Elicitation Of Immune Responses Research Articles

Association of the Single Nucleotide Polymorphism 19216T/C in the TLR2 Gene (rs3804099) with Entamoeba histolytica/Entamoeba dispar/Entamoeba moshkovskii Infection Among Lebanese Children.

Toll-like receptors (TLRs), particularly the TLR2, take part in the elicitation of immune responses against Entamoeba histolytica. This study aimed to investigate the relationship between a specific polymorphism called rs3804099 in the TLR2 gene and E. histolytica/E. dispar/E. moshkovskii infection among Lebanese children. A case-control study encompassed 180 participants including 68 children with amebiasis and 112 matched controls. Blood samples were collected, and genomic DNA was extracted using the classical proteinase K digestion and phenol-chloroform extraction method. The variant rs3804099 was examined using the Amplification Refractory Mutation System Polymerase Chain Reaction. The accuracy of the genotyping was supported by sequencing 5% of samples. The TLR2 rs3804099 polymorphism was identified in the studied population, and the observed genotypic distributions were consistent with Hardy-Weinberg equilibrium (P > 0.05). The frequency of the rare CC genotype was significantly higher in patients compared to the noninfected group (P < 0.01). In controls, the homozygous TT genotype was less frequent than the heterozygous CT genotype. The rare CC genotype was associated with a higher risk of amebiasis among children (odds ratios = 3.27, P = 0.002). These findings provide evidence supporting the association between the rs3804099 SNP in the TLR2 gene and E. histolytica/E. dispar/E. moshkovskii infection among Lebanese children.

Malaria transmission blocking activity of Anopheles stephensi alanyl aminopeptidase N antigen formulated with MPL, CpG, and QS21 adjuvants.

Malaria, a preventive and treatable disease, is still responsible for annual deaths reported in most tropical regions, principally in sub-Saharan Africa. Subunit recombinant transmission-blocking vaccines (TBVs) have been proposed as promising vaccines to succeed in malaria elimination and eradication. Here, a provisional study was designed to assess the immunogenicity and functional activity of alanyl aminopeptidase N (APN1) of Anopheles stephensi, as a TBV candidate, administered with MPL, CpG, and QS21 adjuvants in the murine model. The mouse groups were immunized with recombinant APN1 (rAPN1) alone or formulated with CpG, MPL, QS-21, or a combination of adjuvants (CMQ), and the elicited immune responses were evaluated after the third immunization. The standard membrane feeding assay (SMFA) measured the functional activity of antibodies against bacterial-expressed APN1 protein in adjuvanted vaccine groups on transmission of P. falciparum (NF54) to An. stephensi mosquitoes. Evaluation of mice vaccinated with rAPN1 formulated with distinct adjuvants manifested a significant increase in the high-avidity level of anti-APN1 IgG and IgG subclasses; however, rAPN1 induced the highest level of high-avidity anti-APN1 IgG1, IgG2a, and IgG2b antibodies in the immunized vaccine group 5 (APN1/CMQ). In addition, vaccine group 5 (receiving APN1/CMQ), had still the highest level of anti-APN1 IgG antibodies relative to other immunized groups after six months, on day 180. The SMFA data indicates a trend towards higher transmission-reducing activity in groups 2 and 5, which received the antigen formulated with CpG or a combination of three adjuvants. The results have shown the capability of admixture to stimulate high-affinity and long-lasting antibodies against the target antigen to hinder Plasmodium parasite development in the mid-gut of An. stephensi. The attained results authenticated APN1/CMQ and APN1/CpG as a potent APN1-based TBV formulation which will be helpful in designing a vaccine in the future.

Water-Soluble and Freezable Aluminum Salt Vaccine Adjuvant.

