Bordetella pertussis antigens encapsulated into N-trimethyl chitosan nanoparticulate systems as a novel intranasal pertussis vaccine.

The mucosal immune system serves as the first line of defense against Bordetella pertussis. Intranasal vaccination, due to its potential to induce systemic and mucosal immune responses, appears to prevent the initial adherence and colonization of the bacteria at the first point of contact. In the present study, two B. pertussis antigens, pertussis Toxoid (PTd) and Filamentous hemagglutinin (FHA), which play a very significant role in virulence and protection against pertussis, were encapsulate into N-trimethyl chitosan (TMC) nanoparticulate systems. After preparation of TMC nanoparticles (NPs), the NPs were characterized and their ability to induce efficient immune responses against B. pertussis was studied in a mouse model. Our findings showed that PTd + FHA-loaded TMC NPs have strong ability to induce IL-4, IL-17, IFN-γ, IgG, and IgA in the mouse model. Results from this study suggest that nasal administration of the PTd + FHA-loaded TMC NPs induced not only a systemic immune response but also a local mucosal response, which may improve the efficacy of pertussis prevention through respiratory tract transmission.

Comparison of different immunization routes on the immune responses induced by Mycobacterium tuberculosis ESAT-6/CFP-10 recombinant protein.

According to some difficulties against tuberculosis (TB) vaccination, development of new TB vaccines has been noted in recent years. Selection of proper route for vaccination is one of the most important factors for induction of good immune responses. Hence, in this study, the effects of different administration routes, including intranasal (I.N), subcutaneous (S.C) and intramuscular (I.M) on immune responses against Mycobacterium tuberculosis ESAT-6/CFP-10 recombinant protein has been considered. Recombinant ESAT-6/CFP-10 protein with or without adjuvant (MF59 or cholera toxin B (CTB)) was administered by three routes of I.M, I.N and S.C to mice for three times. Then, the levels of specific antibodies, lymphocyte proliferation and IFN-γ/IL-5 cytokine profile have been carried out to evaluate the humoral and cellular responses. The results showed that the titers of specific antibodies were quickly elevated in S.C and I.M groups after first immunization. Otherwise, the raise of antibody has delay in the I.N immunized animals. The levels of IFN-γ and lymphocyte proliferation have been increased in all of vaccinated groups. However, the I.N immunized mice have lower levels of IL-5 production. Based on our finding, the ESAT-6/CFP-10 recombinant protein is a potent stimulator of immune responses in all of three immunization strategies. However intranasal administration of this antigen has tended to reinforcement of cellular immune responses.

Nasal vaccination with r4M2e.HSP70c antigen encapsulated into N-trimethyl chitosan (TMC) nanoparticulate systems: Preparation and immunogenicity in a mouse model.

In this study, the potential of N-trimethyl chitosan (TMC) nanoparticles as a carrier system for the nasal delivery of the r4M2e.HSP70c, as an M2e-based universal recombinant influenza virus vaccine candidate, was investigated in mice. The anti-M2e specific cellular and humoral immune responses were assessed and the protective efficacy against a 90% lethal dose (LD90) of influenza A/PR/8/34 (H1N1) in a mice model was evaluated. Our results showed that the intranasal immunization of mice with r4M2e.HSP70c+TMC rather than the control groups, r4M2e+TMC, r4M2e and PBS (Phosphate buffer saline), significantly elevated both longevity and serum level of the total M2e-specific IgG antibody with a significant shift in the IgG2a/IgG1 ratio toward IgG2a, induced a Th1 skewed humoral and cellular immune responses, increased IFN-γ, IgG, and IgA in the bronchoalveolar lavage fluid (BALF), and promoted the proliferation of peripheral blood lymphocytes with lower morbidity and mortality rate against viral challenge. In conclusion, based on evidence to our finding, nasal vaccination with r4M2e.HSP70c antigen encapsulated into N-Trimethyl Chitosan (TMC) nanoparticulate system showed to induce a long lasting M2e-specific humoral and cellular immune responses and also provided full protection against a 90% lethal dose (LD90) of the influenza virus A/PR/8/34 (H1N1). It seems, protective immunity following intranasal administration of r4M2e could be resulted by the cooperation of both adjuvants, TMC and HSP70c.

