Homologous or heterologous administration of mRNA or adenovirus-vectored vaccines show comparable immunogenicity and effectiveness against the SARS-CoV-2 Omicron variant.

BACKGROUND: Heterologous prime-boost schedules have been employed in SARS-CoV-2 vaccination, yet additional data on immunogenicity and effectiveness are still needed. RESEARCH DESIGN AND METHODS: Here, we measured the immunogenicity and effectiveness in the real-world setting of the mRNA booster dose in 181 subjects who had completed primary vaccination with ChAdOx1, BNT162b2, or mRNA1273 vaccines (IMMUNO_COV study; protocol code 18,869). The spike-specific antibody and B cell responses were analyzed up to 6 months after boosting. RESULTS: After an initial slower antibody response, the heterologous ChAdOx1/mRNA prime-boost formulation elicited spike-specific IgG titers comparable to homologous approaches, while spike-specific B cells showed a higher percentage of CD21-CD27- atypical cells compared to homologous mRNA vaccination. Mixed combinations of BNT162b2 and mRNA-1273 elicited an immune response comparable with homologous strategies. Non-significant differences in the Relative Risk of infection, calculated over a period of 18 months after boosting, were reported among homologous or heterologous vaccination groups, indicating a comparable relative vaccine effectiveness. CONCLUSIONS: Our data endorse the heterologous booster vaccination with mRNA as a valuable alternative to homologous schedules. This approach can serve as a solution in instances of formulation shortages and contribute to enhancing vaccine strategies for potential epidemics or pandemics.

Trajectory of Spike-Specific B Cells Elicited by Two Doses of BNT162b2 mRNA Vaccine.

The mRNA vaccines for SARS-CoV-2 have demonstrated efficacy and immunogenicity in the real-world setting. However, most of the research on vaccine immunogenicity has been centered on characterizing the antibody response, with limited exploration into the persistence of spike-specific memory B cells. Here we monitored the durability of the memory B cell response up to 9 months post-vaccination, and characterized the trajectory of spike-specific B cell phenotypes in healthy individuals who received two doses of the BNT162b2 vaccine. To profile the spike-specific B cell response, we applied the tSNE and Cytotree automated approaches. Spike-specific IgA+ and IgG+ plasmablasts and IgA+ activated cells were observed 7 days after the second dose and disappeared 3 months later, while subsets of spike-specific IgG+ resting memory B cells became predominant 9 months after vaccination, and they were capable of differentiating into spike-specific IgG secreting cells when restimulated in vitro. Other subsets of spike-specific B cells, such as IgM+ or unswitched IgM+IgD+ or IgG+ double negative/atypical cells, were also elicited by the BNT162b2 vaccine and persisted up to month 9. The analysis of circulating spike-specific IgG, IgA, and IgM was in line with the plasmablasts observed. The longitudinal analysis of the antigen-specific B cell response elicited by mRNA-based vaccines provides valuable insights into our understanding of the immunogenicity of this novel vaccine platform destined for future widespread use, and it can help in guiding future decisions and vaccination schedules.

B cell response after SARS-CoV-2 mRNA vaccination in people living with HIV.

BACKGROUND: Limited longitudinal data are available on immune response to mRNA SARS-CoV-2 vaccination in people living with HIV (PLWHIV); therefore, new evidence on induction and persistence of spike-specific antibodies and B cells is needed. METHODS: In this pilot study we investigated the spike-specific humoral and B cell responses up to six months after vaccination with two doses of mRNA vaccines in 84 PLWHIV under antiretroviral therapy compared to 79 healthy controls (HCs). RESULTS: Spike-specific IgG persisted six months in PLWHIV with no significant differences compared to HCs, even though a significantly lower IgG response was observed in patients with CD4+ T cells < 350/mmc. The frequency of subjects with antibodies capable of inhibiting ACE2/RBD binding was comparable between PLWHIV and HCs a month after the second vaccine dose, then a higher drop was observed in PLWHIV. A comparable percentage of spike-specific memory B cells was observed at month six in PLWHIV and HCs. However, PLWHIV showed a higher frequency of spike-specific IgD- CD27- double-negative memory B cells and a significantly lower rate of IgD- CD27+ Ig-switched memory B cells compared to HCs, suggesting a reduced functionality of the antigen-specific memory B population. CONCLUSIONS: The mRNA vaccination against SARS-CoV-2 elicits humoral and B cell responses quantitatively similar between PLWHIV and HCs, but there are important differences in terms of antibody functionality and phenotypes of memory B cells, reinforcing the notion that tailored vaccination policies should be considered for these patients.

