Humoral immunity induced by mRNA COVID-19 vaccines in Nursing Home Residents previously infected with SARS-CoV-2.

BACKGROUND: Nursing home (NH) residents suffered the greatest impact of the COVID-19 pandemic. Limited data are available on vaccine-induced immunity and on the protection ensured by a prior infection in this population. AIMS: The present study aims to monitor antibody levels and their persistence over a 6-month period in NH residents according to the history of prior SARS-CoV-2 infection. METHODS: We measured anti-trimeric Spike IgG antibody levels in a sample of 395 residents from 25 NHs in 6 Italian Regions at study enrolment (prior to the first dose of vaccine, T0) and then after 2 (T1) and 6 months (T2) following the first vaccine dose. All participants received mRNA vaccines (BNT162b2 or mRNA-1273). Analyses were performed using log-transformed values of antibody concentrations and geometric means (GM) were calculated. RESULTS: Superior humoral immunity was induced in NH residents with previous SARS-CoV-2 infection. (T0: GM 186.6 vs. 6.1 BAU/ml, p < 0.001; T1: GM 5264.1 vs. 944.4 BAU/ml, p < 0.001; T2: GM 1473.6 vs. 128.7 BAU/ml, p < 0.001). Residents with prior SARS-CoV-2 infection receiving two vaccine doses presented significantly higher antibody concentration at T1 and T2. A longer interval between previous infection and vaccination was associated with a better antibody response over time. DISCUSSION: In a frail sample of NH residents, prior SARS-CoV-2 infection was associated with a higher humoral response to vaccination. Number of vaccine doses and the interval between infection and vaccination are relevant parameters in determining humoral immunity. CONCLUSIONS: These findings provide important information to plan future immunization policies and disease prevention strategies in a highly vulnerable population.

CD38 modulates respiratory syncytial virus-driven proinflammatory processes in human monocyte-derived dendritic cells.

Respiratory syncytial virus (RSV) is the most common cause of hospitalization due to bronchiolitis in infants. Although the mechanisms behind this association are not completely elucidated, they appear to involve an excessive immune response causing lung pathology. Understanding the host response to RSV infection may help in the identification of targets for therapeutic intervention. We infected in-vitro human monocyte-derived dendritic cells (DCs) with RSV and analysed various aspects of the cellular response. We found that RSV induces in DCs the expression of CD38, an ectoenzyme that catalyses the synthesis of cyclic ADPR (cADPR). Remarkably, CD38 was under the transcriptional control of RSV-induced type I interferon (IFN). CD38 and a set of IFN-stimulated genes (ISGs) were inhibited by the anti-oxidant N-acetyl cysteine. When CD38-generated cADPR was restrained by 8-Br-cADPR or kuromanin, a flavonoid known to inhibit CD38 enzymatic activity, RSV-induced type I/III IFNs and ISGs were markedly reduced. Taken together, these results suggest a key role of CD38 in the regulation of anti-viral responses. Inhibition of CD38 enzymatic activity may represent an encouraging approach to reduce RSV-induced hyperinflammation and a novel therapeutic option to treat bronchiolitis.

Successful therapeutic vaccination with integrase defective lentiviral vector expressing nononcogenic human papillomavirus E7 protein.

Persistent infection with high risk genotypes of human papillomavirus (HPV) is the cause of cervical cancer, one of most common cancer among woman worldwide, and represents an important risk factor associated with other anogenital and oropharyngeal cancers in men and women. Here, we designed a therapeutic vaccine based on integrase defective lentiviral vector (IDLV) to deliver a mutated nononcogenic form of HPV16 E7 protein, considered as a tumor specific antigen for immunotherapy of HPV-associated cervical cancer, fused to calreticulin (CRT), a protein able to enhance major histocompatibility complex class I antigen presentation (IDLV-CRT/E7). Vaccination with IDLV-CRT/E7 induced a potent and persistent E7-specific T cell response up to 1 year after a single immunization. Importantly, a single immunization with IDLV-CRT/E7 was able to prevent growth of E7-expressing TC-1 tumor cells and to eradicate established tumors in mice. The strong therapeutic effect induced by the IDLV-based vaccine in this preclinical model suggests that this strategy may be further exploited as a safe and attractive anticancer immunotherapeutic vaccine in humans.

