Cas9-specific immune responses compromise local and systemic AAV CRISPR therapy in multiple dystrophic canine models.

Adeno-associated virus (AAV)-mediated CRISPR-Cas9 editing holds promise to treat many diseases. The immune response to bacterial-derived Cas9 has been speculated as a hurdle for AAV-CRISPR therapy. However, immunological consequences of AAV-mediated Cas9 expression have thus far not been thoroughly investigated in large mammals. We evaluate Cas9-specific immune responses in canine models of Duchenne muscular dystrophy (DMD) following intramuscular and intravenous AAV-CRISPR therapy. Treatment results initially in robust dystrophin restoration in affected dogs but also induces muscle inflammation, and Cas9-specific humoral and cytotoxic T-lymphocyte (CTL) responses that are not prevented by the muscle-specific promoter and transient prednisolone immune suppression. In normal dogs, AAV-mediated Cas9 expression induces similar, though milder, immune responses. In contrast, other therapeutic (micro-dystrophin and SERCA2a) and reporter (alkaline phosphatase, AP) vectors result in persistent expression without inducing muscle inflammation. Our results suggest Cas9 immunity may represent a critical barrier for AAV-CRISPR therapy in large mammals.

Expression of tdTomato and luciferase in a murine lung cancer alters the growth and immune microenvironment of the tumor.

Imaging techniques based on fluorescence and bioluminescence have been important tools in visualizing tumor progression and studying the effect of drugs and immunotherapies on tumor immune microenvironment in animal models of cancer. However, transgenic expression of foreign proteins may induce immune responses in immunocompetent syngeneic tumor transplant models and augment the efficacy of experimental drugs. In this study, we show that the growth rate of Lewis lung carcinoma (LL/2) tumors was reduced after transduction of tdTomato and luciferase (tdTomato/Luc) compared to the parental cell line. tdTomato/Luc expression by LL/2 cells altered the tumor microenvironment by increasing tumor-infiltrating lymphocytes (TILs) while inhibiting tumor-induced myeloid-derived suppressor cells (MDSCs). Interestingly, tdTomato/Luc expression did not alter the response of LL/2 tumors to anti-PD-1 and anti-CTLA-4 antibodies. These results suggest that the use of tdTomato/Luc-transduced cancer cells to conduct studies in immune competent mice may lead to cell-extrinsic tdTomato/Luc-induced alterations in tumor growth and tumor immune microenvironment that need to be taken into consideration when evaluating the efficacy of anti-cancer drugs and vaccines in immunocompetent animal models.

A high throughput reporter virus particle microneutralization assay for quantitation of Zika virus neutralizing antibodies in multiple species.

Zika virus is a Flavivirus, transmitted via Aedes mosquitos, that causes a range of symptoms including Zika congenital syndrome. Zika has posed a challenging situation for health, public and economic sectors of affected countries. To quantitate Zika virus neutralizing antibody titers in serum samples, we developed a high throughput plate based Zika virus reporter virus particle (RVP) assay that uses an infective, non-replicating particle encoding Zika virus surface proteins and capsid (CprME) and a reporter gene (Renilla luciferase). This is the first characterization of a Zika virus RVP assay in 384-well format using a Dengue replicon Renilla reporter construct. Serially diluted test sera were incubated with RVPs, followed by incubation with Vero cells. RVPs that have not been neutralized by antibodies in the test sera entered the cells and expressed Renilla luciferase. Quantitative measurements of neutralizing activity were determined using a plate-based assay and commercially available substrate. The principle of limiting the infection to a single round increases the precision of the assay measurements. RVP log10EC50 titers correlated closely with titers determined using a plaque reduction neutralization test (PRNT) (R2>95%). The plate-based Zika virus RVP assay also demonstrated high levels of precision, reproducibility and throughput. The assay employs identical reagents for human, rhesus macaque and mouse serum matrices. Spiking studies indicated that the assay performs equally well in different species, producing comparable titers irrespective of the serum species. The assay is conducted in 384-well plates and can be automated to simultaneously achieve high throughput and high reproducibility.

Single-cell reporters for pathogen responses to antimicrobial host attacks.

