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Summary: Background: Pre-exposure vaccination with MVA-BN has been widely used against mpox to contain the 2022 outbreak. Many countries have defined prioritized strategies, administering a single dose to those historically vaccinated for smallpox, to achieve quickly adequate coverage in front of low supplies. Using epidemiological models, real-life effectiveness was estimated at approximately 36%–86%, but no clinical trials were performed. Few data on MVA-BN immunogenicity are currently available, and there are no established correlates of protection. Immunological response in PLWH in the context of the 2022 outbreak was also poorly described. Methods: Blood samples were collected from participants eligible for pre-exposure MVA-BN vaccination before (T1) receiving a full course of vaccine (single-dose for vaccine-experienced or smallpox-primed and two-dose for smallpox vaccine-naïve or smallpox non-primed) and one month after the last dose (T2 and T3, respectively). MPXV-specific IgGs were measured by in-house immunofluorescence assay, using 1:20 as screening dilution, MPXV-specific nAbs by 50% plaque reduction neutralization test (PRNT50, starting dilution 1:10), and IFN-γ-producing specific T cells to MVA-BN vaccine, by ELISpot assay. Paired or unpaired t-test and Wilcoxon or Mann–Whitney test were used to analyse IgG and nAbs, and T-cell response, as appropriate. The probability of IgG and nAb response in vaccine-experienced vs. vaccine-naïve was estimated in participants not reactive at T1. The McNemar test was used to evaluate vaccination's effect on humoral response both overall and by smallpox vaccination history. In participants who were not reactive at T1, the proportion of becoming responders one month after full-cycle completion by exposure groups was compared by logistic regression and then analysed by HIV status strata (interaction test). The response was also examined in continuous, and the Average Treatment Effect (ATE) of the difference from baseline to schedule completion according to previous smallpox vaccination was estimated after weighting for HIV using a linear regression model. Self-reports of adverse effects following immunization (AEFIs) were prospectively collected after the first MVA-BN dose (T1). Systemic (S-AEFIs: fatigue, myalgia, headache, GI effects, chills) and local (L-AEFIs: redness, swelling, pain) AEFIs were graded as absent (grade 0), mild (1), moderate (2), or severe (3). The maximum level of severity for S-AEFIs and L-AEFIs ever experienced over the 30 days post-dose by vaccination exposure groups were analysed using a univariable multinomial logistic regression model and after adjusting for HIV status; for each of the symptoms, we also compared the mean duration by exposure group using an unpaired t-test. Findings: Among the 164 participants included, 90 (54.8%) were smallpox vaccine-experienced. Median age was 49 years (IQR 41–55). Among the 76 (46%) PLWH, 76% had a CD4 count >500 cells/μL. There was evidence that both the IgG and nAbs titers increased after administration of the MVA-BN vaccine. However, there was no evidence for a difference in the potential mean change in humoral response from baseline to the completion of a full cycle when comparing primed vs. non-primed participants. Similarly, there was no evidence for a difference in the seroconversion rate after full cycle vaccination in the subset of participants not reactive for nAbs at T1 (p = 1.00 by Fisher's exact test). In this same analysis and for the nAbs outcome, there was some evidence of negative effect modification by HIV (interaction p-value = 0.17) as primed people living with HIV (PLWH) showed a lower probability of seroconversion vs. non-primed, and the opposite was seen in PLWoH. When evaluating the response in continuous, we observed an increase in T-cell response after MVA-BN vaccination in both primed and non-primed. There was evidence for a larger increase when using the 2-dose vs. one-dose strategy with a mean difference of −2.01 log2 (p ≤ 0.0001), after controlling for HIV. No evidence for a difference in the risk of developing any AEFIs of any grade were observed by exposure group, except for the lower risk of grade 2 (moderate) fatigue, induration and local pain which was lower in primed vs. non-primed [OR 0.26 (0.08–0.92), p = 0.037; OR 0.30 (0.10–0.88), p = 0.029 and OR 0.19 (0.05–0.73), p = 0.015, respectively]. No evidence for a difference in symptom duration was also detected between the groups. Interpretation: The evaluation of the humoral and cellular response one month after the completion of the vaccination cycle suggested that MVA-BN is immunogenic and that the administration of a two-dose schedule is preferable regardless of the previous smallpox vaccination history, especially in PLWH, to maximize nAbs response. MVA-BN was safe as well tolerated, with grade 2 reactogenicity higher after the first administration in vaccine-naïve than in vaccine-experienced individuals, but with no evidence for a difference in the duration of these adverse effects. Further studies are needed to evaluate the long-term duration of immunity and to establish specific correlates of protection. Funding: The study was supported by the National Institute for Infectious Disease Lazzaro Spallanzani IRCCS “Advanced grant 5 × 1000, 2021” and by the Italian Ministry of Health “Ricerca Corrente Linea 2”.

