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Declined immune response is the main cause of decreased potency of the influenza vaccine in the elderly, regardless of virus mutations. Herein, we hypothesized that the addition of

Uropathogenic E. coli is one of the major agents of urinary tract infection. Today, no effective treatment or vaccine against this infection is exist. Accordingly, in the present study, a genetic constrruct for inducing of cellular immune system was designed. At first, fimH gene from E. coli 35218 was amplified using PCR. PCR product inserted into pET23a expression vector and the recombinant vector was analysed by sequencing. The vector was transformed to E. coli strain Origami and the protein was expressed under the 1 mM IPTG. FimH was purified with Ni-NTA column and the purified protein was used for immunization of BALB/c. Two weeks after the last injection, lymphocyte proliferation assay was carried out. In addition, IL-4 and IFN-γ cytokines, total antibody serum, IgG1 and IgG2a isotypes were quantified. Finally, protection ability of the vaccine in bladder and kidney infection of mice was evaluated.The results indicated that cellular immune response has a main protective role against UTI and FimH, as a vaccine candidate, significantly increase lymphocyte proliferation, IFN-γ response and total antibody amount. Immunization of mice with FimH conferred effective protection of kidney and bladder against urinary tract infection by uropathogenic E. coli (P< 0.002). It can be concluded that, the current FimH will be valuable for more trying to prepare a new vaccine against UTI.

Background: Pseudomonas aeruginosa has an important role in nosocomial infections. Objective: To evaluate biological activity of the detoxified LPS (D-LPS) entrapped into Poly lactic-co-glycolic acid (PLGA) nanoparticles. Materials: LPS was extracted and detoxified from the P. aeruginosa strain PAO1. The D-LPS, conjugated to the PLGA nanoparticles with 1-ethyl-3-dimethyl aminopropyl carbodiimide (EDAC) and N-hydroxy-succinimide (NHS). The connection was evaluated by FTIR (Fourier transform infrared), Zetasizer, and Atomic Force Microscope (AFM). The BALB/c mice injected intramuscularly with the D-LPS-PLGA with two-week intervals and then challenged two weeks after the last immunization. The bioactivity of the induced specific antisera and cytokines responses against D-LPS-PLGA antigen was assessed by ELISA. Results: D-LPS-PLGA conjugation was confirmed by FTIR, Zetasizer, and AFM. The ELISA results showed that D-LPS was successful in the stimulation of the humoral immune response. The immune responses raised against the D-LPS-PLGA, significantly decreased bacterial titer in the spleen of the immunized mice after challenge with PAO1 strain in comparison with the control groups. Conclusion: The conjugation of the bacterial LPS to the PLGA nanoparticle increased their functional activity by decrease in bacterial dissemination and increase the killing of opsonized bacteria.

New strategies to increase the immune response to HIV-1 vaccine using immunological adjuvants such as Toll-like receptor agonists are needed. In this study, HIV-1 p24-Nef and conjugated form of the vaccine candidate to type-A flagellin (FLA) were injected in the BALB/c mice in different routes. Two weeks after the last immunization, lymphocyte proliferation was measured by the BrdU method. The IL-4 and IFN

Several inactivated SARS-CoV-2 vaccines were approved for human use but are not highly potent. Here, different formulations of inactivated SARS-CoV-2 virus in Alum, Montanide 51VG and Montanide ISA720VG adjuvants were developed and then immune responses were assessed. SARS-CoV-2 virus was inactivated with formalin and formulated in the adjuvants. BALB/c mice were immunized subcutaneously with 4µg of experimental vaccines on days 0 and 14 and two weeks after the final immunization, IL-4 and IFN-γ cytokines, CTL activity and specific IgG titer and IgG1, IgG2a, IgG2a/IgG1 ratio and also anti-RBD IgG response were assessed. Immunization with SARS-CoV-2-Montanide ISA51VG showed a significant increase in IFN-γ cytokine versus SARS-CoV-2-Alum, SARS-CoV-2-Montanide ISA720VG and control groups (P

This study aimed to investigate the effects of Montanide ISA-720 and Naloxone (NLX) in Hepatitis B surface antigen (HBsAg) vaccine formulation on cytokine and long-lasting antibody responses.First, the HBsAg was formulated in Montanide ISA-720 adjuvant and Naloxone at 5 and 10 mg/kg. The experimental mice were immunized three times at a 2-week interval, and then IL-4, IL-2, TNF-α, and IFN-γ cytokines; long-lasting IgG antibody responses 220 days after the last shot; and IgG1/IgG2a isotypes were assessed by ELISA.The HBsAg-Alum group exhibited the highest IL-4 cytokine response among the experimental groups, whereas NLX in HBsAg-MON720 vaccine formulation did not affect cytokine responses. In addition, NLX in Alum-based vaccine suppressed IL-4 cytokine response and increased the IL-2/IL-4 cytokine ratio. Moreover, HBsAg-MON720 was more potent than HBsAg-Alum in the induction of antibody responses, and NLX in Alum- and MON720-based vaccines induced long-lasting antibody responses.NLX in Alum-based vaccine decreased IL-4 cytokine response, increased IL-2/IL-4 cytokine ratio, and improved long-lasting humoral immune responses in both vaccine formulations. Therefore, the adjuvant activity of NLX in the vaccine formulation depends on the type of adjuvant and the nature of the antigen in the vaccine formulation.

Toxoplasma gondii (T. gondii) remains as one of the controversial infections in the world. T. gondii is an important obligate intracellular protozoan parasite in the immune-deficient patients and pregnant women, sometimes leading to death and abortion, respectively. Herein, the adjuvant activity of nanocurcumin was assessed in the T. gondii killed vaccine model in BALB/c mice. In this study, 144 BALB/c mice were included in 8 groups and administered with different regimens of the vaccine; vac+30, 20 mg/kg of curcumin and nanocurcumin, vac + Freund's adjuvant, killed vac, vac + Alum adjuvant, and PBS via the subcutaneous route of immunization for three times with two-week intervals. Two weeks after the last immunization, the splenocytes' culture supernatant was evaluated for IL-4, IFN-γ, IL-2 and TNF-α cytokines and IFN-γ/IL-4, IFN-γ/TNF-α, and IL-2/IL-4 cytokine ratios using commercial ELISA kits. Specific total IgG antibodies, IgG1, and IgG2a were assessed with an optimized ELISA. Then the survival rate was determined 10 days after the experimental challenge. The results showed that the vaccine formulation in nanocurcumin at 20 mg/kg significantly increases IFN-γ cytokine and IFN-γ/IL4, IFN-γ/TNFα, and IL-2/IL4 ratios versus the vaccine formulated in curcumin, killed vaccine, and PBS group. In addition, specific total IgG antibody response showed that the vaccine formulated in nanocurcumin was more potent than that formulated in curcumin in the induction of humoral immune responses. Furthermore, results from the experimental challenge showed that nanocurcumin at a dose of 20 mg/kg could promote the life span of mice approximately by 12% versus the killed vaccine group. The present study showed that nanocurcumin in the vaccine formulation not only is more bioactive than curcumin in the modulation of cellular and humoral immune responses, but also provides more protectivity rate in the vaccinated mice on the killed T. gondii vaccine model. It seems that nanocurcumin can be used as an immunomodulator in vaccine formulation or as part of a complex adjuvant.

Control of brucellosis as a worldwide zoonotic disease is based on vaccination of animals and diagnosis of infected cases to be eradicated. Accurate and rapid detection of infected animals is of critical importance for preventing the spread of disease. Current detection of brucellosis is based on whole-cell antigens and investigating serum antibodies against Brucella lipopolysaccharide (LPS). The critical disadvantage is misdiagnosis of vaccinated animals as infected ones and also cross-reactions with other Gram-negative bacteria. Recombinant outer membrane protein 2b (Omp2b) of Brucella abortus was evaluated as a novel serodiagnostic target in comparison to conventional tests which are based on LPS. Recombinant Omp2b (rOmp2b) was expressed in Escherichia coli BL21 and purified by Ni(2+)-based chromatography. rOmp2b was evaluated in an indirect enzyme-linked immunosorbent assay (ELISA) system for diagnosis of brucellosis, with sera from Brucella-infected mice along with negative sera and sera from mice which were inoculated with other Gram-negative species for assurance of specificity. Thereafter, cattle sera collected from different regions were assessed along with known negative and known positive serum samples. We found that Omp2b can discriminate between Brucella-infected animals and non-infected ones. Results for assessment of two hundred and fifty cattle sera by Omp2b-based indirect ELISA which were compared to Rose Bengal plate agglutination test (RBPT) and serum tube agglutination test (SAT) showed that our proposed procedure has the sensitivity of 88.5%, specificity of 100%, and accuracy of 90.8%. We suggest that recombinant Omp2b could be used as a protein antigen for diagnosis of brucellosis in domestic animals and can be evaluated for detection of human brucellosis.

