Pleurotus ferulae water extract enhances the maturation and function of murine bone marrow-derived dendritic cells through TLR4 signaling pathway

Dendritic cells (DCs) play important roles in the regulation of immune system, which link innate and adaptive immune responses. Mature DCs produced interleukin (IL)-12 promote optimal type 1 T helper (Th1) cells and cytotoxic T lymphocytes. The extracts of traditional herbal medicines have been shown to enhance immune responses through promoting the maturation and cytokine production of DCs. Here, we investigated the effects of Pleurotus ferulae water extract (PFWE) on the maturation and function of bone marrow–derived DCs (BM–DCs). Upon PFWE treatment, BM–DCs dose-dependently upregulated the expression of CD40, CD80, CD86 and MHC II and increased the production of IL-12, IL-6 and tumor necrosis factor (TNF)-α but not for IL-10, which is mediated by TLR4 signaling pathway, at least partially. The production of prostaglandin E2 (PGE2) in BM–DCs was decreased by the treatment of PFWE. Moreover, PFWE treatment decreased the expression of active caspase-3 but increased the expression of CCR7. PFWE treated DCs enhanced the proliferation of allogenic CD8⁺ T cells and the capacity of antigen presenting to autologous CD8⁺ T cells. The combination of PFWE and CpG–ODN further enhanced the maturation and function of murine BM–DCs. The results showed that PFWE could enhance the maturation and function of DCs through TLR4 signaling pathway and has additive effect when combined with CpG–ODN, suggesting that PFWE alone or combined with CpG–ODN could be used to enhance the immune responses.     

Modulation of the CD4+ T cell response after acellular pertussis vaccination in the presence of TLR4 ligation

Whole cell pertussis (wP) vaccines are gradually being replaced by aluminum salt-adjuvanted acellular pertussis (aP) vaccines. These promote CD4⁺ T cell responses with a non-protective Th2 component, while protective immune mechanisms to B. pertussis may rather involve long-lived Th1/Th17 type CD4⁺ T cells. Here we asked whether addition of a non-toxic meningococcal LPS derivative, LpxL1, as adjuvant can favorably modulate the aP-induced pertussis-specific CD4⁺ T cell response in mice. To assess the effect of TLR4 ligation, Th type, quantity, and memory potential of pertussis-specific CD4⁺ T cells were determined at the single-cell level after aP and aP+LpxL1 vaccination using intracellular cytokine staining and MHC class II tetramers. Adding LpxL1 to the aP vaccine weakened the Th2 component and strengthened the Th1/Th17 component of the specific CD4⁺ T cell response. Notably, LpxL1 addition also induced higher frequencies of tetramer positive CD4⁺ T cells in draining lymph nodes or blood, depending on the phase after vaccination. Moreover, there was a net profit in the number of CD4⁺ T cells with a central memory phenotype, preferred for long-term immunity. Thus, adding a TLR4 ligand as adjuvant to a current aP vaccine was associated with a more favorable pertussis-specific CD4⁺ T cell response.     

Anti-tumor effect of the alphavirus-based virus-like particle vector expressing prostate-specific antigen in a HLA–DR transgenic mouse model of prostate cancer

The goal of this study was to determine if an alphavirus-based vaccine encoding human Prostate-Specific Antigen (PSA) could generate an effective anti-tumor immune response in a stringent mouse model of prostate cancer. DR2bxPSA F₁ male mice expressing human PSA and HLA-DRB1^*1501 transgenes were vaccinated with virus-like particle vector encoding PSA (VLPV–PSA) followed by the challenge with Transgenic Adenocarcinoma of Mouse Prostate cells engineered to express PSA (TRAMP–PSA). PSA-specific cellular and humoral immune responses were measured before and after tumor challenge. PSA and CD8 reactivity in the tumors was detected by immunohistochemistry. Tumor growth was compared in vaccinated and control groups. We found that VLPV–PSA could infect mouse dendritic cells in vitro and induce a robust PSA-specific immune response in vivo. A substantial proportion of splenic CD8 T cells (19.6 ± 7.4%) produced IFNγ in response to the immunodominant peptide PSA_65–73. In the blood of vaccinated mice, 18.4 ± 4.1% of CD8 T cells were PSA-specific as determined by the staining with H-2D^b/PSA_65–73 dextramers. VLPV–PSA vaccination also strongly stimulated production of IgG2a/b anti-PSA antibodies. Tumors in vaccinated mice showed low levels of PSA expression and significant CD8+ T cell infiltration. Tumor growth in VLPV–PSA vaccinated mice was significantly delayed at early time points (p = 0.002, Gehan–Breslow test). Our data suggest that TC-83-based VLPV–PSA vaccine can efficiently overcome immune tolerance to PSA, mediate rapid clearance of PSA-expressing tumor cells and delay tumor growth. The VLPV–PSA vaccine will undergo further testing for the immunotherapy of prostate cancer.     