Evaluation of specific antibodies against Mycobacterium tuberculosis recombinant antigens for detection of recent infection.

INTRODUCTION: Since an accurate test for detection of Mycobacterium tuberculosis early infection is urgently needed, this study was designed for development of an efficient screening test in diagnosis of tuberculosis infection. MATERIALS AND METHODS: In the present study, two recombinant proteins CFP-10, ESAT-6 were tested as antigens for the diagnosis of recent tuberculosis. The proteins were produced in Escherichia coli, purified and tested in indirect ELISAs with sera from 63 subjects with positive clinical results. Also, 56 sera from healthy persons were tested as controls. The results were compared with molecular and culture. RESULTS: The levels of antibodies against M. tuberculosis antigens in patients with tuberculosis were significantly higher than those in healthy subjects. Among 63 patients, 58 were positive for ESAT-6, 54 for CFP-10. CONCLUSION: Altogether, the role of M. tuberculosis recombinant proteins, as a suitable candidate for early diagnosis of tuberculosis infection was supported in this study. However, these strongly offer the potential of mixture or fusion of these recombinant proteins for better sensitivity and specificity.

A truncated C-terminal fragment of Mycobacterium tuberculosis HSP70 enhances cell-mediated immune response and longevity of the total IgG to influenza A virus M2e protein in mice.

As the importance of virus-specific IgG2a and strong induction of Th1 type immune response for virus clearance was reported, conventional influenza vaccines induce a highly humoral immune response and fail to induce cytotoxic T-lymphocyte (CTL) immunity. Hence, in agreement with heat shock protein 70 (HSP70) acting as Th1 cytokine-like adjuvant, an Escherichia coli-expressed r4M2e.HSP70c fusion protein comprising C-terminus of Mycobacterium tuberculosis HSP70 genetically fused to four tandem repeats of influenza A virus M2e was constructed. Then, the case-control study was carried out to evaluate the humoral and cellular responses elicited against M2e in Balb/C mice by intramuscular immunization with r4M2e.HSP70c alone. Our results showed that r4M2e.HSP70c rather than control groups, r4M2e, r4M2e+Alum, or rHSP70c, significantly elevated both longevity and serum level of the total M2e-specific IgG antibody, induced a Th1 skewed humoral and cellular immune responses, increased the level of IFN-γ in BALF, and promoted the proliferation of peripheral blood lymphocytes. Furthermore, a virus challenge experiment revealed that mice vaccinated with r4M2e.HSP70c limited the severity of influenza A disease by 100% survival rate, less sever body weight loss and delaying the onset of morbidity in mice for 2days rather than other control groups. Here, we used r4M2e.HSP70c to stimulate M2e-specific antibody and cellular immune responses in Balb/C mice. The mHSP70c in the fusion form induced a long lasting Th1 skewed humoral and cellular immune responses against its associated protein. It seems anti-M2e antibodies limit viral replication and ameliorate influenza infection that allows the immune system to induce sterilizing HA-antibody against whole virion that leads to full protection against virulent influenza infection.

Vaccination with recombinant 4 × M2e.HSP70c fusion protein as a universal vaccine candidate enhances both humoral and cell-mediated immune responses and decreases viral shedding against experimental challenge of H9N2 influenza in chickens.

