Recovery of antigen-specific T cell responses from dogs infected with Leishmania (L.) infantum by use of vaccine associated TLR-agonist adjuvant

Visceral leishmaniasis (VL), caused by infection with the obligate intracellular protozoan parasite Leishmania infantum, is a fatal disease of dogs and humans. Protection against VL requires a T helper 1 (Th1) skewed CD4⁺ T response, but despite this knowledge, there are currently no approved-to-market vaccines for humans and only three veterinary-use vaccines globally. As VL progresses from asymptomatic to symptomatic, L. infantum–specific interferon gamma (IFNγ) driven-Th1 responses become dampened and a state of immune exhaustion established. T cell exhaustion and other immunoregulatory processes, starting during asymptomatic disease, are likely to hinder vaccine-induced responses if vaccine is administered to infected, but asymptomatic and seronegative, individuals. In this study we evaluated how immune exhaustion, shown previously by our group to worsen in concert with VL progression, effected the capacity of vaccine candidate antigen/toll-like receptor (TLR) agonist combinations to promote protective CD4⁺ T cell responses during progressive VL. In conjunction with Th1 responses, we also evaluated concomitant stimulation of immune-balanced IL-10 regulatory cytokine production by these vaccine products in progressive VL canine T cells. Vaccine antigen L111f in combination with TLR agonists significantly recovered CD4⁺ T cell IFNγ intracellular production in T cells from asymptomatic VL dogs. Vaccine antigen NS with TLR agonists significantly recovered CD4⁺ T cell production in both endemic control and VL dogs. Combinations of TLR agonists and vaccine antigens overcame L. infantum induced cellular exhaustion, allowing robust Th1 CD4⁺ T cell responses from symptomatic dogs that previously had dampened responses to antigen alone. Antigen-agonist adjuvants can be utilized to promote more robust vaccine responses from infected hosts in endemic areas where vaccination of asymptomatic, L. infantum-infected animals is likely.     

Cryopreservation-related loss of antigen-specific IFNγ producing CD4+ T-cells can skew immunogenicity data in vaccine trials: Lessons from a malaria vaccine trial substudy

Ex vivo functional immunoassays such as ELISpot and intracellular cytokine staining (ICS) by flow cytometry are crucial tools in vaccine development both in the identification of novel immunogenic targets and in the immunological assessment of samples from clinical trials. Cryopreservation and subsequent thawing of PBMCs via validated processes has become a mainstay of clinical trials due to processing restrictions inherent in the disparate location and capacity of trial centres, and also in the need to standardize biological assays at central testing facilities. Logistical and financial requirement to batch process samples from multiple study timepoints are also key. We used ELISpot and ICS assays to assess antigen-specific immunogenicity in blood samples taken from subjects enrolled in a phase II malaria heterologous prime-boost vaccine trial and showed that the freeze thaw process can result in a 3–5-fold reduction of malaria antigen-specific IFNγ-producing CD3⁺CD4⁺ effector populations from PBMC samples taken post vaccination. We have also demonstrated that peptide responsive CD8⁺ T cells are relatively unaffected, as well as CD4⁺ T cell populations that do not produce IFNγ. These findings contribute to a growing body of data that could be consolidated and synthesised as guidelines for clinical trials with the aim of increasing the efficiency of vaccine development pipelines.     

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.     

A modified epitope identified for generation and monitoring of PSA-specific T cells in patients on early phases of PSA-based immunotherapeutic protocols

