Prophylactic Immunization Research Articles

Protecting the vulnerable: SARS-CoV-2 vaccination in immunosuppressed patients with interstitial lung disease

Protecting the vulnerable: SARS-CoV-2 vaccination in immunosuppressed patients with interstitial lung disease

Anti-cancer immunoprotective effects of immunization with hydatid cyst wall antigens in a non-immunogenic and metastatic triple-negative murine mammary carcinoma model

Cancer vaccines have gained lots of attention as the future of cancer treatment. However, poor immunogenicity of tumor-associated antigens often fails to induce an efficient immune response against the tumor. Strange anti-tumor immune responses at the parasite-infected patients due to cross-reactivity have been reported in various studies. Therefore, parasite antigens with significant immunogenicity and high epitope homology with cancer antigens may activate a strong immune response against cancer cells. Herein, the sera of immunized rabbits with the hydatid cyst wall (HCW) antigens were incubated with 4 T1 mammary carcinoma cells to investigate cross-reactivity between the HCW antigens antisera and surface antigens of the breast cancer cells. Also, the SDS-PAGE profile of HCW antigens was prepared and incubated with the breast cancer patients’ sera and considerable reactivity was observed between their sera and a specific band (~27/28 kDa) according to Western blotting analyzes. Then, the protein bands with cross-reactivity with breast cancer patients’ sera were utilized for prophylactic immunizations of Balb/c mice. The immunoprotective effect of immunization with the HCW antigens caused significant inhibition of 4 T1 breast tumor growth, decrease of metastasis, and enlargement of the tumor-bearing mice survival time in comparison with PBS and pure immune adjuvant injected groups. Mass spectrometry analysis showed that the ~ 27/28 kDa band has numbers of proteins/polypeptides with a high degree of homology with cancer cells antigens which can be the reason for this cross-reactivity and anti-tumor immune response. Taking together, immunization with HCW antigens would be a promising approach in cancer immunotherapy after further investigations.

Difluoromethylornithine (DFMO), an Inhibitor of Polyamine Biosynthesis, and Antioxidant N-Acetylcysteine Potentiate Immune Response in Mice to the Recombinant Hepatitis C Virus NS5B Protein

Hepatitis C virus (HCV) is one of the main triggers of chronic liver disease. Despite tremendous progress in the HCV field, there is still no vaccine against this virus. Potential vaccines can be based on its recombinant proteins. To increase the humoral and, especially, cellular immune response to them, more effective adjuvants are needed. Here, we evaluated a panel of compounds as potential adjuvants using the HCV NS5B protein as an immunogen. These compounds included inhibitors of polyamine biosynthesis and urea cycle, the mTOR pathway, antioxidants, and cellular receptors. A pronounced stimulation of cell proliferation and interferon-γ (IFN-γ) secretion in response to concanavalin A was shown for antioxidant N-acetylcysteine (NAC), polyamine biosynthesis inhibitor 2-difluoromethylornithine (DFMO), and TLR9 agonist CpG ODN 1826 (CpG). Their usage during the immunization of mice with the recombinant NS5B protein significantly increased antibody titers, enhanced lymphocyte proliferation and IFN-γ production. NAC and CpG decreased relative Treg numbers; CpG increased the number of myeloid-derived suppressor cells (MDSCs), whereas neither NAC nor DFMO affected MDSC counts. NAC and DFMO suppressed NO and interleukin 10 (IL-10) production by splenocytes, while DFMO increased the levels of IL-12. This is the first evidence of immunomodulatory activity of NAC and DFMO during prophylactic immunization against infectious diseases.

Anti‐fentanyl Vaccines and Monoclonal Antibodies are Effective in Prevention and Reversal of Fentanyl‐induced Overdose in Rats