As cellular immunity is essential for virus clearance, it is commonly accepted that no adequate cellular immunity is achieved by all available inactivated HA-based influenza vaccines. Thus, an improved influenza vaccine to induce both humoral and cell-mediated immune responses is urgently required to control LPAI H9N2 outbreaks in poultry farms. M2e-based vaccines have been suggested and developed as a new generation of universal vaccine candidate against influenza A infection. Our previous study have shown that a prime-boost administration of recombinant 4×M2e.HSP70c (r4M2e/H70c) fusion protein compared to conventional HA-based influenza vaccines provided full protection against lethal dose of influenza A viruses in mice. In the present study, the immunogenicity and protective efficacy of (r4M2e/H70c) was examined in chickens. The data reported herein show that protection against H9N2 viral challenge was significantly increased in chickens by injection of r4M2e/H70c compared with injection of conventional HA-based influenza vaccine adjuvanted with MF59 or recombinant 4×M2e (r4M2e) without HSP70c. Oropharyngeal and cloacal shedding of the virus was detected in all of the r4M2e/H70c vaccinated birds at 2 days after challenge, but the titer was low and decreased rapidly to reach undetectable levels at 7 days after challenge. Moreover, comparison of protective efficacy against LPAI H9N2 in birds intramuscularly immunized with r4M2e/H70c likely represented the ability of the M2e-based vaccine in providing cross-protection against heterosubtypic H9N2 challenge and also allowed the host immune system to induce HA-homosubtype neutralizing antibody against H9N2 challenge. This protective immunity might be attributed to enhanced cell-mediated immunity, which is interpreted as increased lymphocytes proliferation, increased levels of Th1-type (IFN-γ) and Th2-type (IL-4) cytokines production and increased CD4(+) to CD8(+) ratios, resulting from the injection of four tandem repeats of the ectodomain of the conserved influenza matrix protein M2 (4×M2e) genetically fused to C-terminus of Mycobacterium tuberculosis HSP70 (mHSP70c).

Molecular characterization of HAO3, the homologue of the Bm86 tick vaccine antigen, from the Iranian isolate of Hyalomma anatolicum anatolicum.

Hyalomma anatolicum anatolicum tick is widely distributed in many parts of Iran and while the commercial vaccines based on the application of midgut-derived recombinant Bm86 antigen are used for its control, limited information about the efficiency of this vaccination in Iran is available. Herein, with the final aim of evaluation of Bm86-based heterologous vaccination, as the primary step the Bm86 homologue of the H. a. anatolicum (Hao3) from an Iranian isolate was characterized and compared with the commercialized Bm86 and other Bm86 homologoue sequences available in GenBank. Our in silico predictions resulted in the identification of seven epidermal growth factor (EGF)-like domains, one hydrophobic transmembrane region, one leader sequence and several glycosylation sites within the structure of both Hao3 and Bm86 proteins, which suggested the pattern of extracellular membrane-bound glycoproteins with the role of regulation in cell growth for both proteins. Moreover, while the nucleotide and amino acid sequences corresponding to Bm86 homologue showed a high level of conservation among the Iranian isolates (Hao3, Hao3-1 and Hao3-2, more than 99%), the Hao3 amino acid sequence had a homology of around 89%, 64% and 65% with that of Indian, Australian and Argentinean isolates, respectively. This indicated a considerable variation between commercial Bm86 antigen and H. a. anatolicum Bm86-like protein of Iranian and Indian isolates. Taking together, these results imply that the efficiency of commercial Bm86-based vaccine against the Iranian H. a. anatolicum may be under the question and indicates the value of the development of Hao3-based recombinant vaccines and further planning for their in vivo evaluation.

In contrast to conventional inactivated influenza vaccines, 4xM2e.HSP70c fusion protein fully protected mice against lethal dose of H1, H3 and H9 influenza A isolates circulating in Iran.

