Evaluation of the murine immune response to Leishmania meta 1 antigen delivered as recombinant protein or DNA vaccine

The meta 1 gene of Leishmania is conserved across the genus and encodes a protein upregulated in metacyclic promastigotes. Meta 1 constitutive overexpressing mutants show increased virulence to mice. In this paper, both meta 1 recombinant protein and plasmids bearing the meta 1 gene were tested for their antigenicity and potential for inducing protective immunity in mice. Vaccination with the recombinant protein induced a predominant Th2-type of response and did not result in protection upon challenge with live parasites. Surprisingly, the expected reversal to a CD4(+) Th1-type of response upon genetic immunisation by the intramuscular route was not observed. Instead, vaccination with either the meta 1 gene alone or in fusion with the monocyte chemotactic protein (MCP)-3 cDNA induced a Th2-type of response that correlated with lack of protection against infection.     

Immunisation of mice with Mycoplasma hyopneumoniae antigens P37, P42, P46 and P95 delivered as recombinant subunit or DNA vaccines

Porcine enzootic pneumonia (PEP), which is caused by the fastidious bacterium Mycoplasma hyopneumoniae, is one of the most economically important diseases in the pig industry worldwide. Commercial bacterins provide only partial protection; therefore, the development of more efficient vaccines against PEP is necessary. In this study, the cellular and humoral immune responses elicited by DNA and recombinant subunit vaccines based on the P37, P42, P46 and P95 antigens of M. hyopneumoniae were evaluated after the intramuscular inoculation of BALB/c mice. The expression of the cytokines INFγ, TNFα and IL1 was evaluated by real-time RT-PCR in splenocytes from vaccinated mice. All antigens delivered as subunit vaccines, especially P42 and P95, and the pcDNA3/P46 DNA vaccine were able to elicit strong immune responses. These vaccines induced cellular immune responses and the production of antibodies able to react with native M. hyopneumoniae proteins. Because both cellular and humoral immune responses were induced, P42 and P95 are promising candidates for a recombinant subunit vaccine and P46 is a promising candidate for a DNA vaccine against PEP.     

Cysteine proteases as potential antigens in antiparasitic DNA vaccines

Cysteine proteases in parasites are potent inducers of vertebrate host immune responses and may under certain circumstances take part in the pathogen's immune evasion strategies. These capacities place these parasite molecules as interesting candidate antigens in antiparasitic vaccines for use in vertebrates. Parasite cysteine proteases are able to skew the Th1/Th2 profile in mammals towards a response which allows sustainable parasite burdens in the host. DNA vaccines are also able to skew the Th1/Th2 profile by different administration techniques and the use of cysteine proteases in these genetic immunizations open perspectives for manipulation of the host immune response towards higher protection.     

Limitations of plasmid vaccines to complex viruses: selected myxoma virus antigens as DNA vaccines were not protective

Myxoma virus, a poxvirus of the genus Leporipoxvirus, is the causative agent of the disease myxomatosis which is highly lethal in European rabbits (Oryctolagus cuniculus). Current vaccines to protect against myxomatosis are either attenuated live strains of the virus or the antigenically related rabbit fibroma virus. We examined the immune response of outbred domestic rabbits to the individual myxoma virus antigens M055R, M073R, M115L and M121R, delivered as DNA vaccines co-expressing rabbit interleukin-2 or interleukin-4. M115L and M121R were also delivered simultaneously. None of the vaccine constructs were able to protect the rabbits from disease or reduce mortality after challenge with virulent myxoma virus, despite induction of antigen-specific cell-mediated and humoral immune responses.     