SARS-CoV-2 vaccination has been demonstrated to protect people from severe COVID-19 and death. This is achieved through the induction of a specific immune response that recognizes and responds to the virus. Limited data are available on the immune response to SARS-CoV-2 vaccination in people living with HIV (PLWHIV). In this study, we evaluated the immune response up to six months after vaccination with two doses of vaccines in PLWHIV being treated with the standard antiretroviral therapy. We show that the immune response observed in PLWHIV is broadly similar to that in healthy subjects but that there are some differences in the cells induced as part of the immune response. We therefore suggest that specific vaccination policies should be considered for these PLWHIV.

Monitoring Anti-PEG Antibodies Level upon Repeated Lipid Nanoparticle-Based COVID-19 Vaccine Administration.

PEGylated lipids are one of the four constituents of lipid nanoparticle mRNA COVID-19 vaccines. Therefore, various concerns have been raised on the generation of anti-PEG antibodies and their potential role in inducing hypersensitivity reactions following vaccination or in reducing vaccine efficacy due to anti-carrier immunity. Here, we assess the prevalence of anti-PEG antibodies, in a cohort of vaccinated individuals, and give an overview of their time evolution after repeated vaccine administrations. Results indicate that, in our cohort, the presence of PEG in the formulation did not influence the level of anti-Spike antibodies generated upon vaccination and was not related to any reported, serious adverse effects. The time-course analysis of anti-PEG IgG showed no significant booster effect after each dose, whereas for IgM a significant increase in antibody levels was detected after the first and third dose. Data suggest that the presence of PEG in the formulation does not affect safety or efficacy of lipid-nanoparticle-based COVID-19 vaccines.

Intra-Laboratory Evaluation of DNA Extraction Methods and Assessment of a Droplet Digital PCR for the Detection of Xanthomonas citri pv. citri on Different Citrus Species

Xanthomonas citri pv. citri (Xcc) and X. citri pv. aurantifolii (Xca), causal agents of citrus bacterial canker, are both regulated by the European Union to prevent their introduction. Xcc is responsible for severe outbreaks of citrus production worldwide, therefore, a prompt and reliable detection is advisable for the early detection of this bacterium either in symptomatic or asymptomatic plant material. The current EPPO (European and Mediterranean Plant Protection Organization) diagnostic protocol, PM 7/44(1), includes several diagnostic tests even if new assays have been developed in the latter years for which validation data are needed. Recently, a test performance study was organized within the Valitest EU Project to validate Xcc diagnostic methods and provide evidence on the most reliable assays; however, the influence of DNA extraction methods (DEM) on the reliability of the detection has never been assessed. In this study we evaluate four different DEM, by following two different approaches: (i) a comparison by real-time PCR standard curves of bacterial DNA versus bacterial DNA added to plant DNA (lemon, leaves and fruit; orange fruit); and (ii) the evaluation of performance criteria of spiked samples (plant extract added with ten-fold diluted bacterial suspensions at known concentrations). Droplet digital PCR is developed and compared with real-time PCR, as the detection method.

Evidence of SARS-CoV-2-Specific Memory B Cells Six Months After Vaccination With the BNT162b2 mRNA Vaccine.

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.

Human Transcriptomic Response to the VSV-Vectored Ebola Vaccine.

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.

Further In Vitro Assessment and Mid-Term Evaluation of Control Strategy of Xylella fastidiosa subsp. pauca in Olive Groves of Salento (Apulia, Italy).

During recent years; Xylella fastidiosa subsp. pauca (Xfp) has spread in Salento causing relevant damage to the olive groves. Measures to contain the spreading of the pathogen include the monitoring of the areas bordering the so-called "infected" zone and the tree eradication in case of positive detection. In order to provide a control strategy aimed to maintain the tree productivity in the infected areas, we further evaluated the in vitro and in planta mid-term effectiveness of a zinc-copper-citric acid biocomplex. The compound showed an in vitro bactericidal activity and inhibited the biofilm formation in representative strains of X. fastidiosa subspecies, including Xfp isolated in Apulia from olive trees. The field mid-term evaluation of the control strategy assessed by quantitative real-time PCR in 41 trees of two olive groves of the "infected" area revealed a low concentration of Xfp over the seasons upon the regular spraying of the biocomplex over 3 or 4 consecutive years. In particular, the bacterial concentration lowered in July and October with respect to March, after six consecutive treatments. The trend was not affected by the cultivar and it was similar either in the Xfp-sensitive cultivars Ogliarola salentina and Cellina di Nardò or in the Xfp-resistant Leccino. Moreover, the scoring of the number of wilted twigs over the seasons confirmed the trend. The efficacy of the treatment in the management of olive groves subjected to a high pathogen pressure is highlighted by the yielded a good oil production.

