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SARS-CoV-2 mRNA vaccines have demonstrated high efficacy and immunogenicity, but limited information is currently available on memory B cell generation and long-term persistence. Here, we investigated spike-specific memory B cells and humoral responses in 145 subjects, up to 6 months after the BNT162b2 vaccine (Comirnaty) administration. Spike-specific antibodies peaked 7 days after the second dose and significant antibody titers and ACE2/RBD binding inhibiting activity were still observed after 6 months, despite a progressive decline over time. Concomitant to antibody reduction, spike-specific memory B cells, mostly IgG class-switched, increased in the blood of vaccinees and persisted 6 months after vaccination. Following the in vitro restimulation, circulating memory B cells reactivated and produced spike-specific antibodies. A high frequency of spike-specific IgG+ plasmablasts, identified by computational analysis 7 days after boost, positively correlated with the generation of IgG+ memory B cells at 6 months. These data demonstrate that mRNA BNT162b2 vaccine elicits strong B cell immunity with spike-specific memory B cells that still persist 6 months after vaccination, playing a crucial role for a rapid response to SARS-CoV-2 virus encounter.

SUMMARY Objectives : We sought to collate and summarize high-quality data on non-typhoidal Salmonella invasive disease (iNTS) incidence to provide contemporary incidence estimates by location and year. Methods : We systematically searched the databases Embase + MEDLINE, Web of Science, and PubMed for articles published on the incidence of iNTS from inception of the database through 8 May 2020 with no language, country, date, or demographic restrictions applied. A meta-analysis was performed to report pooled iNTS incidence as a rate of cases per 100,000 per year. Results : Among 13 studies eligible for analysis, there were 68 estimates of incidence. Overall pooled incidence (95% CI) was 44.8 (31.5-60.5) per 100,000 persons per year. When stratified by region, pooled incidence was significantly higher in Africa than Asia, 51.0 (36.3-68.0) compared to 1.0 (0.2-2.5), respectively. Incidence was consistently higher in children aged

Adjuvants have been used for decades to enhance the immune response to vaccines, in particular for the subunit-based adjuvants. Physicochemical properties of the adjuvant-protein antigen complexes, such as size, morphology, protein structure and binding, influence the overall efficacy and safety of the vaccine. Here we show how to perform an accurate physicochemical characterization of the nanoaluminum–ovalbumin complex. Using a combination of existing techniques, we developed a multi-staged characterization strategy based on measurements of increased complexity. This characterization cascade has the advantage of being very flexible and easily adaptable to any adjuvant-protein antigen combinations. It will contribute to control the quality of antigen–adjuvant complexes and immunological outcomes, ultimately leading to improved vaccines.

Salmonella Typhimurium (STm) represents the most prevalent cause of invasive non-typhoidal Salmonella (iNTS) disease, and currently no licensed vaccine is available. In this work we characterized the long-term anti-bacterial immunity elicited by a STm vaccine based on Generalized Modules of Membrane Antigens (GMMA) delivering O:4,5 antigen, using a murine model of systemic infection. Subcutaneous immunization of mice with STmGMMA/Alhydrogel elicited rapid, high, and persistent antigen-specific serum IgG and IgM responses. The serum was bactericidal in vitro. O:4,5-specific IgG were also detected in fecal samples after immunization and positively correlated with IgG observed in intestinal washes. Long-lived plasma cells and O:4,5-specific memory B cells were detected in spleen and bone marrow. After systemic STm challenge, a significant reduction of bacterial load in blood, spleen, and liver, as well as a reduction of circulating neutrophils and G-CSF glycoprotein was observed in STmGMMA/Alhydrogel immunized mice compared to untreated animals. Taken together, these data support the development of a GMMA-based vaccine for prevention of iNTS disease.

Characterizing the impact of the vaccination schedule on the induction of B and T cell immune responses is critical for improving vaccine immunogenicity. Here we compare the effect of a short (4 weeks) or a long (18 weeks) interval between priming and boosting in mice, using a model vaccine formulation based on the chimeric tuberculosis vaccine antigen H56 combined with alum. While no significant difference was observed in serum antigen-specific IgG response and the induction of antigen-specific T follicular helper cells into draining lymph nodes after the two immunization schedules, a longer interval between priming and boosting elicited a higher number of germinal center-B cells and H56-specific antibody-secreting cells and modulated the effector function of reactivated CD4+ T cells. These data show that the scheduling of the booster immunization could affect the immune response elicited by vaccination modulating and improving the immunogenicity of the vaccine.