Electroporation of a multivalent DNA vaccine cocktail elicits a protective immune response against anthrax and plague

Electroporation of DNA vaccines represents a platform technology well positioned for the development of multivalent biodefense vaccines. To evaluate this hypothesis, three vaccine constructs were produced using codon-optimized genes encoding Bacillus anthracis Protective Antigen (PA), and the Yersinia pestis genes LcrV and F1, cloned into pVAX1. A/J mice were immunized on a prime-boost schedule with these constructs using the electroporation-based TriGrid Delivery System. Immunization with the individual pDNA vaccines elicited higher levels of antigen-specific IgG than when used in combination. DNA vaccine effectiveness was proven, the pVAX-PA titers were toxin neutralizing and fully protective against a lethal B. anthracis spore challenge when administered alone or co-formulated with the plague pDNA vaccines. LcrV and F1 pVAX vaccines against plague were synergistic, resulting in 100% survival, but less protective individually and when co-formulated with pVAX-PA. These DNA vaccine responses were Th1/Th2 balanced with high levels of IFN-γ and IL-4 in splenocyte recall assays, contrary to complimentary protein Alum vaccinations displaying a Th2 bias with increased IL-4 and low levels of IFN-γ. These results demonstrate the feasibility of electroporation to deliver and maintain the overall efficacy of an anthrax-plague DNA vaccine cocktail whose individual components have qualitative immunological differences when combined.     

Limitations of plasmid vaccines to complex viruses: selected myxoma virus antigens as DNA vaccines were not protective

Myxoma virus, a poxvirus of the genus Leporipoxvirus, is the causative agent of the disease myxomatosis which is highly lethal in European rabbits (Oryctolagus cuniculus). Current vaccines to protect against myxomatosis are either attenuated live strains of the virus or the antigenically related rabbit fibroma virus. We examined the immune response of outbred domestic rabbits to the individual myxoma virus antigens M055R, M073R, M115L and M121R, delivered as DNA vaccines co-expressing rabbit interleukin-2 or interleukin-4. M115L and M121R were also delivered simultaneously. None of the vaccine constructs were able to protect the rabbits from disease or reduce mortality after challenge with virulent myxoma virus, despite induction of antigen-specific cell-mediated and humoral immune responses.     

Distinct immune responses of recombinant plasmid DNA replicon vaccines expressing two types of antigens with or without signal sequences

Here, DNA replicon vaccines encoding the Hc domain of botulinum neurotoxin serotype A (AHc) or the receptor binding domain of anthrax protective antigen (PA4) with or without signal sequences were evaluated in mice. Strong antibody and protective responses were elicited only from AHc DNA vaccines with an Ig κ signal sequence or tissue plasminogen activator signal sequence. Meanwhile, there were no differences in total antibody responses or isotypes, lymphocyte proliferative responses, cytokine profiles and protective immune responses with the PA4 DNA vaccines with or without a signal sequence. Therefore, use of targeting sequences in designing DNA replicon vaccines depends on the specific antigen.     

Evaluation of immune response and protective effect of four vaccines against the tick-borne encephalitis virus

Among three main subtypes of the tick-borne encephalitis virus (TBEV), the Siberian subtype is currently dominant in a majority of the endemic regions of Russia. However, inactivated vaccines are based on TBEV strains of the heterologous Far Eastern or the European subtypes isolated 40–77 years ago. To analyze the efficacy of the available vaccines against currently prevailing TBEV isolates of the Siberian subtype, mice were immunized subcutaneously three times (one group per each vaccine). The expression of seven cytokine genes was determined using RT-PCR. Sera were studied using homologous and heterologous ELISA, hemagglutination inhibition (HI) and neutralization tests with TBEV strains of the Far Eastern, Siberian and European subtypes. Cross-protective efficacy of the vaccines was evaluated with the TBEV strain 2689 of Siberian subtype isolated from an ixodid tick from the Novosibirsk, South-Western Siberia, Russia in 2010. The cytokine gene expression profile indicates a predominantly Th2 response due to exogenous antigen presentation. Titers for homologous combinations of vaccine strain and strain in ELISA, HI and neutralization tests exceeded those for heterologous antigen-antibody pairs. Despite antibody detection by means of ELISA, HI and neutralization tests, the mouse protection afforded by the vaccines differed significantly. Complete protection of mice challenged with 100 LD₅₀ virus of the Siberian subtype was induced by the vaccine “Encevir” (“Microgen”, Tomsk, Russia). The minimal immunization doze (MID₅₀) of “Encevir” protecting 50% of the mice was less than 0.0016 ml. Partial protective effect of vaccines produced in Moscow, Russia and Austria revealed MID₅₀ within recommended intervals (0.001–0.017 ml). However, the MID₅₀ for the vaccine “Encepur” (Novartis, Germany) 0.04 ml exceeded acceptable limits with total loss of mice immunized with vaccine diluted 32, 100 and 320 fold. These results suggest regular evaluation of TBEV vaccines in regions where heterologous virus subtypes prevail.     