Efficacy of vaccination in cancer patients on immunotherapeutic protocols can be difficult to evaluate. The aim of this study was therefore to identify a single natural or modified epitope in prostate-specific antigen (PSA) with the ability to generate high levels of PSA-specific T cells to facilitate monitoring in patients after vaccination against prostate cancer. To the best of our knowledge, this study describes for the first time the peptide specificity of T cells stimulated by endogenously processed PSA antigen. The peptide specificity of HLA-A*0201-restricted CD8⁺ T cells against human and rhesus PSA was investigated both in vivo after DNA vaccination in HLA-A*0201-transgenic mice and in vitro after repetitive stimulation of human T cells with DNA-transfected human dendritic cells (DCs). One of seven native PSA peptides, psa53–61, was able to activate high levels of PSA-specific CD8⁺ T cells in HLA-A*0201-transgenic mice after PSA DNA vaccination. Psa53–61 was also the only peptide that induced human T cells to produce IFNγ after stimulation with PSA transfected DCs, however not in all donors. Therefore, plasmids encoding modified epitopes in predicted HLA-A*0201 sequences were constructed. One of these modified PSA plasmids consistently induced IFNγ producing CD8⁺ T cells to the corresponding modified peptide as well as to the corresponding native peptide, in all murine and human T cell cultures. This study demonstrates a novel concept of introducing a modified epitope within a self-tumor antigen, with the purpose of eliciting a reliable T cell response from the non-tolerized immune repertoire, to facilitate monitoring of vaccine efficacy in cancer patients on immunotherapeutic protocols. The purpose of such a modified epitope is thus not to induce therapeutically relevant T cells but rather to, in case of weak or divergent T cell responses to self antigens/peptides, help answer questions about efficacy of vaccine delivery and about the possibility to induce immune responses in the selected and often immunosuppressed cancer patients.     

A novel lipid nanoparticle adjuvant significantly enhances B cell and T cell responses to sub-unit vaccine antigens

Sub-unit vaccines are primarily designed to include antigens required to elicit protective immune responses and to be safer than whole-inactivated or live-attenuated vaccines. But their purity and inability to self-adjuvant often result in weaker immunogenicity. Emerging evidence suggests that bio-engineered nanoparticles can be used as immunomodulatory adjuvants. Therefore, in this study we explored the potential of novel Merck-proprietary lipid nanoparticle (LNP) formulations to enhance immune responses to sub-unit viral antigens. Immunization of BALB/c and C57BL/6 mice revealed that LNPs alone or in combination with a synthetic TLR9 agonist, immune-modulatory oligonucleotides, IMO-2125 (IMO), significantly enhanced immune responses to hepatitis B virus surface antigen (HBsAg) and ovalbumin (OVA). LNPs enhanced total B-cell responses to both antigens tested, to levels comparable to known vaccine adjuvants including aluminum based adjuvant, IMO alone and a TLR4 agonist, 3-O-deactytaled monophosphoryl lipid A (MPL). Investigation of the quality of B-cell responses demonstrated that the combination of LNP with IMO agonist elicited a stronger Th1-type response (based on the IgG2a:IgG1 ratio) than levels achieved with IMO alone. Furthermore, the LNP adjuvant significantly enhanced antigen specific cell-mediated immune responses. In ELISPOT assays, depletion of specific subsets of T cells revealed that the LNPs elicited potent antigen-specific CD4⁺ and CD8⁺T cell responses. Intracellular FACS analyses revealed that LNP and LNP + IMO formulated antigens led to higher frequency of antigen-specific IFNγ⁺TNFα⁺IL-2⁺, multi-functional CD8⁺T cell responses, than unadjuvanted vaccine or vaccine with IMO only. Overall, our results demonstrate that lipid nanoparticles can serve as future sub-unit vaccine adjuvants to boost both B-cell and T-cell responses in vivo, and that addition of IMO can be used to manipulate the quality of immune responses.     