Viable spores of Coccidioides posadasii Δcps1 are required for vaccination and provide long lasting immunity

Coccidioidomycosis is a systemic fungal infection for which a vaccine has been sought for over fifty years. The avirulent Coccidioides posadasii strain, Δcps1, which is missing a 6 kb gene, showed significant protection in mice. These studies explore conditions of protection in mice and elucidate the immune response. Mice were vaccinated with different doses and viability states of Δcps1 spores, challenged with virulent C. posadasii, and sacrificed at various endpoints, dependent on experimental objectives. Tissues from vaccinated mice were harvested for in vitro elucidation of immune response. Vaccination with viable Δcps1 spores was required for protection from lethal challenge. Viable spore vaccination produced durable immunity, lasting at least 6 months, and prolonged survival (≥6 months). The C. posadasii vaccine strain also protected mice against C. immitis (survival ≥ 6 months). Cytokines from infected lungs of vaccinated mice in the first four days after Cp challenge showed significant increases of IFN-γ, as did stimulated CD4⁺ spleen cells from vaccinated mice. Transfer of CD4⁺ cells, but not CD8⁺ or B cells, reduced fungal burdens following challenge. IFN-γ from CD4⁺ cells in vaccinated mice indicates a Th1 response, which is critical for host control of coccidioidomycosis.     

CD11a and CD49d enhance the detection of antigen-specific T cells following human vaccination

BackgroundDetermining the efficacy of human vaccines that induce antigen-specific protective CD4 T cell responses against pathogens can be particularly challenging to evaluate. Surface expression of CD11a and CD49d has been shown to identify antigen-specific CD4 T cells against viral pathogens in mice. We hypothesized that CD11a and CD49d would also serve as markers of human antigen-specific T cells responding to vaccination.MethodsA phase I vaccine trial enabled us to evaluate a novel gating strategy based on surface expression of CD11a and CD49d as a means of detecting antigen-specific, cytokine producing CD4 and CD8 T cells induced after vaccination of naïve individuals against leishmaniasis. Three study groups received LEISH-F3 recombinant protein combined with either squalene oil-in-water emulsion (SE) alone, SE with the synthetic TLR-4 ligand glucopyranosyl lipid adjuvant (GLA-SE), or SE with Salmonella minnesota-derived monophosphoryl lipid A (MPL-SE). Individuals were given 3 vaccine doses, on days 0, 28 and 168.ResultsStarting after the first vaccine dose, the frequency of both CD11a^hiCD49d⁺ CD4 and CD11a^hiCD49d⁺ CD8 T cells significantly increased over time throughout the 24-week trial. To confirm the role of CD11a^hiCD49d⁺ expression in the identification of the antigen-specific T cells, cytokine production was measured following LEISH-F3 stimulation. All of the IFN-γ, TNF-α, and IL-2 producing cells were found within the CD11a^hiCD49d⁺ population.ConclusionsOur results suggest that the change in the frequency of CD11a^hiCD49d⁺ T cells can be used to track antigen-specific CD4 and CD8 T cell responses following T cell-targeted vaccination.     