The incidence of drug overdose deaths in the United States is an alarming public health threat that has accelerated during the COVID-19 pandemic, with over 80,000 deaths recorded in 2020. A substantial portion of this increase is attributable to the widespread access to fentanyl and other synthetic opioids, either used alone or encountered as adulterants in heroin or psychostimulants such as cocaine or methamphetamine. Vaccines and monoclonal antibodies (mAb) offer prophylactic and therapeutic interventions against opioid overdose by binding to the target opioids in serum, and reducing drug distribution to the brain. Here, the efficacy of anti-fentanyl vaccines and mAb for prevention and reversal of fentanyl-induced overdose was investigated in rats. Rats were immunized with a candidate vaccine consisting of a fentanyl-based hapten (F) conjugated to a diphtheria toxin carrier protein (CRM), or with a control vaccine. Rats were challenged subcutaneously with doses of 0.25 mg/kg fentanyl every 15 minutes to a cumulative dose of 2.25mg/kg, or until cardiac and/or respiratory arrest occurred. Prophylactic immunization with F-CRM was effective in counteracting fentanyl-induced respiratory depression, bradycardia, and antinociception; and reduced the incidence of mortality compared to control. Additionally, a murine anti-fentanyl mAb was isolated from mice immunized against fentanyl. Purified mAb had previously shown pre-exposure efficacy in protecting mice and rats from fentanyl challenges. To evaluate the efficacy of mAb in reversing fentanyl overdose post-exposure, rats were dosed with 0.1-0.5 mg/kg fentanyl, and 15 minutes later received 40 mg/kg mAb, 0.1 mg/kg naloxone, or both intravenously. Within 15 minutes, treatment with mAb reduced fentanyl-induced respiratory depression and bradycardia, and compared favorably to naloxone in terms of the degree and rate of onset of protection. One week later, rats retained mAb-induced protection from the effects of additional doses of fentanyl, as confirmed by a 10-fold reduction of the distribution of fentanyl to the brain compared to control. These results support translation of vaccines and mAb as medical interventions to prevent and reverse opioid overdose related to fentanyl and its analogs.

Modular complement assemblies for mitigating inflammatory conditions

Complement protein C3dg, a key linkage between innate and adaptive immunity, is capable of stimulating both humoral and cell-mediated immune responses, leading to considerable interest in its use as a molecular adjuvant. However, the potential of C3dg as an adjuvant is limited without ways of controllably assembling multiple copies of it into vaccine platforms. Here, we report a strategy to assemble C3dg into supramolecular nanofibers with excellent compositional control, using β-tail fusion tags. These assemblies were investigated as therapeutic active immunotherapies, which may offer advantages over existing biologics, particularly toward chronic inflammatory diseases. Supramolecular assemblies based on the Q11 peptide system containing β-tail-tagged C3dg, B cell epitopes from TNF, and the universal T cell epitope PADRE raised strong antibody responses against both TNF and C3dg, and prophylactic immunization with these materials significantly improved protection in a lethal TNF-mediated inflammation model. Additionally, in a murine model of psoriasis induced by imiquimod, the C3dg-adjuvanted nanofiber vaccine performed as well as anti-TNF monoclonal antibodies. Nanofibers containing only β-tail-C3dg and lacking the TNF B cell epitope also showed improvements in both models, suggesting that supramolecular C3dg, by itself, played an important therapeutic role. We observed that immunization with β-tail-C3dg caused the expansion of an autoreactive C3dg-specific T cell population, which may act to dampen the immune response, preventing excessive inflammation. These findings indicate that molecular assemblies displaying C3dg warrant further development as active immunotherapies.

Microarray patches enable the development of skin-targeted vaccines against COVID-19.

The COVID-19 pandemic is a serious threat to global health and the global economy. The ongoing race to develop a safe and efficacious vaccine to prevent infection by SARS-CoV-2, the causative agent for COVID-19, highlights the importance of vaccination to combat infectious pathogens. The highly accessible cutaneous microenvironment is an ideal target for vaccination since the skin harbors a high density of antigen-presenting cells and immune accessory cells with broad innate immune functions. Microarray patches (MAPs) are an attractive intracutaneous biocargo delivery system that enables safe, reproducible, and controlled administration of vaccine components (antigens, with or without adjuvants) to defined skin microenvironments. This review describes the structure of the SARS-CoV-2 virus and relevant antigenic targets for vaccination, summarizes key concepts of skin immunobiology in the context of prophylactic immunization, and presents an overview of MAP-mediated cutaneous vaccine delivery. Concluding remarks on MAP-based skin immunization are provided to contribute to the rational development of safe and effective MAP-delivered vaccines against emerging infectious diseases, including COVID-19.

EpiMix Based Novel Vaccine Candidate for Shigella: Evidence of Prophylactic Immunity in Balb/c Mice.