Particulate aluminum salts have long occupied a central place worldwide as inexpensive immunostimulatory adjuvants that enable induction of protective immunity for vaccines. Despite their huge benefits and safety, the particulate structures of aluminum salts require transportation and storage at temperatures between 2 °C and 8 °C, and they all have exquisite sensitivity to damage caused by freezing. Here, we propose to solve the critical freezing vulnerability of particulate aluminum salt adjuvants by introducing soluble aluminum salts as adjuvants. The solubility properties of fresh and frozen aluminum chloride and aluminum triacetate, each buffered optimally with sodium acetate, were demonstrated with visual observations and with UV-vis scattering analyses. Two proteins, A244 gp120 and CRM197, adjuvanted either with soluble aluminum chloride or soluble aluminum triacetate, each buffered by sodium acetate at pH 6.5-7.4, elicited murine immune responses that were equivalent to those obtained with Alhydrogel®, a commercial particulate aluminum hydroxide adjuvant. The discovery of the adjuvanticity of soluble aluminum salts might require the creation of a new adjuvant mechanism for aluminum salts in general. However, soluble aluminum salts might provide a practical substitute for particulate aluminum salts as vaccine adjuvants, thereby avoiding the risk of inactivation of vaccines due to accidental freezing of aluminum salt particles.

Phase 3 Safety and Immunogenicity Study of a Three-dose Series of Twenty-valent Pneumococcal Conjugate Vaccine in Healthy Infants and Toddlers.

Global pediatric immunization programs with pneumococcal conjugate vaccines (PCVs) have reduced vaccine-type pneumococcal disease, but a substantial disease burden of non-PCV serotypes remains. This phase 3, randomized (1:1), double-blind study evaluated safety and immunogenicity of 20-valent PCV (PCV20) relative to 13-valent PCV (PCV13) in healthy infants. Participants received 2 infant doses and a toddler dose of PCV20 or PCV13, with diphtheria-tetanus-acellular pertussis combination vaccine at all doses and measles, mumps, rubella and varicella vaccines at the toddler dose. Primary pneumococcal immunogenicity objectives were to demonstrate noninferiority (NI) of PCV20 to PCV13 for immunoglobulin G geometric mean concentrations after infant and toddler doses and percentages of participants with predefined serotype-specific immunoglobulin G concentrations after infant doses. Safety endpoints included local reactions, systemic events and adverse events. Overall, 1204 participants were vaccinated (PCV20, n = 601; PCV13, n = 603). One month after the toddler dose, 19/20 serotypes met NI for immunoglobulin G geometric mean concentrations; serotype 6B narrowly missed NI [PCV20/PCV13 geometric mean ratio: 0.57 (2-sided 95% confidence interval: 0.48-0.67); NI criterion: lower 2-sided 95% confidence interval >0.5]. Sixteen/twenty serotypes met NI for ≥1 primary objective after 2 infant doses. PCV20 induced robust opsonophagocytic activity, and boosting responses were observed for all vaccine serotypes, including those missing statistical NI. The safety/tolerability profile of PCV20 was like that of PCV13. PCV20 3-dose series in infants was safe and elicited robust immune responses. Based on these results and PCV13 experience, PCV20 3-dose series is expected to be protective for all 20 vaccine serotypes. NCT04546425.

Single-cell profiling of African swine fever virus disease in the pig spleen reveals viral and host dynamics

African swine fever, one of the major viral diseases of swine, poses an imminent threat to the global pig industry. The high-efficient replication of the causative agent African swine fever virus (ASFV) in various organs in pigs greatly contributes to the disease. However, how ASFV manipulates the cell population to drive high-efficient replication of the virus in vivo remains unclear. Here, we found that the spleen reveals the most severe pathological manifestation with the highest viral loads among various organs in pigs during ASFV infection. By using single-cell-RNA-sequencing technology and multiple methods, we determined that macrophages and monocytes are the major cell types infected by ASFV in the spleen, showing high viral-load heterogeneity. A rare subpopulation of immature monocytes represents the major population infected at late infection stage. ASFV causes massive death of macrophages, but shifts its infection into these monocytes which significantly arise after the infection. The apoptosis, interferon response, and antigen-presentation capacity are inhibited in these monocytes which benefits prolonged infection of ASFV in vivo. Until now, the role of immature monocytes as an important target by ASFV has been overlooked due to that they do not express classical monocyte marker CD14. The present study indicates that the shift of viral infection from macrophages to the immature monocytes is critical for maintaining prolonged ASFV infection in vivo. This study sheds light on ASFV tropism, replication, and infection dynamics, and elicited immune response, which may instruct future research on antiviral strategies.

