Enhancing antibody responses by multivalent antigen display on thymus-independent DNA origami scaffolds.

Protein-based virus-like particles (P-VLPs) are commonly used to spatially organize antigens and enhance humoral immunity through multivalent antigen display. However, P-VLPs are thymus-dependent antigens that are themselves immunogenic and can induce B cell responses that may neutralize the platform. Here, we investigate thymus-independent DNA origami as an alternative material for multivalent antigen display using the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, the primary target of neutralizing antibody responses. Sequential immunization of mice with DNA-based VLPs (DNA-VLPs) elicits protective neutralizing antibodies to SARS-CoV-2 in a manner that depends on the valency of the antigen displayed and on T cell help. Importantly, the immune sera do not contain boosted, class-switched antibodies against the DNA scaffold, in contrast to P-VLPs that elicit strong B cell memory against both the target antigen and the scaffold. Thus, DNA-VLPs enhance target antigen immunogenicity without generating scaffold-directed immunity and thereby offer an important alternative material for particulate vaccine design.

Characterization of the SARS-CoV-2 BA.5.5 and BQ.1.1 Omicron variants in mice and hamsters.

The continued evolution and emergence of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants have resulted in challenges to vaccine and antibody efficacy. The emergence of each new variant necessitates the need to re-evaluate and refine animal models used for countermeasure testing. Here, we tested a recently circulating SARS-CoV-2 Omicron lineage variant, BQ.1.1, in multiple rodent models including K18-human ACE2 (hACE2) transgenic, C57BL/6J, and 129S2 mice, and Syrian golden hamsters. In contrast to a previously dominant BA.5.5 Omicron variant, inoculation of K18-hACE2 mice with BQ.1.1 resulted in substantial weight loss, a characteristic seen in pre-Omicron variants. BQ.1.1 also replicated to higher levels in the lungs of K18-hACE2 mice and caused greater lung pathology than the BA.5.5 variant. However, in C57BL/6J mice, 129S2 mice, and Syrian hamsters, BQ.1.1 did not cause increased respiratory tract infection or disease compared to animals administered BA.5.5. Moreover, the rates of direct contact or airborne transmission in hamsters were not significantly different after BQ.1.1 and BA.5.5 infections. Taken together, these data suggest that the BQ.1.1 Omicron variant has increased virulence in rodent species that express hACE2, possibly due to the acquisition of unique spike mutations relative to earlier Omicron variants. IMPORTANCE As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, there is a need to rapidly assess the efficacy of vaccines and antiviral therapeutics against newly emergent variants. To do so, the commonly used animal models must also be re-evaluated. Here, we determined the pathogenicity of the BQ.1.1 SARS-CoV-2 variant in multiple SARS-CoV-2 animal models including transgenic mice expressing human ACE2 (hACE2), two strains of conventional laboratory mice, and Syrian hamsters. While BQ.1.1 and BA.5.5 infection resulted in similar levels of viral burden and clinical disease in hamsters and the conventional strains of laboratory mice tested, increases in lung infection were detected in hACE2-expressing transgenic mice, which corresponded with greater levels of pro-inflammatory cytokines and lung pathology. Taken together, our data highlight important differences in two closely related Omicron SARS-CoV-2 variant strains and provide a foundation for evaluating countermeasures.

Characterization of the SARS-CoV-2 BA.5.5 and BQ.1.1 Omicron Variants in Mice and Hamsters.

