Immunogenic Potential Research Articles

Immunotherapies targeting the oncogenic fusion gene CLDN18-ARHGAP in gastric cancer

The CLDN18-ARHGAP fusion gene is an oncogenic driver newly discovered in gastric cancer. It was detected in 9% (8/87) of gastric cancer patients in our center. An immunogenic peptide specifically targeting CLDN18-ARHGAP fusion gene was generated to induce neoantigen-reactive T cells, which was proved to have specific and robust anti-tumor capacity both in in vitro coculture models and in vivo xenograft gastric cancer models. Apart from the immunogenic potential, CLDN18-ARHGAP fusion gene was also found to contribute to immune suppression by inducing a regulatory T (Treg) cell-enriched microenvironment. Mechanistically, gastric cancer cells with CLDN18-ARHGAP fusion activate PI3K/AKT-mTOR-FAS signaling, which enhances free fatty acid production of gastric cancer cells to favor the survival of Treg cells. Furthermore, PI3K inhibition could effectively reverse Treg cells upregulation to enhance anti-tumor cytotoxicity of neoantigen-reactive T cells in vitro and reduce tumor growth in the xenograft gastric cancer model. Our study identified the CLDN18-ARHGAP fusion gene as a critical source of immunogenic neoepitopes, a key regulator of the tumor immune microenvironment, and immunotherapeutic applications specific to this oncogenic fusion.

Development and characterization of dendritic cell internalization and activation assays contributing to the immunogenicity risk evaluation of biotherapeutics.

Immunogenicity refers to the ability of a substance, such as a therapeutic drug, to elicit an immune response. While beneficial in vaccine development, undesirable immunogenicity can compromise the safety and efficacy of therapeutic proteins by inducing anti-drug antibodies (ADAs). These ADAs can reduce drug bioavailability and alter pharmacokinetics, necessitating comprehensive immunogenicity risk assessments starting at early stages of drug development. Given the complexity of immunogenicity, an integrated approach is essential, as no single assay can universally recapitulate the immune response leading to the formation of anti-drug antibodies. To better understand the Dendritic Cell (DC) contribution to immunogenicity, we developed two flow cytometry-based assays: the DC internalization assay and the DC activation assay. Monocyte-derived dendritic cells (moDCs) were generated from peripheral blood mononuclear cells (PBMCs) and differentiated over a five-day period. The internalization assay measured the accumulation rate of therapeutic antibodies within moDCs, while the activation assay assessed the expression of DC activation markers such as CD40, CD80, CD86, CD83, and DC-SIGN (CD209). To characterize these two assays further, we used a set of marketed therapeutic antibodies. The study highlights that moDCs differentiated for 5 days from freshly isolated monocytes were more prone to respond to external stimuli. The internalization assay has been shown to be highly sensitive to the molecule tested, allowing the use of only 4 donors to detect small but significant differences. We also demonstrated that therapeutic antibodies were efficiently taken up by moDCs, with a strong correlation with their peptide presentation on MHC-II. On the other hand, by monitoring DC activation through a limited set of activation markers including CD40, CD83, and DC-SIGN, the DC activation assay has the potential to compare a series of compounds. These two assays provide a more comprehensive understanding of DC function in the context of immunogenicity, highlighting the importance of both internalization and activation processes in ADA development. The DC internalization and activation assays described here address key gaps in existing immunogenicity assessment methods by providing specific and reliable measures of DC function. The assays enhance our ability to pre-clinically evaluate the immunogenic potential of biotherapeutics, thereby improving their safety and efficacy. Future work should focus on further validating these assays and integrating them into a holistic immunogenicity risk assessment framework.

