Vaccination with (1-11)E2 in alum efficiently induces an antibody response to ß-amyloid without affecting brain ß-amyloid load and microglia activation in 3xTg mice.

Immunization against ß-amyloid (Aß) is pursued as a possible strategy for the prevention of Alzheimer's disease (AD). In clinical trials, Aß 1-42 proved poorly immunogenic and caused severe adverse effects; therefore, safer and more immunogenic candidate vaccines are needed. Multimeric protein (1-11)E2 is able to induce an antibody response to Aß, immunological memory, and IL-4 production, with no concomitant anti-Aß T cell response. Antisera recognize Aß oligomers, protofibrils, and fibrils. In this study, we evaluated the effect of prophylactic immunization with three doses of (1-11)E2 in alum in the 3xTg mouse model of AD. Immunization with (1-11)E2 efficiently induced anti-Aß antibodies, but afforded no protection against Aß accumulation and neuroinflammation. The identification of the features of the anti-Aß immune response that correlate with the ability to prevent Aß accumulation remains an open problem that deserves further investigation.

Immunostimulatory Phosphatidylmonogalactosyldiacylglycerols (PGDG) from the Marine Diatom Thalassiosira weissflogii: Inspiration for a Novel Synthetic Toll-Like Receptor 4 Agonist.

An unprecedented phosphatidylmonogalactosyldiacylglycerol pool (PGDG, 1) rich in polyunsaturated fatty acids was isolated from the marine diatoms Thalassiosira weissflogii. Here we report for the first time the NMR characterization of this rare lipid from marine organisms along with a synthetic strategy for the preparation of a PGDG analog (2). PGDG 1 exhibited immunostimulatory activity in human dendritic cells (DCs) and the synthetic PGDG 2 was prepared to explore its mechanism of action. A Toll-like receptor-4 (TLR-4) agonistic activity was evidenced in human and murine DCs underlying the antigen-specific T-cell activation of this class of molecules.

HIV Vaccination: A Roadmap among Advancements and Concerns.

Since the identification of the Human Immunodeficiency Virus type 1 (HIV-1) as the etiologic agent of AIDS (Acquired Immunodeficiency Syndrome), many efforts have been made to stop the AIDS pandemic. A major success of medical research has been the development of the highly active antiretroviral therapy and its availability to an increasing number of people worldwide, with a considerable effect on survival. However, a safe and effective vaccine able to prevent and eradicate the HIV pandemic is still lacking. Clinical trials and preclinical proof-of-concept studies in nonhuman primate (NHP) models have provided insights into potential correlates of protection against the HIV-1 infection, which include broadly neutralizing antibodies (bnAbs), non-neutralizing antibodies targeting the variable loops 1 and 2 (V1V2) regions of the HIV-1 envelope (Env), polyfunctional antibody, and Env-specific T-cell responses. In this review, we provide a brief overview of different HIV-1 vaccine approaches and discuss the current understanding of the cellular and humoral correlates of HIV-1 immunity.

Distinct Antigen Delivery Systems Induce Dendritic Cells' Divergent Transcriptional Response: New Insights from a Comparative and Reproducible Computational Analysis.

Vaccination is the most successful and cost-effective method to prevent infectious diseases. However, many vaccine antigens have poor in vivo immunogenic potential and need adjuvants to enhance immune response. The application of systems biology to immunity and vaccinology has yielded crucial insights about how vaccines and adjuvants work. We have previously characterized two safe and powerful delivery systems derived from non-pathogenic prokaryotic organisms: E2 and fd filamentous bacteriophage systems. They elicit an in vivo immune response inducing CD8+ T-cell responses, even in absence of adjuvants or stimuli for dendritic cells' maturation. Nonetheless, a systematic and comparative analysis of the complex gene expression network underlying such activation is missing. Therefore, we compared the transcriptomes of ex vivo isolated bone marrow-derived dendritic cells exposed to these antigen delivery systems. Significant differences emerged, especially for genes involved in innate immunity, co-stimulation, and cytokine production. Results indicate that E2 drives polarization toward the Th2 phenotype, mainly mediated by Irf4, Ccl17, and Ccr4 over-expression. Conversely, fd-scαDEC-205 triggers Th1 T cells' polarization through the induction of Il12b, Il12rb, Il6, and other molecules involved in its signal transduction. The data analysis was performed using RNASeqGUI, hence, addressing the increasing need of transparency and reproducibility of computational analysis.