Different T cell memory in preadolescents after whole-cell or acellular pertussis vaccination

To better understand vaccine-induced protection and its potential failure in light of recent whooping cough resurgence, we evaluated quantity as well as quality of memory T cell responses in B. pertussis-vaccinated preadolescent children. Using a technique based on flow cytometry to detect proliferation, cytokine production and phenotype of antigen-specific cells, we evaluated residual T cell memory in a cohort of preadolescents who received a whole-cell pertussis (wP; n = 11) or an acellular pertussis vaccine (aP; n = 13) during infancy, and with a median of 4 years elapsed from the last pertussis booster vaccine, which was aP for all children. We demonstrated that B. pertussis-specific memory T cells are detectable in the majority of preadolescent children several years after vaccination. CD4⁺ and CD8⁺ T cell proliferation in response to pertussis toxin and/or filamentous hemagglutinin was detected in 79% and 60% of the children respectively, and interferon-γ or tumor necrosis factor-α producing CD4⁺ T cells were detected in 65% and 53% of the children respectively. Phenotyping of the responding cells showed that the majority of antigen-specific cells, whether defined by proliferation or cytokine production, were CD45RA⁻CCR7⁻ effector memory T cells. Although the time since the last booster vaccine was significantly longer for wP-compared to aP-vaccinated children, their proliferation capacity in response to antigenic stimulation was comparable, and more children had a detectable cytokine response after wP- compared to aP-vaccination. This study supports at the immunological level recent epidemiological studies indicating that infant vaccination with wP induces longer lasting immunity than vaccination with aP-vaccines.     

Recombinant rubella vectors elicit SIV Gag-specific T cell responses with cytotoxic potential in rhesus macaques

Live-attenuated rubella vaccine strain RA27/3 has been demonstrated to be safe and immunogenic in millions of children. The vaccine strain was used to insert SIV gag sequences and the resulting rubella vectors were tested in rhesus macaques alone and together with SIV gag DNA in different vaccine prime-boost combinations. We previously reported that such rubella vectors induce robust and durable SIV-specific humoral immune responses in macaques. Here, we report that recombinant rubella vectors elicit robust de novo SIV-specific cellular immune responses detectable for >10 months even after a single vaccination. The antigen-specific responses induced by the rubella vector include central and effector memory CD4⁺ and CD8⁺ T cells with cytotoxic potential. Rubella vectors can be administered repeatedly even after vaccination with the rubella vaccine strain RA27/3. Vaccine regimens including rubella vector and SIV gag DNA in different prime-boost combinations resulted in robust long-lasting cellular responses with significant increase of cellular responses upon boost. Rubella vectors provide a potent platform for inducing HIV-specific immunity that can be combined with DNA in a prime-boost regimen to elicit durable cellular immunity.     

Vaccination with a piggyBac plasmid with transgene integration potential leads to sustained antigen expression and CD8 T cell responses

DNA vaccination with plasmid has conventionally involved vectors designed for transient expression of antigens in injected tissues. Next generation plasmids are being developed for site-directed integration of transgenes into safe sites in host genomes and may provide an innovative approach for stable and sustained expression of antigens for vaccination. The goal of this study was to evaluate in vivo antigen expression and the generation of cell mediated immunity in mice injected with a non-integrating plasmid compared to a plasmid with integrating potential. Hyperactive piggyBac transposase-based integrating vectors (pmhyGENIE-3) contained a transgene encoding either eGFP (pmhyGENIE-3-eGFP) or luciferase (pmhyGENIE-3-GL3), and were compared to transposase-deficient plasmids with the same transgene and DNA backbone. Both non-integrating and integrating plasmids were equivalent at day 1 for protein expression at the site of injection. While protein expression from the non-integrating plasmid was lost by day 14, the pmhyGENIE-3 was found to exhibit sustained protein expression up to 28 days post-injection. Vaccination with pmhyGENIE-3-eGFP resulted in a robust CD8⁺ T cell response that was three-fold higher than that of non-integrating plasmid vaccinations. Additionally we observed in splenocyte restimulation experiments that only the vaccination with pmhyGENIE-3-eGFP was characterized by IFNγ producing CD8⁺ T cells. Overall, these findings suggest that plasmids designed to direct integration of transgenes into the host genome are a promising approach for designing DNA vaccines. Robust cell mediated CD8⁺ T cell responses generated using integrating plasmids may provide effective, sustained protection against intracellular pathogens or tumor antigens.     