Objectives: To characterize the kinetics of humoral and T-cell responses in rheumatoid arthritis (RA)-patients followed up to 4-6 weeks (T3) after the SARS-CoV-2 vaccine booster dose. Methods: Health care workers (HCWs, n = 38) and patients with RA (n = 52) completing the messenger RNA vaccination schedule were enrolled at T3. In each cohort, 25 subjects were sampled after 5 weeks (T1) and 6 months (T2) from the first vaccine dose. The humoral response was assessed by measuring anti-receptor-binding domain (RBD) and neutralizing antibodies, the T-cell response by interferon-γ-release assay (IGRA), T cell cytokine production, and B cell phenotype at T3 by flow cytometry. Results: Patients with RA showed a significant reduction of antibody titers from T1 to T2 and a significant increase at T3. T-cell response by IGRA persisted over time in patients with RA, whereas it increased in HCWs. Most patients with RA scored positive for anti-RBD, neutralizing antibody and T-cell responses, although the magnitude was lower than HCWs. The spike-specific-cytokine response was mainly clusters of differentiation (CD)4+ T cells restricted in both cohorts and significantly lower with reduced interleukin-2 response and CD4-antigen-responding naïve T cells in patients with RA. Unswitched memory B cells were reduced in patients with RA compared with HCWs independently of vaccination. Conclusion: COVID-19 vaccine booster strengthens the humoral immunity in patients with RA even with a reduced cytokine response.

Abstract Despite the successful deployment of efficacious vaccines and therapeutics, the development of novel vaccines for SARS-CoV-2 remains a major goal to increase vaccine doses availability and accessibility for lower income setting. We report here on the kinetics of Spike-specific humoral and T-cell response in young and old volunteers over 6 months follow-up after a single intramuscular administration of GRAd-COV2, a gorilla adenoviral vector-based vaccine candidate currently in phase-2 of clinical development. At all three tested vaccine dosages, Spike binding and neutralizing antibodies were induced and substantially maintained up to 3 months, to then contract at 6 months. Potent T-cell responses were readily induced and sustained throughout the study period, with only minor decline. No major differences in immune response to GRAd-COV2 vaccination were observed in the two age cohorts. In light of its favorable safety and immunogenicity, GRAd-COV2 is a valuable candidate for further clinical development and potential addition to the COVID-19 vaccine toolbox to help fighting SARS-CoV-2 pandemic.

Summary: We report the follow-up laboratory investigation of three MPXV cases infected in May-June 2022 from diagnosis to disease resolution, monitoring viral shedding in different body fluids and antibody kinetics. Out of 138 non-lesion samples, viral DNA was found in 92.3% saliva, 85.7% semen, 86.2% oropharyngeal swabs, 51.7% plasma, 46.1% stool, and 9.5% urine samples. Viral load quantified by digital PCR widely varied, but tend to be higher in oropharyngeal swabs, saliva, and stool. Replication competent virus was recovered from four out of seventeen samples, including 1 saliva, 1 oropharyngeal swabs, 1 semen, and 1 stool. The analysis of the antibody kinetics revealed that IgM, IgA, and IgG antibodies were detected within two weeks post-symptoms onset for all three patients, with IgG detected early on at day 4-8 and IgM and IgA showing lower titers along the time frame of the study. Antibody levels increased during the second week of illness with IgG reaching high titers.