Surfactants as a means of delivering a reporter genetic construct based on binase suicide gene to tumor cells

Among modern gene therapy methods for combating oncology, suicidal gene therapy based on the delivery of a cytotoxic agent to target cells is of particular importance and promise. As one of such genes, the gene for ribonuclease of Bacillus pumilus 7P, binase, can be considered; the enzyme has a high antitumor potential and low immunogenicity. In addition to the choice of a transgene, another factor influencing the effectiveness of gene therapy is the method of delivering the nucleic acid to target cells. Surfactants have high functional activity and are promising means of delivering therapeutic nucleic acids. The aim of this work was to evaluate the possibility of using geminal surfactants as a means of delivering a genetic construct based on the cytotoxic binase gene into tumor cells. To optimize the transfection conditions, a reporter genetic construct carrying the binase gene fused to the gene for the green fluorescent protein TurboGFP was created, which made it possible to evaluate the delivery efficiency by the fluorescence intensity. To eliminate the toxic effect of binase on recipient cells, the RNase inhibitor gene, barstar, was introduced into the genetic construct. A high complexing ability of geminal surfactants in relation to the reporter system was shown by methods of dynamic light scattering and fluorescence spectroscopy. For surfactant 16-6-16OH, the highest transfecting activity together with a low level of cytotoxicity was found. Thus, the study proved the possibility of using geminal surfactants for the delivery of therapeutic nucleic acids to target cells.

The N and C-terminal deleted variant of the dengue virus NS1 protein is a potential candidate for dengue vaccine development

NS1 is an elusive dengue protein, involved in viral replication, assembly, pathogenesis, and immune evasion. Its levels in blood plasm are positively related to disease severity like thrombocytopenia, hemorrhage, and vascular leakage. Despite its pathogenic roles, NS1 is being used in various vaccine formulations due to its sequence conservancy, ability to produce protective antibodies and low risk for inducing antibody-dependent enhancement. In this study, we have used bioinformatics tools and reported literature to develop an NS1 variant (dNS1). Molecular docking studies were performed to evaluate the receptor-binding ability of the NS1 and dNS1 with TLR4. NS1 and dNS1 (153 to 312 amino acid region) genes were cloned, expressed and protein was purified followed by refolding. Docking studies showed the binding of NS1 and dNS1 with the TLR4 receptor which suggests that N and C-terminal sequences of NS1 are not critical for receptor binding. Antibodies against NS1 and dNS1 were raised in rabbits and binding affinity of anti-dNS1 anti-NS1 sera was evaluated against both NS1 and dNS1. Similar results were observed through western blotting which highlight that N and C-terminal deletion of NS1 does not compromise the immunogenic potential of dNS1 hence, supports its use in future vaccine formulations as a substitute for NS1.

Surface Display of Cholera Toxin B Subunit Recombinant Escherichia coli Ghosts Further Enhances Resistance to Chlamydia abortus Infection in Mice.

Chlamydia abortus (C. abortus) is an important zoonotic pathogen that seriously endangers the development of animal husbandry. Vaccination is the most effective approach to preventing C. abortus infection. We previously reported a recombinant Escherichia coli ghost (rECG)-based C. abortus vaccine that demonstrated outstanding protective efficacy. In this study, we further attempted to fuse the cholera toxin B subunit (CTB), a widely studied potent mucosal immune adjuvant, with macrophage infectivity potentiator (MIP), a candidate antigen of C. abortus, on the surface of the rECG and explore its protective effect against C. abortus infection. The MIP fusion protein was highly expressed in the rECGs, and the CTB-modified rECGs significantly induced the activation of mouse bone marrow-derived dendritic cells in vitro. Intranasal immunization with rECGs induced a Th1-biased cellular immune response. Compared to the rECGs without CTB, the CTB-modified rECGs induced higher concentrations of IgA in the serum and vaginal wash solution. Moreover, in a mouse infection model, the CTB-modified rECGs significantly improved the clearance efficiency of C. abortus and reduced the pathological damage to the uterus. This study demonstrates that incorporating CTB into rECGs significantly enhances the immunogenic potential of the rECG vaccine and can significantly enhance its protective efficacy against a C. abortus challenge.

Impact of the Hydrophilicity of Poly(sarcosine) on Poly(ethylene glycol) (PEG) for the Suppression of Anti-PEG Antibody Binding.

