Mucosal immunization with recombinant heparin-binding haemagglutinin adhesin suppresses extrapulmonary dissemination of Mycobacterium bovis bacillus Calmette-Guérin (BCG) in infected mice

It is generally accepted that cellular immunity plays a critical role in the protection against Mycobacterium tuberculosis, an intracellular pathogen. Recently, however, an increasing number of reports indicate the important contribution of humoral immunity against mycobacterial infection. Since M. tuberculosis establishes its primary lesion in the lung, induction of humoral immunity in the airway tract by mucosal immunization regime could provide protective immunity against tuberculosis. In this study, mycobacterial heparin-binding haemagglutinin adhesin (HBHA) was used as an immunization antigen because HBHA is an essential virulence factor required for the infection of lung epithelial cells and extrapulmonary dissemination of mycobacteria. The effects of intranasal immunization with a yeast-expressed recombinant (r) HBHA co-administered with a mucosal adjuvant cholera toxin (CT) on the induction of humoral and cellular immunity were examined, and its protective efficacy against pulmonary challenge infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG) was evaluated. HBHA-specific antibodies were induced in serum and airway tract of immunized mice, which specifically recognized native HBHA expressed on M. bovis BCG. Th1-type immunity against mycobacterial antigens was also enhanced in the lung of immunized mice after pulmonary BCG infection. Furthermore, the immunization suppressed bacterial load in the spleen after pulmonary BCG infection. These results indicate that systemic and local humoral immunity induced by the HBHA-based mucosal vaccine impairs extrapulmonary dissemination, thus providing immune protection against mycobacterial infection.     

Co-expression of granulocyte-macrophage colony-stimulating factor with antigen enhances humoral and tumor immunity after DNA vaccination

Granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to enhance humoral and tumor immunity resulting from DNA immunization. The genes encoding GM-CSF and antigen were cloned onto the same plasmid backbone, but separate promoters drove expression of each gene. beta-Galactosidase was used as the model antigen to generate antibody responses while the human tumor antigen, MAGE-1, was used to monitor tumor resistance. Immunization with a DNA vaccine co-expressing GM-CSF and beta-gal resulted in higher antigen-specific IgG responses than immunization with antigen encoding plasmid alone or co-inoculated with GM-CSF expressing plasmid. Similarly, DNA vaccines expressing both MAGE-1 antigen and GM-CSF were more effective in protecting against B16-MAGE-1 melanoma. However, both GM-CSF co-expressing DNA vaccines and co-inoculation with plasmids encoding the cytokine or antigen enhanced the generation antigen-specific IFN-gamma and IL-6 responses. These results demonstrate that co-expressing both GM-CSF and antigen on a DNA vaccine enhances humoral and tumor immune responses.     

Immunization with heat-killed Mycobacterium bovis bacille Calmette-Guerin (BCG) in Eurocine L3 adjuvant protects against tuberculosis

The current live attenuated vaccine against tuberculosis, BCG, poses a risk of disseminated infections in immunocompromised subjects. Therefore, in this study we compared the protective effect of a heat-killed bacille Calmette-Guerin (H-kBCG) vaccine given in a new adjuvant (Eurocine L3) with the protection provided by the conventional live attenuated BCG vaccine in mice (C57BL/6 and BALB/c) challenged with virulent Mycobacterium tuberculosis (strain Harlingen). The H-kBCG vaccine alone, in accordance with earlier studies, did not give any or only gave slight protection compared to sham-vaccinated controls. However, the same vaccine given with Eurocine L3 adjuvant, either formulated as a suspension or as an emulsion, afforded significant levels of protection. This protection was at least as good as that of the control live attenuated BCG vaccine. The Eurocine L3 adjuvant is approved for human use as a nasal vaccine adjuvant and a successful phase I trial with nasal immunization with diphtheria vaccine has recently been performed in Sweden. Here we show that, in mice, intranasal priming with H-kBCG in Eurocine L3 adjuvant followed by intranasal booster resulted in the same level of protection as subcutaneous priming followed by intranasal booster. All H-kBCG formulations in the Eurocine L3 adjuvant elicited mycobacterial antigen-specific serum IgG and IFN gamma responses. In general, among the different vaccine formulation(s) in the Eurocine L3 adjuvant those that produced a relatively high Th2 response, as measured by IgG1/IgG2a ratio and IFN gamma production in vitro, were the most protective. In conclusion, H-kBCG in Eurocine L3 adjuvant could represent a safe and a more stable alternative to the conventional live BCG vaccine.     

