Response Of Spleen Cells Research Articles

Orally dissolving film as a potential vaccine delivery carrier to prevent influenza virus infection

Orally dissolving films (ODF) are designed to be dissolved on the tongue and absorbed in the mouth. It offers multiple advantages over the commonly used needle-based vaccines, especially in terms of convenience allowing safe, painless, and easy self-administration. As the efficacy of ODF-encapsulated influenza vaccines has not been demonstrated, we assessed the protection elicited by inactivated influenza virus (A/PR/8/34, H1N1) vaccine delivered using ODFs in mice. Trehalose and pullulan components of the ODF ensured that the HA antigens of the inactivated PR8 virus retained their stability while ensuring the rapid release of the vaccines upon exposure to murine saliva. Mice were immunized thrice by placing the PR8-ODF on the tongues of mice at 4-week intervals, and vaccine-induced protection was evaluated upon lethal homologous challenge infection. The PR8-ODF vaccination elicited virus-specific serum IgG and IgA antibody responses, hemagglutinin inhibition (HAI), and viral neutralization. Upon challenge infection, ODF vaccination showed higher levels of IgG and IgA antibody responses in the lungs and antibody-secreting cell (ASC) responses in both lung and spleen compared to unimmunized controls. These results corresponded with the enhanced T cell and germinal center B cell responses in the lungs and spleens. Importantly, ODF vaccination significantly reduced lung virus titers and inflammatory cytokines (IFN-γ, IL-6) production compared to unvaccinated control. ODF vaccination ensured 100% survival and prevented weight loss in mice. These findings suggest that influenza vaccine delivery through ODFs could be a promising approach for oral vaccine development.

Intradermal Vaccination with PLGA Nanoparticles via Dissolving Microneedles and Classical Injection Needles

PurposeA dissolving microneedle array (dMNA) is a vaccine delivery device with several advantages over conventional needles. By incorporating particulate adjuvants in the form of poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) into the dMNA, the immune response against the antigen might be enhanced. This study aimed to prepare PLGA-NP-loaded dMNA and to compare T-cell responses induced by either intradermally injected aqueous-PLGA-NP formulation or PLGA-NP-loaded dMNA in mice.MethodsPLGA NPs were prepared with microfluidics, and their physicochemical characteristics with regard to encapsulation efficiencies of ovalbumin (OVA) and CpG oligonucleotide (CpG), zeta potentials, polydispersity indexes, and sizes were analysed. PLGA NPs incorporated dMNA was produced with three different dMNA formulations by using the centrifugation method, and the integrity of PLGA NPs in dMNAs was evaluated. The immunogenicity was evaluated in mice by comparing the T-cell responses induced by dMNA and aqueous formulations containing ovalbumin and CpG (OVA/CpG) with and without PLGA NP.ResultsPrepared PLGA NPs had a size of around 100 nm. The dMNA formulations affected the particle integrity, and the dMNA with poly(vinyl alcohol) (PVA) showed almost no aggregation of PLGA NPs. The PLGA:PVA weight ratio of 1:9 resulted in 100% of penetration efficiency and the fastest dissolution in ex-vivo human skin (< 30 min). The aqueous formulation with soluble OVA/CpG and the aqueous-PLGA-NP formulation with OVA/CpG induced the highest CD4 + T-cell responses in blood and spleen cells.ConclusionsPLGA NPs incorporated dMNA was successfully fabricated and the aqueous formulation containing PLGA NPs induce superior CD4+ and CD8+ T-cell responses.