T-Cell Mediated Response after Primary and Booster SARS-CoV-2 Messenger RNA Vaccination in Nursing Home Residents.

OBJECTIVES: Nursing home (NH) residents have been significantly affected by the coronavirus disease 2019 (COVID-19) pandemic. Studies addressing the immune responses induced by COVID-19 vaccines in NH residents have documented a good postvaccination antibody response and the beneficial effect of a third booster vaccine dose. Less is known about vaccine-induced activation of cell-mediated immune response in frail older individuals in the long term. The aim of the present study is to monitor messenger RNA SARS-CoV-2 vaccine-induced T-cell responses in a sample of Italian NH residents who received primary vaccine series and a third booster dose and to assess the interaction between T-cell responses and humoral immunity. DESIGN: Longitudinal cohort study. SETTING AND PARTICIPANTS: Thirty-four residents vaccinated with BNT162b2 messenger RNA SARS-CoV-2 vaccine between February and April 2021 and who received a third BNT162b2 booster dose between October and November 2021 were assessed for vaccine-induced immunity 6 (prebooster) and 12 (postbooster) months after the first BNT162b2 vaccine dose. METHODS: Pre- and postbooster cell-mediated immunity was assessed by intracellular cytokine staining of peripheral blood mononuclear cells stimulated in vitro with peptides covering the immunodominant sequence of SARS-CoV-2 spike protein. The simultaneous production of interferon-γ, tumor necrosis factor-α, and interleukin-2 was measured. Humoral immunity was assessed in parallel by measuring serum concentration of antitrimeric spike IgG antibodies. RESULTS: Before the booster vaccination, 31 out of 34 NH residents had a positive cell-mediated immunity response to spike. Postbooster, 28 out of 34 had a positive response. Residents without a previous history of SARS-CoV-2 infection, who had a lower response prior the booster administration, showed a greater increase of T-cell responses after the vaccine booster dose. Humoral and cell-mediated immunity were, in part, correlated but only before booster vaccine administration. CONCLUSIONS AND IMPLICATIONS: The administration of the booster vaccine dose restored spike-specific T-cell responses in SARS-CoV-2 naïve residents who responded poorly to the first immunization, while a previous SARS-CoV-2 infection had an impact on the magnitude of vaccine-induced cell-mediated immunity at earlier time points. Our findings imply the need for a continuous monitoring of the immune status of frail NH residents to adapt future SARS-CoV-2 vaccination strategies.

Sex differences in the efficacy and safety of SARS-CoV-2 vaccination in residents of long-term care facilities: insights from the GeroCovid Vax study.

Despite the reported sex-related variations in the immune response to vaccination, whether the effects of SARS-CoV-2 vaccination differ by sex is still under debate, especially considering old vulnerable individuals, such as long-term care facilities (LTCFs) residents. This study aimed to evaluate COVID-19 infections, adverse events, and humoral response after vaccination in a sample of LTCF residents. A total of 3259 LTCF residents (71% females; mean age: 83.4 ± 9.2 years) were enrolled in the Italian-based multicenter GeroCovid Vax study. We recorded the adverse effects occurring during the 7 days after vaccine doses and COVID-19 cases over 12 months post-vaccination. In a subsample of 524 residents (69% females), pre- and post-vaccination SARS-CoV-2 trimeric S immunoglobulin G (Anti-S-IgG) were measured through chemiluminescent assays at different time points. Only 12.1% of vaccinated residents got COVID-19 during the follow-up, without any sex differences. Female residents were more likely to have local adverse effects after the first dose (13.3% vs. 10.2%, p = 0.018). No other sex differences in systemic adverse effects and for the following doses were recorded, as well as in anti-S-IgG titer over time. Among the factors modifying the 12-month anti-S-IgG titers, mobility limitations and depressive disorder were more likely to be associated with higher and lower levels in the antibody response, respectively; a significantly lower antibody titer was observed in males with cardiovascular diseases and in females with diabetes or cognitive disorders. The study suggests that, among LTCF residents, SARS-CoV-2 vaccination was effective regardless of sex, yet sex-specific comorbidities influenced the antibody response. Local adverse reactions were more common in females.

