BCG Priming Research Articles

Protection against tuberculosis achieved by dissolving microneedle patches loaded with live Mycobacterium paragordonae in a BCG prime-boost strategy.

Skin vaccination using dissolving microneedle patch (MNP) technology for transdermal delivery is a promising vaccine delivery strategy to overcome the limitations of the existing vaccine administration strategies using syringes. To improve the traditional microneedle mold fabrication technique, we introduced droplet extension (DEN) to reduce drug loss. Tuberculosis remains a major public health problem worldwide, and BCG revaccination had failed to increase the protective efficacy against tuberculosis. We developed an MNP with live Mycobacterium paragordonae (Mpg) (Mpg-MNP) as a candidate of tuberculosis booster vaccine in a heterologous prime-boost strategy to increase the BCG vaccine efficacy. The MNPs were fabricated by the DEN method on a polyvinyl alcohol mask film and hydrocolloid-adhesive sheet with microneedles composed of a mixture of mycobacteria and hyaluronic acid. We assessed the transdermal delivery efficiency by comparing the activation of the dermal immune system with that of subcutaneous injection. A BCG prime Mpg-MNP boost regimen was administered to a mouse model to evaluate the protective efficacy against M. tuberculosis. We demonstrated the successful transdermal delivery achieved by Mpg-MNP compared with that observed with BCG-MNP or subcutaneous vaccination via an increased abundance of MHCII-expressing Langerin+ cells within the dermis that could migrate into draining lymph nodes to induce T-cell activation. In a BCG prime-boost regimen, Mpg-MNP was more protective than BCG-only immunization or BCG-MNP boost, resulting in a lower bacterial burden in the lungs of mice infected with virulent M. tuberculosis. Mpg-MNP-boosted mice showed higher serum levels of IgG than BCG-MNP-boosted mice. Furthermore, Ag85B-specific T-cells were activated after BCG priming and Mpg-MNP boost, indicating increased production of Th1-related cytokines in response to M. tuberculosis challenge, which is correlated with enhanced protective efficacy. The MNP fabricated by the DEN method maintained the viability of Mpg and achieved effective release in the dermis. Our data demonstrate a potential application of Mpg-MNP as a booster vaccine to enhance the efficacy of BCG vaccination against M. tuberculosis. This study produced the first MNP loaded with nontuberculous mycobacteria (NTM) to be used as a heterologous booster vaccine with verified protective efficacy against M. tuberculosis.

Heparin-binding hemagglutinin as a composition antigen of tuberculosis vaccine exerts protective immune effects by inducing IL-17

To explore the protective immune effect induced by mucosal delivery heparin-binding hemagglutinin (HBHA)-a candidate vaccine antigen of Mycobacterium tuberculosis. Female C57BL/6 mice were between 6 and 8 weeks of age before experimental use. Thirty mice received different immunization strategies and were randomly divided into the control group, the early secreting antigen target-6 (ESAT-6) intranasal immunization group, the HBHA intranasal immunization group, the BCG priming PBS control group, or BCG priming HBHA boost group, 6 mice in each group. In order to analyzed the immune effect, the concentrations of plasma Interleukin-17A (IL-17A) and other cytokines were measured by ELISA. Quantitative real-time PCR analyses were performed to detect the relative quantity (RQ) mRNA of IL-17A in the lung. The lung tissue sections were stained to detect the formation of the tertiary lymphoid structures. The chemokines contributed to formation of the tertiary lymphoid structures were also measured. Flow cytometry was used to detect the frequency of Th1 and Th17 cells in the system. Sixty mice in the BCG priming PBS control group and the BCG priming HBHA boost group were sacrificed at different time points after infection to count the lung bacterial burden. The concentrations of plasma IL-17A and relative quantity of lung IL-17A mRNA were highest in the BCG priming HBHA boost group [(14.76±4.73) pg/mL,RQ (12.27±6.71)], which was significantly higher than the control group [(5.57±2.95) pg/mL,RQ (1.30±0.97)] (t=4.213, P<0.001; t=5.984, P<0.001), and also significantly higher than the BCG priming PBS control group [(6.81±2.18) pg/mL,RQ (1.44±1.16)] (t=3.646 P=0.001; t=6.185 P<0.001). Compared with the BCG priming PBS control group (0.38±0.38)% the frequency of spleen Th17 cells were also significantly increased (t=-0.280, P=0.048) in the BCG-primary HBHA boost group (1.02±0.34)%. In addition, HBHA boosting could promote better formation of the tertiary lymphoid structures in the lung, and decrease the bacterial load on the early stage after BCG challenge. Collectively, mucosal delivery of HBHA can effectively enhance the protective effect after BCG vaccination, and it is a potential candidate vaccine component.

