IL-8 correlates with nonresponse to neoadjuvant nivolumab in HPV positive HNSCC via a potential extracellular vesicle miR-146a mediated mechanism.

Therapy using anti-PD-1 immune checkpoint inhibitors (ICI) has revolutionized the treatment of many cancers including head and neck squamous cell carcinomas (HNSCC), but only a fraction of patients respond. To better understand the molecular mechanisms driving resistance, we performed extensive analysis of plasma and tumor tissues before and after a 4-week neoadjuvant trial in which HNSCC patients were treated with the anti-PD-1 inhibitor, nivolumab. Luminex cytokine analysis of patient plasma demonstrated that HPVpos nonresponders displayed high levels of the proinflammatory chemokine, interleukin-8 (IL-8), which decreased after ICI treatment, but remained higher than responders. miRNAseq analysis of tetraspanin-enriched small extracellular vesicles (sEV) purified from plasma of HPVpos nonresponders demonstrated significantly lower levels of seven miRNAs that target IL-8 including miR-146a. Levels of the pro-survival oncoprotein Dsg2, which has been to down-regulate miR-146a, are elevated with HPVpos tumors displaying higher levels than HPVneg tumors. Dsg2 levels decrease significantly following ICI in responders but not in nonresponders. In cultured HPVpos cells, restoration of miR-146a by forced expression or treatment with miR-146a-loaded sEV, reduced IL-8 level, blocked cell cycle progression, and promoted cell death. These findings identify Dsg2, miR-146a, and IL-8 as potential biomarkers for ICI response and suggest that the Dsg2/miR-146a/IL-8 signaling axis negatively impacts ICI treatment outcomes and could be targeted to improve ICI responsiveness in HPVpos HNSCC patients.

Comprehensive peripheral blood immunoprofiling reveals five immunotypes with immunotherapy response characteristics in patients with cancer.

The lack of comprehensive diagnostics and consensus analytical models for evaluating the status of a patient's immune system has hindered a wider adoption of immunoprofiling for treatment monitoring and response prediction in cancer patients. To address this unmet need, we developed an immunoprofiling platform that uses multiparameter flow cytometry to characterize immune cell heterogeneity in the peripheral blood of healthy donors and patients with advanced cancers. Using unsupervised clustering, we identified five immunotypes with unique distributions of different cell types and gene expression profiles. An independent analysis of 17,800 open-source transcriptomes with the same approach corroborated these findings. Continuous immunotype-based signature scores were developed to correlate systemic immunity with patient responses to different cancer treatments, including immunotherapy, prognostically and predictively. Our approach and findings illustrate the potential utility of a simple blood test as a flexible tool for stratifying cancer patients into therapy response groups based on systemic immunoprofiling.

Seasonal quadrivalent mRNA vaccine prevents and mitigates influenza infection.

Annually, seasonal influenza is responsible for millions of infections and hundreds of thousands of deaths. The current method for managing influenza is vaccination using a standardized amount of the influenza virus' primary surface antigen, hemagglutinin (HA), as the intended target of the immune response. This vaccination strategy results in vaccines with variable efficacy year to year due to antigenic drift of HA, which can be further exacerbated by manufacturing processes optimizing growth of vaccine virus in eggs. Due to these limitations, alternative vaccine platforms are actively being explored to improve influenza vaccine efficacy, including cell-based, recombinant protein, and mRNA vaccines. mRNA's rapid, in vitro production makes it an appealing platform for influenza vaccination, and the success of SARS-CoV-2 mRNA vaccines in the clinic has encouraged the development of mRNA vaccines for other pathogens. Here, the immunogenicity and protective efficacy of a quadrivalent mRNA vaccine encoding HA from four seasonal influenza viruses, A/California/07/2009 (H1N1), A/Hong Kong/4801/2014 (H3N2), B/Brisbane/60/2008 (B-Victoria lineage), and B/Phuket/3073/2013 (B-Yamagata lineage), was evaluated. In mice, a 120 µg total dose of this quadrivalent mRNA vaccine induced robust antibody titers against each subtype that were commensurate with titers when each antigen was administered alone. Following A/California/04/2009 challenge, mice were fully protected from morbidity and mortality, even at doses as low as 1 µg of each antigen. Additionally, a single administration of 10 µg of quadrivalent mRNA was sufficient to prevent weight loss caused by A/California/04/2009. These results support the promise of this mRNA vaccine for prevention and mitigation of influenza vaccine.

mRNA Vaccine Mitigates SARS-CoV-2 Infections and COVID-19.

The novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was identified in December of 2019 and is responsible for millions of infections and deaths across the globe. Vaccination against SARS-CoV-2 has proven effective to contain the spread of the virus and reduce disease. The production and distribution of these vaccines occurred at a remarkable pace, largely through the employment of the novel mRNA platform. However, interruptions in supply chain and high demand for clinical grade reagents have impeded the manufacture and distribution of mRNA vaccines at a time when accelerated vaccine deployment is crucial. Furthermore, the emergence of SARS-CoV-2 variants across the globe continues to threaten the efficacy of vaccines encoding the ancestral virus spike protein. Here, we report results from preclinical studies on mRNA vaccines developed using a proprietary mRNA production process developed by GreenLight Biosciences. Two mRNA vaccines encoding the full-length, nonstabilized SARS-CoV-2 spike protein, GLB-COV2-042 and GLB-COV2-043, containing uridine and pseudouridine, respectively, were evaluated in rodents for their immunogenicity and protection from SARS-CoV-2 challenge with the ancestral strain and the Alpha (B.1.1.7) and Beta (B.1.351) variants. In mice and hamsters, both vaccines induced robust spike-specific binding and neutralizing antibodies, and in mice, vaccines induced significant T cell responses with a clear Th1 bias. In hamsters, both vaccines conferred significant protection following challenge with SARS-CoV-2 as assessed by weight loss, viral load, and virus replication in the lungs and nasopharynx. These results support the development of GLB-COV2-042 and GLB-COV2-043 for clinical use. IMPORTANCE SARS-CoV-2 continues to disrupt everyday life and cause excess morbidity and mortality worldwide. Vaccination has been key to quelling the impact of this respiratory pathogen, and mRNA vaccines have led the charge on this front. However, the emergence of SARS-CoV-2 variants has sparked fears regarding vaccine efficacy. Furthermore, SARS-CoV-2 vaccines continue to be unevenly distributed across the globe. For these reasons and despite the success of emergency authorized and licensed SARS-CoV-2 vaccines, additional vaccines are needed to meet public health demands. The studies presented here are significant as they demonstrate robust protective efficacy of mRNA vaccines developed by GreenLight Biosciences against not only wild-type SARS-CoV-2, but also Alpha and Beta variants. These results support the progression of GreenLight Biosciences SARS-CoV-2 mRNA vaccines to clinical trials as another defense against SARS-CoV-2.

Regulation of Peripheral Inflammation by a Non-Viable, Non-Colonizing Strain of Commensal Bacteria.

