Antigen-adjuvant Combinations Research Articles

Farklı Antijen Hazırlama Metotları İle Hazırlanan İnsan Meme Kanseri Hücre Lizatlarının Adjuvan Kombinasyonları İle İmmünostimülan/Sitotoksik Aktivitelerinin İncelenmesi

In last decades, immunotherapies became more and more significant as a cancer treatment method. Many in vivo and in vitro studies have been conducted in the literature in order to determine the appropriate diagnostic and treatment methods for cancer. During the development of therapeutic vaccines, determination of vaccine concentration which is the beginning step of in vivo studies, is critical for the healthy progression of the studies. Breast cancer which occurs by uncontrolled proliferation as a result of exposure of healthy cells in breast tissue to factors such as DNA damage, UV, radiation, carcinogens and, various chemicals has a huge portion among the cancer types. In this study, for the first time, antigens generated by two different antigen preparation methods (autoclaving, freezing-thawing) using AU-565 / MCF-7 cell lines. Then, antigens derived from human breast cancer cell lines were applied alone and with different adjuvants (Polyoxidonium, Freund’s) to the J774 macrophage cell culture system and the L929 fibroblast cell line in order to investigate immunostimulant activity and cytotoxicity via Griess reagent and MTT test respectively. At the end of the 48th hour, the cell viability well will be examined and the most appropriate antigen-adjuvant combination and its concentration for vaccine preparation were determined. Among the antigen preparation methods, it was determined that the freeze-thaw method was more effective than the autoclave method, and the antigens prepared by the freeze-thaw method from the AU-565 and MCF-7 cell lines showed the highest immunostimulatory activity at a concentration of 160 µg/ml. Among the adjuvants, the immunostimulant effect of Freund's adjuvant was found to be higher. In the light of these results, the optimal vaccine concentration against breast cancer and the viability analysis of these concentrations were determined. All in all, it is thought that antibodies obtained from in vivo studies using the most appropriate antigen preparation method and the most appropriate vaccine concentration can be used in the diagnosis and treatment of breast cancer.

Engineering immunity via skin-directed drug delivery devices.

Extensive research is underway to discover a safe and effective vehicle to deliver the vaccines at the desired cutaneous site. These efforts majorly comprise the development of a fit-to-purpose vehicle for in-situ intracutaneous vaccine delivery for achieving the systemic cellular and humoral response to combat infectious diseases. Advancements in nanoscience, bioengineering, and skin science provided much support to vaccine adjuvant development. However, the bench-to-bed side translation of vaccines is still unsatisfactory. A skilfully designed vaccine delivery program aiming to translate the product into market use must address safety, efficacy, scaleup, reproducibility, cost of production, self-administrative potential, and regulatory concerns. This review provides deep insights into skin immunization approaches like mucosal vaccines, cellular/molecular immunological responses, and antigen-adjuvant combinations in modulating immunity. Further, the manuscript discusses distinct vaccine delivery systems used to date for engineering skin immunization, including microparticles, nanoparticles, spherical nucleic acids, STAR particles, niosomes, dendrimers, ethosomes, liposomes, and microneedles. The manuscript will interest researchers working towards developing a next-generation fit-to-purpose vehicle for intracutaneous vaccine delivery.

Highly Thermotolerant SARS-CoV-2 Vaccine Elicits Neutralising Antibodies against Delta and Omicron in Mice.

As existing vaccines fail to completely prevent COVID-19 infections or community transmission, there is an unmet need for vaccines that can better combat SARS-CoV-2 variants of concern (VOC). We previously developed highly thermo-tolerant monomeric and trimeric receptor-binding domain derivatives that can withstand 100 °C for 90 min and 37 °C for four weeks and help eliminate cold-chain requirements. We show that mice immunised with these vaccine formulations elicit high titres of antibodies that neutralise SARS-CoV-2 variants VIC31 (with Spike: D614G mutation), Delta and Omicron (BA.1.1) VOC. Compared to VIC31, there was an average 14.4-fold reduction in neutralisation against BA.1.1 for the three monomeric antigen-adjuvant combinations and a 16.5-fold reduction for the three trimeric antigen-adjuvant combinations; the corresponding values against Delta were 2.5 and 3.0. Our findings suggest that monomeric formulations are suitable for upcoming Phase I human clinical trials and that there is potential for increasing the efficacy with vaccine matching to improve the responses against emerging variants. These findings are consistent with in silico modelling and AlphaFold predictions, which show that, while oligomeric presentation can be generally beneficial, it can make important epitopes inaccessible and also carries the risk of eliciting unwanted antibodies against the oligomerisation domain.