E2 multimeric scaffold for vaccine formulation: immune response by intranasal delivery and transcriptome profile of E2-pulsed dendritic cells.

BACKGROUND: The E2 multimeric scaffold represents a powerful delivery system able to elicit robust humoral and cellular immune responses upon systemic administrations. Here recombinant E2 scaffold displaying the third variable loop of HIV-1 Envelope gp120 glycoprotein was administered via mucosa, and the mucosal and systemic immune responses were analysed. To gain further insights into the molecular mechanisms that orchestrate the immune response upon E2 vaccination, we analysed the transcriptome profile of dendritic cells (DCs) exposed to the E2 scaffold with the aim to define a specific gene expression signature for E2-primed immune responses. RESULTS: The in vivo immunogenicity and the potential of E2 scaffold as a mucosal vaccine candidate were investigated in BALB/c mice vaccinated via the intranasal route. Fecal and systemic antigen-specific IgA antibodies, cytokine-producing CD4(+) and CD8(+) cells were induced assessing the immunogenicity of E2 particles via intranasal administration. The cytokine analysis identified a mixed T-helper cell response, while the systemic antibody response showed a prevalence of IgG1 isotype indicative of a polarized Th2-type immune response. RNA-Sequencing analysis revealed that E2 scaffold up-regulates in DCs transcriptional regulators of the Th2-polarizing cell response, defining a type 2 DC transcriptomic signature. CONCLUSIONS: The current study provides experimental evidence to the possible application of E2 scaffold as antigen delivery system for mucosal immunization and taking advantages of genome-wide approach dissects the type of response induced by E2 particles.

Analysis of SEMA6B gene expression in breast cancer: identification of a new isoform.

BACKGROUND: SEMA6B is a member of the semaphorins axon-guidance family. A growing body of evidence has been accumulated describing the role of semaphorin molecules in cancer development and the involvement of SEMA6B in cancer progression has recently been proposed. METHODS: Our analysis, based on real-time PCR, focused on the expression of SEMA6B in a panel of breast cancer tissues, compared to the normal counterpart. RESULTS: In cancer tissues we found a significantly strong down-modulation of this transcript. Moreover we identified and characterized a novel SEMA6B isoform, named SEMA6Ba. This isoform has a novel splice junction, created by the usage of alternative donor and acceptor splice sites internal to the exon 17. By in silico analysis we found that the new transcript 3' UTR lacks some highly-conserved miRNA binding sites, suggesting possible consequences on both spatial and temporal expression of SEMA6Ba. The translated sequence of SEMA6Ba lacks the cytoplasmic tail, crucial for triggering the reverse signaling described for the transmembrane semaphorins. We also demonstrated, by immunofluorescence analysis of endogenous and overexpressed SEMA6Ba, that the protein clearly localized to the endoplasmic reticulum and plasma membrane. In conclusion, SEMA6B gene products are strongly down modulated in breast cancer tissues and a new isoform named SEMA6Ba has been described and characterized. GENERAL SIGNIFICANCE: Our work states a clear relation among breast cancer and SEMA6B expression; moreover we describe for the first time the SEMA6Ba protein and report here the analysis of SEMA6Ba RNA messenger, the protein expression and the cellular localization.

PLGA microparticle formulations for tunable delivery of a nano-engineered filamentous bacteriophage-based vaccine: in vitro and in silico-supported approach.

