Alessandra Bonci

Approach to discover T- and B-cell antigens of intracellular pathogens applied to the design of Chlamydia trachomatis vaccines

Natural immunity against obligate and/or facultative intracellular pathogens is usually mediated by both humoral and cellular immunity. The identification of those antigens stimulating both arms of the immune system is instrumental for vaccine discovery. Although high-throughput technologies have been applied for the discovery of antibody-inducing antigens, few examples of their application for T-cell antigens have been reported. We describe how the compilation of the immunome, here defined as the pool of immunogenic antigens inducing T- and B-cell responses in vivo, can lead to vaccine candidates against Chlamydia trachomatis. We selected 120 C. trachomatis proteins and assessed their immunogenicity using two parallel high-throughput approaches. Protein arrays were generated and screened with sera from C. trachomatis-infected patients to identify antibody-inducing antigens. Splenocytes from C. trachomatis-infected mice were stimulated with 79 proteins, and the frequency of antigen-specific CD4+/IFN-γ+ T cells was analyzed by flow cytometry. We identified 21 antibody-inducing antigens, 16 CD4+/IFN-γ+–inducing antigens, and five antigens eliciting both types of responses. Assessment of their protective activity in a mouse model of Chlamydia muridarum lung infection led to the identification of seven antigens conferring partial protection when administered with LTK63/CpG adjuvant. Protection was largely the result of cellular immunity as assessed by CD4+ T-cell depletion. The seven antigens provided robust additive protection when combined in four-antigen combinations. This study paves the way for the development of an effective anti-Chlamydia vaccine and provides a general approach for the discovery of vaccines against other intracellular pathogens.

Rapidly produced SAM ® vaccine against H7N9 influenza is immunogenic in mice

The timing of vaccine availability is essential for an effective response to pandemic influenza. In 2009, vaccine became available after the disease peak, and this has motivated the development of next generation vaccine technologies for more rapid responses. The SAM® vaccine platform, now in pre-clinical development, is based on a synthetic, self-amplifying mRNA, delivered by a synthetic lipid nanoparticle (LNP). When used to express seasonal influenza hemagglutinin (HA), a SAM vaccine elicited potent immune responses, comparable to those elicited by a licensed influenza subunit vaccine preparation. When the sequences coding for the HA and neuraminidase (NA) genes from the H7N9 influenza outbreak in China were posted on a web-based data sharing system, the combination of rapid and accurate cell-free gene synthesis and SAM vaccine technology allowed the generation of a vaccine candidate in 8 days. Two weeks after the first immunization, mice had measurable hemagglutinin inhibition (HI) and neutralizing antibody titers against the new virus. Two weeks after the second immunization, all mice had HI titers considered protective. If the SAM vaccine platform proves safe, potent, well tolerated and effective in humans, fully synthetic vaccine technologies could provide unparalleled speed of response to stem the initial wave of influenza outbreaks, allowing first availability of a vaccine candidate days after the discovery of a new virus.

CT043, a protective antigen that induces a CD4+ Th1 response during Chlamydia trachomatis infection in mice and humans.

ABSTRACT Despite several decades of intensive studies, no vaccines against Chlamydia trachomatis, an intracellular pathogen causing serious ocular and urogenital diseases, are available yet. Infection-induced immunity in both animal models and humans strongly supports the notion that for a vaccine to be effective a strong CD4+ Th1 immune response should be induced. In the course of our vaccine screening program based on the selection of chlamydial proteins eliciting cell-mediated immunity, we have found that CT043, a protein annotated as hypothetical, induces CD4+ Th1 cells both in chlamydia-infected mice and in human patients with diagnosed C. trachomatis genital infection. DNA priming/protein boost immunization with CT043 results in a 2.6-log inclusion-forming unit reduction in the murine lung infection model. Sequence analysis of CT043 from C. trachomatis human isolates belonging to the most representative genital serovars revealed a high degree of conservation, suggesting that this antigen could provide cross-serotype protection. Therefore, CT043 is a promising vaccine candidate against C. trachomatis infection.

