Identification of Mycobacterium tuberculosis vaccine candidates using human CD4 T-cells expression cloning

To identify Mycobacterium tuberculosis (Mtb) antigens as candidates for a subunit vaccine against tuberculosis (TB), we have employed a CD4⁺ T-cell expression screening method. Mtb-specific CD4⁺ T-cell lines from nine healthy PPD positive donors were stimulated with different antigenic substrates including autologous dendritic cells (DC) infected with Mtb, or cultured with culture filtrate proteins (CFP), and purified protein derivative of Mtb (PPD). These lines were used to screen a genomic Mtb library expressed in Escherichia coli and processed and presented by autologous DC. This screening led to the recovery of numerous T-cell antigens, including both novel and previously described antigens. One of these novel antigens, referred to as Mtb9.8 (Rv0287), was recognized by multiple T-cell lines, stimulated with either Mtb-infected DC or CFP. Using the mouse and guinea pig models of TB, high levels of IFN-γ were produced, and solid protection from Mtb challenge was observed following immunization with Mtb9.8 formulated in either AS02A or AS01B Adjuvant Systems. These results demonstrate that T-cell screening of the Mtb genome can be used to identify CD4⁺ T-cell antigens that are candidates for vaccine development.     

Towards an immunosense vaccine to prevent toxoplasmosis: protective Toxoplasma gondii epitopes restricted by HLA-A*0201

The ideal vaccine to protect against toxoplasmosis in humans would include antigens that elicit a protective T helper cell type 1 immune response, and generate long-lived IFN-γ-producing CD8⁺ T cells. Herein, we utilized a predictive algorithm to identify candidate HLA-A02 supertype epitopes from Toxoplasma gondii proteins. Thirteen peptides elicited production of IFN-γ from PBMC of HLA-A02 supertype persons seropositive for T. gondii infection but not from seronegative controls. These peptides displayed high-affinity binding to HLA-A02 proteins. Immunization of HLA-A*0201 transgenic mice with these pooled peptides, with a universal CD4⁺ epitope peptide called PADRE, formulated with adjuvant GLA-SE, induced CD8⁺ T cell IFN-γ production and protected against parasite challenge. Peptides identified in this study provide candidates for inclusion in immunosense epitope-based vaccines.     

BCG vaccination-induced long-lasting control of Mycobacterium tuberculosis correlates with the accumulation of a novel population of CD4+IL-17+TNF+IL-2+ T cells

Mycobacterium bovis Bacille Calmette-Guerin (BCG) is the only vaccine in use to prevent Mycobacterium tuberculosis (Mtb) infection. Here we analyzed the protective efficacy of BCG against Mtb challenges 21 or 120 days after vaccination. Only after 120 days post-vaccination were mice able to efficiently induce early Mtb growth arrest and maintain long-lasting control of Mtb. This protection correlated with the accumulation of CD4⁺ T cells expressing IL-17⁺TNF⁺IL-2⁺. In contrast, mice challenged with Mtb 21 days after BCG vaccination exhibited only a mild and transient protection, associated with the accumulation of CD4⁺ T cells that were mostly IFN-γ⁺TNF⁺ and to a lesser extent IFN-γ⁺TNF⁺IL-2⁺. These data suggest that the memory response generated by BCG vaccination is functionally distinct depending upon the temporal proximity to BCG vaccination. Understanding how these responses are generated and maintained is critical for the development of novel vaccination strategies against tuberculosis.     

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.     

Higher human CD4 T cell response to novel Mycobacterium tuberculosis latency associated antigens Rv2660 and Rv2659 in latent infection compared with tuberculosis disease

One third of the world's population is infected with Mycobacterium tuberculosis (M.tb). A vaccine that would prevent progression to TB disease will have a dramatic impact on the global TB burden. We propose that antigens of M.tb that are preferentially expressed during latent infection will be excellent candidates for post-exposure vaccination. We therefore assessed human T cell recognition of two such antigens, Rv2660 and Rv2659. Expression of these was shown to be associated with non-replicating persistence in vitro. After six days incubation of PBMC from persons with latent tuberculosis infection (LTBI) and tuberculosis (TB) disease, Rv2660 and Rv2659 induced IFN-γ production in a greater proportion of persons with LTBI, compared with TB diseased patients. Persons with LTBI also had increased numbers of viable T cells, and greater specific CD4⁺ T cell proliferation and cytokine expression capacity. Persons with LTBI preferentially recognize Rv2659 and Rv2660, compared with patients with TB disease. These results suggest promise of these antigens for incorporation into post-exposure TB vaccines.     