PPD induced in vitro interferon gamma production is not a reliable correlate of protection against Mycobacterium tuberculosis

Correlates of protection against tuberculosis are crucial for the evaluation of new vaccine candidates and for the demonstration of their potential efficacy. Such correlates can be proposed on the basis of animal models. In this study, we hypothesized that protection against tuberculosis (TB) induced by bacillus Calmette-Guerin (BCG) correlates with in vitro TB antigen-specific IFN-gamma production. BCG vaccination, known to provide effective protection against TB in animals, was used to investigate the use of in vitro IFN-gamma production as a marker of BCG-induced protection against TB. Our results show that BCG vaccination does provide substantial protection against challenge with Mycobacterium tuberculosis. However, despite previous compelling evidence that Th1 type immune responses are essential for TB immunity, the magnitude of in vitro purified protein derivative (PPD)-specific IFN-gamma production assessed during the course of TB infection did not correlate with protection. This emphasizes the need to identify further correlates of protection, in addition to IFN-gamma, to be used as markers of protective immunity against M. tuberculosis and/or to identify M. tuberculosis antigens inducing IFN-gamma that correlate with protective immunity.     

Intranasal boosting with MVA encoding secreted mycobacterial proteins Ag85A and ESAT-6 generates strong pulmonary immune responses and protection against M. tuberculosis in mice given BCG as neonates

Bacille-Calmette-Guerin (BCG) has variable efficacy as an adult tuberculosis (TB) vaccine but can reduce the incidence and severity of TB infection in humans. We have engineered modified vaccinia Ankara (MVA) strain vaccine constructs to express the secreted mycobacterial proteins Ag85A and ESAT-6 (MVA-AE) and evaluated their immunogenicity and protective efficacy as mucosal booster vaccines for BCG given subcutaneously in early life. Intranasal delivery of MVA-AE to young adult mice induced CD4⁺ and CD8⁺ T cell responses to both Ag85A and ESAT-6 in lung mucosae. These responses were markedly enhanced in mice that had been primed neonatally with BCG prior to intranasal MVA-AE immunization (BCG/MVA-AE), as evidenced by numbers of pulmonary Ag85A-, ESAT-6-, and PPD-specific CD4⁺ and CD8⁺ T cells and by their capacity to secrete multiple antimicrobial factors, including IFNγ, IL-2 and IL-17. Moreover, MVA-AE boosting generated multifunctional lung CD4⁺ T cells responding to ESAT-6, which were not, as expected, detected in control mice given BCG, and elevated Ag85A-specific circulating antibody responses. After aerosol challenge with M. tuberculosis H37Rv (Mtb), the BCG/MVA-AE group had significantly reduced mycobacterial burden in the lungs, compared with either BCG primed mice boosted with control MVA or mice given only BCG. These data indicate that intranasal delivery of MVA-AE can boost BCG-induced Th1 and Th17-based immunity locally in the lungs and improve the protective efficacy of neonatally-administered BCG against M. tuberculosis infection.     

Protection against an aerogenic Mycobacterium tuberculosis infection in BCG-immunized and DNA-vaccinated mice is associated with early type I cytokine responses