A method of poly(ethylene glycol) (PEG) conjugation is known as PEGylation, which has been employed to deliver therapeutic drugs, proteins, or nanoparticles by considering the intrinsic non- or very low immunogenic property of PEG. However, PEG has its weaknesses, and one major concern is the potential immunogenicity of PEGylated proteins. Because of its hydrophilicity, poly(sarcosine) (P(Sar)) may be an attractive-and superior-substitute for PEG. In the present study, we designed a double hydrophilic diblock copolymer, methoxy-PEG-b-P(Sar) m (m = 5-55) (mPEG-P(Sar) m ), and synthesized a triblock copolymer with hydrophobic poly(l-isoleucine) (P(Ile)). We validated that double hydrophilic mPEG-P(Sar) block copolymers suppressed the specific binding of three monoclonal anti-PEG antibodies (anti-PEG mAbs) to PEG. The results of our indirect ELISAs indicate that P(Sar) significantly helps to reduce the binding of anti-PEG mAbs to PEG. Importantly, the steady suppression of this binding was made possible, in part, thanks to the maximum number of sarcosine units in the triblock copolymer, as evidenced by sandwich ELISA and biolayer interferometry assay (BLI): the intrinsic hydrophilicity of P(Sar) had a clear supportive effect on PEG. Finally, because we used P(Ile) as a hydrophobic block, PEG-P(Sar) might be an attractive alternative to PEG in the search for protein shields that minimize the immunogenicity of PEGylated proteins.

Immunogenicity and Protective Efficacy of an Acellular Pertussis Vaccine Candidate in a Mice Model.

Acellular pertussis vaccines (aPVs) have been developed as an alternative to whole-cell pertussis vaccines (wPVs) due to their similar efficacy but reduced reactogenicity. The aPV contains three or more immunogenic components of BP. We aimed to evaluate the immunogenicity and protective potency of an aPV vaccine produced in our laboratory consisting of pertussis toxin (PT), filamentous hemagglutinin (FHA), and pertactin (PRN) in mice. The aPV components were produced and purified from the supernatant and pellet of the bacterial culture. Two doses of formulated vaccine in parallel with two commercial vaccines, were administered intraperitoneally (IP) in mice at 3-week intervals. Antibody titers against aPV antigens were measured by ELISA after primary and booster vaccinations. To assess the protective efficacy, an intranasal challenge with a live pathogenic BP strain was conducted two weeks after the booster vaccination, and bacterial count (colony-forming unit, CFU) in the lungs was conducted two hours and ten days after the challenge. The results demonstrated a significant increase in antibody titers against all pertussis antigens in the serum of vaccinated groups compared to the negative control group, following both the primary and booster doses. No significant differences were observed between our formulation and the commercial vaccines. Furthermore, the CFU results showed complete eradication of infection 10 days after the challenge in all immunized groups, in contrast to the control group. Our aPV formulation, the first aPV candidate developed in Iran, exhibits immunogenicity and protective efficacy comparable to commercial vaccines. Further investigation in human subjects is warranted.

Self-assembling Peptides Based Fibers: A Pioneering Innovation for Regenerative Medicine

Self-assembling peptides (SAPs) have emerged as a groundbreaking platform in regenerative medicine, offering innovative strategies for tissue engineering and repair. These biomolecules, designed to autonomously organize into nanofibers, mimic the natural extracellular matrix, providing essential structural support and biological cues for tissue regeneration. This review highlights the versatile applications of SAPs in regenerating diverse tissues, including bone, cartilage, cardiac, neural, and dermal structures, showcasing their potential to address a broad spectrum of medical challenges. The unique properties of SAPs, such as biocompatibility, biodegradability and specific cell receptor interaction capabilities, make them ideal for developing advanced therapeutic strategies. We discuss here the molecular design principles that allow for precise control over the nanofibers physical and chemical characteristics, facilitating the creation of scaffolds that promote cell attachment, proliferation, and differentiation. Additionally, the review covers the use of SAPs in drug delivery systems, emphasizing their ability to control the release of therapeutic agents, a critical aspect in enhancing tissue repair processes. Moreover, we address key considerations regarding the safety, biodegradability and potential immunogenicity of SAPs, underscoring the importance of thorough preclinical and clinical evaluations. The future directions and challenges in optimizing SAPs design and functionalization for maximizing regenerative outcomes are also explored.

