Antigen-specific T-cell Immune Responses Research Articles

Orchestrated codelivery of peptide antigen and adjuvant to antigen-presenting cells by using an engineered chimeric peptide enhances antitumor T-cell immunity.

The intrinsic pharmacokinetic limitations of traditional peptide-based cancer vaccines hamper effective cross-presentation and codelivery of antigens and adjuvants, which are crucial for inducing robust antitumor CD8+ T-cell responses. Here, we report the development of a versatile strategy that simultaneously addresses the different pharmacokinetic challenges of soluble subunit vaccines composed of antigens and CpG to modulate vaccine efficacy via translating an engineered chimeric peptide, eTAT, as an intramolecular adjuvant. Linking antigens to eTAT enhanced cytosolic delivery of the antigens. This, in turn, led to improved activation and lymph node-trafficking of antigen-presenting cells and antigen cross-presentation, thus promoting antigen-specific T-cell immune responses. Simple mixing of eTAT-linked antigens and CpG significantly enhanced codelivery of antigens and CpG to the antigen-presenting cells, and this substantially augmented the adjuvant effect of CpG, maximized vaccine immunogenicity and elicited robust and durable CD8+ T-cell responses. Vaccination with this formulation altered the tumor microenvironment and exhibited potent antitumor effects, with generally further enhanced therapeutic efficacy when used in combination with anti-PD1. Altogether, the engineered chimeric peptide-based orchestrated codelivery of antigen and adjuvant may serve as a promising but simple strategy to improve the efficacy of peptide-based cancer vaccines.

Developmental self-reactivity determines pathogenic Tc17 differentiation potential of naive CD8+ T cells in murine models of inflammation

The differentiation of naive CD8+ T cells into effector cells is important for establishing immunity. However, the effect of heterogeneous naive CD8+ T cell populations is not fully understood. Here, we demonstrate that steady-state naive CD8+ T cells are composed of functionally heterogeneous subpopulations that differ in their ability to differentiate into type 17 cytotoxic effector cells (Tc17) in a context of murine inflammatory disease models, such as inflammatory bowel disease and graft-versus-host disease. The differential ability of Tc17 differentiation is not related to T-cell receptor (TCR) diversity and antigen specificity but is inversely correlated with self-reactivity acquired during development. Mechanistically, this phenomenon is linked to differential levels of intrinsic TCR sensitivity and basal Suppressor of Mothers Against Decapentaplegic 3 (SMAD3) expression, generating a wide spectrum of Tc17 differentiation potential within naive CD8+ T cell populations. These findings suggest that developmental self-reactivity can determine the fate of naive CD8+ T cells to generate functionally distinct effector populations and achieve immense diversity and complexity in antigen-specific T-cell immune responses.

Cytomegalovirus antigen-specific T cell immune responses in patients with autoimmune diseases under different cytomegalovirus infection status.

Cytomegalovirus antigen-specific T cell immune responses in patients with autoimmune diseases under different cytomegalovirus infection status.

Distinct tumour antigen-specific T-cell immune response profiles at different hepatocellular carcinoma stages

BackgroundCancer-testis antigens (CTAs) and tumour-associated antigens (TAAs) are frequently expressed in hepatocellular carcinoma (HCC); however, the role of tumour-antigen-specific T cell immunity in HCC progression is poorly defined. We characterized CTA- and TAA-specific T cell responses in different HCC stages and investigated their alterations during HCC progression.MethodsFifty-eight HCC patients, 15 liver cirrhosis patients, 15 chronic hepatitis B patients and 10 heathy controls were enrolled in total. IFN-γ ELSPOT using CTAs, including MAGE-A1, MAGE-A3, NY-ESO-1, and SSX2, and two TAAs, SALL4 and AFP, was performed to characterize the T-cell immune response in the enrolled individuals. The functional phenotype of T cells and the responsive T cell populations were analyzed using short-term T-cell culture.ResultsT cell responses against CTAs and TAAs were specific to HCC. In early-stage HCC patients, the SALL4-specific response was the strongest, followed by MAGE-A3, NY-ESO-1, MAGE-A1 and SSX2. One-year recurrence-free survival after transcatheter arterial chemoembolization plus radiofrequency ablation treatment suggested the protective role of CTA-specific responses. The four CTA- and SALL4-specific T cell responses decreased with the progression of HCC, while the AFP-specific T cell response increased. A higher proportion of CD4+ T cells specific to CTA/SALL4 was observed than AFP-specific T cell responses.ConclusionsThe IFN-γ ELISPOT assay characterized distinct profiles of tumour-antigen-specific T cell responses in HCC patients. CTA- and SALL4-specific T cell responses may be important for controlling HCC in the early stage, whereas AFP-specific T cell responses might be a signature of malignant tumour status in the advanced stage. The application of immunotherapy at an early stage of HCC development should be considered.