The H1N1 influenza virus is known as a serious pandemic threat across the globe. Vaccination is one of the most effective methods of protection against this virus and the way to reduce the seasonal pandemic risk. The commercial vaccine does not adequately respond to pandemic strains. This study examines the potential function of formulated H1N1 hemagglutinin with MF59 adjuvant against A/PR/8/34 (H1N1). To this end, a recombinant hemagglutinin (rHA) gene of influenza A virus was designed and expressed in SF9 cell by the Baculovirus expression system. Four groups of mice were immunized by rHA in combination with MF59, Alum adjuvant, and virus split only. The immunized mice subsequently used for the humoral immune assay and the results compared with untreated mice (negative group). Besides, both treated and control mice groups were challenged with mouse-adapted influenza virus A/PR/8/34(H1N1) through the intranasal drop. Bodyweight, survival, temperature variation, and the medical conditions of the samples were assessed. Mice immunized with the recombinant protein demonstrated a humoral response to the influenza A virus. Upon virus challenging, co-administration of rHA with MF59 adjuvant could lead to 92% survival of the vaccinated mice within 10 days. The MF59-treated group showed slight weight loss and high-temperature body two weeks after infection. This group also displayed a higher hemagglutination inhibition (HI) antibody titer as compared to the group vaccinated with virus split, and Alum adjuvant. Altogether, the results showed that the recombinant protein with the MF59 adjuvant created better safety than the Alum adjuvant, thereby can be considered as a safe and reliable vaccine against the H1N1 virus for further investigations.

Montanide ISA 51VG adjuvant has been approved for human clinical application and stimulates cellular and humoral immune responses. Here, HBsAg was formulated in Montanide ISA51VG adjuvant to compare its potency with the Fendrix and HBsAg-alum vaccines. In particular, the long-term humoral response was assessed up to 220 days after the final immunization. BALB/c mice were allocated into six groups. Treatment groups were injected with HBsAg-Montanide ISA51VG, the Fendrix and commercial HBsAg-alum, respectively. Montanide ISA51 VG, Alum and PBS injected mice were considered as control groups. Mice were immunized three times with 2-week intervals on days 0, 14 and 28 by subcutaneous injection. Lymphocyte proliferation was assessed with the BrdU method. IFN-γ, IL-2 and IL-4 cytokines, specific total IgG and IgG1/IgG2a isotypes were assessed using ELISA. The HBsAg-Montanide ISA51VG vaccine resulted in a significant increase in lymphocyte proliferation versus HBsAg-alum and higher IL-2 cytokine production versus the Fendrix. Comparable IL-4 and IFN-γ cytokines responses were observed for these vaccines. Following the first immunization, IgG increased more in HBs-Montanide 51VG group versus the HBs-alum group, while after the second and third shots comparable responses were observed in comparison to the HBs-alum group. Monitoring for 220 days after the final vaccination showed the superiority of HBsAg-Montanide ISA 51VG vaccine versus HBsAg-alum and even the Fendrix vaccine in the induction of long-term antibody responses. This study suggests that HBsAg-Montanide ISA51VG as a novel vaccine formulation can trigger both cellular and long-lasting humoral immune responses more efficiently than conventional HBsAg vaccines.

BACKGROUND: The potency of a vaccine highly depends upon the nature of the adjuvant used. There are a variety of ineffective vaccines, such as HIV-1 vaccine candidates, that need to be optimized with new adjuvant formulations to improve vaccine potency and efficacy. Studies show the potency of naloxone (NLX)/alum mixture in the induction of Th1/Th2 response for vaccine. However, other immunologic patterns inducing by this adjuvant and its immunoregulatory effect is unclear. In this regard, the aim of the present study was to investigate the effect of the NLX/alum mixture, as an adjuvant, on cytokine networks and immunoregulatory activity for an HIV-1 polytope vaccine. METHODS: BALB/c mice were divided into six groups (n=6) and immunized subcutaneously with 10 mug of the vaccine formulated with NLX/alum, NLX, alum, and Freund's adjuvants. At the same time, the mice in the control groups received an equal volume of PBS or NLX. The lymphocyte proliferation assay was carried out using the BrdU method. ELISA was used to measure the levels of IFN-gamma, IL-2, IL-4, IL-10, IL-12, and IL-17 cytokines, total IgG, as well as IgG1 and IgG2a subtypes in serum samples. RESULTS: Our findings showed that mice receiving the NLX/alum-adjuvanted vaccine exhibited increased antibody levels compared with other groups. In addition, there was a considerable difference in the levels of IgG1, IgG2a, IFN-gamma, IL-2, IL-10, IL-12, and IL-17 in mice receiving the NLX/alum-adjuvanted vaccine as compared with other groups. CONCLUSION: The NLX/alum mixture, as an adjuvant, may have a positive effect on the induction of multi-cytokine responses as well as the increased level of IL-10, showing its higher immunogenicity with a higher immunoregulatory mechanism.

Considering the increased antibiotic resistance of Pseudomonas aeruginosa, the evaluation of immune response against the antigens of this bacterium seems necessary. In this study, the protective efficacy and immunological properties of P. aeruginosa recombinant PilQ (r-PilQ) and type b-flagellin (FLB) proteins was evaluated in the burn mouse model of infection. The inbred BALB/c mice were immunized with r-PilQ and FLB antigens. To investigate the type of induced immune response, sera were analyzed by ELISA for total IgG, IgG1, and IgG2a isotypes. After the final immunization, the IL-4, IFN-γ, and IL-17 cytokines level were examined in the spleen of non-challenged mice. Fifty days after lethal challenge, the survival rate and bacterial burden in the skin and other internal organs of experimental mice were assessed. The in vivo administration of r-PilQ, FLB and combined antigen resulted in a significant increase in the survival of mice (66%, 75%, and 83%, respectively) infected by the PAO1 strain of P. aeruginosa in the burn model of infection. Immunization of mice with r-PilQ and FLB mixture induced high titers of IL-4 and IL-17 cytokines compared to control groups (P 0.05). The high titer of antisera raised against combined antigen was able to inhibit the systemic spread of the PAO1 strain from the site of infection to the internal organs. We concluded that the parallel role of IL-4 and IL-17 is necessary for elimination of the bacteria and promotion of survival in the immunized burn mice.

Methicillin resistant Staphylococcus aureus (MRSA) is a representative pathogen that is responsible for a nosocomial infection and considerable yearly mortality rate. Antibiotic resistance provides a great reason for immunotherapy as an alternative strategy to prevent and/or treat the infection. Herein, following the preparation of recombinant penicillin binding protein 2a (r-PBP2a), rabbit polyclonal IgG was purified. Specificity of IgG to r-PBP2a was evaluated by ELISA and western blotting. IgG fraction was prepared by sulfate ammonium precipitation. In addition opsonophagocytosis assay confirmed bioactivity of purified IgG. Experimental mice were challenged with lethal dose of MRSA (5 × 10 8 ) and mortality rate was recorded in the mice treated with IgG fraction for anti-rPBP2a, normal rabbit IgG, vancomycin therapy, and PBS control group. Bacterial quantity was evaluated by culture of liver, kidney and spleen homogenates. Results showed that passive immunization with anti r-PBP2a resulting in a significant improvement in survival rate as well as vancomycin treatment compared with control groups. Furthermore, anti r-PBP2a IgG enhanced considerably the phagocytosis of the S. aureus COL strain, reduced bacterial load, and inhibited the systemic spread of COL strain to the internal organs. These results confirmed that passive immunization by anti-r-PBP2a plays a considerable role in the control of infections caused by S. aureus similar to that of antibiotic therapy.