Multidrug resistant Shigella is one of the leading causes of mortality in children and infants. Availability of vaccine could prevent the Shigella infection and reduce the mortality. Conventional approaches of vaccine development against shigellosis have not resulted in desirable vaccine. As shigellosis may be caused by multiple strains and serotypes, there is a need to develop a multivalent vaccine, capable of providing protection against multiple Shigella strains. To develop broad spectrum vaccine, we had previously derived a pool of conserved epitopes against Shigella by using multiple immunoinformatic tools. In this study, the identified conserved epitopes derived from the Outer Membrane Proteins A and C of Shigella were chemically synthesized, and the EpiMix made up of 5 epitopes coupled to a carrier protein, ovalbumin was developed and validated for its immunogenicity. The intramuscular immunization with EpiMix in Balb/c mice led to increase in EpiMix specific serum IgG, and significant increase in fecal IgA as well as in IL-4, IL-2and IFN-γ levels. Further, the EpiMix immunized mice showed protection when challenged against S. flexneri ATCC 12022 using the intraperitoneal route. Moreover, the analysis of cytokine profile and IFN-γ/IL4 ratio in post Shigella challenge immunized mice suggested the high levels of IFN-γ levels and possible dominance of Th1 response, playing pivotal role in the elimination of Shigella. Collectively, the results demonstrate the immunogenic potential and protective efficacy of the EpiMix in the murine shigellosis model. However, the detailed study and further optimisation of epitopes would substantiate the prospective use of EpiMix as a prophylactic candidate for vaccination.

Вакцинация детей в рамках национального календаря профилактических прививок в условиях пандемии COVID-19: проблемы и пути решения

Aim: to compare monthly implementation of immunization plan as a part of the National Immunization Schedule in 2019 and 2020 in children aged 0–18 years. Patients and Methods: monthly and annual form No. 5 “Information on prophylactic immunization” (from January 2019 to December 2020) was analyzed. Monthly implementation of immunization plan for each vaccine-preventable disease (whooping cough, poliomyelitis, mumps, rubella, measles, diphtheria tetanus and other) were analyzed separately. Results: in the beginning of the COVID-19 pandemic, a significant (8.8-fold) reduction in the coverage of children with immunization against vaccine-preventable diseases was reported compared to 2019 (р<0.0001). The reduction in the implementation of immunization schedule required urgent solutions to maintain a high level of herd immunity. The recovery of the required implementation of immunization plan against whooping cough and poliomyelitis was achieved by the end of 2020. The recovery of the required implementation of immunization plan against diphtheria and tetanus, measles, rubella, and mumps will be achieved in September 2020 (considering the National Immunization Schedule that includes revaccinations in preschool and school years). Conclusions: timely identification of the drawbacks of the organization of immunization and the prompt development of effective measures and implementation into clinical practice allowed for full implementation of immunization plan in children in 2020, despite the ongoing COVID-19 pandemic. KEYWORDS: vaccination, children, National Immunization Schedule, revaccination, COVID-19 pandemic. FOR CITATION: Girina A.A., Zaplatnikov A.L., Petrovskiy F.I., Tandalova L.P. Childhood vaccination as a part of the National Immunization Schedule during the COVID-19: problems and potential solutions. Russian Journal of Woman and Child Health. 2021;4(1):85–89. DOI: 10.32364/2618-8430-2021-4-1-85-89.

Специфическая иммунизация в профилактике рецидивного нефролитиаза после перкутанной нефролитотрипсии

Aim To study the effect of prophylactic immunization with combined Staphylococcus-Proteus-Pseudomonas vaccine in combination with the probiotic Bacillus subtilis on the development of recurrent nephrolithiasis in patients after percutaneous nephrolithotomy (PCNL). Materials and methods A total of 76 patients with a high infectious risk of stone formation in the early postoperative period after PCNL were included in the study. All patients received antibiotic therapy in accordance with the current guidelines as indicated. The patients were divided into 3 groups. In the comparison group (n=30), patients continued to receive antibiotics on the outpatient basis. In the group A (n=20), in addition to antibiotics, two-stage immunization with Staphylococcus-Proteus-Pseudomonas vaccine was performed. In the group B (n=26), additionally, patients received probiotic preparation Bactisporin dry, which is a lyophilized spore-forming bacteria Bacillus subtilis strain 3N. All participants had urine culture and stone analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. A study of specific antibodies level and factors of cellular and humoral immunity was carried out at specific time points. The duration of follow-up was 36 months. Results The study revealed a difference in the pathogens isolated from urine and infection stones in the same patient. This convincingly suggests that pathogens form biofilms in the stones and are not detected during routine urine culture. The main pathogens isolated from the stones were Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus epidermidis and Klebsiella pneumonia. Microbial associations were found in 25.7% of cases. The Staphylococcus-Proteus-Pseudomonas vaccine demonstrated good immunogenicity, its antigenic components provided protective properties against autologous and opportunistic bacteria. In addition to the ability to induce a specific response to antigens, the vaccine stimulated the phagocytic activity of neutrophils. Immunization with the combined Staphylococcus-Proteus-Pseudomonas vaccine in combination with probiotic results in uncomplicated postoperative course in 84% of patients. Hospital-acquired infectious and inflammatory complications were seen in 16.2% of cases. The recurrence of stone formation in this group of patients within 36 months was 8.2%. In patients receiving only combined Staphylococcus-Proteus-Pseudomonas vaccine after PCNL, the postoperative period was uneventful in 65% of cases. The rate of upper urinary tract infection was 35%, while the inflammatory process was accompanied by mild clinical manifestations and quickly resolved. Recurrence of stone formation during the follow-up was seen in 18% of patients. In the control group, the proportion of uncomplicated cases in patients with nephrostomy tube was 40%, the development of pyelonephritis was noted in 60% of cases, and recurrent stones within 36 months were diagnosed in 46.7% of cases. Conclusion Our results emphasize that the role of an infectious factor in the development of recurrent nephrolithiasis after PCNL is often underestimated. The use of a specific vaccination and the Bacillus subtilis preparation in patients with a high infectious risk of recurrent stone formation allows to achieve a significant reduction in the recurrence rate (more than 5 times) compared to the control group during 36 months of postoperative follow-up.