Abstract PO066: Effect of radiation on oral cancer cell viability and anti-tumor T-cell responses

Abstract Background: Oral cancer is a malignant, neoplastic disorder most commonly presenting as squamous cell carcinoma (HNSCC). It is responsible for nearly 300,000 new diagnoses and 145,000 deaths globally each year, and in the United States alone, approximately one person will die from oral cancer every hour every day in 2020. Although primary surgical resection remains the standard of care for these patients, tumor recurrence is common (40%) and late stage five year survival rate is low (<50%). As a result, ionizing radiation has gained traction as an adjunctive therapy to improve oral cancer clinical outcomes. However, the effects of radiation on oral cancer cell viability and subsequent anti-tumor T cell responses to oral cancer are not fully understood. Objective: To examine the effects of ionizing radiation on HNSCC cell viability and determine the ability of irradiated HNSCC cells to elicit antigen specific T-cell immune responses. Methods: A metastatic mouse HNSCC cell line, LY2, was seeded and exposed to increasing doses of ionizing radiation (0, 20, 50, 100 200 Gy) generated via an RS2000 Irradiator, a ten minute exposure to ultraviolet (UV) light, freeze/thaw cycling, or a combination of these treatments. Following exposure, cells were cultured for 96 hours. Cell viability was assessed by microscopic imaging using a Leica microscope, and analysis was performed in ImageJ. Proteins were extracted from the cells, and protein concentration was quantified using a BCA assay. Next, single cell suspensions from the lymph nodes of LY2 tumor bearing, immunocompetent BALB/c mice were obtained and stimulated using various concentrations (0, 1.6, 8, and 40 µg) of proteins obtained from the LY2 cells. Antigen specific T cell proliferative responses, as determined by Alamar Blue assay, were compared between different concentrations of irradiated LY2 protein as well as the different exposure types. Cell supernatants were also harvested for analysis of the T cell specific cytokines, IL-4, IL-2, and IFNg. Results: LY2—HNSCC showed reduced viability in response ionizing radiation in a dose-dependent manner. The ideal, immunogenic range for harvested protein between 1.6µg and 8µg elicited an optimal T cell immune response. Additionally, Alamar Blue assay analysis demonstrated that LY2 proteins harvested from cells exposed to ionizing radiation or freeze/thaw cycling exhibited a proliferative response that was both dose-dependent and antigen specific, while cells exposed to UV light displayed no significant T cell proliferation. Conclusion: These results confirm a dose-dependent, cytotoxic effect of ionizing radiation on HNSCC cells. Proteins from cells exposed to high doses of ionizing radiation and freeze/thaw cycles can be successfully extracted with preserved antigenic profiles. Future work will allow characterization of the cellular response and enhance the study of the anti-tumor immune mechanisms in new and exciting ways. Citation Format: Travis Pero, Nathan Ryan, Kelvin Anderson, Steve Oghumu. Effect of radiation on oral cancer cell viability and anti-tumor T-cell responses [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO066.

Brucella abortus RB51 lipopolysaccharide influence as an adjuvant on the therapeutic efficacy of HPV16 L1 and HPV16 E7 DNA vaccines.

