Exploring Viral Genome Profile in Mpox Patients during the 2022 Outbreak, in a North-Eastern Centre of Italy.

In 2022, an unprecedented outbreak of mpox raged in several nations. Sequences from the 2022 outbreak reveal a higher nucleotide substitution if compared with the estimated rate for orthopoxviruses. Recently, intra-lesion SNVs (single nucleotide variants) have been described, and these have been suggested as possible sources of genetic variation. Until now, it has not been clear if the presence of several SNVs could represents the result of local mutagenesis or a possible co-infection. We investigated the significance of SNVs through whole-genome sequencing analysis of four unrelated mpox cases. In addition to the known mutations harboured by the circulating strains of virus (MPXV), 7 novel mutations were identified, including SNVs located in genes that are involved in immune evasion mechanisms and/or viral fitness, six of these appeared to be APOBEC3-driven. Interestingly, three patients exhibited the coexistence of mutated and wild-type alleles for five non-synonymous variants. In addition, two patients, apparently unrelated, showed an analogous pattern for two novel mutations, albeit with divergent frequencies. The coexistence of mixed viral populations, harbouring non-synonymous mutations in patients, supports the hypothesis of possible co-infection. Additional investigations of larger clinical cohorts are essential to validating intra-patient viral genome heterogeneity and determining the possibility of co-presence events of slightly divergent MPXV strains.

Changes in the Adaptive Cellular Repertoire after Infection with Different SARS-CoV-2 VOCs in a Cohort of Vaccinated Healthcare Workers.

BACKGROUND: Currently approved vaccines are highly effective in protecting against hospitalization and severe COVID-19 infections. How pre-existing immunity responds to new variants with mutated antigens is crucial information for elucidating the functional interplay between antibodies and B and T cell responses during infection with new SARS-CoV-2 variants. METHODS: In this study, we monitored the dynamics and persistence of the immune response versus different SARS-CoV-2 variants of concern that emerged during the pandemic period (2021-2022) in a cohort of vaccinated healthcare workers, who experienced breakthrough infection in the Pre-Delta, Delta, and Omicron waves. We evaluated both the humoral and cell-mediated responses after infection. We also evaluated the anti-SARS-CoV-2 antibodies levels produced by infection in comparison with those produced after vaccination. RESULTS: Our results highlighted that the immune response against the Delta VOC mainly involved an adaptive humoral and switched memory B cells component, even 3 months after the last vaccine dose, conversely showing a high percentage of depleted adaptive T cells. Omicron infections triggered a consistent production of non-vaccine-associated anti-N antibodies, probably to balance the spike epitope immune escape mechanisms. CONCLUSION: Our results suggest a direct dependence between the VOC and different humoral and B and T cell balances in the post-infection period, despite the administration of a different number of vaccine doses and the elapsed time since the last vaccination.

Urine: A Pitfall for Molecular Detection of Toscana Virus? An Analytical Proof-of-Concept Study.

Toscana virus (TOSV), a sandfly-borne virus, is an important etiological agent in human acute meningitis and meningoencephalitis in the Mediterranean area during the summer. However, the actual number of TOSV infections is underestimated. Laboratory confirmation is necessary because TOSV infection has overlapping clinical features with other neuro-invasive viral infections. Nowadays, the reference test for direct diagnosis in the acute phase of TOSV infection is the PCR based method for detecting TOSV in cerebrospinal fluid and/or plasma, serum, or blood. Although poorly employed, urine is another helpful biological matrix for TOSV detection. Urine is a matrix rich in PCR inhibitors that affect PCR efficiency; consequently, false negatives could be generated. To investigate the potential effect of urine PCR inhibitors on TOSV detection, we compared undiluted and diluted urine using 10-fold series of spiked TOSV. The results showed a significant improvement in TOSV detection performance in diluted urine (1 TCID50 vs. 1 × 104 TCID50 limit of detection and 101.35% vs. 129.62% efficiency, respectively, in diluted and undiluted urine). In conclusion, our data provide preliminary important insights into the use of diluted urine to limit the impact of the inhibitory effects of urine on the detection of TOSV in RT-PCR-based approaches.