Prophylactic and therapeutic HBV vaccination by an HBs-expressing cytomegalovirus vector lacking an interferon antagonist in mice.

Cytomegalovirus (CMV)-based vaccines show promising effects against chronic infections in nonhuman primates. Therefore, we examined the potential of hepatitis B virus (HBV) vaccines based on mouse CMV (MCMV) vectors expressing the small HBsAg. Immunological consequences of vaccine virus attenuation were addressed by either replacing the dispensable gene m157 ("MCMV-HBsȍ) or the gene M27 ("ΔM27-HBs"), the latter encodes a potent IFN antagonist targeting the transcription factor STAT2. M27 was chosen, since human CMV encodes an analogous gene product, which also induced proteasomal STAT2 degradation by exploiting Cullin RING ubiquitin ligases. Vaccinated mice were challenged with HBV through hydrodynamic injection. MCMV-HBs and ΔM27-HBs vaccination achieved accelerated HBV clearance in serum and liver as well as robust HBV-specific CD8+ T-cell responses. When we explored the therapeutic potential of MCMV-based vaccines, especially the combination of ΔM27-HBs prime and DNA boost vaccination resulted in increased intrahepatic HBs-specific CD8+ T-cell responses and HBV clearance in persistently infected mice. Our results demonstrated that vaccines based on a replication competent MCMV attenuated through the deletion of an IFN antagonist targeting STAT2 elicit robust anti-HBV immune responses and mediate HBV clearance in mice in prophylactic and therapeutic immunization regimes.

Induction of Progenitor Exhausted Tissue-Resident Memory CD8+ T Cells Upon Salmonella Typhi Porins Adjuvant Immunization Correlates With Melanoma Control and Anti-PD-1 Immunotherapy Cooperation.

Immunotherapy has improved the clinical response in melanoma patients, although a relevant percentage of patients still cannot be salvaged. The search for the immune populations that provide the best tumor control and that can be coaxed by immunotherapy strategies is a hot topic in cancer research nowadays. Tumor-infiltrating TCF-1+ progenitor exhausted CD8+ T cells seem to grant the best melanoma prognosis and also efficiently respond to anti-PD-1 immunotherapy, giving rise to a TIM-3+ terminally exhausted population with heightened effector activity. We tested Porins from Salmonella Typhi as a pathogen associated molecular pattern adjuvant of natural or model antigen in prophylactic and therapeutic immunization approaches against murine melanoma. Porins induced protection against melanomas, even upon re-challenging of tumor-free mice. Porins efficiently expanded IFN-γ-producing CD8+ T cells and induced central and effector memory in lymph nodes and tissue-resident (Trm) T cells in the skin and tumors. Porins induced TCF-1+ PD-1+ CD8+ Trm T cells in the tumor stroma and the presence of this population correlated with melanoma growth protection in mice. Porins immunization also cooperated with anti-PD-1 immunotherapy to hamper melanoma growth. Importantly, the potentially protective Trm populations induced by Porins in the murine model were also observed in melanoma patients in which their presence also correlated with disease control. Our data support the use of cancer vaccination to sculpt the tumor stroma with efficient and lasting Trm T cells with effector activities, highlighting the use of Porins as an adjuvant. Furthermore, our data place CD8+ Trm T cells with a progenitor exhausted phenotype as an important population for melanoma control, either independently or in cooperation with anti-PD-1 immunotherapy.