Polypharmacy and Antibody Response to SARS-CoV-2 Vaccination in Residents of Long-Term Care Facilities: The GeroCovid Vax Study.

BACKGROUND AND OBJECTIVE: Polypharmacy is common in older adults, particularly among those living in long-term care facilities. This condition represents a marker of clinical complexity and might directly affect the immunological response. However, there are limited data on the association of polypharmacy with vaccine immunogenicity. This study evaluated the immune response to anti-SARS-CoV-2 vaccines in older residents of long-term care facilities as a function of the number of medications used. METHODS: In 478 long-term care facility residents participating in the GeroCovid Vax study, we assessed SARS-CoV-2 trimeric S IgG levels through chemiluminescent assays before the vaccination and after 2, 6, and 12 months. A booster dose was administered between 6- and 12-month assessments. Sociodemographic information and data on chronic diseases and medications were derived from medical records. Based on the number of daily medications, residents were classified into the no polypharmacy (zero to four medications), polypharmacy (five to nine medications), and hyperpolypharmacy (ten or more medications) groups. RESULTS: In the sample (mean age 82.1 years, 69.2% female), 200 (41.8%) residents were taking five or fewer medications/day (no polypharmacy), 229 (47.9%) had polypharmacy, and 49 (10.3%) had hyperpolypharmacy. Using linear mixed models adjusted for potential confounders, we found that hyperpolypharmacy was associated with a steeper antibody decline after 6 months from the first vaccine dose administration (ß = - 0.29, 95% confidence interval - 0.54, - 0.03, p = 0.03) than no polypharmacy, while no significant differences were observed at 12 months. CONCLUSIONS: The humoral immune response to SARS-CoV-2 vaccination of older residents showed only slight changes as a function of the number of medications taken. Although it seemed less durable among older residents with hyperpolypharmacy, the booster dose administration equalized such a difference.

A 12-month follow-up of the immune response to SARS-CoV-2 primary vaccination: evidence from a real-world study.

A real-world population-based longitudinal study, aimed at determining the magnitude and duration of immunity induced by different types of vaccines against COVID-19, started in 2021 by enrolling a cohort of 2,497 individuals at time of their first vaccination. The study cohort included both healthy adults aged ≤65 years and elderly subjects aged >65 years with two or more co-morbidities. Here, patterns of anti-SARS-CoV-2 humoral and cell-mediated specific immune response, assessed on 1,182 remaining subjects, at 6 (T6) and 12 months (T12) after the first vaccine dose, are described. At T12 median anti-Spike IgG antibody levels were increased compared to T6. The determinants of increased anti-Spike IgG were the receipt of a third vaccine dose between T6 and T12 and being positive for anti-Nucleocapside IgG at T12, a marker of recent infection, while age had no significant effect. The capacity of T12 sera to neutralize in vitro the ancestral B strain and the Omicron BA.5 variant was assessed in a subgroup of vaccinated subjects. A correlation between anti-S IgG levels and sera neutralizing capacity was identified and higher neutralizing capacity was evident in healthy adults compared to frail elderly subjects and in those who were positive for anti-Nucleocapside IgG at T12. Remarkably, one third of T12 sera from anti-Nucleocapside IgG negative older individuals were unable to neutralize the BA.5 variant strain. Finally, the evaluation of T-cell mediated immunity showed that most analysed subjects, independently from age and comorbidity, displayed Spike-specific responses with a high degree of polyfunctionality, especially in the CD8 compartment. In conclusion, vaccinated subjects had high levels of circulating antibodies against SARS-CoV-2 Spike protein 12 months after the primary vaccination, which increased as compared to T6. The enhancing effect could be attributable to the administration of a third vaccine dose but also to the occurrence of breakthrough infection. Older individuals, especially those who were anti-Nucleocapside IgG negative, displayed an impaired capacity to neutralize the BA.5 variant strain. Spike specific T-cell responses, able to sustain immunity and maintain the ability to fight the infection, were present in most of older and younger subjects assayed at T12.

Simian immunodeficiency virus-Vpx for improving integrase defective lentiviral vector-based vaccines.