Changes in the Immune Phenotype and Gene Expression Profile Driven by a Novel Tuberculosis Nanovaccine: Short and Long-Term Post-immunization.

Deciphering protection mechanisms against Mycobacterium tuberculosis (Mtb) remains a critical challenge for the development of new vaccines and therapies. We analyze the phenotypic and transcriptomic profile in lung of a novel tuberculosis (TB) nanoparticle-based boosting mucosal vaccine Nano-FP1, which combined to BCG priming conferred enhanced protection in mice challenged with low-dose Mtb. We analyzed the vaccine profile and efficacy at short (2 weeks), medium (7 weeks) and long term (11 weeks) post-vaccination, and compared it to ineffective Nano-FP2 vaccine. We observed several changes in the mouse lung environment by both nanovaccines, which are lost shortly after boosting. Additional boosting at long-term (14 weeks) recovered partially cell populations and transcriptomic profile, but not enough to enhance protection to infection. An increase in both total and resident memory CD4 and CD8 T cells, but no pro-inflammatory cytokine levels, were correlated with better protection. A unique gene expression pattern with differentially expressed genes revealed potential pathways associated to the immune defense against Mtb. Our findings provide an insight into the critical immune responses that need to be considered when assessing the effectiveness of a novel TB vaccine.

An Ebola virus vaccine that leverages helper T cells induced by prior BCG immunization

Abstract Protective antibody responses against the Ebola virus (EBOV) are dependent on the quality of CD4+ helper T (Th) cells. We propose that BCG specific Th cells may provide a source of Th cells that can be recruited to drive antibody responses against EBOV, given that the majority of individuals residing in EBOV endemic regions have been previously vaccinated with BCG. To take advantage of pre-existing BCG Th cells, a vaccine was developed that consists of the EBOV GP fused to the mycobacteria Th cell epitope (P25) of Ag85B (P25-EBOV GP). Presentation of the P25 epitope by B cells recruited cognate interactions with P25 Th cells to promote antibody responses against the EBOV GP. Experiments in mice showed BCG priming induced a broad range of Th cells including Th1, Th2, and Tfh subsets. These P25 Th subsets provided a significant dose sparing effect on the P25-EBOV GP vaccine in the induction of EBOV GP antibodies and resulted in increased titers of high affinity neutralizing IgG1 antibodies. The induction of IgG2c antibodies only occurred in BCG primed mice, where BCG vaccination is known to be a strong inducer of Th1 responses that drive class-switching to IgG2c. Microscopy of sections from secondary lymphoid organs of BCG primed mice showed that the majority of P25 Th cells remained in extrafollicular regions outside germinal centers and were most responsible for the induction of the IgG2c class-switched antibodies that were weakly neutralizing but capable of mediating antibody-dependent cellular toxicity (ADCC). BCG priming followed by vaccination with P25-EBOV GP protected mice against lethal EBOV infection, thus suggesting a potentially useful strategy for vaccination against EBOV in populations with high prevalence of previous BCG vaccination.

