Strong Th1 Response Research Articles

Abstract LB353: Repurposing varicella zoster virus and human papillomavirus vaccines for local immunotherapy against solid tumors

Abstract Local immunotherapy against solid tumors is considered a viable approach to stimulate the tumor microenvironment (TME) and anti-tumor immunity. We assessed in situ vaccination with licensed subunit vaccines to leverage preexisting anti-vaccine immunity in the TC-1 tumor model expressing HPV16 viral oncogenes E6 and E7. We chose Shingrix, a VZV vaccine containing the glycoprotein E (gE), and Gardasil-9, an HPV vaccine containing L1 virus-like particles (VLP), to preferentially induce CD4 and CD8 T cell responses, respectively. Intratumoral (IT) injection of Shingrix in Shingrix prevaccinated mice led to complete regression in 20% to 50% of treated mice and elicited CD8 T cell responses against the viral oncoprotein E7. IT injection of an MHC-II-restricted gE peptide with polyI:C also led to durable remission, suggesting that gE-specific CD4 T cells played a significant role in tumor control. In contrast, IT injection of Gardasil-9 in Gardasi-9 prevaccinated animals did not delay tumor growth. However, IT injection of an MHC-I-restricted L1 peptide with Shingrix consistently led to complete remissions perhaps by overcoming preexisting antibodies against native HPV VLP which appeared to inhibit the cross-presentation of VLP derived antigens. TME analysis showed that IT injection of Shingrix with Gardasil-derived peptide induced IFN-gamma, TNF-alpha and CXCL9. This treatment also dramatically reshaped the immune cell infiltrate causing the recruitment of T cells and neutrophils and the decrease of tumor-associated macrophage and eosinophils. We are investigating now how these changes may contribute to tumor clearance and epitope spreading against tumor-derived antigens. Finally, IT injection of Shingrix and a E7 viral neoantigen peptide led to the eradication of all primary injected and abscopal tumors. Our results indicate that the ability of Shingrix to induce exceptionally strong Th1 responses makes it a versatile component for in-situ vaccination which can be combined with peptides derived from licensed vaccines or tumor antigens. Because this approach relies on relatively low cost existing vaccines and inexpensively manufactured minimal CD8 restricted peptides, it has the potential for a broadly applicable cancer immunotherapy strategy for use in resource-constrained settings. The approach is currently being evaluated in a phase I clinical trial in companion dogs with spontaneously arising cancers. Citation Format: Nicolas Cuburu, Shiv K. Sethi, Claire E. Bradley, Lukas Bialkowski, Cynthia D. Thompson, Douglas R. Lowy, John T. Schiller. Repurposing varicella zoster virus and human papillomavirus vaccines for local immunotherapy against solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB353.

Oral administration of DNA alginate nanovaccine induced immune-protection against Helicobacter pylori in Balb/C mice

BackgroundHelicobacter pylori (H. Pylori), is an established causative factor for the development of gastric cancer and the induction of persistent stomach infections that may lead to peptic ulcers. In recent decades, several endeavours have been undertaken to develop a vaccine for H. pylori, although none have advanced to the clinical phase. The development of a successful H. pylori vaccine is hindered by particular challenges, such as the absence of secure mucosal vaccines to enhance local immune responses, the absence of identified antigens that are effective in vaccinations, and the absence of recognized indicators of protection.MethodsThe DNA vaccine was chemically cloned, and the cloning was verified using PCR and restriction enzyme digestion. The efficacy of the vaccination was investigated. The immunogenicity and immune-protective efficacy of the vaccination were assessed in BALB/c mice. This study demonstrated that administering a preventive Alginate/pCI-neo-UreH Nanovaccine directly into the stomach effectively triggered a robust immune response to protect against H. pylori infection in mice.ResultsThe level of immune protection achieved with this nano vaccine was similar to that observed when using the widely accepted formalin-killed H. pylori Hel 305 as a positive control. The Alginate/pCI-neo-UreH Nanovaccine composition elicited significant mucosal and systemic antigen-specific antibody responses and strong intestinal and systemic Th1 responses. Moreover, the activation of IL-17R signaling is necessary for the defensive Th1 immune responses in the intestines triggered by Alginate/pCI-neo-UreH.ConclusionAlginate/pCI-neo-UreH is a potential Nanovaccine for use in an oral vaccine versus H. pylori infection, according to our findings.

Comparison of Adjuvant Effects of Montanide ISA-720 and Heat Shock Protein 27 in Increasing Immunostimulatory Properties of HIV-1 Nef-Vif Fusion Protein Construct.