A Survey of Preclinical Studies Evaluating Nanoparticle-Based Vaccines Against Non-Viral Sexually Transmitted Infections

A worldwide estimate of over one million STIs are acquired daily and there is a desperate need for effective preventive as well as therapeutic measures to curtail this global health burden. Vaccines have been the most effective means for the control and potential eradication of infectious diseases; however, the development of vaccines against STIs has been a daunting task requiring extensive research for the development of safe and efficacious formulations. Nanoparticle-based vaccines represent a promising platform as they offer benefits such as targeted antigen presentation and delivery, co-localized antigen-adjuvant combinations for enhanced immunogenicity, and can be designed to be biologically inert. Here we discuss promising types of nanoparticles along with outcomes from nanoparticle-based vaccine preclinical studies against non-viral STIs including chlamydia, syphilis, gonorrhea, and recommendations for future nanoparticle-based vaccines against STIs.

Immunochemistry-Based Diagnosis of Extrapulmonary Tuberculosis: A Strategy for Large-Scale Production of MPT64-Antibodies for Use in the MPT64 Antigen Detection Test.

Tuberculosis (TB) is a global health problem. The immunohistochemistry (IHC)-based MPT64 antigen detection test has shown promising results for diagnosing extrapulmonary TB in previous studies. However, the anti-MPT64 antibody currently used in the test is in limited supply, and reproduction of a functional antibody is a prerequisite for further large-scale use. Various antigen-adjuvant combinations and immunisation protocols were tested in mice and rabbits to generate monoclonal and polyclonal antibodies. Antibodies were screened in IHC, and the final new antibody was validated on clinical human specimens. We were not able to generate monoclonal antibodies that were functional in IHC, but we obtained multiple functional polyclonal antibodies through careful selection of antigen-adjuvant and comprehensive screening in IHC of both pre-immune sera and antisera. To overcome the limitation of batch-to-batch variability with polyclonal antibodies, the best performing individual polyclonal antibodies were pooled to one final large-volume new anti-MPT64 antibody. The sensitivity of the new antibody was in the same range as the reference antibody, while the specificity was somewhat reduced. Our results suggest that it possible to reproduce a large-volume functional polyclonal antibody with stable performance, thereby securing stable supplies and reproducibility of the MPT64 test, albeit further validation remains to be done.

Vaccinations in the newborn.

Neonatal immunisation includes vaccination in the first 4 weeks of life (Neonatal period) as well as in high-risk preterm infants in the first few months (until 44 weeks corrected gestational age). Neonates have an immature immune system, which renders them highly susceptible to life-threatening infections. This highlights the importance of vaccination in this vulnerable population; however, at the same time also making it challenging because of their inability to generate a protective immune response. Other challenges include interference from maternal antibodies and excessive skewing towards T Helper Cell Type 2 (Th2) immunity. Despite these challenges, several vaccines have been developed and proven safe and effective at birth. Presently, there are 3 vaccines - Hepatitis B vaccine, Bacillus Calmette-Guerine (BCG) and Oral Polio vaccine (OPV) widely used in neonates, which provides evidence that certain antigen-adjuvant combinations can elicit protective neonatal responses. This review focusses on current vaccinations in neonates, including preterm infants and highlights some novel approaches to enhance neonatal vaccination.

Multi-functional nanocomplex codelivery of Trp2 and R837 to activate melanoma-specific immunity.

