A SARS-CoV-2 Vaccination Strategy Focused on Population-Scale Immunity.

COVID-19 vaccinations in patients with inflammatory bowel disease

BackgroundParents' reported ambivalence toward large-scale vaccination programs for childhood diseases may be related to their perception of the risks of side-effects or safety of vaccination and the risk of contracting the disease. The aim of this study is to evaluate parents' perceptions of their child's risk contracting a Meningococcal C infection and parents' perceived control in preventing infection in relation to their evaluation of the safety, effectiveness and usefulness of vaccination.MethodsIn a large-scale interview study, a random sample of parents was interviewed after their children had received vaccination against Meningococcal C in a catch-up campaign. Questions were asked about the perceived relative vulnerability of their child contracting an infection, perceived control in preventing an infection, and parents' evaluation of the safety, usefulness and effectiveness of vaccination.Results61% of 2910 (N = 1763) parents who were approached participated. A higher perceived relative vulnerability of their own child contracting the disease was related to a more positive evaluation of the vaccination campaign, while a lower perceived vulnerability did not result in a more negative evaluation. A higher perceived control in being able to prevent an infection was, however, related to a more critical attitude toward the safety, usefulness and effectiveness of vaccination.ConclusionPerceived relative vulnerability contracting an infection and parents' perceived control in preventing an infection seem to influence parents' evaluation of the vaccination programme. Future studies should determine if, and under which circumstances, these perceptions also affect parents' vaccination behaviour and would be relevant to be taken into account when educating parents about vaccination.

BackgroundSeveral arenaviruses cause severe hemorrhagic fever and aseptic meningitis in humans for which no licensed vaccines are available. A major obstacle for vaccine development is pathogen heterogeneity within the Arenaviridae family. Evidence in animal models and humans indicate that T cell and antibody-mediated immunity play important roles in controlling arenavirus infection and replication. Because CD4+ T cells are needed for optimal CD8+ T cell responses and to provide cognate help for B cells, knowledge of epitopes recognized by CD4+ T cells is critical to the development of an effective vaccine strategy against arenaviruses. Thus, the goal of the present study was to define and characterize CD4+ T cell responses from a broad repertoire of pathogenic arenaviruses (including lymphocytic choriomeningitis, Lassa, Guanarito, Junin, Machupo, Sabia, and Whitewater Arroyo viruses) and to provide determinants with the potential to be incorporated into a multivalent vaccine strategy.ResultsBy inoculating HLA-DRB1*0101 transgenic mice with a panel of recombinant vaccinia viruses, each expressing a single arenavirus antigen, we identified 37 human HLA-DRB1*0101-restricted CD4+ T cell epitopes from the 7 antigenically distinct arenaviruses. We showed that the arenavirus-specific CD4+ T cell epitopes are capable of eliciting T cells with a propensity to provide help and protection through CD40L and polyfunctional cytokine expression. Importantly, we demonstrated that the set of identified CD4+ T cell epitopes provides broad, non-ethnically biased population coverage of all 7 arenavirus species targeted by our studies.ConclusionsThe identification of CD4+ T cell epitopes, with promiscuous binding properties, derived from 7 different arenavirus species will aid in the development of a T cell-based vaccine strategy with the potential to target a broad range of ethnicities within the general population and to protect against both Old and New World arenavirus infection.