Effective T-cell-mediated immunity has emerged as an essential component of human immunodeficiency virus-1 (HIV-1) vaccination. Thus, inducing an immune response against HIV proteins such as Nef and Vif, two major accessory proteins with critical roles in HIV pathogenesis and immune evasion, may lead to an effective approach. Our goal is to evaluate and compare Montanide ISA-720 and heat shock protein 27 in increasing immunostimulatory properties of HIV-1 Nef-Vif fusion protein as a vaccine candidate. In this study, the nef-vif fusion gene with and without the heat shock protein 27 (hsp27) gene was cloned in the prokaryotic pET24a (+) vector. Then, the recombinant Nef-Vif and Hsp27-Nef- Vif proteins were generated in the E. coli system. Finally, their immunostimulatory properties were evaluated in mice. Indeed, the potency of Hsp27 as an endogenous natural adjuvant was investigated to enhance HIV-1 Nef-Vif antigen-specific immunity compared to Montanide ISA-720 as a commercial adjuvant in protein-based immunization strategy. Our results approved the role of Hsp27 as an effective adjuvant in the stimulation of B- and T-cell immunity. The linkage of Hsp27 to antigen could elicit higher levels of IgG1, IgG2a, IFN-γ, IL- 5 and Granzyme B than antigen mixed with Montanide ISA-720. Moreover, the ratios of IFN-γ/IL-5 and IgG2a/IgG1 were significantly increased in groups receiving Nef-Vif protein + Montanide ISA- 720 and Hsp27-Nef-Vif protein indicating the direction of the immune response pathway toward strong Th1 response. These ratios were higher in the group receiving Hsp27-Nef-Vif protein than in the group receiving Nef-Vif protein + Montanide ISA-720. Our findings suggest that Hsp27 can be used as an effective adjuvant to enhance antigenspecific immune responses in HIV-1 infectious models for therapeutic vaccine development.

SARS-CoV-2 Delta requires human ACE2 but not human TMPRSS2 to infect mice and elicits greater lung injury and adaptive immune response than Omicron in human ACE2 knock-in mice

Abstract SARS-CoV-2 pathogenesis remains poorly understood in large part due to lack of knowledge about the mechanisms of entry and behaviors of SARS-CoV-2 variants in vivo. We generated single and double knock-in (KI) mice expressing human ACE2 (hACE2) and/or human TMPRSS2 (hTMPRSS2) under endogenous promoters in place of murine ACE2 (mACE2) and TMPRSS2 on the C57BL/6 and BALAB/c genetic backgrounds to evaluate Delta vs Omicron BA.1 infection, disease, and immune response. Delta replication was observed in lungs of mice expressing hACE2 but not hTMPRSS2 or mACE2 following intranasal inoculation, and similar levels of Delta replication were observed in lungs of hACE2-KI and hACE2xhTMPRSS2-KI mice. Thus, Delta requires hACE2 but not hTMPRSS2 to infect mice. In contrast, BA.1 established similar levels of replication in lungs of single and double KI mice, demonstrating that BA.1 requires neither hACE2 nor hTMPRSS2 to infect mice. Although no significant differences in viral burden were observed in hACE2-KI mice infected with Delta vs BA.1, Delta-infected hACE2-KI mice exhibited increased histopathologic lung injury and higher SARS-CoV-2-specific CD4+ and CD8+ T cell responses (spleen) and anti-SARS-CoV-2 spike IgG titers (serum). Additionally, hACE2-KI mice on the C57BL/6 background showed more severe lung disease and stronger Th1 response than BALB/c. These results associate the severity of lung disease with the magnitude of Th1-dominant immune responses, and set a foundation for dissecting mechanisms of COVID-19 pathogenesis in hACE2-KI mice representing Th1- vs Th2-dominant genetic backgrounds.

In silico design and in vivo evaluation of two multi-epitope vaccines containing build-in adjuvant with chitosan nanoparticles against uropathogenic Escherichia coli.