Boosting BCG with proteins or rAd5 does not enhance protection against tuberculosis in rhesus macaques

Tuberculosis (TB) is the leading cause of death from infection worldwide. The only approved vaccine, BCG, has variable protective efficacy against pulmonary TB, the transmissible form of the disease. Therefore, improving this efficacy is an urgent priority. This study assessed whether heterologous prime-boost vaccine regimens in which BCG priming is boosted with either (i) protein and adjuvant (M72 plus AS01E or H56 plus CAF01) delivered intramuscularly (IM), or (ii) replication-defective recombinant adenovirus serotype 5 (Ad5) expressing various Mycobacterium tuberculosis (Mtb) antigens (Ad5(TB): M72, ESAT-6/Ag85b, or ESAT-6/Rv1733/Rv2626/RpfD) administered simultaneously by IM and aerosol (AE) routes, could enhance blood- and lung-localized T-cell immunity and improve protection in a nonhuman primate (NHP) model of TB infection. Ad5(TB) vaccines administered by AE/IM routes following BCG priming elicited ~10–30% antigen-specific CD4 and CD8 T-cell multifunctional cytokine responses in bronchoalveolar lavage (BAL) but did not provide additional protection compared to BCG alone. Moreover, AE administration of an Ad5(empty) control vector after BCG priming appeared to diminish protection induced by BCG. Boosting BCG by IM immunization of M72/AS01E or H56:CAF01 elicited ~0.1–0.3% antigen-specific CD4 cytokine responses in blood with only a transient increase of ~0.5–1% in BAL; these vaccine regimens also failed to enhance BCG-induced protection. Taken together, this study shows that boosting BCG with protein/adjuvant or Ad-based vaccines using these antigens, by IM or IM/AE routes, respectively, do not enhance protection against primary infection compared with BCG alone, in the highly susceptible rhesus macaque model of tuberculosis.

The effects of immunity induced by BCG priming and tuberculosis fusion protein AMM vaccine boosting

Objective To investigate the boosting efficiency of a subunit vaccine consisting of the fusion protein Ag85B-Mpt64190-198-Mth8.4 (AMM) , dimethyl-dioctyldecyl ammonium bromide (DDA) and BCG polysaceharide nucleic acid (BCG-PSN) on the primed inoculation with BCG. Methods The AMM subunit vaccine was composed of fusion protein AMM, adjuvant DDA and BCG-PSN. The first mouse experi-mental group was immunized with BCG first, then boosted with the AMM subanit vaccine in the 10th week. The second experimental group was boosted with the AMM subunit vaccine in the 8th week and the 10th week respectively with a two weeks interval after the primed with BCG. Two control groups were treated re-spectively with physiological saline alone and BCG alone. After the primed inoculation, ELISPOT and ELISA were used for the detection of the cell-mediated and humoral immune response in week 14 and week 22 re-spectively. Furthermore, the immunized mice were challenged with live BCG to mimic tuberculosis infection in the 22nd week after the primed inoculation. Subsequently the T cell typing and humoral response were de-tected by flow cytometry and ELISA, respectively. Results ( 1 ) The level of secreting IFN-γ: 14 weeks af-ter the primed inoculation,with the stimulation of the specific antigen-Ag85B, the number of cells secreting IFN-γ in the second experimental group (135±14) was more than BCG alone immunized group (19±16), t = 10. 98, P < 0.01. In the 22nd week, the number of cells secreting IFN-γ in the second experimental group (208±11) was still more than BCG alone group (57±18), t =6.43, P <0.01. (2) The level of humoral immune response: the IgG1 antibody titer in the second experimental group was obviously higher than that in the first experimental group. However, the ratio of IgG2a to IgG1, as the index reflecting the Thl-type immune response, in the experimental group 2 was lower than that in the experimental group 1. (3) The contents of CD4+ CD25+ T cells after challenged with live BCG strain: the first and the second ex-perimental groups were both higher than the BCG alone group (t1 = 3.08, t2 = 3.16, P < 0.05 ). Conclu-sion Boosting the BCG-pfimed mice with tuberculosis AMM subunit vaccine twice can induce higher level of cell-mediated and humoral immune response than BCG alone, which could activate the regulative immune response at the same time. Key words: Subunit vaccine; Fusion protein; BCG; Immunization strategy; Prime-boost

Enhanced effect of BCG vaccine against pulmonary Mycobacterium tuberculosis infection in mice with lung Th17 response to mycobacterial heparin-binding hemagglutinin adhesin antigen.