From Bivariate to Multivariate Analysis of Cytometric Data: Overview of Computational Methods and Their Application in Vaccination Studies.

Flow and mass cytometry are used to quantify the expression of multiple extracellular or intracellular molecules on single cells, allowing the phenotypic and functional characterization of complex cell populations. Multiparametric flow cytometry is particularly suitable for deep analysis of immune responses after vaccination, as it allows to measure the frequency, the phenotype, and the functional features of antigen-specific cells. When many parameters are investigated simultaneously, it is not feasible to analyze all the possible bi-dimensional combinations of marker expression with classical manual analysis and the adoption of advanced automated tools to process and analyze high-dimensional data sets becomes necessary. In recent years, the development of many tools for the automated analysis of multiparametric cytometry data has been reported, with an increasing record of publications starting from 2014. However, the use of these tools has been preferentially restricted to bioinformaticians, while few of them are routinely employed by the biomedical community. Filling the gap between algorithms developers and final users is fundamental for exploiting the advantages of computational tools in the analysis of cytometry data. The potentialities of automated analyses range from the improvement of the data quality in the pre-processing steps up to the unbiased, data-driven examination of complex datasets using a variety of algorithms based on different approaches. In this review, an overview of the automated analysis pipeline is provided, spanning from the pre-processing phase to the automated population analysis. Analysis based on computational tools might overcame both the subjectivity of manual gating and the operator-biased exploration of expected populations. Examples of applications of automated tools that have successfully improved the characterization of different cell populations in vaccination studies are also presented.

Computational Analysis of Multiparametric Flow Cytometric Data to Dissect B Cell Subsets in Vaccine Studies.

The generation of the B cell response upon vaccination is characterized by the induction of different functional and phenotypic subpopulations and is strongly dependent on the vaccine formulation, including the adjuvant used. Here, we have profiled the different B cell subsets elicited upon vaccination, using machine learning methods for interpreting high-dimensional flow cytometry data sets. The B cell response elicited by an adjuvanted vaccine formulation, compared to the antigen alone, was characterized using two automated methods based on clustering (FlowSOM) and dimensional reduction (t-SNE) approaches. The clustering method identified, based on multiple marker expression, different B cell populations, including plasmablasts, plasma cells, germinal center B cells and their subsets, while this profiling was more difficult with t-SNE analysis. When undefined phenotypes were detected, their characterization could be improved by integrating the t-SNE spatial visualization of cells with the FlowSOM clusters. The frequency of some cellular subsets, in particular plasma cells, was significantly higher in lymph nodes of mice primed with the adjuvanted formulation compared to antigen alone. Thanks to this automatic data analysis it was possible to identify, in an unbiased way, different B cell populations and also intermediate stages of cell differentiation elicited by immunization, thus providing a signature of B cell recall response that can be hardly obtained with the classical bidimensional gating analysis. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Lipid Profile of Xylella fastidiosa Subsp. pauca Associated With the Olive Quick Decline Syndrome.

Lipids, components of the plasma and intracellular membranes as well as of droplets, provide different biological functions related to energy, carbon storage, and stress responses. Bacterial species display diverse membrane composition that changes in response to the different environmental conditions. During plant-pathogen interactions, lipids might have roles in several aspects such as recognition, signal transduction, and downstream responses. Among lipid entities, free fatty acids (FFAs) and their oxidized form, the oxylipins, represent an important class of signaling molecules in host-pathogen perception, especially related to virulence and defense. In bacteria, FFAs (e.g., diffusible signaling factors) and oxylipins have a crucial role in modulating motility, biofilm formation, and virulence. In this study, we explore by LC-TOF and LC-MS/MS the lipid composition of Xylella fastidiosa subsp. pauca strain De Donno in pure culture; some specific lipids (e.g., ornithine lipids and the oxylipin 7,10-diHOME), characteristic of other pathogenic bacteria, were revealed. Nicotiana tabacum was used for testing the ability of this pathogen in producing such lipids in the host. Different lipid compounds present a clear distribution pattern within the infected plant tissues compared to the uninfected ones.

Heterologous Prime-Boost Combinations Highlight the Crucial Role of Adjuvant in Priming the Immune System.

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 adjuvant (o/w squalene). 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 analyzed 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 upregulated 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 primary T and B cell responses that can be efficiently reactivated by booster immunization also performed with antigen alone.