A Modified Recombinant DNA-Based SARS-CoV-2 Vaccine Expressing Stabilized Uncleavable Spike Protein Elicits Humoral and Cellular Immunity against Various SARS-CoV-2 Variants of Concern

The appearance of several variants of concern (VOCs) of SARS-CoV-2 affects the efficacy of currently available vaccines and causes continuous spread and reinfection between humans. These variants possess different spike (S) protein mutations, which could affect viral pathogenicity, transmission, and immune escape. Herein, we develop a synthetic codon-optimized DNA vaccine (VIU-1007) expressing full-length S protein. The developed vaccine is stabilized by two K986P and V987P proline substitutions and resistant to cleavage by proteases such as furin by deletion of arginine residues (R682, R683, and R685) in multibasic furin cleavage site (RRAR). Additionally, it carries K417N, E484K, N501Y, and D614G substitutions in the receptor binding domain (RBD) derived from the beta VOC. Following the validation and characterization of the in vitro S protein expression, the humoral and cellular immunogenicity of VIU-1007 was assessed in immunized Balb/c mice. While both regimens elicited a Th-1-biased immune response based on S1-specific binding IgG isotypes, three vaccine doses significantly enhanced IgG levels. Furthermore, CD4+ and CD8+ memory T cell responses in spleens and draining inguinal lymph nodes were significantly higher in mice received three doses of VIU-1007 when compared to those received two doses only. Importantly, sera from mice immunized with three doses showed broad neutralization breadth against several SARS-CoV-2 variants, including alpha, beta, gamma, delta, and omicron VOCs. Moreover, the sera showed limited neutralization capacity against SARS-CoV-1, Bat SARS-like coronavirus WIV1, and MERS-CoV. Together, while these data suggest the presence of common neutralizing-rich epitopes between SARS-CoV-2 variants and some other betacoronaviruses, the ongoing evolution of SARS-CoV-2 could result in escape from vaccine-induced immunity, which requires a continuous update of vaccines.

Wild mouse gut microbiota limits initial tuberculosis infection in BALB/c mice.

Mouse models are critical tools in tuberculosis (TB) research. Recent studies have demonstrated that the wild mouse gut microbiota promotes host fitness and improves disease resistance. Here we examine whether the wild mouse gut microbiota alters the immunopathology of TB in BALB/c mice. Conventional BALB/c mice (LabC) and mice born to germ-free BALB/c mothers reconstituted with the wild mouse gut microbiota (WildR) were used in our studies. WildR mice controlled initial TB infection better than LabC mice. The microbial gut communities of LabC mice and WildR mice had similar richness but significantly different composition prior to infection. TB reduced the gut community richness in both cohorts while differences in community composition remained indicating a general TB-induced dysbiosis. The wild mouse gut microbiota did not alter the typical lung histopathology of TB in the BALB/c model that includes unstructured immune cell infiltrates with infected foamy macrophages invading alveolar spaces. Animals of both cohorts mounted robust T cell responses in lungs and spleen with lower absolute counts of CD4 and CD8 T cells in lungs of WildR mice during acute infection, corresponding with observed differences in pathogen load. In summary, LabC mice and WildR mice showed largely overlapping TB immunopathology and pathogen kinetics, with WildR mice controlling early acute infection better than LabC mice.

Comparison of the effects of probiotic strains (Lactobacillus gasseri, Lactiplantibacillus plantarum, Lactobacillus acidophilus, and Limosilactobacillus fermentum) isolated from human and food products on the immune response of CT26 tumor-bearing mice.

This study aimed to compare the effects of the probiotic bacteria, L. gasseri (52b), L. plantarum (M11), L. acidophilus (AC2), and L. fermentum (19SH), isolated from human source and traditional food products on the modulation of the immune system and inflammatory response on BALB/c mouse model bearing CT26 tumor. Five groups of female inbred BALB/c mice were orally administered with the probiotics and their mixes (MIX, at a 1:1 ratio) at varying dosages (1.5 × 108cfu/ml and 1.2 × 109cfu/ml) before and after the injection of a subcutaneous CT26 tumor over the course of 38days via gavage. Finally, their effects on the tumor apoptosis and the cytokine levels in spleen cell cultures were analyzed and compared. M11, MIX, and 52b groups had the greatest levels of interleukin-12 (IL-12) and interferon gamma (IFN-γ) production. The highest production level of granzyme B (GrB) was related to the MIX and 52b groups. Moreover, these groups showed the lowest production level of (IL-4) and transforming growth factor beta (TGF-β). Furthermore, the groups of MIX and 52b demonstrated the greatest amount of lymphocyte proliferation of spleen cells in response to the tumor antigen. The delayed-type hypersensitivity (DTH) response significantly increased in the groups of MIX and 52b compared with the control (p < 0.05). The findings demonstrated that the oral treatment of the human strain (52b) and the combination of these bacteria generated strong T helper type 1 (Th1) immune responses in the tumor tissue of the tumor-bearing mice, which led to the suppression of the tumor development.