Although the BCG vaccine can prevent tuberculosis (TB) in infants, its ability to prevent adult pulmonary TB is reportedly limited. Therefore, development of a novel effective vaccine against pulmonary TB has become an international research priority. We have previously reported that intranasal vaccination of mice with a mycobacterial heparin-binding hemagglutinin adhesin (HBHA) plus mucosal adjuvant cholera toxin (CT) enhances production of IFN-γ and anti-HBHA antibody and suppresses extrapulmonary bacterial dissemination after intranasal infection with BCG. In the present study, the effects of intranasal HBHA + CT vaccine on murine pulmonary Mycobacterium tuberculosis (Mtb) infection were examined. Intranasal HBHA + CT vaccination alone failed to reduce the bacterial burden in the infected lung. However, a combination vaccine consisting of s.c. BCG priming and an intranasal HBHA + CT booster significantly enhanced protective immunity against pulmonary Mtb infection on day 14 compared with BCG vaccine alone. Further, it was found that intranasal HBHA + CT vaccine enhanced not only IFN-γ but also IL-17A production by HBHA-specific T cells in the lung after pulmonary Mtb infection. Therefore, this combination vaccine may be a good candidate for a new vaccine strategy against pulmonary TB.

IL-28B down-regulates regulatory T cells but does not improve the protective immunity following tuberculosis subunit vaccine immunization.

Regulatory T cells (Tregs), which could be down-regulated by IL-28B, were reported to suppress T-cell-mediated immunity. The aim of this study was to investigate the role of IL-28B on the immune responses and protective efficacy of a tuberculosis (TB) subunit vaccine. First, a recombinant adenoviral vector expressing mouse IL-28B (rAd-mIL-28B) was constructed; then C57BL/6 mice were immunized with subunit vaccine ESAT6-Ag85B-Mpt64(190-198)-Mtb8.4-HspX (EAMMH) and rAd-mIL-28B together thrice or primed with Mycobacterium bovis bacillus Calmette-Gue'rin (BCG) and boosted by EAMMH and rAd-mIL-28B twice. At last the immune responses were evaluated, and the mice primed with BCG and boosted by subunit vaccines were challenged with virulent Mycobacterium tuberculosis H37Rv to evaluate the protective efficacy. The results showed that rAd-mIL-28B treatment significantly down-regulated the frequency of Tregs at 4 weeks after the last immunization but did not increase the Th1-type immune responses. Moreover, in the regimen of BCG priming and EAMMH boosting, rAd-mIL-28B treatment did not increase the antigen-specific cellular and humoral immune responses, and consequently did not reduce the bacteria load following H37Rv challenge. Instead, it induced more serious pathology reaction. In conclusion, IL-28B down-regulates Tregs following EAMMH vaccination but does not improve the protective immune responses.

AdHu5Ag85A Respiratory Mucosal Boost Immunization Enhances Protection against Pulmonary Tuberculosis in BCG-Primed Non-Human Primates.