Host-pathogen interactions are often heterogeneous involving individual encounters between host and pathogen cells with diverse molecular mechanisms, response networks, and diverging outcomes. Single-cell reporters can identify the various types of interactions and participating pathogen subsets, help to unravel underlying molecular mechanism, and determine individual outcomes and their impact on disease progression. In this review, we discuss reporters-based on fluorescent proteins. We present different types of reporters and their experimental advantages and challenges, and describe how different strategies can interrogate exposure to antimicrobial host mechanism, pathogen response, inflicted damage, and impact on pathogen fitness at the single-cell level. We find many gaps in available tools but also exciting avenues to address these issues.

A mouse model that is immunologically tolerant to reporter and modifier proteins.

Reporter proteins have become an indispensable tool in biomedical research. However, exogenous introduction of these reporters into mice poses a risk of rejection by the immune system. Here, we describe the generation, validation and application of a multiple reporter protein tolerant 'Tol' mouse model that constitutively expresses an assembly of shuffled reporter proteins from a single open reading frame. We demonstrate that expression of the Tol transgene results in the deletion of CD8+ T cells specific for a model epitope, and substantially improves engraftment of reporter-gene transduced T cells. The Tol strain provides a valuable mouse model for cell transfer and viral-mediated gene transfer studies, and serves as a methodological example for the generation of poly-tolerant mouse strains.

Novel, Non-Gene-Destructive Knock-In Reporter Mice Refute the Concept of Monoallelic Gata3 Expression.

Accurately tuned expression levels of the transcription factor GATA-3 are crucial at several stages of T cell and innate lymphoid cell development and differentiation. Moreover, several lines of evidence suggest that Gata3 expression might provide a reliable molecular marker for the identification of elusive progenitor cell subsets at the earliest stages of T lineage commitment. To be able to faithfully monitor Gata3 expression noninvasively at the single-cell level, we have generated a novel strain of knock-in reporter mice, termed GATIR, by inserting an expression cassette encoding a bright fluorescent marker into the 3'-untranslated region of the endogenous Gata3 locus. Importantly, in contrast to three previously published strains of Gata3 reporter mice, GATIR mice preserve physiological Gata3 expression on the targeted allele. In this study, we show that GATIR mice faithfully reflect endogenous Gata3 expression without disturbing the development of GATA-3-dependent lymphoid cell populations. We further show that GATIR mice provide an ideal tool for noninvasive monitoring of Th2 polarization and straightforward identification of innate lymphoid cell 2 progenitor populations. Finally, as our reporter is non-gene-destructive, GATIR mice can be bred to homozygosity, not feasible with previously published strains of Gata3 reporter mice harboring disrupted alleles. The availability of hetero- and homozygous Gata3 reporter mice with an exceptionally bright fluorescent marker, allowed us to visualize allelic Gata3 expression in individual cells simply by flow cytometry. The unambiguous results obtained provide compelling evidence against previously postulated monoallelic Gata3 expression in early T lineage and hematopoietic stem cell subsets.

Cre-loxP Reporter Mouse Reveals Stochastic Activity of the Foxp3 Promoter.

Mouse models that combine specific loxP-flanked gene sequences with Cre recombinase expressed from cell-regulated promoters have become important tools to investigate gene function. Critically however, expression of Cre recombinase may not always be restricted to the target cell or tissue of interest due to promiscuous activity of the driving promoter. Expression of Cre recombinase and, by extension, excision of the loxP-flanked gene may occur in non-target cells and may not be readily apparent. Here we report on the fidelity of Cre recombinase expressed from the il17a or Foxp3 promoters by combining them with a constitutively expressed floxed-stopped tdTomato reporter gene. Foxp3-driven Cre recombinase in F1 mice induced tdTomato red fluorescent protein in Treg cells but also in a range of other immune cells. Frequency of tdTomato expression was variable but positively correlated (p < 0.0001) amongst lymphoid (B cells and CD8 T cells) and blood-resident myeloid cells (dendritic cells, monocytes, neutrophils) suggesting stochastic activity of the Foxp3 promoter rather than developmental regulation in common ancestral progenitors. Interestingly, frequency of tdTomato+ dendritic cells, monocytes and neutrophils did not correlate with the tdTomato+ fraction in eosinophils, indicating that activity of the Foxp3 promoter in eosinophils occurred after the split from a common multipotent progenitor. When these F1 mice were crossed to achieve homozygosity of the promoter and reporter gene, a novel visually red phenotype was observed segregating amongst littermates. The red coloration was widespread and prevalent in non-immune tissues. Thymocytes examined from these red mice showed that all four subsets of immature thymocytes (CD4- CD8-) based on differential expression of CD25 and CD44 were expressing tdTomato. Finally, we show evidence of Foxp3 Cre recombinase independent tdTomato expression, suggesting germ line transmission of an activated tdTomato reporter gene. Our data highlights potential issues with conclusions drawn from using specifically the B6.129(Cg)-Foxp3tm4(YFP/Cre)Ayr/J mice.