Antigen-adjuvant combination could induce a protective and long-lasting anti-tumor immune response. However, exploiting system which could co-deliver melanoma antigen peptide Trp2 (Tyrosinase-related protein 2) and Toll-like-receptor-7 (TLR7) agonists imiquimod (R837) both are poor aqueous solubility is still challenging. Our new nanocomplex was explored for specific delivery of Trp2 and R837 into antigen-presenting cells (APCs). R837 was loaded into mannosylated-β-cyclodextrin (Man-CD) to target dendritic cells (DCs) by binding mannose receptors (MR) on DCs. A fusion peptide (WT) was constructed by incorporating the amino acid region of TAT (cell-penetrating peptide) into Trp2 to improve the TAT-mediated intracellular efficiency. Negatively charged sodium alginate (SA), a biocompatible polymer, which can induce adjuvant responses by affecting the functions of DCs, was complexed with Man-CD/R837 and WT via physical adsorption. The optimized nanocomplex promoted the cellular uptake and showed remarkable efficacy to enhance the secreting of Th1-cytokines. This multi-functional nanocomplex system may allow effective targeting-codelivery of multi-hydrophobic drugs and be a promising subunit vaccine candidate as a potential prevention action of tumor.

Ginseng Stem-and-Leaf Saponin (GSLS)-Enhanced Protective Immune Responses Induced by Toxoplasma gondii Heat Shocked Protein 70 (HSP70) Against Toxoplasmosis in Mice.

Toxoplasma gondii is an obligate intracellular protozoan parasite and is able to infect birds and mammals including humans. In order to find effective antigen-adjuvant combinations that can boost the immunogenicity and protection of antigen vaccines against toxoplasmosis, we examined the protective efficacy in mice immunized with recombinant protein HSP70 when co-administered with ginseng stem-and-leaf saponins (GSLS) isolated from Panax ginseng . All immunized mice produced significantly high levels of specific antibodies against rTgHSP70, and splenocytes from mice presented strong proliferative immune responses. Vaccinated mice displayed a significantly increased percentage of CD4+ and CD8+ T cells, indicating a strong immune response was triggered. The cellular and humoral immune responses were enhanced, which could be reflected of the increased mRNA levels of IFN-γ and IL-4, respectively. Immunization with rTgHSP70 and GSLS prolonged survival time of the treated mice compared to the controls, which died within 6 days after challenge with the virulent T. gondii RH strain. Our data demonstrate that by addition with GSLS, rTgHSP70 induced a strong immune response and provided partial protection against T. gondii ; therefore GSLS could be used as a promising vaccine adjuvant against acute toxoplasmosis.

Anti-PD-1 inhibits Foxp3+ Treg cell conversion and unleashes intratumoural effector T cells thereby enhancing the efficacy of a cancer vaccine in a mouse model.

The co-inhibitory molecule PD-1 suppresses T cell responses and has been targeted in the treatment of cancer. Here, we examined the role of PD-1 in regulating the balance between regulatory and effector T cells and whether blocking PD-1 could enhance tumour vaccine-induced protective immunity. A significantly higher proportion of tumour-resident T cells expressed PD-1 and Foxp3 compared with T cells in the tumour circulation or draining lymph nodes, and this correlated with a lower frequency of IFN-γ- and TNF-secreting CD8 T cells. Blocking PD-1 with a specific antibody reduced Foxp3+ regulatory T (Treg) cell induction and enhanced proliferation, cytokine production, and tumour killing by CD8 T cells. Treatment of CT26 tumour-bearing mice with anti-PD-1 in combination with a vaccine, comprising heat-shocked irradiated tumour cells and a TLR 7/8 agonist, significantly reduced tumour growth and enhanced survival. Furthermore, surviving mice resisted tumour re-challenge. The rejection of tumours in mice treated with the anti-PD-1 vaccine combination was associated with a reduction in tumour-infiltrating Treg cells and enhancement of IFN-γ-secreting CD8 T cells. Our findings demonstrate that high PD-1 expression correlates with increased tumour-infiltrating Treg cells and reduced effector T cells and that when combined with a potent antigen-adjuvant combination, blocking PD-1 effectively enhances anti-tumour immunity.