Co-localization of a CD1d-binding glycolipid with an adenovirus-based malaria vaccine for a potent adjuvant effect

A CD1d-binding, invariant (i) natural killer T (NKT)-cell stimulatory glycolipid, α-Galactosylceramide (αGalCer), has been shown to act as an adjuvant. We previously identified a fluorinated phenyl ring-modified αGalCer analog, 7DW8-5, displaying a higher binding affinity for CD1d molecule and more potent adjuvant activity than αGalCer. In the present study, 7DW8-5 co-administered intramuscularly (i.m.) with a recombinant adenovirus expressing a Plasmodium yoelii circumsporozoite protein (PyCSP), AdPyCS, has led to a co-localization of 7DW8-5 and a PyCSP in draining lymph nodes (dLNs), particularly in dendritic cells (DCs). This occurrence initiates a cascade of events, such as the recruitment of DCs to dLNs and their activation and maturation, and the enhancement of the ability of DCs to prime CD8+ T cells induced by AdPyCS and ultimately leading to a potent adjuvant effect and protection against malaria.     

Human innate responses and adjuvant activity of TLR ligands in vivo in mice reconstituted with a human immune system

TLR ligands (TLR-Ls) represent a class of novel vaccine adjuvants. However, their immunologic effects in humans remain poorly defined in vivo. Using a humanized mouse model with a functional human immune system, we investigated how different TLR-Ls stimulated human innate immune response in vivo and their applications as vaccine adjuvants for enhancing human cellular immune response. We found that splenocytes from humanized mice showed identical responses to various TLR-Ls as human PBMCs in vitro. To our surprise, various TLR-Ls stimulated human cytokines and chemokines differently in vivo compared to that in vitro. For example, CpG-A was most efficient to induce IFN-α production in vitro. In contrast, CpG-B, R848 and Poly I:C stimulated much more IFN-α than CpG-A in vivo. Importantly, the human innate immune response to specific TLR-Ls in humanized mice was different from that reported in C57BL/6 mice, but similar to that reported in nonhuman primates. Furthermore, we found that different TLR-Ls distinctively activated and mobilized human plasmacytoid dendritic cells (pDCs), myeloid DCs (mDCs) and monocytes in different organs. Finally, we showed that, as adjuvants, CpG-B, R848 and Poly I:C can all enhance antigen specific CD4⁺ T cell response, while only R848 and Poly I:C induced CD8⁺ cytotoxic T cells response to a CD40-targeting HIV vaccine in humanized mice, correlated with their ability to activate human mDCs but not pDCs. We conclude that humanized mice serve as a highly relevant model to evaluate and rank the human immunologic effects of novel adjuvants in vivo prior to testing in humans.     

Monitoring the efficacy of infant hepatitis B vaccination and revaccination in 0- to 8-year-old children: Protective anti-HBs levels and cellular immune responses

Vaccination against hepatitis B virus (HBV) is recommended worldwide. The aim of this study was to assess the efficacy of infant hepatitis B vaccination and revaccination in 0- to 8-year-old children in the context of protective anti-HBs levels and cellular immune responses. Using a random questionnaire survey, 1695 pre-school children were recruited as research subjects during January 2015 to June 2017. Blood samples were obtained to measure HBV serological markers as well as peripheral immunocytes. The children were divided into non-, low- and hyper- responsive groups (NR, LR, and HR) based on the vaccination efficacy. Additionally, the effect of revaccination on the NR group was evaluated at 1 month after completion of the vaccination course. Among a total of 1695 children, 1591 (93.86%) were infants who were followed while undergoing their primary course of hepatitis B vaccination at the 0-1-6 month schedule, and 1249 (79.30%) of them developed antibodies against HBsAg (anti-HBs) titers greater than 10 IU/L. The results of immunocyte studies indicated that the CD8⁺ T cells, CD4⁺CD45RO⁺ T cells, CD8⁺CD45RA⁺ T cells, and T follicular helper (Tfh) cells increased significantly in NR compared with HR. However, lymphocytes, CD4⁺ T cells, and CD4⁺CD45RA⁺ T cells in NR were lower than that in HR. 96 of the non-response cases showed seroprotection after revaccination among 103 cases. Therefore, most of the preschool children who received hepatitis B vaccine in infancy achieved significant seroprotection. Seroconversion rates of individuals revaccinated after initial vaccination failure were significantly higher than those after primary vaccination. Different vaccination efficacy groups showed significant changes in circulating immunocytes, which might be a factor affecting the recombinant HBV vaccine’s immune effectiveness.     