Hepatitis B virus (HBV) infection is limited through vaccination against HBsAg formulated in the Alum adjuvant. However, this alum-formulated vaccine fails to be preventive in some cases, also known as non-responders. Recent studies have shown the immunomodulatory effect of α-tocopherol in various models. Here, we developed a new formulation for HBsAg using α-tocopherol, followed by assessment of immune responses. Experimental BALB/c mice were immunized with a commercial alum-based vaccine or the one formulated in α-tocopherol at different doses. Mice were immunized subcutaneously with 5 μg of HBsAg with different formulations three times with 2-week intervals. Specific total IgG, IgG1, and IgG2a isotypes of antibodies were measured by ELISA. Immunologic cytokines, such as IFN-γ, IL-4, IL-2, and TNF-α, were also evaluated through commercial ELISA kits. Our results showed that the new α-tocopherol-formulated vaccine had the ability to reinforce specific total IgG responses. Moreover, α-tocopherol in the HBsAg vaccine increased IFN-γ, IL-2, and TNF-α cytokines at higher concentrations; however, the vaccine suppressed IL-4 cytokine release. At a lower concentration of α-tocopherol, the IL-4 cytokine response increased without a positive effect on IFN-γ and TNF-α cytokine response. It seems that α-tocopherol can change the immune responses against HBsAg; however, the type of response depends on the dose of α-tocopherol used in the vaccine formulation.

Methicillin resistant Staphylococcus aureus is one of the most common causes of nosocomial infections. Current therapeutic approaches are not always effective in treatment of nosocomial infections, thus, there is a global demand for the development of novel therapeutic strategies. Staphylococcus aureus possesses various systems to uptake iron. One of the most important of them is iron regulated surface determinant (Isd) which can be an excellent candidate for immunization. Here, following the preparation of recombinant IsdE protein, 20 μg of r-IsdE prepared in various formulations were subcutaneously injected in different groups of mice. Two booster vaccinations were administered in two-week intervals, then, blood samples were collected two weeks after each injection. ELISA was used for the evaluation of total IgG and its isotypes (IgG1 and IgG2a) as well as quantity of IFN-γ, IL-4, IL-17, IL-2 and TNF-α cytokines on the serum samples. Meanwhile, the immunized mice were intraperitoneally inoculated with 5 × 108 CFU of bacteria then, their mortality rate and bacterial load were assessed. Our results showed that immunization with the r-IsdE in various formulations raised total IgG and isotypes (IgG1 and IgG2a) compared with the control groups. Moreover, r-IsdE formulation with MF59 and Freund adjuvants raised production of IFN-γ, IL-4, IL-17, IL-2 and TNF-α cytokines and provided an acceptable protection against Staphylococcus aureus infections. Results of present study suggest that r-IsdE which can easily be expressed by Escherichia coli BL21 system shows a great potential to develop a protective immunity against infections caused by Methicillin resistant Staphylococcus aureus.

The methicillin-resistant Staphylococcus aureus infection is a hot topic area in microbiology research. Here a novel vaccine candidate consisting of recombinant PBP2a and autolysin proteins were used. The proteins over expressed in E.coli BL21 (DE3) cells, and purified by the Ni-NTA affinity column and conjugated using EDAC and ADH as a linker and spacer, respectively. To investigate the immunogenicity and protective effects of recombinant proteins, 5 and 20μg of proteins in various formulations were subcutaneously injected in different groups. Two booster vaccinations were carried out in three-week intervals and blood samples were collected three weeks after each injection. To evaluate the immune response, total IgG, IgG1, IgG2a, and IgG2b were analyzed. Immunization of mice with r-autolysin and r-autolysin-PBP2a mixture raised total IgGantibody. Additionally, both IgG1 and IgG2a responses induced. Opsonophagocytosis assay showed that anti r-PBP2a and r-autolysin IgG not only promoted phagocytosis of S.aureus, but also decreased the number of viable bacterial cells. Furthermore, survival rate of experimental mice increased in the bacteremia infection. Our results demonstrated that active vaccination with a mixture of r-PBP2a/r-autolysin and conjugate form vaccine reduced the mortality rate and protected mice against lethal MRSA challenge as well as single proteins.

Vaccines currently available for AIDS show poor efficiency, demonstrating the need for new strategies to increase their immunogenicity. In this study, the HIV-1P24-Nef peptide was used as a model vaccine, followed by utilization of a novel strategy to increase its immunogenicity. There is a growing interest in using TLR agonists for vaccine formulations. Such molecules bind to their receptors on immune cells, especially the cell surface of antigen presenting cells, thereby activating these cells and inflammatory responses. In the present study, FLiC (flagellin molecule sequence from Pseudomonas aeruginosa) was used as a TLR5 agonist. In addition, PLGA nanoparticles were used as a transmitter system to enhance vaccine efficiency and its effective transfer to immune systems. In light of this, the P24-Nef peptide was conjugated to FLiC through chemical reactions. The HIV-1P24-Nef/FLiC conjugate was constructed as a nano-vaccine using PLGA particles. Subsequently, mice were immunized intradermally three times with three-week intervals with HIV-p24-Nef/FLiC/PLGA, HIV-p24-Nef/PLGA, FLiC/PLGA, PLGA, and PBS in two doses (20 and 5μg). Three weeks after the last booster injection, cell proliferation was assessed using the Brdu/ELISA assay, and cytotoxicity was evaluated by CFSE and splenocyte cytokine secretion (IL-4 and IFN-γ); in addition, IgG1 and IgG2a antibody isotype titers were determined using a commercial ELISA kit. Our results showed that Co-utilization of TLR5 and nano-particles not only improves vaccine immunogenicity but also decreases the immunogenic dose of vaccine candidate required. We showed that the immune system was effectively stimulated via the nano-vaccination strategy using the TLR5 agonists. The effect of this strategy showed variations in different parameters of the immune system; in this regard, cellular immune responses had a higher stimulation level, compared with humoral immune responses.

Cytokines are mediators for polarization of immune response in vaccines. Studies show that co-immunization of DNA vaccines with granulocyte-macrophage colony-stimulating factor (GM-CSF) can increase immune responses. Here, experimental mice were immunized with HIV-1tat/pol/gag/env DNA vaccine with GM-CSF and boosted with recombinant vaccine. Lymphocyte proliferation with Brdu and CTL activity, IL-4, IFN-γ, IL-17 cytokines, total antibody, and IgG1 and IgG2a isotypes were assessed with ELISA. Results show that GM-CSF as adjuvant in DNA immunization significantly increased lymphocyte proliferation and IFN-γ cytokines, but CTL response was tiny increased. Also GM-CSF as adjuvant decreased IL-4 cytokine vs mere vaccine group. IL-17 in the group that immunized with mixture of DNA vaccine/GM-CSF was significantly increased vs DNA vaccine group. Result of total antibody shows that GM-CSF increased antibody response in which both IgG1 and IgG2a increased. Overall, results confirmed the beneficial effect of GM-CSF as adjuvant to increase vaccine immunogenicity. The hallmark result of this study was to increase IL-17 cytokine with DNA vaccine/GM-CSF immunized group. This study for the first time provides the evidence of the potency of GM-CSF in the induction of IL-17 in response to a vaccine, which is important for control of infection such as HIV-1.

Mucoid strains of Pseudomonas aeruginosa are closely associated with chronic pulmonary infections. In this report we describe a straightforward approach to conjugate high molecular weight alginate to type b-flagellin (FLB) and investigation of its bioactivity. The conjugation process was performed by using ADH and EDAC. The endotoxin was eliminated from the candidate vaccine by LPS removal resin followed by LAL test. The bioconjugate molecules were verified by simultaneously determination of polysaccharide/protein content followed by gel filtration chromatography and FTIR spectroscopy. Groups of eight BALB/c mice were injected intranasally with 5 μg (per each nostril) of purified alginate, FLB and conjugated alginate-FLB with two week intervals. The functional activity of the vaccine was evaluated by ELISA and opsonophagocytosis tests. Vaccination with the alginate-FLB conjugate induced a significant (P = 0.0033) rise in alginate specific IgG in mice. At all dilution ranges, the opsonic activity of the conjugate vaccine antisera was significantly higher than alginate alone (61.9% vs. 17.3% at 1:4 dilution; P = 0.0067). The alginate-FLB conjugate could elicit high specific antibodies titer against alginate by improving its immunogenicity. In addition, the antisera raised against conjugate vaccine act as a suitable opsonin for phagocytosis of the mucoid strains of P. aeruginosa.

