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Vaccination is the best strategy against COVID-19. We aimed to determine antibodies against SARS-CoV-2 in breastmilk and serum of mothers vaccinated with the mRNA vaccine.This prospective study included 18 lactating women vaccinated with the BNT162b2 vaccine. Serum and breastmilk were collected before the first dose (T0), at the second dose (T1), 3 weeks after the second dose (T2), and 6 months after the first dose (T3). Serum anti-SARS-CoV-2 Spike (S) Immunoglobulin G (IgG) and Immunoglobulin A (IgA) were measured using a semi-quantitative enzyme-linked immunosorbent assay (ELISA) and secretory antibody (s) IgG and IgA in breastmilk using quantitative analysis.We detected serum anti-S IgG and IgA in all women after vaccination. Specific IgG and IgA were higher at T1, T2, and T3 compared with T0 (P 0.0001). Higher antibody levels were observed at T2 and lower values at T3 versus T2 (P = 0.007). After 6 months, all patients had serum IgG, but three of 18 (16%) had serum IgA. In breastmilk, sIgA was present at T1 and T2 and decreased after 6 months at T3 (P = 0.002). Breastmilk sIgG levels increased at T1 and T2 and peaked at T3 (P = 0.008).Secretory antibodies were transmitted through breastmilk until 6 months after anti-COVID-19 mRNA vaccination. Protection of the newborn through breastfeeding needs to be addressed.

Currently approved COVID-19 vaccines prevent symptomatic infection, hospitalization, and death of the disease. However, the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants raises concerns of reduced vaccine effectiveness and increased risk of infection. Repeated homologous booster in elderly individuals and immunocompromised patients is considered to solve severe form of disease caused by new SARS-CoV-2 variants but cannot protect completely against breakthrough infection. In our previous study we assessed the immunogenicity of an adenovirus-based vaccine expressing SARS-CoV-2-S1 (Ad5.S1) in mice, resulting in that a single immunization with Ad5.S1, via subcutaneously injection or intranasal delivery, induced robust humoral and cellular immune responses [1]. As a follow up study, here we showed that vaccinated mice had high titers of anti-S1 antibodies at one year after vaccination compared to PBS immunized mice. Furthermore, one booster dose of non-adjuvanted recombinant S1Beta (rS1Beta) subunit vaccine was effective in stimulating strong long-lived S1-specific immune responses and inducing significantly high neutralizing antibodies against the Wuhan, Beta, and Delta strain with 3.6- to 19.5-fold change increases. Importantly, the booster dose elicits cross-reactive antibody responses resulting in ACE2 binding inhibition against spike of SARS-CoV-2 variants (Wuhan, Alpha, Beta, Gamma, Delta, Zeta, Kappa, New York, India) as early as two-week post-boost injection, persisting over 28 weeks after a booster vaccination. Interestingly, levels of neutralizing antibodies were correlated with not only level of S1-binding IgG but also level of ACE2 inhibition in the before- and after-booster serum samples. Our findings show that S1 recombinant protein subunit vaccine candidate as a booster has potential to offer cross-neutralization against broad variants, and has important implications for vaccine control of new emerging breakthrough SARS-CoV-2 variants in elderly individuals primed with adenovirus-based vaccine like AZD1222 and Ad26.COV2.S.

Background Human Cytomegalovirus (HCMV) still represents a crucial concern in solid organ transplant recipients (SOTRs) and the use of antiviral therapy are limited by side effects and the selection of viral mutations conferring antiviral drug resistance. Case presentation Here we reported the case of an HCMV seronegative patient with common variable immunodeficiency (CVID), multiple hepatic adenomatosis, hepatopulmonary syndrome and portal hypertension who received a liver transplant from an HCMV seropositive donor. The patient was treated with Valganciclovir (vGCV) and then IV Ganciclovir (GCV) at 5 week post-transplant for uncontrolled HCMV DNAemia. However, since mutation A594V in UL97 gene conferring resistance to ganciclovir was reported, GCV therapy was interrupted. Due to the high toxicity of Foscarnet (FOS) and Cidofovir (CDV), Letermovir (LMV) monotherapy at the dosage of 480 mg per day was administered, with a gradual viral load reduction. However, a relapse of HCMV DNAemia revealed the presence of mutation C325Y in HCMV UL56 gene conferring resistance to LMV. Conclusions In conclusion, even if LMV is an effective and favorable safety molecule it might have a lower genetic barrier to resistance. A warning on the use of LMV monotherapy as rescue treatments for HCMV GCV-resistant infections in transplant recipients is warranted.