Detection of anti-adalimumab antibodies in a RA responsive cohort of patients using three different techniques.

Reliable monitoring of clinical relevant anti-drug antibodies is fundamental in the follow-up of patients under adalimumab treatment. The aim of this study is to compare anti-adalimumab antibodies by using three methods based on different technologies. A cross-sectional study was performed in 50 patients with rheumatoid arthritis (RA) treated with adalimumab. Anti-adalimumab antibodies were detected in patients' sera by different techniques: bridging ELISA, reporter gene assay (RGA), and surface plasmon resonance (SPR). Results showed that all methods recognized anti-adalimumab antibodies and the percentage of positives fluctuated among the assays. Five (10%) of the 50 patients were positive in ELISA, 4 (8%) in RGA, and 6 (12%) in SPR. Among positive patients, 4 were positive in the three assays, one patient uniquely in ELISA, and two in SPR. Spearman correlation between ELISA and RGA showed good agreement (Spearman r = 0.800). No correlation between RGA and SPR was observed (Spearman r = 0.108). Similar results were obtained between ELISA and SPR (Spearman r = - 0.241). Summarizing, ELISA, RGA and SPR recognized anti-adalimumab antibodies in few RA patients, showing good agreement among the methodology employed. On the other hand, differences observed between SPR and ELISA or RGA highlight the relevance of the employed technologies in anti-drug antibody identification.

Monitoring C5aR2 Expression Using a Floxed tdTomato-C5aR2 Knock-In Mouse.

The biological significance of C5a receptor [(C5aR)2/C5L2], a seven-transmembrane receptor binding C5a and C5adesArg, remains ill-defined. Specific ligation of C5aR2 inhibits C5a-induced ERK1/2 activation, strengthening the view that C5aR2 regulates C5aR1-mediated effector functions. Although C5aR2 and C5aR1 are often coexpressed, a detailed picture of C5aR2 expression in murine cells and tissues is still lacking. To close this gap, we generated a floxed tandem dye (td)Tomato-C5aR2 knock-in mouse that we used to track C5aR2 expression in tissue-residing and circulating immune cells. We found the strongest C5aR2 expression in the brain, bone marrow, and airways. All myeloid-derived cells expressed C5aR2, although with different intensities. C5aR2 expression in blood and tissue neutrophils was strong and homogeneous. Specific ligation of C5aR2 in neutrophils from tdTomato-C5aR2 mice blocked C5a-driven ERK1/2 phosphorylation, demonstrating functionality of C5aR2 in the reporter mice. In contrast to neutrophils, we found tissue-specific differences in C5aR2 expression in eosinophils, macrophages, and dendritic cell subsets. Naive and activated T cells stained negative for C5aR2, whereas B cells from different tissues homogeneously expressed C5aR2. Also, NK cell subsets in blood and spleen strongly expressed C5aR2. Activation of C5aR2 in NK cells suppressed IL-12/IL-18-induced IFN-γ production. Intratracheal IL-33 challenge resulted in decreased C5aR2 expression in pulmonary eosinophils and monocyte-derived dendritic cells. In summary, we provide a detailed map of murine C5aR2 immune cell expression in different tissues under steady-state conditions and upon pulmonary inflammation. The C5aR2 knock-in mouse will help to reliably track and conditionally delete C5aR2 expression in experimental models of inflammation.

Effects of transgene expression level per cell in mice livers on induction of transgene-specific immune responses after hydrodynamic gene transfer.