Multi-epitope vaccines might cause immunity against multiple antigenic targets. Four immunodominant epitopes of HIV-1 genome were used to construct a polytope vaccine, formulated by dendrimer. Two regimens of polytopes mixture with dendrimer were utilized to immunize BALB/c mice. Adjuvants were also used to boost immune responses. The conjugated polytope could arouse significant cellular immune responses (P 0.05) and Th1 response showed higher intensity compared to Th2 (P 0.05). Our study depicted that conjugated dendrimer with multi-epitopic rHIVtop4 would efficiently induce cell-mediated immune responses and might be considered as promising delivery system for vaccines formulation.

Cancer cachexia is characterized by inflammation, loss of skeletal muscle and adipose tissue mass, and functional impairment. Oxidative stress and inflammation are believed to regulate pathways controlling skeletal muscle wasting. The aim of this study was to determine the effects of aerobic interval training and the purported antioxidant treatment, selenium nanoparticle supplementation, on expression of IL-15 and inflammatory cytokines in 4T1 breast cancer-bearing mice with cachexia. Selenium nanoparticle supplementation accelerated cachexia symptoms in tumor-bearing mice, while exercise training prevented muscle wasting in tumor-bearing mice. Also, aerobic interval training enhanced the anti-inflammatory indices IL-10/TNF-α ratio and IL-15 expression in skeletal muscle in tumor-bearing mice. However, combining exercise training and antioxidant supplementation prevented cachexia and muscle wasting and additionally decreased tumor volume in 4T1 breast cancer mice. These finding suggested that combining exercise training and antioxidant supplementation could be a strategy for managing tumor volume and preventing cachexia in breast cancer.

Summary: Hepatitis B virus (HBV) infection is known as a life-threatening liver infection and leads to chronic liver disease if left untreated. Nevertheless, the prevalence of HBV infection has been reduced by an approved vaccination approach using recombinant Hepatitis B surface Antigen (HBsAg) and Alum, known as the HBV vaccine. Alum can be used as an adjuvant to increase HBsAg immunogenicity as a strong Th2 stimulator. There is a vital need to stimulate Th1 immunity by HBsAg vaccination; however, the present vaccine does not induce a prophylactic immune response in some groups. The main aim of the present study was to induce a Th1 cytokine pattern and stimulate an immune response after HBsAg vaccination. Experimental mice were fed selenium nanoparticles (SeNPs) and were later immunized with 5 μg of Hepatitis B Vaccine. After a period of 30 days, the experimental animals were given two booster doses of SeNPs during their vaccination course. Group one, i.e., the control vaccine group, was only administered the HBsAg vaccine. The two treated groups, Groups 2 and 3, were daily fed different doses of SeNPs (100 μg and 200 μg, respectively) via gavage. Group four was considered the control group and was only given phosphate buffered saline (PBS). Lymphocyte proliferation, IFN-γ and IL-4 levels, total antibody and the isotypes of IgG1, IgG2a, IgG2b, and IgM were measured by Enzyme Linked Immunosorbent Assay (ELISA). The administration of SeNPs and the HBs antigen vaccine affected the lymphocyte proliferation; moreover, the total antibody responses also increased the IFN-γ level and induced a Th1 response. Conclusions: The present study proposed that the administration of SeNPs with a conventional HBs antigen vaccine induces a better immune response with a Th1 bias. Keywords: Selenium nanoparticles, Hepatitis B vaccine, IFN-γ, Th1 immune response

Several vaccine candidates have been introduced for immunization against Pseudomonas aeruginosa strains. Despite extensive efforts in recent decades, there is no accurate immunogenic candidate against this pathogen in the market yet. Due to the rapid increase in several drug-resistant strains, P. aeruginosa has caused various health concerns worldwide. It encodes many specific virulence features, which can be used as an appropriate vaccine candidate. The primary stage of the pathogenesis of P. aeruginosa is the expression of many dynamic adhesive molecules, such as type IV pili (T4P), which acts as a principal colonization factor. It has been confirmed that three different subtypes of T4P, including type IVa (T4aP), type IVb (T4bP) and tight adherence (Tad) pili are expressed by P. aeruginosa. The IVa fimbriae type is almost the main cause of challenges to design an effective pili based-immunotherapy method. Nevertheless, in terms of heterogeneity, variability and hidden conserved binding site of T4aP, this attitude has been remained controversial and there is no permitted human study based on IVa pilin commercially. The engineered synthetic peptide-based vaccines are highly talented to mimic the target. In this research, for the first time, some dominant immunogenic features of the Flp protein, such as both B- and T-cell-associated epitopes, presence of IgE-associated epitopes, solvent-accessible surface area were evaluated by analytical immunoinformatics methods. In addition, we designed the engineered Flp pilin as an effective immunogenic substance against several clinically important P. aeruginosa strains. Moreover, by practical active immunization approaches, the humoral and cellular immune response against the extremely conserved region of the engineered synthetic Flp (EFlp) formulated in Montanide ISA 266 compared to the control group. The results of active immunization against EFlp significantly signified that EFlp-Montanide ISA 266 (EFLP-M) strongly could induce both humoral and cellular immune responses. We concluded that Flp pilin has therapeutic potential against numerous clinically significant P. aeruginosa strains and can be served as a novel immunogen for further investigations for development of effective immunotherapy methods against P. aeruginosa as a dexterous pathogen.

Pseudomonas aeruginosa is the major infectious agent of concern for cystic fibrosis (CF) patients. Therefore, it is necessary to develop appropriate strategies for preventing colonization by this bacterium and/or neutralizing virulence factors. In this study, we formulated the encapsulation of exotoxin A into PLGA nanoparticles. The biological activities of the nanovaccine candidate were also characterized. Based on the results, ETA-PLGA can act as a suitable immunogen to stimulate the humoral and cellular immune response. The antibodies raised against ETA-PLGA significantly decreased bacterial titer in the spleens of the immunized mice after challenge with PAO1 strain, compared to the control groups. The encapsulation of PLGA into ETA led to a significantly higher production of INF-γ, TNF-α, IL-4, and IL-17A cytokine responses compared to the ETA group. ETA-PLGA enhanced IgG responses in immunized mice compared to ETA antigen. We concluded that encapsulation of Pseudomonas aeruginosa ETA to PLGA nanoparticles can increase its functional activity by decreasing the bacterial dissemination.

Staphylococcus aureus is one of the most common pathogens in the hospital and the community. The emergence of broad-spectrum antibiotic resistance in S. aureus has made the treatment process more difficult. Therefore, it is obvious that an effective prevention strategy against the pathogen could significantly reduce costs related to care in hospitals. In this report, we describe a simple approach to conjugate S. aureus capsular polysaccharide 5 (CP5) from S. aureus Reynolds strain and 8 (CP8) from S. aureus Becker strain to a fusion protein (Hla-MntC-SACOL0723) and investigation of its bioactivity. The conjugation was done by using ADH (as a bridge) and EDAC (as a coupling agent). The immunoconjugates were characterized by routine polysaccharide/protein contents assays followed by reverse phase chromatography and FTIR spectroscopy. The groups of mice were immunized with conjugate vaccines, capsular polysaccharides, and phosphate-buffered saline (PBS) as a control group. The functional activity of the vaccine candidates was evaluated by ELISA, opsonophagocytosis tests, and determination of bacterial load in challenge study. The results showed that the specific antibody (total IgG) titers raised against conjugate molecules were higher than those of the nonconjugated capsular polysaccharides. The opsonic activity of the conjugate vaccines antisera was significantly higher than polysaccharides alone (58% reduction in the number of bacteria versus 16.3% at 1:2 dilution, p < .05), Further, the conjugate vaccine group had a significant reduction in bacterial load after challenge with S. aureus COL strain cells as compared to the PBS and nonconjugated controls. In conclusion, the immunoconjugates could be developed as a potential vaccine candidate against S. aureus.