BACKGROUND: Integrase defective lentiviral vectors (IDLV) represent a promising delivery system for immunization purposes. Human dendritic cells (DC) are the main cell types mediating the immune response and are readily transduced by IDLV, allowing effective triggering of in vitro expansion of antigen-specific primed CD8+ T cells. However, IDLV expression in transduced DC is at lower levels than those of the integrase (IN) competent counterpart, thus requiring further improvement of IDLV for future use in the clinic. RESULTS: In this paper we show that the addition of simian immunodeficiency (SIV)-Vpx protein in the vector preparation greatly improves transduction of human and simian DC, but not of murine DC, thus increasing the ability of transduced DC to act as functional antigen presenting cells, in the absence of integrated vector sequences. Importantly, the presence of SIV-Vpx allows for using lower dose of input IDLV during in vitro transduction, thus further improving the IDLV safety profile. CONCLUSIONS: These results have significant implications for the development of IDLV-based vaccines.

Evolution of SARS-CoV-2 IgG Seroprevalence in Children and Factors Associated with Seroconversion: Results from a Multiple Time-Points Study in Friuli-Venezia Giulia Region, Italy.

Data on the effective burden of the SARS-CoV-2 pandemic in the pediatric population are limited. We aimed at assessing the prevalence of SARS-CoV-2 IgG antibodies in children at three subsequent time-points. The study was conducted between January 2021 and July 2021 among children referring to the Research Institute for Maternal and Child Health "Burlo Garofolo" in Trieste, a referral regional hospital in Friuli Venezia Giulia, Italy. A multivariate analysis was conducted to assess factors independently associated with seroconversion. A total of 594 children were included. Anti-SARS-CoV-2 trimeric Spike protein IgG antibodies were found in 32 (15.4%) children tested in April-May and in 20 (11.8%) in June-July 2021, compared with 24 (11.1%) of those tested in January-February 2021 (p = 0.37, Armitage exact test for trend over time p = 0.76). A subgroup analysis and a multivariate logistic regression analysis were performed considering sociodemographic, clinical, and historical variables. Three categories of children showed statistically significant increased odds of positive anti-SARS-CoV-2 IgG antibodies: children previously positive to a nasopharyngeal swab (AdjOR 15.41, 95%CI 3.44-69.04, p < 0.001), cohabitant with a person with an history of a previous positive nasopharyngeal swab (AdjOR 9.95, 95%CI 5.35-18.52, p < 0.001), and children with a foreign citizenship (AdjOR 2.4, 95%CI 1.05-5.70, p = 0.002). The study suggests that seroprevalence studies may be of limited help in estimating the prevalence of the COVID-19 pandemic in children. Further studies are needed to identify other markers of previous SARS-CoV-2 infection in children, such as CD4+ T cells or memory B-cells.

Early IgG / IgA response in hospitalized COVID-19 patients is associated with a less severe disease.

We determined the kinetics of anti-SARS-CoV-2 antibody response in fifteen hospitalized COVID-19 patients. Patients were divided into mild/moderate (mild, n = 1; moderate, n = 4) or severe (n = 10) and virus-specific anti-Nucleocapsid IgM, anti-Spike IgG and anti-Spike IgA were measured in serial serum samples collected 0 to 15 days after hospital admission. Surrogate neutralization assays were performed by testing inhibition of ACE-2 binding to Spike. In 3 patients (2 severe and 1 moderate case), serum antibodies and T-cell memory were monitored 6 months after baseline. Although IgM response tended to appear first, patients affected by less severe disease were more prone to an early IgG/IgA response. Neutralization of Spike binding to ACE2 correlated with anti-Spike IgG and IgA. IgG and IgA antibody response persisted at the 6 months follow-up. A recall T-cell response to the Spike antigen was observed in 2 out of 3 patients, not related to disease severity.

Diabetes Affects Antibody Response to SARS-CoV-2 Vaccination in Older Residents of Long-term Care Facilities: Data From the GeroCovid Vax Study.