Persisting high global tuberculosis (TB) morbidity and mortality and poor efficacy of BCG vaccine emphasizes an urgent need for developing effective novel boost vaccination strategies following parenteral BCG priming in humans. Most of the current lead TB vaccine candidates in the global pipeline were developed for parenteral route of immunization. Compelling evidence indicates respiratory mucosal delivery of vaccine to be the most effective way to induce robust local mucosal protective immunity against pulmonary TB. However, despite ample supporting evidence from various animal models, there has been a lack of evidence supporting the safety and protective efficacy of respiratory mucosal TB vaccination in non-human primates (NHP) and humans. By using a rhesus macaque TB model we have evaluated the safety and protective efficacy of a recombinant human serotype 5 adenovirus-based TB vaccine (AdHu5Ag85A) delivered via the respiratory mucosal route. We show that mucosal AdHu5Ag85A boost immunization was safe and well tolerated in parenteral BCG-primed rhesus macaques. A single AdHu5Ag85A mucosal boost immunization in BCG-primed rhesus macaques enhanced the antigen–specific T cell responses. Boost immunization significantly improved the survival and bacterial control following M.tb challenge. Furthermore, TB-related lung pathology and clinical outcomes were lessened in BCG-primed, mucosally boosted animals compared to control animals. Thus, for the first time we show that a single respiratory mucosal boost immunization with a novel TB vaccine enhances protection against pulmonary TB in parenteral BCG-primed NHP. Our study provides the evidence for the protective potential of AdHu5Ag85A as a respiratory mucosal boost TB vaccine for human application.

Immunogenic potential of latency associated antigens against Mycobacterium tuberculosis

Tuberculosis remains a great health threat to the world among infectious diseases particularly with the advent of human immunodeficiency virus and emergence of drug resistant strains. In the light of the inconsistent efficacy imparted by the only currently available pre-exposure vaccine bacillus Calmette–Guerin BCG, the development of an improved TB vaccine is a very high international research priority. Vaccine candidates currently in clinical trials are also pre-exposure vaccines that aim to prevent active tuberculosis during an individual's lifetime. According to World Health Organization approximately a third of the world's population is latently infected with Mycobacterium tuberculosis. Dormancy or latency of Mycobacteria is associated with the formation of granuloma with poorly perfused interior leading to expression of genes which help them survive in this hostile environment. A group of about 50 genes belonging to the DosR regulon also known as latency antigens are expressed by Mycobacteria when they are persisting in the immuno-competent host. An understanding of the immunological effects produced by products of these latency induced genes may help in making a more potent vaccine. Incorporation of latency antigens into improved (live or subunit) vaccines may enhance the impact of these vaccines in which BCG priming can be followed by multisubunit protein boosting. These vaccines could act as post exposure vaccines for containment and prevention of latent TB activation. This heterologous boosting of BCG-primed immunity will be able to stimulate the known immune correlates of protective immunity against M. tuberculosis i.e. TH1 cells (CD4+ and CD8+ T cells) mediated immune responses with cytokines such as IFN-γ and TNF-α⋅ In our review we have analysed and compared the immunogenic potential of various latency-associated antigens of the DosR regulon in line with the current strategy of developing a recombinant post exposure booster vaccine.

BCG and BCG/DNAhsp65 Vaccinations Promote Protective Effects without Deleterious Consequences for Experimental Autoimmune Encephalomyelitis

A prime-boost strategy conserving BCG is considered the most promising vaccine to control tuberculosis. A boost with a DNA vaccine containing the mycobacterial gene of a heat shock protein (pVAXhsp65) after BCG priming protected mice against experimental tuberculosis. However, anti-hsp65 immunity could worsen an autoimmune disease due to molecular mimicry. In this investigation, we evaluated the effect of a previous BCG or BCG/pVAXhsp65 immunization on experimental autoimmune encephalomyelitis (EAE) development. Female Lewis rats were immunized with BCG or BCG followed by pVAXhsp65 boosters. The animals underwent EAE induction and were daily evaluated for weight loss and clinical score. They were euthanized during recovery phase to assess immune response and inflammatory infiltration at the central nervous system. Previous immunization did not aggravate or accelerate clinical score or weight loss. In addition, this procedure clearly decreased inflammation in the brain. BCG immunization modulated the host immune response by triggering a significant reduction in IL-10 and IFN-γ levels induced by myelin basic protein. These data indicated that vaccination protocols with BCG or BCG followed by boosters with pVAXhsp65 did not trigger a deleterious effect on EAE evolution.