Although vaccination against tuberculosis (TB) was initiated more than 80 years ago, the correlates of protective immunity against infection by Mycobacterium tuberculosis have still not been well defined. To investigate the vaccine-induced immune responses against TB, we evaluated the early pulmonary cytokine responses elicited by a low dose M. tuberculosis aerogenic challenge in mice that had been immunized with either BCG or a TB DNA vaccine cocktail, two vaccine preparations that induce long-term protection in the mouse model of pulmonary TB. Using three different assays, we showed that specific cytokine responses were elevated in the lungs of vaccinated mice (relative to na?ve controls) during the second week post-challenge. By measuring cytokine levels in the bronchoalveolar lavage fluid (BAL) and cytokine mRNA concentrations in pulmonary cells, the levels of IFN-gamma, IL-12, and RANTES were shown to be elevated from days 7-14 post-challenge in the lungs. By intracellular cytokine staining (ICS), increased numbers of lung CD4 and CD8 cells expressing IFN-gamma were also seen at days 10 and 14 after the infection. Moreover, increased post-challenge IFN-gamma levels were detected using the ICS and cytokine mRNA assays in aging BCG-immunized mice that had been effectively boosted with a TB DNA vaccine. Taken together, these data suggest that the post-infection induction of early type 1 cytokine responses correlate with the induction of long-term protective immunity in vaccinated mice.     

Protection of mice from Mycobacterium tuberculosis by ID87/GLA-SE, a novel tuberculosis subunit vaccine candidate

Tuberculosis is a major health concern. Non-living tuberculosis (TB) vaccine candidates may not only be safer than the current vaccine (BCG) but could also be used to boost BCG to enhance or elongate protection. No subunit vaccines, however, are currently available for TB. To address this gap and to improve the global TB situation, we have generated a defined subunit vaccine by genetically fusing the genes of 3 potent protein Mtb antigens, Rv2875, Rv3478 and Rv1886, into a single product: ID87. When delivered with a TLR4 agonist-based adjuvant, GLA-SE, ID87 immunization reduced Mtb burden in the lungs of experimentally infected mice. The reduction in bacterial burden of ID87/GLA-SE immunized mice was accompanied by an early and significant leukocyte infiltration into the lungs during the infectious process. ID87/GLA-SE appears to be a promising new vaccine candidate that warrants further development.     

Fusion protein Ag85B-MPT64190–198-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.     

The antigen 85 complex vaccine against experimental Mycobacterium leprae infection in mice

The proteins in culture filtrate derived from Bacillus Calmette-Guérin (BCG) were examined for protection against infection by Mycobacterium leprae. Immunization with the major secreted proteins, antigen 85 complex (Ag 85) A, B and C, induced effective protective immunity against multiplication of M. leprae in the foot pads of mice. The most effective protection was observed when mice were immunized with Ag 85A. A single immunization with Ag 85 could induce antigen-specific interferon gamma (IFNgamma) synthesis and more effective protection than live BCG vaccine. This study demonstrates that Ag 85 is an important immunoprotective molecule against leprosy infection.     

Protective immunity against Mycobacterium bovis induced by vaccination with Rv3109c--a member of the esat-6 gene family

In a number of clinical studies the current TB vaccine, Mycobacterium bovis bacille Calmette-Guerin (BCG), has provided little or no protection against pulmonary tuberculosis in cattle and man. A new generation of vaccines is therefore required to replace or supplement BCG. Safety concerns surrounding a number of strategies make protein subunits an attractive approach. Moreover, novel prime-boost strategies based on primary immunisations with BCG are not only showing promise but also present a clear strategy for testing new TB vaccines in clinical studies. We report the evaluation of six protein vaccine candidates for their ability to induce protective immunity in a murine virulent M. bovis challenge model. One protein (Rv3019c) induced reproducibly significant protection in the spleen and lungs approaching that induced by BCG. Detailed analysis of antigen-specific T cell responses revealed that despite robust responses in the spleen and lungs of vaccinated mice, there was no correlation between these responses and the protective efficacy of the vaccine. Significantly, Rv3019c also stimulated IFN-gamma responses in PBMC from BCG vaccinated cattle, indicating its potential for use in a heterologous prime-boost strategy in conjunction with BCG in the target species.     