Objective(s):Human papillomavirus (HPV) is a primary contributing agent of cervical cancer. Eradication of HPV-related infections requires therapeutic strategies. We used Brucella abortus RB51 rough lipopolysaccharide (R-LPS) as an adjuvant along with two HPV16 therapeutic DNA vaccines, pcDNA3-E7 and pcDNA3-L1, for improving DNA vaccine efficacy. Materials and Methods:For evaluation of the B. abortus LPS adjuvant efficacy in combination with DNA vaccines to induce cellular immune responses, C57BL/6 mice were immunized with the DNA vaccines, with or without R-LPS adjuvant. IFN-γ and IL-4 cytokines assay was carried out for assessment of cellular and humoral immune responses. Results:Findings indicated that vaccination with pcDNA3-E7 or pcDNA3-L1 alone could induce strong cellular immune responses, but stronger antigen-specific T-cell immune responses were shown by co-administration of HPV16 E7 and HPV16 L1 DNA vaccines along with R-LPS adjuvant. Conclusion:Overall, B. abortus R-LPS through enhancement of T-cell immune responses can be considered an efficient vaccine adjuvant in future studies and trials.

Self-assembled particulate vaccine elicits strong immune responses and reduces Mycobacterium avium subsp. paratuberculosis infection in mice

Mycobacterium avium subspecies paratuberculosis (MAP) causes chronic progressive granulomatous enteritis leading to diarrhoea, weight loss, and eventual death in ruminants. Commercially available vaccines provide only partial protection against MAP infection and can compromise the use of bovine tuberculosis diagnostic tests. Here, we report the development of a protein-particle-based vaccine containing MAP antigens Ag85A202–347-SOD1–72-Ag85B173–330-74F1–148+669–786 as a fusion (‘MAP fusion protein particle’). The fusion antigen displayed on protein particles was identified using mass spectrometry. Surface exposure and accessibility of the fusion antigen was confirmed by flow cytometry and ELISA. The MAP fusion protein particle vaccine induced strong antigen-specific T-cell immune responses in mice, as indicated by increased cytokine (IFN-γ and IL-17A) and costimulatory signals (CD40 and CD86) in these animals. Following MAP-challenge, a significant reduction in bacterial burden was observed in multiple organs of the mice vaccinated with the MAP fusion protein particle vaccine compared with the PBS group. The reduction in severity of MAP infection conferred by the MAP fusion protein particle vaccine was similar to that of Silirum and recombinant protein vaccines. Overall, the results provide evidence that MAP antigens can be engineered as a protein particulate vaccine capable of inducing immunity against MAP infection. This utility offers an attractive platform for production of low-cost particulate vaccines against other intracellular pathogens.

Evaluation of Duration of Immunogenicity and Protective Efficacy of Improved Influenza Viral Vector-Based Brucella abortus Vaccine Against Brucella melitensis Infection in Sheep and Goats.

In this study, we first evaluated the duration of a protective immune response against Brucella melitensis infection in non-pregnant sheep and goats immunized with an improved (by vaccine formulation and route of administration) commercial Brucella abortus vaccine based on influenza viral vectors expressing Brucella immunodominant Omp16, L7/L12, Omp19, or Cu-Zn superoxide dismutase (SOD) proteins (Flu-BA_Omp19-SOD). Sheep and goats in the vaccinated group were immunized thrice concurrently via the subcutaneous and conjunctival routes of administration at an interval of 21 days. Animals in the control group were administered with 20% Montanide Gel01 adjuvant in phosphate-buffered saline in the same way. We showed that the Flu-BA_Omp19-SOD vaccine in sheep and goats induces antigen-specific Th1-biased [immunoglobulin G2a (IgG2a) over IgG1] antibody response and T-cell and interferon γ responses lasting over a period of 1 month post–last vaccination (PLV). The levels of protection against B. melitensis 16M infection (vaccination efficacy) in vaccinated sheep for a period of 6 months were 0–20% and in goats 20–40% compared to control challenge group. But the severity of B. melitensis 16M infection in the Flu-BA_Omp19-SOD–vaccinated sheep and goats during the entire period of observation revealed the infection index (P = 0.001–P < 0.0001) and Brucella colonization in lymph nodes and organs (P = 0.04–P < 0.0001) were significantly lower than those in the control group. To conclude, the Flu-BA_Omp19-SOD vaccine using improved formulation and administration method in sheep and goats provides augmented antigen specific humoral and T-cell immune response lasting only for 1 month PLV and partial protection for 6 months against B. melitensis 16M infection.