Immunogenicity and Pre-Clinical Efficacy of an OMV-Based SARS-CoV-2 Vaccine.

The vaccination campaign against SARS-CoV-2 relies on the world-wide availability of effective vaccines, with a potential need of 20 billion vaccine doses to fully vaccinate the world population. To reach this goal, the manufacturing and logistic processes should be affordable to all countries, irrespective of economical and climatic conditions. Outer membrane vesicles (OMVs) are bacterial-derived vesicles that can be engineered to incorporate heterologous antigens. Given the inherent adjuvanticity, such modified OMVs can be used as vaccines to induce potent immune responses against the associated proteins. Here, we show that OMVs engineered to incorporate peptides derived from the receptor binding motif (RBM) of the spike protein from SARS-CoV-2 elicit an effective immune response in vaccinated mice, resulting in the production of neutralizing antibodies (nAbs) with a titre higher than 1:300. The immunity induced by the vaccine is sufficient to protect the animals from intranasal challenge with SARS-CoV-2, preventing both virus replication in the lungs and the pathology associated with virus infection. Furthermore, we show that OMVs can be effectively decorated with the RBM of the Omicron BA.1 variant and that such engineered OMVs induce nAbs against Omicron BA.1 and BA.5, as measured using the pseudovirus neutralization infectivity assay. Importantly, we show that the RBM438-509 ancestral-OMVs elicited antibodies which efficiently neutralize in vitro both the homologous ancestral strain, the Omicron BA.1 and BA.5 variants with a neutralization titre ranging from 1:100 to 1:1500, suggesting its potential use as a vaccine targeting diverse SARS-CoV-2 variants. Altogether, given the convenience associated with the ease of engineering, production and distribution, our results demonstrate that OMV-based SARS-CoV-2 vaccines can be a crucial addition to the vaccines currently available.

Immunogenicity and pre-clinical efficacy of an OMV-based SARS-CoV-2 vaccine.

The vaccination campaign against SARS-CoV-2 relies on the world-wide availability of effective vaccines, with a potential need of 20 billion vaccine doses to fully vaccinate the world population. To reach this goal, the manufacturing and logistic processes should be affordable to all countries, irrespectively of economical and climatic conditions. Outer membrane vesicles (OMV) are bacterial-derived vesicles that can be engineered to incorporate heterologous antigens. Given the inherent adjuvanticity, such modified OMV can be used as vaccine to induce potent immune responses against the associated protein. Here we show that OMVs engineered to incorporate peptides derived from the receptor binding motif (RBM) of the spike protein from SARS-CoV-2 elicit an effective immune response in vaccinated mice, resulting in the production of neutralizing antibodies (nAbs). The immunity induced by the vaccine is sufficient to protect the animals from intranasal challenge with SARS-CoV-2, preventing both virus replication in the lungs and the pathology associated with virus infection. Furthermore, we show that OMVs can be effectively decorated with the RBM of the Omicron BA.1 variant and that such engineered OMVs induced nAbs against Omicron BA.1 and BA.5, as judged by pseudovirus infectivity assay. Importantly, we show that the RBM438-509 ancestral-OMVs elicited antibodies which efficiently neutralized in vitro both the homologous ancestral strain, the Omicron BA.1 and BA.5 variants, suggesting its potential use as a pan SARS-CoV-2 vaccine. Altogether, given the convenience associated with ease of engineering, production and distribution, our results demonstrate that OMV-based SARS-CoV-2 vaccines can be a crucial addition to the vaccines currently available.

Functional CVIDs phenotype clusters identified by the integration of immune parameters after BNT162b2 boosters.