Urinary pathogenic Escherichia coli (UPEC) is one of the most important bacterial causes of urinary tract infections (UTIs). Rising antimicrobial resistance and serious clinical challenges such as persistent and recurrent UTIs make it a serious public health concern. Therefore, preventative approaches such as vaccinations are required. In this study, we selected three conserve and protective antigens (FdeC, Hma and UpaB) and also subunit B of cholera toxin (as build-in adjuvant) to design two multi-epitope vaccines (construct B containing B cell epitopes and construct T containing T epitopes) using different bioinformatics methods. The expression of the recombinant protein was performed using the BL21(DE3)/pET28 expression system and purified through a Ni-NTA column. Vaccine proteins were encapsulated in chitosan nanoparticles (CNP) based on ionic gelation via a microfluidic system. Mice were immunized intranasally with different vaccine formulations. Antibody responses and also cytokine expression (IFN-γ and IL-4) were measured by ELISA and real-time PCR respectively. The effectiveness of immune responses was assessed by bladder challenge. Based on the in silico study, construct B and construct T have high confidence value and stable structure in vivo. High yield expression of both constructs was confirmed by SDS-PAGE and western blot assay. Immunization of mice with construct B induced strong Th2 (IgG1 and IL4) responses and construct T shift immune responses to Th1 (IFNγ and IgG2a). Vaccine protein-encapsulated CNP elicited higher levels of antibodies and cell-mediated responses than the vaccine proteins alone. The results of this study suggest that intranasal administration of the construct B has the potential to enhance humoral immunity and construct T has the potential to stimulate cellular immunity. In addition, the combination of CTB as a build-in adjuvant and CNP can be proposed as a potent adjuvant for the development of a novel vaccine against UTI.

Immunogenicity of a spike protein subunit-based COVID-19 vaccine with broad protection against various SARS-CoV-2 variants in animal studies.

SARS-CoV-2 pandemic has profound impacts on human life and global economy since the outbreak in 2019. With the new variants continue to emerge with greater immune escaping capability, the protectivity of the available vaccines is compromised. Therefore, development a vaccine that is capable of inducing immunity against variants including omicron strains is in urgent need. In this study, we developed a protein-based vaccine BCVax that is consisted of antigen delta strain spike protein and QS21-based adjuvant AB801 in nanoparticle immune stimulation complex format (AB801-ISCOM). Results from animal studies showed that high level of anti-S protein IgG was induced after two doses of BCVax and the IgG was capable of neutralizing multiple variants of pseudovirus including omicron BA.1 or BA.2 strains. In addition, strong Th1 response was stimulated after BCVax immunization. Furthermore, BCvax with AB801-ISCOM as the adjuvant showed significant stronger immunity compared with the vaccine using aluminum hydroxide plus CpG 1018 as the adjuvant. BCVax was also evaluated as a booster after two prior vaccinations, the IgG titers and pseudovirus neutralization activities against BA.2 or BA.4/BA.5 were further enhanced suggesting BCVax is a promising candidate as booster. Taken together, the pre-clinical data warrant BCVax for further development in clinic.

The Problem of Host and Pathogen Genetic Variability for Developing Strategies of Universally Efficacious Vaccination against and Personalised Immunotherapy of Tuberculosis: Potential Solutions?

Rational vaccination against and immunotherapy of any infectious disease requires knowledge of how protective and non-protective immune responses differ, and how immune responses are regulated, so their nature can be controlled. Strong Th1 responses are likely protective against M tuberculosis. Understanding how immune class regulation is achieved is pertinent to both vaccination and treatment. I argue that variables of infection, other than PAMPs, primarily determine the class of immunity generated. The alternative, non-PAMP framework I favour, allows me to propose strategies to achieve efficacious vaccination, transcending host and pathogen genetic variability, to prevent tuberculosis, and personalised protocols to treat disease.

Multiple Orientia clusters and Th1-skewed chemokine profile: a cross-sectional study in patients with scrub typhus from Nepal

ObjectivesScrub typhus is an emerging infectious disease in Asia caused by Orientia tsutsugamushi (Ot). From Nepal, only scant data on the genetic epidemiology of this agent is available, and determinants of immunoregulation are poorly understood. MethodsPatients (n = 238) referred to the National Public Health Laboratory (Kathmandu, Nepal) from all over Nepal for suspected scrub typhus were enrolled upon positive immunoglobulin (Ig)M testing between July and October 2015. From Ot 16S and 47 kD polymerase chain reaction (PCR)-positive samples, the variable domain I of the 56 kD gene was sequenced and phylogenetically analyzed. T helper (Th) cell-associated cytokines (n = 13) and chemokines (n = 12) were quantified by multiplex bead arrays. ResultsIn 93/238 (39.1%) IgM-positive samples, Ot DNA was detected by quantitative PCR. Phylogenetic analysis of 56 kD sequences revealed seven distinct clusters, six of them with high homologies to strains detected in other countries. The Th1-related cytokines interferon-γ and C-X-C motif chemokine ligand 10 were strongly upregulated and correlated with bacteremia, while levels of Th2-associated chemokines were reduced. Bacteremia also correlated with concentrations of interleukin (IL)-6 and IL-10 but not tumor necrosis factor-α. ConclusionWe identified a considerable genetic heterogeneity of human-pathogenic Ot strains circulating in Nepal. Acute Nepalese scrub typhus patients showed strong Th1 but impaired Th2 responses, especially on the chemokine level.