OBJECTIVE: Type 2 diabetes may affect the humoral immune response after vaccination, but data concerning coronavirus disease 19 (COVID-19) vaccines are scarce. We evaluated the impact of diabetes on antibody response to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination in older residents of long-term care facilities (LTCFs) and tested for differences according to antidiabetic treatment. RESEARCH DESIGN AND METHODS: For this analysis, 555 older residents of LTCFs participating in the GeroCovid Vax study were included. SARS-CoV-2 trimeric S immunoglobulin G (anti-S IgG) concentrations using chemiluminescent assays were tested before the first dose and after 2 and 6 months. The impact of diabetes on anti-S IgG levels was evaluated using linear mixed models, which included the interaction between time and presence of diabetes. A second model also considered diabetes treatment: no insulin therapy (including dietary only or use of oral antidiabetic agents) and insulin therapy (alone or in combination with oral antidiabetic agents). RESULTS: The mean age of the sample was 82.1 years, 68.1% were women, and 25.2% had diabetes. In linear mixed models, presence of diabetes was associated with lower anti-S IgG levels at 2 (ß = -0.20; 95% CI -0.34, -0.06) and 6 months (ß = -0.22; 95% CI -0.37, -0.07) after the first vaccine dose. Compared with those without diabetes, residents with diabetes not using insulin had lower IgG levels at 2- and 6-month assessments (ß = -0.24; 95% CI -0.43, -0.05 and ß = -0.30; 95% CI -0.50, -0.10, respectively), whereas no differences were observed for those using insulin. CONCLUSIONS: Older residents of LTCFs with diabetes tended to have weaker antibody response to COVID-19 vaccination. Insulin treatment might buffer this effect and establish humoral immunity similar to that in individuals without diabetes.

The BNT162b2 vaccine induces humoral and cellular immune memory to SARS-CoV-2 Wuhan strain and the Omicron variant in children 5 to 11 years of age.

SARS-CoV-2 mRNA vaccines prevent severe COVID-19 by generating immune memory, comprising specific antibodies and memory B and T cells. Although children are at low risk of severe COVID-19, the spreading of highly transmissible variants has led to increasing in COVID-19 cases and hospitalizations also in the youngest, but vaccine coverage remains low. Immunogenicity to mRNA vaccines has not been extensively studied in children 5 to 11 years old. In particular, cellular immunity to the wild-type strain (Wuhan) and the cross-reactive response to the Omicron variant of concern has not been investigated. We assessed the humoral and cellular immune response to the SARS-CoV-2 BNT162b2 vaccine in 27 healthy children. We demonstrated that vaccination induced a potent humoral and cellular immune response in all vaccinees. By using spike-specific memory B cells as a measurable imprint of a previous infection, we found that 50% of the children had signs of a past, undiagnosed infection before vaccination. Children with pre-existent immune memory generated significantly increased levels of specific antibodies, and memory T and B cells, directed against not only the wild type virus but also the omicron variant.

Evaluation of humoral and cellular response to four vaccines against COVID-19 in different age groups: A longitudinal study.

To date there has been limited head-to-head evaluation of immune responses to different types of COVID-19 vaccines. A real-world population-based longitudinal study was designed with the aim to define the magnitude and duration of immunity induced by each of four different COVID-19 vaccines available in Italy at the time of this study. Overall, 2497 individuals were enrolled at time of their first vaccination (T0). Vaccine-specific antibody responses induced over time by Comirnaty, Spikevax, Vaxzevria, Janssen Ad26.COV2.S and heterologous vaccination were compared up to six months after immunization. On a subset of Comirnaty vaccinees, serology data were correlated with the ability to neutralize a reference SARS-CoV-2 B strain, as well as Delta AY.4 and Omicron BA.1. The frequency of SARS-CoV-2-specific CD4+ T cells, CD8+ T cells, and memory B cells induced by the four different vaccines was assessed six months after the immunization. We found that mRNA vaccines are stronger inducer of anti-Spike IgG and B-memory cell responses. Humoral immune responses are lower in frail elderly subjects. Neutralization of the Delta AY.4 and Omicron BA.1 variants is severely impaired, especially in older individuals. Most vaccinees display a vaccine-specific T-cell memory six months after the vaccination. By describing the immunological response during the first phase of COVID-19 vaccination campaign in different cohorts and considering several aspects of the immunological response, this study allowed to collect key information that could facilitate the implementation of effective prevention and control measures against SARS-CoV-2.

Early IgG / IgA response in hospitalized COVID-19 patients is associated with a less severe disease.