We previously showed that high and sustained transgene expression of antigenic proteins induced transgene-specific immune responses. In the present study, a detailed relationship between the level of transgene expression per cell and immune response after hydrodynamic gene transfer was investigated. Cypridina luciferase (cLuc), a secretory antigenic reporter protein, was selected as a model antigen, and pROSA-cLuc, a plasmid expressing cLuc, was constructed. A fixed dose (30 µg) of pROSA-cLuc was delivered to mice by a single hydrodynamic injection or three injections at 24-h intervals because the number of cells transfected with plasmids is dependent on the number of hydrodynamic injections. Serum cLuc activity, an indicator of the total amount of cLuc transgene expression, was almost equal between these two groups. In contrast, the high-dose single injection induced higher levels of cLuc-specific humoral and cellular immune responses than the three low-dose injections. Moreover, the serum cLuc activity of the high-dose single injection group began to decline ~10 days after injection, whereas the activity remained constant in the three low-dose injection group. These results indicate that it is preferable to reduce the level of transgene expression per cell to avoid induction of the transgene-specific immune response after hydrodynamic gene transfer.

Current status of gene therapy for cancer.

PURPOSE OF REVIEW: In recent years, remarkable progress has been made in the development of cancer gene therapy into an applicable treatment modality for immunogene, suicide, gene correction and oncolytic therapies. New exciting developments for gene suppression or miRNA therapies are under way. The efforts are focused on more efficient and specific attack at known and novel targets, improvement of vector delivery and therapeutic efficacy. In this review, promising and new gene therapy approaches and clinical studies are briefly discussed to highlight important future directions of preclinical and clinical efforts. RECENT FINDINGS: Apart from progress for vector development and even more important, improvements for suicide, T-cell-based, oncolytic virus therapies were achieved. In addition, new emerging therapies are successfully developed, which are particularly promising for siRNA-based technologies applied to gene suppression therapy. Novel approaches, such as transcription factor ODN-based decoy, complement the spectrum of current cancer gene therapy. SUMMARY: In summary, cancer gene therapy has made remarkable progress in the improvement/refinement of existing strategies and delivery systems. The field is moving toward a therapeutic option, which will also be applicable for the treatment of disseminated metastases. Furthermore, numerous new approaches are about to be translated in clinical trials.

Complete TCR-α gene locus control region activity in T cells derived in vitro from embryonic stem cells.

Locus control regions (LCRs) are cis-acting gene regulatory elements with the unique, integration site-independent ability to transfer the characteristics of their locus-of-origin's gene expression pattern to a linked transgene in mice. LCR activities have been discovered in numerous T cell lineage-expressed gene loci. These elements can be adapted to the design of stem cell gene therapy vectors that direct robust therapeutic gene expression to the T cell progeny of engineered stem cells. Currently, transgenic mice provide the only experimental approach that wholly supports all the critical aspects of LCR activity. In this study, we report the manifestation of all key features of mouse TCR-α gene LCR function in T cells derived in vitro from mouse embryonic stem cells. High-level, copy number-related TCR-α LCR-linked reporter gene expression levels are cell type restricted in this system, and upregulated during the expected stage transition of T cell development. We also report that de novo introduction of TCR-α LCR-linked transgenes into existing T cell lines yields incomplete LCR activity. These data indicate that establishing full TCR-α LCR activity requires critical molecular events occurring prior to final T lineage determination. This study also validates a novel, tractable, and more rapid approach for the study of LCR activity in T cells, and its translation to therapeutic genetic engineering.

Screening for potential effects of endocrine-disrupting chemicals in peri-urban creeks and rivers in Melbourne, Australia using mosquitofish and recombinant receptor-reporter gene assays.

Sexually mature male mosquitofish (Gambusia holbrooki) were collected from various sites around Melbourne in 2009 to evaluate the performance of gonopodial indices as a biomarker for endocrine disruption in Melbourne's waterways. The mosquitofish indices assessed were body length (BL), gonopodial length (GL)/BL ratio, ray 4:6 ratio and the absence or presence of hooks and serrae, and these varied between sites. The study was complemented by measurements of estrogenic, retinoid, thyroid and aryl hydrocarbon (AhR) receptor activities of the water. Male mosquitofish were 16.3-21.5 mm in length, and although there was a statistically significant positive relationship showing that bigger fish had longer gonopodia than small fish (r2 = 0.52, p < 0.001), there were few significant differences in GL/BL ratio of fish between sites. Measured estrogenic activity was mostly in the range 0.1-1.7 ng/L EEQ, with one site having much higher levels (~12 ng/L EEQ). Aryl hydrocarbon (AhR) receptor activity was observed in all water samples (7-180 ng/L ßNF EQ), although there was no consistent pattern in the level of AhR activity observed, i.e., 'clean' sites were as likely to return a high AhR activity response as urban or wastewater treatment plant (WWTP)-impacted sites. There was no correlation between measurements of receptor actvity and gonopodial length (GL):BL ratio and BL. We conclude that the mosquitofish gonopodia only fulfills part of the criteria for biomarker selection for screening. The mosquitofish indices assessed were cheap and easy-to-perform procedures; however, there is no baseline data from the selected sites to evaluate whether differences in the morpholical indices observed at a site were a result of natural selection in the population or due to estrogenic exposure.