Influence of Type I Interferons in Gammaherpesvirus-68 and Its Influence on EAE Enhancement.

Epstein-Barr virus (EBV) has been identified as a putative trigger of multiple sclerosis (MS). Previously, we reported that mice latently infected with murine gammaherpesvirus 68 (γHV-68), the murine homolog to EBV, and induced for experimental autoimmune encephalomyelitis (EAE), developed an enhanced disease more reminiscent of MS. These prior results showed that expression of CD40 on CD11b+CD11c+ cells in latently infected mice was required to prime the strong Th1 response driving disease as well as decreasing Treg frequencies in the periphery and CNS. Subsequent work demonstrated that transfer of B cells from latently infected mice was sufficient to enhance disease. Herein, we show that B cells from infected mice do not need type I IFN signaling to drive a strong Th1 response, yet are important in driving infiltration of the CNS by CD8+ T cells. Given the importance of type I IFNs in MS, we used IFNARko mice in order to determine if type I IFN signaling was important in the enhancement of EAE in latently infected mice. We found that while type I IFNs are important for the control of γHV-68 infection and maintenance of latency, they do not have a direct effect in the development of enhanced EAE.

Abstract CT205: A phase I/Ib trial of intratumoral Poly-ICLC in resectable malignant pleural mesothelioma

Abstract Background: Malignant pleural mesothelioma (MPM) is usually fatal, though multimodality therapy— now including immunotherapy— has improved survival. Recurrence after surgery is close to 100%, even with adjuvant chemotherapy and radiation. Our collaborators have performed deep immunophenotyping of treatment-naïve MPM lesions using mass cytometry (CyTOF) and single-cell RNA sequencing (scRNAseq) to define the tumor microenvironment. A population of rare CD141+ dendritic cells (DC1) is disproportionately represented in some MPM lesions analyzed. These DC1 cells— which express high levels of Toll-like receptor 3 (TLR3)— are among the most potent cross-presenters of antigen and are key to priming anti-tumor CD4+ and CD8+ T cell responses. Polyinosinic-polycytidylic acid stabilized with polylysine and carboxymethylcellulose (poly-ICLC), is a double-stranded RNA host-targeted therapeutic viral-mimic. Poly-ICLC activates multiple innate immune receptors including TLR3 and melanoma differentiation-associated gene 5 (MDA5), leading to cross-presentation of antigen to T cells and induction of strong Th1 response. We hypothesize that injection of poly-ICLC prior to surgical resection may activate intratumoral (IT) DC1s, increase tumor antigen presentation to cytotoxic T cells, and induce tumor-specific immune surveillance. Methods: This is a phase I/Ib study to evaluate the safety of IT poly-ICLC prior to surgical resection for patients with MPM (NCT04525859). The primary endpoint is safety as assessed by frequency and severity of toxicities by CTCAE 5.0. Secondary endpoints are objective response as measured by RECIST 1.1 and recurrence free survival measured from the time of first poly-ICLC injection. Exploratory endpoints include evaluation of circulating immune cells (including regulatory T cells and NK cells), evaluation of immune cell infiltration in pre-injection tumor biopsy and surgically resected tissue, as well as characterization of immune parameters such as local B cell specificity. The protocol features a Simon’s two-stage design, with six patients enrolled in a phase I safety cohort, proceeding to a phase Ib expansion cohort (additional 13 patients) if no more than 1 dose limiting toxicity occurs. Eligible patients must have MPM deemed operable by the treating thoracic surgeon. Eligible subjects may not have uncontrolled immunocompromised states or autoimmune disorders. After enrollment, patients undergo biopsies at which time 2mg poly-ICLC is injected across two sites in the tumor. Patients then undergo resection of the tumor (pleurectomy/decortication or extra pleural pneumonectomy per standard of care) at day 21+/- 7 after poly-ICLC injection. Blood is drawn at three points (prior to poly-ICLC injection, at time of surgery, and at a post-operative visit) for immune profiling. At the time of submission six patients have been treated and phase Ib accrual is continuing as planned. Interim analysis of phase I safety and exploratory endpoints will be reported in late 2022. Citation Format: Bailey G. Fitzgerald, Thomas U. Marron, Robert Sweeney, Jorge Gomez, Nicole Hall, Daniel O'Grady, Christian Rolfo, Raj Veluswamy, Deborah Doroshow, John Mandeli, David Yankelevitz, Nina Bhardwaj, Sacha Gnjatic, Fred R. Hirsch, Miriam Merad, Alexander Tsankov, Raja Flores, Andrea Wolf. A phase I/Ib trial of intratumoral Poly-ICLC in resectable malignant pleural mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT205.