Bacteriophages have attracted great attention in the bioengineering field in diverse research areas from tissue engineering to therapeutic and clinical applications. Recombinant filamentous bacteriophage, carrying multiple copies of foreign peptides on protein capsid has been successfully used in the vaccine delivery setting, even if their plasma instability and degradation have limited their use on the pharmaceutical market. Encapsulation techniques in polymeric materials can be applied to preserve bacteriophage activity, extend its half-life, and finely regulate their release in the target environment. The main goal of this study was to provide tunable formulations of the bacteriophage encapsulated in polymeric microparticles (MPs). We used poly (lactic-co-glycolic-acid) as a biocompatible and biodegradable polymer with ammonium bicarbonate as a porogen to encapsulate bacteriophage expressing OVA (257-264) antigenic peptide. We demonstrate that nano-engineered fdOVA bacteriophages encapsulated in MPs preserve their structure and are immunologically active, inducing a strong immune response towards the delivered peptide. Moreover, MP encapsulation prolongs bacteriophage stability over time also at room temperature. Additionally, in this study, we show the ability of in silico-supported approach to predict and tune the release of bacteriophages. These results lay the framework for a versatile bacteriophage-based vaccine delivery system that could successfully generate robust immune responses in a sustained manner, to be used as a platform against cancer and new emerging diseases. Graphical abstract: Synopsis: administration of recombinant bacteriophage-loaded PLGA microparticles for antigen delivery. PLGA microparticles release the bacteriophages, inducing activation of dendritic cells and enhancing antigen presentation and specific T cell response. Bacteriophage-encapsulated microneedles potentially can be administered into human body and generate robust immune responses.

Co-Delivery of the Human NY-ESO-1 Tumor-Associated Antigen and Alpha-GalactosylCeramide by Filamentous Bacteriophages Strongly Enhances the Expansion of Tumor-Specific CD8+ T Cells.

Tumor-associated antigens (TAAs) represent attractive targets in the development of anti-cancer vaccines. The filamentous bacteriophage is a safe and versatile delivery nanosystem, and recombinant bacteriophages expressing TAA-derived peptides at a high density on the viral coat proteins improve TAA immunogenicity, triggering effective in vivo anti-tumor responses. To enhance the efficacy of the bacteriophage as an anti-tumor vaccine, we designed and generated phage particles expressing a CD8+ peptide derived from the human cancer germline antigen NY-ESO-1 decorated with the immunologically active lipid alpha-GalactosylCeramide (α-GalCer), a potent activator of invariant natural killer T (iNKT) cells. The immune response to phage expressing the human TAA NY-ESO-1 and delivering α-GalCer, namely fdNY-ESO-1/α-GalCer, was analyzed either in vitro or in vivo, using an HLA-A2 transgenic mouse model (HHK). By using NY-ESO-1-specific TCR-engineered T cells and iNKT hybridoma cells, we observed the efficacy of the fdNY-ESO-1/α-GalCer co-delivery strategy at inducing activation of both the cell subsets. Moreover, in vivo administration of fdNY-ESO-1 decorated with α-GalCer lipid in the absence of adjuvants strongly enhances the expansion of NY-ESO-1-specific CD8+ T cells in HHK mice. In conclusion, the filamentous bacteriophage delivering TAA-derived peptides and the α-GalCer lipid may represent a novel and promising anti-tumor vaccination strategy.

Booster Dose of SARS-CoV-2 mRNA Vaccine in Kidney Transplanted Patients Induces Wuhan-Hu-1 Specific Neutralizing Antibodies and T Cell Activation but Lower Response against Omicron Variant.

Kidney transplanted recipients (KTR) are at high risk of severe SARS-CoV-2 infection due to immunosuppressive therapy. Although several studies reported antibody production in KTR after vaccination, data related to immunity to the Omicron (B.1.1.529) variant are sparse. Herein, we analyzed anti-SARS-CoV-2 immune response in seven KTR and eight healthy controls after the second and third dose of the mRNA vaccine (BNT162b2). A significant increase in neutralizing antibody (nAb) titers were detected against pseudoviruses expressing the Wuhan-Hu-1 spike (S) protein after the third dose in both groups, although nAbs in KTR were lower than controls. nAbs against pseudoviruses expressing the Omicron S protein were low in both groups, with no increase after the 3rd dose in KTR. Reactivity of CD4+ T cells after boosting was observed when cells were challenged with Wuhan-Hu-1 S peptides, while Omicron S peptides were less effective in both groups. IFN-γ production was detected in KTR in response to ancestral S peptides, confirming antigen-specific T cell activation. Our study demonstrates that the 3rd mRNA dose induces T cell response against Wuhan-Hu-1 spike peptides in KTR, and an increment in the humoral immunity. Instead, humoral and cellular immunity to Omicron variant immunogenic peptides were low in both KTR and healthy vaccinated subjects.

Vaccination with filamentous bacteriophages targeting DEC-205 induces DC maturation and potent anti-tumor T-cell responses in the absence of adjuvants.