Dexamethasone premedication suppresses vaccine-induced immune responses against cancer

ABSTRACT Glucocorticosteroids (GCS) have an established role in oncology and are administered to cancer patients in routine clinical care and in drug development trials as co-medication. Given their strong immune-suppressive activity, GCS may interfere with immune-oncology drugs. We are developing a therapeutic cancer vaccine, which is based on a liposomal formulation of tumor-antigen encoding RNA (RNA-LPX) and induces a strong T-cell response both in mice as well as in humans. In this study, we investigated in vivo in mice and in human PBMCs the effect of the commonly used long-acting GCS Dexamethasone (Dexa) on the efficacy of this vaccine format, with a particular focus on antigen-specific T-cell immune responses. We show that Dexa, when used as premedication, substantially blunts RNA-LPX vaccine-mediated immune effects. Premedication with Dexa inhibits vaccine-dependent induction of serum cytokines and chemokines and reduces both the number and activation of splenic conventional dendritic cells (cDC) expressing vaccine-encoded antigens. Consequently, priming of functional effector T cells and therapeutic activity is significantly impaired. Interestingly, responses are less impacted when Dexa is administered post-vaccination. Consistent with this observation, although many inflammatory cytokines are reduced, IFNα, a key cytokine in T-cell priming, is less impacted and antigen expression by cDCs is intact. These findings warrant special caution when combining GCS with immune therapies relying on priming and activation of antigen-specific T cells and suggest that careful sequencing of these treatments may preserve T-cell induction.

In vitro Mycobacterial Growth Inhibition in South Korean Adults With Latent TB Infection.

Background: It is important to understand the ability to inhibit mycobacterial growth in healthy adults who would have been Bacillus Calmette-Guérin (BCG) vaccinated in childhood as this group will be the potential target population for novel booster TB vaccine trials. In this study we investigated not only the long-term immunity induced by childhood BCG vaccination but also protective immunity in terms of the ability to inhibit mycobacterial growth in those who were BCG vaccinated in childhood, with evidence of recent or remote TB infection.Methods: We measured the baseline immune response using a functional mycobacterial growth inhibition assay (MGIA) as a novel approach and an intracellular cytokine staining (ICS) assay as a reference approach in healthy adults, with different status of Mycobacterium tuberculosis (Mtb) infection.Results: Based on MGIA responses in historically BCG-vaccinated healthy adults, demographical characteristics including age, and gender did not affect mycobacterial growth inhibition in PBMC. However, the uninfected healthy control (HC) group showed a greater ability to inhibit mycobacterial growth compared with the latent TB infection (LTBI) group (P = 0.0005). In terms of the M. tuberculosis antigen-specific T-cell immune response in diluted whole blood quantitated using an ICS assay, the LTBI group had a higher frequency of polyfunctional CD 4+ T cells compared with the HC group (P = 0.0002), although there was no correlation between ICS and the MGIA assay.Conclusion: The Mtb infection status had a significant impact on mycobacterial growth inhibition in PBMC from healthy adults in South Korea, a country with an intermediate burden of tuberculosis, with healthy controls showing the greatest mycobacterial growth inhibition.