Enhanced immune responses to E2 protein and DNA formulated with ISA 61 VG administered as a DNA prime–protein boost regimen against bovine viral diarrhea virus

The aim of this study was to develop and test an optimal vaccination strategy against bovine viral diarrhea virus (BVDV) based on the E2 glycoprotein of the BJ1305 strain. To achieve higher E2-specific antibody titers and to broaden the cellular immune response, a plasmid encoding the E2 protein (pcDNA3.1-E2) was constructed and a purified recombinant E2 protein was generated. The E2 protein was emulsified in the adjuvant ISA 61 VG prior to administration. We immunized mice three times with pcDNA3.1-E2 or the recombinant E2 protein or primed twice with pcDNA3.1-E2 and boosted once with the E2 protein. To evaluate the protection against BVDV conferred by the vaccines, the mice were challenged with BVDV strain Oregon C24V after the third immunization. Although all immunized mice developed humoral and cellular immune responses, the E2-specific antibody titers in the DNA prime–protein boost group were significantly higher than those elicited by either the DNA or the protein vaccine. In addition, vaccination with the E2 DNA vaccine induced higher percentages of CD4⁺IFN-γ⁺ T cells and CD8⁺IFN-γ⁺ T cells among total CD3⁺ T cells than the other regimens. The predominant antibody subclass in the vaccinated mice was IgG1. Serum tumor necrosis factor alpha (TNF-α) levels in the DNA prime–protein boost group were significantly higher after the third immunization than in the other groups. Moreover, the mice treated with the DNA prime–protein boost vaccination regimen acquired protection against BVDV challenge, as shown by a significant reduction of viremia, only minor pathological changes, and a lower viral antigen burden than in the control and solo vaccinated mice. These results demonstrate the potential advantage of a DNA prime–protein boost vaccination approach over a solo vaccination for the prevention of BVDV. The ability of this vaccine strategy to control and eradicate BVD in herds warrants further investigation.     

Rescue of CD8+ T cell vaccine memory following sublethal γ irradiation

Sublethal γ irradiation eliminates CD8+ T cell mediated memory responses. In this work, we explored how these memory responses could be rescued in the aftermath of such exposure. We utilized two models of CD8+ T cell mediated immunity: a mouse model of Listeria monocytogenes (LM) infection in which CD8+ T cells specific for LM expressed antigens (Listeriolysin O, LLO) can be tracked, and a murine skin graft model in which CD8+ T cells mediate rejection across a MHC class I (D^d) disparity. In the LM immunized mice, LL0 specific CD8+ T memory cells were lost on irradiation, preserved with rapid revaccination with an attenuated strain 1–3 days post-irradiation (PI), and these mice survived a subsequent wild type LM challenge. A genetic “signature of rescue” identified a group of immune-associated mRNA maintained or upregulated following irradiation and rescue. A number of these factors, including IL-36γ, dectin-2 (Clec4n), and mir101c are upregulated rapidly after exposure of mice to sublethal γ radiation alone and are sustained by early, but not later rescue. Such factors will be evaluated as potential therapeutics to replace individual vaccines for global rescue of CD8+ T memory cell responses following sublethal γ irradiation. The skin allograft model mirrored that of the LM model in that the accelerated D^d skin allograft rejection response was lost in mice exposed to sublethal γ radiation, but infusion of allogeneic D^d expressing bone marrow cells 1–4 days PI preserved the CD8+ T memory mediated accelerated rejection response, further suggesting that innate immune responses may not always be essential to rescue of CD8+ memory T cells following γ irradiation.     

