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Tumors with microsatellite instability (MSI) are caused by a defective DNA mismatch repair system that leads to the accumulation of mutations within microsatellite regions. Indels in microsatellites of coding genes can result in the synthesis of frameshift peptides (FSP). FSPs are tumor-specific neoantigens shared across patients with MSI. In this study, we developed a neoantigen-based vaccine for the treatment of MSI tumors. Genetic sequences from 320 MSI tumor biopsies and matched healthy tissues in The Cancer Genome Atlas database were analyzed to select shared FSPs. Two hundred nine FSPs were selected and cloned into nonhuman Great Ape Adenoviral and Modified Vaccinia Ankara vectors to generate a viral-vectored vaccine, referred to as Nous-209. Sequencing tumor biopsies of 20 independent patients with MSI colorectal cancer revealed that a median number of 31 FSPs out of the 209 encoded by the vaccine was detected both in DNA and mRNA extracted from each tumor biopsy. A relevant number of peptides encoded by the vaccine were predicted to bind patient HLA haplotypes. Vaccine immunogenicity was demonstrated in mice with potent and broad induction of FSP-specific CD8 and CD4 T-cell responses. Moreover, a vaccine-encoded FSP was processed in vitro by human antigen-presenting cells and was subsequently able to activate human CD8 T cells. Nous-209 is an “off-the-shelf” cancer vaccine encoding many neoantigens shared across sporadic and hereditary MSI tumors. These results indicate that Nous-209 can induce the optimal breadth of immune responses that might achieve clinical benefit to treat and prevent MSI tumors.Significance:These findings demonstrate the feasibility of an “off-the-shelf” vaccine for treatment and prevention of tumors harboring frameshift mutations and neoantigenic peptides as a result of microsatellite instability.

List of 1087 FSPs suitable for the inclusion in NOUS-209 vaccine.

The emergence of escape-mutants of influenza hemagglutinin (HA) following vaccination compels the yearly re-formulation of flu vaccines. Since binding the sialic acid receptor remains in all cases essential for infection, small-molecule inhibitors of HA binding to sialic acid could be interesting therapeutic complements or alternatives to immuno-prophylaxis in the control of flu epidemics. In this work, we made use of NMR spectroscopy to study the interaction between a derivative of sialic acid (the Neu5Ac-α-(2,6)-Gal-β-(1-4)-GlcNAc trisaccharide) and HAs (H1 and H5) from human and avian strains of influenza virus, directly expressed on the surface of stable transfected 293 T human cells. The HAs were shown to retain their native trimeric conformation and binding properties. Exploiting the magnetization transfer between the proteins and the ligand, we obtained evidence of the binding event and mapped the (non-identical) sugar epitopes recognized by the two HA species. The rapid and reliable method for screening sialic acid-related HA ligands we have developed could yield useful information for an efficient drug design.

Tumors with microsatellite instability (MSI) are caused by a defective DNA mismatch repair system that leads to the accumulation of mutations within microsatellite regions. Indels in microsatellites of coding genes can result in the synthesis of frameshift peptides (FSP). FSPs are tumor-specific neoantigens shared across patients with MSI. In this study, we developed a neoantigen-based vaccine for the treatment of MSI tumors. Genetic sequences from 320 MSI tumor biopsies and matched healthy tissues in The Cancer Genome Atlas database were analyzed to select shared FSPs. Two hundred nine FSPs were selected and cloned into nonhuman Great Ape Adenoviral and Modified Vaccinia Ankara vectors to generate a viral-vectored vaccine, referred to as Nous-209. Sequencing tumor biopsies of 20 independent patients with MSI colorectal cancer revealed that a median number of 31 FSPs out of the 209 encoded by the vaccine was detected both in DNA and mRNA extracted from each tumor biopsy. A relevant number of peptides encoded by the vaccine were predicted to bind patient HLA haplotypes. Vaccine immunogenicity was demonstrated in mice with potent and broad induction of FSP-specific CD8 and CD4 T-cell responses. Moreover, a vaccine-encoded FSP was processed in vitro by human antigen-presenting cells and was subsequently able to activate human CD8 T cells. Nous-209 is an “off-the-shelf” cancer vaccine encoding many neoantigens shared across sporadic and hereditary MSI tumors. These results indicate that Nous-209 can induce the optimal breadth of immune responses that might achieve clinical benefit to treat and prevent MSI tumors. Significance: These findings demonstrate the feasibility of an “off-the-shelf” vaccine for treatment and prevention of tumors harboring frameshift mutations and neoantigenic peptides as a result of microsatellite instability.