Immunogenicity and efficacy of a novel multi-patch SARS-CoV-2/COVID-19 vaccine candidate.

While there has been considerable progress in the development of vaccines against SARS-CoV-2, largely based on the S (spike) protein of the virus, less progress has been made with vaccines delivering different viral antigens with cross-reactive potential. In an effort to develop an immunogen with the capacity to induce broad antigen presentation, we have designed a multi-patch synthetic candidate containing dominant and persistent B cell epitopes from conserved regions of SARS-CoV-2 structural proteins associated with long-term immunity, termed CoV2-BMEP. Here we describe the characterization, immunogenicity and efficacy of CoV2-BMEP using two delivery platforms: nucleic acid DNA and attenuated modified vaccinia virus Ankara (MVA). In cultured cells, both vectors produced a main protein of about 37 kDa as well as heterogeneous proteins with size ranging between 25-37 kDa. In C57BL/6 mice, both homologous and heterologous prime/boost combination of vectors induced the activation of SARS-CoV-2-specific CD4 and CD8 T cell responses, with a more balanced CD8+ T cell response detected in lungs. The homologous MVA/MVA immunization regimen elicited the highest specific CD8+ T cell responses in spleen and detectable binding antibodies (bAbs) to S and N antigens of SARS-CoV-2. In SARS-CoV-2 susceptible k18-hACE2 Tg mice, two doses of MVA-CoV2-BMEP elicited S- and N-specific bAbs as well as cross-neutralizing antibodies against different variants of concern (VoC). After SARS-CoV-2 challenge, all animals in the control unvaccinated group succumbed to the infection while vaccinated animals with high titers of neutralizing antibodies were fully protected against mortality, correlating with a reduction of virus infection in the lungs and inhibition of the cytokine storm. These findings revealed a novel immunogen with the capacity to control SARS-CoV-2 infection, using a broader antigen presentation mechanism than the approved vaccines based solely on the S antigen.

Regulation of Staphylococcal Enterotoxin-Induced Inflammation in Spleen Cells from Diabetic Mice by Polyphenols.

Patients with diabetes are known to be more susceptible to infections following the establishment of Staphylococcus aureus in their nasal passages and on their skin. The present study evaluated the effects of staphylococcal enterotoxin A (SEA) on the immune responses of spleen cells derived from diabetic mice, and examined the effects of polyphenols, catechins, and nobiletin on inflammation-related gene expression associated with the immune response. (-)-Epigallocatechin gallate (EGCG), possessing hydroxyl groups, interacted with SEA, whereas nobiletin, possessing methyl groups, did not interact with SEA. The exposure of spleen cells derived from diabetic mice to SEA enhanced the expression of interferon gamma, suppressor of cytokine signaling 1, signal transducer and activator of transcription 3, interferon-induced transmembrane protein 3, Janus kinase 2, and interferon regulatory factor 3, suggesting that SEA sensitivity is variable in the development of diabetes. Both EGCG and nobiletin changed the expression of genes related to SEA-induced inflammation in spleen cells, suggesting that they inhibit inflammation through different mechanisms. These results may lead to a better understanding of the SEA-induced inflammatory response during diabetogenesis, and the establishment of methods to control these effects with polyphenols.