A recombinant chimera composed of R1 repeat region of Mycoplasma hyopneumoniae P97 adhesin with Escherichia coli heat-labile enterotoxin B subunit elicits immune response in mice

Swine mycoplasmal pneumonia (SMP), caused by fastidious bacterium Mycoplasma hyopneumoniae, is the most important respiratory disease in swine breeding. The commonly used vaccines to control this disease consist of inactivated whole cells (bacterins), whose production cost is high and the efficiency is limited. The objective of this study was to develop and to evaluate in BALB/c mice a recombinant subunit vaccine (rLTBR1) containing the R1 region of P97 adhesin of M. hyopneumoniae (R1) fused to the B subunit of the heat-labile enterotoxin of Escherichia coli (LTB). rLTBR1 formed functional oligomers that presented high affinity to GM1 ganglioside. Mice inoculated with rLTBR1 by intranasal (IN) or intramuscular (IM) route produced high levels of anti-R1 systemic and mucosal antibodies (IgA), which recognized the native P97. On the other hand, mice inoculated with the inactivated whole cell vaccine did not produce anti-R1 antibodies. The administration route influenced the modulation of the immune response by LTB, showing that IM rLTBR1 induced Th2-biased immune responses and IN rLTBR1 induced Th1-biased immune responses. rLTBR1 administrated by IN route also induced IFN-gamma secretion by lymphocytes. rLTBR1 may constitute a new strategy for preventing infection by M. hyopneumoniae and may have potential for developing vaccines against other infectious diseases as well.     

基因重组壳质酶融合蛋白抗原用于丝虫病血清学诊断

目的　应用基因重组壳质酶融合蛋白抗原酶联免疫吸附试验方法检测微丝蚴（Ｍｆ）血症沙鼠、正常沙鼠和班氏丝虫微丝蚴,探讨该方法在丝虫病血清学诊断中的价值及在现场推广前景。方法　用丝虫性单克隆抗体Ｍｆ１ 识别的微丝蚴壳质酶融合蛋白与祖系克隆的核苷酸系列基础引物合成寡核苷酸并进行ＰＣＲ扩增,以Ｐｍａｌｃ 为载体表达、亲和层析纯化。试验动物分别用壳质酶抗原和布鲁马来丝虫感染期幼虫接种免疫和感染,用ＳＤＳ聚丙烯酰胺凝胶电泳,免疫印渍技术分析壳质酶抗原的功能性蛋白分子。以酶联免疫吸附试验方法检测试验血清特异性抗体水平,并与Ｍｆｘｔ 抗原进行比较。结果　壳质酶抗原的特异功能性蛋白相对分子量为６９ ０００ 。壳质酶抗原检测微丝蚴血症长爪沙鼠和微丝蚴血症患者血清抗体阳性率为１００％ 。Ｍｆｘｔ 抗原检测的血清抗体阳性率为８０％ 。壳质酶抗原检测的正常沙鼠和正常人血清标本结果均为阴性,Ｍｆｘｔ 抗原在正常人血清中出现５％ 假阳性。流行区非微丝蚴血症者壳质酶抗原和Ｍｆｘｔ 抗原的假阳性率分别为５％ 和２０％ 。结论　基因重组壳质酶抗原酶联免疫吸附试验方法用于丝虫病血清学诊断敏感性高,特异性强,操作简便易行,具有实用价值和推广前景。     