Ideal vaccines against influenza viruses should elicit not only a humoral response, but also a cellular response. Mycobacterium tuberculosis HSP70 (mHSP70) have been found to promote immunogenic APCs function, elicit a strong cytotoxic T lymphocyte (CTL) response, and prevent the induction of tolerance. Moreover, it showed linkage of antigens to the C-terminus of mHSP70 (mHSP70c) can represent them as vaccines resulted in more potent, protective antigen specific responses in the absence of adjuvants or complex formulations. Hence, recombinant fusion protein comprising C-terminus of mHSP70 genetically fused to four tandem repeats of the ectodomain of the conserved influenza matrix protein M2 (M2e) was expressed in Escherichia coli, purified under denaturing condition, refolding, and then confirmed by SDS-PAGE, respectively. The recombinant fusion protein, 4xM2e.HSP70c, retained its immunogenicity and displayed the protective epitope of M2e by ELISA and FITC assays. A prime-boost administration of 4xM2e.HSP70c formulated in F105 buffer by intramuscular route in mice (Balb/C) provided full protection against lethal dose of mouse-adapted H1N1, H3N2, or H9N2 influenza A isolates from Iran compared to 0-33.34% survival rate of challenged unimmunized and immunized mice with the currently in use conventional vaccines designated as control groups. However, protection induced by immunization with 4xM2e.HSP70c failed to prevent weight loss in challenged mice; they experienced significantly lower weight loss, clinical symptoms and higher lung viral clearance in comparison with protective effects of conventional influenza vaccines in challenged mice. These data demonstrate that C-terminal domain of mHSP70 can be a superior candidate to deliver the adjuvant function in M2e-based influenza A vaccine in order to provide significant protection against multiple influenza A virus strains.

In vivo electroporation enhances immunogenicity and protection against influenza A virus challenge of an M2e-HSP70c DNA vaccine.

There is a growing concern regarding continuous risk of emerging a new influenza pandemic. It is highlighted the need for novel vaccination techniques that quickly and effectively employed to respond to such threats. Although, DNA vaccine is a simple and effective approach to induce antigen specific immune responses, their potency requires further improvement. DNA vaccine encoding conserved antigen of influenza virus could provide protection in various animal models. Therefore, we constructed a plasmid vector encoding M2e-HSP70c sequences, pcDNA/MHc, as a candidate for universal influenza vaccine. The expression of newly constructed vectors was verified by transient transfection of mammalian cells (HEK293T cell line) and western blot analysis using commercial antibodies. Mice were injected subcutaneously (s.c.) by the help of electroporation (IEP) in the footpad area and boosted without IEP with 100 µg of constructed vector. Furthermore, the potency of this construct to provoke humoral immune responses and its protectivity against lethal dose of viral challenge were evaluated. Based on our study, the fusion construct was immunogenic in mice and was able to confer both protection against lethal challenge of H1N1 virus and reduce viral load in lung homogenates of the infected mice.

Expression of recombinant HAO3 from an Iranian isolate of Hyalomma anatolicum anatolicum in Pichia pastoris and evaluation of its antigenicity.

Hyalomma anatolicum anatolicum tick is considered as one of the main problem of ruminants' productivity in endemic countries such as parts of Africa, the Middle East and India. The disease is economically important and hence, its control and eradication is a priority. This problem reinforces the need for alternative approach like vaccine to control tick infestations instead of continuous application of acaricide which led to the natural selection of the acaricide-resistant ticks. Therefore, the present study provided evidence for the construction of transformant containing the chromosomally integrated multi-copy expression cassettes of HAO3, its successful and efficient expression in Pichia pastoris yeast and purification of the secreted protein by ultrafiltration (UF) system in a high level yield and purity. The result of antigenicity assay for the rHAO3 protein pointed well toward its capability for the elicitation of antibody response in immunized rabbits. Interestingly, the results indicated that the expressed HAO3 protein reacted well with mid gut antigen (MGAg) and rBm86 (Gavac) antisera in ELISA and western blot assays making it evident that the epitopes present in expressed protein are well recognized by the antibodies against MGAg and rBm86 proteins. Moreover, the presence of cross-reactive epitopes between rHAO3 protein with its native antigen from mid gut cells was also determined.

Study of infection with an Iranian field-isolated H9N2 avian influenza virus in vaccinated and unvaccinated Japanese quail.