Differential cytotoxic effects of fenbendazole on mouse lymphoma EL-4 cells and spleen cells

Fenbendazole (FBZ) is a benzimidazole anthelmintic widely used to treat parasitic infections. The anticancer effect of FBZ has been recently highlighted leading to its consideration as a potential anticancer agent. Although previous studies have demonstrated the effect of FBZ on cancer cells, there is a paucity of studies on the effect of FBZ on lymphoma cells and normal immune cells. Herein, we investigated the effects of FBZ on a mouse lymphoma cell line, EL-4 cells, and spleen cells, using vincristine as a positive control. The cellular metabolic activity of EL-4 cells was decreased by FBZ, but that of the spleen cells was not decreased. Moreover, FBZ reduced the mitochondrial membrane potential and induced reactive oxygen species production in EL-4 cells, but not in spleen cells. FBZ induced G2/M phase arrest and increased the sub G0/G1 phase ratio, indicating apoptosis. Furthermore, compared to the control cells, the reactivity of spleen cells pretreated with FBZ to lipopolysaccharide was maintained. In summary, FBZ is cytotoxic to EL-4 cells, but not to spleen cells. This study provides experimental evidence that FBZ exerts an anticancer effect, and less cytotoxic effects and functional damage to normal spleen cells.

Study of immunogenicity and safety of a candidate rotavirus vaccine based on a recombinant hybrid protein

BACKGROUND: Rotaviruses are the main cause of acute gastroenteritis in children in both developed and developing countries. Vaccination is the only way to prevent severe and fatal course of this disease. Live attenuated viruses-based vaccines currently available can have a number of side effects. A candidate rotavirus vaccine reported is based on a hybrid recombinant protein FliCVP6VP8, which includes a VP6 protein fragment, a rotavirus A VP8 protein fragment, and S. typhimurium FliC flagellin components. AIM: The aim was to evaluate the immunogenicity and safety of а preparation Rotavirus vaccine, recombinant in preclinical studies. MATERIALS AND METHODS: The immunogenicity of vaccine (blood antibody titers, antigen-specific proliferative response of spleen cells) was evaluated in BALB/c mice. The acute and subchronic toxicity, the possible irritating effect, pyrogenicity and the anaphylactic effect and delayed type hypersensitivity were evaluated in laboratory mice, rats, Guinea pigs, and rabbits. RESULTS: Double immunization of mice with the candidate vaccine demonstrated a significant increase in antibody titers in mouse sera compared to that in control mice. Evaluation of antigen-specific proliferative response after double immunization with a candidate vaccine demonstrated a significant increase in the values of stimulated proliferation. Evaluation of safety through acute and chronic toxicity studies demonstrated no toxicity. The immunostimulatory effect of vaccine was demonstrated when evaluating the number of antibody-producing cells with sheep red blood cells as antigens. The number of white blood cells was demonstrated to increase after the prolonged vaccine administration. CONCLUSIONS: The preclinical studies have demonstrated safety of the candidate rotavirus vaccine and its capability to produce the immune response.

Intranasal immunization with chitosan microparticles enhances LACK-DNA vaccine protection and induces specific long-lasting immunity against visceral leishmaniasis

Development of a protective vaccine against Leishmania depends on antigen formulation and adjuvants that induce specific immunity and long-lasting immune responses. We previously demonstrated that BALB/c mice intranasally vaccinated with a plasmid DNA encoding the p36/LACK leishmanial antigen (LACK-DNA) develop a protective immunity for up to 3 months after vaccination, which was linked with the systemic expression of vaccine mRNA in peripheral organs. In this study, LACK-DNA vaccine was associated with biocompatible chitosan microparticles cross-linked with glyceraldehyde (CMC) to boost the long-lasting immunity against the late Leishmania infantum challenge. Infection at 7 days, 3 or 6 months after vaccination resulted in significantly lower parasite loads when compared with non-vaccinated controls. Besides, LACK-DNA-chitosan vaccinated mice showed long-time protection observed after the late time point challenge. The achieved protection was correlated with an enhanced spleen cell responsiveness to parasite antigens, marked by increased proliferation and IFN-γ as well as decreased IL-10 production. Moreover, we found diminished systemic levels of TNF-α that was compatible with the better health condition observed in LACK-DNA/CMC vaccinated-infected mice. Together, our data indicate the feasibility of chitosan microparticles as a delivery system tool to extend the protective immunity conferred by LACK-DNA vaccine, which may be explored in vaccine formulations against Leishmania parasite infections.