When the Mpox virus (MPXV) began spreading globally in 2022, it became critical to evaluate whether residual immunity from smallpox vaccination provided cross-protection. To assess the cross-immune response to MPXV, we collected serum samples (n = 97) and PBMCs (n = 30) from healthy-donors, either born before 1974 and reporting smallpox vaccination during childhood or born after 1975 and not vaccinated with Vaccinia virus (VACV)-based vaccines. We evaluated the levels of anti-MPXV IgG and neutralizing antibodies (Nabs) and the presence of a T cell response against MPXV. We found anti-MPXV IgG and Nabs in 60 (89.6%) and 40 (70.1%) vaccinated individuals, respectively. We observed a T cell response to Orthopoxviruses and MPXV peptide pools in 30% of vaccinated individuals. We thus show that a high proportion of subjects who received the smallpox vaccine 40 to 60 years ago have humoral cross-immunity, while the T-cell-specific response against MPXV was observed in a smaller group (30%) of vaccinated individuals. This study, combined with information on immunity developed during natural infection or the administration of current vaccines, will contribute to a better understanding of humoral and cellular responses against MPXV.

Ebola is a highly pathogenic virus, which in humans reaches a mortality rate above 50%. Due to a lack of laboratories in territories where Ebola viruses are endemic and the limited number of surveillance programmes, tests for the confirmation of suspected cases of Ebola are often performed in Reference Laboratories. While this provides guarantees regarding the accuracy of results, the shipment of samples to a centralized facility where the diagnostic test can be performed and the time required to achieve the results takes several days, which increases costs and entails delays in the isolation of positive subjects and therapeutic intervention with negative consequences both for patients and the community. Molecular tests have been the most frequently used tool in Ebola diagnosis in recent outbreaks. One of the most commonly used molecular tests is the Real-Star Altona, which targets a conserved area of the L gene. This assay showed different sensitivities depending on the Ebola virus: 471 copies/mL (EBOV) and 2871 copies/ml (SUDAN virus). The Cepheid system also showed good sensitivity (232 copies/mL). The LAMP platform is very promising because, being an isothermal reaction, it does not require high-precision instrumentation and can be considered a Point of Care (PoC) tool. Its analytical sensitivity is 1 copy/reaction. However, since data from real life studies are not yet available, it is premature to give any indications on its feasibility. Moreover, in November 2014, the WHO recommended the development of rapid diagnostic tests (RDT) according to ASSURED criteria. Several RDT assays have since been produced, most of which are rapid tests based on the search for antibody anti-Ebola viral proteins with immunochromatographic methods. Several viral antigens are used for this purpose: VP40, NP and GP. These assays show different sensitivities according to the protein used: VP40 57.4–93.1%, GP 53–88.9% and 85% for NP compared to reference molecular assays. From these results, it can be deduced that no RDT reaches the 99% sensitivity recommended by the WHO and therefore any RDT negative results in suspected cases should be confirmed with a molecular test.

IntroductionImmunocompromised patients have been shown to have an impaired immune response to COVID-19 vaccines.MethodsHere we compared the B-cell, T-cell and neutralizing antibody response to WT and Omicron BA.2 SARS-CoV-2 virus after the fourth dose of mRNA COVID-19 vaccines in patients with hematological malignancies (HM, n=71), solid tumors (ST, n=39) and immune-rheumatological (IR, n=25) diseases. The humoral and T-cell responses to SARS-CoV-2 vaccination were analyzed by quantifying the anti-RBD antibodies, their neutralization activity and the IFN-γ released after spike specific stimulation.ResultsWe show that the T-cell response is similarly boosted by the fourth dose across the different subgroups, while the antibody response is improved only in patients not receiving B-cell targeted therapies, independent on the pathology. However, 9% of patients with anti-RBD antibodies did not have neutralizing antibodies to either virus variants, while an additional 5.7% did not have neutralizing antibodies to Omicron BA.2, making these patients particularly vulnerable to SARS-CoV-2 infection. The increment of neutralizing antibodies was very similar towards Omicron BA.2 and WT virus after the third or fourth dose of vaccine, suggesting that there is no preferential skewing towards either virus variant with the booster dose. The only limited step is the amount of antibodies that are elicited after vaccination, thus increasing the probability of developing neutralizing antibodies to both variants of virus.DiscussionThese data support the recommendation of additional booster doses in frail patients to enhance the development of a B-cell response directed against Omicron and/or to enhance the T-cell response in patients treated with anti-CD20.