Efficacy of a trans-sialidase-ISCOMATRIX subunit vaccine candidate to protect against experimental Chagas disease

Recombinant protein vaccines are safe but elicit low immunological responses. The new generation of adjuvants is currently reversing this situation. Here, a new antigen–adjuvant combination for protection against experimental Chagas disease was assessed. The antigen used in the formulation was a glycosylated mutant inactive trans-sialidase (mTS) that was previously proven to be highly protective against Trypanosoma cruzi infection; here, we show that it can be produced in large quantities and high quality using Pichia pastoris. The adjuvant used in the formulation was ISCOMATRIX (IMX), which was found to be effective and safe in human clinical trials of vaccines designed to control other intracellular infections. Fifteen days after the third immunization, mice immunized with mTS-IMX showed a TS-specific IgG response with titers >106 and high avidity, an increased IgG2a/IgG1 ratio, significant delayed-type hypersensitivity (DTH) reactivity, a balanced production of IFN-γ and IL-10 by splenocytes and a strong IFN-γ secretion by CD8+ T lymphocytes. When these mice where challenged with 1000 trypomastigotes of T. cruzi, all mTS-IMX immunized mice survived, whereas mice immunized with mTS alone, IMX or PBS exhibited high mortality. Remarkably, during acute infection, when the parasitemia is highest in this infection model (day 21), mTS-IMX immunized mice had ∼50 times less parasitemia than non-immunized mice. At this moment and also in the chronic phase, 100 days after infection, tissue presented ∼4.5 times lower parasite load and associated inflammatory infiltrate and lesions. These results indicate that protection against Chagas disease can be achieved by a protein antigen–adjuvant mTS formulation that is compatible with human medicine. Therefore, the current formulation is a highly promising T. cruzi vaccine candidate to be tested in clinical trials.

In vivo monitoring of the immune response to mIgG conjugated to iron oxide nanoparticles using the pulp (dermis) of growing feathers as a test-tissue in chickens, (IRC8P.495)

Abstract Biomedical applications of nanoparticles (NPs) are growing. Data on the biological activities of NPs are, however, limited. We are evaluating the growing feather (GF) in chickens as a test-site (US-Patent 8,216,551; GF Erf) to monitor in vivo activities of NPs. In this study, the immune response to mouse (m) IgG initiated with different antigen-adjuvant combinations was examined. Specifically, 8-week-old male chickens were immunized i.m. with 0.1 mL of mIgG alone, a mixture of Alum adjuvant & mIgG, or mIgG conjugated to iron oxide NP (IONP-mIgG, 10 nm) (n=6 birds/treatment). To monitor the immune response, 0.01 mL mIgG was injected into the pulp (dermis) of 20 GF/bird ten days post-immunization. GF were collected before (0 h) and at 0.25, 1, 2, 3, 5, and 7 d post-GF-injection for flow cytometry, mRNA and histological analysis. Flow-cytometric cell-population analysis of pulp cell suspensions revealed higher levels (%, P<0.001) of lymphocyte infiltration [due to B cells (P<0.001), alpha-beta TCR+ T cells (P<0.005) and CD4+ T cells (P<0.05)] in birds immunized with IONP-mIgG compared to Alum & mIgG or mIgG. These data demonstrate the effectiveness of GF as a test-site to monitor qualitative and quantitative aspects of immune activities in vivo and the usefulness of this minimally invasive procedure as a window into biological activities of NP. Moreover, the results suggest effective adjuvant activities of IONP in stimulating a primary immune response to protein antigen.

Enhancement of protective immune response to recombinant Toxoplasma gondii ROP18 antigen by ginsenoside Re

Toxoplasma gondii, the etiological agent of toxoplasmosis, is an obligate intracellular protozoan parasite that infects a variety of mammals including humans. In an attempt to find new antigen-adjuvant combinations that enhance the immunogenicity of antigen candidates for toxoplasma vaccines, we analyzed the potent protection in mice immunized with recombinant protein ROP18 when co-administered with ginsenoside Re, a most important component isolated from Panax ginseng. All immunized mice produced specific anti-rROP18 immunoglobulins, with high levels of IgG antibody and a mixed IgG1/IgG2a response, with predominance of IgG1 production. The cellular and humoral immune responses were associated with the production of IFN-γ and IL-4 cytokines respectively. Vaccinated mice displayed a significantly increased survival time compared with control mice which died within 6days of challenge with RH strain. Our data demonstrate that by addition of ginsenoside Re, the rROP18 triggered a stronger humoral and cellular response against T. gondii, and that Re is a promising vaccine adjuvant against toxoplasmosis, deserves further evaluation and development.