IRX-2 increases the T cell-specific immune response to protein/peptide vaccines

Therapeutic cancer vaccines are attractive due to the prospect of specificity and their lack of toxicity; however, their clinical development has been hampered by several biologic and clinical challenges. One of the most important biologic challenges is the relative lack of effective cellular immune adjuvants. Effective physiologic immune responses are characterized by the local generation of a complex cytokine environment that activates and regulates multiple immune cell types. IRX-2 is a primary cell-derived biologic with physiological levels of multiple active cytokine components, produced under pharmaceutical standards. The hypothesis that IRX-2 amplifies the T cell response to defined antigens was assessed in mice by measuring the T cell-specific peptide response to a dominant mouse peptide (NFT) derived from human prostate-specific membrane antigen (PSMA). IRX-2 enhances the T cell response to NFT when antigens were delivered either via irradiated cells expressing human PSMA, NFT peptide in Incomplete Freund's adjuvant (IFA) or NFT peptide conjugated to KLH. The T cell-specific activity was measured in spleen or lymph nodes cells by IFN-γ ELISpot and/or IFN-γ secretion over 6 days or in vivo by peptide-specific delayed-type hypersensitivity reaction (DTH). Further more, a single administration of IRX-2 with the antigen was not active as compared to 4 or 9 additional administrations which were sufficient to enhance the T cell response to antigens. The influence of IRX-2 on the B cell response to ovalbumin when it was used as a carrier protein was measured by ELISA. IRX-2 was compared to a commercially available combination adjuvant (MPL + TDM in squalene/Tween 80) which based on the literature is a potent adjuvant in murine systems. In the T cell assay IRX-2 was superior to the commercially available combination adjuvant and while IRX-2 also increased antibody titer, it was not as potent as the combination adjuvant. Mice immunized with IRX-2 and antigen also exhibited delayed tumor progression following challenge with PSMA-expressing tumor cells. These studies demonstrate that IRX-2 is an immunomodulator with adjuvant activity which preferentially enhances the T cell-specific responses to tumor associated antigens. Based on these studies, IRX-2 is a candidate for evaluation as a T cell adjuvant in a variety of preclinical vaccine delivery systems as well as in human clinical trials with cancer vaccine candidates.     

Co-administration of attenuated Mycoplasma hyopneumoniae 168 strain with bacterial DNA enhances the local and systemic immune response after intranasal vaccination in pigs

Mycoplasma hyopneumoniae, the primary pathogen of enzootic pneumonia, occurs worldwide and causes major economic losses to the pig industry. M. hyopneumoniae infects pigs at mucosal surfaces of respiratory tract. The aim of the present study was to investigate if the protection rate against M. hyopneumoniae infection following intranasal immunization with attenuated M. hyopneumoniae 168 strain is improved by administration of bacterial DNA containing CpG motifs. Thirty pigs were immunized intranasally or intramuscularly and the levels of local respiratory tract and systemic immune responses were detected. The results showed that the number of intraepithelial lymphocytes in the tracheal fork, the levels of cytokine IL-6, and M. hyopneumoniae specific SIgA in local nasal cavity increased respectively after intranasal vaccination with the attenuated M. hyopneumoniae 168 strain alone. However, the levels of IL-10 and IFN-γ in local nasal cavity, the number of intraepithelial lymphocytes in trachea, CD4(+) and CD8(+) T lymphocytes in the lung and hilar lymph nodes, the specific IgG antibody level in serum on 35 day post immunization were all increased significantly after intranasal vaccination of the attenuated M. hyopneumoniae 168 strain adjuvanted with bacterial DNA. We concluded that intranasal administration of attenuated M. hyopneumoniae 168 strain adjuvanted with bacterial DNA may be effective in evoking the local cellular and humoral immune response in the respiratory tract and the systemic immune response. Intranasal vaccination will be effective in prevention of the transmission and prevalence of MPS.     

Factors influencing the immune response to foreign antigen expressed in recombinant BCG vaccines

A wide range of recombinant BCG vaccine candidates containing foreign viral, bacterial, parasite or immunomodulatory genetic material have been developed and evaluated, primarily in animal models, for immune response to the foreign antigen. This review considers some of the factors that may influence the immunogenicity of these vaccines. The influence of levels and timing of expression of the foreign antigen and the use of targeting sequences are considered in the first section. Genetic and functional stability of rBCG is reviewed in the second section. In the last section, the influence of dose and route of immunization, strain of BCG and the animal model used are discussed.     