In the present study, we examined the mortality rate, egg production, and clinical signs of quail experimentally infected with a field isolate of A/Chicken/Iran/339/02 (H9N2) avian influenza virus obtained from an infected commercial layer farm with severe morbidity and mortality. A total of 120 quail at 14 days old were randomly divided into four groups of vaccinated (B and C) and unvaccinated (A and D) birds. Vaccination was done on days 20 and 32, and viral inoculation of birds in groups C and D was then carried out on day 43. For evaluation of viral transmission, at 24 hr postinoculation additional unvaccinated birds were placed in direct contact with challenged birds. All the birds were evaluated for clinical signs, egg production, antibody production, viral titration in lung homogenates, and viral transmission following inoculation. All unvaccinated-challenged birds were infected and showed clinical signs, whereas the infection rate along with clinical signs of vaccinated-challenged birds reached 30%-40%. Although vaccination induced high antibody titers, reduction in food and water consumption was evident in this vaccinated-challenged group compared with the unchallenged control group. These results could indicate that inactivated vaccine did not fully prevent the infection, although it was capable of protecting birds against clinical signs and significantly decreased viral titers in lungs after intranasal challenge.

Cloning, expression, and immunogenicity of novel fusion protein of Mycobacterium tuberculosis based on ESAT-6 and truncated C-terminal fragment of HSP70.

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.

Influenza A viruses: why focusing on M2e-based universal vaccines.

The threat of highly virulent avian influenza, such as H5N1 and swine-origin H1N1 influenza viruses, bring out an urgent need to develop a universal influenza vaccine, which may provide cross-protection against different strain of influenza A viruses. The extra-domain of influenza M2 protein (M2e), which is almost completely conserved among all subtypes of influenza A viruses, is considered as a promising candidate target for the development of a broad-spectrum recombinant influenza A vaccine. The results of several preclinical studies with M2e protein, with or without carriers, have already proved the successful protection of M2e-based vaccinated animal model against lethal challenge of heterologous and homologous influenza A viruses. Recently, the results of Phase I/II clinical trail studies with M2e-based vaccines have raised hopes for considering these vaccines against seasonal and pandemic influenza A strains. Hence, it is expected that more and more effective and safe universal influenza vaccines based on M2e will be developed for prevention of seasonal and pandemic influenza in the near future.

Invasive aspergillosis promotes tumor growth and severity in a tumor-bearing mouse model.

Invasive aspergillosis increases in chronic immunosuppressive diseases such as cancer. There is little information about the mechanisms by which Aspergillus infection affects the immune regulation and microenvironment of cancer cells. Hence, this study was aimed at investigating the effect of invasive aspergillosis on immunosurveillance, metastasis, and prognosis of cancer in tumor-bearing mice. After implantation of mouse mammary tumor in BALB/c mice, they were infected with Aspergillus conidia intravenously. For comparison, groups of mice were experimentally infected with Aspergillus conidia or implanted with tumor cells separately. Seven days after Aspergillus infection, the serum levels of tissue inhibitor of metalloproteinase-1 (TIMP-1) were measured by ELISA, and subsequently regulatory T lymphocytes were analyzed by flow cytometry. The survival of animals and mean tumor size were then determined. Our results indicated that tumor sizes in mice increased significantly after infection with Aspergillus conidia. Moreover, invasive aspergillosis enhanced the population of regulatory lymphocytes and level of TIMP-1. This study supports the idea that massive Aspergillus infection could stimulate tumor growth and increases the possibility of a bad prognosis. As a result, treatment of Aspergillus infection could be considered an important issue for efficient cancer therapy.

Prokaryotic expression and characterization of avian influenza A virus M2 gene as a candidate for universal recombinant vaccine against influenza A subtypes; specially H5N1 and H9N2.