SARS-CoV-2 mRNA vaccines have demonstrated high efficacy and immunogenicity, but limited information is currently available on memory B cells generation and long-term persistence. Here, we investigated Spike-specific memory B cells and humoral responses in 145 subjects, up to six months after the BNT162b2 vaccine (Comirnaty) administration. Spike-specific antibody titers peaked 7 days after the second dose and significant titers and neutralizing activity were still observed after six months, despite a progressive decline over time. Concomitant to antibody reduction, Spike-specific memory B cells, mostly IgG class-switched, increased in blood of vaccinees and persisted six months after vaccination. Following in vitro restimulation, circulating memory B cells reactivated and produced Spike-specific antibodies. A high frequency of Spike-specific IgG+ plasmablasts, identified by computational analysis 7 days after boost, positively correlated with the generation of IgG+ memory B cells at six months.These data demonstrate that mRNA BNT162b2 vaccine elicits strong B cell immunity with Spike-specific memory B cells that still persist six months after vaccination, playing a crucial role for rapid response to SARS-CoV-2 virus encounter.One Sentence SummarymRNA BNT162b2 vaccine elicits persistent spike-specific memory B cells crucial for rapid response to SARS-CoV-2 virus encounter

Immunization with mRNA SARS-CoV-2 vaccines has been highly recommended and prioritized in fragile categories with higher risk of mortality after COVID-19 disease compared to healthy people, including patients with myelofibrosis (MF). Available data on the vaccine immune response developed by MF patients, and the impact of the treatment with the inhibitor of JAK-STAT signaling ruxolitimib, are still fragmented to support an informed decision for a third dose for this category of subjects. Here, we show that 76% of MF patients develop spike-specific IgG after the second vaccine dose, but the response has a slower kinetic compared to healthy subjects, suggesting a reduced capability of their immune system to promptly react to vaccination. A reduced ACE2/RBD inhibition binding activity of spike-specific antibodies was also observed, especially in ruxolitimib treated patients. Our results contribute to answer the open question on the induction of the antibody responses in MF patients following vaccination with COVID-19 mRNA vaccines, showing a slow kinetic that support the need for a third dose of SARS-CoV-2 mRNA vaccines.

Ebolavirus Disease (EVD) is a severe haemorrhagic fever that occurs in epidemic outbreaks, with a high fatality rate and no specific therapies available. rVSVΔG-ZEBOV-GP (Ervebo®), a live-attenuated recombinant vesicular stomatitis virus vector expressing the glycoprotein G of Zaire Ebolavirus, is the first vaccine approved for prevention of EVD. Both innate and adaptive responses are deemed to be involved in vaccine-induced protection, yet the mechanisms are not fully elucidated. A global transcriptomic approach was used to profile the blood host-response in 51 healthy volunteers enrolled in a phase 1/2 clinical trial. Signatures of the host responses were investigated assessing the enrichment in differentially expressed genes (DEGs) of specific “blood transcription modules” (BTM). Comparison of gene-expression levels showed that vaccination produces a peak of 5469 DEGs at day one, representing 38.6% of the expressed genes. Out of 346 BTMs, 144 were significantly affected by vaccination. Innate immunity pathways were induced from day 1 to day 14. At days 2 and 3, neutrophil modules were downregulated and complement-related modules upregulated. T-cell and cell-cycle associated modules were upregulated at days 7 and 14, while at day 28, no modules remained activated. At day 14, a direct correlation was observed between ZEBOV glycoprotein-specific antibody titres and activation of seven BTMs, including two related to B-cell activation and B cell receptor signalling. Transcriptomic analysis identified an rVSVΔG-ZEBOV-GP-induced signature and demonstrated a direct correlation of blood transcriptomic changes with ZEBOV glycoprotein-specific antibody titres.

The rVSV-ZEBOV Ebolavirus vaccine confers protection within days after immunization, suggesting the contribution of innate immune responses. We report modulation of rVSV-ZEBOV vaccinee blood CD56+ NK cell numbers, NKG2D or NKp30 surface receptor expression, Killer Immunoglobulin-like Receptor (KIR)+ cell percentages and NK-cell-related genes on day 1 post immunization. Inverse correlations existed between the concentration of several plasma cytokines and inhibitory KIR+ CD56dim or cytokine-responsive CD56bright NK cells. Thus, NK cells may contribute to the early protective efficacy of rVSV-ZEBOV in humans.