Effect of a combination DNA vaccine for the prevention and therapy of Trypanosoma cruzi infection in mice: Role of CD4 and CD8 T cells

Chagas disease is a major public health problem, with about 10 million infected people, and DNA vaccines are a promising alternative for the control of Trypanosoma cruzi, the causing agent of the disease. We tested here a new DNA vaccine encoding a combination of two leading parasite antigens, TSA-1 and Tc24, for the prevention and therapy of T. cruzi infection. Immunized Balb/c mice challenged by T. cruzi presented a significantly lower parasitemia and inflammatory cell density in the heart compared to control mice. Similarly, the therapeutic administration of the DNA vaccine was able to significantly reduce the parasitemia and inflammatory reaction in acutely infected Balb/c and C57BL/6 mice, and reduced cardiac tissue inflammation in chronically infected ICR mice. Therapeutic vaccination induced a marked increase in parasite-specific IFNγ producing CD4⁺ and CD8⁺ T cells in the spleen as well as an increase in CD4⁺ and CD8⁺ T cells in the infected cardiac tissue. In addition, some effect of the DNA vaccine could still be observed in CD4-knockout C57BL/6 mice, which presented a lower parasitemia and inflammatory cell density, but not in CD8-deficient mice, in which the vaccine had no effect. These results indicate that the activation of CD8⁺ T cells plays a major role in the control of the infection by the therapeutic DNA vaccine, and to a somewhat lesser extent CD4⁺ T cells. This observation opens interesting perspectives for the potentiation of this DNA vaccine candidate by including additional CD8⁺ T cell antigens/epitopes in future vaccine formulations.     

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.     

Differential effects of IL-15 on the generation,maintenance and cytotoxic potential of adaptive cellular responses induced by DNA vaccination

IL-15 is an important cytokine for the regulation of lymphocyte homeostasis. However, the role of IL-15 in the generation, maintenance and cytotoxic potential of antigen specific T cells is not fully understood. Because the route of antigenic delivery and the vaccine modality could influence the IL-15 requirement for mounting and preserving cytotoxic T cell responses, we have investigated the immunogenicity of DNA-based vaccines in IL-15 KO mice. DNA vaccination with SIV Gag induced antigen-specific CD4⁺ and CD8⁺ T cells in the absence of IL-15. However, the absolute number of antigen-specific CD8⁺ T cells was decreased in IL-15 KO mice compared to WT animals, suggesting that IL-15 is important for the generation of maximal number of antigen-specific CD8⁺ T cells. Interestingly, antigen-specific memory CD8 cells could be efficiently boosted 8 months after the final vaccination in both WT and KO strains of mice, suggesting that the maintenance of antigen-specific long-term memory T cells induced by DNA vaccination is comparable in the absence and presence of IL-15. Importantly, boosting by DNA 8-months after vaccination revealed severely reduced granzyme B content in CD8⁺ T cells of IL-15 KO mice compared to WT mice. This suggests that the cytotoxic potential of the long-term memory CD8⁺ T cells is impaired. These results suggest that IL-15 is not essential for the generation and maintenance of adaptive cellular responses upon DNA vaccination, but it is critical for the preservation of maximal numbers and for the activity of cytotoxic CD8⁺ T cells.     

Antigenic peptide nanofibers elicit adjuvant-free CD8 T cell responses

Vaccines that elicit robust CD8⁺ T cell responses are desirable for protection against infectious diseases and cancers. However, most vaccine adjuvants fail to elicit robust CD8⁺ T cell responses without inflammation and associated toxicity. We recently reported that self-assembling peptides that form nanofibers in physiological buffers elicited strong adjuvant-free and antigen-specific antibody responses in mice. However, whether or not such nanofibers likewise can elicit strong CD8⁺ T cell responses is unknown. Here, we demonstrate that the self-assembling peptide Q11 conjugated to a CD8⁺ T cell epitope of ovalbumin (Q11-OVA), elicits strong antigen-specific primary and recall responses, and in a vaccination regimen protects against subsequent infection. Importantly, we show that these antigenic peptide nanofibers do not persist as an inflammatory antigen depot at the injection site. Our results demonstrate for the first time that self-assembling peptides may be useful as carriers for vaccines where CD8⁺ T cell-mediated protection is needed.     