Induction of broad-based immunity and protective efficacy by self-amplifying mRNA vaccines encoding influenza virus hemagglutinin

ABSTRACT Seasonal influenza is a vaccine-preventable disease that remains a major health problem worldwide, especially in immunocompromised populations. The impact of influenza disease is even greater when strains drift, and influenza pandemics can result when animal-derived influenza virus strains combine with seasonal strains. In this study, we used the SAM technology and characterized the immunogenicity and efficacy of a self-amplifying mRNA expressing influenza virus hemagglutinin (HA) antigen [SAM(HA)] formulated with a novel oil-in-water cationic nanoemulsion. We demonstrated that SAM(HA) was immunogenic in ferrets and facilitated containment of viral replication in the upper respiratory tract of influenza virus-infected animals. In mice, SAM(HA) induced potent functional neutralizing antibody and cellular immune responses, characterized by HA-specific CD4 T helper 1 and CD8 cytotoxic T cells. Furthermore, mice immunized with SAM(HA) derived from the influenza A virus A/California/7/2009 (H1N1) strain (Cal) were protected from a lethal challenge with the heterologous mouse-adapted A/PR/8/1934 (H1N1) virus strain (PR8). Sera derived from SAM(H1-Cal)-immunized animals were not cross-reactive with the PR8 virus, whereas cross-reactivity was observed for HA-specific CD4 and CD8 T cells. Finally, depletion of T cells demonstrated that T-cell responses were essential in mediating heterologous protection. If the SAM vaccine platform proves safe, well tolerated, and effective in humans, the fully synthetic SAM vaccine technology could provide a rapid response platform to control pandemic influenza. IMPORTANCE In this study, we describe protective immune responses in mice and ferrets after vaccination with a novel HA-based influenza vaccine. This novel type of vaccine elicits both humoral and cellular immune responses. Although vaccine-specific antibodies are the key players in mediating protection from homologous influenza virus infections, vaccine-specific T cells contribute to the control of heterologous infections. The rapid production capacity and the synthetic origin of the vaccine antigen make the SAM platform particularly exploitable in case of influenza pandemic.

Relatedness and Phylogeny Within the Family of Periplasmic Chaperones Involved in the Assembly of Pili or Capsule-Like Structures of Gram-Negative Bacteria

Abstract. The structure of a Salmonella enterica serovar typhi gene located within the fim gene cluster and encoding a putative periplasmic chaperone-like protein involved in the assembly of type 1 pili was determined. This gene, named fimC, has the ability to encode a 26-kDa polypeptide which is similar, at the sequence level, to the PapD periplasmic chaperonin mediating the assembly of P pili of Escherichia coli, as well as to other periplasmic chaperone-like proteins involved in the biogenesis of pili or capsule-like structures of various Gram-negative bacteria. A comprehensive search through the literature and sequence databases identified 31 (putative) bacterial proteins that can be included in this protein family on the basis of sequence similarity. Results of a multiple sequence comparison analysis showed that several residues, including most of those known to be critical in maintaining the three-dimensional structure of PapD, are either conserved or conservatively substituted in all these proteins, suggesting an overall similar folding for all of them. It was also evident that members of this family are clustered into different subfamilies according to structural and phyletic data.

An Innovative Pseudotypes-Based Enzyme-Linked Lectin Assay for the Measurement of Functional Anti-Neuraminidase Antibodies.

