Selection of vaccine-candidate peptides from Mycobacterium avium subsp. paratuberculosis by in silico prediction, in vitro T-cell line proliferation, and in vivo immunogenicity.

Mycobacterium avium subspecies paratuberculosis (MAP) is a global concern in modern livestock production worldwide. The available vaccines against paratuberculosis do not offer optimal protection and interfere with the diagnosis of bovine tuberculosis. The aim of this study was to identify immunogenic MAP-specific peptides that do not interfere with the diagnosis of bovine tuberculosis. Initially, 119 peptides were selected by either (1) identifying unique MAP peptides that were predicted to bind to bovine major histocompatibility complex class II (MHC-predicted peptides) or (2) selecting hydrophobic peptides unique to MAP within proteins previously shown to be immunogenic (hydrophobic peptides). Subsequent testing of peptide-specific CD4+ T-cell lines from MAP-infected, adult goats vaccinated with peptides in cationic liposome adjuvant pointed to 23 peptides as being most immunogenic. These peptides were included in a second vaccine trial where three groups of eight healthy goat kids were vaccinated with 14 MHC-predicted peptides, nine hydrophobic peptides, or no peptides in o/w emulsion adjuvant. The majority of the MHC-predicted (93%) and hydrophobic peptides (67%) induced interferon-gamma (IFN-γ) responses in at least one animal. Similarly, 86% of the MHC-predicted and 89% of the hydrophobic peptides induced antibody responses in at least one goat. The immunization of eight healthy heifers with all 119 peptides formulated in emulsion adjuvant identified more peptides as immunogenic, as peptide specific IFN-γ and antibody responses in at least one heifer was found toward 84% and 24% of the peptides, respectively. No peptide-induced reactivity was found with commercial ELISAs for detecting antibodies against Mycobacterium bovis or MAP or when performing tuberculin skin testing for bovine tuberculosis. The vaccinated animals experienced adverse reactions at the injection site; thus, it is recommend that future studies make improvements to the vaccine formulation. In conclusion, immunogenic MAP-specific peptides that appeared promising for use in a vaccine against paratuberculosis without interfering with surveillance and trade tests for bovine tuberculosis were identified by in silico analysis and ex vivo generation of CD4+ T-cell lines and validated by the immunization of goats and cattle. Future studies should test different peptide combinations in challenge trials to determine their protective effect and identify the most MHC-promiscuous vaccine candidates.

A Mycobacterium tuberculosis-specific subunit vaccine that provides synergistic immunity upon co-administration with Bacillus Calmette-Guérin.

Given the encouraging clinical results of both candidate subunit vaccines and revaccination with Bacillus Calmette-Guérin (BCG) against tuberculosis (TB), there is support for combining BCG and subunit vaccination for increased efficacy. BCG and Mycobacterium tuberculosis (Mtb) share ~98% of their genome and current subunit vaccines are almost exclusively designed as BCG boosters. The goal of this study is to design a TB subunit vaccine composed of antigens not shared with BCG and explore the advantages of this design in a BCG + subunit co-administration vaccine strategy. Eight protective antigens are selected to create an Mtb-specific subunit vaccine, named H107. Whereas traditional vaccines containing BCG-shared antigens exhibit in vivo cross-reactivity to BCG, H107 shows no cross-reactivity and does not inhibit BCG colonization. Instead, co-administering H107 with BCG leads to increased adaptive responses against both H107 and BCG. Importantly, rather than expanding BCG-primed T cells, H107 broadens the overall vaccine repertoire with new T cell clones and introduces 'adjuvant-imprinted' qualities including Th17 responses and less-differentiated Th1 cells. Collectively, these features of H107 are associated with a substantial increase in long-term protection.

In Vivo Antigen Expression Regulates CD4 T Cell Differentiation and Vaccine Efficacy against Mycobacterium tuberculosis Infection.