In vitro immunoregulatory activity and anti-inflammatory effect of Echinococcus granulosus laminated layer

The Laminated layer of Echinococcus granulosus (LL) is the outer layer of the hydatic cyst. It plays a pivotal role in protecting the metacestode from host immunity. In our current study, we investigated the immunomodulatory effect of the LL on mouse spleen cells in presence of Lipopolysaccharide (LPS). Mouse spleen cells were cultured with or without LL in presence of LPS. After 24 h, the nitrites level representative of Nitric oxide (NO) production was measured in the culture supernatant by Griess-modified method. In addition, the mRNA expression levels of cytokines (IFN-γ, IL-1β, TGF-β, IL-10), Foxp3, and CTLA-4 were measured by quantitative Real-Time Polymerase chain reaction (qRT-PCR). Interestingly, our results showed a significant decrease (p< 0.01) in NO production and IFN-γ mRNA level (p< 0.001) from LPS- induced spleen cells in response to LL after 24h of culture. Moreover, LPS induced high level of IL-1β that was significantly (p<0.05) down regulated by LL. Importantly, mRNA levels of TGF-β (p< 0.01), Foxp3 and IL-10 (p< 0.05) were significantly upregulated by LL. In conclusion, our data indicated the in vitro immuno-regulatory and anti-inflammatory effects of the hydatic Laminated Layer on mouse spleen cells. These effects are related to an innate response implicating up-regulation of Foxp3, IL-10 and TGF-β expression and down-regulation of IFN-γ and IL-1β expression. LL could constitute a potential candidate for controlling inflammation during inflammatory disease.

Recombinant Live Attenuated Influenza Virus Expressing Conserved G-Protein Domain in a Chimeric Hemagglutinin Molecule Induces G-Specific Antibodies and Confers Protection against Respiratory Syncytial Virus.

Respiratory syncytial virus (RSV) is one of the most important pathogens causing significant morbidity and mortality in infants and the elderly. Live attenuated influenza vaccine (LAIV) is a licensed vaccine platform in humans and it is known to induce broader immune responses. RSV G attachment proteins mediate virus binding to the target cells and they contain a conserved central domain with neutralizing epitopes. Here, we generated recombinant LAIV based on the attenuated A/Puerto Rico/8/1934 virus backbone, expressing an RSV conserved G-domain in a chimeric hemagglutinin (HA) fusion molecule (HA-G). The attenuated phenotypes of chimeric HA-G LAIV were evident by restricted replication in the upper respiratory tract and low temperature growth characteristics. The immunization of mice with chimeric HA-G LAIV induced significant increases in G-protein specific IgG2a (T helper type 1) and IgG antibody-secreting cell responses in lung, bronchioalveolar fluid, bone marrow, and spleens after RSV challenge. Vaccine-enhanced disease that is typically caused by inactivated-RSV vaccination was not observed in chimeric HA-G LAIV as analyzed by lung histopathology. These results in this study suggest a new approach of developing an RSV vaccine candidate while using recombinant LAIV, potentially conferring protection against influenza virus and RSV.