We reported the long-term kinetics of immune response after vaccination and evaluated the immunogenicity after a third dose of mRNA vaccine in 86 healthcare workers. Humoral response was analyzed by measuring anti-spike IgG and SARS-CoV-2 NTAbs titer; cell-mediated response was measured as frequency of IFN-γ producing T-cells and cell proliferation. Memory B cells secreting SARS-CoV-2 RBD-IgG were measured by B-spot assay. At three weeks after the third dose (T4), the frequency of subjects showing NT-Abs titer at the upper detection limit (≥640) was significantly higher than that observed at three weeks after the second dose (26/77; 33.7% vs. 9/77; 11.6%; p = 0.0018). Additionally, at T4, all the subjects reached positive levels of T-cell mediated response (median 110 SFU/106 PBMC, IQR 73-231). While the number of IFNγ-producing T-cells decreased between second and third dose administration, the T-cell proliferative response did not decrease but was sustained during the follow-up. Among T-cell subsets, a higher proliferative response was observed in CD4+ than in CD8+ population. Moreover, even if a decline in antibody response was observed between the second and third dose, a sustained persistence of memory B cells was observed. Subsequently, the third dose did not affect the frequency of memory B cells, while it restored or increased the peak antibody levels detected after the second dose.

COVID-19 is a global health threat with a huge number of confirmed cases and deaths all over the world. Human-to-human transmission via respiratory droplets and contact with aerosol-infected surfaces are the major routes of virus spread. Because SARS-CoV-2 can remain in the air and on surfaces from several hours to several days, disinfection of frequently touched surfaces and critical rooms, in addition to observing individual hygiene tips, is required to reduce the virus spreading. Here we report on an investigation into the use of gaseous ozone as a potentially effective sanitizing method against the new coronavirus.

Highlights • EBV reactivation was observed in patients with COVID-19. • NK + and CD8 + T cell reduction was related to the severity of COVID-19 infection. • An increase of B cells in patients with severe symptoms was documented., Objectives The immunologic profile and opportunistic viral DNA increase were monitored in Italian patients with COVID-19 in order to identify markers of disease severity. Methods A total of 104 patients infected with SARS-CoV-2 were evaluated in the study. Of them, 42/104 (40.4%) were hospitalized in an intensive care unit (ICU) and 62/104(59.6%) in a sub-intensive care unit (SICU). Human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV), Parvovirus B19 and Human Herpesvirus 6 virus reactivations were determined by real-time PCR, and lymphocyte subpopulation counts were determined by flow cytometry. Results Among opportunistic viruses, only EBV was consistently detected. EBV DNA was observed in 40/42 (95.2%) of the ICU patients and in 51/61 (83.6%) of the SICU patients. Comparing the two groups of patients, the EBV DNA median level among ICU patients was significantly higher than that observed in SICU patients. In parallel, a significant reduction of CD8 T cell and NK count in ICU patients as compared with SICU patients was observed (p

The human papillomavirus (HPV) can cause different cancers in both men and women. The virus interferes with functions of the cervix, vulva, vagina, anus in the anogenital area, breast, and head and neck cancer due to the local lesions. The tumors lead to death if not treated as a result of distant metastasis to internal organs and brain. Moreover, HPV attenuates the immune system during chronic infection and releases viral antigens into the tumor microenvironment. The tumors know how difficult is to win the battle with a strong united army of immune cells that are equipped with cytokines and enzymes. They confuse the immune cells with secreting viral antigens. The immune system is equipped with cytokines, a complement system, antibodies, and other secretory proteins to overcome the foreign invaders and viral antigens. However, the majority of the time, tumors win the battle without having all the equipment of the immune cells. Thus, in this review, we describe the recent progression in cellular and humoral immunity studies during the progression of HPV-related cancers. First of all, we describe the role of B, plasmoid cells, and B regulatory cells (Breg) in their functions in the tumor microenvironment. Then, different subtypes of T cells such as T CD8, CD4, T regulatory (Treg) cells were studied in recently published papers. Furthermore, NK cells and their role in tumor progression and prevention were studied. Finally, we indicate the breakthroughs in immunotherapy techniques for HPV-related cancers.