New vaccines are urgently needed against Mycobacterium tuberculosis (Mtb), which kills more than 1.4 million people each year. CD4 T cell differentiation is a key determinant of protective immunity against Mtb, but it is not fully understood how host-pathogen interactions shape individual antigen-specific T cell populations and their protective capacity. Here, we investigated the immunodominant Mtb antigen, MPT70, which is upregulated in response to gamma interferon (IFN-γ) or nutrient/oxygen deprivation of in vitro-infected macrophages. Using a murine aerosol infection model, we compared the in vivo expression kinetics of MPT70 to a constitutively expressed antigen, ESAT-6, and analyzed their corresponding CD4 T cell phenotype and vaccine protection. For wild-type Mtb, we found that in vivo expression of MPT70 was delayed compared to ESAT-6. This delayed expression was associated with induction of less differentiated MPT70-specific CD4 T cells but, compared to ESAT-6, also reduced protection after vaccination. In contrast, infection with an MPT70-overexpressing Mtb strain promoted highly differentiated KLRG1+CX3CR1+ CD4 T cells with limited lung-homing capacity. Importantly, this differentiated phenotype could be prevented by vaccination, and against the overexpressing strain, vaccination with MPT70 conferred protection similar to vaccination with ESAT-6. Together, our data indicate that high in vivo antigen expression drives T cells toward terminal differentiation and that targeted vaccination with adjuvanted protein can counteract this phenomenon by maintaining T cells in a protective less differentiated state. These observations shed new light on host-pathogen interactions and provide guidance on how future Mtb vaccines can be designed to tip the immune balance in favor of the host.IMPORTANCE Tuberculosis, caused by Mtb, constitutes a global health crisis of massive proportions and the impact of the current coronavirus disease 2019 (COVID-19) pandemic is expected to cause a rise in tuberculosis-related deaths. Improved vaccines are therefore needed more than ever, but a lack of knowledge on protective immunity hampers their development. The present study shows that constitutively expressed antigens with high availability drive highly differentiated CD4 T cells with diminished protective capacity, which could be a survival strategy by Mtb to evade T cell immunity against key antigens. We demonstrate that immunization with such antigens can counteract this phenomenon by maintaining antigen-specific T cells in a state of low differentiation. Future vaccine strategies should therefore explore combinations of multiple highly expressed antigens and we suggest that T cell differentiation could be used as a readily measurable parameter to identify these in both preclinical and clinical studies.

The subunit vaccine H65 + CAF01 increased the BCG- protection against Mycobacterium bovis infection in a mouse model of bovine tuberculosis.

H65, a fusion protein of three pairs of ESX-secreted antigens of Mycobacterium tuberculosis and Mycobacterium bovis, formulated with the liposomal adjuvant CAF01 has been shown to confer protection against M. tuberculosis infection in mice. In this study, we evaluated the impact of combining BCG with H65 + CAF01 immunization in a M. bovis mouse model of infection. We found that a BCG-H65 + CAF01/ H65 + CAF01 prime-boost scheme induced higher protection than BCG and H65 + CAF01 alone. Altogether, H65 antigen formulated in liposomal adjuvant improved the BCG-induced immune protection, thus making this vaccine strategy a promising tool to control bovine tuberculosis.

In vivo antigen expression regulates CD4 T cell differentiation and vaccine efficacy against Mycobacterium tuberculosis infection.

New vaccines are urgently needed against Mycobacterium tuberculosis (Mtb), which kills more than 1.4 million people each year. CD4 T cell differentiation is a key determinant of protective immunity against Mtb, but it is not fully understood how host-pathogen interactions shape individual antigen-specific T cell populations and their protective capacity. Here, we investigated the immunodominant Mtb antigen, MPT70, which is upregulated in response to IFN-γ or nutrient/oxygen deprivation of in vitro infected macrophages. Using a murine aerosol infection model, we compared the in vivo expression kinetics of MPT70 to a constitutively expressed antigen, ESAT-6, and analysed their corresponding CD4 T cell phenotype and vaccine-protection. For wild-type Mtb, we found that in vivo expression of MPT70 was delayed compared to ESAT-6. This delayed expression was associated with induction of less differentiated MPT70-specific CD4 T cells but, compared to ESAT-6, also reduced protection after vaccination. In contrast, infection with an MPT70-overexpressing Mtb strain promoted highly differentiated KLRG1+CX3CR1+ CD4 T cells with limited lung-homing capacity. Importantly, this differentiated phenotype could be prevented by vaccination and, against the overexpressing strain, vaccination with MPT70 conferred similar protection as ESAT-6. Together our data indicate that high in vivo antigen expression drives T cells towards terminal differentiation and that targeted vaccination with adjuvanted protein can counteract this phenomenon by maintaining T cells in a protective less-differentiated state. These observations shed new light on host-pathogen interactions and provide guidance on how future Mtb vaccines can be designed to tip the immune-balance in favor of the host.