Vaccine Adjuvant Systems containing monophosphoryl lipid A and QS-21 induce strong humoral and cellular immune responses against hepatitis B surface antigen which persist for at least 4 years after vaccination

BackgroundRecombinant hepatitis B surface antigen (HBsAg) was used as a model antigen to evaluate persistence of cellular and humoral immune responses when formulated with three different Adjuvant Systems containing 3-O-desacyl-4′-monophosphoryl lipid A (MPL) and QS-21, in an oil-in-water emulsion (AS02_B and AS02_V), or with liposomes (AS01_B).MethodsThis is an open, 4-year follow-up of a previous randomised, double-blind study. Healthy subjects aged 18–40 years received three vaccine doses on a month 0, 1, 10 schedule and were initially followed for 18 months. A total of 93 subjects (AS02_B: n = 30; AS02_V: n = 28; AS01_B: n = 35) were enrolled in this follow-up and had an additional blood sample taken at Year 4 (NCT02153320). The primary endpoint was the frequency of HBsAg-specific CD4⁺ and CD8⁺ T-cells expressing cytokines upon short-term in vitro stimulation of peripheral blood mononuclear cells with HBsAg-derived peptides. Secondary endpoints were anti-HBs antibody titres and frequency of HBsAg-specific memory B-cells.ResultsA strong and persistent specific CD4⁺ T-cell response was observed at Year 4 in all groups. HBsAg-specific CD4⁺ T-cells expressed mainly CD40L and IL-2, and to a lesser extent TNF-α and IFN-γ. HBsAg-specific CD8⁺ T-cells were not detected in any group. A high, persistent HBsAg-specific humoral immune response was observed in all groups, with all subjects seroprotected (antibody titre ≥10 mIU/mL) at Year 4. The geometric mean antibody titre at Year 4 was above 100,000 mIU/mL in all groups. A strong memory B-cell response was observed post-dose 2, which tended to increase post-dose 3 and persisted at Year 4 in all groups.ConclusionThe MPL/QS-21/HBsAg vaccine formulations induced persistent immune responses up to 4 years after first vaccination. These Adjuvant Systems offer potential for combination with recombinant, synthetic or highly purified subunit vaccines, particularly for vaccination against challenging diseases, or in specific populations, although additional studies are needed.     

Vaccination of sheep with Quil-A® adjuvant expands the antibody repertoire to the Fasciola MF6p/FhHDM-1 antigen and administered together impair the growth and antigen release of flukes

Fasciolosis continues to be a major cause of economic losses in the livestock industry and a growing threat to humans. The limited spectrum of effective anthelmintics and the appearance of resistances urge the need for developing an effective vaccine. Most studies have been focused on the use of TH₁-polarizing adjuvants and the use of recombinant Fasciola critical molecules and, despite the efforts, no reproducible protections have been achieved. The F. hepatica MF6p/FhHDM-1 protein is a heme-binding protein also reported to have immunomodulatory properties, constituting a promising target for vaccination and/or as target for the development of new flukicides. Thus, in this study, we investigated the effects of the TH₁-polarizing adjuvant Quil A® on sheep immune response to MF6p/FhHDM-1, and the vaccine potential of both native and synthetic forms of this protein against ovine fasciolosis. Subcutaneous injection of Quil A® alone, i.e., without co-injecting any antigen, expands the antibody repertoire to MF6p/FhHDM-1 triggered by a subsequent primoinfection with metacercariae. This effect was not observed with aluminum hydroxide, the most frequently adjuvant used in commercial vaccines. On the other hand, vaccination with synthetic MF6p/FhHDM-1 in Quil A® prompted a 2–4-week delay in the antibody response induced in sheep by a challenge experimental infection. Moreover, fluke populations stablished showed stunted growth and low antigen release probably due to reduced metabolic activity. These observations suggest that primary circulating antibodies induced by the immunization had harmful effects on fluke development. Such effects could not be demonstrated to be associated to TH₁ immune response linked events (production of IgG₂ isotype antibodies and IFN-γ).     

Whole inactivated avian Influenza H9N2 viruses induce nasal submucosal dendritic cells to sample luminal viruses via transepithelial dendrites and trigger subsequent DC maturation