Introduction: Assessing the response to vaccinations is one of the diagnostic criteria for Common Variable Immune Deficiencies (CVIDs). Vaccination against SARS-CoV-2 offered the unique opportunity to analyze the immune response to a novel antigen. We identify four CVIDs phenotype clusters by the integration of immune parameters after BTN162b2 boosters. Methods: We performed a longitudinal study on 47 CVIDs patients who received the 3rd and 4th vaccine dose of the BNT162b2 vaccine measuring the generation of immunological memory. We analyzed specific and neutralizing antibodies, spike-specific memory B cells, and functional T cells. Results: We found that, depending on the readout of vaccine efficacy, the frequency of responders changes. Although 63.8% of the patients have specific antibodies in the serum, only 30% have high-affinity specific memory B cells and generate recall responses. Discussion: Thanks to the integration of our data, we identified four functional groups of CVIDs patients with different B cell phenotypes, T cell functions, and clinical diseases. The presence of antibodies alone is not sufficient to demonstrate the establishment of immune memory and the measurement of the in-vivo response to vaccination distinguishes patients with different immunological defects and clinical diseases.

Evaluation of a ddPCR Commercial Assay for the Absolute Quantification of the Monkeypox Virus West Africa in Clinical Samples.

BACKGROUND: Monkeypox virus (MPXV) is a double-stranded DNA virus belonging to the orthopoxvirus genus in the family Poxviridae. Distinct clades are identified: the clade I belonging to the Central African (or Congo Basin) clade and the subclades IIa and IIb belonging to the West African clade. Here, a commercial droplet digital PCR (ddPCR) assay was evaluated for the quantification of the MPXV West Africa clade in clinical samples. METHODS: The ddPCR reaction was assessed as a duplex assay using RPP30 as an internal amplification control. A total of 60 clinical specimens were tested, 40 positives (skin lesions, n=10; rectal swabs, n = 10; pharyngeal swabs, n = 10; and whole blood, n = 10), and 20 negatives (n = 5 for each biological matrix) were found at the routine molecular diagnostics (orthopoxvirus qPCR followed by confirmation with Sanger sequencing). To evaluate the analytical sensitivity, the ddPCR reaction was first analyzed on serial dilutions of synthetic DNA spiked in water and in negative biological matrices, achieving a limit of detection of 3.5 copy/µL. RESULTS: Regarding the clinical samples, compared to routine molecular diagnostics, the ddPCR duplex assay showed 100% of specificity for all biological matrices and 100% sensitivity (10/10) for lesions, 100% (10/10) for rectal swabs, 90% (9/10) for pharyngeal swabs, and 60% (6/10) for whole blood. CONCLUSION: Overall, our data showed that the commercial ddPCR assay allowed the DNA detection of MPXV in 87.5% (35/40) of our cohort, highlighting useful technical indications for the different specimens with a potential greatest performance for skin lesions and rectal swabs.

Targeting Muscle-Resident Single Cells Through in vivo Electro-Enhanced Plasmid Transfer in Healthy and Compromised Skeletal Muscle.

Skeletal muscle is composed of syncytial muscle fibers, and by various mononucleated cellular types, such as muscle stem cells, immune cells, interstitial and stromal progenitors. These cell populations play a crucial role during muscle regeneration, and alterations of their phenotypic properties have been associated with defective repair and fibrosis in aging and dystrophic muscle. Studies involving in vivo gene modulation are valuable to investigate the mechanisms underlining cell function and dysfunction in complex pathophysiological settings. Electro-enhanced transfer of plasmids using square-wave generating devices represents a cost-effective approach that is widely used to transport DNA to muscle fibers efficiently. Still, it is not clear if this method can also be applied to mononuclear cells present in muscle. We demonstrate here that it is possible to efficiently deliver DNA into different muscle-resident cell populations in vivo. We evaluated the efficiency of this approach not only in healthy muscle but also in muscles of aging and dystrophic animal models. As an exemplificative application of this method, we used a strategy relying on a reporter gene-based plasmid containing regulatory sequences from the collagen 1 locus, and we determined collagen expression in various cell types reportedly involved in the production of fibrotic tissue in the dystrophic settings. The results enclosed in this manuscript reveal the suitability in applying electro-enhanced transfer of plasmid DNA to mononucleated muscle-resident cells to get insights into the molecular events governing diseased muscle physiology.