Targeting Antigens to Different Receptors on Conventional Type 1 Dendritic Cells Impacts the Immune Response

Targeting Ag to surface receptors on conventional type 1 dendritic cells can enhance induction of Ab and T cell responses. However, it is unclear to what extent the targeted receptor influences the resulting responses. In this study, we target Ag to Xcr1, Clec9A, or DEC-205, surface receptors that are expressed on conventional type 1 dendritic cells, and compare immune responses in BALB/c and C57BL/6 mice in vitro and in vivo after intradermal DNA vaccination. Targeting hemagglutinin from influenza A to Clec9A induced Ab responses with higher avidity that more efficiently neutralized influenza virus compared with Xcr1 and DEC-205 targeting. In contrast, targeting Xcr1 resulted in higher IFN-γ+CD8+ T cell responses in spleen and lung and stronger cytotoxicity. Both Clec9A and Xcr1 targeting induced Th1-polarized Ab responses, although the Th1 polarization of CD4+ T cells was more pronounced after Xcr1 targeting. Targeting DEC-205 resulted in poor Ab responses in BALB/c mice and a more mixed Th response. In an influenza challenge model, targeting either Xcr1 or Clec9A induced full and long-term protection against influenza infection, whereas only partial short-term protection was obtained when targeting DEC-205. In summary, the choice of targeting receptor, even on the same dendritic cell subpopulation, may strongly influence the resulting immune response, suggesting that different targeting strategies should be considered depending on the pathogen.

NK Cells Negatively Regulate CD8 T Cells to Promote Immune Exhaustion and Chronic Toxoplasma gondii Infection.

NK cells regulate CD4+ and CD8+ T cells in acute viral infection, vaccination, and the tumor microenvironment. NK cells also become exhausted in chronic activation settings. The mechanisms causing these ILC responses and their impact on adaptive immunity are unclear. CD8+ T cell exhaustion develops during chronic Toxoplasma gondii (T. gondii) infection resulting in parasite reactivation and death. How chronic T. gondii infection impacts the NK cell compartment is not known. We demonstrate that NK cells do not exhibit hallmarks of exhaustion. Their numbers are stable and they do not express high PD1 or LAG3. NK cell depletion with anti-NK1.1 is therapeutic and rescues chronic T. gondii infected mice from CD8+ T cell exhaustion dependent death, increases survival after lethal secondary challenge and alters cyst burdens in brain. Anti-NK1.1 treatment increased polyfunctional CD8+ T cell responses in spleen and brain and reduced CD8+ T cell apoptosis in spleen. Chronic T. gondii infection promotes the development of a modified NK cell compartment, which does not exhibit normal NK cell characteristics. NK cells are Ly49 and TRAIL negative and are enriched for expression of CD94/NKG2A and KLRG1. These NK cells are found in both spleen and brain. They do not produce IFNγ, are IL-10 negative, do not increase PDL1 expression, but do increase CD107a on their surface. Based on the NK cell receptor phenotype we observed NKp46 and CD94-NKG2A cognate ligands were measured. Activating NKp46 (NCR1-ligand) ligand increased and NKG2A ligand Qa-1b expression was reduced on CD8+ T cells. Blockade of NKp46 rescued the chronically infected mice from death and reduced the number of NKG2A+ cells. Immunization with a single dose non-persistent 100% protective T. gondii vaccination did not induce this cell population in the spleen, suggesting persistent infection is essential for their development. We hypothesize chronic T. gondii infection induces an NKp46 dependent modified NK cell population that reduces functional CD8+ T cells to promote persistent parasite infection in the brain. NK cell targeted therapies could enhance immunity in people with chronic infections, chronic inflammation and cancer.

Virus-like particle vaccine displaying Toxoplasma gondii apical membrane antigen 1 induces protection against T. gondii ME49 infection in mice