Nasal mucosal barrier is a key impediment for the absorption of influenza whole inactivated virus (WIV) intranasal vaccine. Yet it is still unclear how WIV cross the epithelial cells (ECs) in nasal cavity. Here, in vitro, a coculture system was well established, consisting of surrogate nasal ECs (Calu-3) and dendritic cells (DCs). After adding H9N2 WIV on the apical side of ECs, we found that submucosal DCs extended their transepithelial dendrites (TEDs) and sampled luminal viruses. However, ECs were not involved in the transepithelial transport of viruses. Subsequently, the phenotypic and functional maturation of DCs were also enhanced, whereas they were attenuated after blocking of TED formation by anti-JAM1 antibody. In vivo, we confirmed that H9N2 WIV were capable of inducing nasal submucosal DCs to sample luminal viruses via TEDs in the nasal passage but not nasal-associated lymphoid tissue (NALT). CD103⁺ and CD103⁻ DC subsets participated in this process. Of note, chemokine CCL20, released from the H9N2 WIV-induced ECs, played a vital role in DC recruitment and TED formation. Taken together, our findings indicated that TEDs played a critical role in facilitating viral transport across the epithelial barrier, which may guide the design of novel nasal mucosal vaccine strategies.     

Vaccination of pigs with attenuated Lawsonia intracellularis induced acute phase protein responses and primed cell-mediated immunity without reduction in bacterial shedding after challenge

BackgroundLawsonia intracellularis causes porcine proliferative enteropathy and is one of the most economically important diseases in modern pig production worldwide. The Enterisol^® Ileitis vaccine have been shown to reduce clinical disease and to increase weight gain, however, while the natural infection with L. intracellularis can provide complete protection against re-infection, this has not been achieved by this vaccine. We therefore undertook a detailed characterization of immune responses to L. intracellularis infection in vaccinated pigs (VAC) compared to previously infected pigs (RE) in order to pinpoint immunological determinants of protection.ResultsThe VAC pigs shed L. intracellularis to the same extent as non-vaccinated pigs after challenge, however less L. intracellularis in ileum and lymph nodes was seen post mortem. In the RE group, challenge did not lead to L. intracellularis shedding and no challenge bacteria were found post mortem. In both VAC and RE the acute phase haptoglobin response was diminished and L. intracellularis specific IgG responses were delayed and reduced compared to non-vaccinated pigs. On the other hand L. intracellularis specific IFN-γ responses tended to develop faster in the VAC group compared to controls.ConclusionAlthough vaccinated and non-vaccinated pigs shed L. intracellularis at similar levels after challenge, a lower number of intestinal L. intracellularis was observed in the vaccinated pigs at post mortem inspection. This might be due to the observed faster CMI responses upon challenge in vaccinated pigs. Complete protection against infection without L. intracellularis shedding, however, was only seen after a previous infection resulting in IFN-γ production predominantly by CD8⁺ and CD4⁺ CD8⁺ cells. Improved protective vaccines against L. intracellularis should therefore target stimulation of these T cell subsets.     

Polyfunctional CD4 T-cells correlate with in vitro mycobacterial growth inhibition following Mycobacterium bovis BCG-vaccination of infants

BackgroundVaccination with Bacillus Calmette Guerin (BCG) protects infants against childhood tuberculosis however the immune mechanisms involved are not well understood. Further elucidation of the infant immune response to BCG will aid with the identification of immune correlates of protection against tuberculosis and with the design of new improved vaccines. The purpose of this study was to investigate BCG-induced CD4+ T-cell responses in blood samples from infants for cytokine secretion profiles thought to be important for protection against tuberculosis and compare these to PBMC-mediated in vitro mycobacterial growth inhibition.MethodsBlood from BCG-vaccinated or unvaccinated infants was stimulated overnight with Mycobacterium tuberculosis (M. tb) purified protein derivative (PPD) or controls and intracellular cytokine staining and flow cytometry used to measure CD4+ T-cell responses. PBMC cryopreserved at the time of sample collection were thawed and incubated with live BCG for four days following which inhibition of BCG growth was determined.ResultsPPD-specific IFNγ+TNFα+IL-2+CD4+ T-cells represented the dominant T-cell response at 4 months and 1 year after infant BCG. These responses were undetectable in age-matched unvaccinated infants. IL-17+ CD4+ T-cells were significantly more frequent in vaccinated infants at 4 months but not at 1-year post-BCG. PBMC-mediated inhibition of mycobacterial growth was significantly enhanced at 4 months post-BCG as compared to unvaccinated controls. In an analysis of all samples with both datasets available, mycobacterial growth inhibition correlated significantly with the frequency of polyfunctional (IFNγ+TNFα+IL-2+) CD4+ T-cells.ConclusionsThese data suggest that BCG vaccination of infants induces specific polyfunctional T-helper-1 and T-helper-17 responses and the ability, in the PBMC compartment, to inhibit the growth of mycobacteria in vitro. We also demonstrate that polyfunctional T-helper-1 cells may play a role in growth inhibition as evidenced by a significant correlation between the two.     