Rabies virus glycoprotein and immune response pattern using recombinant protein or recombinant RNA viral vectors

The present study shows the humoral and cellular aspects of immune response generated by a recombinant rabies virus glycoprotein (rRVGP) as compared to those generated by viral vector carrying the RNA coding for this protein (RVGP-RNA). The rRVGP was synthesized by stably transfected Drosophila melanogaster Schneider 2 (S2) cells and the RVGP-RNA was carried by a recombinant Semiliki Forest Virus (SFV-RVGP). The data show that protein as well as the RNA vaccine was capable of inducing reasonably acceptable levels of antibodies as compared to the optimized commercial whole virus vaccine. As expected, the RNA vaccine was clearly more effective than the protein vaccines in inducing a cellular immune response, as evaluated by the IgG2a/IgG1 ratio and synthesis of interferon gamma (IFNγ) and interleukin 2 (IL2). Our study supports the importance of vaccine designing taking into consideration the concept of DNA/RNA ability to induce an effective cell immune response.     

The humoral immune response and protective efficacy of vaccination with inactivated split and whole influenza virus vaccines in BALB/c mice

Recently the urgency of developing a pandemic influenza vaccine has lead to the re-evaluation of the use of whole virus vaccine. We have compared the humoral immune response and the protective efficacy of whole and split influenza virus vaccines in mice. Whole virus vaccine was more immunogenic particularly after the first dose of vaccine, generally eliciting higher numbers of systemic antibody secreting cells and an earlier and higher neutralising antibody response. Immunisation with one dose of whole virus vaccine more effectively reduced viral shedding upon non-lethal homologous viral challenge, but two doses of split virus vaccine was most effective at limiting viral replication and this was correlated with high influenza specific serum IgG concentrations. The two vaccine formulations induced different T helper profiles particularly after one dose of vaccine; split virus vaccine induced a type 2 bias response, whereas whole virus vaccine elicited a dominant type 1 response.     

In vivo protein expression and immune responses generated by DNA vaccines expressing mycobacterial antigens fused with a reporter protein

We cloned six mycobacterial antigens into a mammalian expression vector as fusion proteins with the enhanced green fluorescent protein (EGFP). Plasmid DNA was injected intramuscularly, and the injection sites were examined 1 week later. Expression of each antigen-EGFP fusion protein was visualized as green fluorescence in muscle tissue sections. A plasmid expressing EGFP alone and a plasmid with a frameshift mutation served as positive and negative controls. Visualization of fluorescent protein in vivo was 100% specific when compared to in vitro results. In vivo sensitivity was only 37% based on individual injection sites, but increased to 100% when results from multiple injection sites were combined for each plasmid. EGFP alone was expressed in a higher proportion of myocytes than the antigen-EGFP fusion proteins (P < 0.001). There was a trend toward an inverse correlation between protein size and the proportion of myocytes with visible fluorescence (r = -0.68; P = 0.09). We compared antibody subtypes generated to Mycobacterium bovis antigen 85A, when it was expressed alone or as a fusion protein. Inclusion of EGFP modified the immune response toward a Th1 response, as indicated by the ratio of antigen 85A-specific IgG2a to IgG1 generated by each plasmid (antigen 85A alone 0.73 +/- 0.18 versus antigen 85A-EGFP 1.82 +/- 0.57, mean +/- S.D.; P < 0.01), though the magnitude of the antibody isotype shift was modest. Direct visualization of antigen-EGFP fusion proteins provided a simple and rapid method to confirm in vivo antigen expression.     

Evaluation of the immune response and protective efficacy of Schistosoma mansoni Cathepsin B in mice using CpG dinucleotides as adjuvant

Schistosomiasis is the most important human helminth infection due to its impact on public health. Worldwide, schistosomiasis is estimated to infect at least 200 million individuals while 700 million are at risk. The clinical manifestations are chronic and significantly decrease an individual's quality of life. Infected individuals suffer from long-term organ pathologies including fibrosis which eventually leads to organ failure. The development of a vaccine against this parasitic disease would contribute to a long-lasting decrease in disease spectrum and transmission. Our group has chosen to target Schistosoma mansoni Cathepsin B as a prospective vaccine candidate. The recombinant protein was tested in the presence of synthetic oligodeoxynucleotides containing unmethylated CpG dinucleotides, which are Toll-like receptor 9 agonists known to stimulate a Th1 response. This formulation conferred a 59% decrease in worm burden as well as a reduction in egg burden. Hepatic egg burden and intestinal egg burden were decreased by 56% and 54% respectively. Immunizations with the formulation elicited robust production of Sm-Cathepsin B specific antibodies, both IgG1 and IgG2c but with the latter predominating. Furthermore, splenocytes isolated from the immunized animals, compared to control animals, had increased secretion levels of key Th1 cytokines, IFN-γ and TNF-α, as well as the chemokine CCL5 when stimulated with recombinant Sm-Cathepsin B. These results highlight the potential of Sm-Cathepsin B/CpG as a vaccine candidate against schistosomiasis.     