The continued evolution and emergence of novel SARS-CoV-2 variants has resulted in challenges to vaccine and antibody efficacy. The emergence of each new variant necessitates the need to re-evaluate and refine animal models used for countermeasure testing. Here, we tested a currently circulating SARS-CoV-2 Omicron lineage variant, BQ.1.1, in multiple rodent models including K18-hACE2 transgenic, C57BL/6J, and 129S2 mice, and Syrian golden hamsters. In contrast to a previously dominant BA.5.5 Omicron variant, inoculation of K18-hACE2 mice with BQ.1.1 resulted in a substantial weight loss, a characteristic seen in pre-Omicron variants. BQ.1.1 also replicated to higher levels in the lungs of K18-hACE2 mice and caused greater lung pathology than the BA.5.5 variant. However, C57BL/6J mice, 129S2 mice, and Syrian hamsters inoculated with BQ.1.1 showed no differences in respiratory tract infection or disease compared to animals administered BA.5.5. Airborne or direct contact transmission in hamsters was observed more frequently after BQ.1.1 than BA.5.5 infection. Together, these data suggest that the BQ.1.1 Omicron variant has increased virulence in some rodent species, possibly due to the acquisition of unique spike mutations relative to other Omicron variants. IMPORTANCE: As SARS-CoV-2 continues to evolve, there is a need to rapidly assess the efficacy of vaccines and antiviral therapeutics against newly emergent variants. To do so, the commonly used animal models must also be reevaluated. Here, we determined the pathogenicity of the circulating BQ.1.1 SARS-CoV-2 variant in multiple SARS-CoV-2 animal models including transgenic mice expressing human ACE2, two strains of conventional laboratory mice, and Syrian hamsters. While BQ.1.1 infection resulted in similar levels of viral burden and clinical disease in the conventional laboratory mice tested, increases in lung infection were detected in human ACE2-expressing transgenic mice, which corresponded with greater levels of pro-inflammatory cytokines and lung pathology. Moreover, we observed a trend towards greater animal-to-animal transmission of BQ.1.1 than BA.5.5 in Syrian hamsters. Together, our data highlight important differences in two closely related Omicron SARS-CoV-2 variant strains and provide a foundation for evaluating countermeasures.

Human induced pluripotent stem cell engineering establishes a humanized mouse platform for pediatric low-grade glioma modeling.

A major obstacle to identifying improved treatments for pediatric low-grade brain tumors (gliomas) is the inability to reproducibly generate human xenografts. To surmount this barrier, we leveraged human induced pluripotent stem cell (hiPSC) engineering to generate low-grade gliomas (LGGs) harboring the two most common pediatric pilocytic astrocytoma-associated molecular alterations, NF1 loss and KIAA1549:BRAF fusion. Herein, we identified that hiPSC-derived neuroglial progenitor populations (neural progenitors, glial restricted progenitors and oligodendrocyte progenitors), but not terminally differentiated astrocytes, give rise to tumors retaining LGG histologic features for at least 6 months in vivo. Additionally, we demonstrated that hiPSC-LGG xenograft formation requires the absence of CD4 T cell-mediated induction of astrocytic Cxcl10 expression. Genetic Cxcl10 ablation is both necessary and sufficient for human LGG xenograft development, which additionally enables the successful long-term growth of patient-derived pediatric LGGs in vivo. Lastly, MEK inhibitor (PD0325901) treatment increased hiPSC-LGG cell apoptosis and reduced proliferation both in vitro and in vivo. Collectively, this study establishes a tractable experimental humanized platform to elucidate the pathogenesis of and potential therapeutic opportunities for childhood brain tumors.

RNA sequence analysis reveals ITGAL/CD11A as a stromal regulator of murine low-grade glioma growth.

BACKGROUND: Emerging insights from numerous laboratories have revealed important roles for nonneoplastic cells in the development and progression of brain tumors. One of these nonneoplastic cellular constituents, glioma-associated microglia (GAM), represents a unique population of brain monocytes within the tumor microenvironment that have been reported to both promote and inhibit glioma proliferation. To elucidate the role of GAM in the setting of low-grade glioma (LGG), we leveraged RNA sequencing meta-analysis, genetically engineered mouse strains, and human biospecimens. METHODS: Publicly available disease-associated microglia (DAM) RNA-seq datasets were used, followed by immunohistochemistry and RNAScope validation. CD11a-deficient mouse microglia were used for in vitro functional studies, while LGG growth in mice was assessed using anti-CD11a neutralizing antibody treatment of Neurofibromatosis type 1 (Nf1) optic glioma mice in vivo. RESULTS: We identified Itgal/CD11a enrichment in GAM relative to other DAM populations, which was confirmed in several independently generated murine models of Nf1 optic glioma. Moreover, ITGAL/CD11A expression was similarly increased in human LGG (pilocytic astrocytoma) specimens from several different datasets, specifically in microglia from these tumors. Using CD11a-knockout mice, CD11a expression was shown to be critical for murine microglia CX3CL1 receptor (Cx3cr1) expression and CX3CL1-directed motility, as well as glioma mitogen (Ccl5) production. Consistent with an instructive role for CD11a+ microglia in stromal control of LGG growth, antibody-mediated CD11a inhibition reduced mouse Nf1 LGG growth in vivo. CONCLUSIONS: Collectively, these findings establish ITGAL/CD11A as a critical microglia regulator of LGG biology relevant to future stroma-targeted brain tumor treatment strategies.