As a vaccination vector, MVA has been widely investigated both in animal models and humans. The construction of recombinant MVA (rMVA) relies on homologous recombination between an acceptor virus and a donor plasmid in infected/transfected permissive cells. Our construction strategy “Red-to-Green gene swapping” – based on the exchange of two fluorescent markers within the flanking regions of MVA deletion ΔIII, coupled to fluorescence activated cell sorting – is here extended to a second insertion site, within the flanking regions of MVA deletion ΔVI. Exploiting this strategy, both double and triple rMVA were constructed, expressing as transgenes the influenza A proteins HA, NP, M1, and PB1. Upon validation of the harbored transgenes co-expression, double and triple recombinants rMVA(ΔIII)-NP-P2A-M1 and rMVA(ΔIII)-NP-P2A-M1-(ΔVI)-PB1 were assayed for in vivo immunogenicity and protection against lethal challenge. In vivo responses were identical to those obtained with the reported combinations of single recombinants, supporting the feasibility and reliability of the present improvement and the extension of Red-to-Green gene swapping to insertion sites other than ΔIII.

Vaccination offers protection against influenza, although current vaccines need to be reformulated each year. The development of a broadly protective influenza vaccine would guarantee the induction of heterosubtypic immunity also against emerging influenza viruses of a novel subtype. Vaccine candidates based on the stalk region of the hemagglutinin (HA) have the potential to induce broad and persistent protection against diverse influenza A viruses.Modified vaccinia virus Ankara (MVA) expressing a headless HA (hlHA) of A/California/4/09 (CA/09) virus was used as a vaccine to immunize C57BL/6 mice. Specific antibody and cell-mediated immune responses were determined, and challenge experiments were performed by infecting vaccinated mice with CA/09 virus.Immunization of mice with CA/09-derived hlHA, vectored by MVA, was able to elicit influenza-specific broad cross-reactive antibodies and cell-mediated immune responses, but failed to induce neutralizing antibodies and did not protect mice against virus challenge.Although highly immunogenic, our vaccine was unable to induce a protective immunity against influenza. A misfolded and unstable conformation of the hlHA molecule may have affected its capacity of inducing neutralizing antiviral, conformational antibodies. Design of stable hlHA-based immunogens and their delivery by recombinant MVA-based vectors has the potential of improving this promising approach for a universal influenza vaccine.

The emergence of novel strains of influenza A viruses with hemagglutinins (HAs) that are antigenically distinct from those circulating in humans, and thus have pandemic potential, pose concerns and call for the development of more broadly protective influenza vaccines. In the present study, modified vaccinia virus Ankara (MVA) encoding internal influenza antigens were evaluated for their immunogenicity and ability to protect HLA-A2.1 transgenic (AAD) mice from infection with influenza viruses.MVAs expressing NP (MVA-NP), M1 (MVA-M1) or polymerase PB1 (MVA-PB1) of A/California/4/09 (CA/09) virus were generated and used to immunize AAD mice. Antibodies and CD8+T cell responses were assessed by ELISA and ELISPOT, respectively, and challenge experiments were performed by infecting vaccinated mice with CA/09 virus.CD8+T cells specific to immunodominant and subdominant epitopes on the internal influenza proteins were elicited by MVA-based vectors in AAD mice, whereas influenza-specific antibodies were detected only in MVA-NP-immunized mice. Both M1- and NP-based MVA vaccines, regardless of whether they were applied individually or in combination, conferred protection against lethal influenza virus challenge.Our data further emphasize the promising potential of MVA vector expressing internal antigens toward the development of a universal influenza vaccine.