A phase 3 randomized study to evaluate safety and immunogenicity of 20-valent pneumococcal conjugate vaccine in healthy Japanese infants

ObjectivesSafety and immunogenicity evaluation of a 4-dose series with 20-valent pneumococcal conjugate vaccine (PCV20). MethodsThis phase 3, double-blind study randomized healthy Japanese infants to receive 4 doses (3 infant doses, 1 toddler dose) of PCV20 by subcutaneous (SC) or intramuscular (IM) injection or 13-valent PCV (PCV13) SC. A primary immunogenicity objective was to demonstrate noninferiority of PCV20 SC to PCV13 SC for percentages of participants meeting predefined serotype-specific immunoglobulin G concentrations 1 month after Dose 3. The 7 additional PCV20 serotypes were compared with the lowest vaccine serotype result in the PCV13 group. Safety and tolerability were assessed as the primary safety objective. ResultsOverall, 668 participants were randomized (PCV20 SC, n = 226; PCV13 SC, n = 224; PCV20 IM, n = 218). The primary noninferiority objective for PCV20 SC to PCV13 SC was met for 11/13 matched and 5/7 additional serotypes. Additional data showed PCV20 SC and IM elicited robust functional opsonophagocytic activity and boosting responses to all 20 vaccine serotypes. PCV20 had a similar safety/tolerability profile to PCV13, although local reactions were less frequent with PCV20 IM. ConclusionsA 4-dose series of PCV20 SC or IM elicited immune responses expected to be protective against all 20 serotypes in Japanese infants. NCT04530838

VAX SAVVY: EXPLORING KNOWLEDGE, AWARENESS, AND PERCEPTIONS OF COVID-19 VACCINES AMONG VACCINATED STUDENTS

Abstract: Efforts to control COVID-19 continue to be a global priority, with ongoing research and public health measures aimed at minimizing the impact of the disease. In response to the COVID-19 pandemic, vaccines have emerged as a vital tool for mitigating its impact. This cross-sectional study involved the distribution of structured questionnaires to 165 vaccinated pharmacy students at a Malaysian public university. The study aimed to investigate the knowledge, awareness, and perceptions of COVID-19 vaccines among this specific cohort. The findings underscore a commendable level of knowledge (between 88.5% and 98.9%) as well as awareness and perceptions among the respondents regarding COVID-19 and its vaccines. They exhibited a clear understanding of the importance of vaccination in curbing the disease and expressed a willingness to educate others on its significance. Moreover, the respondents held positive perceptions about vaccine safety, the elicited immune response, and the protective efficacy conferred by vaccination. The study's outcomes reveal the pharmacy students' robust grasp of the importance, safety, and effectiveness of COVID-19 vaccines. Their positive attitude towards vaccination emphasizes its role in controlling the pandemic and underscores the importance of disseminating this knowledge. This study offers valuable insights into the knowledge and perceptions of a specific population group, contributing to our broader understanding of various aspects of COVID-19 vaccine.&#x0D; Keywords: Awareness; COVID-19; Knowledge; Perception; Students; Vaccine

Trivalent inactivated influenza vaccine response and immunogenicity assessment after one week and three months in repeatedly vaccinated adults