Hepatitis C virus (HCV) is a major health problem all over the world. Among HCV proteins, nonstructural protein 3 (NS3) is one of the most promising target for anti-HCV therapy and a candidate for vaccine design. DNA vaccine is an efficient approach to stimulate antigen-specific immunity but the main problem with that is less immunogenic efficiency in comparison with traditional vaccines. Several approaches have been applied to enhance the immunogenicity of DNA. Recently, bacteria-derived substances are considered as one of the most attractive adjuvants for vaccines, which among them, Listeriolysin O (LLO) of Listeria monocytogenes is a toxin with an extremely immunogenic feature. We investigated detoxified form of LLO gene as genetic adjuvant to modulate NS3 DNA vaccine potency. Immunogenic truncated NS3 gene sequence of HCV (1095-1380aa) and detoxified LLO gene region (5-441aa) were amplified by PCR and cloned into the pcDNA3.1 plasmid separately. The expression of recombinant proteins (pc-NS3, pLLO) was confirmed in HEK293T cell line by western blotting. BALB/c mice models received three doses of different formula of plasmids in two-week intervals and two weeks after the final immunization, the immune responses were evaluated by specific total antibody level, lymphocyte proliferation, cytotoxicity, and cytokine levels assays. To evaluate in vivo cytotoxic activity, tumor challenge was performed. The recombinant plasmids were successfully expressed in mammalian cell line, and coadministration of pc-NS3 with pLLO induced the highest titer of total IgG against NS3 antigen compared with other controls. Determination of IgG subclasses confirmed the efficient increase in mixed responses with Th1 dominancy. Furthermore, significant levels of cytokines (p < .05) and lymphocyte proliferation responses (p < .05) indicated the superiority of this regimen. The findings may have important implication for LLO gene application as genetic adjuvant in immune response against HCV.

Staphylococcus aureus is a powerful pathogen that causes a wide range of infectious diseases and results in a high mortality rate in humans. Treating S. aureus-related infections is extremely difficult because of its ability to resist many antibiotics; therefore, developing an effective vaccine against this infection can be an alternative and promising approach. In this study, we evaluated the protective effects of a Hla-MntC-SACOL0723 multi-epitope protein (HMS) compared with HMS conjugated to polysaccharides 5 and 8 (CP5 and CP8) of S. aureus and CP5 and CP8 in a mouse sepsis model. To evaluate the type of induced immune response, specific IgG, and antibody isotypes (IgG1 and IgG2a) were determined using the ELISA method. The functional activity of these vaccine candidates was assessed by opsonophagocytosis. Mice were infected with S. aureus COL strain and evaluated for bacterial load in the kidney and spleen homogenates. Th1, Th2, and Th17-related cytokines in the spleen cell supernatants were assessed by flow cytometry. The therapeutic effect of specific anti-HMS protein IgG antibodies against S. aureus COL strain infection was evaluated by passive immunization. HMS recombinant protein induced a higher level of Th1, Th2, and Th17-related cytokines compared with conjugated molecules. Also, mice immunized with the HMS protein reduced the bacterial load in the kidney and spleen more than the one that received the conjugated molecules. Our study suggests that the HMS fusion protein and conjugate molecule vaccine candidates could be suitable candidates for the removal of S. aureus in the mouse sepsis model but HMS protein can be a more effective candidate.

There is still no human vaccine against Toxoplasma gondii (T. gondii) , as one of the most successful parasites. In present study, we designed a subunit vaccine composed of recombinant SAG1 (rSAG1) and recombinant GRA2 (rGRA2) proteins. In order to improve the induced immune responses, rSAG1 and rGRA2 were adsorbed on Poly (DL-lactide-co-glycolide) (PLGA) microspheres (MS) prepared by double emulsion solvent evaporation method. BALB/c mice were subcutaneously vaccinated by rSAG1-adsorbed PLGA MS (rSAG1-PLGA), rGRA2-adsorbed PLGA MS (rGRA2-PLGA), and the mixture of both formulations (rSAG1/rGRA2-PLGA), twice with a 3-week interval. PLGA MS characteristics, protein release, cellular and humoral immune responses, and protection against acute toxoplasmosis were evaluated. All vaccinated mice induced significantly partial protection and longer survival times associated with higher IFN-γ/IL-10 ratio and higher amount of Toxoplasma -specific IgG antibodies compared to control groups. Interestingly, the synergistic effect of rSAG1 and rGRA2 in eliciting more potent cellular and humoral responses and consequently higher protection in comparison to single antigen was confirmed. This study introduces the mixture of rSAG1 and rGRA2 (derived from different stages of Toxoplasma life-cycle) formulated in PLGA MS as a promising candidate in vaccine development against T. gondii .

Today's, vaccination is the most cost-effective approaches for preventing infectious diseases. In this strategy, adjuvants play an important role. Propolis from honey bee can stimulate the immune system and several studies have shown the modulating effects of Propolis on the immune responses. Here, the adjuvant effects of aqueous and alcoholic extracts of Propolis were studied on the multi-epitope vaccines against HIV-1. A recombinant vaccine against HIV-1 was prepared and BALB/c mice were immunized. subcutaneously on day 0 with 100 μl of candidate vaccine (10 μg) formulated in an alcoholic extract of Propolis. The second group of mice was immunized with the vaccine (10 μg) formulated in aqueous extract of Propolis. Also, candidate vaccine was formulated in Freund's and Alum adjuvants in the third and fourth groups. Experimental mice were immunized three times with two week intervals under the same conditions and suitable control groups. After final injection, lymphocyte proliferation was measured by BrdU method, IL-4 and IFN-γ cytokines, specific total IgG antibodies, IgG1 and IgG2a isotypes were evaluated using ELISA. The results show that the aqueous and alcoholic extracts were able to enhance lymphocyte proliferation, IL-4 and IFN-γ cytokines and antibody responses with dominant IgG1 pattern and comparable to Freund's and Alum adjuvants. It seems that aqueous and alcoholic extracts of Propolis show adjuvant activity and may be useful for vaccine formulation.

Staphylococcus aureus is an important cause of both hospital and community acquired infections worldwide. S.aureus can develop multidrug resistance; thus, immunotherapy can be a rational alternative. High level β-lactam resistance of S. aureus has been attributed to the penicillin binding protein 2a (PBP2a). In this study, we assessed the immunogenicity and protectivity of PBP2a formulated in Montanide ISA266 and Alum adjuvants. Recombinant PBP2a with a molecular weight of approximately 13 kDa was expressed and purified by nickel-nitrilotriacetic acid (NI-NTA) affinity chromatography and characterized by SDS-PAGE and Western blot. To investigate the immunogenicity and protective effects of recombinant protein, 20 μg of r-PBP2a in various formulations were subcutaneously injected in different groups. Two booster vaccinations were carried out in two-week intervals and blood samples were collected two weeks after each injection. To determine the type of induced immune response, sera and splenocytes were analyzed by ELISA for total IgG and isotypes (IgG1 and IgG2a) and cytokine secretion (IFN-γ, IL-4, IL-17 and TNF-α), respectively. Three weeks following the last immunization, experimental mice were challenged with 5 × 108 CFU of bacteria intraperitoneally and mortality rate and bacterial load were assessed. Interestingly, analysis of humoral immune responses revealed that administration of r-PBP2a with Montanide ISA266 significantly increased specific IgG responses and also IgG1 isotype compared to alum-adjuvanted vaccine group. Also, r-PBP2a formulation with alum and MontanideISA266 adjuvants raised IFN-γ, IL-4, IL-17 cytokines secretion, and protectivity following experimental challenge. The results of the present study provide evidences for immunogenicity and protectivity of PBP2a protein as a vaccine candidate.

Background: A licensed vaccine against hepatitis C virus (HCV) has not become available to date. The stability and antigenicity of a targeted synthesized recombinant fusion protein consisting of a truncated core and NS3 (rC/N) of HCV had been predicted. Although safe antigens, recombinant proteins are not efficacious vaccines without adjuvants. The present study evaluated the immunogenicity of rC/N as a bipartite antigen accompanied by Neisseria meningitidis serogroup B outer membrane vesicles (NMB OMVs) in BALB/c mice. Methods: The NMB OMVs were produced and evaluated accurately. The administrations were as follows: rC/N-OMV, rC/N-Freund’s complete/incomplete adjuvant (CIA), rC/N-MF59, rC/N, OMV, MF59, and PBS. The production of Th1 (IFN-γ, IL-2)/Th2 (IL-4)/Th17 (IL-17) cytokines and granzyme B (cytotoxic indicator) by splenic mononuclear cells and the humoral concentration of total IgG/IgG1 (Th2)/IgG2a (Th1) in sera of mice were measured using mouse ELISA kits. Results: Concentrations of Th1/Th2/Th17 cytokines, granzyme B, and immunoglobulins in the spleens and sera of immunized mice, which had received antigen plus each adjuvant (rC/N-OMV, rC/N-Freund’s CIA, and rC/N-MF59), significantly raised compared to the controls (rC/N, OMV, MF59, and PBS). Th1-type responses were dominant over Th2-type responses in vaccinated mice with rC/N-OMV, and Th2 type responses increased dominantly in vaccinated mice with rC/N-MF59 (p < 0.05). Conclusion: NMB OMVs were able to increase Th1 immune responses dramatically more than MF59 and Freund’s CIA. The formulation of rC/N with NMB OMVs showed its ability to induce Th1, Th2, and Th17 immune responses. rC/N-NMB OMVs is a promising approach for the development of an HCV therapeutic vaccine.