Antigen encoded by vaccine vectors derived from human adenovirus serotype 5 is preferentially presented to CD8+ T lymphocytes by the CD8α+ dendritic cell subset

Different subsets of dendritic cells (DC) elicit qualitatively different immune responses. In mice, two lymphoid tissue-resident subsets, CD8α⁺ and CD8α⁻, have been implicated in the induction of T helper 1 (Th1) or Th2 responses, respectively. Moreover, CD8α⁺ DC appear to play a major role in priming CD8⁺ T lymphocyte responses to viral antigens in the course of diverse viral infections. These considerations have been less extensively explored for vaccine vectors derived from viruses. Despite inefficient ex vivo transduction of DC, vectored vaccines derived from human adenoviruses of serotype 5 (Ad5) elicit robust immune responses, predominantly of the Th1 orientation, in humans and mice. At present it is unknown whether Ad5 interacts with DC subsets in a differential manner, thereby influencing the quality of the elicited IR. To address this issue, successive steps (attachment, transgene expression, MHC class I antigen presentation and activation of antigen-specific T lymphocytes) involved in induction of immune responses by Ad5-based vectors have been examined in CD8α⁺ and CD8α⁻ murine DC subsets. Although in both ex vivo and in vivo experiments CD8α⁺ and CD8α⁻ DC subsets captured an Ad5-based vector to a similar extent, transgene expression and subsequent MHC class I display of a transgene-encoded antigen were more efficient in CD8α⁺ DC. Moreover, following in vivo and ex vivo transduction with an Ad5-based vaccine, antigen-specific CD8⁺ T lymphocytes were more efficiently activated by CD8α⁺ DC than by CD8α⁻ DC. Thus, superior antigen expression and MHC class I display in CD8α⁺ DC may contribute to preferred priming of antigen-specific CD8⁺ lymphocytes by Ad5-transduced CD8α⁺ DC.     

First-in-human trial of the post-exposure tuberculosis vaccine H56:IC31 in Mycobacterium tuberculosis infected and non-infected healthy adults

BackgroundH56:IC31 is a candidate tuberculosis vaccine comprising a fusion protein of Ag85B, ESAT-6 and Rv2660c, formulated in IC31 adjuvant. This first-in-human, open label phase I trial assessed the safety and immunogenicity of H56:IC31 in healthy adults without or with Mycobacterium tuberculosis (M.tb) infection.MethodsLow dose (15 μg H56 protein in 500 nmol IC31) or high dose (50 μg H56, 500 nmol IC31) vaccine was administered intramuscularly thrice, at 56-day intervals. Antigen-specific T cell responses were measured by intracellular cytokine staining and antibody responses by ELISA.ResultsOne hundred and twenty-six subjects were screened and 25 enrolled and vaccinated. No serious adverse events were reported. Nine subjects (36%) presented with transient cardiovascular adverse events. The H56:IC31 vaccine induced antigen-specific IgG responses and Th1 cytokine-expressing CD4⁺ T cells. M.tb-infected vaccinees had higher frequencies of H56-induced CD4⁺ T cells than uninfected vaccinees. Low dose vaccination induced more polyfunctional (IFN-γ⁺TNF-α⁺IL-2⁺) and higher frequencies of H56-specific CD4⁺ T cells compared with high dose vaccination. A striking increase in IFN-γ-only-expressing CD4⁺ T cells, displaying a CD45RA⁻CCR7⁻ effector memory phenotype, emerged after the second high-dose vaccination in M.tb-infected vaccinees. TNF-α⁺IL-2⁺ H56-specific memory CD4⁺ T cells were detected mostly after low-dose H56 vaccination in M.tb-infected vaccinees, and predominantly expressed a CD45RA⁻CCR7⁺ central memory phenotype. Our results support further clinical testing of H56:IC31.     