ABSTRACTBackgroundThe influenza vaccine administrated every year is a recommended infection control procedure for individuals above the age of six months. However, the effectiveness of repeated annual vaccination is still an active research topic. Therefore, we investigated the vaccine immunogenicity in two independent groups: previously vaccinated versus non-vaccinated individuals at three time points; prior vaccination, one week and three months post vaccination. The assessment enabled us to evaluate the elicited immune responses and the durability of the induced protection in both groups.Research Design and MethodsA research study was conducted to assess the immunogenicity of a single dose of Trivalent Inactivated Influenza Vaccine (A/H1N1, A/H3N2, and B) in 278 healthy adults aged between 32 and 66 years. Almost half of the participants, 140 (50·36%), received influenza vaccination at least once precursor to past influenza seasons. One blood sample was taken prior to vaccination for complete blood analysis and baseline immunogenicity assessment. The selected study participants received a single vaccine dose on the first day, and then followed up for three months. Two blood samples were taken after one week and three months post vaccination, respectively, for vaccine immunogenicity assessment.ResultsBefore vaccination, the seroprotection, defined as a hemagglutination-inhibiting titer of =>1:40, was detected for the three vaccine virus strains in 20 previously vaccinated participants (14·29%) [8·95%, 21·2%]. We compared the overall vaccine response for the three virus strains using a normalized response score calculated from linearly transformed titer measurements; the score before vaccination was 84% higher in the previously vaccinated group and the mean difference between the two groups was statistically significant. Three months post-vaccination, we didn’t find a significant difference in vaccine responses; the number of fully seroprotected individuals became 48 (34·29%) [26·48%, 42·77%] in the previously vaccinated group and 59 (42·75%) [34·37%, 51·45%] in the non-vaccinated group. The calculated response score was almost equal in both groups and the mean difference was no longer statistically significant.ConclusionOur findings suggest that a single dose of influenza vaccine is equally protective after three months for annually vaccinated adults and first-time vaccine receivers.

Salmonella Gallinarum mgtC mutant shows a delayed fowl typhoid progression in chicken

Salmonella Gallinarum (SG) provokes fowl typhoid, an infectious disease of acute clinical course that affects gallinaceous of any age and leads to high mortality rates. During the typhoid-like systemic infection of S. Typhimurium (STM) in mice, the bacterium expresses the mgtC gene, which is encoded in the Salmonella Pathogenecity Island – 3 (SPI-3). In this serovar, the function is linked to bacterial replication within macrophages, and its absence attenuates the pathogen. We hypothesized that deleting mgtC from SG genome would alter the microorganism pathogenicity in susceptible commercial poultry in a similar manner. Thus, the present study sought to elucidate the importance of mgtC on SG pathogenicity. For this, a mgtC-mutant lacking S. Gallinarum mutant was constructed (SG ΔmgtC). Its ability to replicate in medium that mimicries the mgtC-related intracellular environment of macrophages as well as in primary macrophages from chicken was evaluated. Moreover, the infection of susceptible chickens was performed to elucidate its pathogenicity and the elicited immune responses by measuring key interleukins by qRT-PCR and the population of macrophages and lymphocytes T CD4+ and CD8+ by means of immunohistochemistry. It was observed that mgtC was required for S. Gallinarum replication in acidified low-Mg2+ media and survival within macrophages. However, unlike its requirement for initial phase of STM infection in mice, lower bacterial counts were only observed at the late stage of macrophage infection without affecting the citotoxicity. Experiments showed that knocking-out the mgtC gene neither altered bacterial uptake by macrophages nor affects bacterial counts in liver and spleen and total chicken mortality. However, plotting a survival curve and analyzing the clinical-pathologic conditions, it was observed a slower progression of the disease in chickens infected by SG ΔmgtC compared to those challenged by the wild-type strain. Furthermore, the mRNA expression of IFN-γ and LITAF were similar between the infected chickens, but higher than in the uninfected group. The same was observed in macrophages and lymphocytes T CD4+ populations. On the other hand, the presence of lymphocytes T CD8+ was increased in the initial phase of the disease provoked by the wild-type strain over the mutant strain. We concluded that the role of mgtC in Fowl Typhoid in susceptible chickens differs from the role in typhoid-like infections in mammals. Thus, the deletion of mgtC gene from S. Gallinarum genome does not affect the overall pathogenicity, but slightly alters the pathogenesis.

Single-dose VSV-based vaccine protects against Kyasanur Forest disease in nonhuman primates.

Kyasanur Forest disease virus (KFDV) is an endemic arbovirus in western India mainly transmitted by hard ticks of the genus Haemaphysalis. KFDV causes Kyasanur Forest disease (KFD), a syndrome including fever, gastrointestinal symptoms, and hemorrhages. There are no approved treatments, and the efficacy of the only vaccine licensed in India has recently been questioned. Here, we studied the protective efficacy of a vesicular stomatitis virus (VSV)-based vaccine expressing the KFDV precursor membrane and envelope proteins (VSV-KFDV) in pigtailed macaques. VSV-KFDV vaccination was found to be safe and elicited strong humoral and cellular immune responses. A single-dose vaccination reduced KFDV loads and pathology and protected macaques from KFD-like disease. Furthermore, VSV-KFDV elicited cross-reactive neutralizing immune responses to Alkhurma hemorrhagic fever virus, a KFDV variant found in Saudi Arabia.