The gastrointestinal tract represents one of the largest body surfaces that is exposed to the outside world. It is the only mucosal surface that is required to simultaneously recognize and defend against pathogens, while allowing nutrients containing foreign antigens to be tolerated and absorbed. It differentiates between these foreign substances through a complex system of pattern recognition receptors expressed on the surface of the intestinal epithelial cells as well as the underlying immune cells. These immune cells actively sample and evaluate microbes and other particles that pass through the lumen of the gut. This local sensing system is part of a broader distributed signaling system that is connected to the rest of the body through the enteric nervous system, the immune system, and the metabolic system. While local tissue homeostasis is maintained by commensal bacteria that colonize the gut, colonization itself may not be required for the activation of distributed signaling networks that can result in modulation of peripheral inflammation. Herein, we describe the ability of a gut-restricted strain of commensal bacteria to drive systemic anti-inflammatory effects in a manner that does not rely upon its ability to colonize the gastrointestinal tract or alter the mucosal microbiome. Orally administered EDP1867, a gamma-irradiated strain of Veillonella parvula, rapidly transits through the murine gut without colonization or alteration of the background microbiome flora. In murine models of inflammatory disease including delayed-type hypersensitivity (DTH), atopic dermatitis, psoriasis, and experimental autoimmune encephalomyelitis (EAE), treatment with EDP1867 resulted in significant reduction in inflammation and immunopathology. Ex vivo cytokine analyses revealed that EDP1867 treatment diminished production of pro-inflammatory cytokines involved in inflammatory cascades. Furthermore, blockade of lymphocyte migration to the gut-associated lymphoid tissues impaired the ability of EDP1867 to resolve peripheral inflammation, supporting the hypothesis that circulating immune cells are responsible for promulgating the signals from the gut to peripheral tissues. Finally, we show that adoptively transferred T cells from EDP1867-treated mice inhibit inflammation induced in recipient mice. These results demonstrate that an orally-delivered, non-viable strain of commensal bacteria can mediate potent anti-inflammatory effects in peripheral tissues through transient occupancy of the gastrointestinal tract, and support the development of non-living bacterial strains for therapeutic applications.

Bacterial-Derived Outer Membrane Vesicles are Potent Adjuvants that Drive Humoral and Cellular Immune Responses.

Discovery and development of novel adjuvants that can improve existing or next generation vaccine platforms have received considerable interest in recent years. In particular, adjuvants that can elicit both humoral and cellular immune responses would be particularly advantageous because the majority of licensed vaccines are formulated with aluminum hydroxide (alum) which predominantly promotes antibodies. We previously demonstrated that bacterial-derived outer membrane vesicles (OMV) possess inherent adjuvanticity and drive antigen-specific antibody and cellular immune responses to OMV components. Here, we investigated the ability of OMVs to stimulate innate and adaptive immunity and to function as a stand-alone adjuvant. We show that OMVs are more potent than heat-inactivated and live-attenuated bacteria in driving dendritic cell activation in vitro and in vivo. Mice immunized with OMVs admixed with heterologous peptides generated peptide-specific CD4 and CD8 T cells responses. Notably, OMV adjuvant induced much greater antibody and B cell responses to co-delivered ovalbumin compared to the responses elicited by the adjuvants alum and CpG DNA. Additionally, pre-existing antibodies raised against the OMVs did not impair OMV adjuvanticity upon repeat immunization. These results indicate that vaccines adjuvanted with OMVs elicit robust cellular and humoral immune responses, supporting further development of OMV adjuvant for use in next-generation vaccines.

Burkholderia pseudomallei OMVs derived from infection mimicking conditions elicit similar protection to a live-attenuated vaccine.

Burkholderia pseudomallei is a Gram-negative, facultative intracellular bacillus that causes the disease melioidosis. B. pseudomallei expresses a number of proteins that contribute to its intracellular survival in the mammalian host. We previously demonstrated that immunization with OMVs derived from B. pseudomallei grown in nutrient-rich media protects mice against lethal disease. Here, we evaluated if OMVs derived from B. pseudomallei grown under macrophage-mimicking growth conditions could be enriched with intracellular-stage proteins in order to improve the vaccine. We show that OMVs produced in this manner (M9 OMVs) contain proteins associated with intracellular survival yet are non-toxic to living cells. Immunization of mice provides significant protection against pulmonary infection similar to that achieved with a live attenuated vaccine and is associated with increased IgG, CD4+, and CD8+ T cells. OMVs possess inherent adjuvanticity and drive DC activation and maturation. These results indicate that M9 OMVs constitute a new promising vaccine against melioidosis.

An oral alpha-galactosylceramide adjuvanted Helicobacter pylori vaccine induces protective IL-1R- and IL-17R-dependent Th1 responses.