Toxoplasmosis is an intracellular parasitic disease caused by the protozoa Toxoplasma gondii, which affects about half of the world's population. In spite of the strenuous endeavors, a T. gondii vaccine for clinical use remains unreported to date. In the present study, we generated virus-like particles (VLPs) containing T. gondii apical membrane antigen 1 (AMA1) and assessed its efficacy in a murine model. VLPs were characterized using western blot and TEM. T. gondii-specific IgG and IgA antibody responses in sera, germinal center B cell responses in spleen, brain cyst counts and their sizes were determined. Elevated T. gondii-specific IgG and IgA antibody responses were observed from the sera of AMA1 VLP-immunized mice. Immunization with AMA1 VLPs enhanced T. gondii-specific antibody-secreting cell responses and germinal center B cell responses upon antigen stimulation. Brain tissue analysis revealed that AMA1 VLP-immunization reduced cyst formation and its size compared to control. Also, VLP-immunized mice were less susceptible to body weight loss and displayed enhanced survival rate compared to the control group. Our results demonstrated that the immune response induced by T. gondii AMA1 VLPs confer partial protection against T. gondii infection and provides important insight into potential T. gondii vaccine design strategy.

Prevention of tuberculosis in macaques after intravenous BCG immunization

Mycobacterium tuberculosis (Mtb) is the leading cause of death from infection worldwide1. The only available vaccine, BCG (Bacillus Calmette–Guérin), is given intradermally and has variable efficacy against pulmonary tuberculosis, the major cause of mortality and disease transmission1,2. Here we show that intravenous administration of BCG profoundly alters the protective outcome of Mtb challenge in non-human primates (Macaca mulatta). Compared with intradermal or aerosol delivery, intravenous immunization induced substantially more antigen-responsive CD4 and CD8 T cell responses in blood, spleen, bronchoalveolar lavage and lung lymph nodes. Moreover, intravenous immunization induced a high frequency of antigen-responsive T cells across all lung parenchymal tissues. Six months after BCG vaccination, macaques were challenged with virulent Mtb. Notably, nine out of ten macaques that received intravenous BCG vaccination were highly protected, with six macaques showing no detectable levels of infection, as determined by positron emission tomography–computed tomography imaging, mycobacterial growth, pathology and granuloma formation. The finding that intravenous BCG prevents or substantially limits Mtb infection in highly susceptible rhesus macaques has important implications for vaccine delivery and clinical development, and provides a model for defining immune correlates and mechanisms of vaccine-elicited protection against tuberculosis.

De‑O‑acylated lipooligosaccharide of E.coli B reduces the number of metastatic foci via downregulation of myeloid cell activity.

Lipopolysaccharides are the main surface antigens and virulence factors of gram‑negative bacteria. Removal of four ester‑bound fatty acid residues from hexaacyl lipidA of Escherichiacoli lipooligosaccharide (LOS) resulted in the de‑O‑acylated derivative E.coli LOS‑OH (LOS‑OH). This procedure caused a significant reduction in the toxicity of this compound compared to the native molecule. We investigated the effect of such a structural LOS modification on its biological activity using invitro assays with monocytic cells of the RAW264.7 line, dendritic cells of the JAWS II line, bone marrow‑derived dendritic cells (BM‑DCs), and spleen cells. Furthermore, in invivo experiments with a melanoma B16 metastasis model, the anti‑metastatic activity of the compounds and spleen cell reactivity mediated by them representing a systemic response were analyzed. The results revealed that LOS‑OH demonstrated weaker ability than LOS to stimulate and polarize an immune response both invitro and invivo. It induced lower cytokine production by cells of myeloid lines. Multiple applications of LOS‑OH into mice injected intravenously with B16 cells significantly (P<0.05; P<0.01) reduced the number of metastatic foci in the lungs, presumably via silencing of myeloid cell reactivity as well as the inability to stimulate lymphoid cells both directly and indirectly. These findings suggest that LOS‑OH maintained in the body of metastasis‑bearing mice appears to modulate or downregulate the innate response, leading to the inability of blood myeloid cells to support the migration of melanoma cells to lung tissue.

Influenza M2 virus-like particle vaccination enhances protection in combination with avian influenza HA VLPs.