Leveraging Senescent Cancer Cell Membrane to Potentiate Cancer Immunotherapy Through Biomimetic Nanovaccine.

Senescent cancer cells are endowed with high immunogenic potential that has been leveraged to elicit antitumor immunity and potentially complement anticancer therapies. However, the efficacy of live senescent cancer cell-based vaccination is limited by interference from immunosuppressive senescence-associated secretory phenotype and pro-tumorigenic capacity of senescent cells. Here, a senescent cancer cell-based nanovaccine with strong immunogenicity and favorable potential for immunotherapy is reported. The biomimetic nanovaccine integrating a senescent cancer cell membrane-coated nanoadjuvant outperforms living senescent cancer cells in enhancing dendritic cells (DCs) internalization, improving lymph node targeting, and enhancing immune responses. In contrast to nanovaccines generated from immunogenic cell death-induced tumor cells, senescent nanovaccines facilitate DC maturation, eliciting superior antitumor protection and improving therapeutic outcomes in melanoma-challenged mice with fewer side effects when combined with αPD-1. The study suggests a versatile biomanufacturing approach to maximize immunogenic potential and minimize adverse effects of senescent cancer cell-based vaccination and advances the design of biomimetic nanovaccines for cancer immunotherapy.

Immunogenic and diagnostic potential of recombinant apical membrane antigen-1 from Plasmodium malariae

The apical membrane antigen-1 (AMA-1) is a crucial target for malaria management and prevention strategies. While the immunogenicity of AMA-1 has been extensively studied for Plasmodium falciparum and Plasmodium vivax, there is a notable scarcity of information for Plasmodium malariae. In this study, recombinant PmAMA-1 was expressed in Escherichia coli, and its integrity was confirmed via western blotting and indirect immunofluorescence assays. Immunization of BALB/c mice with rPmAMA-1 emulsified in Freund's adjuvant resulted in significantly elevated specific IgG antibodies, predominantly IgG1. The immune response exhibited Th1, Th2, and Th17 phenotypes, with a notable Th1 bias. Antisera from immunized mice effectively recognized native PmAMA-1 on P. malariae. These results suggest that PmAMA-1 is a promising target for both vaccine development and diagnostic applications for P. malariae infections, offering dual preventive and diagnostic benefits in malaria control.

Semisynthetic Glycoconjugates as Potential Vaccine Candidates Against Haemophilus influenzae Type a.

Glycoconjugate vaccines are based on chemical conjugation of pathogen-associated carbohydrates with immunogenic carrier proteins and are considered a very cost-effective way to prevent infections. Most of the licensed glycoconjugate vaccines are composed of saccharide antigens extracted from bacterial sources. However, synthetic oligosaccharide antigens have become a promising alternative to natural polysaccharides with the advantage of being well-defined structures providing homogeneous conjugates. Haemophilus influenzae (Hi) is responsible for a number of severe diseases. In recent years, an increasing rate of invasive infections caused by Hi serotype a (Hia) raised some concern, because no vaccine targeting Hia is currently available. The capsular polysaccharide (CPS) of Hia is constituted by phosphodiester-linked 4-β-d-glucose-(1→4)-d-ribitol-5-(PO4→) repeating units and is the antigen for protein-conjugated polysaccharide vaccines. To investigate the antigenic potential of the CPS from Hia, we synthesized related saccharide fragments containing up to five repeating units. Following the synthetic optimization of the needed disaccharide building blocks, they were assembled using the phosphoramidite approach for the installation of the phosphodiester linkages. The resulting CPS-based Hia oligomers were conjugated to CRM197 carrier protein and evaluated in vivo for their immunogenic potential, showing that all glycoconjugates were capable of raising antibodies recognizing Hia synthetic fragments.

A Structural Comparison of Oral SARS-CoV-2 Drug Candidate Ibuzatrelvir Complexed with the Main Protease (Mpro) of SARS-CoV-2 and MERS-CoV.