Development of a self-assembling protein nanoparticle vaccine targeting Plasmodium falciparum Circumsporozoite Protein delivered in three Army Liposome Formulation adjuvants

We have developed FMP014, a vaccine candidate against Plasmodium falciparum malaria, which is comprised of 60 identical monomer protein chains that form an icosahedral shaped self-assembling protein nanoparticle (SAPN). Each monomer contains selected P. falciparum Circumsporozoite Protein (PfCSP) CD4+ and CD8+ epitopes, universal T_H epitopes, portions of the α-TSR domain, and 6 repeats of the NANP motifs of the PfCSP. Here we describe the conditions that are required for successful scale-up and cGMP manufacturing of FMP014 with a yield of ≈1.5 g of drug substance per 100 g of wet bacterial paste. When adjuvanted with an Army Liposomal Formulation (ALF) based adjuvant, the nanoparticle vaccine is highly immunogenic and prevents infection of mice by an otherwise lethal dose of transgenic P. berghei sporozoites expressing the full-length PfCSP.     

Strategies to enhance immunogenicity of cDNA vaccine encoded antigens by modulation of antigen processing

Most vaccines are based on protective humoral responses while for intracellular pathogens CD8⁺ T cells are regularly needed to provide protection. However, poor processing efficiency of antigens is often a limiting factor in CD8⁺ T cell priming, hampering vaccine efficacy. The multistage cDNA vaccine H56, encoding three secreted Mycobacterium tuberculosis antigens, was used to test a complete strategy to enhance vaccine’ immunogenicity. Potential CD8⁺ T cell epitopes in H56 were predicted using the NetMHC3.4/ANN program. Mice were immunized with H56 cDNA using dermal DNA tattoo immunization and epitope candidates were tested for recognition by responding CD8⁺ T cells in ex vivo assays. Seven novel CD8⁺ T cell epitopes were identified. H56 immunogenicity could be substantially enhanced by two strategies: (i) fusion of the H56 sequence to cDNA of proteins that modify intracellular antigen processing or provide CD4⁺ T cell help, (ii) by substitution of the epitope’s hydrophobic C-terminal flanking residues for polar glutamic acid, which facilitated their proteasome-mediated generation. We conclude that this whole strategy of in silico prediction of potential CD8⁺ T cell epitopes in novel antigens, followed by fusion to sequences with immunogenicity-enhancing properties or modification of epitope flanking sequences to improve proteasome-mediated processing, may be exploited to design novel vaccines against emerging or ‘hard to treat’ intracellular pathogens.     

Fibroblast-stimulating lipopeptide-1 as a potential mucosal adjuvant enhances mucosal and systemic immune responses to enterovirus 71 vaccine

To prevent viral infection at the site of entry, mucosal vaccines are potent tools for inducing IgA secretion for defense. Because Toll-like receptor (TLR) ligands serve as strong adjuvants, two ligands that mimic the structure of mycoplasmal and bacterial lipopeptides represent interesting vaccine candidates. Pam3CSK4, a synthetic triacylated lipopeptide, interacts with TLR2/1. Because fibroblast-stimulating lipopeptide-1 (FSL-1), a synthetic diacylated lipopeptide, is recognized by TLR2/6, we targeted the potential immuno-inducibility of Pam3CSK4 and FSL-1 as adjuvants of an enterovirus 71 (EV71) mucosal vaccine. Naïve BALB/c mice were used for intranasal immunization three times over a 3-week interval, with results showing that EV71-specific IgG and IgA in serum, nasal washes, bronchoalveolar lavage fluid, and feces from the EV71 + FSL-1 group were significantly higher than levels observed in mice treated with EV71 + Pam3CSK4, EV71 alone, or the control group treated with phosphate-buffered saline. Furthermore, we observed more EV71-specific IgG and IgA-producing cells in treatments using EV71 formulated with FSL-1. Additionally, T cell-proliferative responses and interferon-γ and interleukin-17 secretion were significantly increased when inactivated EV71 was formulated using FSL-1. Moreover, serum from immunized mice was capable of neutralizing the infectivity of EV71 (C2 genotype) and was able to cross-neutralize the B4 and B5 genotypes of EV71. Our data suggested that FSL-1 could be used as an efficient adjuvant for intranasal EV71-vaccine immunization.     