Despite the ability to induce a broad range of cross protection, M2e5x virus-like particles (VLPs) alone provide limited vaccine efficacy and confer low efficacy of protection against highly pathogenic avian influenza virus (HPAIV) infection in chickens. Avian influenza hemagglutinin (HA) has been a major antigenic target that enhances humoral immunity, but the efficacy of avian HA-based vaccines against HPAIV needs to be further improved. In this study, we evaluated the vaccine efficacy induced by combination of conserved tandem repeat M2e5x VLPs and HA VLPs against an avian influenza virus. We found that combinatorial vaccine elicited higher levels of reassortant H5N1 (rgH5N1) virus-specific IgG, IgG1, IgG2a and IgA antibody responses compared to M2e5x VLPs in sera and lungs of mice. Combinatorial VLPs vaccination induced higher levels of CD8+ T cell and germinal center B cell responses in lung and spleen compared to M2e5x VLPs. Combinatorial VLPs vaccination showed significantly reduced inflammatory responses and lung viral loads upon rgH5N1 virus challenge infection, resulting in less body weight loss compared to M2e5x VLPs alone. These results indicate that immune responses to both M2e and HA might provide a strategy of vaccination inducing enhanced protection against avian influenza virus.

Recombinant live attenuated influenza virus expressing G protein fragment in a chimeric hemagglutinin molecule induces G-specific antibodies and confers protection against respiratory syncytial virus

Abstract Respiratory syncytial virus (RSV) is one of the most important pathogens associated with significant morbidity and mortality in infants and the elderly. Live attenuated influenza vaccine (LAIV) is a licensed platform for vaccination in humans and known to induce broader immune responses locally and systemically. RSV G attachment proteins mediate virus binding to the target cells and contain a conserved central domain with neutralizing epitopes. Here, we generated recombinant LAIV based on the attenuated A/PR8/34 virus backbone, expressing a RSV G conserved domain in a chimeric hemagglutinin (HA) fusion molecule (HA-G). The attenuated phenotypes of chimeric HA-G LAIV were evident by restricted replication in the upper respiratory (nose) but not in the lower respiratory track lung tissues of the mice. Chimeric HA-G LAIV grew better at low temperature 33□. Prime and boost intranasal vaccination of mice with HA-G LAIV induced G-protein specific IgG and IgG2a (T helper type 1) isotype antibodies. Immunization of mice with chimeric HA-G LAIV showed significant increases in G-protein specific IgG and antibody-secreting cell responses in lung, bronchioalveolar fluid, bone marrow, and spleens after challenge. Vaccine-enhanced disease typically caused by inactivated-RSV vaccination was not observed in chimeric HA-G LAIV as analyzed by lung histopathology and infiltrating innate and adaptive immune cells after RSV challenge. Immunity of chimeric HA-G LAIV against influenza virus was not compromised. These results in this study suggest a novel approach of developing RSV vaccine candidates using LAIV, potentially dual vaccines conferring protection against both pathogens.

Biochemical characterisation of lectin from wild chickpea (Cicer reticulatum L.) with potential inhibitory action against human cancer cells.

A wild chickpea lectin (WCL) from the seeds of Cicer reticulatum L. was chromato-purified using DEAE-Cellulose and SP-Sephadex ion exchange chromatography. WCL was thermostable upto 60°C with broad pH optima (pH 5-9) and various divalent metal ions did not influence its activity. WCL demonstrated DNA protection in a dose-dependent manner. The lectin exerted antifungal activity against diverse fungal pathogens. WCL augmented the mitogenic response of mouse spleen cells at 10µg/ml concentration and showed an inhibition of HIV-1 reverse transcriptase at IC50 of 200µM. Against human cancer cell lines, lectin demonstrated anticancer potential. The cell viability assay in HepG2, Ishikawa, MCF-7 and MDA-MB-231 cell line demonstrated IC50 values of 61.8, 54.4, 37.5 and 44.2µg/ml respectively. PRACTICAL APPLICATIONS: WCL exhibited distinct medicinal properties vis-à-vis antiproliferative, mitogenic, antifungal/bacterial and HIV-1 reverse transcriptase inhibiting activities. The potential of WCL can be a subject of exploration from a pharmacological standpoint.