Helicobacter pylori causes chronic gastric infection that can lead to peptic ulcers and is an identified risk factor for gastric cancer development. Although much effort has been put into the development of a Helicobacter pylori vaccine over the last three decades, none has yet reached clinical application. Specific challenges pertaining to effective H. pylori vaccine development include the lack of proven vaccine-effective antigens and safe mucosal adjuvants to enhance local immune responses as well as the lack of accepted correlates of protection. Herein, we demonstrate that prophylactic intragastric immunisation with a whole-cell killed H. pylori antigen administered together with the non-toxic oral adjuvant α-galactosylceramide (α-GalCer) induced effective immune protection against H. pylori infection in mice, which was of similar magnitude as when using the "gold standard" cholera toxin as adjuvant. We further describe that this α-GalCer-adjuvanted vaccine formulation elicited strong intestinal and systemic Th1 responses as well as significant antigen-specific mucosal and systemic antibody responses. Finally, we report that the protective intestinal Th1 responses induced by α-GalCer are dependent on CD1d, IL-1R as well as IL-17R signalling. In summary, our results show that α-GalCer is a promising adjuvant for inclusion in an oral vaccine against H. pylori infection.

Alpha-galactosylceramide enhances mucosal immunity to oral whole-cell cholera vaccines.

Cholera is a severe diarrheal disease caused by the bacterium Vibrio cholerae (V. cholerae) that results in 3-4 million cases globally with 100,000-150,000 deaths reported annually. Mostly confined to developing nations, current strategies to control the spread of cholera include the provision of safe drinking water and improved sanitation and hygiene, ideally in conjunction with oral vaccination. However, difficulties associated with the costs and logistics of these strategies have hampered their widespread implementation. Specific challenges pertaining to oral cholera vaccines (OCVs) include a lack of safe and effective adjuvants to further enhance gut immune responses, the complex and costly multicomponent vaccine manufacturing, limitations of conventional liquid formulation and the lack of an integrated delivery platform. Herein we describe the use of the orally active adjuvant α-Galactosylceramide (α-GalCer) to strongly enhance intestinal bacterium- and toxin-specific IgA responses to the OCV, Dukoral® in C57BL/6 and BALB/c mice. We further demonstrate the mucosal immunogenicity of a novel multi-antigen, single-component whole-cell killed V. cholerae strain and the enhancement of its immunogenicity by adding α-GalCer. Finally, we report that combining these components and recombinant cholera toxin B subunit in the SmPill® minisphere delivery system induced strong intestinal and systemic antigen-specific antibody responses.

A Burkholderia pseudomallei Outer Membrane Vesicle Vaccine Provides Cross Protection against Inhalational Glanders in Mice and Non-Human Primates.

Burkholderia mallei is a Gram-negative, non-motile, facultative intracellular bacillus and the causative agent of glanders, a highly contagious zoonotic disease. B. mallei is naturally resistant to multiple antibiotics and there is concern for its potential use as a bioweapon, making the development of a vaccine against B. mallei of critical importance. We have previously demonstrated that immunization with multivalent outer membrane vesicles (OMV) derived from B. pseudomallei provide significant protection against pneumonic melioidosis. Given that many virulence determinants are highly conserved between the two species, we sought to determine if the B. pseudomallei OMV vaccine could cross-protect against B. mallei. We immunized C57Bl/6 mice and rhesus macaques with B. pseudomallei OMVs and subsequently challenged animals with aerosolized B. mallei. Immunization with B. pseudomallei OMVs significantly protected mice against B. mallei and the protection observed was comparable to that achieved with a live attenuated vaccine. OMV immunization induced the production of B.mallei-specific serum IgG and a mixed Th1/Th17 CD4 and CD8 T cell response in mice. Additionally, immunization of rhesus macaques with B. pseudomallei OMVs provided protection against glanders and induced B.mallei-specific serum IgG in non-human primates. These results demonstrate the ability of the multivalent OMV vaccine platform to elicit cross-protection against closely-related intracellular pathogens and to induce robust humoral and cellular immune responses against shared protective antigens.