An attenuated Salmonella-vectored vaccine elicits protective immunity against Mycobacterium tuberculosis

There is a need to develop protective vaccines against tuberculosis (TB) that elicit full immune responses including mucosal immunity. Here, a live attenuated Salmonellatyphimurium aroA SL7207 vector TB vaccine, namely SL(E6-85B), harboring the Mycobacterium tuberculosis (M. tb) H37Rv ESAT6-Ag85B fusion gene was developed. The experimental data demonstrated that this SL(E6-85B) vaccine, or when it is combined with BCG vaccination, induced the strongest TB Ag-specific mucosal, humoral, and cellular immune responses comprised of increased proliferation of T cells, IFN-gamma expression, granzyme B production, as well as the greatest IFN-gamma production of effector-memory T (T_EM) or effector CD8⁺ T cell responses and exerted high protective efficacy in mice against virulent M. tb H37Rv challenge compared to the other vaccinated groups (mice immunized with SL(Ag85B), a DNA vaccine or BCG only). This strategy may represent a novel promising mucosal vaccine candidate for the prevention of TB which are inexpensive to produce, efficacious, and able to be given orally rather than by injection.     

RLJ-NE-299A: a new plant based vaccine adjuvant

Alum has been in use since long as an adjuvant for vaccines. However, its use as a vaccine adjuvant offers limitation in supporting cell mediated response. Therefore, a new plant based product RLJ-NE-299A from Picrorhiza kurroa reported for its immunostimulatory activity, has been explored for its potential as an alternative adjuvant. In order to compare the adjuvant activity with alum, antigen-specific immune responses were evaluated following immunization with a formulation containing hepatitis B surface antigen (HBsAg) adjuvanted with RLJ-NE-299A and alum in mice. The adjuvant RLJ-NE-299A up-regulated remarkably the expression of Th1 cytokines IL-2, IL-12, IFN-gamma, TNF alpha and Th2 cytokine IL-4 in lymph node cell cultures after 2 weeks of primary immunization with HBsAg. Further, the levels of both immunoglobulins IgG2a (Th1) and IgG1 (Th2) subtypes increased profoundly in blood sera of mice immunized with HBsAg/RLJ-NE-299A. The results indicated that RLJ-NE-299A has strong potential to increase both cell mediated and humoral immune responses and is capable of sustaining the total antigen-specific antibody response. Besides, the RLJ-NE-299A provides a signal to gear up both CD4 helper cells (Th1 and Th2) and CD8 cells populations, which may have important implications for vaccination against hepatitis B virus. Variable doses of RLJ-NE-299A (0.312-40 microg) containing vaccine antigen (HBsAg) were well tolerated with optimum T cell response at 2.5 microg/ml. Not only this, the adjuvant was also able to induce cellular immune responses to HBsAg as evidenced by Th1 and Th2 cytokines upregulation, which enabled mice to overcome the unresponsiveness to antigen HBsAg encountered with alum-adjuvanted vaccine in otherwise non-responding mice population. The study presents evidence that the HPLC standardized fraction RLJ-NE-299A, is an adjuvant of choice over alum in improving and maintaining the improved immune status against HBsAg, and may also prove useful adjuvant candidate with other vaccine antigens, too.     

A combined DNA vaccine enhances protective immunity against Mycobacterium tuberculosis and Brucella abortus in the presence of an IL-12 expression vector

We examined the immunogenicity and protective efficacy of a combined DNA vaccine that included six genes encoding immunodominant antigens from Mycobacterium tuberculosis and Brucella abortus. The IL-12 adjuvant system was used for immunization in combination with the combined DNA vaccine (DNA-IL-12(+)). Mice immunized with DNA-IL-12(+) had significantly reduced CFU counts for M. tuberculosis and B. abortus in lung and spleen, respectively (P<0.001), and DNA-IL-12(+) elicited better protection than the combined DNA vaccine alone (DNA-IL-12(-)) or with the positive control groups after challenge with a virulent M. tuberculosis strain and B. abortus 2308 infection. The DNA-IL-12(+) group had stronger antigen-specific IFN-gamma ELISPOT activities and higher levels of antigen-specific CD4(+) and CD8(+) T cell responses than either the DNA-IL-12(-) or positive control groups. Likewise, antigen-specific IgG titers were also much higher than in other immunized groups. Moreover, DNA-IL-12(+) gave a stronger IgG2a-skewed response than did DNA-IL-12(-). In addition, its mean concentrations of IFN-gamma and IL-2 were about 2.5- to 4.5-fold higher than those observed in the DNA-IL-12(-)-treated mice, and were significantly higher than control groups (P<0.01 or P<0.001), whereas IL-4 and IL-10 secretion were lower. These results suggest that IL-12 acts as an adjuvant to enhance protective immunity against M. tuberculosis and B. abortus through the induction of stronger Th1-associated immune responses. This is the first report to show that a single combined DNA vaccine protects animals against two infectious diseases.     