An accelerated rabies vaccine schedule based on toll-like receptor 3 (TLR3) agonist PIKA adjuvant augments rabies virus specific antibody and T cell response in healthy adult volunteers

BackgroundRabies is a fatal disease where post-exposure prophylaxis (PEP) is crucial in preventing infection. However, deaths even after appropriate PEP, have been reported. The PIKA Rabies vaccine adjuvant is a TLR3 agonist that activates B and T cells leading to a robust immune response.MethodsWe conducted a phase I, open label, randomized study in healthy adults to assess the safety and immunogenicity of the PIKA Rabies vaccine and an accelerated vaccine regimen. Thirty-seven subjects were randomized into 3 groups: control vaccine classic regimen, PIKA vaccine classic regimen and PIKA vaccine accelerated regimen. Subjects were followed up for safety, rabies virus neutralizing antibodies (RVNA) and T cell responses.ResultsBoth the control and PIKA Rabies vaccine were well tolerated. All adverse events (AEs) were mild and self-limiting. Seventy-five percent of subjects in the PIKA accelerated regimen achieved a RVNA titer ⩾0.5 IU/mL on day 7, compared to 53.9% in the PIKA classic regimen (p = 0.411) and 16.7% in control vaccine classic regimen (p = 0.012). The PIKA rabies vaccine elicited multi-specific rabies CD4 mediated T cell response already detectable ex vivo at day 7 after vaccination and that was maintained at day 42.ConclusionThe investigational PIKA rabies vaccine was well tolerated and more immunogenic than the commercially available vaccine in healthy adults.Clinical trial registry: The study was registered with clinicaltrials.gov NCT02657161.     

Co-delivery of a CD4 T cell helper epitope via covalent liposome attachment with a surface-arrayed B cell target antigen fosters higher affinity antibody responses

Liposomal vaccines incorporating adjuvant and CD4 T cell helper peptides enhance antibody responses against weakly immunogenic B cell epitopes such as found in the membrane proximal external region (MPER) of the HIV-1 gp41 subunit. While the inclusion of exogenous helper peptides in vaccine formulations facilitates stronger and more durable antibody responses, the helper peptide incorporation strategy per se may influence the overall magnitude and quality of B cell target antigen immunogenicity. Both variability in individual peptide encapsulation as well as the potential for liposome surface-associated helper peptides to misdirect the humoral response are potential parameters impacting outcome. In this study, we used MPER/liposome vaccines as a model system to examine how the mode of the potent LACK T helper peptide formulation modulates antibody responses against the MPER antigen. We directly compared liposome surface-arrayed palmitoyl LACK (pLACK) versus soluble LACK (sLACK) encapsulated in the liposomes and free in solution. Independent of LACK formulation methods, dendritic cell activation and LACK presentation were equivalent in vivo. The frequency of MPER-specific GC B cells promoted by sLACK was higher than that stimulated by pLACK formulation, a finding associated with a significantly greater frequency of LACK-specific GC B cells induced by pLACK. While there were no significant differences in the quantity of MPER-specific serological responses, the MPER-specific antibody titer trended higher with sLACK formulated vaccines at the lower dose of LACK. However, pLACK generated relatively greater MPER-specific antibody affinities than those induced by sLACK-formulated vaccines. Overall, the results suggest that liposomal surface-associated LACK enhances immunogenicity of LACK through better engagement of LACK-specific B cells. Of note, this is not detrimental to the induction of MPER-specific immune responses; rather, the elicitation of higher affinity anti-MPER antibodies benefits from augmented help delivered via covalent linkage of the pLACK CD4 T cell epitope in conjunction with MPER/liposome presentation.