B lymphocyte-induced maturation protein-1 contributes to intestinal mucosa homeostasis by limiting the number of IL-17-producing CD4+ T cells.

The transcription factor B lymphocyte-induced maturation protein-1 (Blimp-1) plays important roles in embryonic development and immunity. Blimp-1 is required for the differentiation of plasma cells, and mice with T cell-specific deletion of Blimp-1 (Blimp-1CKO mice) develop a fatal inflammatory response in the colon. Previous work demonstrated that lack of Blimp-1 in CD4(+) and CD8(+) T cells leads to intrinsic functional defects, but little is known about the functional role of Blimp-1 in regulating differentiation of Th cells in vivo and their contribution to the chronic intestinal inflammation observed in the Blimp1CKO mice. In this study, we show that Blimp-1 is required to restrain the production of the inflammatory cytokine IL-17 by Th cells in vivo. Blimp-1CKO mice have greater numbers of IL-17-producing TCRß(+)CD4(+)cells in lymphoid organs and in the intestinal mucosa. The increase in IL-17-producing cells was not restored to normal levels in wild-type and Blimp-1CKO-mixed bone marrow chimeric mice, suggesting an intrinsic role for Blimp-1 in constraining the production of IL-17 in vivo. The observation that Blimp-1-deficient CD4(+) T cells are more prone to differentiate into IL-17(+)/IFN-γ(+) cells and cause severe colitis when transferred to Rag1-deficient mice provides further evidence that Blimp-1 represses IL-17 production. Analysis of Blimp-1 expression at the single cell level during Th differentiation reveals that Blimp-1 expression is induced in Th1 and Th2 but repressed by TGF-ß in Th17 cells. Collectively, the results described here establish a new role for Blimp-1 in regulating IL-17 production in vivo.

On the role of regulatory T cells during viral-induced inflammatory lesions.

Ocular HSV-1 infection can result in stromal keratitis, a blinding immunoinflammatory lesion that represents an immunopathological response to the infection. CD4(+) T cells are the main orchestrators, and lesions are more severe if the regulatory T cell (Treg) response is compromised from the onset of infection. Little is known about the role of Foxp3(+)CD4(+) Tregs during ongoing inflammatory reactions, which is the topic of this article. We used DEREG mice and depleted Tregs at different times postinfection. We show that lesions became more severe even when depletion was begun in the clinical phase of the disease. This outcome was explained both by Tregs' influence on the activity of inflammatory effector T cells at the lesion site and by an effect in lymphoid tissues that led to reduced numbers of effectors and less trafficking of T cells and neutrophils to the eye. Our results demonstrate that Tregs can beneficially influence the impact of ongoing tissue-damaging responses to a viral infection and imply that therapies boosting Treg function in the clinical phase hold promise for controlling a lesion that is an important cause of human blindness.

IFN-γ elicits macrophage autophagy via the p38 MAPK signaling pathway.

Autophagy is a major innate immune defense pathway in both plants and animals. In mammals, this cascade can be elicited by cytokines (IFN-γ) or pattern recognition receptors (TLRs and nucleotide-binding oligomerization domain-like receptors). Many signaling components in TLR- and nucleotide-binding oligomerization domain-like receptor-induced autophagy are now known; however, those involved in activating autophagy via IFN-γ remain to be elucidated. In this study, we engineered macrophages encoding a tandem fluorescently tagged LC3b (tfLC3) autophagosome reporter along with stably integrated short hairpin RNAs to demonstrate IFN-γ-induced autophagy required JAK 1/2, PI3K, and p38 MAPK but not STAT1. Moreover, the autophagy-related guanosine triphosphatase Irgm1 proved dispensable in both stable tfLC3-expressing RAW 264.7 and tfLC3-transduced Irgm1(-/-) primary macrophages, revealing a novel p38 MAPK-dependent, STAT1-independent autophagy pathway that bypasses Irgm1. These unexpected findings have implications for understanding how IFN-γ-induced autophagy is mobilized within macrophages for inflammation and host defense.