Bacillus Calmette-Guérin vaccination using a microneedle patch

Tuberculosis (TB) caused by Mycobacterium tuberculosis continues to be a leading cause of mortality among bacterial diseases, and the bacillus Calmette-Guérin (BCG) is the only licensed vaccine for human use against this disease. TB prevention and control would benefit from an improved method of BCG vaccination that simplifies logistics and eliminates dangers posed by hypodermic needles without compromising immunogenicity. Here, we report the design and engineering of a BCG-coated microneedle vaccine patch for a simple and improved intradermal delivery of the vaccine. The microneedle vaccine patch induced a robust cell-mediated immune response in both the lungs and the spleen of guinea pigs. The response was comparable to the traditional hypodermic needle based intradermal BCG vaccination and was characterized by a strong antigen specific lymphocyte proliferation and IFN-γ levels with high frequencies of CD4⁺IFN-γ⁺, CD4⁺TNF-α⁺ and CD4⁺IFN-γ⁺TNF-α⁺ T cells. The BCG-coated microneedle vaccine patch was highly immunogenic in guinea pigs and supports further exploration of this new technology as a simpler, safer, and compliant vaccination that could facilitate increased coverage, especially in developing countries that lack adequate healthcare infrastructure.     

Recombinant pro-apoptotic Mycobacterium tuberculosis generates CD8 T cell responses against human immunodeficiency virus type 1 Env and M. tuberculosis in neonatal mice

Mycobacterium bovis BCG is an attractive vaccine vector against breast milk HIV transmission because it elicits Th1-type responses in newborns. However, BCG causes disease in HIV-infected infants. Genetically attenuated Mycobacterium tuberculosis (Mtb) mutants represent a safer alternative for immunocompromised populations. In the current study, we compared the immunogenicity in mice of three different recombinant attenuated Mtb strains expressing an HIV envelope (Env) antigen construct. Two of these strains (ΔlysA ΔpanCD Mtb and ΔRD1 ΔpanCD Mtb) failed to induce significant levels of HIV Env-specific CD8⁺ T cell responses. In striking contrast, an HIV-1 Env-expressing attenuated ΔlysA Mtb containing a deletion in secA2, which encodes a virulence-related secretion system involved in evading adaptive immunity, generated consistently measurable Env-specific CD8⁺ T cell responses that were significantly greater than those observed after immunization with BCG expressing HIV Env. Similarly, another strain of ΔlysA ΔsecA2 Mtb expressing SIV Gag induced Gag- and Mtb-specific CD8⁺ T cells producing perforin or IFNγ, and Gag-specific CD4⁺ T cells producing IFNγ within 3 weeks after immunization in adult mice; in addition, IFNγ-producing Gag-specific CD8⁺ T cells and Mtb-specific CD4⁺ T cells were observed in neonatal mice within 1 week of immunization. We conclude that ΔlysA ΔsecA2 Mtb is a promising vaccine platform to construct a safe combination HIV-TB vaccine for use in neonates.     

Nonlytic Fc-fused IL-7 synergizes with Mtb32 DNA vaccine to enhance antigen-specific T cell responses in a therapeutic model of tuberculosis