The unprecedented increase of life expectancy challenges society to protect the elderly from morbidity and mortality making vaccination a crucial mean to safeguard this population. Indeed, infectious diseases, such as influenza and pneumonia, are among the top killers of elderly people in the world. Elderly individuals are more prone to severe infections and less responsive to vaccination prevention, due to immunosenescence combined with the progressive increase of a proinflammatory status characteristic of the aging process (inflammaging). These factors are responsible for most age-related diseases and correlate with poor response to vaccination. Therefore, it is of utmost interest to deepen the knowledge regarding the role of inflammaging in vaccination responsiveness to support the development of effective vaccination strategies designed for elderly. In this review we analyse the impact of age-associated factors such as inflammaging, immunosenescence and immunobiography on immune response to vaccination in the elderly, and we consider systems biology approaches as a mean for integrating a multitude of data in order to rationally design vaccination approaches specifically tailored for the elderly.

A clear vision for vaccines research and development (R&D) is needed if Europe is to continue to lead the discovery of next generation vaccines. Innovation Partnership for a Roadmap on Vaccines in Europe (IPROVE) is a collaboration between leading vaccine experts to develop a roadmap setting out how Europe can best invest in the science and technology essential for vaccines innovation. This FP7 project, started in December 2013, brought together more than 130 key public and private stakeholders from academia, public health institutes, regulators, industry and small and medium-sized enterprises to determine and prioritise the gaps and challenges to be addressed to bolster innovation in vaccines and vaccination in Europe. The IPROVE consultation process was structured around seven themes: vaccine R&D, manufacturing and quality control, infrastructure, therapeutic vaccines, needs of small and medium-sized enterprises, vaccines acceptance and training needs. More than 80 recommendations were made by the consultation groups, mainly focused on the need for a multidisciplinary research approach to stimulate innovation, accelerated translation of scientific knowledge into technological innovation, and fostering of real collaboration within the European vaccine ecosystem. The consultation also reinforced the fact that vaccines are only as good as their vaccine implementation programmes, and that more must be done to understand and address vaccination hesitancy of both the general public and healthcare professionals. Bringing together a wide range of stakeholders to work on the IPROVE roadmap has increased mutual understanding of their different perspectives, needs and priorities. IPROVE is a first attempt to develop such a comprehensive view of the vaccine sector. This prioritisation effort, aims to help policy-makers and funders identify those vaccine-related areas and technologies where key investment is needed for short and medium-long term success.

The rVSV-ZEBOV vaccine is currently the only Ebola vaccine with demonstrated clinical efficacy in a ring-vaccination clinical trial. It has been shown to be reactogenic but immunogenic and safe in several Phase I clinical studies. However, its mechanisms of protection are unknown and available immunogenicity data are mostly limited to classical serological analysis; it is now of paramount importance to apply cutting-edge technologies, including transcriptomic and metabolomic analyses, and to perform integrative analyses with standard serology and clinical data to comprehensively profile the rVSV-ZEBOV immune signature.

The SARS-CoV-2 pandemic urgently calls for the development of effective preventive tools. COVID-19 hits greatly the elder and more fragile fraction of the population boosting the evergreen issue of the vaccination of older people. The development of a vaccine against SARS-CoV-2 tailored for the elderly population faces the challenge of the poor immune responsiveness of the older population due to immunosenescence, comorbidities, and pharmacological treatments. Moreover, it is likely that the inflammaging phenotype associated with age could both influence vaccination efficacy and exacerbate the risk of COVID-19-related “cytokine storm syndrome” with an overlap between the factors which impact vaccination effectiveness and those that boost virulence and worsen the prognosis of SARS-CoV-2 infection. The complex and still unclear immunopathological mechanisms of SARS-CoV-2 infection, together with the progressive age-related decline of immune responses, and the lack of clear correlates of protection, make the design of vaccination strategies for older people extremely challenging. In the ongoing effort in vaccine development, different SARS-CoV-2 vaccine candidates have been developed, tested in pre-clinical and clinical studies and are undergoing clinical testing, but only a small fraction of these are currently being tested in the older fraction of the population. Recent advances in systems biology integrating clinical, immunologic, and omics data can help to identify stable and robust markers of vaccine response and move towards a better understanding of SARS-CoV-2 vaccine responses in the elderly.