Objective The effect of candidemia on immunologic parameters in breast tumor bearing patients is not well studied. Here, we hypothesised that candidemia in the tumor background may change the outcome of immunologic parameters and tumor condition. Method Mice were divided into four groups, including normal, tumor, Candida infected (only Candidiasis) and tumor/Candidiasis groups. Tumor changes were recorded daily after tumor transplantation and induction of candidemia. Splenocytes of mice were harvested, cultured, and stimulated with PHA; afterwards, IL-4, IL-10, IFN-γ, TNF-α and TGF-β cytokines were assessed using ELISA kits. We also evaluated the population of CD4+CD25+Foxp3+ regulatory T cells in the tumor infiltrated and splenocytes. Results The results showed that infection with C. albicans decreased the IFN-γ/IL-4 ratio in tumor/candidiasis and candidiasis groups versus their non-infected controls. IL-10, TGF-β and TNF-α levels increased in the candidiasis group. In addition, Candidemia led to an increase in the Treg population in tumor microenvironment and splenocytes of experimental groups compared with non-infected controls. Finally, candidemia increased tumor growth of tumor/Candidiasis group compared with the tumor group. Conclusion It seems that systemic infection with C. albicans could not only induce regulatory T cells but also result in dysregulation of cytokine network and thereby facilitate tumor growth.

Objectives Physical exercise decreases the incidence of breast cancer and also improves survival in breast cancer patients. However, the mechanistic basis of these protective effects of exercise is not well known. Changes in tumor cytokines, such as oncostatin-M (OSM), have been associated with modulation of antitumor immune responses in breast cancer. Exercise and antioxidants such as selenium affect both antitumor immune responses as well as tumor cytokine expression. Thus, the aim of this study was to determine the effects of aerobic exercise training (AET) and selenium nanoparticle (SeNP) administration on T-helper 1 and 2 and tumor tissue cytokines in mice bearing the 4 T1 mammary carcinoma. Methods We examined the effects of 6 wk of AET and SeNP administration (100 μg three times/wk) on tumor size, concentration of tumor necrosis factor (TNF)-α, interleukin (IL)-6, interferon (IFN)-γ, IL-4 and OSM in tumor tissue and INF-γ and IL-4 in splenocytes of 64 mice bearing the 4 T1 mammary carcinoma. Results AET increased OSM levels in tumor tissue. Moreover, AET increased levels of TNF-α in tumor tissue, whereas SeNP supplementation decreased IL-4 levels tumor tissue. Also, the combination of AET and SeNP administration decreased tumor volume and increased T-helper 1 cytokines in the splenocytes of tumor-bearing mice. Conclusion These findings suggest that the combination of AET and SeNP supplementation effects antitumor immune responses in splenocytes, whereas AET induced antitumor cytokines, such as OSM and TNF-α in tumor tissue.

The modification of tumor-associated antigen-based vaccine to elicit a more robust immune response has been addressed in several ways. In the present work, we aimed to investigate the immunomodulatory effect of selenium nanoparticles as an immunoadjuvant in formulation of a tumor-associated antigen-based vaccine in a preventive form. Fortyfive female inbred BALB/c mice five-to-seven weeks old were used and divided into three groups of test and control, each containing fifteen mice. Group one injected by PBS and used as a control. Group two injected by breast tumor cell lysate alone as vaccine. Group three injected by SeNPs with tumor cell lysate as vaccine. All injections were carried out on day fourteen, twentyone and twentyeight of the study. Tumor induction was done at day thirty. Twenty days after tumor induction serum samples were gathered to measure the cytokine assay. Tumor growth and weight of mice as well as delayed type hyper sensitivity (DTH) response were monitored during the study. Results of the present work showed a significant increase in the level of serum IFN-γ, IL-2, IL-12 and decreased TGF-β in SeNPs/vaccine injected mice as well as lower tumor volume, more potent DTH responses and longer survival rate in comparison to control and tumor lysate vaccine. Taken together, it can be deduced from this work that SeNPs can be considered as an adjuvant in vaccine in triggering robust immune response against breast cancer. But further evaluations are still needed to find the best formula for this agent in antitumor vaccines.

In the present study, the adjuvant activity of flagellin was compared, in the conjugated and mixed forms, against a peptide vaccine from human immunodeficiency virus type 1 (HIV-1) p24-Nef vaccine candidate. Mice were immunized with the HIV-1 p24-Nef peptide with flagellin in both conjugated and mixed forms. Lymphocyte proliferation, CTL activity, and IL-4 and IFN-γ cytokines were evaluated by bromodeoxyuridine, carboxyfluorescein succinimidyl ester and ELISA methods, respectively. At the same time, the frequency of IFN-γ-producing T-lymphocytes, as well as total and isotype-specific antibodies, were assessed by ELISPOT and indirect ELISA, respectively. Our experimental results showed that the immunized mice with the HIV-1 p24-Nef conjugated or mixed forms of flagellin led to increases in the proliferative responses and Th1 cytokine pattern. The conjugated form of vaccine led to increased CTL activity and a Th1 cytokine pattern of immune responses, as well as an IgM isotype of humoral responses in comparison with the mixed form. The flagellin-conjugated vaccine seems to be more potent in increasing vaccine immunogenicity.

Propranolol (Pro), a non-specific β-adrenergic blocking drug, competitively prevents the binding of catecholamines to receptors and suppresses cancer cells. The anti-tumor activity of propranolol has been proved in different kinds of cancers. In this study, we assessed the adjuvant activity of propranolol combined with a tumor vaccine model on the immunological parameters of breast tumor-bearing mice. Breast tumor pieces were implanted into the flank of inbred BALB/C female mice from stock mice. Tumor-bearing mice were treated with tumor antigen lysate vaccine and propranolol/Vaccine (Pro/Vac) combination (as treatment groups), propranolol and PBS (as control groups) for 5 consecutive days, every 12 h. Moreover, all experimental groups received vaccine for three times with one-week interval via s.c injection. After immunization courses, spleens of tumor-bearing mice were removed and dissected, cell suspension was stimulated in vitro, and the cytokine levels in supernatant of splenocytes were measured via commercial ELISA kits. Compared with the vaccine group, immunization with tumor lysate in combination with propranolol significantly increased IL-2, IL-4, IL-12, IL-17, and IFN-γ cytokines. Considering the suppression of tumor growth, propranolol seems to be a potent immunomodulator capable of inducing cellular immune responses against breast cancer.

To evaluate the combined effects of CpG oligodeoxynucleotides (CpG-ODNs) adjuvant and subcutaneous injection route on efficacy of a HIV-1-tat DNA vaccine candidate using BALB/c mice as an animal model. Evaluation of cellular and humoral immunity of mice injected subcutaneously with HIV-1-tat gene cloned into a pcDNA3.1 vector indicated that significant levels of IFN-γ cytokine secretion (900 pg/ml), lymphocyte proliferation (2.5 stimulation index) and IgG2a (1.45 absorbance 450 nm) production could be achieved. These indicators of stimulated cellular immunity were elicited 2 weeks after the last injection (P

Background Brucella is a Gram-negative and facultative intracellular organism that causes brucellosis, a common zoonotic disease. Over 500,000 people are annually affected by brucellosis. Brucella is highly infectious through inhalation route; for this reason it is used for biological warfare aims. This study aimed to study the serum bactericidal and splenic activity of Total-OMP-r CagA immunogens from Brucella abortus and Helicobacter pylori in a BALB/c mouse model. Methods Immunization of BALB/c mice was performed with immunogenic proteins three times subcutaneously (S.C.) at 14-day intervals. The protective effects of two component vaccines with CpG adjuvant were evaluated after mice were challenged with H. pylori ss1 and Brucella abortus strain 544. The specific IgG1 and IgG2a antibodies in sera were assessed using ELISA test. For measuring the antigen-specific IL-4, IL-12 and IFN-γ responses in sera of immunized mice after challenge, RT-PCR technique was applied. Twenty days after the challenge, mice were killed then gastric, splenic and serum samples were assessed and bacterial colony count was measured based on the pour plate count agar. Results The results indicated that rCagA + OMP decreased bacterial colonization in these tissues, and significant difference was observed between test and control groups (p value˂0.001). Conclusion Our results showed that the combination vaccine was effective against an oral exposure and the bacterial burden in the spleen, serum and gastric tissues were reduced in mice immunized with the Total- OMP-CagA.