Vaccine targeting antigens associated with cancer stem cells/epithelial to mesenchymal transition inhibits triple negative breast cancer growth

Abstract Triple negative breast cancer (TNBC) is characterized by higher relapse and poor survival rates compared to other breast cancer types and is marked by a cancer stem cell (CSC)/epithelial to mesenchymal transition (EMT) signature. We questioned whether a vaccine targeting CSC/EMT associated proteins could prevent development of TNBC. We identified five overexpressed proteins found in TNBC and associated with different CSC/EMT pathways. We used a scoring system derived from three common algorithms for predicting class II MHC binding to select Th epitopes for CD105, CDH3, MDM2, SOX-2 and YB-1. Those epitopes that induced a selective Th1 (IFN-gamma) immune response in human population based screening were included in a multi-antigen vaccine. Epitopes were highly homologous between mouse and man. Immunization with the multi-antigen vaccine induced Th1 specific for all five antigens compared to adjuvant control (p&amp;lt;0.05 for all). Immunity persisted 3 months after the final vaccine (p&amp;lt;0.05 for all). Vaccination generated an antigen-specific cytotoxic T-cell (CTL) immune response to three antigens (p&amp;lt;0.05). The vaccine increased effector memory T-cells by 2-fold compared to control (p=0.03). Tumor growth in vaccinated FVB-Tg(C3-1-TAg) mice, a basal breast cancer model, was inhibited by over 80% compared to control mice (p&amp;lt;0.0001). We observed significantly more CTL (p=0.04) and Th1 (p&amp;lt;0.001) infiltrating the tumor with a concomitant reduction in cells expressing the stem cell marker Sca-1 (p=0.003) after immunization with vaccine compared to control. This multi-antigen vaccine targeting CSC/EMT antigens is currently being investigated in a Phase I clinical trial (NCT02157051).

Mycobacterium tuberculosis-associated synthetic mycolates differentially exert immune stimulatory adjuvant activity.

Mycolic acids (MAs) are highly hydrophobic long-chain α-alkyl β-hydroxy fatty acids present in the cell wall of Mycobacterium tuberculosis (Mtb) as a complex mixture of molecules with a common general structure but with variable functional groups in the meromycolate chain. In this study, we addressed the relationship between the MA molecular structure and their contribution to the development of T-cell immune responses. Hereto, we used the model antigen ovalbumin and single synthetic MAs, differing in oxygenation class and cis versus trans proximal cyclopropane configuration, as immune stimulatory agents. Subcutaneous delivery of liposome-formulated MAs with a proximal cis cyclopropane elicited antigen-specific Th1 and cytotoxic T-cell immune responses, whereas intratracheal immunization elicited pulmonary Th17 responses. These immune stimulatory activities depended not only on the cis versus trans proximal cyclopropane configuration but also on the MA oxygenation class. Our study thus shows that both the presence and nature of the functional groups in the meromycolate chain affect the immune stimulatory adjuvant activity of Mtb mycolates and suggests that Mtb bacilli may impact on the host protective immune response by modulating the cis versus trans stereochemistry of its mycolates as well as by altering the oxygenation class of the meromycolate functional group.

Prime-booster vaccination of cattle with an influenza viral vector Brucella abortus vaccine induces a long-term protective immune response against Brucella abortus infection