An ESAT-6:CFP10 DNA vaccine administered in conjunction with Mycobacterium bovis BCG confers protection to cattle challenged with virulent M. bovis

The potency of genetic immunization observed in the mouse has demonstrated the utility of DNA vaccines to induce cell-mediated and humoral immune responses. However, it has been relatively difficult to generate comparable responses in non-rodent species. The use of molecular adjuvants may increase the magnitude of these suboptimal responses. In this study, we demonstrate that the co-administration of plasmid-encoded GM-CSF and CD80/CD86 with a novel ESAT-6:CFP10 DNA vaccine against bovine tuberculosis enhances antigen-specific cell-mediated immune responses. ESAT-6:CFP10+GM-CSF+CD80/CD86 DNA vaccinated animals exhibited significant (p<0.01) antigen-specific proliferative responses compared to other DNA vaccinates. Increased expression (p< or =0.05) of CD25 on PBMC from ESAT-6:CFP10+GM-CSF+CD80/CD86 DNA vaccinates was associated with increased proliferation, as compared to control DNA vaccinates. Significant (p<0.05) numbers of ESAT-6:CFP10-specific IFN-gamma producing cells were evident from all ESAT-6:CFP10 DNA vaccinated animals compared to control DNA vaccinates. However, the greatest increase in IFN-gamma producing cells was from animals vaccinated with ESAT-6:CFP10+GM-CSF+CD80/CD86 DNA. In a low-dose aerosol challenge trial, calves vaccinated as neonates with Mycobacterium bovis BCG and ESAT-6:CFP10+GM-CSF+CD80/CD86 DNA exhibited decreased lesion severity in the lung and lung-associated lymph nodes following viruluent M. bovis challenge compared to other vaccinated animals or non-vaccinated controls. These data suggest that a combined vaccine regimen of M. bovis BCG and a candidate ESAT-6:CFP10 DNA vaccine may offer greater protection against tuberculosis in cattle than vaccination with BCG alone.     

Construction and evaluation of a multistage Mycobacterium tuberculosis subunit vaccine candidate Mtb10.4-HspX

To search for more effective vaccines to enhance the immunogenicity and protective efficacy of Mycobacterium bovis Bacille Calmette-Guerin (BCG) and to control or even eradicate Mycobacterium tuberculosis (M. tuberculosis) in all stages of infection including the persister bacteria, antigens of Mtb10.4 (Rv0288) expressed in replicating bacilli and HspX (also called Acr, Hsp16.3, Rv2031c) highly expressed in dormant bacilli were fused together to construct a multistage fusion protein Mtb10.4-HspX (MH for short) without affinity tag with potential advantage for clinical use. The human T-cell responses to MH were evaluated for its immunogenicity. Furthermore, MH was emulsified in an adjuvant composed of N,N′-dimethyl-N,N′-dioctadecylammonium bromide (DDA) and mycobacterial cord factor trehalose-6,6-dimycolate (TDM) to construct subunit vaccine, whose immunogenicity and potency to boost BCG primed immunity against M. tuberculosis infection were evaluated in mice. The results showed that the fusion protein MH without affinity tag was stably produced in Escherichia coli and was successfully purified by chromatography. MH was strongly recognized by human T cells from TB patients and persons latently infected with M. tuberculosis. In conclusion, MH in adjuvant DDA-TDM generated strong antigen-specific humoral and cell-mediated immunity, and had the capability to enhance BCG-primed immunity and the protective efficacy against M. tuberculosis in mice. These findings suggest that MH in DDA-TDM have the potential to be a good multistage tuberculosis vaccine candidate.     