Asthma reduces glioma formation by T cell decorin-mediated inhibition of microglia.

To elucidate the mechanisms underlying the reduced incidence of brain tumors in children with Neurofibromatosis type 1 (NF1) and asthma, we leverage Nf1 optic pathway glioma (Nf1OPG) mice, human and mouse RNAseq data, and two different experimental asthma models. Following ovalbumin or house dust mite asthma induction at 4-6 weeks of age (WOA), Nf1OPG mouse optic nerve volumes and proliferation are decreased at 12 and 24 WOA, indicating no tumor development. This inhibition is accompanied by reduced expression of the microglia-produced optic glioma mitogen, Ccl5. Human and murine T cell transcriptome analyses reveal that inhibition of microglia Ccl5 production results from increased T cell expression of decorin, which blocks Ccl4-mediated microglia Ccl5 expression through reduced microglia NFκB signaling. Decorin or NFκB inhibitor treatment of Nf1OPG mice at 4-6 WOA inhibits tumor formation at 12 WOA, thus establishing a potential mechanistic etiology for the attenuated glioma incidence observed in children with asthma.

Midkine activation of CD8+ T cells establishes a neuron-immune-cancer axis responsible for low-grade glioma growth.

Brain tumors (gliomas) are heterogeneous cellular ecosystems, where non-neoplastic monocytic cells have emerged as key regulators of tumor maintenance and progression. However, relative to macrophages/microglia, comparatively less is known about the roles of neurons and T cells in glioma pathobiology. Herein, we leverage genetically engineered mouse models and human biospecimens to define the axis in which neurons, T cells, and microglia interact to govern Neurofibromatosis-1 (NF1) low-grade glioma (LGG) growth. NF1-mutant human and mouse brain neurons elaborate midkine to activate naïve CD8+ T cells to produce Ccl4, which induces microglia to produce a key LGG growth factor (Ccl5) critical for LGG stem cell survival. Importantly, increased CCL5 expression is associated with reduced survival in patients with LGG. The elucidation of the critical intercellular dependencies that constitute the LGG neuroimmune axis provides insights into the role of neurons and immune cells in controlling glioma growth, relevant to future therapeutic targeting.

Comparative evaluation of Salmonella Typhimurium vaccines derived from UK-1 and 14028S: Importance of inherent virulence.

The initial virulence and invasiveness of a bacterial strain may play an important role in leading to a maximally efficacious attenuated live vaccine. Here we show that χ9909, derived from Salmonella Typhimurium UK-1 χ3761 (the most virulent S. Typhimurium strain known to us), is effective in protecting mice against lethal UK-1 and 14028S (less virulent S. Typhimurium strain) challenge. As opposed to this, 14028S-derived vaccine χ12359 induces suboptimal levels of protection, with survival percentages that are significantly lower when challenged with lethal UK-1 challenge doses. T-cell assays have revealed that significantly greater levels of Th1 cytokines IFN-γ and TNF-α were secreted by stimulated T-lymphocytes obtained from UK-1(ΔaroA) immunized mice than those from mice immunized with 14028S(ΔaroA). In addition, UK-1(ΔaroA) showed markedly higher colonizing ability in the spleen, liver, and cecum when compared to 14028S(ΔaroA). Enumeration of bacteria in fecal pellets has also revealed that UK-1(ΔaroA) can persist in the host for over 10 days whereas 14028S(ΔaroA) titers dropped significantly by day 10. Moreover, co-infection of parent strains UK-1 and 14028S resulted in considerably greater recovery of the former in multiple mucosal and gut associated lymphatic tissues. Mice immunized with UK-1(ΔaroA) were also able to clear UK-1 infection remarkably more efficiently from the target organs than 14028S(ΔaroA). Together, these results provide ample evidence to support the hypothesis that attenuated derivatives of parent strains with higher initial virulence make better vaccines.

Multiple antigens of Yersinia pestis delivered by live recombinant attenuated Salmonella vaccine strains elicit protective immunity against plague.