Plasmid DNA vaccination is a promising vaccine platform for prevention and treatment of infectious disease. Enhancement of the DNA vaccine potency by co-inoculation of immunoadjuvant has been shown to be an effective strategy. Modulation of dendritic cells and T-cells locomotion and trafficking to prime an immune response is mediated by distinct chemokines. The recent study was designed to elucidate the adjuvant activity of plasmid expressing CC-chemokine ligand 20 (pCCL20) in co-inoculation with hepatitis C virus (HCV) core DNA vaccine immunization. pCCL20 was constructed and evaluated for its functional expression. Sub-cutaneous inoculation of pCCL20 with HCV core DNA vaccine was performed via electroporation in BALB/c mice on day 0 and 14 and a HCV core protein booster was applied on day 28. On week after final immunization, both humoral and cell-mediated immune responses were assessed by indirect ELISA for core specific antibodies, lymphocyte proliferation, cytokine ELISA/ELISpot and cytotoxic Grenzyme B (GrzB) release assays. Mice were co-immunized with pCCL20 developed higher levels of core specific IFN-γ/IL-4 ratio and IL-2 release, IFN-γ producing cells, lymphocyte proliferation and cytotoxic Grenzyme B release in both draining lymph nodes and spleen cells of immunized mice. The core-specific serum total IgG and IgG2a/IgG1 ratio were significantly higher when the pCCL20 was co-inoculated. These results suggest the potential of CCL20 chemokine as vaccine adjuvant to enhance Th1 mediated cellular and humoral immune responses in HCV core DNA immunization.

Despite a huge number of studies towards vaccine development against human immunodeficiency virus-1, no effective vaccine has been approved yet. Thus, new vaccines should be provided with new formulations. Herein, a new DNA vaccine candidate encoding conserved and immunogenic epitopes from HIV-1 antigens of tat, pol, gag and env is designed and constructed. After bioinformatics analyses to find the best epitopes and their tandem, nucleotide sequence corresponding to the designed multiepitope was synthesized and cloned into pcDNA3.1+ vector. Expression of pcDNA3.1-tat/pol/gag/env plasmid was evaluated in HEK293T cells by RT-PCR and western-blotting. Seven groups of BALB/c mice were intramuscularly immunized three times either with 50, 100, 200 µg of plasmid in 2-week intervals or with similar doses of insert-free plasmid. Two weeks after the last injection, proliferation of T cells and secretion of IL4 and IFN-γ cytokines were evaluated using Brdu and ELISA methods, respectively. Results showed the proper expression of the plasmid in protein and mRNA levels. Moreover, the designed multiepitope plasmid was capable of induction of both proliferation responses as well as IFN-γ and IL-4 cytokine production in a considerable level compared to the control groups. Overall, our primary data warranted further detailed studies on the potency of this vaccine.

Prevention of hepatitis B requires a vaccine that stimulates the humoral and cellular immune responses in a balanced manner, particularly those associated with Th1 and cytotoxic T cells. Alum adjuvant is currently used in the hepatitis B vaccine formulations but it lacks the efficiency of establishing such immune responses. Therefore, for accessing a suitable vaccine to prevent this fatal disease, it is essential to design and construct a new adjuvant which can overcome the limitations of the alum adjuvant and can stimulate a strong Th1 response as used along with it. In the present study, the adjuvant effect of Hep-c, the first nano-complex which was synthesized by nanochelating technology to improve the immunogenicity of the vaccine against hepatitis B, had been evaluated. Female Balb/c mice were divided into 7 groups and were injected with 10 μg/ml of the hepatitis B vaccine and different doses of Hep-c for 3 times. Groups merely treated with the vaccine, Hep-c or phosphate buffered solution were used as control. Total specific antibody, IgG1, IgG2a, IgG2b, IgM, interleukin-4 (IL-4) and interferon-gamma (IFN-γ) levels were examined by the ELISA method. The proliferative response of the splenocytes was evaluated using bromodeoxyuridine assay. Results showed that immunization with hepatitis B vaccine and Hep-c increased the lymphocyte proliferation and specific IgM and IgG2a compared to the hepatitis B vaccine immunized group. Also, this nano-complex significantly increased the IFN-γ and IL-4 cytokine levels compared to the hepatitis B vaccine immunized group. Our findings show that Hep-c can not only preserve the alum capacity to effectively stimulate production of the antibodies but also cover its inefficiency in inducing Th1 response and prompting cellular immunity. Thus, by boosting the performance of the hepatitis B vaccine, it seemed that this nano-adjuvant has the suitable potential to be used in the commercial HBS vaccine formulation.

Methicillin-resistant Staphylococcus aureus infections are focal and development of an effective vaccine can help to control this infection. Here, recombinant PBP2a was studied in mouse model. Following the preparation of recombinant PBP2a, Balb/c mice were injected subcutaneously with 20 μg of r-PBP2a formulated in Freund's adjuvant three times with three weeks intervals with proper control group. Total and specific isotype antibodies were evaluated on sera by ELISA. Opsonophagocytic activity was also investigated on the sera samples. Intraperitonealchallenge with a sub-lethal dose of MRSA (5 × 108 CFU) was done in experimental mice. Following that, the number of bacteria from kidneys of experimental mice were determined. Survival rate was recorded for 60 days. Significant increase of antibody with high level of IgG1, IgG2a and IgG2b isotypes was demonstrated in vaccinated mice versus the control group (P

Staphylococcus aureus (MRSA) is an opportunistic pathogen which causes a variety of clinical diseases and leads to high rates of morbidity and mortality. Development of an effective vaccine appears to be a useful strategy to control the infection. Here, the internal region of atl was cloned into the pET24a plasmid and expressed in E. coli BL21 (DE3). Cloning of atl was confirmed by colony-PCR, enzymatic digestion and sequencing. Protein expressed in E coli, BL21 DE3 and was confirmed with SDS-PAGE and western blot analysis. Subsequently, BALB/c mice were injected subcutaneously three times with 20μg of the recombinant autolysin. After Bleeding, autolysin-specific total IgG antibodies and isotypes were evaluated using ELISA. Opsonophagocytic killing assay was performed and experimental challenge was done by intraperitoneal injection with sub lethal doses of MRSA in mice and also survival rate was regularly monitored. Results showed that vaccinated mice could exhibit higher levels of autolysin-specific antibodies (P

In the present study we used a fusion peptide from HIV-1 p24 and Nef as vaccine model and adjuvant activity of Naloxone/alum mixture was evaluated in a peptide vaccine model. HIV-1 p24-Nef fusion peptide was synthesized. Female BALB/c mice were divided into five groups. The first group immunized subcutaneously with the p24-Nef fusion peptide adjuvanted with Naloxone/alum mixture and boosted with same protocol. The second was immunized with fusion peptide adjuvanted in alum. The control groups were injected with NLX (Group 3), Alum (Group 4), or PBS (Groups 5) under the same conditions. To determine the type of induced immune response, sera and splenocytes were analyzed by commercial ELISA method for total IgG and isotypes and cytokine secretion (IL-4 & IFN-γ), respectively. We have also used the ELISPOT assay to monitor changes in the frequency of IFN-γ-producing T cells. The proliferation of T cells was assessed using Brdu method and T-cell cytotoxicity was assessed with CFSE method. Immunization of mice with HIV-1 p24-Nef fusion peptide formulated in Naloxone/alum mixture significantly increased lymphocyte proliferation and shifted cytokine responses toward Th1 profile compared to all other groups. Analysis of humoral immune responses revealed that administration of HIV-1 p24-Nef fusion peptide with Naloxone/alum mixture significantly increased specific IgG responses and also increased IgG1,IgG2a, IgG2b, IgG3, and IgM vs. alum-adjuvanted vaccine groups. Naloxone/alum mixture as an adjuvant could improve cellular and humoral immune response for HIV vaccine model and this adjuvant maybe useful for HIV vaccine model in human clinical trial.