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

IntroductionImmunocompromised patients with B-cell depletion agents are at risk for persistence and/or severe SARS-COV-2 infection. We describe a case series of 21 COVID-19 patients under B cell depletion therapy, mostly treated with a combined therapy based on intravenous remdesevir (RDV) and steroid associated with SARS-CoV-2 monoclonal antibodies against Spike glycoprotein and/or hyper-immune convalescent plasma.MethodsThis is a single-center longitudinal study. We retrospectively enrolled a total number of 21 B-cell depleted consecutive hospitalized patients with COVID-19 at the Lazzaro Spallanzani National Institute for Infectious Diseases, Rome, Italy, from November 2020 to December 2021. Demographic characteristics, medical history, clinical presentation, treatment, adverse drug reactions, and clinical and virological outcome were collected for all patients. In a subgroup, we explore immune T cells activation, T cells specific anti-SARS-COV-2 response, and neutralizing antibodies.ResultsTwenty-one inpatients with B-cell depletion and SARS-COV-2 infection were enrolled. A median of 1 B cells/mm3 was detected. Eighteen patients presented hypogammaglobulinemia. All patients presented interstitial pneumonia treated with intravenous RDV and steroids. Sixteen patients were treated with monoclonal antibodies against SARS-CoV-2 Spike protein, four patients were treated with SARS-CoV-2 hyper-immune convalescent plasma infusion, and three patients received both treatments. A variable kinetic of T cell activation returning to normal levels at Day 30 after immunotherapy infusion was observed. All treated patients recovered.ConclusionIn COVID-19 immunosuppressed subjects, it is mandatory to establish a prompt, effective, and combined multi-target therapy including oxygen, antiviral, steroid, and antibody-based therapeutics, tailored to the patient’s clinical needs.

ObjectivesComparative analysis between different monoclonal antibodies (mAbs) against SARS-CoV-2 are lacking. We present an emulation trial from observational data to compare effectiveness of Bamlanivimab/Etesevimab (BAM/ETE) and Casirivimab/Imdevimab (CAS/IMD) in outpatients with early mild-to-moderate COVID-19 in a real-world scenario of variants of concern (VoCs) from Alpha to Delta.MethodsAllocation to treatment was subject to mAbs availability, and the measured factors were not used to determine which combination to use. Patients were followed through day 30. Viral load was measured by cycle threshold (CT) on D1 (baseline) and D7.Primary outcome was time to COVID-19-related hospitalization or death from any cause over days 0-30. Weighted pooled logistic regression and marginal structural Cox model by inverse probability weights were used to compare BAM/ETE vs. CAS/IMD. ANCOVA was used to compare mean D7 CT values by intervention. Models were adjusted for calendar month, MASS score and VoCs. We evaluated effect measure modification by VoCs, vaccination, D1 CT levels and enrolment period.ResultsCOVID19-related hospitalization or death from any cause occurred in 15 of 237 patients in the BAM/ETE group (6.3%) and in 4 of 196 patients in the CAS/IMD group (2.0%) (relative risk reduction [1 minus the relative risk] 72%; p=0.024). Subset analysis carried no evidence that the effect of the intervention was different across stratification factors. There was no evidence in viral load reduction from baseline through day 7 across the two groups (+0.17, 95% -1.41;+1.74, p=0.83). Among patients who experienced primary outcome, none showed a negative RT-PCR test in nasopharyngeal swab (p=0.009) and 82.4% showed still high viral load (p