Survivin protein is an attractive candidate for cancer immunotherapy since it is abundantly expressed in most common human cancers and mostly absent in normal adult tissues. Malignant mesothelioma (MM) is a deadly cancer associated with asbestos or erionite exposure for which no successful therapies are currently available. In this study, we evaluated the therapeutic efficacy of a novel survivin-based vaccine by subcutaneous or intraperitoneum injection of BALB/c mice with murine fiber-induced MM tumor cells followed by vaccination with recombinant Fowlpox virus replicons encoding survivin. Vaccination generated significant immune responses in both models, leading to delayed tumor growth and improved animal survival. Flow cytometry and immunofluorescence analyses of tumors from vaccinated mice showed CD8 1 T-cell infiltration, and real-time PCR demonstrated increased mRNA and protein levels of immunostimulatory cytokines. Analyses of survivin peptide-pulsed spleen and lymph node cells from vaccinated mice using ELISPOT and intracellular cytokine staining confirmed antigen-specific, interferon-c-producing CD8 1 T-cell responses. In addition pentamer-based flow cytometry showed that vaccination generated survivin-specific CD8 1 T cells. Importantly, vaccination did not affect fertility or induce autoimmune abnormalities in mice. Our results demonstrate that vaccination with recombinant Fowlpox expressing survivin improves T-cell responses against aggressive MM tumors and may form the basis for promising clinical applications.

The IgE-mediated immune system activation can be redirected to combat tumors. Mouse and human IgE have been shown to provide a potent adjuvant effect in antitumor vaccination, with a crucial role played by FcεRI. This effect results from T cell-mediated adaptive immune response. Modified vaccinia virus Ankara (MVA) has been used to infect IgE-loaded tumor cells. These results led to a shift toward a highly safe protocol employing membrane IgE (mIgE), thus eliminating any possible anaphylactogenicity caused by circulating IgE. Evidence that human mIgE and a truncated version lacking IgE Fabs (tmIgE) bind and activate FcεRI has been fundamental and forms the core of this report. Human tmIgE has been engineered into a recombinant MVA (rMVA-tmIgE), and the expression of tmIgE and its transport to the surface of rMVA-tmIgE–infected cells has been detected by Western blot and cytofluorimetry, respectively. FcεRI activation by tmIgE has been confirmed by the release of β-hexosaminidase in a cell-to-cell contact assay using human FcεRI-transfected RBL-SX38 cells. The rMVA-tmIgE antitumor vaccination strategy has been investigated in FcεRIα−/− human FcεRIα+ mice, with results indicating a level of protection comparable to that obtained using soluble human IgE tumor cell loading. The rMVA-tmIgE vector represents a device that suits safe IgE-based antitumor vaccines, harboring the possibility to couple tmIgE with other gene insertions that might enhance the antitumor effect, thus bringing the field closer to the clinics.

Pairs of recombinant MVA (Modified Vaccinia Ankara) and FPV (Fowlpox Virus) expressing the same transgene are reasonable candidates for prime/boost regimens, because cross-reacting immune responses between the two vectors, both non-replicative in mammalian hosts, are very limited. The acceptor virus FPD-Red, a derivative of FPV, carrying a red fluorescent protein gene flanked by the homology regions of MVA deletion III, was constructed. The same MVA Transfer Plasmid Green, designed to insert transgenes into the MVA deletion III locus, can therefore be used to transfer transgenes into both acceptor viruses MVA-Red and FPD-Red with the described recently Red-to-Green gene swapping method. Cells infected by either recombinant virus can be sorted differentially by a simple and reliable FACS-based purification protocol. The procedure is carried out in primary chick embryo fibroblasts grown in serum-free media and was applied to the production of three rMVA/rFPV pairs expressing the H5N1 avian influenza antigens M1, M2 and NP. The viral genes were human codon-optimized and expressed at high levels in both chick and mammalian cells. Both single-step and multiple-step growth analyses showed no significant differences in growth due to the transgenes in either rMVA or rFPV derivatives.

The hypothesis that open conformers of HLA-C on target cells might directly exert an effect on their infectability by human immunodeficiency virus (HIV) has been suggested previously. This was tested by exploiting the peculiar specificity of monoclonal antibody (mAb) L31 for HLA-C open conformers to show that normal levels of Env-driven fusion were restored in HLA-C transfectants of a major histocompatibility complex-deleted (fusion-incompetent) cell line. The physiological relevance of this finding is now confirmed in this report, where small interfering RNA (siRNA) technology was used to silence HLA-C expression in peripheral blood lymphocytes (PBLs) from 11 healthy donors. Infectability by HIV (strains IIIB and Bal and primary isolates) was significantly reduced (P=0.016) in silenced cells compared with cells that maintained HLA-C expression in 10 of the 11 PBL donors. Normal infectability was resumed, together with HLA-C expression, when the effect of siRNA interference waned after several days in culture. Additional confirmation of the HLA-C effect was obtained in several assays employing HLA-C-positive and -negative cell lines, a number of HIV strains and also pseudoviruses. In particular, viruses pseudotyped with env genes from HIV strains AC10 and QH0692.42 were assayed on siRNA-silenced lymphocytes from three healthy donors: the differences in infection with pseudoviruses were even higher than those observed in infections with normal viruses.

