycolic) acid (PLGA) polymer matrix, and adjuvanted with Alum to enhance immune activation. Following vaccination, serum, mucosal, and tissue samples were analyzed to evaluate humoral and cellular immune responses against the model antigen, as well as other variants such as Alpha and Beta variants, to understand the cross-reactive response. Result: In vitro evaluations confirmed the vaccine’s safety and its ability to stimulate immune responses. On administering microparticulate oral dissolving films to mice, whole inactivated delta and omicron variant-specific antibodies were observed in serum samples along with neutralizing titers in terminal week. The formulated vaccine showed significant secretory IgA antibody levels in mucosal samples. Moreover, CD4⁺ and CD8a cellular responses were observed in tissue samples of spleen and lymph nodes, along with antibodies (IgG, IgA, and IgM) detected in lung supernatant samples. Humoral and cellular cross-reactive antibodies were observed in the samples. Conclusions: This approach offers a promising platform for developing next-generation vaccines capable of inducing broad immunity. Full article