Safety and Immunogenicity of the BNT162b2 Vaccine Coadministered with Seasonal Inactivated Influenza Vaccine in Adults.

Vaccination is a critical tool for preventing coronavirus disease 2019 (COVID-19) and influenza illnesses. Coadministration of the COVID-19 vaccine, BNT162b2, with seasonal inactivated influenza vaccine (SIIV) can provide substantial benefits, including streamlining vaccine delivery. In this phase 3 study, healthy 18- to 64-year-olds who had received three previous doses of BNT162b2 were randomized (1:1) to the coadministration group (month0, BNT162b2 + SIIV; month1, placebo) or the separate-administration group (month0, placebo + SIIV; month1, BNT162b2). The primary immunogenicity objective was to demonstrate that the immune responses elicited by BNT162b2 and SIIV [measured by full-length S-binding immunoglobulin G (IgG) levels and strain-specific hemagglutination inhibition assay (HAI) titers against four influenza strains 1month post-vaccination, respectively] when coadministered were noninferior to those elicited by either vaccine administered alone, based on a prespecified 1.5-fold noninferiority margin [lower bound 95% CI for geometric mean ratio (GMR) > 0.67]. Reactogenicity and adverse event (AE) rates were evaluated. Randomized participants who received study vaccination (N = 1128; coadministration group, n = 564; separate-administration group, n = 564) had a median age of 39years. Model-adjusted GMRs for coadministration to separate administration were 0.83 (95% CI 0.77, 0.89) for full-length S-binding IgG levels and 0.89-1.00 (lower bound of all 95% CIs > 0.67) for the four influenza strain-specific HAI titers, with all endpoints achieving the prespecified noninferiority criterion. Reactogenicity events were mostly mild or moderate when BNT162b2 was coadministered with SIIV. Serious AEs were reported in < 1% of participants within 1month after any vaccination; none were considered vaccine-related. BNT162b2 coadministered with SIIV elicited immune responses that were noninferior to those elicited by BNT162b2 alone and SIIV alone, and BNT162b2 had an acceptable safety profile when coadministered with SIIV. The results of this study support the coadministration of BNT162b2 and SIIV in adults. ClinicalTrials.gov registration: NCT05310084.

Particulate antigens administrated by intranasal and intravaginal routes in a prime-boost strategy improve HIV-specific TFH generation, high-quality antibodies and long-lasting mucosal immunity

Mucosal surfaces serve as the primary entry points for pathogens such as SARS- CoV-2 coronavirus or HIV in the human body. Mucosal vaccination plays a crucial role to successfully induce long-lasting systemic and local immune responses to confer sterilizing immunity. However, antigen formulations and delivery methods must be properly selected since they are decisive for the quality and the magnitude of the elicited immune responses in mucosa. We investigated the significance of using particulate antigen forms for mucosal vaccination by comparing VLP- or protein- based vaccines in a mouse model.Based on a mucosal prime-boost immunization protocol combining (i) HIV- pseudotyped recombinant VLPs (HIV-VLPs) and (ii) plasmid DNA encoding HIV- VLPs (pVLPs), we demonstrated that combination of intranasal primes and intravaginal boosts is optimal to elicit both humoral and cellular memory responses in mucosa. Interestingly, our results show that in contrast to proteins, particulate antigens induce high-quality humoral responses characterized by a high breadth, long-term neutralizing activity and cross-clade reactivity, accompanying with high T follicular helper cell (TFH) response.These results underscore the potential of a VLP-based vaccine in effectively instigating long-lasting, HIV-specific immunity and point out the specific role of particulate antigen form in driving high-quality mucosal immune responses.