Age-dependent rise in IFN-γ competence undermines effective type 2 responses to nematode infection

The efficient induction of type 2 immune responses is central to the control of helminth infections. Previous studies demonstrated that strong Th1 responses driven by intracellular pathogens as well as a bias for type 1 activity in senescent mice impedes the generation of Th2 responses and the control of intestinal nematode infections. Here, we show that the spontaneous differentiation of Th1 cells and their expansion with age restrains type 2 immunity to infection with the small intestinal nematode H. polygyrus much earlier in life than previously anticipated. This includes the more extensive induction of IFN-γ competent, nematode-specific Th2/1 hybrid cells in BALB/c mice older than three months compared to younger animals. In C57BL/6 mice, Th1 cells accumulate more rapidly at steady state, translating to elevated Th2/1 differentiation and poor control of parasite fitness in primary infections experienced at a young age. Blocking of early IFN-γ and IL-12 signals during the first week of nematode infection leads to sharply decreased Th2/1 differentiation and promotes resistance in both mouse lines. Together, these data suggest that IFN-γ competent, type 1 like effector cells spontaneously accumulating in the vertebrate host progressively curtail the effectiveness of anti-nematode type 2 responses with rising host age.

Setting the pace: CD4 T cell-intrinsic Arginase 1 orchestrates Th1 induction and contraction

Abstract CD4 T cell-intrinsic engagement of the complement receptor CD46 controls nutrient influx and the metabolic reprogramming events that are essential for both the initiation and contraction of human Th1 responses, characterized by IFN-g and IL-10 production respectively. Here, we demonstrate that CD46 also orchestrates T cell arginine metabolism by upregulating the arginine transporter CAT-1 and, unexpectedly, Arginase 1 (Arg1). Arg1 has been well characterized in macrophages where it is associated with the IL-10 secreting M2 type, but its expression or function in T cells has not been described. Surprisingly, in CD4 T cell, Arg1 seems to restrain IL-10 production and contraction: T cells isolated from four patients with rare Arg1 deficiency mount strong Th1 responses but display significantly increased IL-10 switching and early contraction when compared to healthy control cells. Similarly, Arg1fl/fl CD4-cre+ mice infected with influenza virus are characterized by an enhanced Th1 response that contracts more rapidly, resulting in viral control and significantly reduced lung pathology. Unexpectedly, both Arg1-deficient mouse and human T cells produce normal levels of nitric oxide (NO) and polyamines. Metabolic profiling rather revealed that T cells lacking Arg1 have enhanced glycolysis, reduced TCA-cycle intermediates, and engage an “alternative” glutamine pathway often utilized by cancer cells. Normalization of these metabolic perturbations, through the targeting of specific metabolic enzymes, restored typical Th1 induction/contraction. Overall, these data unveil an unexpected intrinsic role for Arginase 1 as a pacemaker of the Th1 lifecycle, which could be harnessed for the amelioration Th1-driven pathologies.

The Host Peritoneal Cavity Harbors Prominent Memory Th2 and Early Recall Responses to an Intestinal Nematode.

Intestinal parasitic nematodes affect a quarter of the world’s population, typically eliciting prominent effector Th2-driven host immune responses. As not all infected hosts develop protection against reinfection, our current understanding of nematode-induced memory Th2 responses remains limited. Here, we investigated the activation of memory Th2 cells and the mechanisms driving early recall responses to the enteric nematode Heligmosomoides polygyrus in mice. We show that nematode-cured mice harbor memory Th2 cells in lymphoid and non-lymphoid organs with distinct transcriptional profiles, expressing recirculation markers like CCR7 and CD62-L in the mesenteric lymph nodes (mLN), and costimulatory markers like Ox40, as well as tissue homing and activation markers like CCR2, CD69 and CD40L in the gut and peritoneal cavity (PEC). While memory Th2 cells persist systemically in both lymphoid and non-lymphoid tissues following cure of infection, peritoneal memory Th2 cells in particular displayed an initial prominent expansion and strong parasite-specific Th2 responses during early recall responses to a challenge nematode infection. This effect was paralleled by a significant influx of dendritic cells (DC) and eosinophils, both also appearing exclusively in the peritoneal cavity of reinfected mice. In addition, we show that within the peritoneal membrane lined by peritoneal mesothelial cells (PeM), the gene expression levels of cell adhesion markers VCAM-1 and ICAM-1 decrease significantly in response to a secondary infection. Overall, our findings indicate that the host peritoneal cavity in particular harbors prominent memory Th2 cells and appears to respond directly to H. polygyrus by an early recall response via differential regulation of cell adhesion markers, marking the peritoneal cavity an important site for host immune responses to an enteric pathogen.