Our previous work involved the development of a recombinant fowlpox virus encoding survivin (FP-surv) vaccine that was evaluated for efficacy in mesothelioma mouse models. Results showed that FP-surv vaccination generated significant immune responses, which led to delayed tumor growth and improved animal survival. We have extended those previous findings in the current study, which involves the pre-clinical development of an optimized version of FP-surv designed for human immunization (HIvax). Survivin-derived peptides for the most common haplotypes in the human population were identified and their immunogenicity confirmed in co-culture experiments using dendritic cells and T cells isolated from healthy donors. Peptides confirmed to induce CD8(+) and CD4(+) T cells activation in humans were then included in 2 transgenes optimized for presentation of processed peptides on MHC-I (HIvax1) and MHC-II (HIvax2). Fowlpox vectors expressing the HIvax transgenes were then generated and their efficacy was evaluated with subsequent co-culture experiments to measure interferon-γ and granzyme B secretion. In these experiments, both antigen specific CD4(+) and CD8(+) T cells were activated by HIvax vaccines with resultant cytotoxic activity against survivin-overexpressing mesothelioma cancer cells. These results provide a rationale for clinical testing of HIvax1 and HIvax2 vaccines in patients with survivin-expressing cancers.

We previously reported that enfuvirtide (ENF) treatment is accompanied by a selective increase of serum IgE. We asked whether ENF had intrinsic capability to direct B-lymphocytes to switch to IgE and/or if it could drive CD4 T cells to a Th2 phenotype. ENF was added in vitro: (a) to B-lymphocytes stimulated with IgE-switch inducing stimuli; (b) to peripheral blood mononuclear cells. Total IgE production by B cells and IL4 and IFN-gamma production by CD4 T lymphocytes were evaluated, respectively. ENF had no measurable effect on the IgE production by B-lymphocytes. In contrast, it sharply increased the IL4 to IFN-gamma (a correlate of the Th2 phenotype) when added in vitro to T cells from healthy donors or from single ENF-treated patients. The hyper-IgE production in ENF-treated patients is associated with the in vitro induction of a type-2 phenotype in CD4 T cells.

Interaction of secretory IgE with FcεRI is the prerequisite for allergen-driven cellular responses, fundamental events in immediate and chronic allergic manifestations. Previous studies reported the binding of soluble FcεRIα to membrane IgE exposed on B cells. In this study, the functional interaction between human membrane IgE and human FcεRI is presented. Four different IgE versions were expressed in mouse B cell lines, namely: a truncation at the Cε2-Cε3 junction of membrane IgE isoform long, membrane IgE isoform long (without Igα/Igβ BCR accessory proteins), and both εBCRs (containing membrane IgE isoforms short and long). All membrane IgE versions activated a rat basophilic leukemia cell line transfected with human FcεRI, as detected by measuring the release of both preformed and newly synthesized mediators. The interaction led also to Ca2+ responses in the basophil cell line, while membrane IgE-FcεRI complexes were detected by immunoprecipitation. FcεRI activation by membrane IgE occurs in an Ag-independent manner. Noteworthily, human peripheral blood basophils and monocytes also were activated upon contact with cells bearing membrane IgE. In humans, the presence of FcεRI in several cellular entities suggests a possible membrane IgE-FcεRI-driven cell-cell dialogue, with likely implications for IgE homeostasis in physiology and pathology.