Shortwave-infrared-light-emitting probes for the in vivo tracking of cancer vaccines and the elicited immune responses

Tracking and imaging immune cells in vivo non-invasively would offer insights into the immune responses induced by vaccination. Here we report a cancer vaccine consisting of polymer-coated NaErF4/NaYF4 core–shell down-conversion nanoparticles emitting luminescence in the near-infrared spectral window IIb (1,500–1,700 nm in wavelength) and with surface-conjugated antigen (ovalbumin) and electrostatically complexed adjuvant (class-B cytosine–phosphate–guanine). Whole-body wide-field imaging of the subcutaneously injected vaccine in tumour-bearing mice revealed rapid migration of the nanoparticles to lymph nodes through lymphatic vessels, with two doses of the vaccine leading to the complete eradication of pre-existing tumours and to the prophylactic inhibition of tumour growth. The abundance of antigen-specific CD8+ T lymphocytes in the tumour microenvironment correlated with vaccine efficacy, as we show via continuous-wave imaging and lifetime imaging of two intravenously injected near-infrared-emitting probes (CD8+-T-cell-targeted NaYbF4/NaYF4 nanoparticles and H-2Kb/ovalbumin257-264 tetramer/PbS/CdS quantum dots) excited at different wavelengths, and by volumetrically visualizing the three nanoparticles via light-sheet microscopy with structured illumination. Nanoparticle-based vaccines and imaging probes emitting infrared light may facilitate the design and optimization of immunotherapies.

A prophage tail-like protein facilitates the endophytic growth of Burkholderia gladioli and mounting immunity in tomato.

A prophage tail-like protein (Bg_9562) of Burkholderia gladioli strain NGJ1 possesses broad-spectrum antifungal activity, and it is required for the bacterial ability to forage over fungi. Here, we analyzed whether heterologous overexpression of Bg_9562 or exogenous treatment with purified protein can impart disease tolerance in tomato. The physiological relevance of Bg_9562 during endophytic growth of NGJ1 was also investigated. Bg_9562 overexpressing lines demonstrate fungal and bacterial disease tolerance. They exhibit enhanced expression of defense genes and activation of mitogen-activated protein kinases. Treatment with Bg_9562 protein induces defense responses and imparts immunity in wild-type tomato. The defense-inducing ability lies within 18-51 aa region of Bg_9562 and is due to sequence homology with the bacterial flagellin epitope. Interaction studies suggest that Bg_9562 is perceived by FLAGELLIN-SENSING 2 homologs in tomato. The silencing of SlSERK3s (BAK1 homologs) prevents Bg_9562-triggered immunity. Moreover, type III secretion system-dependent translocation of Bg_9562 into host apoplast is important for elicitation of immune responses during colonization of NGJ1. Our study emphasizes that Bg_9562 is important for the endophytic growth of B. gladioli, while the plant perceives it as an indirect indicator of the presence of bacteria to mount immune responses. The findings have practical implications for controlling plant diseases.

Unmodified rabies mRNA vaccine elicits high cross-neutralizing antibody titers and diverse B cell memory responses

Licensed rabies virus vaccines based on whole inactivated virus are effective in humans. However, there is a lack of detailed investigations of the elicited immune response, and whether responses can be improved using novel vaccine platforms. Here we show that two doses of a lipid nanoparticle-formulated unmodified mRNA vaccine encoding the rabies virus glycoprotein (RABV-G) induces higher levels of RABV-G specific plasmablasts and T cells in blood, and plasma cells in the bone marrow compared to two doses of Rabipur in non-human primates. The mRNA vaccine also generates higher RABV-G binding and neutralizing antibody titers than Rabipur, while the degree of somatic hypermutation and clonal diversity of the response are similar for the two vaccines. The higher overall antibody titers induced by the mRNA vaccine translates into improved cross-neutralization of related lyssavirus strains, suggesting that this platform has potential for the development of a broadly protective vaccine against these viruses.