Serial sampling of rectal tumors during radiotherapy: A proof-of-concept study.

153 Background: Treatment response to neoadjuvant therapy in locally advanced rectal cancer (LARC) remains heterogenous. Clinicians are guided by pre-treatment clinical assessment alone in determining neoadjuvant strategy. More must be done to uncover biological mechanisms underpinning response and resistance. We have developed a biospecimen collection protocol in LARC performing serial sampling of tumors and peripheral blood samples prior to, during and after treatment to characterize the biological evolution of this heterogenous response. Here we present early proof of concept results with a focus on the intra-tumoral immune response relating to radiotherapy (RT). Methods: Patients receiving standard-of-care neoadjuvant RT were recruited to an ethically approved study between Dec 2018 - Aug 2021. The protocol consisted of a baseline biopsy and blood sample prior to RT followed by repeat sampling at 2, 6 and 12wks after Day 1 of RT. Standard immunohistochemistry (IHC) was performed for markers of immune activity. Target capture sequencing was performed using RNA baits extracted from serial biopsies to target a 276 genes panel. Paired tumor-normal sequencing was performed. Bulk 3’ RNA seq (Lexogen Illumina Quantseq) characterized immune and inflammatory gene expression. A multiplex bead array (Luminex xPONENT) of 24 cytokines and chemokines was performed using serial plasma samples. Results: 17pts were recruited, 3 with stage IV disease. 14pts received chemoradiation and 3 pts had short-course based regimens. Treatment responses were evaluable in 14pts: graded complete in 2pts; good/ near complete in 7pts and partial/ poor in 5pts. All tumors were MSS, and most frequently mutated genes were APC (75%), KRAS (38%), NRAS (25%) , NOTCH1 (25%) and PIK3CA (25%) (n = 8). Quantseq demonstrated that the immune/ inflammatory response, as measured by interferon-gamma response and IL-6/ JAK-STAT signaling, was significantly elevated up to 12wks after Day 1 RT, with a peak at around 6wks (n = 3). Correlative IHC showed an increase in innate immune cells in pts with a favorable response at 6wks (n = 8). Cytokine/ chemokine analysis suggested patients with a favorable response demonstrated strong inflammatory (MCP1 & IL-17a) responses 2 and 6wks post-RT and strong CTL (Granzyme B) and Th1 (GM-CSF & IP-10) responses 12wks post-RT (n = 10). Conclusions: We show acquisition of meaningful genomic and transcriptomic material from serial biopsies in rectal cancer is possible. Early data suggest that dynamic profiling of rectal tumors demonstrates transcriptomic evolution during treatment. Specifically, we show that the immune response to radiotherapy peaks at around 6wks after initiation of RT and persists to 12wks. This supports ongoing trials of immunomodulatory treatments in combination with, and following, RT in rectal cancer. Further work is required to define differences between responders and non-responders.

ASCVac-1, a Multi-Peptide Chimeric Vaccine, Protects Mice Against Ascaris suum Infection

Control of human ascariasis, the most prevalent neglected tropical disease globally affecting 450 million people, mostly relies on mass drug administration of anthelmintics. However, chemotherapy alone is not efficient due to the high re-infection rate for people who live in the endemic area. The development of a vaccine that reduces the intensity of infection and maintains lower morbidity should be the primary target for infection control. Previously, our group demonstrated that immunization with crude Ascaris antigens in mice induced an IgG-mediated protective response with significant worm reduction. Here, we aimed to develop a multipeptide chimera vaccine based on conserved B-cell epitopes predicted from 17 common helminth proteomes using a bioinformatics algorithm. More than 480 B-cell epitopes were identified that are conserved in all 17 helminths. The Ascaris-specific epitopes were selected based on their reactivity to the pooled sera of mice immunized with Ascaris crude antigens or infected three times with A. suum infective eggs. The top 35 peptides with the strongest reactivity to Ascaris immune serum were selected to construct a chimeric antigen connected in sequence based on conformation. This chimera, called ASCVac-1, was produced as a soluble recombinant protein in an Escherichia coli expression system and, formulated with MPLA, was used to immunize mice. Mice immunized with ASCVac-1/MPLA showed around 50% reduced larvae production in the lungs after being challenged with A. suum infective eggs, along with significantly reduced inflammation and lung tissue/function damage. The reduced parasite count and pathology in infected lungs were associated with strong Th2 immune responses characterized by the high titers of antigen-specific IgG and its subclasses (IgG1, IgG2a, and IgG3) in the sera and significantly increased IL-4, IL-5, IL-13 levels in lung tissues. The reduced IL-33 titers and stimulated eosinophils were also observed in lung tissues and may also contribute to the ASCVac-1-induced protection. Taken together, the preclinical trial with ASCVac-1 chimera in a mouse model demonstrated its significant vaccine efficacy associated with strong IgG-based Th2 responses, without IgE induction, thus reducing the risk of an allergic response. All results suggest that the multiepitope-based ASCVac-1 chimera is a promising vaccine candidate against Ascaris sp. infections.