Rescuing ESAT-6 Specific CD4 T Cells From Terminal Differentiation Is Critical for Long-Term Control of Murine Mtb Infection.

In most cases, Mycobacterium tuberculosis (Mtb) causes life-long chronic infections, which poses unique challenges for the immune system. Most of the current tuberculosis (TB) subunit vaccines incorporate immunodominant antigens and at this point, it is poorly understood how the CD4 T cell subsets recognizing these antigens are affected during long-term infection. Very little is known about the requirements for sustainable vaccine protection against TB. To explore this, we screened 62 human-recognized Mtb antigens during chronic murine Mtb infection and identified the four most immunodominant antigens in this setting (MPT70, Rv3020c, and Rv3019c and ESAT-6). Combined into a subunit vaccine, this fusion protein induced robust protection both in a standard short-term model and in a long-term infection model where immunity from BCG waned. Importantly, replacement of ESAT-6 with another ESAT-6-family antigen, Rv1198, led to similar short-term protection but a complete loss of bacterial control during chronic infection. This observation was further underscored, as the ESAT-6 containing vaccine mediated sustainable protection in a model of post-exposure vaccination, where the ESAT-6-replacement vaccine did not. An individual comparison of the CD4 T cell responses during Mtb infection revealed that ESAT-6-specific T cells were more terminally differentiated than the other immunodominant antigens and immunization with the ESAT-6 containing vaccine led to substantially greater reduction in the overall T cell differentiation status. Our data therefore associates long-term bacterial control with the ability of a vaccine to rescue infection-driven CD4T cell differentiation and future TB antigen discovery programs should focus on identifying antigens with the highest accompanying T cell differentiation, like ESAT-6. This also highlights the importance of long-term readouts in both preclinical and clinical studies with TB vaccines.

Immunization with Mycobacterium tuberculosis-Specific Antigens Bypasses T Cell Differentiation from Prior Bacillus Calmette-Guérin Vaccination and Improves Protection in Mice.

Despite the fact that the majority of people in tuberculosis (TB)-endemic areas are vaccinated with the Bacillus Calmette-Guérin (BCG) vaccine, TB remains the leading infectious cause of death. Data from both animal models and humans show that BCG and subunit vaccines induce T cells of different phenotypes, and little is known about how BCG priming influences subsequent booster vaccines. To test this, we designed a novel Mycobacterium tuberculosis-specific (or "non-BCG") subunit vaccine with protective efficacy in both mice and guinea pigs and compared it to a known BCG boosting vaccine. In naive mice, this M. tuberculosis-specific vaccine induced similar protection compared with the BCG boosting vaccine. However, in BCG-primed animals, only the M. tuberculosis-specific vaccine added significantly to the BCG-induced protection. This correlated with the priming of T cells with a lower degree of differentiation and improved lung-homing capacity. These results have implications for TB vaccine design.

Targeting the Mincle and TLR3 receptor using the dual agonist cationic adjuvant formulation 9 (CAF09) induces humoral and polyfunctional memory T cell responses in calves.

There is a need for the rational design of safe and effective vaccines to protect against chronic bacterial pathogens such as Mycobacterium tuberculosis and Mycobacterium avium subsp. paratuberculosis in a number of species. One of the main challenges for vaccine development is the lack of safe adjuvants that induce protective immune responses. Cationic Adjuvant Formulation 01 (CAF01)-an adjuvant based on trehalose dibehenate (TDB) and targeting the Mincle receptor-has entered human trials based on promising pre-clinical results in a number of species. However, in cattle CAF01 only induces weak systemic immune responses. In this study, we tested the ability of three pattern recognition receptors, either alone or in combination, to activate bovine monocytes and macrophages. We found that addition of the TLR3 agonist, polyinosinic:polycytidylic acid (Poly(I:C)) to either one of the Mincle receptor agonists, TDB or monomycoloyl glycerol (MMG), enhanced monocyte activation, and calves vaccinated with CAF09 containing MMG and Poly(I:C) had increased cell-mediated and humoral immune response compared to CAF01 vaccinated animals. In contrast to the highly reactogenic Montanide ISA 61 VG, CAF09-primed T cells maintained a higher frequency of polyfunctional CD4+ T cells (IFN-γ+ TNF-α+ IL-2+). In conclusion, CAF09 supports the development of antibodies along with a high-quality cell-mediated immune response and is a promising alternative to oil-in-water adjuvant in cattle and other ruminants.