Low-dose influenza vaccine Grippol Quadrivalent with adjuvant Polyoxidonium induces a T helper-2 mediated humoral immune response and increases NK cell activity

The influenza vaccine Grippol® Quadrivalent (GQ) is a new vaccine, containing the adjuvant Polyoxidonium® and recombinant hemagglutinins from 4 strains of the influenza virus in amount of 5–6 μg of each hemagglutinin per human dose. These doses of antigens are about 3 times less than the standard dose recommended by WHO. We sought to characterize the immune response to the GQ vaccine and to determine the contribution of the adjuvant in this response. BALB/c mice were vaccinated with GQ or with adjuvant-free antigen mixtures (AGs). Then, the antibody response, the number of memory T cells in the spleen, and the functional properties of splenocytes were determined. The vaccine GQ has been shown to induce antibodies to all 4 influenza hemagglutinins. The vaccination with GQ caused a strong increase in the AG-induced proliferation and production of Th2 cytokines ex vivo. These effects were equal to effect achieved by standard dose of antigens. Vaccination also caused the accumulation of CD4⁺ large lymphocytes with the phenotype of central and effector memory T cells in the spleen. The GQ vaccine enhanced the cytolytic activity of natural killer (NK) cells, whereas the adjuvant-free mixture of AGs in lowered and standard doses did not affect NK activity. We did not find a noticeable response of Th1 and CD8⁺ T cells to vaccination. In vitro, the GQ vaccine stimulated the maturation of human monocyte-derived dendritic cells (DCs) enhancing the expression of HLA-DR, CD80, CD83, CD86 and ICOSL molecules. Polyoxidonium without AGs also induced expression of ICOSL, which plays an important role in T-dependent humoral immune response. In summary, the low-dose influenza vaccine GQ with Polyoxidonium adjuvant is immunogenic, induces a Th2-polarized T-cell response and CD4⁺ memory T cells maturation, activates the production of antibodies to influenza hemagglutinins, and increases the activity of NK cells.     

Phenotype of the anti-Rickettsia CD8 T cell response suggests cellular correlates of protection for the assessment of novel antigens

The obligately intracellular bacteria Rickettsia infect endothelial cells and cause systemic febrile diseases that are potentially lethal. No vaccines are currently available and current knowledge of the effective immune response is limited. Natural and experimental rickettsial infections provide strong and cross-protective cellular immunity if the infected individual survives the acute infection. Although resistance to rickettsial infections is attributed to the induction of antigen-specific T cells, particularly CD8⁺ T cells, the identification and validation of correlates of protective cellular immunity against rickettsial infections, an important step toward vaccine validation, remains a gap in this field. Here, we show that after a primary challenge with Rickettsia typhi in the C3H mouse model, the peak of anti-Rickettsia CD8⁺ T cell-mediated responses occurs 7 days post-infection (dpi), which coincides with the beginning of rickettsial clearance. At this time point, both effector-type and memory-type CD8⁺ T cells are present, suggesting that 7 dpi is a valid time point for the assessment of CD8⁺ T cell responses of mice previously immunized with protective antigens. Based on our results, we suggest four correlates of cellular protection for the assessment of protective rickettsial antigens: (1) production of IFN-γ by antigen-experienced CD3⁺CD8⁺CD44^high cells, (2) production of Granzyme B by CD27^lowCD43^low antigen-experienced CD8⁺ T cells, (3) generation of memory-type CD8⁺ T cells [Memory Precursor Effector Cells (MPECs), as well as CD127^highCD43^low, and CD27^highCD43^low CD8⁺ T cells], and (4) generation of effector-like memory CD8⁺ T cells (CD27^lowCD43^low). We propose that these correlates could be useful for the general assessment of the quality of the CD8⁺ T cell immune response induced by novel antigens with potential use in a vaccine against Rickettsia.     