This study analyzed the duration of the antigen-specific humoral and T-cell immune responses and protectiveness of a recently-developed influenza viral vector Brucella abortus (Flu-BA) vaccine expressing Brucella proteins Omp16 and L7/L12 and containing the adjuvant Montadine Gel01 in cattle. At 1 month post-booster vaccination (BV), both humoral (up to 3 months post-BV; GMT IgG ELISA titer 214±55 to 857±136, with a prevalence of IgG2a over IgG1 isotype antibodies) and T-cell immune responses were observed in vaccinated heifers (n=35) compared to control animals (n=35, injected with adjuvant/PBS only). A pronounced T-cell immune response was induced and maintained for 12 months post-BV, as indicated by the lymphocyte stimulation index (2.7±0.4 to 10.1±0.9cpm) and production of IFN-γ (13.7±1.7 to 40.0±3.0ng/ml) at 3, 6, 9, and 12 months post-BV. Prime-boost vaccination provided significant protection against B. abortus infection at 3, 6, 9 and 12 months (study duration) post-BV (7 heifers per time point; alpha=0.03–0.01 vs. control group). Between 57.1 and 71.4% of vaccinated animals showed no signs of B. abortus infection (or Brucella isolation) at 3, 6, 9 and 12 months post-BV; the severity of infection, as indicated by the index of infection (P=0.0003 to <0.0001) and rates of Brucella colonization (P=0.03 to <0.0001), was significantly lower for vaccinated diseased animals than appropriate control animals. Good protection from B. abortus infection was also observed among pregnant vaccinated heifers (alpha=0.03), as well as their fetuses and calves (alpha=0.01), for 12 months post-BV. Additionally, 71.4% of vaccinated heifers calved successfully whereas all pregnant control animals aborted (alpha=0.01). Prime-boost vaccination of cattle with Flu-BA induces an antigen-specific humoral and pronounced T cell immune response and most importantly provides good protectiveness, even in pregnant heifers, for at least 12 months post-BV.

A randomized clinical trial in adults and newborns in South Africa to compare the safety and immunogenicity of bacille Calmette-Guérin (BCG) vaccine administration via a disposable-syringe jet injector to conventional technique with needle and syringe

IntroductionIntradermal bacille Calmette-Guérin (BCG) vaccination by needle-free, disposable-syringe jet injectors (DSJI) is an alternative to the Mantoux method using needle and syringe (NS). We compared the safety and immunogenicity of BCG administration via the DSJI and NS techniques in adults and newborn infants at the South African Tuberculosis Vaccine Initiative (SATVI) research site in South Africa. MethodThirty adults and 66 newborn infants were randomized 1:1 to receive intradermal BCG vaccine (0.1mL in adults; 0.05mL in infants) via DSJI or NS. Wheal diameter (mm) and skin fluid deposition at the site of injection (SOI) were measured immediately post-vaccination. Adverse events and SOI reactogenicity data were collected 30min and 1, 2, 4, and 12 weeks after vaccination for adults and at 30min and 4, 10, and 14 weeks for infants. Blood was collected in infants at 10 and 14 weeks to assess BCG-specific T-cell immune responses. ResultsMore infant BCG vaccinations by DSJI deposited >5μL fluid on the skin surface, compared to NS (49% versus 9%, p=0.001). However, all 12 infant vaccinations that did not produce any SOI wheal occurred in the NS group (36%, p<0.001). Median wheal diameter, in participants for which an SOI wheal formed, did not differ significantly between groups in infants (combined 3.0mm IQR 2.0 to 4.0, p=0.59) or in adults (combined 9.0mm IQR 7.0 to 10.0, p=0.13). Adverse events were similar between study arms. Proportion of participants with BCG scars after three months did not differ in adults (combined 97%, p=0.67) or infants (combined 62%, p=0.13). Frequencies of BCG-specific clusters of differentiation 4 (CD4) and clusters of differentiation 8 (CD8) T-cells co-expressing IFN-γ, TNF-α, IL-2, and/or IL-17 were not different in the DSJI and NS groups. ConclusionBCG vaccination of newborn infants via DSJI was more likely to deliver an appropriate intradermal wheal at the SOI as compared to NS, despite leaving more fluid on the surface of the skin. Safety, reactogenicity, and antigen-specific T-cell immune responses did not differ between DSJI and NS techniques.