Simultaneous detection and characterization of antigen-specific B cells and CD4 +and CD8 +T cell responses upon natural infection and vaccination

Abstract An important lesson learned from the COVID-19 pandemic is the need for a more comprehensive assessment of the immune status upon a natural infection and vaccination. In addition to measuring antibody titers, monitoring the status of antigen-specific B and T cell must be taken into consideration to gain a more complete understanding of the elicited immune response and its durability. The objective of this study was to develop an immune monitoring assay based on the Dextramer ®technology for simultaneous monitoring of antigen-specific B and T cells (CD4 and CD8). The assay can be applied in combination with flow cytometry and in single cell RNA-seq analysis. The assay builds on Dextramer ®reagents displaying full-length spike protein (specific B cell assessment) or MHC-peptide complexes displaying virus peptides (detecting specific CD8 or CD4 T cells). The assay was evaluated by longitudinal assessment of immunity in individuals vaccinated against SARS-CoV-2 by combining the assay with flow cytometry (n=5) and the Chromium 10× RNA-seq platform (n=2). The latter allows an in-depth view of the immune status by simultaneous investigation of the transcriptome, surface proteins and receptor sequences of antigen-specific cells. Our results show that by using the virus-specific Dextramer ®assay we could monitor changes in SARS-CoV-2-specific B- and T-cell responses before and after primary vaccination and booster doses using flow cytometry and single cell RNA-seq analysis. These results show that the Dextramer ®assay can aid in understanding the elicitation and persistence of antigen-specific responses. Moreover, the assay can easily be tailored to any antigen-specificity by replacing the ligands coupled to the Dextramer ®reagents.

CRYOPRESERVATION OF PRIMARY SPLENOCYTES FROM ANTIBODY PRODUCED MICE AGAINST Bitis arientans SNAKE VENOM

Bitis Arientans are associated with the highest number of morbidity and mortality in Nigeria.The most effective treatments against snakebite is the administration of antivenom.Antibodies that is specific for a single epitope of an antigen are obtain by isolating antibody-secreting lymphocytes. These lymphocytes are found within the Splenocytes cells which can be used for a wide variety of immunology-based applications. However, development of hybridoma cell is rare and time consuming. Therefore, valuable time and cell materials can be saved through cryopreservation. This research aimed at Cryopreservation of primary splenocytes from Antibody produced mice against Bitis arientans snake venom. The LD50 of Bitis Arientans venom toxicity was determined in mice according to World Health Organization guidelines. Antibody production was achieved using six mice after immunization for six weeks and antibody titer were determined by indirect ELISA method. Myeloma cells line X63 Ag8.653 were cultured and Mouse Splenocytes with the highest immune response were removed aseptically by mechanical method and cells viability was determined then the isolated splenocytes were cryopreserved in cryovial tube stored at -80C.The LD50 was found to be 1.8mg/kg and Elisa analysis showed mice 2 and 6 to elicited highest immune response with IgG Concentration 3.1µg/ml and 4.6µg/ml. isolated splenocytes were counted to be 6.5x10 and 1.2x10 cells for mice 2 and 6 and myeloma cells to be 1.95x105 cells.In this finding antibody against Bitis Arientans venom were produced and mouse splenocytes were isolated and cryopreserved. Thus, cryopreserved splenocytes and myeloma can be used for the generation of monoclonal antibody

Next-Generation Leishmanization: Revisiting Molecular Targets for Selecting Genetically Engineered Live-Attenuated Leishmania.

Despite decades of research devoted to finding a vaccine against leishmaniasis, we are still lacking a safe and effective vaccine for humans. Given this scenario, the search for a new prophylaxis alternative for controlling leishmaniasis should be a global priority. Inspired by leishmanization-a first generation vaccine strategy where live L. major parasites are inoculated in the skin to protect against reinfection-live-attenuated Leishmania vaccine candidates are promising alternatives due to their robust elicited protective immune response. In addition, they do not cause disease and could provide long-term protection upon challenge with a virulent strain. The discovery of a precise and easy way to perform CRISPR/Cas-based gene editing allowed the selection of safer null mutant live-attenuated Leishmania parasites obtained by gene disruption. Here, we revisited molecular targets associated with the selection of live-attenuated vaccinal strains, discussing their function, their limiting factors and the ideal candidate for the next generation of genetically engineered live-attenuated Leishmania vaccines to control leishmaniasis.