Improvement to the immunogenicity of DNA vaccines was evaluated in a Mycobacterium tuberculosis (MTB) infection mouse model examining the combined effects of nonlytic Fc-fused IL-7 DNA (IL-7-nFc) and Flt3-ligand fused Mtb32 (F-Mtb32) DNA. Mice were treated with conventional chemotherapy for 6 weeks from 4 weeks after aerosol infection of MTB. Following the start of chemotherapy, DNA immunizations were administered five times with 2-week intervals. Coadministration of IL-7-nFc and F-Mtb32 DNA given during chemotherapy synergistically enhanced the magnitude of Mtb32-specific T cell responses and sustained for one-year after the last immunization assessed by IFN-γ ELISPOT assay. After dexamethasone treatment, a significantly reduced MTB reactivation was observed in mice received both IL-7-nFc and F-Mtb32 DNA, compared with F-MTb32 DNA alone or with control mice. In addition, mice treated with IL-7-nFc and F-Mtb32 DNA together showed improved lung pathology and reduced pulmonary inflammation values relative to F-Mtb32 DNA or saline injected mice. Intracellular cytokine staining revealed that the protection levels induced by combination therapy with IL-7-nFc and F-Mtb32 DNA was associated with enhanced Mtb32-specific IFN-γ secreting CD4⁺ T cell responses and CD8⁺ T cell responses stimulated with CTL epitope peptide in the lungs and spleens. These data suggest that IL-7-nFc as a novel TB adjuvant may facilitate therapeutic TB DNA vaccine to the clinics through significant enhancement of codelivered DNA vaccine-induced T cell immunity.     

Vaccine-induced anti-tuberculosis protective immunity in mice correlates with the magnitude and quality of multifunctional CD4 T cells

The development of improved vaccines against Mycobacterium tuberculosis has been hindered by a limited understanding of the immune correlates of anti-tuberculosis protective immunity. In this study, we examined the relationship between long-term anti-tuberculosis protection and the mycobacterial-specific CD4 multifunctional T (MFT) cell responses induced by five different TB vaccines (live-attenuated, subunit, viral vectored, plasmid DNA, and combination vaccines) in a mouse model of pulmonary tuberculosis. In a 14-month experiment, we showed that TB vaccine-induced CD4 T cell responses were heterogenous. Antigen-specific monofunctional CD4 T cells expressing single cytokines and MFT CD4 T cells expressing multiple cytokines (IFN-γ and TNF-α, IFN-γ and IFN-γ, TNF-α, and IL-2, and all three cytokines) were identified after the immunizations. Interestingly, compared to the monofunctional cells, significantly higher median fluorescent intensities (MFIs) for IFN-γ and TNF-α were detected for triple-positive MFT CD4 T cells induced by the most protective vaccines while modest differences in relative MFI values were seen for the less protective preparations. Most importantly during the 14-month study, the levels of vaccine-induced pulmonary and splenic protective immunity correlated with the frequency and the integrated MFI (iMFI, frequency × MFI) values of triple-positive CD4 T cells that were induced by the same vaccines. These data support efforts to use MFT cell analyses as a measure of TB vaccine immunogenicity in human immunization studies.     

Memory CD8 T cells elicited by HIV-1 lipopeptide vaccines display similar phenotypic profiles but differences in term of magnitude and multifunctionality compared with FLU- or EBV-specific memory T cells in humans

Differentiation marker, multifunctionality and magnitude analyses of specific-CD8⁺ memory T cells are crucial to improve development of HIV vaccines designed to generate cell-mediated immunity. Therefore, we fully characterized the HIV-specific CD8⁺ T cell responses induced in volunteers vaccinated with HIV lipopeptide vaccines for phenotypic markers, tetramer staining, cytokine secretion, and cytotoxic activities. The frequency of ex vivo CD8⁺ T cells elicited by lipopeptide vaccines is very rare and central-memory phenotype and functions of these cells were been shown to be important in AIDS immunity. So, we expanded them using specific peptides to compare the memory T cell responses induced in volunteers by HIV vaccines with responses to influenza (FLU) or Epstein Barr virus (EBV). By analyzing the differentiation state of IFN-γ-secreting CD8⁺ T cells, we found a CCR7⁻CD45RA⁻CD28^+int/CD28⁻ profile (>85%) belonging to a subset of intermediate-differentiated effector T cells for HIV, FLU, and EBV. We then assessed the quality of the response by measuring various T cell functions. The percentage of single IFN-γ T cell producers in response to HIV was 62% of the total of secreting T cells compared with 35% for FLU and EBV, dual and triple (IFN-γ/IL-2/CD107a) T cell producers could also be detected but at lower levels (8% compared with 37%). Finally, HIV-specific T cells secreted IFN-γ and TNF-α, but not the dual combination like FLU- and EBV-specific T cells. Thus, we found that the functional profile and magnitude of expanded HIV-specific CD8⁺ T precursors were more limited than those of to FLU- and EBV-specific CD8⁺ T cells. These data show that CD8⁺ T cells induced by these HIV vaccines have a similar differentiation profile to FLU and EBV CD8⁺ T cells, but that the vaccine potency to induce multifunctional T cells needs to be increased in order to improve vaccination strategies.     