Low antigen-specific CD4 T-cell immune responses despite normal absolute CD4 counts after long-term antiretroviral therapy an African cohort

BackgroundCD4 counts guide antiretroviral therapy (ART) initiation and prophylaxis for opportunistic infections. It is unclear whether normal CD4 counts translate to normalized immune responses among ART-treated adults. We compared antigen-specific CD4 T-cell immune responses among ART-treated adults with CD4≥500cells/μl, optimal immune responders (O-IR), and their age-matched healthy HIV-negative counterparts. MethodsIn a sample-based case–control study, cryopreserved peripheral blood mononuclear cells from 15 O-IR after 7 years of ART and 15 healthy controls, were analyzed for CD4+ T-cell proliferation using CFSE dye and cytokine production. ResultsCD4 T-cell proliferation, upon stimulation with PPD and pneumococcal polysaccharide antigen, was lower among O-IR relative to HIV-negative controls; p=0.016 and p=0.016 respectively. CD4 T-cell production of IL-2 was lower among O-IR relative to HIV-negative control p=0.002. CD4 T-cell proliferation upon stimulation with SEB and CMV antigens was similar among O-IR and HIV-negative controls p=0.971 and p=0.480, respectively, and so was IL-4 and IFN γ production; p=0.528 and p=0.892, respectively. ConclusionSeven years of suppressive ART caused partial CD4 T-cell function recovery in an African HIV treatment cohort, despite restoration of CD4 T-cell counts to levels≥500cells/μl. The role innate immunity in the recovery of immune function during long-term ART should be investigated to guide decisions on continued prophylaxis against opportunistic infections.

Novel influenza virus vectors expressing Brucella L7/L12 or Omp16 proteins in cattle induced a strong T-cell immune response, as well as high protectiveness against B. abortus infection

This paper presents the results of a study of the immunogenicity and protectiveness of new candidate vector vaccine against Brucella abortus – a bivalent vaccine formulation consisting of a mixture of recombinant influenza A subtype H5N1 or H1N1 (viral constructs vaccine formulation) viruses expressing Brucella ribosomal protein L7/L12 and Omp16, in cattle. To increase the effectiveness of the candidate vaccine, adjuvants such as Montanide Gel01 or chitosan were included in its composition. Immunization of cattle (heifers aged 1–1.5 years, 5 animals per group) with the viral constructs vaccine formulation only, or its combination with adjuvants Montanide Gel01 or chitosan, was conducted via the conjunctival method using cross prime (influenza virus subtype H5N1) and booster (influenza virus subtype H1N1) vaccination schedules at an interval of 28 days. Vaccine candidates were evaluated in comparison with the positive (B. abortus S19) and negative (PBS) controls. The viral constructs vaccine formulations, particularly in combination with Montanide Gel01 adjuvant promoted formation of IgG antibodies (with a predominance of antibodies of isotype IgG2a) against Brucella L7/L12 and Omp16 proteins in ELISA. Moreover, these vaccines in cattle induced a strong antigen-specific T-cell immune response, as indicated by a high number of CD4+ and CD8+ cells, as well as the concentration of IFN-γ, and most importantly provided a high level of protectiveness comparable to the commercial B. abortus S19 vaccine and superior to the B. abortus S19 vaccine in combination with Montanide Gel01 adjuvant. Based on these findings, we recommended the bivalent vaccine formulation containing the adjuvant Montanide Gel01 for practical use in cattle.

Maraba Virus as a Potent Oncolytic Vaccine Vector

The rhabdovirus Maraba has recently been characterized as a potent oncolytic virus. In the present study, we engineered an attenuated Maraba strain, defined as MG1, to express a melanoma-associated tumor antigen. Its ability to mount an antitumor immunity was evaluated in tumor-free and melanoma tumor-bearing mice. Alone, the MG1 vaccine appeared insufficient to prime detectable adaptive immunity against the tumor antigen. However, when used as a boosting vector in a heterologous prime-boost regimen, MG1 vaccine rapidly generated strong antigen-specific T-cell immune responses. Once applied for treating syngeneic murine melanoma tumors, our oncolytic prime-boost vaccination protocol involving Maraba MG1 dramatically extended median survival and allowed complete remission in more than 20% of the animals treated. This work describes Maraba virus MG1 as a potent vaccine vector for cancer immunotherapy displaying both oncolytic activity and a remarkable ability to boost adaptive antitumor immunity.