The efficacy of a new vaccine-delivery vector, based on the filamentous bacteriophage fd displaying a single-chain antibody fragment known to bind the mouse DC surface molecule DEC-205, is reported. We demonstrate both in vitro and in vivo an enhanced receptor-mediated uptake of phage particles expressing the anti-DEC-205 fragment by DCs. We also report that DCs targeted by fd virions in the absence of other stimuli produce IFN-α and IL-6, and acquire a mature phenotype. Moreover, DC-targeting with fd particles double-displaying the anti-DEC-205 fragment on the pIII protein and the OVA(257-264) antigenic determinant on the pVIII protein induced potent inhibition of the growth of the B16-OVA tumor in vivo. This protection was much stronger than other immunization strategies and similar to that induced by adoptively transferred DCs. Since targeting DEC-205 in the absence of DC activation/maturation agents has previously been described to result in tolerance, the ability of fd bacteriophages to induce a strong tumor-specific immune response by targeting DCs through DEC-205 is unexpected, and further validates the potential employment of this safe, versatile and inexpensive delivery system for vaccine formulation.

Filamentous bacteriophage fd as an antigen delivery system in vaccination.

Peptides displayed on the surface of filamentous bacteriophage fd are able to induce humoral as well as cell-mediated immune responses, which makes phage particles an attractive antigen delivery system to design new vaccines. The immune response induced by phage-displayed peptides can be enhanced by targeting phage particles to the professional antigen presenting cells, utilizing a single-chain antibody fragment that binds dendritic cell receptor DEC-205. Here, we review recent advances in the use of filamentous phage fd as a platform for peptide vaccines, with a special focus on the use of phage fd as an antigen delivery platform for peptide vaccines in Alzheimer's Disease and cancer.

Design and characterization of a peptide mimotope of the HIV-1 gp120 bridging sheet.

The Bridging Sheet domain of HIV-1 gp120 is highly conserved among the HIV-1 strains and allows HIV-1 binding to host cells via the HIV-1 coreceptors. Further, the bridging sheet domain is a major target to neutralize HIV-1 infection. We rationally designed four linear peptide epitopes that mimic the three-dimensional structure of bridging sheet by using molecular modeling. Chemically synthesized peptides BS3 and BS4 showed a fair degree of antigenicity when tested in ELISA with IgG purified from HIV(+) broadly neutralizing sera while the production of synthetic peptides BS1 and BS2 failed due to their high degree of hydrophobicity. To overcome this limitation, we linked all four BS peptides to the COOH-terminus of GST protein to test both their antigenicity and immunogenicity. Only the BS1 peptide showed good antigenicity; however, no envelope specific antibodies were elicited upon mice immunization. Therefore we performed further analyses by linking BS1 peptide to the NH2-terminus of the E2 scaffold from the Geobacillus Stearothermophylus PDH complex. The E2-BS1 fusion peptide showed good antigenic results, however only one immunized rabbit elicited good antibody titers towards both the monomeric and oligomeric viral envelope glycoprotein (Env). In addition, moderate neutralizing antibodies response was elicited against two HIV-1 clade B and one clade C primary isolates. These preliminary data validate the peptide mimotope approach as a promising tool to obtain an effective HIV-1 vaccine.

Comparative analysis of the neutralizing activity against SARS-CoV-2 Wuhan-Hu-1 strain and variants of concern: Performance evaluation of a pseudovirus-based neutralization assay.