Bursicon mediates antimicrobial peptide gene expression to enhance crowded larval prophylactic immunity in the oriental armyworm, Mythimna separata

It has been reported that a high population density alters insect prophylactic immunity. Bursicon plays a key role in the prophylactic immunity of newly emerged adults. In this paper, full-length cDNAs encoding the alpha and beta subunits of bursicon in Mythimna separata larvae (Msburs α and Msburs β) were identified. The cDNAs of Msburs α and Msburs β contain open reading frames (ORFs) encoding 145- and 139-amino acid residue proteins, respectively. Multiple alignment sequences and phylogenetic analysis indicated that Msbursicons (Msburs α and Msburs β) are orthologous to bursicons in other lepidopterans. The Msbursicons were expressed throughout all developmental states with higher relative expression during the egg, pupae, and adult stages. Msbursicons (Msburs α and Msburs β) were highly expressed in the ventral nerve cord and brain relative to other tested tissues. Msbursicon expression of larvae subject to high-density treatment (10 larvae per jar) was significantly increased compared with that of the larvae subject to low-density treatment (1 larva per jar) in the whole fourth and fifth instar stages. The trend in the expression of the antimicrobial peptide (AMP) genes cecropin C and defensin in the test stage was accorded and delayed with increased expression of bursicons. Silencing Msburs α (or Msburs β) expression by dsRNA injection in larvae subject to high-density treatment significantly decreased the expression levels of the cecropin C and defensin genes. Recombinant Msbursicon homodimers significantly induced the expression of the cecropin C and defensin genes. There was a notable decrease in the survival rate of the Msburs α (or Msburs β or Mscecropin C or Msdefensin) knockdown larvae infected by Beauveria thuringiensis. Our findings provide the first insights into how larval density mediates AMP gene expression, which subsequently affects the prophylactic immunity of insects under high-density conditions.

Progress in L2-Based Prophylactic Vaccine Development for Protection against Diverse Human Papillomavirus Genotypes and Associated Diseases.

The human papillomaviruses (HPVs) are a family of small DNA tumor viruses including over 200 genotypes classified by phylogeny into several genera. Different genera of HPVs cause ano-genital and oropharyngeal cancers, skin cancers, as well as benign diseases including skin and genital warts. Licensed vaccines composed of L1 virus-like particles (VLPs) confer protection generally restricted to the ≤9 HPV types targeted. Here, we examine approaches aimed at broadening the protection against diverse HPV types by targeting conserved epitopes of the minor capsid protein, L2. Compared to L1 VLP, L2 is less immunogenic. However, with appropriate presentation to the immune system, L2 can elicit durable, broadly cross-neutralizing antibody responses and protection against skin and genital challenge with diverse HPV types. Such approaches to enhance the strength and breadth of the humoral response include the display of L2 peptides on VLPs or viral capsids, bacteria, thioredoxin and other platforms for multimerization. Neither L2 nor L1 vaccinations elicit a therapeutic response. However, fusion of L2 with early viral antigens has the potential to elicit both prophylactic and therapeutic immunity. This review of cross-protective HPV vaccines based on L2 is timely as several candidates have recently entered early-phase clinical trials.

Immunological regulation by a β-adrenergic-like octopamine receptor gene in crowded larvae of the oriental Armyworm, Mythmina separata

Recent reports demonstrate that octopamine plays an important immunological role in crowded larvae of the Oriental Armyworm, Mythmina separata. We identified an octopamine receptor, the β-adrenergic-like gene (designated MsOctβ2R), with a 1191 bp open reading frame that encodes 396 amino acids and contains seven conserved hydrophobic transmembrane domains. Multiple sequence alignments and a phylogenetic analysis indicated that MsOctβ2R was orthologous to Octβ2R that is present in other lepidopterans. MsOctβ2R was expressed throughout all developmental stages with higher relative expression during the fourth instar and adult stages. MsOctβ2R was highly expressed in the ventral nerve cord and the fat body relative to other examined tissues. Elevated MsOctβ2R expression was observed in larvae that were under higher-density conditions (7 and 10 larvae per jar). Silencing MsOctβ2R expression via dsRNA injections in larvae from higher-density conditions significantly decreased phenoloxidase (PO) and lysozyme activity, total haemocyte counts, and survival rates against Beauveria bassiana infections (54.06%, 9.91%, 36.22%, and 23.53%, respectively) when compared with control larvae. These results suggest that high-density conditions might alter prophylactic immunity in larvae by regulating the MsOctβ2R gene in M. separara and provide new insights into density-dependent prophylaxis in insects.