Antibodies (Ab) to neuraminidase (NA) play a role in limiting influenza infection and might help reduce the disease impact. The most widely used serological assay to measure functional anti-NA immune responses is the Enzyme-Linked Lectin Assay (ELLA) which relies on hemagglutinin (HA) mismatched virus reassortants, or detergent treated viruses as the NA source to overcome interference associated with steric hindrance of anti-HA Ab present in sera. The difficulty in producing and handling these reagents, which are not easily adapted for screening large numbers of samples, limits the routine analysis of functional anti-NA Ab in clinical trials. In this study, we produced influenza lentiviral pseudoparticles (PPs) containing only the NA antigen (NA-PPs) with a simple two-plasmid co-transfection system. NA-PPs were characterized and tested as an innovative source of NA in the NA inhibition (NI) assay. Both swine A/California/07/2009 (H1N1) and avian A/turkey/Turkey/01/2005 (H5N1) N1s within NA-PPs retained their sialidase activity and were specifically inhibited by homologous and N1 subtype-specific, heterologous sheep sera. Moreover, A/California/07/2009 N1-PPs were a better source of NA compared to whole live and detergent treated H1N1 viruses in ELLA, likely due to lack of interference by anti-HA Ab, and absence of possible structural modifications caused by treatment with detergent. This innovative assay is safer and applicable to all NAs. Taken together, these results highlight the potential of NA-PPs-based NI assays to be developed as sensitive, flexible, easy to handle and scalable serological tests for routine NA immune response analysis.

Positive Contribution of Adjuvanted Influenza Vaccines to the Resolution of Bacterial Superinfections

BACKGROUND Most preclinical studies assess vaccine effectiveness in single-pathogen infection models. This is unrealistic given that humans are continuously exposed to different commensals and pathogens in sequential and mixed infections. Accordingly, complications from secondary bacterial infection are a leading cause of influenza-associated morbidity and mortality. New vaccination strategies are needed to control infections on simultaneous fronts. METHODS We compared different anti-influenza vaccines for their protective potential in a model of viral infection with bacterial superinfection. Mice were immunized with H1N1/A/California/7/2009 subunit vaccines, formulated with different adjuvants inducing either T-helper type 1 (Th1) (MF59 plus CpG)-, Th1/2 (MF59)-, or Th17 (LTK63)-prone immune responses and were sequentially challenged with mouse-adapted influenza virus H1N1/A/Puerto Rico/8/1934 and Staphylococcus aureus USA300, a clonotype emerging as a leading contributor in postinfluenza pneumonia in humans. RESULTS Unadjuvanted vaccine controlled single viral infection, yet mice had considerable morbidity from viral disease and bacterial superinfection. In contrast, all adjuvanted vaccines efficiently protected mice in both conditions. Interestingly, the Th1-inducing formulation was superior to Th1/2 or Th17 inducers. CONCLUSIONS Our studies should help us better understand how differential immunity to influenza skews immune responses toward coinfecting bacteria and discover novel modes to prevent bacterial superinfections in the lungs of persons with influenza.

NMR STRUCTURAL INVESTIGATION OF THE BIOTIN-AVIDIN COMPLEX: A13C NMR PARAMAGNETIC RELAXATION STUDY

Abstract The paramagnetic contributions to the spin-lattice relaxation rates of biotin 13C nuclei, induced by the presence in the water/DMSO solution of the TEMPOL nitroxide, have been analysed in the interaction with avidin. The paramagnetic relaxation data, obtained at different temperatures, indicate that the average solvent/spin-label exposure of biotin carbons is consistent with the conformational features previously observed for the complex in the crystal. The analysis of the paramagnetic perturbation profiles, derived from 13C spin lattice relaxation measurements, seems to be highly informative of the sterical aspects of interaction processes of large biopolymers with their ligands.

A 1H paramagnetic relaxation study on the interaction of peptides with aminoxyl spin labels in apolar environments

Abstract The use of stable free radicals such as aminoxyl spin-labels has been recently exploited for obtaining information on solution structures of peptides (1,2) and proteins (3,4) from multidimensional NMR spectra. In those reports, it has been suggested that an efficient spin-labelling of water and dimethylsulphoxide (DMSO) solvents may be achieved and the paramagnetic solvent yields relaxation effects on backbone proton nuclei which are directly driven by the molecular surface accessibility. Upon the addition of the paramagnetic probe, the observation of absence in chemical shifts changes and of nuclear relaxation effects which are fully rationalised in terms of the solution structure, support a dynamic model for the interaction between the chemical probe and the biomolecule where weak collisional adducts favour the dipolar coupling of the unpaired electron with the outer nuclei of the investigated molecule.