The main focus of this thesis is on immunomodulation in cancer therapy. We describe different therapeutic strategies in metastatic renal cell cancer (mRCC) and breast cancer (BC) and their effect within the context of the cancer-immunity cycle (CIC). In the past era when there were few effective treatment options for mRCC, we sought to improve the at that time standard second-line treatment with everolimus. Everolimus was demonstrated to affect multiple immune cell subsets, especially Tregs, and altogether tip the balance in favor of immunosuppression (step 5 CIC). By the administration of metronomic cyclophosphamide Tregs can be selectively depleted, most probably by causing a DNA repair defect. As there was controversy on the optimal Treg depletion dose and schedule of cyclophosphamide, we performed a phase 1 clinical trial in which everolimus was combined with several schedules of metronomic oral cyclophosphamide in patients with mRCC. Selective and significant Treg depletion in peripheral blood could be achieved with a standard once daily therapeutic dose of 10mg everolimus combined with 50mg cyclophosphamide once daily. This dose was further evaluated in the phase 2 clinical trial. Treg modulating effects of metronomic cyclophosphamide were comparable to those observed in the phase 1 part of the study, however the phase 2 trial was abrogated at the predefined interim analysis after inclusion of 24 patients, since the PFS did not improve from 50% to the pre-set 70% at 4 months. The immunomodulatory effects of the combination of metronomic cyclophosphamide and everolimus did not translate into an altered clinical outcome, possible due to the fact that the Tregs that persisted in the peripheral blood were proliferating and had strong inhibitory functions, implicating some sort of recruitment of these cells, perhaps as a feedback mechanism. In the phase 3 Spinoza trial we tried to boost dendritic cells (DC) to create a more robust immune response in the treatment of patients with locally advanced BC (LABC) by the addition of growth factor GM-CSF to neoadjuvant chemotherapy (NAC). The role of DC in the tumor mico environment (TME) is not limited to the transfer of antigens from tumor to tumor draining lymph nodes (TDLN), but also to ensure the attraction, activation and expansion of antigen-specific T cells in the tumor itself (step 3 and substeps 5A-C CIC). BC is generally considered immune excluded or an immune desert and could therefore benefit from activation of DCs. Indeed we showed an enhanced systemic DC differentiation in patients with LABC upon NAC, supported by either GM-CSF or G-CSF. Notably, GM-CSF appeared to be more potent in stimulating maturation of DC in TDLN. GM-CSF supported NAC could thus possibly create a more robust anti-tumor immune response than current G-CSF based NAC strategies. In patients with Her2+ metastatic BC (mBC) and brain metastasis, a systematic review and meta-analysis showed Trastuzumab-deruxtecan (T-Dxd) to be the most effective treatment modality leading to highest objective response rates, although this meta-analysis should be interpreted with caution due to heterogeneity of included studies and a related serious risk of bias. T-Dxd specifically binds to Her2+ (cancer) cells, leading to a targeted effect of the cytostatic deruxtecan component, thus leading to the selective death of targeted tumor cells and the subsequent release of cancer antigens (step 1 CIC). BC cells exhibiting high Her2 expression have down-regulation of MHC class I expression, inhibiting CD8+ T cell recognition. Blockade of the Her2 receptor by T-Dxd could restore MHC class 1 expression and therefor promote T cell recognition (Step 6 CIC). Also, the trastuzumab compound of T-Dxd is known to have an effect through antibody-dependent cell-mediated cytotoxicity (step 7 CIC).

Synthetic peptide vaccines targeting SVA elicit robust protective immune responses in swine.

Still Seeking an Effective “One–Two” Malaria Vaccine Punch

Murine cytomegalovirus (MCMV) is an important animal model of human cytomegalovirus (HCMV), a β-Herpesvirus that infects the majority of the world's population and causes disease in neonates and immunocompromised adults. CD8+ T cells are a major part of the immune response to MCMV and HCMV. Processing of peptides for presentation to CD8+ T cells may be critically dependent on the immunoproteasome, expression of which is affected by MCMV. However, the overall importance of the immunoproteasome in the generation of immunodominant peptides from MCMV is not known. We therefore examined the role of the immunoproteasome in stimulation of CD8+ T cell responses to MCMV – both conventional memory responses and those undergoing long-term expansion or “inflation”. We infected LMP7−/− and C57BL/6 mice with MCMV or with newly-generated recombinant vaccinia viruses (rVVs) encoding the immunodominant MCMV protein M45 in either full-length or epitope-only minigene form. We analysed CD8+ T cell responses using intracellular cytokine stain (ICS) and MHC Class I tetramer staining for a panel of MCMV-derived epitopes. We showed a critical role for immunoproteasome in MCMV affecting all epitopes studied. Interestingly we found that memory “inflating” epitopes demonstrate reduced immunoproteasome dependence compared to non-inflating epitopes. M45-specific responses induced by rVVs remain immunoproteasome-dependent. These results help to define a critical restriction point for CD8+ T cell epitopes in natural cytomegalovirus (CMV) infection and potentially in vaccine strategies against this and other viruses.