With SARS-CoV-2 infection, pregnant women may be at a high risk of severe disease and adverse perinatal outcomes. A COVID-19 vaccination campaign represents the key strategy to combat the pandemic; however, public acceptance of maternal immunization has to be improved, which may be achieved by highlighting the promising mechanism of passive immunity as a strategy for protecting newborns against SARS-CoV-2 infection. We tested the anti-SARS-CoV-2 antibody response following COVID-19 full-dose vaccination in the serum and amniotic fluid of two pregnant women who presented between April and June 2021, at the Center for the Treatment and Prevention of Infections in Pregnancy of the National Institute for Infectious Diseases “L. Spallanzani”, for antenatal consultancy. Anti-SARS-CoV-2 IgG was found in residual samples of amniotic fluid collected from both women at the 18th week of gestation (63 and 131 days after the second dose’s administration). Titers in amniotic fluid mirrored the levels detected in serum and were inversely linked to the time from vaccination. Our results suggest that antibodies elicited by COVID-19 vaccination can cross the placenta and reach the fetus; therefore, they may offer passive immunity at birth. It is critical to fully understand the kinetics of the maternal response to vaccination, the efficiency of IgG transfer, and the persistence of antibodies in infants to optimize maternal immunization regimens.

The new Omicron variant of SARS-CoV-2, first identified in November 2021, is rapidly spreading all around the world. Omicron has become the dominant variant of SARS-CoV-2. There are many ongoing studies evaluating the effectiveness of existing vaccines. Studies on the neutralizing activity of vaccinated sera against the Omicron variant are currently being carried out in many laboratories. In this study, we have shown the neutralizing activity of sera against the SARS-CoV-2 Omicron variant compared to the reference Wuhan D614G variant in individuals vaccinated with two doses of Sputnik V up to 6 months after vaccination and in individuals who experienced SARS-CoV-2 infection either before or after vaccination. As a control to our study we also measured neutralizing antibody titers in individuals vaccinated with two doses of BNT162b2. The decrease in NtAb titers to the Omicron variant was 8.1-fold for the group of Sputnik V-vaccinated individuals. When the samples were stratified for the time period after vaccination, a 7.6-fold or 8.8-fold decrease in NtAb titers was noticed after up to 3 and 3-to-6 months after vaccination. We observed a 6.7- and 5-fold decrease in Sputnik V-vaccinated individuals experiencing asymptomatic or symptomatic infection, respectively. These results highlight the observation that the decrease in NtAb to the SARS-CoV-2 Omicron variant compared to the Wuhan variant occurs for different COVID-19 vaccines in use, with some showing no neutralization at all, confirming the necessity of a third booster vaccination.

To investigate the dynamic association among binding and functional antibodies in health-care-workers receiving two doses of BNT162b2 mRNA COVID-19-vaccine, SARS-CoV-2 anti-RBD IgG, anti-Trimeric-S IgG, and neutralizing antibodies (Nabs) were measured in serum samples collected at 2 weeks, 3 months, and 6 months from full vaccination. Despite the high correlation, results for anti-RBD and anti-Trimeric S IgG were numerically different even after recalculation to BAU/mL following WHO standards indications. Moreover, after a peak response at 2 weeks, anti-RBD IgG levels showed a 4.5 and 13 fold decrease at 3 and 6 months, respectively, while the anti-Trimeric S IgG presented a less pronounced decay of 2.8 and 4.7 fold. Further different dynamics were observed for Nabs titers, resulting comparable at 3 and 6 months from vaccination. We also demonstrated that at NAbs titers ≥40, the area under the receiver operating characteristic curve and the optimal cutoff point decreased with time from vaccination for both anti-RBD and anti-Trimeric S IgG. The mutating relation among the anti-RBD IgG, anti-Trimeric S IgG, and neutralizing antibodies are indicative of antibody maturation upon vaccination. The lack of standardized laboratory procedures is one factor interfering with the definition of a correlate of protection from COVID-19.