SARS-CoV-2 vaccination elicits unconventional IgM specific responses in naïve and previously COVID-19-infected individuals.

BACKGROUND: Currently, evaluation of the IgG antibodies specific for the SARS-CoV-2 Spike protein following vaccination is used worldwide to estimate vaccine response. Limited data are available on vaccine-elicited IgM antibodies and their potential implication in immunity to SARS-CoV-2. METHODS: We performed a longitudinal study to quantify anti-S SARS-CoV-2 IgG and IgM (IgG-S and IgM-S) in health care worker (HCW) recipients of the BNT162b2 vaccine. Samples were collected before administration (T0), at the second dose (T1) and three weeks after T1 (T2). The cohort included 1584 immunologically naïve to SARS-CoV-2 (IN) and 289 with history of previous infection (PI). FINDINGS: IN showed three patterns of responses: (a) IgG positive/IgM negative (36.1%), (b) coordinated IgM-S/IgG-S responses appearing at T1 (37.4%) and (c) IgM appearing after IgG (26.3%). Coordinated IgM-S/IgG-S responses were associated with higher IgG titres. In IgM-S positive PI, 64.5% were IgM-S positive before vaccination, whereas 32% and 3.5% developed IgM-S after the first and second vaccine dose, respectively. IgM-S positive sera had higher pseudovirus neutralization titres compared to the IgM-S negative. INTERPRETATION: Coordinated expression of IgG-S and IgM-S after vaccination was associated with a significantly more efficient response in both antibody levels and virus-neutralizing activity. The unconventional IgG-S positive/IgM-S negative responses may suggest a recruitment of cross coronaviruses immunity by vaccination, warranting further investigation. FUNDING: Italian Ministry of Health under "Fondi Ricerca Corrente"- L1P5 and "Progetto Ricerca Finalizzata COVID-2020-12371675"; FUR 2020 Department of Excellence 2018-2022, MIUR, Italy; The Brain Research Foundation Verona.

Serology study after BTN162b2 vaccination in participants previously infected with SARS-CoV-2 in two different waves versus naïve.

Background: The antibody response to SARS-CoV-2 mRNA vaccines in individuals with waning immunity generated by a previous SARS-CoV-2 infection, as well as the patterns of IgA and IgM responses in previously infected and in naïve individuals are still poorly understood. Methods: We performed a serology study in a cohort of BTN162b2 mRNA vaccine recipients who were immunologically naïve (N, n = 50) or had been previously infected with SARS-CoV-2 (P.I., n = 51) during the first (n = 25) or second (n = 26) pandemic waves in Italy, respectively. We measured IgG, IgM and IgA antibodies against the SARS-CoV-2 Spike (S) and IgG against the nucleocapsid (N) proteins, as well as the neutralizing activity of sera collected before vaccination, after the first and second dose of vaccine. Results: Most P.I. individuals from the first pandemic wave who showed declining antibody titres responded to the first vaccine dose with IgG-S and pseudovirus neutralization titres that were significantly higher than those observed in N individuals after the second vaccine dose. In all recipients, a single dose of vaccine was sufficient to induce a potent IgA response that was not associated with serum neutralization titres. We observed an unconventional pattern of IgM responses that were elicited in only half of immunologically naïve subjects even after the second vaccine dose. Conclusions: The response to a single dose of vaccine in P.I. individuals is more potent than that observed in N individuals after two doses. Vaccine-induced IgA are not associated with serum neutralization.