Transcutaneous immunization with a novel lipid-based adjuvant protects against Chlamydia genital and respiratory infections

Mucosal adjuvants are important to overcome the state of immune tolerance normally associated with mucosal delivery and to enhance adaptive immunity to often-weakly immunogenic subunit vaccine antigens. Unfortunately, adverse side effects of many experimental adjuvants limit the number of adjuvants approved for vaccination. Lipid C is a novel, non-toxic, lipid oral vaccine-delivery formulation, developed originally for oral delivery of the live Mycobacterium bovis Bacille Calmette-Guerin (BCG) vaccine. In the present study, murine models of chlamydial respiratory and genital tract infections were used to determine whether transcutaneous immunization (TCI) with Lipid C-incorporated protein antigens could elicit protective immunity at the genital and respiratory mucosae. BALB/c mice were immunized transcutaneously with Lipid C containing the chlamydial major outer membrane protein (MOMP), with and without addition of cholera toxin and CpG-ODN 1826 (CT/CpG). Both vaccine combinations induced mixed cell-mediated and mucosal antibody immune responses. Immunization with Lipid C-incorporated MOMP (Lipid C/MOMP), either alone or with CT/CpG resulted in partial protection following live challenge with Chlamydia muridarum as evidenced by a significant reduction in recoverable Chlamydia from both the genital secretions and lung tissue. Protection induced by immunization with Lipid C/MOMP alone was not further enhanced by the addition of CT/CpG. These results highlight the potential of Lipid C as a novel mucosal adjuvant capable of targeting multiple mucosal surfaces following TCI. Protection at both the respiratory and genital mucosae was achieved without the requirement for potentially toxic adjuvants, suggesting that Lipid C may provide a safe effective mucosal adjuvant for human vaccination.     

Resiquimod is a modest adjuvant for HIV-1 gag-based genetic immunization in a mouse model

DNA vaccines have been effective at generating useful immune responses in many animal species. However, it is clearly desirable to increase their potency. The identification of adjuvants that increase their cell-mediated immune (CMI) response is therefore an important goal. Resiquimod is an imiquimod analog proven to activate dendritic cells through TLR-7. The adjuvant capacity of resiquimod has not, to our knowledge, been studied in the context of genetic immunization. Here, we studied resiquimod as an adjuvant for plasmid vaccine therapy by intra-muscular immunization of BALB/c mice with HIV-1 gag DNA vaccine without and with several concentrations of resiquimod (ranging from 5-100nM). We observed that resiquimod moderately enhanced IFN-gamma production as measured by a peptide-based ELISPOT assay compared to that obtained in mice immunized with DNA gag only. Antigen-specific T-cell proliferation studies showed a several-fold increase in the stimulation index in mice immunized with DNA gag +50 nM of resiquimod as compared to mice receiving DNA gag alone. Antibody titer also increased, while the antibody isotyping data showed a strong Th1 biased type response. Analysis of cytokine production in serum samples demonstrated a stronger Th1 cytokine bias in the presence of resiquimod. Furthermore, relevant increase in IL-4 production, as measured by ELISPOT assay, was not observed. Our results show that resiquimod can have modest adjuvant activity, in a DNA formulation, driving the immune system towards a cell-mediated immune response. Additional studies involving this adjuvant for DNA vaccines are underway.     

Intranasal immunization with liposome-formulated Yersinia pestis vaccine enhances mucosal immune responses

The induction of mucosal immune responses by a liposome-formulated Y. pestis vaccine (formaldehyde-killed whole cell vaccine; KWC) was evaluated. We demonstrated that intranasal immunization of mice with Y. pestis KWC vaccine, formulated with liposomes, significantly enhanced mucosal immune responses in the lung when compared to the responses induced with KWC vaccine alone. These immune responses were characterized by increased titres of specific IgA and IgG in mucosal secretions (lung and nasal washes), and an increased frequency of specific antibody-secreting cells in the lungs. In addition, antigen-specific proliferative responses and IFN-gamma-secreting cells were also significantly enhanced in the spleens of mice immunized with the KWC vaccine formulated in liposomes. Animals that were immunized intranasally with the KWC vaccine showed significant protection against an intranasal challenge with Y. pestis. These results highlight the importance of mucosal administration of vaccine antigens to stimulate immunity in the respiratory tract and demonstrate that liposome formulations can improve the effectiveness of conventional vaccines.     