Ibuzatrelvir (1) was recently disclosed and patented by Pfizer for the treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It has received fast-track status from the USA Food and Drug Administration (FDA) and has entered phase III clinical trials as a possible replacement for Paxlovid. Like nirmatrelvir (2) in Paxlovid, this orally active drug candidate is designed to target viral main proteases (Mpro) through reversible covalent interaction of its nitrile warhead with the active site thiol of the chymotrypsin-like cysteine protease (3CL protease). Inhibition of Mpro hinders the processing of the proteins essential for viral replication in vivo. However, ibuzatrelvir apparently does not require ritonavir (3), which is coadministered in Paxlovid to block human oxidative metabolism of nirmatrelvir. Here, we report the crystal structure of the complex of ibuzatrelvir with the active site of SARS-CoV-2 Mpro at 2.0 Å resolution. In addition, we show that ibuzatrelvir also potently inhibits the Mpro of Middle East respiratory syndrome-related coronavirus (MERS-CoV), which is fortunately not widespread but can be dangerously lethal (∼36% mortality). Co-crystal structures show that the binding mode of the drug to both active sites is similar and that the trifluoromethyl group of the inhibitor fits precisely into a critical S2 substrate binding pocket of the main proteases. However, our results also provide a rationale for the differences in potency of ibuzatrelvir for these two proteases due to minor differences in the substrate preferences leading to a weaker H-bond network in MERS-CoV Mpro. In addition, we examined the reversibility of compound binding to both proteases, which is an important parameter in reducing off-target effects as well as the potential immunogenicity. The crystal structures of the ibuzatrelvir complexes with Mpro of SARS-CoV-2 and of MERS-CoV will further assist drug design for coronaviral infections in humans and animals.

Bacterial surface derived extracellular vesicles: A ground‐breaking approach in cancer therapy

AbstractConventional cancer therapies are often limited by inherent and acquired drug resistance, non‐specific toxicity, and suboptimal drug delivery. Bacterial extracellular vesicles (BEVs), including outer membrane vesicles and membrane vesicles, present a novel therapeutic strategy for cancer treatment. BEVs were found in both Gram‐negative and Gram‐positive bacteria. This article provides a classified investigation of the therapeutic potential of BEVs in cancer treatment. We discuss the molecular mechanisms underlying their observed anti‐tumor effects, including the induction of apoptosis in cancer cells, suppression of angiogenesis within the tumor microenvironment, and stimulation of both innate and adaptive anti‐tumor immune responses. Moreover, BEVs being biocompatible opens avenue for targeted drug delivery systems, potentially improving the therapeutic index of conventional chemotherapeutics. Challenges to clinical translation, such as BEV heterogeneity and potential immunogenicity, are addressed. We also explore future directions for research and development, comprehensively highlighting how BEVs transform the landscape of precision oncology and cancer treatment, eventually improving patient outcomes.

Conserved linear B-cell peptides among the influenza A viral neuraminidases enhance the cross-protective potential of inactivated whole-virion influenza vaccine

Introduction. Influenza is a disease caused by a widespread virus with pandemic potential. Frequently, individuals vaccinated against seasonal influenza virus are still susceptible to the disease, indicating the need to improve the immunogenic potential of existing vaccines. To assess the efficacy of influenza virus vaccines, immune response only to a single viral antigen — hemagglutinin molecule, is taken into consideration. However, according to preclinical and clinical studies, neuraminidase (NA) stimulates cross-protective immunity, which is effective against not only homologous but also drifted variants of influenza A virus. Materials and methods. In the present study, we investigated the ability of previously selected conserved linear B-cell NA epitopes (SGYSGK, SWPDGK, EECSCYPK, VELIRGRK) to enhance the immunogenicity of an inactivated whole-virion influenza vaccine based on the model strain PR8 (iPR8). BALB/c mice were injected with iPR8 in combination with one of the peptides intramuscularly three times at two-week intervals. Blood samples were collected 14 days after the last immunization, after which the mice were challenged with heterosubtypic influenza viruses H1N1pdm09 and H3N2. Results. All immunized mice showed induction of H1N1 (PR8)-specific IgG antibodies two weeks after the third immunization. The group of mice immunized with the iPR8 vaccine preparation in combination with VELIRGRK peptide showed the most pronounced induction of IgG antibodies to the H6N1 reassortant strain, the NA of which corresponds to the iPR8 virus, indicating the ability of the NA peptide to stimulate the production of NA-specific antibodies. However, the antibodies produced after immunization were not capable to inhibit the NA enzymatic activity. Despite this, mice immunized with iPR8 in combination with anti-NA peptides showed a higher survival rate after infection with heterologous virulent influenza viruses: A/California/07/09 (H1N1pdm09) and A/Philippines/2/82 (H3N2) compared to the PBS and iPR8 groups. Conclusion. Thus, the study demonstrated the immune-potentiating effect of individual peptides corresponding to conservative linear epitopes of the NA molecule in combination with a standard inactivated influenza vaccine, which made it possible to improve the protective effect of the vaccine against heterosubtypic influenza viruses.