Based on our improved novel Salmonella vaccine delivery platform, we optimized the recombinant attenuated Salmonella typhimurium vaccine (RASV) χ12094 to deliver multiple Yersinia pestis antigens. These included LcrV196 (amino acids, 131-326), Psn encoded on pYA5383 and F1 encoded in the chromosome, their synthesis did not cause adverse effects on bacterial growth. Oral immunization with χ12094(pYA5383) simultaneously stimulated high antibody titers to LcrV, Psn and F1 in mice and presented complete protection against both subcutaneous (s.c.) and intranasal (i.n.) challenges with high lethal doses of Y. pestis CO92. Moreover, no deaths or other disease symptoms were observed in SCID mice orally immunized with χ12094(pYA5383) over a 60-day period. Therefore, the trivalent S. typhimurium-based live vaccine shows promise for a next-generation plague vaccine.

Oral administration of a recombinant attenuated Yersinia pseudotuberculosis strain elicits protective immunity against plague.

A Yersinia pseudotuberculosis PB1+ (Yptb PB1+) mutant strain combined with chromosome insertion of the caf1R-caf1A-caf1M-caf1 operon and deletions of yopJ and yopK, χ10068 [pYV-ω2 (ΔyopJ315 ΔyopK108) ΔlacZ044::caf1R-caf1M-caf1A-caf1] was constructed. Results indicated that gene insertion and deletion did not affect the growth rate of χ10068 compared to wild-type Yptb cultured at 26 °C. In addition, the F1 antigen in χ10068 was synthesized and secreted on the surface of bacteria at 37 °C (mammalian body temperature), not at ambient culture temperature (26 °C). Immunization with χ10068 primed antibody responses and specific T-cell responses to F1 and YpL (Y. pestis whole cell lysate). Oral immunization with a single dose of χ10068 provided 70% protection against a subcutaneous (s.c.) challenge with ∼ 2.6 × 10(5) LD50 of Y. pestis KIM6+ (pCD1Ap) (KIM6+Ap) and 90% protection against an intranasal (i.n.) challenge with ∼ 500 LD50 of KIM6+Ap in mice. Our results suggest that χ10068 can be used as an effective precursor to make a safe vaccine to prevent plague in humans and to eliminate plague circulation among humans and animals.

LcrV delivered via type III secretion system of live attenuated Yersinia pseudotuberculosis enhances immunogenicity against pneumonic plague.

Here, we constructed a Yersinia pseudotuberculosis mutant strain with arabinose-dependent regulated and delayed shutoff of crp expression (araC P(BAD) crp) and replacement of the msbB gene with the Escherichia coli msbB gene to attenuate it. Then, we inserted the asd mutation into this construction to form χ10057 [Δasd-206 ΔmsbB868::P(msbB) msbB(EC) ΔP(crp21)::TT araC P(BAD) crp] for use with a balanced-lethal Asd-positive (Asd(+)) plasmid to facilitate antigen synthesis. A hybrid protein composed of YopE (amino acids [aa]1 to 138) fused with full-length LcrV (YopE(Nt138)-LcrV) was synthesized in χ10057 harboring an Asd(+) plasmid (pYA5199, yopE(Nt138)-lcrV) and could be secreted through a type III secretion system (T3SS) in vitro and in vivo. Animal studies indicated that mice orally immunized with χ10057(pYA5199) developed titers of IgG response to whole-cell lysates of Y. pestis (YpL) and subunit LcrV similar to those seen with χ10057(pYA3332) (χ10057 plus an empty plasmid). However, only immunization of mice with χ10057(pYA5199) resulted in a significant secretory IgA response to LcrV. χ10057(pYA5199) induced a higher level of protection (80% survival) against intranasal (i.n.) challenge with ~240 median lethal doses (LD50) (2.4 × 10(4) CFU) of Y. pestis KIM6+(pCD1Ap) than χ10057(pYA3332) (40% survival). Splenocytes from mice vaccinated with χ10057(pYA5199) produced significant levels of gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin-17 (IL-17) after restimulation with LcrV and YpL antigens. Our results suggest that it is possible to use an attenuated Y. pseudotuberculosis strain delivering the LcrV antigen via the T3SS as a potential vaccine candidate against pneumonic plague.