Prospects for Improving Quality Evaluation of the Live Brucellosis Vaccine in Terms of Specific Activity

Prophylactic immunisation against brucellosis is part of the National Immunisation Schedule for Epidemic Settings. The immunisation is performed with a live vaccine—a lyophilized suspension of the Brucella abortus strain 19 BА in a stabilizing medium. The paper presents the results of quality evaluation of 9 batches of live brucellosis vaccine that were submitted to the Testing Centre for Evaluation of Medicinal Immunobiological Products’ Quality of the Federal State Budgetary Institution “Scientific Centre for Expert Evaluation of Medicinal Products” of the Ministry of Health of the Russian Federation for assessment of the product’s compliance with the established specifications. The paper also presents the results of evaluation of the passport information provided by the manufacturer for these batches. There is no doubt about the need for objective quality evaluation of brucellosis vaccines as well as about the significance of its improvement.The aim of study was to assess the prospects for improving quality evaluation of live brucellosis vaccines in terms of Specific activity (concentration of microbial cells, number of living microbial cells, number of cutaneous doses).Materials and methods: specific activity (concentration of microbial cells and number of living microbial cells) was determined by visual and microbiological methods using the industrial reference standard of brucellosis vaccine OSO 42-28-396-2018, batch 6 and the bacterial suspension of the Brucella abortus strain 19 BА acquired from the joint stock company Scientific and Production Association “Microgen” in 2016. The number of cutaneous doses in the brusellosis vaccine was determined by the calculation method. Statistical processing of the results was performed using Microsoft Excel.Results: there was a mismatch between the brucella concentration coefficient of 1.7×109 microbial cells/mL determined by comparison with the industrial reference standard of bacterial suspension turbidity, 10 IU and the actual concentration of microbial cells obtained in the study. According to preliminary results, the brucella concentration coefficient corresponding to the industrial reference standard of bacterial suspension turbidity, 10 IU can reach 3.0×109 microbial cells/mL.Conclusions: the obtained results can serve as a basis for amending the data on the brucella concentration coefficient in the Passport and the Instructions for use of the industrial reference standard of bacterial suspension turbidity, 10 IU, as well as the Specific activity section (concentration of microbial cells, number of living microbial cells, number of cutaneous doses) of the established specifications for the brucellosis vaccine. Before amending the information on the brucella concentration corresponding to 10 IU in the Passport and the Instructions for use of the reference standard of bacterial suspension turbidity (OSO 42-28-85P), additional studies should be performed with other types of brucella.

The influence of the anti-stress pharmacological complex “SPAO” on the effectiveness of preventive immunization of chickens against Newcastle disease

Methodology. The assessment of the appropriateness of using the anti-stress pharmacological complex of SPAO for prophylactic immunization of hens with egg productivity against Newcastle disease was started at 45 days old before intramuscular vaccination against Newcastle disease with Gallimune ND vaccine at 47–48 days old. SPAO-complex — stress protector antioxidant — pharmacological complex for birds was developed at the Department of Morphology, Physiology and Pharmacology of the South Ural State Agrarian University, which was used at a dose of 185 mg/kg of body weight according to the scheme with water through a system of medications two days before, on the day of exposure and within two days after exposure to stress factors. Antibodies to the Newcastle disease virus were tested by hem agglutination inhibition test. Results. As a result, it was proved that vaccinations by parenteral administration of vaccines cause the development of non-specific adaptive reactions in chickens. The level of development of reactions corresponds to the formation of stress. The use of the SPAO-complex reduces the immunosuppressive effect of the hypothalamic-pituitaryadrenal system during the development of adaptive reactions in birds under industrial stresses. In the prophylaxis of Newcastle disease chickens of the egg productivity direction on the background of stress prevention by the pharmacological complex of SPAO antibody production is more uniform throughout the herd. In the experimental group, titers are in the range of 9.40–13.40 log2, in the control group — 8.52–12.36. The level of statistical differences in the intensity of antibody production log2 is at the level of P = 0.0277. After transferring the hens to the production shop in the experimental group a higher level of productivity is noted compared to the control: egg production in the experimental group is 2.04% higher, the livestock safety over the period from 105 to 250 days of life of hens reached the highest values in the experimental group and amounted to 99.72%, in the control group — 99.44%.