Analysis of multifunctional CD4+ T cells is fundamental for characterizing the immune responses to vaccination or infection. Major histocompatibility complex (MHC)/peptide tetramers represent a powerful technology for the detection of antigen-specific T cells by specific binding to their T-cell receptor, and their combination with functional assays is fundamental for characterizing the antigen-specific immune response. Here we optimized a protocol for the detection of multiple intracellular cytokines within epitope-specific CD4+ T cells identified by the MHC class II tetramer technology. The optimal procedure for assessing the functional activity of tetramer-binding CD4+ T cells was based on the simultaneous intracellular staining with both MHC tetramers and cytokine-specific antibodies upon in vitro restimulation of cells with the vaccine antigen. The protocol was selected among procedures that differently combine the steps of cellular restimulation and tetramer staining with intracellular cytokine labeling. This method can be applied to better understand the complex functional profile of CD4+ T-cell responses upon vaccination or infection.

The rVSV-ZEBOV Ebolavirus vaccine confers protection within days after immunization, suggesting the contribution of innate immune responses. We report modulation of rVSV-ZEBOV vaccinee blood CD56

Current influenza vaccines manufactured using eggs have considerable limitations, both in terms of scale up production and the potential impact passaging through eggs can have on the antigenicity of the vaccine virus strains. Alternative methods of manufacture are required, particularly in the context of an emerging pandemic strain. Here we explore the production of recombinant influenza haemagglutinin using the ciliated protozoan Tetrahymena thermophila. For the first time we were able to produce haemagglutinin from both seasonal influenza A and B strains. This ciliate derived material was immunogenic, inducing an antibody response in both mice and non-human primates. Mice immunized with ciliate derived haemagglutinin were protected against challenge with homologous influenza A or B viruses. The antigen could also be combined with submicron particles containing a Nod2 ligand, significantly boosting the immune response and reducing the dose of antigen required. Thus, we show that Tetrahymena can be used as a manufacturing platform for viral vaccine antigens.

Transcriptomic profiling of the immune response induced by vaccine adjuvants is of critical importance for the rational design of vaccination strategies. In the present study, transcriptomics was employed to profile the effect of the vaccine adjuvant used for priming on the immune response following re-exposure to the vaccine antigen alone. mRNA sequencing was performed on blood samples collected (1, 2 and 7 days after priming and after boosting) from mice primed with the chimeric vaccine antigen H56 of Mycobacterium tuberculosis administered alone or with the CAF01 adjuvant and boosted with the antigen alone. Gene expression analysis at day 2 after priming showed that the CAF01 adjuvanted vaccine induced a stronger upregulation of the innate immunity modules compared to the unadjuvanted formulation. The immunostimulant effect of the CAF01 adjuvant, used in the primary immunization, was clearly seen after a booster immunization with a low dose of antigen alone. One day after boost, we observed a strong upregulation of multiple genes in blood of mice primed with H56 + CAF01 compared to mice primed with the H56 alone. In particular, blood transcription modules related to innate immune response, such as monocyte and neutrophil recruitment, activation of antigen presenting cells and interferon response were activated. Seven days after boost, differential expression of innate response genes faded while a moderate differential expression of T cell activation modules was appreciable. Indeed, immunological analysis showed a higher frequency of H56-specific CD4+ T cells and germinal center B cells in draining lymph nodes, a strong H56-specific humoral response and a higher frequency of antibody secreting cells in spleen of mice primed with H56 + CAF01. Taken together, these data indicate that the adjuvant used for priming strongly re-programs the immune response that, upon boosting, results in a stronger recall innate response essential for shaping the downstream adaptive response.

The induction and modulation of the immune response to vaccination can be rationally designed by combining different vaccine formulations for priming and boosting. Here, we investigated the impact of heterologous prime-boost approaches on the vaccine-specific cellular and humoral responses specific for a mycobacterial vaccine antigen. C57BL/6 mice were primed with the chimeric vaccine antigen H56 administered alone or with the CAF01 adjuvant, and boosted with H56 alone, or combined with CAF01 or with the squalene-based oil-in-water emulsion (o/w Squalene) adjuvant. A strong secondary H56-specific CD4+ T cell response was recalled by all the booster vaccine formulations when mice had been primed with H56 and CAF01, but not with H56 alone. The polyfunctional nature of T helper cells was analysed and visualized with the multidimensional flow cytometry FlowSOM software, implemented as a package of the R environment. A similar cytokine profile was detected in groups primed with H56 + CAF01 and boosted with or without adjuvant, except for some clusters of cells expressing high level of IL-17 together with TNF-α, IL-2 and IFN-γ, that were significantly up-regulated only in groups boosted with the adjuvants. On the contrary, the comparison between groups primed with or without the adjuvant, showed a completely different clusterization of cells, strengthening the impact of the formulation used for primary immunization on the profiling of responding cells. The presence of the CAF01 adjuvant in the priming formulation deeply affected also the secondary humoral response, especially in groups boosted with H56 alone or o/w squalene. In conclusion, the presence of CAF01 adjuvant in the primary immunization is crucial for promoting a primary T and B cell response that can be efficiently reactivated by booster immunization also performed with antigen alone; the presence of the adjuvant in the booster formulation can be useful for modulating the secondary response of functional T cells expressing specific pattern of cytokines.