Chlamydia vaccines: recent developments and the role of adjuvants in future formulations

Bacteria of the genus Chlamydia cause a plethora of ocular, genital and respiratory diseases that continue to pose a considerable public health challenge worldwide. The major diseases are conjunctivitis and blinding trachoma, non-gonococcal urethritis, cervicitis, pelvic inflammatory disease, ectopic pregnancy, tubal factor infertility and interstitial pneumonia. The rampart asymptomatic infections prevent timely and effective antibiotic treatments, and quite often clinical presentation of sequelae is the first evidence of an infection. Besides, significant broad coverage in population screening and treatment is economically and logistically impractical, and mass education for public awareness has been ineffective. The current medical opinion is that an efficacious prophylactic vaccine is the best approach to protect humans from chlamydial infections. Unfortunately, a human vaccine has yet to be realized despite successful veterinary vaccines. Fortunately, recent advances in chlamydial immunobiology, cell biology, molecular pathogenesis, genomics, antigen discovery and animal models of infections are hastening progress toward an efficacious vaccine. Thus, it is established that Chlamydia immunity is mediated by T cells and a complementary antibody response, and several potential vaccine candidates have been identified. However, further advances are needed in effective vaccine delivery systems and safe potent adjuvants to boost and sustain immune responses for long-lasting protective immunity. This article focuses on the current status of human chlamydial vaccine research, specifically how application of new delivery systems and human compatible adjuvants could lead to a timely achievement of efficacious Chlamydia vaccines. The ranking of the candidate vaccine antigens for human vaccine development will await the availability of results from studies in which the antigens are tested by comparable experimental standards, such as antigen–adjuvant combination, route of delivery and possible toxicity.

Combination of CpG-oligodeoxynucleotides with recombinant ROP2 or GRA4 proteins induces protective immunity against Toxoplasma gondii infection

Synthetic oligodeoxynucleotides containing unmethylated CpG motifs (CpG-ODN) have been characterized as Th1-promoting immunopotentiators, an adjuvant activity desirable for vaccination against intracellular parasites like Toxoplasma gondii. In an attempt to find new antigen–adjuvant combinations that enhance the immunogenicity of antigen candidates for toxoplasma vaccines, we analyzed the extent of protection in mice immunized with ROP2 and GRA4 recombinant proteins when co-administered with CpG-ODN. Both GRA4+CpG-ODN and ROP2+CpG-ODN formulations were shown to induce a strong humoral Th1-biased response characterized by a high IgG2a to IgG1 antibody ratio. Both vaccination regimens led to increased secretion of IFN-γ and IL-10, and negligible amounts of IL-4, upon specific re-stimulation of spleen cells from these groups of mice. After a non-lethal challenge with tissue cysts of a moderately virulent strain, only the brains from mice vaccinated with ROP2 or GRA4 in combination with CpG-ODN showed a significant reduction (63% and 62%, respectively) in their parasite load compared to the controls. The rate of protection obtained with GRA4+ROP2+CpG-ODN resulted equivalent (66%) to those achieved with the single antigens plus CpG-ODN. Taken together, these results indicate that CpG-ODN is an important candidate adjuvant for use in potential multicomponent anti-T. gondii vaccines for animals and humans.

Safety and efficacy of neonatal vaccination

Newborns have an immature immune system that renders them at high risk for infection while simultaneously reducing responses to most vaccines, thereby posing challenges in protecting this vulnerable population. Nevertheless, certain vaccines, such as BCG and Hepatitis B vaccine, do demonstrate safety and some efficacy at birth, providing proof of principal that certain antigen-adjuvant combinations are able to elicit protective neonatal responses. Moreover, birth is a major point of healthcare contact globally meaning that effective neonatal vaccines achieve high population penetration. Given the potentially significant benefit of vaccinating at birth, availability of a broader range of more effective neonatal vaccines is an unmet medical need and a public health priority. This review focuses on safety and efficacy of neonatal vaccination in humans as well as recent research employing novel approaches to enhance the efficacy of neonatal vaccination.