A small molecule based immunotherapy targeting IL-10/STAT3 praxis to augment the potential of rBCG30 vaccine against experimental tuberculosis

Abstract Several recent studies have contributed to our understanding of the need for host directed immunotherapeutic strategies against Mycobacterium tuberculosis (Mtb), either concomitantly with classical chemotherapy or as standalone intervention. Nevertheless, most of these studies were focused on enhancing chemotherapeutic potential of standard anti-tuberculosis chemotherapy and only few were aimed towards improving prophylactic immunity offered either by BCG or its potential replacements. In the present study we tried to address the potential gap with the aim to improve anti-TB immunity offered by 2nd generation rBCG30 vaccine. We attempted to bolster rBCG30 invoked anti-TB immunity in mice through modulation of the IL-10/STAT3 interaction mediated anti inflammatory program. An immunomodulator of IL-10/STAT3 signaling axis was administered in immunized mice to reinforce the anti-pathogen capabilities of mononuclear phagocyte system with expectation to improve T cell mediated secondary immune responses. We envisage that modulation of this crucial praxis may result in reduced expansion of pathogen permissive AAMs/AADCs (Alternatively Activated Monocytes/Macrophages/DCs) and augmented abundance of pro-inflammatory CAMs/CADCs (Classically Activated Monocytes/Macrophages/DCs), the cells that resist establishment of successful Mtb infection. The study was also aimed towards generating optimum activated APCs early either after vaccination or infection, which can process and present mycobacterial antigens more efficiently to T cells.

Design and adjuvant formulation of a mutant HPV-E7 protein devoid of transforming/oncogenic properties but retaining high anti-tumor cellular activities as a candidate immunotherapeutic vaccine - Bioinformatics and in vivo analyses

Background & Aim Infection with Human papillomavirus (HPV) is major cause of the cervical carcinoma. Approved vaccines based on induction of antibodies (Abs) against HPV-L1 capsid protein provide prophylactic immunity against infection but no therapeutic effect on already infected individuals who might develop cancer. Alternatively, for anti-tumor immunotherapy purposes, induction of cellular immune responses against HPV oncogenic proteins, especially E7 is required. The oncogenic potential of HPV-E7 antigen (Ag) however, promoted us to search for a modified (mutated/deleted) form of this Ag with eliminated transforming properties but retained antigenicity. Methods, Results & Conclusion Herein, we first present application of several immune-informatics tools to evaluate the changes in the antigenicity, MHC binding/processing and cellular responses of the reported HPV16-E7 mutations responsible for its main transformation capabilities including del 21-24, C24G, L67R, C58G,C91G, M84S, V69A, L79A, QKP96-98EEA and their various combinations. Finally, the HPV16-E7 with triple substitutions of C24G/L67R/C91G (mE7) which showed the maximal antigenic similarity profile to that of the wild type was synthesized and inserted in pET28a vector for expression of protein in E.coli. SDS-PAGE and WB analyses confirmed the expression of the 17 kDa recombinant mE7 protein which was purified by NI-NTA chromatography using the vector encoded 6xHis-taq. The E.coli-derived Ag in combination with human compatible adjuvants including: Montanaide ISA 50 (M50), ISA 720 (M720), Pluronic acid (F127) in mixture with Immunostimulatory CpG ODN 1826 (mE7+M50/CpG; mE7+M720/CpG; mE7+F127/CpG, respectively) was evaluated in immunized C57BL/6 mice. Evaluation of the lymphocytes replication by the Brdu method and cytokine measurements by ELISA indicated that all groups developed significant immune responses, but mice immunized with mE7+M50/CpG showed the highest stimulation index (0.4) and INF-γ levels (150 pg/ml) comparable to those immunized by mE7+complete/incomplete Freund adjuvants (C/IFA). Accordingly, the IL-4 cytokine levels were almost similar and lower (20 pg/ml) for all groups indicating a Th1 polarization. Results of the tumor-challenge studies on TC1 cells in tumor bearing mice showed complete reduction/inhibition of tumor growth for mE7+M50/CpG immunized mice, indicating the potency of this formulation as a candidate immunotherapeutic vaccine against HPV infection-induced cancer.