Refereed/Peer-reviewed Breast cancer is the leading cause of cancer death in women. Chemotherapy is regarded as the most essential strategy in inhibiting the proliferation of tumor cells. Paclitaxel is a widely used taxane; however, the side effects of available Cremophor-based formulations and also the limitations of passive targeting uncovered an essential need to develop tumor-specific targeted nanocarriers.A hyaluronic acid targeted liposomal formulation of paclitaxel was prepared in which, hyaluronic acid was electrostatistically attracted to the surface of liposomes. Liposomes, had a particle size of 106.4 +/- 3.2 nm, a weakly negative zeta potential of -9.7 +/- 0.8 mV and an acceptable encapsulation efficiency of 92.1 +/- 1.7%. The release profile of liposomes in buffer showed that 95% of PTX was released during 40 h. Confocal laser scanning microscopy and flow cytometry analysis showed the greater cellular internalization of coumarin-loaded liposomes compared to free coumarin. MTT assay on 4T1 and T47D cells demonstrated the stronger cytotoxic activity of liposomes in comparison to free paclitaxel. Cell cycle analysis showed that cells were mainly blocked at G2/M phases after 48 h treatment with liposomes. In vivo real time imaging on 4T1 tumor-bearing mice revealed that the liposomal formulation mainly accumulated in the tumor area. Liposomes also had better antitumor efficacy against Cremophor-based formulation. In conclusion, hyaluronic acid targeted paclitaxel liposome can serve as a promising targeted formulation of paclitaxel for future cancer chemotherapy.

The immunomodulatory effects of lactic acid bacteria have been demonstrated previously. In this study, a Lactobacillus plantarum strain was selected and enriched with selenium nanoparticles for use as a new immunomodulating agent in a breast cancer murine model. 30 female inbred BALB/c mice were equally divided into a test group and a control group. For 2 weeks prior to tumor induction, each mouse received a daily oral administration of 2.5×108 CFU/ml of L. plantarum enriched with selenium nanoparticles (SeNPs), and then 1×106 4T1 cells were injected subcutaneously. After tumor induction, daily SeNP administration was repeated for 3 cycles of 7 days on/3 days off. Immunological parameters such as levels of cytokines, NK cell activity, tumor growth, and mouse survival were evaluated. The production of the pro-inflammatory cytokines IFN-γ, TNF-α, and IL-2 in spleen cell cultures was increased in test mice administered SeNP-enriched L. plantarum. The test mice also showed significant increases in NK cell activity. The tumor volumes of treated mice were decreased and their survival rate notably increased when compared to mice that received L. plantarum alone or control mice. Administration of SeNP-enriched L. plantarum can induce an efficient immune response through the elevation of the pro-inflammatory cytokines IFN-γ, TNF-α and IL-2 levels and increased NK cell activity. Therefore, this treatment may result in better cancer prognosis.

Background Development of agents that specifically kill cancer cells and simultaneously elicit antitumor immune response is a step forward in cancer therapy. In the present study, we investigated whether the administration of artemether contributes to the augmentation of antitumor immunity and the regression of tumor tissues in a mouse model of breast cancer. Methods An optimal immunostimulatory dose of artemether (ART) was defined by DTH reaction and antibody production in sRBC-challenged mice. Subsequent experiments were carried out on tumor-bearing BALB/c mice. In the first group of tumor-bearing mice, the dose of 10 mg/kg/day of artemether were intraperitoneally administered to each animal for six times. The second group was treated with 20 mg/kg/day of cyclophosphamide as a positive control, and the last group (negative control) received the ART diluents. Tumor size was measured during the 10-day experiment; on the last day, mice were sacrificed and their splenocytes and tumor infiltrating lymphocytes were harvested. The concentration of IL-4 and IFN-γ cytokines (using ELISA assay) and the percentage of splenic and tumor Treg cells (using Flowcytometry analysis) were measured. Results Artemether could increase both DTH reaction and the production of hemagglutinating antibody in normal mice. Administration of ART profoundly suppressed the progression of tumor tissues. As well, it was significantly effective in the depletion of splenic CD4+ CD25+ Foxp3+ Treg cells (p-value > 0.05). ART also increased the production of IL-4 (p-value 0.05). As a conclusion, the cytotoxic and immunomodulatory properties of artemether were acknowledged in vivo

Genetic adjuvants have potential role in improvement of immune responses against DNA vaccines. GM-CSF as a genetic adjuvant can recruit and augment dendritic cell numbers in the site of immune responses and thereby induce cellular and humoral immune responses. Here we show that co-immunization of a DNA vaccine from HIV-1P24-Nef with GM-CSF in DNA priming and peptide boost strategy increases the immunogenicity of our candidate vaccine. Analysis of immune response shows that co-immunization with GM-CSF boosts cellular immune responses through increasing proliferation activity and CTL function. Results of cytokine profile studies show that both IL-4 and IFN-γ levels were augmented. Also, co-immunization with GM-CSF resulted in a higher level of total IgG, comprising approximately equal levels of both specific IgG1 and IgG2a subtypes. Monitoring of cellular and humoral immune responses for 20 weeks after final immunization revealed the aptitude of GM-CSF for inducing long-lived humoral and cell mediated immune responses. Overall, our results suggest that GM-CSF is able to induce long term memory for the HIV-1 P24-Nef vaccine candidate while the exact mechanisms involved remained to be clarified.

Tuberculosis (TB) remains as a major public health problem worldwide. Identification and selection of immunodominant antigens of Mycobacterium tuberculosis (MTB), capable of efficiently inducing a protective immune response is the ultimate goal of TB vaccine development studies. Accordingly, this study was designed to produce a novel M. tuberculosis fusion protein consisted of MTB ESAT-6 (early secreted antigenic target-6 kDa), as a potent immunogenic protein, fused to C-terminus of MTB HSP70 (HSP70 359–610 ), as an appropriate carrier and adjuvant. The constructed gene was inserted into a prokaryotic expression vector (pQE30); consequently, the recombinant fusion protein with a 6xHis-tag was successfully over expressed in Escherichia coli M15. Inclusion bodies from bacterial cell lysates were solubilized and the recombinant fusion protein was easily purified by Ni-NTA affinity chromatography under denaturing conditions followed by urea gradient dialysis. The purified and refolded protein was then applied for immunization of mice that resulted in the detection of high titers of specific antibodies, high level of IFN-γ and cell proliferation. The results of our study could confirm the capability of E6H70C fusion protein, as a potential tuberculosis vaccine candidate, for the efficient induction of specific immune responses in a mouse model. However, further investigation need to evaluate the protectivity of this recombinant protein in host model.

Background Artemisinin (ART) is a sesquiterpene lactone. Possessing an endoperoxide bridge is unique among ant-imalarial drugs, and now much attention is focused on the anti-cancer properties of ART. In this study we aimed at the immunomodulatory effects of artemisinin in the treatment of breast cancer in comparison to the conventional anti-cancer drug, cyclophasphamide (CTX). Methods We examined delayed-type hypersensitivity, antibody and IL-4 and IFNγ production, tumour volume, tumour infiltrated regulatory T cells (Treg) and spleen lymphocyte proliferation assay. Briefly three groups of five 4–6 week old female Balb/c tumour-bearing mice (mouse mammary tumour) were treated with 2.8 mg/kg ART and 20 mg/kg CTX intraperitoneally for 20 consecutive days. Tumour volume was measured using a digital vernier calliper (with accuracy of 0.01). Mice were sacrificed and percentage of tumour infiltrating Tregs were obtained using flow cytometry (BD, USA). Proliferation of splenocytes was obtained using BrdU proliferation assay (Roche). Results Our results showed that ART can reduce the number of Tregs in tumour stroma (P-value ≤ 0.047) as compared to CTX (P-value ≥ 0.05) and control. Furthermore ART increased IFNγ/IL-4 ratio produced in splenocyte culture (P-value ≤ 0.001). Proliferation assay did not show any significant difference (P-value ≥ 0.05). Discussion Cancer is a multi-factorial disease which needs a multi-approach treatment. Early accumulation of Treg cells in the tumour tissue correlates with tumour progression and is an indication of bad prognosis. According to the obtained results, ART can reduce the number of Tregs. We suggest using artemisinin, with its dual action mechanism. It can effectively kill cancer cells along with reducing the suppressive microenvironment.