Salmonella enterica serovars Typhimurium and Enteritidis are the predominant causes of invasive non-typhoidal Salmonella (iNTS) disease. Considering the co-endemicity of S. Typhimurium and S. Enteritidis, a bivalent vaccine formulation against both pathogens is necessary for protection against iNTS disease, thus investigation of glycoconjugate combination is required. In the present work, we investigated the immune responses induced by S. Typhimurium and S. Enteritidis monovalent and bivalent glycoconjugate vaccines adjuvanted with aluminum hydroxide (alum) only or in combination with cytosine-phosphorothioate-guanine oligodeoxynucleotide (CpG). Humoral and cellular, systemic and local, immune responses were characterized in two different mouse strains. All conjugate vaccines elicited high levels of serum IgG against the respective O-antigens (OAg) with bactericidal activity. The bivalent conjugate vaccine induced systemic production of antibodies against both S. Typhimurium and S. Enteritidis OAg. The presence of alum or alum + CpG adjuvants in vaccine formulations significantly increased the serum antigen-specific antibody production. The alum + CpG bivalent vaccine formulation triggered the highest systemic anti-OAg antibodies and also a significant increase of anti-OAg IgG in intestinal washes and fecal samples, with a positive correlation with serum levels. These data demonstrate the ability of monovalent and bivalent conjugate vaccines against S. Typhimurium and S. Enteritidis to induce systemic and local immune responses in different mouse strains, and highlight the suitability of a bivalent glycoconjugate formulation, especially when adjuvanted with alum + CpG, as a promising candidate vaccine against iNTS disease.

Adjuvants contribute to enhancing and shaping the vaccine immune response through different modes of action. Since the primary immune response can influence the overall quality of the response generated, here we investigate early biomarkers of adjuvanticity after primary immunization with four different adjuvants combined with the chimeric tuberculosis vaccine antigen H56. C57BL/6 mice were immunized by the subcutaneous route with different vaccine formulations, and the modulation of primary CD4+ T cell and B cell responses was assessed within draining lymph nodes, blood and spleen, 7 and 12 days after priming. Vaccine formulations containing the liposome system CAF01 or a squalene-based oil-in-water emulsion (o/w Squalene), but not aluminum hydroxide (Alum) or CpG ODN 1826, elicited a significant primary antigen-specific CD4+ T cell response compared to antigen alone, 7 days after immunization. The effector function of activated CD4+ T cells was skewed towards a Th1/Th17 response by CAF01, while a Th1/Th2 response was elicited by o/w Squalene. Differentiation of B cells in short-lived plasma cells, and subsequent early H56-specific IgG secretion, was observed in mice immunized with o/w Squalene or CpG adjuvants. Tested adjuvants promoted the germinal centre reaction with different magnitude. These results show that the immunological activity of different adjuvants can be characterized by profiling early immunization biomarkers after primary immunization. These data and this approach could give an important contribution to the rational development of heterologous prime-boost vaccine immunization protocols.

The 2014-2015 Ebola epidemic affected several African countries, claiming more than 11,000 lives and leaving thousands with ongoing sequelae. Safe and effective vaccines could prevent or limit future outbreaks. The recombinant vesicular stomatitis virus-vectored Zaire Ebola (rVSV-ZEBOV) vaccine has shown marked immunogenicity and efficacy in humans but is reactogenic at higher doses. To understand its effects, we examined plasma samples from 115 healthy volunteers from Geneva who received low-dose (LD) or high-dose (HD) vaccine or placebo. Fifteen plasma chemokines/cytokines were assessed at baseline and on days 1, 2 to 3, and 7 after injection. Significant increases in monocyte-mediated MCP-1/CCL2, MIP-1β/CCL4, IL-6, TNF-α, IL-1Ra, and IL-10 occurred on day 1. A signature explaining 68% of cytokine/chemokine vaccine-response variability was identified. Its score was higher in HD versus LD vaccinees and was associated positively with vaccine viremia and negatively with cytopenia. It was higher in vaccinees with injection-site pain, fever, myalgia, chills, and headache; higher scores reflected increasing severity. In contrast, HD vaccinees who subsequently developed arthritis had lower day 1 scores than other HD vaccinees. Vaccine dose did not influence the signature despite its influence on specific outcomes. The Geneva-derived signature associated strongly (ρ = 0.97) with that of a cohort of 75 vaccinees from a parallel trial in Lambaréné, Gabon. Its score in Geneva HD vaccinees with subsequent arthritis was significantly lower than that in Lambaréné HD vaccinees, none of whom experienced arthritis. This signature, which reveals monocytes' critical role in rVSV-ZEBOV immunogenicity and safety across doses and continents, should prove useful in assessments of other vaccines.