Subcutaneous immunization with the fusion protein ΔA146Ply-SP0148 confers protection against Streptococcus pneumoniae infection

Pneumococcal SP0148 and pneumolysin (Ply) derivatives are important vaccine candidates. SP0148 is a conserved lipoprotein with high immunogenicity produced by Streptococcus pneumoniae. We have previously demonstrated that SP0148 can confer protection against fatal infections caused by S. pneumoniae. ΔA146Ply is a noncytotoxic mutant of Ply that retains the TLR4 agonistic effect and has mucosal and subcutaneous adjuvant activities suggested to induce protective immunity against S. pneumoniae infection. In this study, we constructed the fusion protein ΔA146Ply-SP0148, composed of ΔA146Ply and SP0148, and evaluated the immunoprotective effect of the fusion protein. When mice were subcutaneously immunized with the fusion protein ΔA146Ply-SP0148, high levels of anti-ΔA146Ply and anti-SP0148 IgG antibodies were induced in the serum. Specific antibodies can bind to a variety of different serotypes of S. pneumoniae. Compared with mice immunized with ΔA146Ply and SP0148 alone, mice immunized subcutaneously with the fusion protein ΔA146Ply-SP0148 with Al(OH)3 had a higher survival rate when challenged by a lethal dose of S. pneumoniae, and they also had significantly lower lung bacterial loads and milder lung inflammation. In addition, mice immunized subcutaneously with the fusion protein ΔA146Ply-SP0148 stimulated strong Th1, Th2, and Th17 cell responses. In summary, these results suggest that subcutaneous immunization with the ΔA146Ply-SP0148 fusion protein can protect mice against fatal pneumococcal infection and lung infection. The fusion protein ΔA146ply-SP0148 can be a new pneumococcal vaccine target.

Anti-SARS-CoV-2 T-cell Responses After mRNA Vaccination in Belatacept-treated Renal Transplant Patients.

Dear Editor, We read with interest the recent letter by Chavarot et al1 on the humoral and T-cell responses after 2 injections of mRNA vaccine in kidney transplant recipients. The authors reported a low sero-conversion rate at day 60 and a T-cell response in only 30.4% of the patients measured. Importantly, the T-cell response was measured by IFNγ-EliSpot after stimulation with overlapping peptide pools for SARS-CoV-2 spike protein. We would like to further discuss the determination of the T-cell response, as it has important implications for the interpretation of the findings. The safety and efficacy of mRNA vaccines have been demonstrated in numerous clinical studies, and, in most of these studies, vaccine-specific T-cell responses were reported.2-4 In these studies, next to IFNγ EliSpot, T cells were detected in addition by flow cytometry to characterize the cytokine profile. Sahin et al3 found in a phase I/II study that BNT161b1 elicited a strong Th1 response. In most of the experimental groups, IL-2 producing T-cells dominated over IFNγ producing T-cells or even IFNγ+IL-2+ T cells. A similar observation was made in a phase I/II trial on BNT162b2 as reported in preprint by Sahin et al.4 In a phase I study on the mRNA-1273 vaccine, Anderson et al2 demonstrated that vaccine-specific T-helper-cell responses measured on day 43 were of the Th1 type. However, the IL-2 response by T helper cells was greater than the IFNγ response but lower than the TNFα response.2 This effect was consistent over 2 different age groups ranging from 56 to 70 y of age and ≥71 y of age. Thus, vaccine-specific T-cell responses are not restricted to IFNγ producing Th1 cells. We would like to point out that the lack of T-cell response reported by Chavarot et al may not give a comprehensive information on the overall T-cell response to vaccination in renal transplant patients as an IFNγ EliSpot was the only readout.1 IL-2 or TNFα single positive T helper cells are not detected in this assay but usually evolve after vaccination in healthy people. Thus, renal transplant patients may well harbor vaccine-specific T-cell immunity upon vaccination. Additionally, it remains unclear whether the impaired Th1 response is a mainly belatacept-dependent effect as suggested by the authors. From our point of view, this is not exclusively related to the inhibition of costimulation but to the high-grade immunosuppressive regimen after kidney transplantation. In summary, the measurement of vaccine-specific T-cell responses is complex and a comprehensive characterization of vaccine-specific T-cell responses is necessary to draw conclusions on the immunogenicity of BNT162b2 in renal transplant patients. Nevertheless, the present study provides substantial evidence that vaccine responses in kidney transplant patients should be monitored and individualized vaccination strategies might be needed in this vulnerable cohort.