Optimisation of a murine splenocyte mycobacterial growth inhibition assay using virulent Mycobacterium tuberculosis.

In the absence of a validated correlate of protection or robust animal models for human tuberculosis, Mycobacterial growth inhibition assays (MGIAs) aim to assess vaccines ability to inhibit mycobacterial growth in-vitro. We optimised a reproducible murine splenocyte MGIA based on in-vitro infection with virulent Mycobacterium tuberculosis (M.tb) Erdman. We identified splenocyte viability as a problem in state-of-art MGIA protocols, which can be improved by simple changes in culture conditions (viability increase from 21% to 46% at last day of culture). The growth inhibitory potential in mice immunised with either BCG, H56:CAF01 or H56:CAF01 administered side-by-side with BCG was significantly better compared to placebo in all groups (0.3 log10 CFU [±0.2, p = 0.049], 0.5 [±0.2, p = 0.016] and 0.6 [±0.1, p = 0.0007], respectively) corresponding to the levels of in-vivo protection. Unexpectedly the CAF01 adjuvant control group also induced significant growth inhibition of 0.3 log10 CFU (±0.2, p = 0.047). Finally, we explored vaccine-associated T cell effector functions. Despite presence of high levels of vaccine-specific T cells, we found no increase in CD4+ T cell number or cytokine expression profile, nor a difference in cytokine levels in the supernatant after four days culture with or without M.tb. Spontaneous IFN-γ release correlated with growth inhibition levels (p = 0.02), however the cellular source was not found.

An attenuated Mycobacterium tuberculosis clinical strain with a defect in ESX-1 secretion induces minimal host immune responses and pathology.

Although Mycobacterium tuberculosis (M.tb) DK9897 is an attenuated strain, it was isolated from a patient with extrapulmonary tuberculosis and vaccination with a subunit vaccine (H56) induced poor protection against it. Both attenuation and lack of protection are because M.tb DK9897 cannot secrete the EsxA virulence factor nor induce a host response against it. Genome sequencing identified a frameshift mutation in the eccCa1 gene. Since the encoded EccCa1 protein provides energy for ESX-1 secretion, it suggested a defect in the ESX-1 type VII secretion system. Genetic complementation with a plasmid carrying the M.tb H37Rv sequence of eccCa1-eccCb1-pe35 re-established EsxA secretion, host specific EsxA T-cell responses, and increased strain virulence. The ESX-1 secretion defect prevents several virulence factors from being functional during infection and therefore attenuates M.tb. It precludes specific T-cell responses against strong antigens and we found very little in vivo cytokine production, gross pathology or granuloma formation in lungs from M.tb DK9897 infected animals. This coincides with M.tb DK9897 being unable to disrupt the phagosome membrane and make contact to the cytosol.

High-frequency vaccine-induced CD8⁺ T cells specific for an epitope naturally processed during infection with Mycobacterium tuberculosis do not confer protection.

Relatively few MHC class I epitopes have been identified from Mycobacterium tuberculosis, but during the late stage of infection, CD8(+) T-cell responses to these epitopes are often primed at an extraordinary high frequency. Although clearly available for recognition during infection, their role in resistance to mycobacterial infections still remain unclear. As an alternative to DNA and viral vaccination platforms, we have exploited a novel CD8(+) T-cell-inducing adjuvant, cationic adjuvant formulation 05 (dimethyldioctadecylammonium/trehalose dibehenate/poly (inositic:cytidylic) acid), to prime high-frequency CD8 responses to the immunodominant H2-K(b) -restricted IMYNYPAM epitope contained in the vaccine Ag tuberculosis (TB)10.4/Rv0288/ESX-H (where ESX is mycobacterial type VII secretion system). We report that the amino acid C-terminal to this minimal epitope plays a decisive role in proteasomal cleavage and epitope priming. The primary structure of TB10.4 is suboptimal for proteasomal processing of the epitope and amino acid substitutions in the flanking region markedly increased epitope-specific CD8(+) T-cell responses. One of the optimized sequences was contained in the closely related TB10.3/Rv3019c/ESX-R Ag and when recombinantly expressed and administered in the cationic adjuvant formulation 05 adjuvant, this Ag promoted very high CD8(+) T-cell responses. This abundant T-cell response was functionally active but provided no protection against challenge, suggesting that CD8(+) T cells play a limited role in protection against M. tuberculosis in the mouse model.