Implantable Silk Composite Microneedles for Programmable Vaccine Release Kinetics and Enhanced Immunogenicity in Transcutaneous Immunization

Microneedle vaccines mimic several aspects of cutaneous pathogen invasion by targeting antigen to skin-resident dendritic cells and triggering local inflammatory responses in the skin, which are correlated with enhanced immune responses. Here, we tested whether control over vaccine delivery kinetics can enhance immunity through further mimicry of kinetic profiles present during natural acute infections. An approach for the fabrication of silk/poly(acrylic acid) (PAA) composite microneedles composed of a silk tip supported on a PAA base is reported. On brief application of microneedle patches to skin, the PAA bases rapidly dissolved to deliver a protein subunit vaccine bolus, while also implanting persistent silk hydrogel depots into the skin for a low-level sustained cutaneous vaccine release over 1-2 weeks. Use of this platform to deliver a model whole-protein vaccine with optimized release kinetics resulted in >10-fold increases in antigen-specific T-cell and humoral immune responses relative to traditional parenteral needle-based immunization.

Sortase-mediated modification of αDEC205 affords optimization of antigen presentation and immunization against a set of viral epitopes

A monoclonal antibody against the C-type lectin DEC205 (αDEC205) is an effective vehicle for delivery of antigens to dendritic cells through creation of covalent αDEC205-antigen adducts. These adducts can induce antigen-specific T-cell immune responses or tolerance. We exploit the transpeptidase activity of sortase to install modified peptides and protein-sized antigens onto the heavy chain of αDEC205, including linkers that contain nonnatural amino acids. We demonstrate stoichiometric site-specific labeling on a scale not easily achievable by genetic fusions (49 distinct fusions in this report). We conjugated a biotinylated version of a class I MHC-restricted epitope to unlabeled αDEC205 and monitored epitope generation upon binding of the adduct to dendritic cells. Our results show transfer of αDEC205 heavy chain to the cytoplasm, followed by proteasomal degradation. Introduction of a labile dipeptide linker at the N terminus of a T-cell epitope improves proteasome-dependent class I MHC-restricted peptide cross-presentation when delivered by αDEC205 in vitro and in vivo. We also conjugated αDEC205 with a linker-optimized peptide library of known CD8 T-cell epitopes from the mouse γ-herpes virus 68. Animals immunized with such conjugates displayed a 10-fold reduction in viral load.

Tumor-Targeted Delivery of IL-2 by NKG2D Leads to Accumulation of Antigen-Specific CD8+ T Cells in the Tumor Loci and Enhanced Anti-Tumor Effects

Interleukin-2 (IL-2) has been shown to promote tumor-specific T-cell proliferation and differentiation but systemic administration of IL-2 results in significant toxicity. Therefore, a strategy that can specifically deliver IL-2 to the tumor location may alleviate concerns of toxicity. Because NKG2D ligands have been shown to be highly expressed in many cancer cells but not in healthy cells, we reason that a chimeric protein consisting of NKG2D linked to IL-2 will lead to the specific targeting of IL-2 to the tumor location. Therefore, we created chimeric proteins consisting of NKG2D linked to Gaussia luciferase (GLuc; a marker protein) or IL-2 to form NKG2D-Fc-GLuc and NKG2D-Fc-IL2, respectively. We demonstrated that NKG2D linked to GLuc was able to deliver GLuc to the tumor location in vivo. Furthermore, we showed that TC-1 tumor-bearing mice intramuscularly injected with DNA encoding NKG2D-Fc-IL2, followed by electroporation, exhibited an increased number of luciferase-expressing E7-specific CD8+ T cells at the tumor location. More importantly, treatment with the DNA construct encoding NKG2D-Fc-IL2 significantly enhanced the therapeutic anti-tumor effects generated by intradermal vaccination with therapeutic HPV DNA in tumor-bearing mice. Therefore, by linking NKG2D to IL2, we are able to specifically deliver IL-2 to the tumor location, enhancing antigen-specific T-cell immune response and controlling tumor growth. Our approach represents a platform technology to specifically deliver proteins of interest to tumor loci.