Broad heparin‐binding haemagglutinin‐specific cytokine and chemokine response in infants following Mycobacterium bovis BCG vaccination

Heparin-binding haemagglutinin (HBHA)-specific immune responses have been linked to protection against tuberculosis (TB). We investigated the hypothesis that BCG vaccination of human infants primes an HBHA-specific response, using multiplex to measure secreted cytokines and chemokines following HBHA and Mycobacterium tuberculosis purified protein derivative (PPD) stimulation of diluted whole blood samples from BCG-vaccinated or -unvaccinated infants. Of 42 analytes measured, 24 and 32 significant, BCG-associated increases were detected in response to HBHA and PPD, respectively. Both response profiles included Th-1, Th-2, Th-17 and inflammatory cytokines and chemokines (e.g. IFN-γ, TNF-α, IL-5, IL-10, IL-13, IL-17, MIP-1α and MIP-1β). We also found that six of the seven responses most closely correlated with IFN-γ were common to both HBHA and PPD. Notably, all HBHA-specific secretion of cytokines and chemokines from infant samples was dependent on previous BCG vaccination. Also, long-term persistence of HBHA-specific responses was found in adolescents with evidence of infant BCG vaccination. This study demonstrates for the first time BCG priming of an HBHA-specific immune response in infants that is characterised by a broad cytokine and chemokine signature. It also suggests a number of BCG vaccination associated, HBHA-induced responses that should be useful for future studies of biomarkers of protection against TB.

Nasal associated lymphoid tissue (NALT) contributes little to protection against aerosol challenge with Mycobacterium tuberculosis after immunisation with a recombinant adenoviral vaccine

Intra-nasal administration of a recombinant adenovirus expressing Mycobacterium tuberculosis antigen 85A (Ad85A) has been shown to provide protection against challenge with M. tuberculosis. However the role of the upper respiratory tract associated lymphoid tissue, specifically the nasal associated lymphoid tissue (NALT), in providing protection has yet to be elucidated. Here we administered Ad85A to BALB/c mice alone or following BCG priming, using intranasal inocula targeting the whole respiratory tract or only the NALT, to show that Ad85A induces an immune response in the NALT insufficient to provide protection. Rather, Ad85A delivered through the respiratory tract must induce a deep lung immune response in order to protect against M. tuberculosis.

Neonatal BCG Immunization Followed by DNAhsp65 Boosters: Highly Immunogenic but not Protective Against Tuberculosis - a Paradoxical Effect of the Vector?

A new tuberculosis vaccine is urgently needed. Prime-boost strategies are considered very promising and the inclusion of BCG is highly desirable. In this investigation, we tested the protective efficacy of BCG delivered in the neonatal period followed by boosters in the adult phase with a DNA vaccine containing the hsp65 gene from Mycobacterium leprae (pVAXhsp65). Immune responses were characterized by serum anti-hsp65 antibody levels and IFN-gamma and IL-5 production by the spleen. Amounts of these cytokines were also determined in lung homogenates. Protective efficacy was established by the number of colony-forming units (CFU) and histopathological analysis of the lungs after challenge with Mycobacterium tuberculosis. Immunization with BCG alone triggered a significant reduction of CFU in the lungs and also clearly preserved the pulmonary parenchyma. BCG priming also increased the immunogenicity of pVAXhsp65. However, boosters with pVAXhsp65 or the empty vector abolished the protective efficacy of BCG. Also, higher IL-5 levels were produced by spleen and lungs after DNA boosters. These results demonstrated that neonatal BCG immunization followed by DNAhsp65 boosters is highly immunogenic but is not protective against tuberculosis.