We determined the kinetics of anti-SARS-CoV-2 antibody response in fifteen hospitalized COVID-19 patients. Patients were divided into mild/moderate (mild, n = 1; moderate, n = 4) or severe (n = 10) and virus-specific anti-Nucleocapsid IgM, anti-Spike IgG and anti-Spike IgA were measured in serial serum samples collected 0 to 15 days after hospital admission. Surrogate neutralization assays were performed by testing inhibition of ACE-2 binding to Spike. In 3 patients (2 severe and 1 moderate case), serum antibodies and T-cell memory were monitored 6 months after baseline. Although IgM response tended to appear first, patients affected by less severe disease were more prone to an early IgG/IgA response. Neutralization of Spike binding to ACE2 correlated with anti-Spike IgG and IgA. IgG and IgA antibody response persisted at the 6 months follow-up. A recall T-cell response to the Spike antigen was observed in 2 out of 3 patients, not related to disease severity.

Anti-SARS-CoV-2 antibodies persistence after natural infection: a repeated serosurvey in Northern Italy.

INTRODUCTION: To evaluate the decline of antibodies induced by SARS-CoV-2 infection, the individuals resident in 5 municipalities of the Autonomous Province of Trento, Northern Italy, who resulted IgG positive for anti-SARS-CoV-2 nucleocapsid (NC) in May 2020, were tested four months later. METHODS: Anti-SARS-CoV-2 NC antibodies were detected using the Abbott SARS-CoV-2 IgG assay. Samples that gave a negative result were re-tested using the Liaison SARS-CoV-2 IgG assay to assess anti-spike (S) S1/S2 antibodies. The fifty-percent tissue culture infective dose (TCID50) neutralizing assay was performed on a subgroup of formerly positive sera. Statistical analysis was performed by STATA version 16.1 (STATA Corp., College Station, Texas, USA). RESULTS: Overall, 480 out of 1159 participants became seronegative for anti-NC IgG antibodies. Age above 70 years and cough were associated with persistent anti-NC IgG levels. Most anti-NC IgG negative sera were positive for anti-S IgG (77.9%). The neutralization assay showed high concordance with anti-S antibodies positivity. CONCLUSION: In conclusion, a decline of anti-NC IgG values was recorded four months after the first evaluation. A high proportion of anti-NC seronegative individuals were positive for anti-spike IgG antibodies, which appear to persist longer and to better correlate with neutralization activity.

Prevalence of SARS-CoV-2 IgG antibodies in an area of northeastern Italy with a high incidence of COVID-19 cases: a population-based study.

OBJECTIVES: A seroprevalence study of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was conducted in a high-incidence area located in northeastern Italy. METHODS: All citizens above 10 years of age resident in five municipalities of the Autonomous Province of Trento, with the highest incidence of coronavirus disease 2019 (COVID-19) cases, were invited to participate in the study. Among 6098 participants, 6075 sera and a standardized questionnaire administered face-to-face were collected between 5 May and 15 May 2020 and examined. Symptomatic individuals and their family contacts were tested by RT-PCR. Anti-SARS-CoV-2 antibodies were detected using an Abbott SARS-CoV-2 IgG assay, which was performed on the Abbott Architect i2000SR automated analyser. Seroprevalence was calculated as the proportion of positive results among the total number tested. A multivariable logistic regression model was performed to assess the relationship between seropositive versus seronegative individuals for a set of explanatory variables. RESULTS: A total of 1402 participants were positive for IgG antibodies against SARS-CoV-2, with a prevalence of 23.1% (1402/6075). The highest prevalence was found in the age class 40-49 years. Overall, 34.4% (2096/6098) of the participants reported at least one symptom. The ratio between reported cases identified by molecular test and those with seropositive results was 1:3, with a maximum ratio of about 1:7 in the age group <20 years and a minimum around 1:1 in those >70 years old. The infection fatality rate was 2.5% (35/1402). Among the symptoms, anosmia and ageusia were strongly associated with seropositivity. CONCLUSIONS: The estimated seroprevalence of 23% was three-fold higher than the number of cases reported in the COVID-19 Integrated Surveillance data in the study area. This may be explained in part by a relatively high number of individuals presenting mild or no illness, especially those of younger age, and people who did not seek medical care or testing, but who may contribute to virus transmission in the community.

Nonintegrating Lentiviral Vector-Based Vaccine Efficiently Induces Functional and Persistent CD8+ T Cell Responses in Mice.