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. T_H1 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.     

Identification of non-CSP antigens bearing CD8 epitopes in mice immunized with irradiated sporozoites

Immunization of BALB/c mice with irradiated sporozoites (IrSp) of Plasmodium yoelii can lead to sterile immunity. The circumsporozoite protein (CSP) plays a dominant role in protection. Nevertheless after hyper-immunization with IrSp, complete protection is obtained in CSP-transgenic BALB/c mice that are T-cell tolerant to the CSP and cannot produce antibodies [CSP-Tg/JhT(−/−)]. This protection is mediated exclusively by CD8⁺ T cells [1]. To identify the non-CSP protective T cell antigens, we studied the properties of 34 P. yoelii sporozoite antigens that are predicted to be secreted and to contain strong Kd-restricted CD8⁺ T cell epitopes. The synthetic peptides corresponding to the epitopes were used to screen for the presence of peptide-specific CD8⁺ T cells secreting interferon-γ (IFN-γ) in splenocytes from CSP-Tg/JhT(−/−) BALB/c mice hyper immunized with IrSp. However, the numbers of IFN-γ-secreting splenocytes specific for the non-CSP antigen-derived peptides were 20-100 times lower than those specific for the CSP-specific peptide. When mice were immunized with recombinant adenoviruses expressing selected non-CSP antigens, the animals were not protected against challenge with P. yoelii sporozoites although large numbers of CD8⁺ specific T cells were generated.     

A T cell epitope-based vaccine protects against chlamydial infection in HLA-DR4 transgenic mice

Vaccination with recombinant chlamydial protease-like activity factor (rCPAF) has been shown to provide robust protection against genital Chlamydia infection. Adoptive transfer of IFN-γ competent CPAF-specific CD4⁺ T cells was sufficient to induce early resolution of chlamydial infection and reduction of subsequent pathology in recipient IFN-γ-deficient mice indicating the importance of IFN-γ secreting CD4⁺ T cells in host defense against Chlamydia. In this study, we identify CD4⁺ T cell reactive CPAF epitopes and characterize the activation of epitope-specific CD4⁺ T cells following antigen immunization or Chlamydia challenge. Using the HLA-DR4 (HLA-DRB1*0401) transgenic mouse for screening overlapping peptides that induced T cell IFN-γ production, we identified at least 5 CPAF T cell epitopes presented by the HLA-DR4 complex. Immunization of HLA-DR4 transgenic mice with a rCPAFep fusion protein containing these 5 epitopes induced a robust cell-mediated immune response and significantly accelerated the resolution of genital and pulmonary Chlamydia infection. rCPAFep vaccination induced CPAF-specific CD4⁺ T cells in the spleen were detected using HLA-DR4/CPAF-epitope tetramers. Additionally, CPAF-specific CD4⁺ clones could be detected in the mouse spleen following Chlamydia muridarum and a human Chlamydia trachomatis strain challenge using these novel tetramers. These results provide the first direct evidence that a novel CPAF epitope vaccine can provide protection and that HLA-DR4/CPAF-epitope tetramers can detect CPAF epitope-specific CD4⁺ T cells in HLA-DR4 mice following C. muridarum or C. trachomatis infection. Such tetramers could be a useful tool for monitoring CD4⁺ T cells in immunity to Chlamydia infection and in developing epitope-based human vaccines using the murine model.     