Survivin protein is an attractive candidate for cancer immunotherapy since it is abundantly expressed in most common human cancers and mostly absent in normal adult tissues. Malignant mesothelioma (MM) is a deadly cancer associated with asbestos or erionite exposure for which no successful therapies are currently available. In this study, we evaluated the therapeutic efficacy of a novel survivin-based vaccine by subcutaneous or intraperitoneum injection of BALB/c mice with murine fiber-induced MM tumor cells followed by vaccination with recombinant Fowlpox virus replicons encoding survivin. Vaccination generated significant immune responses in both models, leading to delayed tumor growth and improved animal survival. Flow cytometry and immunofluorescence analyses of tumors from vaccinated mice showed CD8(+) T-cell infiltration, and real-time PCR demonstrated increased mRNA and protein levels of immunostimulatory cytokines. Analyses of survivin peptide-pulsed spleen and lymph node cells from vaccinated mice using ELISPOT and intracellular cytokine staining confirmed antigen-specific, interferon-γ-producing CD8(+) T-cell responses. In addition pentamer-based flow cytometry showed that vaccination generated survivin-specific CD8(+) T cells. Importantly, vaccination did not affect fertility or induce autoimmune abnormalities in mice. Our results demonstrate that vaccination with recombinant Fowlpox expressing survivin improves T-cell responses against aggressive MM tumors and may form the basis for promising clinical applications.

Background HIV protective downregulating CCR5-ECL1 IgA have been found in mucosal fluids from uninfected sexual partners of HIV-positive individuals and in a subset of HIV-seropositve HIV-controlling subjects. This finding concurred to the hypothesis that lack of expression of CCR5 might play a role in HIV protection. We induced and reproduced this immune status in animal models such as mouse and characterized the immune responses in Peyer's patches.

Working with C57BL/6 mouse tumor models, we had previously demonstrated that vaccination with IgE-coated tumor cells can protect against tumor challenge, an observation that supports the involvement of IgE in antitumor immunity. The adjuvant effect of IgE was shown to result from eosinophil-dependent priming of the T cell-mediated adaptive immune response. The protective effect is likely to be mediated by the interaction of tumor cell-bound IgE with receptors, which then trigger the release of mediators, recruitment of effector cells, cell killing and tumor Ag cross-priming. It was therefore of utmost importance to demonstrate the strict dependence of the protective effect on IgE receptor activation. First, the protective effect of IgE was confirmed in a BALB/c tumor model, in which IgE-loaded modified VV Ankara-infected tumor cells proved to be an effective cellular vaccine. However, the protective effect was lost in Fc(epsilon)RIalpha(-/-) (but not in CD23(-/-)) knockout mice, showing the IgE-Fc(epsilo)nRI interaction to be essential. Moreover, human IgE (not effective in BALB/c mice) had a protective effect in the humanized knockin mouse (Fc(epsilon)RIalpha(-/-) hFc(epsilon)RIalpha(+)). This finding suggests that the adjuvant effect of IgE could be exploited for human therapeutics.

Modified vaccinia virus Ankara (MVA) is employed as a human vaccine vector for the high expression of heterologous genes and the lack of replication in mammalian cells. This study demonstrates that cells infected by recombinant viruses can be obtained by fluorescence-activated cell sorting. Recombinant viruses are generated by a swapping event between a red fluorescent protein gene in the acceptor virus and a plasmid cassette coding for both a green fluorescent marker and a transgene. To prevent the carry-over of parental virus, due to superinfection of the cells harbouring recombinant viruses, the sorting is performed on cells infected at low m.o.i. in the presence of a reversible inhibitor of viral particle release. Terminal dilution cloning is then used to isolate both green and marker-free recombinant viruses, which can be identified by whole-plate fluoroimaging. The differential visualization of all the viral types involved allows a stepwise monitoring of all recombinations and leads to a straightforward and efficient flow cytometry-based cell sorting purification protocol. As an example of the efficacy of this sorting procedure, the construction of rMVA's coding for the rat nuclear protein HMGB1 and H5N1 influenza A virus hemagglutinin is reported. The entire recombinant MVA production process is carried out in serum-free media employing primary chicken embryo fibroblasts (CEF), which are certified for the preparation of human vaccines. This rMVA production method is faster, simpler and more reliable than any other available procedure for obtaining safe vaccine stocks for human use.