Oral immunization of BALB/c mice with recombinant Helicobacter pylori antigens and double mutant heat-labile toxin (dmLT) induces prophylactic protective immunity against H. pylori infection

Helicobacter pylori infection and associated diseases remain a major public health problem worldwide. Much effort has been made over the last several decades in vaccine development, but there is no licensed vaccine on the market. We have previously reported that oral immunization with H. pylori lysates and double mutant heat-labile toxin (dmLT) affords prophylactic protection against H. pylori infection in mice. In the present study, we investigated the effects of oral immunization with recombinant H. pylori protein antigens (NAP/UreA/UreB) and dmLT on H. pylori challenge in BALB/c mice. We found that oral immunization with candidate antigens and dmLT significantly reduced the gastric colonization of H. pylori 6 weeks after challenge, as compared to unimmunized mice. Moreover, the subunit vaccine appeared to provide a better protection than the bacterial lysate vaccine. The immunized mice showed enhanced antigen-specific lymphocyte proliferation, and serum IgG and mucosal IgA responses. Furthermore, the immunization increased the proportion of CD4+ IL-17+ lymphocytes in spleen and mesenteric lymph nodes, and enhanced the production of IL-17, IL-16, IL-6 and TNF-α in lymphocyte culture supernatants. Taken together, our results suggest that oral vaccination with recombinant H. pylori antigens (NAP/UreA/UreB) and dmLT confers more effective prophylactic protection against H. pylori infection than whole bacterial lysates in BALB/c mice. The reduction of H. pylori colonization was associated with the induction of antigen-specific Th17 and local mucosal IgA immune responses.

Abstract A14: Irreversible electroporation is an “in situ vaccine” and induces antitumor immune responses in pancreatic cancer

Abstract The pancreatic cancer (PC) microenvironment is notoriously immunosuppressive and responds poorly to conventional checkpoint blockade immunotherapy, necessitating a need to overcome this challenge. Irreversible electroporation (IRE) is a nonthermal method of inducing cell death that is currently being used clinically for selected patients with locally advanced PC. Using a robust syngeneic mouse model of PC using cells established from a tumor arising in an LSL-KrasG12D/+; LSL-Trp53R172H/+; PDX1Cre/+; LSL-ROSA26 Luc/+ mouse (KPC), we have demonstrated that IRE alone can serve as an “in situ vaccine” and induce systemic immune responses that prevent secondary tumor growth (prophylactic immunity). We have furthermore demonstrated that these effects are augmented by combination with innate immune activators and PD-1 checkpoint blockade. We observed a significant decrease in immunosuppressive myeloid-derived suppressor cells (MDSCs) (CD11b+, CD11c-, GR1+) at one week in the IRE and drug alone groups with an additive effect in the combination groups. We also saw a profound shift in the ratio of immunostimulatory M1 (MHC II+, CD206 lo): immunosuppressive M2 (MHC II -, CD206 hi) macrophages in the combination groups and a modest increase in CD8+ T cells with IRE alone that was almost doubled in the combination groups with a concomitant decrease in T regulatory cells. Using exome and RNA sequencing of the KPC tumors expressed, nonsynonymous mutations in these tumors were identified and 101 long peptides (20 mer) were generated harboring these mutations at positions 9 and 15. When the CD8+ T-cells post-IRE were incubated along with these mutant peptides and bone marrow-derived dendritic cells, we were able to observe that these T cells were reactive (Interferon gamma secretion) to these neoantigenic peptide pools. Further, we were able to identify specific mutant peptides against which T-cell response was stimulated post IRE that were similar or even better than vaccinating the mice with irradiated KPC cells, suggesting that IRE induced tumor neoantigen-specific prophylactic immune response. Specifically, local delivery of toll-like receptor 7 (TLR7) agonist (1V270) and agonistic CD40 antibody resulted in complete regression of treated primary tumors in 7/10 mice in the experimental group. Increased presence of CD8+ dendritic cells (CD45+ CD11C+) in the draining lymph node suggested that the agonistic CD40 antibody could have triggered increased dendritic cell maturation and cross-presentation. Moreover, it resulted in the complete elimination of the untreated secondary tumors in the group, which IRE as a monotherapy failed to achieve, showing that with appropriate combination strategies, successful therapeutic systemic antitumor immunity can also be achieved with IRE to be used for the management of metastatic PC in the near future. Citation Format: Jayanth S. Shankara Narayanan, Aaron Miller, Tomoko Hayashi, Partha Ray, Stephen Schoenberger, Dennis Carson, Rebekah White. Irreversible electroporation is an “in situ vaccine” and induces antitumor immune responses in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A14.