Evaluation of Mycobacterium avium subsp. paratuberculosis isocitrate lyase (IcL) and ABC transporter (BacA) knockout mutants as vaccine candidates.

There has been little success in controlling Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis, due to suboptimal diagnostics and the ineffectiveness of available vaccines. By knocking out BacA and IcL, genes required for MAP survival in dairy calves, two live-attenuated vaccine candidates were created. This study evaluated the host-specific attenuation of MAP IcL and BacA mutants in mouse and calf models, as well as the elicited immune responses. Deletion mutants were generated in MAP strain A1-157 through specialized transduction and found viable in vitro. First, the mutants' attenuation and elicited cytokine secretion were assessed in a mouse model, 3 weeks after intraperitoneal inoculation with MAP strains. Later, vaccine strains were assessed in a natural host infection model where calves received 109CFU oral dose of MAP wild-type or mutant strains at 2 weeks old. Transcription levels of cytokines in PBMCs were evaluated at 12-, 14-, and 16-weeks post-inoculation (WPI) and MAP colonization in tissue was assessed at 4.5 months after inoculation. Whereas both vaccine candidates colonized mouse tissues similarly to wild-type strain, both failed to persist in calf tissues. In either mouse or calf models, gene deletion did not reduce immunogenicity. Instead, inoculation with ΔBacA induced a greater upregulation of proinflammatory cytokines than ΔIcL and wild-type in both models and a greater expansion of cytotoxic and memory T-cells than uninfected control in calves. ΔBacA and wild-type strains significantly increased secretion of IP-10, MIG, TNFα, and RANTES in mice serum compared to uninfected control. This agreed with upregulation of IL-12, IL-17, and TNFα in calves inoculated with ΔBacA at all time points. The ΔBacA also gave rise to greater populations of CD4+CD45RO+, and CD8+ cells than uninfected control calves at 16 WPI. Low survival rate of MAP in macrophages co-incubated with PBMCs isolated from the ΔBacA group indicated that these cell populations are capable of killing MAP. Overall, the immune response elicited by ΔBacA is stronger compared to ΔIcL and it is maintained over two different models and over time in calves. Further investigation is warranted to evaluate the BacA mutant's protection against MAP infection as a live attenuated vaccine candidate.

Therapeutic potential of a novel IP-10-(anti-HER2 scFv) fusion protein for the treatment of HER2-positive breast cancer.

Interferon-γ-inducible protein 10 (IP-10) is a potent antitumor agent and acts by its angiostatic and immunomodulatory properties. IP-10 can target to tumor site by linking with single chain variable fragment (scFv) that recognized specific tumor antigen. In this study, we evaluated biological activity of the fusion protein including IP-10 and anti-HER2 scFv (IP-10-(anti-HER2 scFv)). The HER2- and cell-based ELISA as well as the flow cytometry analysis demonstrated that the fusion protein specifically binds to HER2 antigen. In addition, competitive ELISA demonstrated that the fusion protein recognized the same epitope of HER2 antigen as trastuzumab. The results of MTT assay demonstrated that the growth of HER2-enriched SK-BR3 cells was inhibited in the presence of the fusion protein. Moreover, the cytotoxic effect of the fusion protein was not significantly different from that of trastuzumab. However, no significant cytotoxic effect compared to trastuzumab and anti-HER2 scFv was observed in HER2-low-expressing MDA-MB-231 cells. The obtained findings demonstrated that IP-10-(anti-HER2 scFv) can selectively reduce the cell viability in HER2+ cells. Moreover, similar inhibitory effect on growth of both SK-BR-3 and MDA-MB-231 cell lines was observed in the presence of anti-HER2 scFv protein even at high concentration after 72h. The chemotaxis properties of the fusion protein were also analyzed by a chemotaxis assay. It was demonstrated that the fusion protein induced migration of activated T cell similar to recombinant IP-10 protein. Our findings suggested that IP-10-(anti-HER2 scFv) fusion protein can specifically direct IP-10 to the HER2-expressing tumor cells and may act as an adjuvant along with HER2-based vaccine to gather the elicited immune response at the site of HER2-overexpressimg tumors.