Fusion protein Ag85B-MPT64 190–198-Mtb8.4 has higher immunogenicity than Ag85B with capacity to boost BCG-primed immunity against Mycobacterium tuberculosis in mice

Tuberculosis (TB) remains a major infectious disease worldwide despite chemotherapy and BCG vaccine. The efficacy of the current TB vaccine BCG varies from 0 to 80%. New vaccines that have better protection than BCG or have the capability to boost BCG-primed immunity are urgently needed. We have previously constructed a fusion protein Ag85B-MPT64 190–198-Mtb8.4 (AMM). In this study, we investigated the immunogenicity of the fusion protein AMM in a novel adjuvant of dimethyl-dioctyldecyl ammonium bromide and BCG polysaccharide nucleic acid (DDA–BCG PSN), and its capacity to boost BCG-primed immunity. The anti-Ag85B antibodies IgG1 and IgG2a were determined using ELISA and the number of spleen cells secreting IFN-γ was determined by ELISPOT. In addition, the ability of the subunit vaccine AMM to boost BCG-primed immunity against Mycobacterium tuberculosis was analyzed. The fusion protein AMM induced more effective humoral and cell-mediated immune responses in mice than Ag85B alone. Mice primed with BCG vaccination followed by boosting with AMM produced a stronger immune response and afforded a better protection against M. tuberculosis infection than mice immunized with BCG alone or BCG priming followed by boosting with Ag85B. These findings suggest that AMM is a promising candidate subunit vaccine to enhance the protective efficiency of BCG.

Subcutaneous boosting with heparin binding haemagglutinin increases BCG-induced protection against tuberculosis

Pulmonary tuberculosis remains a major health problem. Effective vaccination strategies are urgently needed. It was previously demonstrated that purified Mycobacterium bovis BCG Heparin Binding Haemagglutinin (HBHA) is able to induce in BALB/c mice protection levels against a Mycobacterium tuberculosis infection that are similar to those offered by BCG. Here we developed a heterologous prime/boost immunisation approach using a combination of BCG and HBHA in order to increase the protective immune response. We show that the time period between BCG priming and HBHA boosting strongly influences the efficacy of the boost. The optimized vaccine protocol consisting of a BCG administration followed 8 months later by boosting with HBHA resulted in an increase in the level of protection of up to 0.7 log when compared to BCG alone. These results suggest an immunisation strategy where BCG is administered to neonates and is followed by subcutaneous HBHA boosting in young adults.

Improved efficacy of DNA vaccination against prostate carcinoma by boosting with recombinant protein vaccine and by introduction of a novel adjuvant epitope

DNA vaccine represents an attractive approach for cancer treatment by inducing active immune-deprivation of gastrin-releasing peptide (GRP) from tumor cells, the growth of which is dependent on the stimulation of GRP. In this study, we developed a DNA vaccine using a plasmid vector to deliver the immunogen of six copies of the B cell epitope GRP 18–27 (GRP6). In order to increase the potency of this DNA vaccine, multiple strategies have been applied including DNA-prime protein-boost immunization and introduction of a foreign T-helper epitope into DNA vaccine. Mice vaccinated DNA vaccine boosting with HSP65-GRP6 protein induced high titer and relatively high avidity of anti-GRP antibodies as well as inhibition effect on the growth of murine prostate carcinoma, superior to the treatment using DNA alone or BCG priming HSP65-GRP6 protein boosting. Furthermore, the introduction of a novel foreign T-helper epitope into the GRP DNA vaccine showed a markedly stronger humoral immune response against GRP and tumor rejection even than the DNA-prime protein-boost strategy. No further stronger immunogenicity of this foreign T-helper epitope modified DNA vaccine was observed even using the strategy of modified DNA vaccine-priming and HSP65-GRP6 boosting method. The data presented demonstrate that improvement of potency of anti-GRP DNA vaccine with the above two feasible approaches should offer useful methods in the development of new DNA vaccine against growth factors for cancer immunotherapy.