Controlling the cross-presentation of exogenous Ags to CD8+ T cells represents a major step for designing new vaccination strategies. Whereas several recombinant pseudo-viral particles have been used as delivery systems for triggering potent CTL responses to heterologous exogenous Ags, the adjuvant properties of virus-like particles (VLPs) themselves were little questioned. Here, we analyzed the contribution of the porcine parvovirus (PPV)-VLPs to the induction of protective cellular responses to exogenous Ags carried by an independent delivery system. Microspheres, which are known to transfer exogenous Ags into the MHC class I pathway, were chosen for delivering the immunodominant OVA(257-264) CD8+ T cell epitope (B-OVAp). This delivery system fulfills the requirements in terms of cross-presentation, but fails to induce cross-priming of specific CD8+ T cells. Coinjection of PPV-VLPs with B-OVAp results in the priming of potent CTL responses and type 1-biased immunity in a CD4- and CD40-independent manner, as efficiently as the recombinant PPV-VLPs carrying the same epitope (PPV-OVAp). Furthermore, vaccination with PPV-VLPs and B-OVAp was fully efficient to protect mice against the development of OVA-bearing melanoma. These findings indicate that PPV-VLPs act not only as a delivery system but also as a strong adjuvant when independently provided with exogenous Ag. Thus, dissociation between delivery system and adjuvant would provide a more flexible and reliable system to induce potent and protective CTL.

Bordetella pertussis adenylate cyclase (CyaA) is an invasive bacterial toxin that delivers its N-terminal catalytic domain into the cytosol of eukaryotic cells bearing the alpha(M)beta(2) integrin (CD11b/CD18), such as myeloid dendritic cells. This allows use of engineered CyaA for targeted delivery of CD8(+) T cell epitopes into the MHC class I pathway of APC and induction of robust and protective cytotoxic responses. In this study, we demonstrate that CyaA can efficiently codeliver both a CD8(+) T cell epitope (OVA(257-264)) and a CD4(+) T cell epitope (MalE(100-114)) into, respectively, the conventional cytosolic or endocytic routes of processing of murine bone marrow-derived dendritic cells. Upon CyaA delivery, a strong potentiation of the MalE(100-114) CD4(+) T cell epitope presentation is observed as compared with the MalE protein, which depends on CyaA interaction with its CD11b receptor and its subsequent clathrin-mediated endocytosis. In vivo, CyaA induces strong and specific Th1 CD4(+) and CD8(+) T cell responses against, respectively, the MalE(100-114) and OVA(257-264) epitopes. These results underscore the potency of CyaA for design of new vaccines.

Designing a multi-epitopes subunit vaccine against human herpes virus 6A based on molecular dynamics and immune stimulation

The live attenuated yellow fever vaccine 17D (YF-17D) is one of the most effective vaccines. Despite its excellent safety record, some cases of viscerotropic adverse events develop, which are sometimes fatal. The mechanisms underlying such events remain a mystery. Here, we present an analysis of the immunologic and genetic factors driving disease in a 64-year-old male who developed viscerotropic symptoms. We obtained clinical, serologic, virologic, immunologic and genetic data on this case patient. Viral RNA was detected in the blood 33 days after vaccination, in contrast to the expected clearance of virus by day 7 after vaccination in healthy vaccinees. Vaccination induced robust antigen-specific T and B cell responses, which suggested that persistent virus was not due to adaptive immunity of suboptimal magnitude. The genes encoding OAS1, OAS2, TLR3, and DC-SIGN, which mediate antiviral innate immunity, were wild type. However, there were heterozygous genetic polymorphisms in chemokine receptor CCR5, and its ligand RANTES, which influence the migration of effector T cells and CD14+CD16bright monocytes to tissues. Consistent with this, there was a 200-fold increase in the number of CD14+CD16bright monocytes in the blood during viremia and even several months after virus clearance. In this patient, viscerotropic disease was not due to the impaired magnitude of adaptive immunity but instead to anomalies in the innate immune system and a possible disruption of the CCR5-RANTES axis.

Respiratory syncytial virus (RSV) is an important cause of respiratory tract disease in infants and the elderly. Currently, no licensed vaccine against RSV is available. Here we describe the development of a safe and effective intranasal subunit vaccine that is based on recombinant fusion (F) protein bound to the surface of immunostimulatory bacterium-like particles (BLPs) derived from the food-grade bacterium Lactococcus lactis. Different variants of F were analyzed with respect to their conformation and reactivity with neutralizing antibodies, assuming that F proteins mimicking the metastable prefusion form of RSV F expose a more extensive and relevant epitope repertoire than F proteins corresponding to the postfusion structure. Our results indicate that the recombinant soluble ectodomain of RSV F readily adopts a postfusion conformation, generation of which cannot be prevented by C-terminal addition of a trimerization motif, but whose formation is prevented by mutation of the two furin cleavage sites in F. While the putative postfusion form of F is recognized well by the monoclonal antibody Palivizumab, this is much less so for the more potently neutralizing, prefusion-specific antibodies D25 and AM22. Both addition of the trimerization motif and mutation of the furin cleavage sites increased the reactivity of F with D25 and AM22, with the highest reactivity being observed for F proteins in which both these features were combined. Intranasal vaccination of mice or cotton rats with BLPs loaded with this latter prefusion-like F protein (BLP-F), resulted in the potent induction of F-specific immunoglobulins and in significantly decreased virus titers in the lungs upon RSV challenge. Moreover, and in contrast to animals vaccinated with formalin-inactivated RSV, animals that received BLP-F exhibited high levels of F-specific secretory IgA in the nose and RSV-neutralizing antibodies in sera, but did not show symptoms of enhanced disease after challenge with RSV.