Working with C57BL/6 mouse tumor models, we had previously demonstrated that vaccination with IgE-coated tumor cells can protect against tumor challenge, an observation that supports the involvement of IgE in antitumor immunity. The adjuvant effect of IgE was shown to result from eosinophil-dependent priming of the T cell-mediated adaptive immune response. The protective effect is likely to be mediated by the interaction of tumor cell-bound IgE with receptors, which then trigger the release of mediators, recruitment of effector cells, cell killing and tumor Ag cross-priming. It was therefore of utmost importance to demonstrate the strict dependence of the protective effect on IgE receptor activation. First, the protective effect of IgE was confirmed in a BALB/c tumor model, in which IgE-loaded modified VV Ankara-infected tumor cells proved to be an effective cellular vaccine. However, the protective effect was lost in FcεRIα−/− (but not in CD23−/−) knockout mice, showing the IgE-FcεRI interaction to be essential. Moreover, human IgE (not effective in BALB/c mice) had a protective effect in the humanized knockin mouse (FcεRIα−/− hFcεRIα+). This finding suggests that the adjuvant effect of IgE could be exploited for human therapeutics.

Human Th2 cytokines (interleukins 4 and 13) induce co-expression of IgE and IgG4 through sequential switching. The regulation of IgG4 responses and the role of these responses in the pathogenesis of allergy have not been characterized. We are addressing these issues by comparing and contrasting the expression of allergen-specific IgE and IgG4 in a population of European children thoroughly defined for lifestyle, environmental exposures and allergic phenotypes. The current analysis focused exclusively on children from non-farming families (n=493) in order to avoid potential effects of exposure to microbial products abundant in farming environments. We found that allergens induce Th2-mediated IgG4 and/or IgE responses in the majority of the population. Approximately two-thirds of the children had allergen-specific IgG4 but not IgE, only a minority had both IgG4 and IgE, only a few were negative for both, and virtually none had only IgE. The prevalence of asthma and hay fever was dramatically higher in children with high IgG4 and IgE compared to children who only mounted IgG4 or low IgG4 and IgE responses. These results appear to recapitulate different stages of in vivo Th2-dependent sequential switching from IgG4 to IgE. These patterns of Th2-induced antibody responses may warrant a redefinition of the notion of allergen sensitization.

Modified Vaccinia Virus Ankara (MVA) is employed widely as an experimental and human vaccine vector for its lack of replication in mammalian cells and high expression of heterologous genes. Recombinant MVA technology can be improved greatly by combining transient host-range selection (based on the restoration in MVA of the deleted vaccinia gene K1L) with the differential expression of fluorescent proteins. Recombinant virus results from swapping a red protein gene (in the acceptor virus) with a cassette of the transfer plasmid comprising the transgene and the green marker K1Lgfp (a chimeric gene comprising K1L and EGFP). Recombinant selection is performed in the selective host RK13. Finally, in the non-selective host BHK-21, a single crossover between identical flanking regions excises the marker gene. The three types of viruses involved (red parental, green intermediate and colourless final recombinant) are visualized differentially by fluorescence microscopy or fluoro-imaging of terminal dilution microcultures, leading to a straightforward and efficient purification protocol. This method (Red-to-Green gene swapping) reduces greatly the time needed to obtain marker-free recombinant MVA and increases the reliability of the construction process.

Interaction of secretory IgE with FcepsilonRI is the prerequisite for allergen-driven cellular responses, fundamental events in immediate and chronic allergic manifestations. Previous studies reported the binding of soluble FcepsilonRIalpha to membrane IgE exposed on B cells. In this study, the functional interaction between human membrane IgE and human FcepsilonRI is presented. Four different IgE versions were expressed in mouse B cell lines, namely: a truncation at the Cepsilon2-Cepsilon3 junction of membrane IgE isoform long, membrane IgE isoform long (without Igalpha/Igbeta BCR accessory proteins), and both epsilonBCRs (containing membrane IgE isoforms short and long). All membrane IgE versions activated a rat basophilic leukemia cell line transfected with human FcepsilonRI, as detected by measuring the release of both preformed and newly synthesized mediators. The interaction led also to Ca(2+) responses in the basophil cell line, while membrane IgE-FcepsilonRI complexes were detected by immunoprecipitation. FcepsilonRI activation by membrane IgE occurs in an Ag-independent manner. Noteworthily, human peripheral blood basophils and monocytes also were activated upon contact with cells bearing membrane IgE. In humans, the presence of FcepsilonRI in several cellular entities suggests a possible membrane IgE-FcepsilonRI-driven cell-cell dialogue, with likely implications for IgE homeostasis in physiology and pathology.