Background Healthcare workers (HCWs) represent a tuberculosis (TB) risk group for a wide range of tasks in healthcare, even in countries with low TB incidence, like Italy. Latent Tuberculosis Infection (LTBI) screening programs are an important tool for TB prevention in these setting. Methods A retrospective study under a LTBI screening program among HCWs at the Siena University Hospital (Italy), was conducted between September 2011 and July 2015. Tuberculin Skin Test (TST) was used as a first level examination; all TST-positive cases were tested with QuantiFERON-TB Gold In-Tube (QFT-GIT) test, together with a group of TST-negative subjects. Results Among the 2136 HCWs screened, 144 (6.7 %) were TST-positive and therefore tested with QFT-GIT, confirming a positive result in 36 cases (25 %). Agreement between two tests was poor (k = 0.092; 95 %, Confidence Interval [CI]- 0.048–0.136, p = 0.002). Among TST-positive cases, discordant results occurred more frequently in BCG vaccinated than unvaccinated HCWs (86.3 %, p

Generation of primed T cells is crucial for the development of optimal vaccination strategies. Using a TCR-transgenic CD4(+) and CD8(+) T cell adoptive transfer model, we demonstrate that a single nasal immunization with recombinant Streptococcus gordonii induces antigen-specific primed T cells in lymph nodes draining the genital and intestinal tracts with about 80% of CD4(+) and 50% of CD8(+) proliferating cells. T cell clonal expansion was also observed in cervical lymph nodes, draining the immunization site, and in the spleen. The modulation of CD44 and CD45RB marker expression indicated that proliferating T cells were activated. Proliferation in distal mesenteric and iliac lymph nodes and in the spleen was observed 5 days after nasal immunization, while in draining cervical lymph nodes proliferation peaked already at day 3. The division profile of transgenic T cells observed in iliac and mesenteric lymph nodes was discontinuous, showing the lack of early cell divisions. The kinetics of T cell clonal expansion, the discontinuous division profile and the modulation of migration markers such as CD62L suggest that activated antigen-specific T cells disseminate from the immunization site to distal intestinal and genital tracts. These data demonstrate the efficacy of nasal immunization with recombinant S. gordonii in eliciting CD4(+) and CD8(+) T cell priming not only in draining sites, but also in the genital and intestinal tracts and in the spleen.

The primary activation of T-helper and T-cytotoxic cells following mucosal immunization with recombinantStreptococcus gordoniiwas studied in vivo by adoptive transfer of ovalbumin (OVA)-specific transgenic CD8+(OT-I) and CD4+(OT-II) T cells. A recombinant strain, expressing on the surface the vaccine antigen Ag85B-ESAT-6 fromMycobacterium tuberculosisfused to OVA T-helper and T-cytotoxic epitopes (peptides 323 to 339 and 257 to 264), was constructed and used to immunize C57BL/6 mice by the intranasal route. Recombinant, but not wild-type, bacteria induced OVA-specific CD4+and CD8+T-cell clonal expansion in cervical lymph nodes, lung, and spleen. OVA-specific CD4+and CD8+T-cell proliferation appeared first in cervical lymph nodes and later in the spleen, suggesting a possible migration of activated cells from the inductive site to the systemic district. A significant correlation between the percentages of CD4+and CD8+proliferating T cells was observed for each animal. The expression of CD69, CD44, and CD45RB on proliferating T lymphocytes changed as a function of the cell division number, confirming T-cell activation following the antigen encounter. These data indicate that intranasal immunization with recombinantS. gordoniiis capable of inducing primary activation of naive antigen-specific CD4+and CD8+T cells, both locally and systemically.