P38 MAPK activation by Toxoplasma gondii secretory effector protein GRA24: Role in cytokine regulation and induction of protective immunity

Abstract The apicomplexan Toxoplasma gondii induces strong protective immunity in part dependent upon recognition by Toll-like receptors (TLR) 11 and 12 operating in conjunction with MyD88 in the murine host. However, these TLR are not present in humans, prompting us to investigate MyD88-independent signaling pathways of resistance. We show here that parasite dense granule protein GRA24 induces p38 MAPK activation and subsequent IL-12 production in bone marrow derived mouse macrophages (BMDM). Furthermore, Toxoplasma triggers two waves of p38 MAPK phosphorylation in MyD88+/+ and MyD88−/− BMDM. Using the uracil auxotrophic Type I T. gondii strain cps1-1, we demonstrate that the late response does not require active parasite proliferation, but strictly depends upon GRA24. By i. p. inoculation with cps1-1 and cps1-1:Δgra24, we identified unique subsets of chemokines and cytokines that were up and down regulated by GRA24. We demonstrate that cps1-1 triggers a strong host-protective GRA24-dependent Th1 response in the absence of MyD88. To gain deeper insight into genes regulated by GRA24, we employed qPCR to evaluate expression of transcripts for 84 cytokine and chemokine genes in BMDM infected with GRA24-positive compared to GRA24-negative parasites. Our results identify a specific signature of genes regulated by this dense granule protein. In conjunction, using qPCR we evaluated the impact of GRA24 on a panel of macrophage long noncoding RNA (lncRNA) regulated by Toxoplasma infection. Taken together, our data identify GRA24 as a major mediator of p38 MAPK activation, induction of IL-12 and other immune mediators, as well as protective immunity that operates independently of the TLR/MyD88 cascade.

Cationic polymer-modified Alhagi honey polysaccharide PLGA nanoparticles as an adjuvant to induce strong and long-lasting immune responses

Alhagi honey polysaccharide (AHP) exhibit an excellent immune adjuvant effect, but low bioavailability in the body limits its application. Cationic polymer-modified poly (lactic-co-glycolic acid) (PLGA) nanoparticles have been widely investigated as vaccine delivery systems owing to their excellent antigen-loading efficiency. In this study, three kinds of cationic polymer were used to coat AHP-encapsulated PLGA nanoparticles (AHPP) to build positively charged antigen carriers. Among them, H5N1-loaded PEI-AHPP formulation could induce highest hemagglutination inhibition (HI) titer, IgG-subtype, and cytokines, activated dendritic cells (DCs) in lymph nodes, and CD3e+CD4+ and CD3e+CD8a+ T cells in the spleen of immunized mice. PEI-AHPP could stimulate DCs to highly express MHCI and MHCII molecules and had good antigen slow-release effect at the injected site along with lymph node targeting. These findings demonstrate that PEI-AHPP has the potential to be an effective adjuvant to induce strong and long-lasting Th1 and Th2 mixed immune responses.

A single-dose live attenuated chimeric vaccine candidate against Zika virus

The mosquito-borne Zika virus is an emerging pathogen from the Flavivirus genus for which there are no approved antivirals or vaccines. Using the clinically validated PDK-53 dengue virus vaccine strain as a backbone, we created a chimeric dengue/Zika virus, VacDZ, as a live attenuated vaccine candidate against Zika virus. VacDZ demonstrates key markers of attenuation: small plaque phenotype, temperature sensitivity, attenuation of neurovirulence in suckling mice, and attenuation of pathogenicity in interferon deficient adult AG129 mice. VacDZ may be administered as a traditional live virus vaccine, or as a DNA-launched vaccine that produces live VacDZ in vivo after delivery. Both vaccine formulations induce a protective immune response against Zika virus in AG129 mice, which includes neutralising antibodies and a strong Th1 response. This study demonstrates that VacDZ is a safe and effective vaccine candidate against Zika virus.