Novel recombinant BCG expressing perfringolysin O and the over-expression of key immunodominant antigens; pre-clinical characterization, safety and protection against challenge with Mycobacterium tuberculosis

Mycobacterium tuberculosis ( Mtb), the causative agent of tuberculosis (TB), has infected approximately two billion individuals worldwide with approximately 9.2 million new cases and 1.6 million deaths annually. Current efforts are focused on making better BCG priming vaccines designed to induce a comprehensive and balanced immunity followed by booster(s) targeting a specific set of relevant antigens in common with the BCG prime. We describe the generation and immunological characterization of recombinant BCG strains with properties associated with lysis of the endosome compartment and over-expression of key Mtb antigens. The endosome lysis strain, a derivative of BCG SSI-1331 (BCG 1331) expresses a mutant form of perfringolysin O (PfoA G137Q), a cytolysin normally secreted by Clostridium perfringens. Integration of the PfoA G137Q gene into the BCG genome was accomplished using an allelic exchange plasmid to replace ureC with pfoA G137Q under the control of the Ag85B promoter. The resultant BCG construct, designated AERAS-401 (BCG 1331 Δ ureC::Ω pfoA G137Q) secreted biologically active Pfo, was well tolerated with a good safety profile in immunocompromised SCID mice. A second rBCG strain, designated AFRO-1, was generated by incorporating an expression plasmid encoding three mycobacterial antigens, Ag85A, Ag85B and TB10.4, into AERAS-401. Compared to the parental BCG strain, vaccination of mice and guinea pigs with AFRO-1 resulted in enhanced immune responses. Mice vaccinated with AFRO-1 and challenged with the hypervirulent Mtb strain HN878 also survived longer than mice vaccinated with the parental BCG. Thus, we have generated improved rBCG vaccine candidates that address many of the shortcomings of the currently licensed BCG vaccine strains.

Improve protective efficacy of a TB DNA-HSP65 vaccine by BCG priming

Vaccines are considered by many to be one of the most successful medical interventions against infectious diseases. But many significant obstacles remain, such as optimizing DNA vaccines for use in humans or large animals. The amount of doses, route and easiness of administration are also important points to consider in the design of new DNA vaccines. Heterologous prime-boost regimens probably represent the best hope for an improved DNA vaccine strategy. In this study, we have shown that heterologous prime-boost vaccination against tuberculosis (TB) using intranasal BCG priming/DNA-HSP65 boosting (BCGin/DNA) provided significantly greater protection than that afforded by a single subcutaneous or intranasal dose of BCG. In addition, BCGin/DNA immunization was also more efficient in controlling bacterial loads than were the other prime-boost schedules evaluated or three doses of DNA-HSP65 as a naked DNA. The single dose of DNA-HSP65 booster enhanced the immunogenicity of a single subcutaneous BCG vaccination, as evidenced by the significantly higher serum levels of anti-Hsp65 IgG2a Th1-induced antibodies, as well as by the significantly greater production of IFN-γ by antigen-specific spleen cells. The BCG prime/DNA-HSP65 booster was also associated with better preservation of lung parenchyma.The improvement of the protective effect of BCG vaccine mediated by a DNA-HSP65 booster suggests that our strategy may hold promise as a safe and effective vaccine against TB.

Intranasal Boosting with an Adenovirus-Vectored Vaccine Markedly Enhances Protection by Parenteral Mycobacterium bovis BCG Immunization against Pulmonary Tuberculosis

Parenterally administered Mycobacterium bovis BCG vaccine confers only limited immune protection from pulmonary tuberculosis in humans. There is a need for developing effective boosting vaccination strategies. We examined a heterologous prime-boost regimen utilizing BCG as a prime vaccine and our recently described adenoviral vector expressing Ag85A (AdAg85A) as a boost vaccine. Since we recently demonstrated that a single intranasal but not intramuscular immunization with AdAg85A was able to induce potent protection from pulmonary Mycobacterium tuberculosis challenge in a mouse model, we compared the protective effects of parenteral and mucosal booster immunizations following subcutaneous BCG priming. Protection by BCG prime immunization was not effectively boosted by subcutaneous BCG or intramuscular AdAg85A. In contrast, protection by BCG priming was remarkably boosted by intranasal AdAg85A. Such enhanced protection by intranasal AdAg85A was correlated to the numbers of gamma interferon-positive CD4 and CD8 T cells residing in the airway lumen of the lung. Our study demonstrates that intranasal administration of AdAg85A represents an effective way to boost immune protection by parenteral BCG vaccination.