Protective CD4 T cells targeting cryptic epitopes of Mycobacterium tuberculosis resist infection-driven terminal differentiation.

CD4 T cells are crucial to the control of Mycobacterium tuberculosis infection and are a key component of current vaccine strategies. Conversely, immune-mediated pathology drives disease, and recent evidence suggests that adaptive and innate responses are evolutionarily beneficial to M. tuberculosis. We compare the functionality of CD4 T cell responses mounted against dominant and cryptic epitopes of the M. tuberculosis 6-kDa early secreted Ag (ESAT-6) before and postinfection. Protective T cells against cryptic epitopes not targeted during natural infection were induced by vaccinating mice with a truncated ESAT-6 protein, lacking the dominant epitope. The ability to generate T cells that recognize multiple cryptic epitopes was MHC-haplotype dependent, including increased potential via heterologous MHC class II dimers. Before infection, cryptic epitope-specific T cells displayed enhanced proliferative capacity and delayed cytokine kinetics. After aerosol M. tuberculosis challenge, vaccine-elicited CD4 T cells expanded and recruited to the lung. In chronic infection, dominant epitope-specific T cells developed a terminal differentiated KLRG1(+)/PD-1(lo) surface phenotype that was significantly reduced in the cryptic epitope-specific T cell populations. Dominant epitope-specific T cells in vaccinated animals developed into IFN-γ- and IFN-γ,TNF-α-coproducing effector cells, characteristic of the endogenous response. In contrast, cryptic epitope-specific CD4 T cells maintained significantly greater IFN-γ(+)TNF-α(+)IL-2(+) and TNF-α(+)IL-2(+) memory-associated polyfunctionality and enhanced proliferative capacity. Vaccine-associated IL-17A production by cryptic CD4 T cells was also enhanced, but without increased neutrophilia/pathology. Direct comparison of dominant/cryptic epitope-specific CD4 T cells within covaccinated mice confirmed the superior ability of protective cryptic epitope-specific T cells to resist M. tuberculosis infection-driven T cell differentiation.

Tuberculosis vaccine with high predicted population coverage and compatibility with modern diagnostics.

A central goal in vaccine research is the identification of relevant antigens. The Mycobacterium tuberculosis chromosome encodes 23 early secretory antigenic target (ESAT-6) family members that mostly are localized as gene pairs. In proximity to five of the gene pairs are ESX secretion systems involved in the secretion of the ESAT-6 family proteins. Here, we performed a detailed and systematic investigation of the vaccine potential of five possible Esx dimer substrates, one for each of the five ESX systems. On the basis of gene transcription during infection, immunogenicity, and protective capacity in a mouse aerosol challenge model, we identified the ESX dimer substrates EsxD-EsxC, ExsG-EsxH, and ExsW-EsxV as the most promising vaccine candidates and combined them in a fusion protein, H65. Vaccination with H65 gave protection at the level of bacillus Calmette-Guérin, and the fusion protein exhibited high predicted population coverage in high endemic regions. H65 thus constitutes a promising vaccine candidate devoid of antigen 85 and fully compatible with current ESAT-6 and culture filtrate protein 10-based diagnostics.

ESAT-6 (EsxA) and TB10.4 (EsxH) based vaccines for pre- and post-exposure tuberculosis vaccination.