Electroporation enhances protective immune response of a DNA vaccine against Japanese encephalitis in mice and pigs

Japanese encephalitis virus (JEV) is a pathogenic cause of Japanese Encephalitis (JE), which is a zoonotic disease transmitted by mosquitoes and amplified by pigs. Infection of JEV may lead to severe neurological sequelae, even death in humans and reproductive disorders in pigs. Vaccination is the only way to control JEV infection in humans. For pigs play important role in the JEV transmission cycle, developing a new veterinary vaccine is considered as a useful strategy for cutting off the transmission route of JEV. We have previously reported that DNA vaccine pCAG-JME, expressing prM-E proteins of JEV, is effective in mice through intramuscular injection (IM). However, the poor immunogenicity, due to low expression of immunogen, is the major obstacle for the development of DNA vaccine in large animals. In the present study, therefore, we immunized mice and pigs with pCAG-JME intramuscularly accompanied with electroporation (EP) stimulation, the attractive gene delivery approach. As compared with IM, EP-mediated vaccination markedly increased the expression of immunogen in the injection site and induced a dose- and time-dependent immune response. 100% survival rate was observed in groups vaccinated with doses ranged from 10 to 100μg, indicating that 10μg of DNA with EP for individual was enough for inducing effective protection in mice. Surprisingly, survival rate and end-point titers of anti-JEV antibodies were higher in mice even at lower dose of DNA (5μg) than that in mice inoculated 100μg through IM. Notably, the prM-E antigens also induced high antibody response in pig, while the neutralizing antibody titer achieved 1:320. Our results suggested that EP-mediated DNA immunization might act as an effective strategy against JEV, at least in pig, and that EP has a potential application prospect in DNA vaccination.     

Evaluation of protective and immune responses following vaccination with recombinant MIP and CPAF from Chlamydia abortus as novel vaccines for enzootic abortion of ewes

MIP and CPAF from Chlamydia have been shown to be effective in inducing immune responses important in clearing chlamydial infections. This study evaluates the protection conferred by MIP and CPAF as novel vaccines in pregnant C. abortus challenged ewes. Fifty C. abortus sero-negative sheep were randomly allocated into 5 groups of 10 according to the treatment they were to receive (1) 100 µg of MBP-MIP (2) 100 µg CPAF (3) 50 µg MBP-MIP and 50 µg CPAF (4) Tris-buffer (negative control) (5) Enzovax (positive control). Booster inoculations were administered 3 weeks after primary inoculations. Blood samples were taken pre-vaccination and weekly for 5 weeks. Five months after vaccination the ewes were mated. Pregnant ewes were then challenged on day 90 of gestation. Blood samples taken at four time-points post challenge were analysed for IFNγ levels, TNFα and IL-10 expression and anti-chlamydial antibody levels. Vaginal swabs, placental and foetal tissue and bacterial shedding were analysed using qPCR to quantify levels of C. abortus. Enzovax was 100% effective with no abortions occurring. The MIP/CPAF combined vaccine offered the greatest protection of the novel vaccines with 67% of ewes giving birth to one or more live lambs equating to a 50% vaccine efficacy rate. MIP and CPAF administered singly did not confer protection. Enzovax and MIP/CPAF vaccinated ewes had longer gestations and lambs with higher birth weights than negative control ewes. Aborting ewes shed higher numbers of C. abortus than ewes that had live lambs, all vaccinated ewes demonstrated lower levels of bacterial shedding than negative control ewes with Enzovax ewes shedding significantly fewer bacteria. Ewes that went on to abort had significantly higher levels of IFNγ and IL-10 at day 35 post challenge and significantly higher levels of anti-chlamydial antibodies at 24 h post lambing compared to ewes that had live lambs.