CD8+ T cells are an essential component of an effective host immune response to tumors and viral infections. Genetic immunization is particularly suitable for inducing CTL responses, because the encoded proteins enter the MHC class I processing pathway through either transgene expression or cross-presentation. In order to compare the efficiency and persistence of immune response induced by genetic vaccines, BALB/c mice were immunized either twice intramuscularly with DNA plasmid expressing a codon-optimized HIV-1 gp120 Envelope sequence together with murine GM-CSF sequence or with a single immunization using an integrase defective lentiviral vector (IDLV) expressing the same proteins. Results strongly indicated that the schedule based on IDLV vaccine was more efficient in inducing specific immune response, as evaluated three months after the last immunization by IFNgamma ELISPOT in both splenocytes and bone marrow- (BM-) derived cells, chromium release assay in splenocytes, and antibody detection in sera. In addition, IDLV immunization induced high frequency of polyfunctional CD8+ T cells able to simultaneously produce IFNgamma, TNFalpha, and IL2.

Increased T-helper Cell 2 Response in Infants With Respiratory Syncytial Virus Bronchiolitis Hospitalized Outside Epidemic Peak.

AIM: To test the hypothesis that the balance of type-1/type-2 immune response differs between infants hospitalized with respiratory syncytial virus (RSV) bronchiolitis during the peak months and those during the nonpeak months. METHODS: We prospectively enrolled 90 unrelated full-term previously healthy infants hospitalized during the first year of life for RSV sole bronchiolitis over 2 epidemics (November 2016 to April 2017 and October 2017 to April 2018). We stratified infants as follows: hospitalized during the peak months (n: 71) and during the nonpeak months (n: 19). The frequencies of CD4+ producing interferon (IFN)-γ and interleukin (IL)-4 and of CD8+ producing IFN-γ T cells were measured by flow cytometry from infant peripheral whole blood. The T-helper cell (Th2) polarization index was calculated as the ratio between CD4+ T cells producing IL-4 and CD4+ T cells producing IFN-γ. RESULTS: Infants hospitalized during nonpeak months were significantly less frequently breast-fed, had a higher eosinophils count, a significantly higher percentage of CD4+ T cells producing IL-4 and higher Th2 polarization index than infants hospitalized during the peak months. CONCLUSIONS: We elucidated the presence of different endotypes in infants with RSV sole bronchiolitis. Previously healthy full-term infants hospitalized during the nonpeak months seem to be more likely those with a possible predisposition to atopy.

Multicentre Harmonisation of a Six-Colour Flow Cytometry Panel for Naïve/Memory T Cell Immunomonitoring.

BACKGROUND: Personalised medicine in oncology needs standardised immunological assays. Flow cytometry (FCM) methods represent an essential tool for immunomonitoring, and their harmonisation is crucial to obtain comparable data in multicentre clinical trials. The objective of this study was to design a harmonisation workflow able to address the most effective issues contributing to intra- and interoperator variabilities in a multicentre project. METHODS: The Italian National Institute of Health (Istituto Superiore di Sanità, ISS) managed a multiparametric flow cytometric panel harmonisation among thirteen operators belonging to five clinical and research centres of Lazio region (Italy). The panel was based on a backbone mixture of dried antibodies (anti-CD3, anti-CD4, anti-CD8, anti-CD45RA, and anti-CCR7) to detect naïve/memory T cells, recognised as potential prognostic/predictive immunological biomarkers in cancer immunotherapies. The coordinating centre distributed frozen peripheral blood mononuclear cells (PBMCs) and fresh whole blood (WB) samples from healthy donors, reagents, and Standard Operating Procedures (SOPs) to participants who performed experiments by their own equipment, in order to mimic a real-life scenario. Operators returned raw and locally analysed data to ISS for central analysis and statistical elaboration. RESULTS: Harmonised and reproducible results were obtained by sharing experimental set-up and procedures along with centralising data analysis, leading to a reduction of cross-centre variability for naïve/memory subset frequencies particularly in the whole blood setting. CONCLUSION: Our experimental and analytical working process proved to be suitable for the harmonisation of FCM assays in a multicentre setting, where high-quality data are required to evaluate potential immunological markers, which may contribute to select better therapeutic options.