The ESX systems from Mycobacterium tuberculosis are responsible for the secretion of highly immunogenic proteins of key importance for bacterial survival and growth. The two prototypic proteins, ESAT-6 (EsxA from ESX-1) and TB10.4 (EsxH from ESX-3) share a lot of characteristics regarding genome organization, size, antigenic properties, and vaccine potential but the two molecules clearly have very different roles in bacterial physiology. To further investigate the role of ESAT-6 and TB10.4 as preventive and post-exposure tuberculosis vaccines, we evaluated four different fusion-protein vaccines; H1, H4, H56 and H28, that differ only in these two components. We found that all of these vaccines give rise to protection in a conventional prophylactic vaccination model. In contrast, only the ESAT-6-containing vaccines resulted in significant protection against reactivation, when administered post-exposure. This difference in post-exposure activity did not correlate with a difference in gene expression during infection or a differential magnitude or quality of the vaccine-specific CD4 T cells induced by ESAT-6 versus TB10.4-containing vaccines. The post-exposure effect of the ESAT-6 based vaccines was found to be influenced by the infectious load at the time-point of vaccination and was abolished in chronically infected animals with high bacterial loads at the onset of vaccination. Our data demonstrate that there are specific requirements for the immune system to target an already established tuberculosis infection and that ESAT-6 has a unique potential in post-exposure vaccination strategies.

Comparing adjuvanted H28 and modified vaccinia virus ankara expressingH28 in a mouse and a non-human primate tuberculosis model.

Here we report for the first time on the immunogenicity and protective efficacy of a vaccine strategy involving the adjuvanted fusion protein "H28" (consisting of Ag85B-TB10.4-Rv2660c) and Modified Vaccinia Virus Ankara expressing H28. We show that a heterologous prime-boost regimen involving priming with H28 in a Th1 adjuvant followed by boosting with H28 expressed by MVA (H28/MVA28) induced the highest percentage of IFN-γ expressing T cells, the highest production of IFN-γ per single cell and the highest induction of CD8 T cells compared to either of the vaccines given alone. In contrast, in mice vaccinated with adjuvanted recombinant H28 alone (H28/H28) we observed the highest production of IL-2 per single cell and the highest frequency of antigen specific TNF-α/IL-2 expressing CD4 T cells pre and post infection. Interestingly, TNF-α/IL-2 expressing central memory-like CD4 T cells showed a significant positive correlation with protection at week 6 post infection, whereas the opposite was observed for post infection CD4 T cells producing only IFN-γ. Moreover, as a BCG booster vaccine in a clinically relevant non-human primate TB model, the H28/H28 vaccine strategy induced a slightly more prominent reduction of clinical disease and pathology for up to one year post infection compared to H28/MVA28. Taken together, our data showed that the adjuvanted subunit and MVA strategies led to different T cell subset combinations pre and post infection and that TNF-α/IL-2 double producing but not IFN-γ single producing CD4 T cell subsets correlated with protection in the mouse TB model. Moreover, our data demonstrated that the H28 vaccine antigen was able to induce strong protection in both a mouse and a non-human primate TB model.

Broadening of the T-cell repertoire to HIV-1 Gag p24 by vaccination of HLA-A2/DR transgenic mice with overlapping peptides in the CAF05 adjuvant.

Induction of broad T-cell immune responses is regarded as critical for vaccines against the human immunodeficiency virus type 1 (HIV-1) which exhibit high diversity and, therefore, focus has been on inducing cytotoxic CD8 T-cell responses against the more conserved parts of the virus, such as the Gag protein. Herein, we have used the p24 protein which contains a range of conserved T-cell epitopes. We demonstrate that a vaccine of HIV-1 subtype B consensus group-specific antigen (Gag) p24 protein with the CD8-inducing liposomal cationic adjuvant formulation (CAF) 05, induces both CD4 and CD8 T-cell responses in CB6F1 mice. The adjuvanted vaccine also induced functional antigen-specific cytotoxicity in vivo. Furthermore, we found that when fragmenting the Gag p24 protein into overlapping Gag p24 peptides, a broader T-cell epitope specificity was induced in the humanized human leukocyte antigen (HLA)-A2/DR-transgenic mouse model. Thus, combining overlapping Gag p24 peptides with CAF05 appears to be a promising and simple strategy for inducing broader T-cell responses to multiple conserved epitopes which will be relevant for both prophylactic and therapeutic HIV-1 vaccines.

Cell-mediated and humoral immune responses after immunization of calves with a recombinant multiantigenic Mycobacterium avium subsp. paratuberculosis subunit vaccine at different ages.