CD8 T cell response in HLA-A*0201 transgenic mice is elicited by epitopes from SARS-CoV S protein

Cytotoxic CD8⁺ T lymphocytes (CTLs) play an important role in antiviral immunity. Several human HLA-A*0201 restricted CTL epitopes of severe acute respiratory syndrome (SARS) spike (S) protein have been identified in HLA-A*0201 transgenic (Tg) mice, but the mechanisms and properties of immune responses are still not well understood. In this study, HLA-A*0201 Tg mice were primed intramuscularly with SARS S DNA and boosted subcutaneously with HLA-A*0201 restricted peptides. The lymphocytes from draining lymph nodes, spleens and lungs were stimulated with the cognate peptides. Three different methods (ELISA, ELISPOT and FACS) were used to evaluate the immune responses during short and long periods of time after immunization. Results showed that peptide-specific CD8⁺ T cells secreted IFN-γ, TNF-α and IL-2 and expressed CD107a/b on cell surface. IFN-γ⁺CD8⁺ T cells and CD107a/b⁺CD8⁺ T cells distributed throughout the lymphoid and non-lymphoid tissues, but the frequency of peptide-specific CD8⁺ T cells was higher in lungs than in spleens and lymph nodes. The phenotype of the CD8⁺ T cells was characterized based on the expression of IFN-γ. Most of the HLA-A*0201 restricted peptide-specific CD8⁺ T cells represented a memory subset with CD45RB^high and CD62L^low. Taken together, these data demonstrate that immunization with SARS S DNA and HLA-A*0201 restricted peptides can elicit antigen-specific CD8⁺ T cell immune responses which may have a significant implication in the long-term protection. We provide novel information in cellular immune responses of SARS S antigen-specific CD8⁺ T cells, which are important in the development of vaccine against SARS-CoV infection.     

Flagellin is a Th1 polarizing factor for human CD4+ T cells and induces protection in a murine neonatal vaccination model of rotavirus infection

Neonates have an increased susceptibility to infections, particularly those caused by intracellular pathogens, leading to high morbidity and mortality rates. This is partly because of a poor response of neonatal CD4⁺ T cells, leading to deficient antibody production and a low production of IFN-γ, resulting in deficient elimination of intracellular pathogens. The poor memory response of human neonates has underpinned the need for improving vaccine formulations. Molecular adjuvants that improve the response of neonatal lymphocytes, such as the ligands of toll-like receptors (TLRs), are attractive candidates. Among them, flagellin, the TLR5 ligand, is effective at very low doses; prior immunity to flagellin does not impair its adjuvant activity. Human CD4⁺ and CD8⁺ T cells express TLR5. We found that flagellin induces the expression of IFN-γ, IL-1β and IL-12 in mononuclear cells from human neonate and adult donors. When human naïve CD4⁺ T cells were activated in the presence of flagellin, there was high level of expression of IFN-γ in both neonates and adults. Furthermore, flagellin induced IFN-γ production in Th1 cells obtained from adult donors; in the Th2 population, it inhibited IL-4 cytokine production. Flagellin also promoted expression of the IFN-γ receptor in naive CD4⁺ T cells from neonates and adults. To test the adjuvant capacity of flagellin in vivo, we used a murine neonate vaccination model for infection with rotavirus, a pathogen responsible for severe diarrhea in young infants. Using the conserved VP6 antigen, we observed an 80% protection against rotavirus infection in the presence of flagellin, but only in those mice previously primed in the neonatal period. Our data suggest that flagellin could be an attractive adjuvant for achieving a Th1 response.     

Predicting promiscuous antigenic T cell epitopes of Mycobacterium tuberculosis mymA operon proteins binding to MHC Class I and Class II molecules