Streptococcus pneumoniae is the leading cause of bacterial meningitis. Pneumococcal meningitis is a life-threatening disease with high rates of mortality and neurological sequelae. Immune targeting of S. pneumoniae is essential for clearance of infection; however, within the brain, the induced inflammatory response contributes to pathogenesis. In this study we investigate the local inflammatory response and the role of IFN-γ in a murine model of pneumococcal meningitis induced by intracranial injection of type 4 S. pneumoniae. Lymphoid and myeloid cell populations involved in meningitis, as well as cytokine gene expression, were investigated after infection. Animals were treated with a monoclonal antibody specific for murine IFN-γ to evaluate its role in animal survival. Intracranial inoculation of 3 × 10(4) colony-forming units of type 4 strain TIGR4 caused 75% of mice to develop meningitis within 4 days. The amount of lymphocytes, NK cells, neutrophils, monocytes and macrophages in the brain increased 48 h post infection. IFN-γ mRNA levels were about 240-fold higher in brains of infected mice compared to controls. Pro-inflammatory cytokines such as IL-1β and TNF-α, and TLR2 were also upregulated. In vivo treatment with anti-IFN-γ antibody increased survival of infected mice. This study shows that IFN-γ produced during meningitis by type 4 S. pneumoniae enhances bacterial pathogenesis exerting a negative effect on the disease outcome.

The design of heterologous prime-boost vaccine combinations that optimally shape the immune response is of critical importance for the development of next generation vaccines. Here, we tested different prime-boost combinations using the tuberculosis vaccine antigen H56 with CAF01 or CpG ODN 1826 adjuvants, administered by the parenteral and nasal routes. Using peptide-MHC class II tetramers, antigen-specific CD4(+) T cells were tracked following primary and booster immunizations. Both parenteral priming with H56 plus CAF01 and nasal priming with H56 plus CpG elicited significant expansion of CD4(+) tetramer-positive T cells in the spleen; however, only parenterally primed cells responded to booster immunization. Subcutaneous (SC) priming with H56 and CAF01 followed by nasal boosting with H56 and CpG showed the greater expansion of CD4(+) tetramer-positive T cells in the spleen and lungs compared to all the other homologous and heterologous prime-boost combinations. Nasal boosting exerted a recruitment of primed CD4(+) T cells into lungs that was stronger in subcutaneously than nasally primed mice, in accordance with different chemokine receptor expression induced by primary immunization. These data demonstrate that SC priming is fundamental for eliciting CD4(+) T cells that can be efficiently boosted by the nasal route and results in the recruitment of antigen-experienced cells into the lungs. Combination of different vaccine formulations and routes of delivery for priming and boosting is a strategic approach for improving and directing vaccine-induced immune responses.

Tetanus toxin fragment C (TTFC) was expressed on the surface of the vaccine vector Streptococcus gordonii, a Gram-positive commensal bacterium of the human oral cavity. The immunogenicity of recombinant S. gordonii expressing TTFC was assayed in mice immunized by the parenteral and mucosal routes. High serum TTFC-specific IgG responses were induced in both BALB/c and C57BL/6 mice immunized subcutaneously. A total of 82% of vaccinated BALB/c mice were protected from the lethal challenge with 50 LD(50) of tetanus toxin (TT) and a direct correlation between the serum TTFC-specific IgG concentration and survival time of unprotected animals was observed. Intranasal immunization of BALB/c mice was also effective in inducing TTFC-specific serum IgG and local IgA in lung washes. Furthermore, 38% of animals immunized intranasally were protected from the lethal challenge with 10 LD(50) of TT while all control animals died within 24 h. Analysis of the serum IgG subclasses showed that the IgG1 subclass was predominant after parenteral immunization in BALB/c mice (IgG1/IgG2a ratio congruent with6) while following mucosal immunization a mixed IgG1 and IgG2a pattern (IgG1/IgG2a ratio congruent with1) was observed. These data show that TTFC expressed on the surface of S. gordonii is immunogenic by the subcutaneous and mucosal routes and the immune response induced is capable of conferring protection from the lethal challenge with TT.

Five different V3 domains of HIV-1 gp120 were expressed on the surface of the gram-positive bacterium Streptococcus gordonii, a model live vector for vaccine delivery. Sera of HIV-1-infected individuals and human monoclonal antibodies specifically recognized the gp120 sequences on the bacterial surface. Recombinant V3 from the reference HIV-1 strain MN was also shown to retain a conformation that allowed reaction with a conformation-specific monoclonal antibody. A V3-specific serum antibody response was detected in mice immunized both by subcutaneous injection and by vaginal colonization. V3-specific IgG2a antibodies, suggestive of a Th1 response, were found in the sera of mice colonized by recombinant bacteria.