Cytokine reporter mice: the special case of IL-10.

IL-10 is one of the key cytokines preventing inflammation-mediated tissue damage. In an attempt to identify IL-10-producing cells in vivo, several groups have recently developed IL-10 reporter mouse strains. Up until now, in total, eight IL-10 reporter strains have been published. This incomparable interest in IL-10 reporter mice emphasizes the importance and difficulties in tracking and subsequently investigating the role of IL-10-producing cells in infectious, inflammatory, autoimmune and cancer diseases. In this review, I summarize and compare the properties of those published IL-10 reporter mouse models. I also discuss the necessity to develop new strategies to generate 'multi-cytokine' reporter mouse models enabling highly sensitive in/ex vivo detection of many cytokines in the same single cell. Such 'multi-cytokine' reporter mice will enable to reconsider the dichotomy 'T-effector versus T-regulatory' paradigm and to provide an accurate revised model for cellular sources of cytokines. Finally, I propose to launch cooperative, international initiatives to promote and coordinate the generation of accurate, combinatorial, reporter mice for every individual murine cytokine.

Convergent and divergent development among M cell lineages in mouse mucosal epithelium.

M cells are specialized epithelial cells mediating immune surveillance of the mucosal lumen by transepithelial delivery of Ags to underlying dendritic cells (DC). At least three M cell phenotypes are known in the airways and intestine, but their developmental relationships are unclear. We used reporter transgenic mouse strains to follow the constitutive development of M cell subsets and their acute induction by cholera toxin (CT). M cells overlying intestinal Peyer's patches (PPs), isolated lymphoid follicles, and nasal-associated lymphoid tissue are induced by distinct settings, yet show convergent phenotypes, such as expression of a peptidoglycan recognition protein-S (PGRP-S) transgene reporter. By contrast, though PP, isolated lymphoid follicle, and villous M cells are all derived from intestinal crypt stem cells, their phenotypes were clearly distinct; for example, PP M cells frequently appeared to form M cell-DC functional units, whereas villous M cells did not consistently engage underlying DC. B lymphocytes are critical to M cell function by forming a basolateral pocket and possible signaling through CD137; however, initial commitment to all M cell lineages is B lymphocyte and CD137 independent. CT causes induction of new M cells in the airway and intestine without cell division, suggesting transdifferentiation from mature epithelial cells. In contrast with intestinal PP M cells, CT-induced nasal-associated lymphoid tissue M cells appear to be generated from ciliated Foxj1(+)PGRP-S(+) cells, indicative of a possible precommitted progenitor. In summary, constitutive and inducible differentiation of M cells is toward strictly defined context-dependent phenotypes, suggesting specialized roles in surveillance of mucosal Ags.

Intranasal administration of adeno-associated virus type 12 (AAV12) leads to transduction of the nasal epithelia and can initiate transgene-specific immune response.

A critical aspect in defining the utility of a vector for gene therapy applications is the cell tropism and biodistribution of the vector. Adeno-associated virus type 12 (AAV12) has several unique biological and immunological properties that could be exploited for gene therapy purposes, including a unique cell surface receptor, transduction of epithelial cells, and limited neutralization by pooled human antibodies. However, little is known about its cell tropism and biodistribution in vivo. In vivo biodistribution studies with AAV12 vectors encoding a cytomegalovirus promoted luciferase transgene indicated preferential transduction of the nasal epithelia which was not observed with AAV2-based vectors. Expression peaked 2 weeks postadministration, before decreasing to a persistent level. The level of neutralizing antibodies (Nab) induced was sevenfold lower for AAV12 than for AAV2, an advantage for use in repeat administration. Furthermore, vectors encoding influenza A nucleoprotein (NP), an antigen which has previously been shown to induce immune protection against challenge, resulted in generation of both anti-A/NP antibodies and lung anti-A/NP T cells. Our findings suggest further evaluation of AAV12 as a vector for gene therapy and as a potential nasal vaccine.