A Micronemal Protein, Scot1, Is Essential for Apicoplast Biogenesis and Liver Stage Development in Plasmodium berghei.

Plasmodium sporozoites invade hepatocytes, transform into liver stages, and replicate into thousands of merozoites that infect erythrocytes and cause malaria. Proteins secreted from micronemes play an essential role in hepatocyte invasion, and unneeded micronemes are subsequently discarded for replication. The liver-stage parasites are potent immunogens that prevent malarial infection. Late liver stage-arresting genetically attenuated parasites (GAPs) exhibit greater protective efficacy than early GAP. However, the number of late liver-stage GAPs for generating GAPs with multiple gene deletions is limited. Here, we identified Scot1 (Sporozoite Conserved Orthologous Transcript 1), which was previously shown to be upregulated in sporozoites, and by endogenous tagging with mCherry, we demonstrated that it is expressed in the sporozoite and liver stages in micronemes. Using targeted gene deletion in Plasmodium berghei, we showed that Scot1 is essential for late liver-stage development. Scot1 KO sporozoites grew normally into liver stages but failed to initiate blood-stage infection in mice due to impaired apicoplast biogenesis and merozoite formation. Bioinformatic studies suggested that Scot1 is a metal-small-molecule carrier protein. Remarkably, supplementation with metals in the culture of infected Scot1 KO cells did not rescue their phenotype. Immunization with Scot1 KO sporozoites in C57BL/6 mice confers protection against malaria via infection. These proof-of-concept studies will enable the generation of P. falciparum Scot1 mutants that could be exploited to generate GAP malaria vaccines.

Chimeric lipoproteins for leptospirosis vaccine: immunogenicity and protective potential

Leptospirosis, a neglected zoonotic disease, is caused by pathogenic spirochetes belonging to the genus Leptospira and has one of the highest morbidity and mortality rates worldwide. Vaccination stands out as one of the most effective preventive measures for susceptible populations. Within the outer membrane of Leptospira spp., we find the LIC12287, LIC11711, and LIC13259 lipoproteins. These are of interest due to their surface location and potential immunogenicity. Thorough examination revealed the conservation of these proteins among pathogenic Leptospira spp.; we mapped the distribution of T- and B-cell epitopes along their sequences and assessed the 3D structures of each protein. This information aided in selecting immunodominant regions for the development of a chimeric protein. Through gene synthesis, we successfully constructed a chimeric protein, which was subsequently expressed, purified, and characterized. Hamsters were immunized with the chimeric lipoprotein, formulated with adjuvants aluminum hydroxide, EMULSIGEN®-D, Sigma Adjuvant System®, and Montanide™ ISA206VG. Another group was vaccinated with an inactivated Escherichia coli bacterin expressing the chimeric protein. Following vaccination, hamsters were challenged with a virulent L. interrogans strain. Our evaluation of the humoral immune response revealed the production of IgG antibodies, detectable 28 days after the second dose, in contrast to pre-immune samples and control groups. This demonstrates the potential of the chimeric protein to elicit a robust humoral immune response; however, no protection against challenge was achieved. While this study provides valuable insights into the subject, further research is warranted to identify protective antigens that could be utilized in the development of a leptospirosis vaccine.Key points• Several T- and B-cell epitopes were identified in all the three proteins.• Four different adjuvants were used in vaccine formulations.• Immunization stimulated significant levels of IgG2/3 in vaccinated animals.