Combinations of different delivery routes for priming and boosting represent vaccination strategies that can modulate magnitude, quality, and localization of the immune response. A murine model was used to study T cell clonal expansion following intranasal (IN) or subcutaneous (SC) priming, and secondary immune responses after boosting by either homologous or heterologous routes. T cell primary activation was studied by using the adoptive transfer model of ovalbumin-specific transgenic CD4(+) T cells. Both IN and SC immunization efficiently elicited, in the respective draining lymph nodes, primary clonal expansion of antigen-specific CD4(+) T cells that disseminated toward distal lymph nodes (mesenteric and iliac) and the spleen. After boosting, a significant serum IgG response was induced in all groups independent of the combination of immunization routes used, while significant levels of local IgA were detected only in mice boosted by the IN route. Mucosal priming drove a stronger Th1 polarization than the systemic route, as shown by serum IgG subclass analysis. IFN-gamma production was observed in splenocytes of all groups, while prime-boost vaccine combinations that included the mucosal route, yielded higher levels of IL-17. Memory lymphocytes were identified in both spleen and draining lymph nodes in all immunized mice, with the highest number of IL-2 producing cells detected in mice primed and boosted by the nasal route. This work shows the critical role of immunization routes in modulating quality and localization of immune responses in prime-boost vaccine strategies.

T cell priming is a critical event in the initiation of the immune response to vaccination since it deeply influences both the magnitude and the quality of the immune response induced. CD4(+) T cell priming, required for the induction of high-affinity antibodies and immune memory, represents a key target for improving and modulating vaccine immunogenicity. A major challenge in the study of in vivo T cell priming is due to the low frequency of antigen-specific T cells. This review discusses the current knowledge on antigen-specific CD4(+) T cell priming in the context of vaccination, as well as the most advanced tools for the characterization of the in vivo T cell priming and the opportunities offered by the application of systems biology.

Enteric fevers remain a common and serious disease, affecting mainly children and adolescents in developing countries. Salmonella enterica serovar Typhi was believed to cause most enteric fever episodes, but several recent reports have shown an increasing incidence of S. Paratyphi A, encouraging the development of a bivalent vaccine to protect against both serovars, especially considering that at present there is no vaccine against S. Paratyphi A. The O-specific polysaccharide (O:2) of S. Paratyphi A is a protective antigen and clinical data have previously demonstrated the potential of using O:2 conjugate vaccines. Here we describe a new conjugation chemistry to link O:2 and the carrier protein CRM(197), using the terminus 3-deoxy-D-manno-octulosonic acid (KDO), thus leaving the O:2 chain unmodified. The new conjugates were tested in mice and compared with other O:2-antigen conjugates, synthesized adopting previously described methods that use CRM(197) as carrier protein. The newly developed conjugation chemistry yielded immunogenic conjugates with strong serum bactericidal activity against S. Paratyphi A.

Non-pathogenic Gram-positive oral commensal bacteria expressing recombinant fusion proteins on their cell surface have been successfully used to raise both a mucosal and a systemic immune response to foreign antigens while colonizing the oropharynx. In this system, fusion-protein vaccines are delivered and anchored to the surface of a commensal, which occupies the mucosal niche invaded by a particular pathogen. Surface expression of these foreign proteins is achieved by exploiting the common mechanism employed by Gram-positive bacteria for translocating and anchoring proteins to the cell surface. The process offers a safe alternative to the use of engineered pathogens as live vaccine delivery vehicles.

A key gap in the development and evaluation of HIV-1 vaccines is insufficient knowledge with regard to sampling techniques and assessment of mucosal immune responses required for early prevention and inhibition of viral dissemination. In an attempt to start bridging this gap, the EUROPRISE network of scientists working on HIV-1 vaccine and microbicide research organized a workshop with the aim to review the types of mucosal responses/biomarkers currently measured in mucosal immunology and to define how the mucosal responses/biomarkers are measured and/or the assays and sampling methods used. The Workshop addressed two critical questions: first whether, with current knowledge, it would be possible to define a consensus set of mucosal sampling methods to facilitate cross-species comparisons and ensure standardized implementation in clinical trials; second to determine the remaining challenges (technical and logistical) and their possible solutions for assessing mucosal responses to HIV-1 vaccines.

While mucosal vaccines have the potential to solve a number of high impact diseases such as HIV and TB, the development of mucosal vaccines has so far been a low priority on the global health agenda. The European Commission through the MUVAPRED program has been able to focus diverse academic and industrial partners on the development of mucosal vaccines and to move the field considerably, proving that mucosal vaccines against poverty-related diseases can work in humans.