Neonates and juvenile ruminants are very susceptible to paratuberculosis infection. This is likely due to a high degree of exposure from their dams and an immature immune system. To test the influence of age on vaccine-induced responses, a cocktail of recombinant Mycobacterium avium subsp. paratuberculosis proteins (MAP0217, MAP1508, MAP3701c, MAP3783, and MAP1609c/Ag85B) was formulated in a cationic liposome adjuvant (CAF01) and used to vaccinate animals of different ages. Male jersey calves were divided into three groups that were vaccinated at 2, 8, or 16 weeks of age and boosted twice at weeks 4 and 12 relative to the first vaccination. Vaccine-induced immune responses, the gamma interferon (IFN-γ) cytokine secretion and antibody responses, were followed for 20 weeks. In general, the specific responses were significantly elevated in all three vaccination groups after the first booster vaccination with no or only a minor effect from the second booster. However, significant differences were observed in the immunogenicity levels of the different proteins, and it appears that the older age group produced a more consistent IFN-γ response. In contrast, the humoral immune response is seemingly independent of vaccination age as we found no difference in the IgG1 responses when we compared the three vaccination groups. Combined, our results suggest that an appropriate age of vaccination should be considered in vaccination protocols and that there is a possible interference of vaccine-induced immune responses with weaning (week 8).

Vaccine-induced th17 cells are maintained long-term postvaccination as a distinct and phenotypically stable memory subset.

Th17 cells are increasingly being recognized as an important T helper subset for immune-mediated protection, especially against pathogens at mucosal ports of entry. In several cases, it would thus be highly relevant to induce Th17 memory by vaccination. Th17 cells are reported to exhibit high plasticity and may not stably maintain their differentiation program once induced, questioning the possibility of inducing durable Th17 memory. Accordingly, there is no consensus as to whether Th17 memory can be established unless influenced by continuous Th17 polarizing conditions. We have previously reported (T. Lindenstrøm, et al., J. Immunol. 182:8047-8055, 2009) that the cationic liposome adjuvant CAF01 can prime both Th1 and Th17 responses and promote robust, long-lived Th1 memory. Here, we demonstrate that subunit vaccination in mice with CAF01 leads to establishment of bona fide Th17 memory cells. Accordingly, Th17 memory cells exhibited lineage stability by retaining both phenotypic and functional properties for nearly 2 years. Antigen-specific, long-term Th17 memory cells were found to be mobilized from lung-draining lymph nodes to the lung following an aerosol challenge by Mycobacterium tuberculosis nearly 2 years after their induction and proliferated at levels comparable to those of Th1 memory cells. During the infection, the vaccine-induced Th17 memory cells expanded in the lungs and adapted Th1 characteristics, implying that they represent a metastable population which exhibits plasticity when exposed to prolonged Th1 polarizing, inflammatory conditions such as those found in the M. tuberculosis-infected lung. In the absence of overt inflammation, however, stable bona fide Th17 memory can indeed be induced by parenteral immunization.

Correlation of antigen-specific IFN-γ responses of fresh blood samples from Mycobacterium avium subsp. paratuberculosis infected heifers with responses of day-old samples co-cultured with IL-12 or anti-IL-10 antibodies.

Paratuberculosis is a chronic infection of the intestine of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Early stage MAP infection can be detected by measuring cell-mediated immune responses using the interferon gamma (IFN-γ) assay. Whole blood samples are cultured overnight with specific MAP antigens followed by quantification of IFN-γ by ELISA. It is recommended that the time interval from sampling to culture does not exceed eight hours but addition of the co-stimulating cytokine interleukin 12 (IL-12) or anti-IL-10 antibodies to culture have been demonstrated to enhance IFN-γ responses of cultures stimulated with Johnin purified protein derivative (PPDj). Here we examined the correlation of IFN-γ production in response to PPDj and 15 recombinant antigens in day-old blood samples from heifers 10-21 months of age from a MAP infected herd with addition of either recombinant bovine IL-12 or anti-bovine IL-10 antibody with IFN-γ production in sample day samples. IFN-γ responses of sample day samples showed high correlation with responses to some antigens in day-old samples with addition of IL-12 or anti-IL-10 antibodies to cultures, indicating that day-old protocols can be applied as an alternative to the conventional IFN-γ protocol. Immunogenicity of the novel antigens was generally low for day-old samples. The most promising antigen using the day-old protocol with addition of IL-12 was latency protein LATP-2 as correlations, immunogenicity and diagnostic specificity collectively was high. The latency protein LATP-1 was the most promising antigen in the day-old protocol with addition of anti-IL-10 antibodies.