CpG oligodeoxynucleotide enhances immunity against blood-stage malaria infection in mice parenterally immunized with a yeast-expressed 19 kDa carboxyl-terminal fragment of Plasmodium yoelii merozoite surface protein-1 (MSP1(19)) formulated in oil-based Montanides

The 19kDa carboxyl-terminal fragment of Plasmodium yoelii merozoite surface protein-1 (MSP1(19)), an analog of the leading falciparum malaria vaccine candidate, induces protective immunity to challenge infection when formulated with complete/incomplete Freund's adjuvant (CFA/IFA), an adjuvant unsuitable for use in humans. In this study, we investigate Montanide ISA51 and Montanide ISA720 as well as CpG oligodeoxynucleotide (ODN) as adjuvants for induction of immunity to MSP1(19). Mice immunized with MSP1(19) adjuvanted with Montanide ISA51 were protected even though some mice experienced low-grade parasitemia before resolving the infection. Mice immunized with MSP1(19) adjuvanted with Montanide ISA720 showed delayed patent parasitemia with all mice ultimately succumbing to infection. Interestingly, when the synthetic CpG ODN 1826 was included in either Montanide formulation, mice were completely protected with no parasites detected in the blood. MSP1(19)-specific antibodies in MSP1(19)-immunized mice adjuvanted with Montanide ISA51 or Montanide ISA720 showed predominantly IgG1 antibody and low levels of IgG2a. CpG ODN 1826 significantly enhanced both IgG1 and IgG2a antibody responses in Montanide ISA51-adjuvanted mice but significantly enhanced only the IgG2a antibody response in Montanide ISA720-adjuvanted mice. To investigate the relative roles of antibody and CD4(+) T cells in protection, MSP1(19)-immunized mice adjuvanted with Montanide ISA720 and CpG ODN 1826 were depleted of CD4(+) T cells just prior to challenge. Results showed that three of nine immunized/T cell depleted mice died following infection. These results suggest that antibody and CD4(+) T cells are critical for protection following immunization with MSP1(19) adjuvanted with Montanide and CpG ODN and that the formulation of a human malaria vaccine candidate in Montanide ISA720 or ISA51 together with human compatible CpG ODN would be useful for improving efficacy.     

Oral vaccination of mice with lipid-encapsulated Mycobacterium bovis BCG: Effect of reducing or eliminating BCG load on cell-mediated immunity

Oral delivery of lipid-encapsulated BCG represents an effective method for vaccination against tuberculosis (Tb). This method establishes live, replicating BCG in the lymphatic tissues of the alimentary tract, and promotes systemic-level cell-mediated immunity (CMI) and consequent protection against virulent mycobacterial challenge. Here, we investigated the effects of reducing or eliminating the BCG load on CMI responses in mice. Mice receiving a standard immunising dose of approximately 10(7) BCG (range, 1-5 x 10(7)) developed mycobacterial antigen-specific lymphocyte transformation (LT) responses, as well as interleukin-2 (IL-2) and gamma-interferon (IFN-gamma) secretion, at 8 and 18 weeks post-oral vaccination. These responses were concurrent with establishment of viable, replicating BCG in the alimentary tract lymphatics in over 90% of cases. Reducing the immunising dose by 10-fold reduced the magnitude of CMI, concurrent with abridged establishment of BCG in the lymphatics; reducing the dose 100-fold ablated BCG establishment, and diminished the production of IFN-gamma by antigen-stimulated lymphocytes of these mice. In mice immunised using the standard dose, replicating BCG were eliminated from the alimentary tract lymphatics using selective antibiotics. Interestingly, while lymphocyte transformation and interleukin-2 responses remained largely unaltered in these mice, levels of IFN-gamma produced by antigen-stimulated lymphocytes were shown to be reduced significantly. This study identifies a dosage threshold for effective oral vaccination using lipid-encapsulated BCG, and furthermore highlights the requirement of on-going intra-lymphatic BCG replication for the maintenance of strong IFN-gamma production, above other indicator CMI responses.