Fingolimod can act as a facilitator to establish the primary T-cell response with reduced need of adjuvants

The CD8⁺ T-cell response is an essential part of the adaptive immunity. Adjuvants are routinely required for priming of T cells against antigens encountered in lymph nodes (LNs) to generate antigen-specific immunity but may concomitantly trigger unexpected inflammatory responses. Sphingosine-1-phosphate (S1P) induces transient desensitization of S1P receptors on LN T cells and temporarily blocks their egress, leading to prolonged intranodal retention that allows effective immunosurveillance and increases the chance of priming. In light of the regulatory role of S1P in T-cell migration, we here develop a strategic approach to the T-cell priming with protein vaccine containing low-dose TLR-based adjuvants (LDAV) to induce antigen-specific CD8⁺ T cell responses as efficiently as using regular dose adjuvants in vaccine (RDAV). We found that when combined with one low dose of the S1P analog fingolimod administered into the same vaccination site posteriorly at a specific time, LDAV can elicit a primary response that reaches the level of that induced by RDAV with respect to the response magnitude and functionality. Time-course studies indicate that LDAV and fingolimod in combination act to mimic the expansion kinetics of RDAV-primed antigen-specific CD8⁺ T cells. Further, intranodal accumulation of cDC1 is markedly enhanced in mice receiving the combination vaccination despite the decrease in adjuvant use. Of particular note is the marginal cutaneous inflammation at the injection site, indicating an added benefit of using fingolimod. Therefore, fingolimod as a nonadjuvant agent essentially facilitates antigen-specific T-cell priming with reduced need of adjuvants and minimized adverse reactions.     

Enhancement of humoral and cellular responses to HBsAg DNA vaccination by immunization with praziquantel through inhibition TGF-β/Smad2,3 signaling

Praziquantel (PZQ), which is used to treat all forms of schistosomiasis, has been shown to induce strong T cell activities and decrease T regulatory cell levels. In our study, we investigated whether PZQ may be used as an adjuvant for a hepatitis B surface antigen (HBsAg) DNA vaccine (pcD-S2) in eliciting strong humoral and cellular responses. Our data demonstrate that PZQ as an adjuvant increased T cell proliferation and an HBsAg-specific antibody response that was characterized by a higher ratio of IgG2a/IgG1. Moreover, a higher level of IFN-γ in CD4⁺ and CD8⁺ T cells were elicited. In addition, a significantly antigen-specific cytotoxic T lymphocyte response was also observed. The expression of TGF-β can be induced by HBsAg, while PZQ as an adjuvant can inhibit the expression of TGF-β and TGF-β/Smad2,3 signaling. The frequency of CD4⁺CD25⁺Foxp3⁺ Treg cells was reduced. Importantly, the regulatory function of CD4⁺CD25⁺ Treg cells was correspondingly impaired. Together, these results suggest that PZQ can enhance humoral and cellular responses to HBsAg DNA vaccination through inhibition TGF-β/Smad2,3 signaling.     

Circulating regulatory T cells (CD4CD25FOXP3) decrease in breast cancer patients after vaccination with a modified MHC class II HER2/neu (AE37) peptide

Regulatory T cells (T_Reg), CD4⁺CD25⁺FOXP3⁺, are implicated in suppressing tumor immune responses. We analyzed peripheral blood lymphocytes (PBL) from breast cancer patients receiving a modified HLA class II HER2/neu peptide (AE37) vaccine for T_Reg cells and correlated their levels with vaccine-specific immune responses. The mean CD4⁺CD25⁺FOXP3⁺ T_Reg cells decreased in patients with vaccination with no significant difference in serum TGF-β levels. IFN-γ ELISPOT and DTH increased after vaccination with a good correlation between T_Reg cell reduction and size of DTH to AE37. The T_Reg cell reduction and associated immune response suggest that AE37 may be clinically useful.