Objectives: Emergence of new variants of SARS-CoV-2 might affect vaccine efficacy. Therefore, assessing the capacity of sera to neutralize variants of concern (VOCs) in BSL-2 conditions will help evaluating the immune status of population following vaccination or infection. Methods: Pseudotyped viruses bearing SARS-CoV-2 spike protein from Wuhan-Hu-1/D614G strains (wild type, WT), B.1.617.2 (Delta), or B.1.1.529 (Omicron) VOCs were generated to assess the neutralizing antibodies (nAbs) activity by a pseudovirus-based neutralization assay (PVNA). PVNA performance was assessed in comparison to the micro-neutralization test (MNT) based on live viruses. Sera collected from COVID-19 convalescents and vaccinees receiving mRNA (BNT16b2 or mRNA-1273) or viral vector (AZD1222 or Ad26.COV2.S) vaccines were used to measure nAbs elicited by two-dose BNT16b2, mRNA-1273, AZD1222 or one-dose Ad26.CO2.S, at different times from completed vaccination, ~ 1.5 month and ~ 4-6 months. Sera from pre-pandemic and unvaccinated individuals were analyzed as controls. Neutralizing activity following booster vaccinations against VOCs was also determined. Results: PVNA titers correlated with the gold standard MNT assay, validating the reliability of PVNA. Sera analyzed late from the second dose showed a reduced neutralization activity compared to sera collected earlier. Ad26.CO2.S vaccination led to very low or absent nAbs. Neutralization of Delta and Omicron BA.1 VOCs showed significant reduction of nAbs respect to WT strain. Importantly, booster doses enhanced Omicron BA.1 nAbs, with persistent levels at 3 months from boosting. Conclusions: PVNA is a reliable tool for assessing anti-SARS-CoV-2 nAbs helping the establishment of a correlate of protection and the management of vaccination strategies.

Cell loaded hydrogel containing Ag-doped bioactive glass-ceramic nanoparticles as skin substitute: Antibacterial properties, immune response, and scarless cutaneous wound regeneration.

An ideal tissue-engineered dermal substitute should possess angiogenesis potential to promote wound healing, antibacterial activity to relieve the bacterial burden on skin, as well as sufficient porosity for air and moisture exchange. In light of this, a glass-ceramic (GC) has been incorporated into chitosan and gelatin electrospun nanofibers (240-360 nm), which MEFs were loaded on it for healing acceleration. The GC was doped with silver to improve the antibacterial activity. The bioactive nanofibrous scaffolds demonstrated antibacterial and superior antibiofilm activities against Gram-negative and Gram-positive bacteria. The nanofibrous scaffolds were biocompatible, hemocompatible, and promoted cell attachment and proliferation. Nanofibrous skin substitutes with or without Ag-doped GC nanoparticles did not induce an inflammatory response and attenuated LPS-induced interleukin-6 release by dendritic cells. The rate of biodegradation of the nanocomposite was similar to the rate of skin regeneration under in vivo conditions. Histopathological evaluation of full-thickness excisional wounds in BALB/c mice treated with mouse embryonic fibroblasts-loaded nanofibrous scaffolds showed enhanced angiogenesis, and collagen synthesis as well as regeneration of the sebaceous glands and hair follicles in vivo.

A multimeric immunogen for the induction of immune memory to beta-amyloid.

The development of active immunotherapy for Alzheimer's disease (AD) requires the identification of immunogens that can ensure a high titer antibody response toward beta-amyloid, whereas minimizing the risks of a cell-mediated adverse reaction. We describe here two novel anti-beta-amyloid vaccines that consist of 'virus like particles' formed by a domain of the bacterial protein E2 that is able to self-assemble into a 60-mer peptide. Peptides 1-11 and 2-6 of beta-amyloid were displayed as N terminal fusions on the surface of the E2 particles. E2-based vaccines induced a fast-rising, robust and persistent antibody response to beta-amyloid in all vaccinated mice. The immune memory induced by a single administration of vaccine (1-11) E2 can be rapidly mobilized by a single booster injection, leading to a very high serum concentration of anti-beta-amyloid antibodies (above 1 mg ml(-1)). E2 vaccination polarizes the immune response toward the production of the anti-inflammatory cytokine interleukin-4 and does not induce a T cell response to beta-amyloid. Thus, E2-based vaccines are promising candidates for the development of immunotherapy protocols for AD.

Exploiting viral sensing mediated by Toll-like receptors to design innovative vaccines.

Toll-like receptors (TLRs) are transmembrane proteins belonging to the family of pattern-recognition receptors. They function as sensors of invading pathogens through recognition of pathogen-associated molecular patterns. After their engagement by microbial ligands, TLRs trigger downstream signaling pathways that culminate into transcriptional upregulation of genes involved in immune defense. Here we provide an updated overview on members of the TLR family and we focus on their role in antiviral response. Understanding of innate sensing and signaling of viruses triggered by these receptors would provide useful knowledge to prompt the development of vaccines able to elicit effective and long-lasting immune responses. We describe the mechanisms developed by viral pathogens to escape from immune surveillance mediated by TLRs and finally discuss how TLR/virus interplay might be exploited to guide the design of innovative vaccine platforms.