A novel adjuvanted capsule based strategy for oral vaccination against infectious diarrhoeal pathogens.

Diarrhoeal infections are a major cause of morbidity and mortality with enterotoxigenic Escherichia coli (ETEC) and cholera imposing a significant global burden. There is currently no licensed vaccine for ETEC. Development of new nonliving oral vaccines has proven difficult due to the physicochemical and immunological challenges associated with the oral route. This demands innovative delivery solutions to protect antigens, control their release and build in immune-stimulatory activity. We describe the Single Multiple Pill® (SmPill®) vaccine formulation which combines the benefits of enteric polymer coating to protect against low gastric pH, a dispersed phase to control release and aid the solubility of non-polar components and an optimized combination of adjuvant and antigen to promote mucosal immunity. We demonstrate the effectiveness of this system with whole cell killed E. coli overexpressing colonization factor antigen I (CFA/I), JT-49. Alpha-galactosylceramide was identified as a potent adjuvant within SmPill® that enhanced the immunogenicity of JT-49. The bacteria associated with the dispersed phase were retained within the capsules at gastric pH but released at intestinal pH. Vaccination with an optimized SmPill® formulation promoted CFA/I-specific immunoglobulin A (IgA) responses in the intestinal mucosa in addition to serum IgG and a solubilized adjuvant was indispensable for efficacy.

Delivery strategies to enhance oral vaccination against enteric infections.

While the majority of human pathogens infect the body through mucosal sites, most licensed vaccines are injectable. In fact the only mucosal vaccine that has been widely used globally for infant and childhood vaccination programs is the oral polio vaccine (OPV) developed by Albert Sabin in the 1950s. While oral vaccines against Cholera, rotavirus and Salmonella typhi have also been licensed, the development of additional non-living oral vaccines against these and other enteric pathogens has been slow and challenging. Mucosal vaccines can elicit protective immunity at the gut mucosa, in part via antigen-specific secretory immunoglobulin A (SIgA). However, despite their advantages over the injectable route, oral vaccines face many hurdles. A key challenge lies in design of delivery strategies that can protect antigens from degradation in the stomach and intestine, incorporate appropriate immune-stimulatory adjuvants and control release at the appropriate gastrointestinal site. A number of systems including micro and nanoparticles, lipid-based strategies and enteric capsules have significant potential either alone or in advanced combined formulations to enhance intestinal immune responses. In this review we will outline the opportunities, challenges and potential delivery solutions to facilitate the development of improved oral vaccines for infectious enteric diseases.

Harnessing the antibacterial and immunological properties of mucosal-associated invariant T cells in the development of novel oral vaccines against enteric infections.

Enteric infections are a major cause of mortality and morbidity with significant social and economic implications worldwide and particularly in developing countries. An attractive approach to minimizing the impact of these diseases is via the development of oral vaccination strategies. However, oral vaccination is challenging due to the tolerogenic and hyporesponsive nature of antigen presenting cells resident in the gastrointestinal tract. The inclusion of adjuvants in oral vaccine formulations has the potential to overcome this challenge. To date no oral adjuvants have been licenced for human use and thus oral adjuvant discovery remains a key goal in improving the potential for oral vaccine development. Mucosal-associated invariant T (MAIT) cells are a recently discovered population of unconventional T cells characterized by an evolutionarily conserved αß T cell receptor (TCR) that recognizes antigens presented by major histocompatibility complex (MHC) class I-related (MR1) molecule. MAIT cells are selected intra-thymically by MR1 expressing double positive thymocytes and enter the circulation with a naïve phenotype. In the circulation they develop a memory phenotype and are programmed to home to mucosal tissues and the liver. Once resident in these tissues, MAIT cells respond to bacterial and yeast infections through the production of chemokines and cytokines that aid in the induction of an adaptive immune response. Their abundance in the gastrointestinal tract and ability to promote adaptive immunity suggests that MAIT cell activators may represent attractive novel adjuvants for use in oral vaccination.