The conserved M2 protein of influenza A virus is considered as a promising candidate target for a broad-spectrum, recombinant influenza A vaccine. In the present study, the open reading frame (ORF) of avian influenza A/chicken/Iran/101/1998 (H9N2) M2 gene was amplified then cloned in pAED4, prokaryotic expression vector. M2 protein was produced through the expression of this recombinant expression vector (pAED4-M2) in E. coli BL21 (DE3) strain. The expressed M2 protein was analyzed on SDS-PAGE. Western blot assay was used to examine the immunoreaction of the expressed protein using commercial polyclonal anti-M2 antibody. The antigenicity and biological activity of the recombinant protein was also qualitatively detected on infected MDCK cells surface by immunofluorescence assay using rabbit's immunized antiserum. So, according to the sequence alignment based on the mentioned isolate and the result of immunoassay reaction, it seems recombinant vaccine based on A/chicken/Iran/101/1998(H9N2) M2 protein isolate might cover majority of influenza A virus strains specially H5 and H9 circulating in Iran and neighbor regions significantly.

Heterologous expression, purification and characterization of the influenza A virus M2e gene fused to Mycobacterium tuberculosis HSP70(359-610) in prokaryotic system as a fusion protein.

One of the concerns about influenza A vaccine based on M2e protein is their limited potency; hence, optimal approaches to enhance immunogenicity of M2e protein immunization remain to be established. It seems by linking this M2e-peptide to an appropriate carrier such as mycobacterium tuberculosis C-terminal 28-kDa domain of HSP70 (HSP70(359-610)), we can render it very immunogenic. According to previous reports, this study was designed to produce a novel influenza A virus recombinant fusion protein consisted of M2e, a potent immunogenic protein from influenza A virus, fused to C-terminal domain of mycobacterium tuberculosis HSP70, HSP70(359-610), as a carrier and adjuvant. We fused the genes of M2e and HSP70 ( 359-610 ) then inserted in pQE-60, prokaryotic expression vector. This recombinant fusion protein with a 6xHis-tag was successfully over expressed in Escherichia coli M-15. The recombinant fusion protein was purified by Ni-NTA affinity chromatography under denaturing conditions, followed by urea gradient dialysis. The purified fusion protein was analyzed on SDS-PAGE. Western blot assay was used to examine the immunoreaction of the expressed protein using commercial penta-His HRP conjugate antibody. The antigenicity and biological activity of the recombinant protein was also qualitatively detected on the infected MDCK cells surface by immunofluorescence and cell-ELISA assay using rabbit's immunized antiserum. This observation suggest that the expressed fusion protein is useful as a universal recombinant vaccine for overcoming highly mutational influenza virus, but more immunological study in animal lab remains to be evaluated.

Cloning, expression and purification of the influenza A (H9N2) virus M2e antigen and truncated Mycobacterium tuberculosis HSP70 as a fusion protein in Pichia pastoris.

Due to its conservation, the extracellular domain of the influenza A M2 protein (M2e) has the potential for being applied as a recombinant vaccine candidate against a wide range of strains, though its immunogenicity may need to be improved. The occurrence of several post-translational modifications within the structure of M2 protein may affect its immunopotency for the induction of humoral immune response. Herein, to construct a recombinant M2e-based vaccine candidate with the appropriate structural conformation and immunogenicity the corresponding nucleotide sequence from an H9N2 influenza strain was fused to the N-terminus of the truncated Mycobacterium tuberculosis HSP70(359-610), as a potent adjuvant, and following its cloning into the pPICZ alpha A plasmid the fusion gene was expressed in Pichia pastoris KM71H yeast. The secreted protein was then easily purified from the culture media, based on the presence of polyhistidine tag and used for the production of rabbit polyclonal antisera. This raised antisera could recognize the native M2e protein on the surface of H9N2 influenza virus-infected MDCK cells at a comparable level with the commercial H2N2-specific anti-M2 antibody, which was evidenced with immunofluorescence and cell-ELISA assays. These results not only re-emphasized on the conservancy of the M2e antigen, but also pointed towards the applicability of the M2e-HSP70(359-610) fusion protein for the induction of specific antibodies capable of binding to the native M2e antigen on the infected cells. Collectively, this study implied that purified M2e-HSP70(359-610) represents a promising vaccine candidate; however, its in vivo potency for the induction of protection remains to be evaluated.