Self-assembled peptide and protein nanostructures for anti-cancer therapy: Targeted delivery, stimuli-responsive devices and immunotherapy.

Self-assembled peptides and proteins possess tremendous potential as targeted drug delivery systems and key applications of these well-defined nanostructures reside in anti-cancer therapy. Peptides and proteins can self-assemble into nanostructures of diverse sizes and shapes in response to changing environmental conditions such as pH, temperature, ionic strength, as well as host and guest molecular interactions; their countless benefits include good biocompatibility and high loading capacity for hydrophobic and hydrophilic drugs. These self-assembled nanomaterials can be adorned with functional moieties to specifically target tumor cells. Stimuli-responsive features can also be incorporated with respect to the tumor microenvironment. This review sheds light on the growing interest in self-assembled peptides and proteins and their burgeoning applications in cancer treatment and immunotherapy.

Triggering DTH and CTL activity by fd filamentous bacteriophages: role of CD4+ T cells in memory responses.

The ability of fd bacteriophage particles to trigger different arms of the immune system has been previously shown by us with particular emphasis on the ability of phages to raise CTL responses in vitro and in vivo. Here we show that fd virions in the absence of adjuvants are able to evoke a DTH reaction mediated by antigen specific CD8+ T cells. In addition, we analyzed the induction of CTL responses in mice depleted of CD4+ T cells, and we observed that short-term secondary CTL responses were induced in the absence of CD4+ T cells while induction of long-term memory CTLs required the presence of CD4+ T lymphocytes. These results examine the cellular mechanism at the basis of fd efficiency and provide new elements to further validate the use of fd particles for eliciting and monitoring antigen-specific CTLs.

Analysis of the Consolidation Phase of Immunological Memory within the IgG Response to a B Cell Epitope Displayed on a Filamentous Bacteriophage.

Immunological memory can be defined as the ability to mount a response of greater magnitude and with faster kinetics upon re-encounter of the same antigen. We have previously reported that a booster dose of a protein antigen given 15 days after the first dose interferes with the development of memory, i.e., with the ability to mount an epitope-specific IgG response of greater magnitude upon re-encounter of the same antigen. We named the time-window during which memory is vulnerable to disruption a "consolidation phase in immunological memory", by analogy with the memory consolidation processes that occur in the nervous system to stabilize memory traces. In this study, we set out to establish if a similar memory consolidation phase occurs in the IgG response to a B cell epitope displayed on a filamentous bacteriophage. To this end, we have analyzed the time-course of anti-ß-amyloid IgG titers in mice immunized with prototype Alzheimer's Disease vaccine fdAD(2-6), which consists of a fd phage that displays the B epitope AEFRH of ß -amyloid at the N-terminus of the Major Capsid Protein. A booster dose of phage fdAD(2-6) given 15 days after priming significantly reduced the ratio between the magnitude of the secondary and primary IgG response to ß-amyloid. This analysis confirms, in a phage vaccine, a consolidation phase in immunological memory, occurring two weeks after priming.

Viral Emerging Diseases: Challenges in Developing Vaccination Strategies.

In the last decades, a number of infectious viruses have emerged from wildlife or re-emerged, generating serious threats to the global health and to the economy worldwide. Ebola and Marburg hemorrhagic fevers, Lassa fever, Dengue fever, Yellow fever, West Nile fever, Zika, and Chikungunya vector-borne diseases, Swine flu, Severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and the recent Coronavirus disease 2019 (COVID-19) are examples of zoonoses that have spread throughout the globe with such a significant impact on public health that the scientific community has been called for a rapid intervention in preventing and treating emerging infections. Vaccination is probably the most effective tool in helping the immune system to activate protective responses against pathogens, reducing morbidity and mortality, as proven by historical records. Under health emergency conditions, new and alternative approaches in vaccine design and development are imperative for a rapid and massive vaccination coverage, to manage a disease outbreak and curtail the epidemic spread. This review gives an update on the current vaccination strategies for some of the emerging/re-emerging viruses, and discusses challenges and hurdles to overcome for developing efficacious vaccines against future pathogens.