Evaluation of YadC protein delivered by live attenuated Salmonella as a vaccine against plague.

Yersinia pestis YadB and YadC are two new outer membrane proteins related to its pathogenicity. Here, codon-optimized yadC, yadC810 (aa 32-551), or yadBC antigen genes delivered by live attenuated Salmonella strains are evaluated in mice for induction of protective immune responses against Y. pestis CO92 through subcutaneous or intranasal challenge. Our findings indicate that mice immunized with Salmonella synthesizing YadC, YadC810, or YadBC develop significant serum IgG responses to purified recombinant YadC protein. For subcutaneous challenge (approximately 230 LD50 of Y. pestis CO92), mice immunized with Salmonella synthesizing YadC or YadC810 are afforded 50% protection, but no protection by immunization with the Salmonella strain synthesizing YadBC. None of these antigens provided protection against intranasal challenge (approximately 31 LD50 of Y. pestis CO92). In addition, subcutaneous immunization with purified YadC810 protein emulsified with alum adjuvant does not elicit a protective response against Y. pestis administered by either challenge route.

Perforin- and granzyme-mediated cytotoxic effector functions are essential for protection against Francisella tularensis following vaccination by the defined F. tularensis subsp. novicida ΔfopC vaccine strain.

A licensed vaccine against Francisella tularensis is currently not available. Two Francisella tularensis subsp. novicida (herein referred to by its earlier name, Francisella novicida) attenuated strains, the ΔiglB and ΔfopC strains, have previously been evaluated as potential vaccine candidates against pneumonic tularemia in experimental animals. F. novicida ΔiglB, a Francisella pathogenicity island (FPI) mutant, is deficient in phagosomal escape and intracellular growth, whereas F. novicida ΔfopC, lacking the outer membrane lipoprotein FopC, which is required for evasion of gamma interferon (IFN-γ)-mediated signaling, is able to escape and replicate in the cytosol. To dissect the difference in protective immune mechanisms conferred by these two vaccine strains, we examined the efficacy of the F. novicida ΔiglB and ΔfopC mutants against pulmonary live-vaccine-strain (LVS) challenge and found that both strains provided comparable protection in wild-type, major histocompatibility complex class I (MHC I) knockout, and MHC II knockout mice. However, F. novicida ΔfopC-vaccinated but not F. novicida ΔiglB-vaccinated perforin-deficient mice were more susceptible and exhibited greater bacterial burdens than similarly vaccinated wild-type mice. Moreover, perforin produced by natural killer (NK) cells and release of granzyme contributed to inhibition of LVS replication within macrophages. This NK cell-mediated LVS inhibition was enhanced with anti-F. novicida ΔfopC immune serum, suggesting antibody-dependent cell-mediated cytotoxicity (ADCC) in F. novicida ΔfopC-mediated protection. Overall, this study provides additional immunological insight into the basis for protection conferred by live attenuated F. novicida strains with different phenotypes and supports further investigation of this organism as a vaccine platform for tularemia.

Non-FcεR bearing mast cells secrete sufficient interleukin-4 to control Francisella tularensis replication within macrophages.

Mast cells have classically been implicated in the triggering of allergic and anaphylactic reactions. However, recent findings have elucidated the ability of these cells to selectively release a variety of cytokines leading to bacterial clearance through neutrophil and dendritic cell mobilization, and suggest an important role in innate host defenses. Our laboratory has established a primary bone marrow derived mast cell-macrophage co-culture system and found that mast cells mediated a significant inhibition of Francisella tularensis live vaccine strain (LVS) uptake and replication within macrophages through contact and the secreted product interleukin-4 (IL-4). In this study, we utilized P815 mast cells and J774 macrophages to further investigate whether mast cell activation by non-FcεR driven signals could produce IL-4 and control intramacrophage LVS replication. P815 supernatants collected upon activation by the mast cell activating peptide MP7, as well as P815 cells co-cultured with J774 macrophages, exhibited marked inhibition of bacterial uptake and replication, which correlated with the production of IL-4. The inhibition noted in vitro was titratable and preserved at ratios relevant to cellular infiltration events following pulmonary challenge. Collectively, our data suggest that both primary mast cell and P815 mast cell (lacking FcεR) secreted IL-4 can control intramacrophage Francisella replication.