Advances in Nanomedicine for Precision Insulin Delivery.

Diabetes mellitus, which comprises a group of metabolic disorders affecting carbohydrate metabolism, is characterized by improper glucose utilization and excessive production, leading to hyperglycemia. The global prevalence of diabetes is rising, with projections indicating it will affect 783.2 million people by 2045. Insulin treatment is crucial, especially for type 1 diabetes, due to the lack of β-cell function. Intensive insulin therapy, involving multiple daily injections or continuous subcutaneous insulin infusion, has proven effective in reducing microvascular complications but poses a higher risk of severe hypoglycemia. Recent advancements in insulin formulations and delivery methods, such as ultra-rapid-acting analogs and inhaled insulin, offer potential benefits in terms of reducing hypoglycemia and improving glycemic control. However, the traditional subcutaneous injection method has drawbacks, including patient compliance issues and associated complications. Nanomedicine presents innovative solutions to these challenges, offering promising avenues for overcoming current drug limitations, enhancing cellular uptake, and improving pharmacokinetics and pharmacodynamics. Various nanocarriers, including liposomes, chitosan, and PLGA, provide protection against enzymatic degradation, improving drug stability and controlled release. These nanocarriers offer unique advantages, ranging from enhanced bioavailability and sustained release to specific targeting capabilities. While oral insulin delivery is being explored for better patient adherence and cost-effectiveness, other nanomedicine-based methods also show promise in improving delivery efficiency and patient outcomes. Safety concerns, including potential toxicity and immunogenicity issues, must be addressed, with the FDA providing guidance for the safe development of nanotechnology-based products. Future directions in nanomedicine will focus on creating next-generation nanocarriers with precise targeting, real-time monitoring, and stimuli-responsive features to optimize diabetes treatment outcomes and patient safety. This review delves into the current state of nanomedicine for insulin delivery, examining various types of nanocarriers and their mechanisms of action, and discussing the challenges and future directions in developing safe and effective nanomedicine-based therapies for diabetes management.

Evaluation of a novel 177Lu-labelled therapeutic Affibody molecule with a deimmunized ABD domain and improved biodistribution profile

PurposeFusion of Affibody molecules with an albumin-binding domain (ABD) provides targeting agents, which are suitable for radionuclide therapy. To facilitate clinical translation, the low immunogenic potential of such constructs with targeting properties conserved is required.MethodsThe HER2-targeting Affibody molecule ZHER2:2891 was fused with a deimmunized ABD variant and DOTA was conjugated to a unique C-terminal cysteine. The novel construct, PEP49989, was labelled with 177Lu. Affinity, specificity, and in vivo targeting properties of [177Lu]Lu-PEP49989 were characterised. Experimental therapy in mice with human HER2-expressing xenografts was evaluated.ResultsThe maximum molar activity of 52 GBq/µmol [177Lu]Lu-PEP49989 was obtained. [177Lu]Lu-PEP49989 bound specifically to HER2-expressing cells in vitro and in vivo. The HER2 binding affinity of [177Lu]Lu-PEP49989 was similar to the affinity of [177Lu]Lu-ABY-027 containing the parental ABD035 variant. The renal uptake of [177Lu]Lu-PEP49989 was 1.4-fold higher, but hepatic and splenic uptake was 1.7-2-fold lower than the uptake of [177Lu]Lu-ABY-027. The median survival of xenograft-bearing mice treated with 21 MBq [177Lu]Lu-PEP49989 (> 90 days) was significantly longer than the survival of mice treated with vehicle (38 days) or trastuzumab (45 days). Treatment using a combination of [177Lu]Lu-PEP49989 and trastuzumab increased the number of complete tumour remissions. The renal and hepatic toxicity was minimal to mild.ConclusionIn preclinical studies, [177Lu]Lu-PEP49989 demonstrated favourable biodistribution and a strong antitumour effect, which was further enhanced by co-treatment with trastuzumab.