Chemical glycosylation in the synthesis of glycoconjugate antitumour vaccines

Therapeutic vaccines derived from carbohydrate antigen-adjuvant combinations are a promising approach for cancer immunotherapy. One of the critical limitations in this area is access to sufficient quantities of tumour-associated carbohydrate antigens and glycoconjugate adjuvants. At present, availability of the complex oligosaccharide constructs that are needed for the systematic design and evaluation of novel vaccine formulations relies on de novo chemical synthesis. The use of both state-of-the-art and emerging glycosylation technologies has led to significant advances in this field, allowing the clinical exploration of carbohydrate-based antigens in the treatment of cancer.

PRELIMINARY OBSERVATIONS ON THE EFFICACY OF A RECOMBINANT MULTISTAGE PLASMODIUM FALCIPARUM VACCINE IN AOTUS NANCYMAI MONKEYS

A vaccine trial was conducted to determine the efficacy of a multicomponent candidate vaccine, FALVAC-1, against Plasmodium falciparum in Aotus nancymai monkeys. After two immunizations, animals were challenged intravenously with parasites of the Vietnam Oak Knoll (FVO) strain of P. falciparum. The primary outcome was to determine the protective response of the monkeys to immunization with the FALVAC-1 antigen produced in baculovirus when combined with different adjuvants (alum, QS-21, ASO2a, CRL1005/oil, and CRL1005/saline) as compared with FALVAC-1 with FCA/FIA and antigen alone. When compared with the monkeys immunized with FALVAC-1 alone, FALVAC-1 with FCA/FIA reduced the mean parasite count (to Day 11), reduced the mean accumulated parasitemia (through Day 11), and extended the number of days to treatment. None of the other 5 antigen-adjuvant combinations were able to provide discernable levels of protection based on log(parasitemia) and log(cumulative parasitemia) to Day 11.

Cytokines and antibody subclass associated with protective immunity against blood-stage malaria in mice vaccinated with the C terminus of merozoite surface protein 1 plus a novel adjuvant.

A blood-stage malaria antigen comprising the C terminus of merozoite surface protein 1 fused to glutathione S-transferase, combined with an adjuvant formulation containing squalane, Tween 80, and pluronic L121 (AF), administered subcutaneously protected mice against death from a lethal Plasmodium yoelii infection. The protection induced by this antigen-adjuvant combination was compared with that induced by the antigen plus saponin in terms of survival from the lethal infection and clearance of parasitemia. The levels of gamma interferon and interleukin-4 in spleens were measured as indicators of Th1 and Th2 cell activation, and antibody classes and subclasses were determined by immunofluorescence. With a 10-micrograms dose of antigen and AF as adjuvant, all mice recovered, but with saponin as the adjuvant, there were only a few survivors. With 30 micrograms of antigen plus AF, the peak parasitemias were 10-fold lower than those with 10 micrograms; with saponin, survival was slightly improved. The levels of both gamma interferon and interleukin-4 rose more rapidly and to higher levels with AF as the adjuvant than with saponin, and the same was true for immunoglobulin G1 (IgG1), IgG2a, and IgG2b subclasses. Thus, in terms of both cytokine production and antibody levels, AF is a more potent adjuvant for a malaria vaccine than is saponin.

Production of guinea pig IgG1 homocytotropic antibodies to hapten-conjugated homologous serum albumin with different adjuvant combinations.

The effects of various antigen-adjuvant combinations on the production of IgG1 antibody was investigated with p-aminobenzoate conjugated to guinea pig serum albumin. The results demonstrate the importance that initial exposure of antigen-adjuvant combination has on IgG1 production. Further, there is a synergistic effect when antigen adsorbed to alum and antigen with complete Freund's adjuvant were used in conjunction with one another. The result of this effect is discussed within the context of T and B cell activation by antigen-adjuvant combinations.