SARS-CoV-2 elicits robust adaptive immune responses regardless of disease severity.

Subunit vaccines can have excellent safety profiles, but their ability to give rise to robust immune responses is often compromised. For glycan-based vaccines, insufficient understanding of B and T cell epitope combinations that yield optimal immune activation hinders optimization. To determine which antigen features promote desired IgG responses, we synthesized epitope-functionalized polymers using ring-opening metathesis polymerization (ROMP) and assessed the effect of B and T cell epitope loading. The most robust responses were induced by polymers with a high valency of B and T cell epitopes. Additionally, IgG responses were greater for polymers with T cell epitopes that are readily liberated upon endosomal processing. Combining these criteria, we used ROMP to generate a nontoxic, polymeric antigen that elicited stronger antibody responses than a comparable protein conjugate. These findings highlight principles for designing synthetic antigens that elicit strong IgG responses against inherently weak immune targets such as glycans.

BackgroundThe use of proteins as immunogens is attractive for the development of vaccines, but requires efficient adjuvants to overcome their weak immunogenicity. Recently, we investigated the potential of the TLR2/4 agonist hyaluronan (HA) as an immunological adjuvant for protein-based vaccines.1 2 Conjugation of HA to antigens strongly increased their immunogenicity and promoted their rapid translocation to draining lymph nodes, resulting in robust and long-lasting humoral responses.1 On these bases, we investigated the potentiality of HA-based technology in the design of cancer vaccines. To this aim, HA was conjugated to the extracellular domain of rat HER2/neu (rHER2/neu) and validated in the preventive and therapeutic vaccination settings.Materials and MethodsFemale BALB/c or BALB-neuT mice were immunized with rHER2/neu-HA. In vivo depletion of CD4+, CD8+ T and B cells was performed, and sera and spleens were collected to characterized antigen-specific humoral and cellular responses. Vaccinated BALB/c mice were challenged and re-challenged with rHER2/neu-overexpressing TUBO cells to assess the protective or therapeutic activity of rHER2/neu-HA vaccination strategy, as well as immunological memory.ResultsHA performed efficiently as robust and long-lasting humoral (IgG1, IgG2a, and IgG2b) and cellular responses were detected using very low antigen doses and number of boosters. Outstandingly, at 1-year post-vaccination, anti-rHER2/neu specific antibodies showed even improved effector functions (maturation of affinity for the receptor and increased complement-derived cytotoxicity functions). HA vaccination turned out effective in both the prophylactic (100% mice survived) and therapeutic (tumor regression in 2/12 mice) settings, and broke tolerance against rHER2/neu, delaying spontaneous tumor growth in BALB-neuT mice. Both humoral and cellular responses contributed to the success of HA-based vaccination, but CD8+ T cells played only a marginal role.ConclusionsCancer vaccines have not yet achieved significant clinical efficacy due to their poor immunogenicity, and the validation of more effective adjuvants occurred sometimes at the expense of safety. HA combines the unique immunomodulatory features of a TLR agonist with the tolerability of a fully natural polymer, proving to be a promising adjuvant for the creation of effective and safe cancer vaccines with the potential for rapid clinical translation.ReferencesDalla Pietà A, Carpanese D, et al. Hyaluronan is a natural and effective immunological adjuvant for protein-based vaccines. Cell Mol Immunol 2021;18(5):1197–1210.Rosato A, Montagner IM, Carpanese D, Dalla Pietà A. Hyaluronic acid as a natural adjuvant for protein and peptide-based vaccines. 30.04.2020. WO/2020/084558, PCT/IB2019/059122.Disclosure InformationD. Carpanese: None. I. Montagner: None. A. Dalla Pietà: None. V. Rossi: None. A. Penna: None. G. Zuccolotto: None. G. Pasut: None. A. Grigoletto: None. A. Rosato: None.