Immunogenicity and vaccine potential of clinical isolate Mycobacterium kansasii strain against Mycobacterium tuberculosis infection.

Mycobacterium kansasii is a bacterium included in non-tuberculous mycobacteria (NTM) that can cause lung disease. It shares a significant number of antigens with Mycobacterium tuberculosis (Mtb), suggesting that it has the potential to be used as a tuberculosis (TB) vaccine. Therefore, we subcutaneously vaccinated mice with reference strain, M. kansasii-ATCC12478 [M. kansasii-American Type Culture Collection (ATCC)], and clinically isolated strain, M. kansasii-SM-1 to evaluate potential as a TB vaccine by comparing with bacille Calmette-Guerin (BCG) vaccine. Ten weeks after vaccination, we evaluated immunogenicity of M. kansasii-ATCC and M. kansasii-SM-1, and M. kansasii-SM-1 immunization induces potent Mtb antigen-specific IFN-γ-producing CD4+ T cells than M. kansasii-ATCC. Upon Mtb infection, M. kansasii-SM-1 provided better protection than M. kansasii-ATCC, which was comparable to the efficacy of BCG. These results showed that the clinical strain M. kansasii-SM-1, which exhibits an enhanced Mtb antigen-specific Th1 response, shows greater vaccine efficacy compared to M. kansasii-ATCC. In this study, we demonstrated that vaccine efficacy can vary depending on the strain of M. kansasii and that its efficacy can be comparable to BCG. This suggests that M. kansasii has the potential to be a live TB vaccine candidate.IMPORTANCEMycobacterium kansasii, a non-tuberculous mycobacteria (NTM) species causing lung disease, shares key antigens with Mycobacterium tuberculosis (Mtb), indicating its potential for TB vaccine development. Subcutaneous vaccination of mice with M. kansasii strains reference strain M. kansasii-ATCC12478 [(M. kansasii-American Type Culture Collection (ATCC)] and clinically isolated strain M. kansasii-SM-1 revealed differences in immunogenicity. M. kansasii-SM-1 induced a robust Mtb antigen-specific IFN-γ-producing CD4+ T cell response compared to M. kansasii-ATCC. Additionally, M. kansasii-SM-1 conferred better protection against Mtb infection than M. kansasii-ATCC, which is comparable to bacille Calmette-Guerin (BCG). These findings underscore the variable vaccine efficacy among M. kansasii strains, with M. kansasii-SM-1 exhibiting promising potential as a live TB vaccine candidate, suggesting its comparative effectiveness to BCG.

Immunogenic Comparison of Nucleic Acid-Based Vaccines Administered by Pyro-Drive Jet Injector.

mRNA vaccines were successfully developed and approved for emergency use to fight coronavirus disease 2019. However, the effect of DNA vaccines against SARS-CoV-2 is considerably lower than that of mRNA vaccines. A pyro-drive jet injector (PJI) efficiently delivers plasmid DNA intradermally into animal models. Here, we compared the immunogenic potential of DNA and mRNA vaccines in mice using the same platform. PJI was used to deliver naked mRNA and pDNA and their efficacy in inducing antigen expression and immune responses was assessed. Our results showed that PJI efficiently delivered mRNA into the skin, and a smaller effective dose than that of pDNA injection was required to achieve similar levels of antigen expression. The PJI-delivered CpG-free pDNA vaccine efficiently induced antigen-specific antibody production and a cell-mediated IFN-γ response compared to the mRNA vaccine, as well as the upregulation of inflammatory cytokines (IL-6, IFN-γ, and IL-1β) in the skin and lymph nodes. However, the intradermal mRNA vaccine was significantly less immunogenic than the standard intramuscular mRNA-lipid nanoparticle vaccine, despite equivalent mRNA dosages. Improvements in lipid nanoparticle and mRNA technology have revolutionized mRNA vaccines, and DNA vaccines can be similarly modified for higher clinical efficacy.