Limited efficacy of Bacillus Calmette–Guérin vaccine has raised the need to explore other immunogenic candidates to develop an effective vaccine against Mycobacterium tuberculosis (Mtb). Both CD4 + and CD8 + T cells play a critical role in host immunity to Mtb. Infection of macrophages with Mtb results in upregulation of mymA operon genes thereby suggesting their importance as immune targets. In the present study, after exclusion of self-peptides mymA operon proteins of Mtb were analyzed in silico for the presence of Human Leukocyte Antigen (HLA) Class I and Class II binding peptides using Bioinformatics and molecular analysis section, NetMHC 3.4, ProPred and Immune epitope database software. Out of 56 promiscuous epitopes obtained, 41 epitopes were predicted to be antigenic for MHC Class I. In MHC Class II, out of 336 promiscuous epitopes obtained, 142 epitopes were predicted to be antigenic. The comparative bioinformatics analysis of mymA operon proteins found Rv3083 to be the best vaccine candidate. Molecular docking was performed with the most antigenic peptides of Rv3083 (LASGAASVV with alleles HLA-B51:01, HAATSGTLI with HLA-A02, IVTATGLNI and EKIHYGLKVNTA with HLA-DRB1_01:01) to study the structural basis for recognition of peptides by various HLA molecules. The software binding prediction was validated by the obtained molecular docking score of peptide-HLA complex. These peptides can be further investigated for their immunological relevance in patients of tuberculosis using major histocompatibility complex tetramer approach.     

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.     

Peptide-pulsed dendritic cells induce the hepatitis C viral epitope-specific responses of naïve human T cells

Hepatitis C virus (HCV) is a major cause of liver disease. Spontaneous resolution of infection is associated with broad, MHC class I- (CD8⁺) and class II-restricted (CD4⁺) T cell responses to multiple viral epitopes. Only 20% of patients clear infection spontaneously, however, most develop chronic disease. The response to chemotherapy varies; therapeutic vaccination offers an additional treatment strategy. To date, therapeutic vaccines have demonstrated only limited success in clinical trials. Vector-mediated vaccination with multi-epitope-expressing DNA constructs provides an improved approach. Highly-conserved, HLA-A2-restricted HCV epitopes and HLA-DRB1-restricted immunogenic consensus sequences (ICS, each composed of multiple overlapping and highly conserved epitopes) were predicted using bioinformatics tools and synthesized as peptides. HLA binding activity was determined in competitive binding assays. Immunogenicity and the ability of each peptide to stimulate naïve human T cell recognition and IFN-γ production were assessed in cultures of total PBMCs and in co-cultures composed of peptide-pulsed dendritic cells (DCs) and purified T lymphocytes, cell populations derived from normal blood donors. Essentially all predicted HLA-A2-restricted epitopes and HLA-DRB1-restricted ICS exhibited HLA binding activity and the ability to elicit immune recognition and IFN-γ production by naïve human T cells. The ability of DCs pulsed with these highly-conserved HLA-A2- and -DRB1-restricted peptides to induce naïve human T cell reactivity and IFN-γ production ex vivo demonstrates the potential efficacy of a multi-epitope-based HCV vaccine targeted to dendritic cells.     

Cellular immune responses of pigs induced by vaccination with either a whole cell sonicate or pepsin-digested Brachyspira (Serpulina) hyodysenteriae bacterin

Brachyspira (Serpulina) hyodysenteriae infection of pigs (swine dysentery) causes a mucohemorrhagic diarrhea resulting in significant economic losses for producers. A commercial vaccine consisting of a proteinase-digested bacterin has shown efficacy in the reduction of disease due to B. hyodysenteriae. Vaccines consisting of whole cell bacterins, however, generally fail to protect pigs from disease. In the present study, cellular immune responses induced by a proteinase-digested bacterin were compared to responses induced by a whole cell sonicate antigen preparation. In addition, usage of either squalene or Freund's incomplete adjuvants in combination with each antigen preparation was also compared. Both antigen preparations induced significant cellular immune responses as measured by in vitro (IFN-γ production and T cell proliferation) and in vivo methods (DTH responses). No significant differences were detected in proliferative, interferon-γ (IFN-γ), or delayed type hypersensitivity (DTH) responses by pigs receiving either adjuvant or antigen preparation. T cells (CD3⁺) but not B cells from vaccinated animals proliferated in response to in vitro stimulation with B. hyodysenteriae antigen. CD8⁺ (single positive and CD4/CD8 double positive) and γδ⁺ T cells were particularly responsive. In addition, high percentages of both CD8 single positive and CD4/CD8 double positive cells were detected in antigen-stimulated cultures. These findings demonstrate the unique sensitivity of porcine CD8⁺ T cells to priming for recall response by vaccination with a proteinase-digested B. hyodysenteriae bacterin.