Objectives To assess the humoral and cell-mediated response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) elicited by the mRNA BNT162b2 vaccine in SARS-CoV-2-experienced and -naive subjects against a reference strain and SARS-CoV-2 variants. Methods The humoral response (including neutralizing antibodies) and T-cell-mediated response elicited by BNT162b2 vaccine in 145 healthcare workers (both naive and positive for previous SARS-CoV-2 infection) were evaluated. In a subset of subjects, the effect of SARS-CoV-2 variants on antibody level and cell-mediated response was also investigated. Results Overall, 125/127 naive subjects (98.4%) developed both neutralizing antibodies and specific T cells after the second dose of vaccine. Moreover, the antibody and T-cell responses were effective against viral variants since SARS-CoV-2 NT Abs were still detectable in 55/68 (80.9%) and 25/29 (86.2%) naive subjects when sera were challenged against β and δ variants, respectively. T-cell response was less affected, with no significant difference in the frequency of responders (p 0.369). Of note, two doses of vaccine were able to elicit sustained neutralizing antibody activity against all the SARS-CoV-2 variants tested in SARS-CoV-2-experienced subjects. Conclusions BNT162b2 vaccine elicited a sustained humoral and cell-mediated response in immunocompetent subjects after two-dose administration of the vaccine, and the response seemed to be less affected by SARS-CoV-2 variants, the only exceptions being the β and δ variants. Increased immunogenicity, also against SARS-CoV-2 variant strains, was observed in SARS-CoV-2-experienced subjects. These results suggest that triple exposure to SARS-CoV-2 antigens might be proposed as valuable strategy for vaccination campaigns.

We aimed to explore whether variants of SARS-CoV-2 (Chinese-derived strain (D614, lineage A), Italian strain PV10734 (D614G, lineage B.1.1) and Alpha strain (lineage B.1.1.7)) were able to infect monocytes (MN) and monocyte-derived macrophages (MDM) and whether these infected cells may, in turn, be vectors of infection. For this purpose, we designed an in vitro study following the evolution of MN and MDM infection at different time points in order to confirm whether these cells were permissive for SARS-CoV-2 replication. Finally, we investigated whether, regardless of viral replication, the persistent virus can be transferred to non-infected cells permissive for viral replication. Thus, we co-cultured the infected MN/MDM with permissive VERO E6 cells verifying the viral transmission. This is a further in vitro demonstration of the important role of MN and MDM in the dissemination of SARS-CoV-2 and evolution of the COVID-19 disease.

Solid organ transplant recipients, due to the administration of post-transplant immunosuppressive therapies, are at greater risk of viral reactivation episodes, mainly from herpes viruses, including varicella-zoster virus (VZV). The aim of this pilot study was to develop functional immunological assays (VZV-ELISpot) for the quantification and characterization of the VZV-specific effector-memory and central-memory responses in healthy subjects and transplanted patients. Glycoprotein gE and immediate-early 63 (IE-63) were used as antigens for in vitro stimulation. VZV-seropositive healthy subjects showed higher responses in respect to seronegative subjects. Even if differences were observed between VZV-seropositive healthy subjects and transplanted subjects at pre-transplant, the VZV-specific T-cell response was reduced at 60 days after transplant, mainly for the high level of immunosuppression. Phenotypical characterization revealed that response against VZV was mainly mediated by CD4 T cells. The results obtained in this study might be useful for the definition of personalized follow-up of the transplanted patients, providing useful information on the status of the patient potentially at risk of viral reactivation or other opportunistic infections.

Optimal vaccines are needed for sustained suppression of SARS‐CoV‐2 and other novel coronaviruses. Here, we developed a recombinant type 5 adenovirus vector encoding the gene for the SARS‐CoV‐2 S1 subunit antigen (Ad5.SARS‐CoV‐2‐S1) for COVID‐19 immunization and evaluated its immunogenicity in mice. A single immunization with Ad5.SARS‐CoV‐2‐S1 via S.C. injection or I.N delivery induced robust antibody and cellular immune responses. Vaccination elicited significant S1‐specific IgG, IgG1, and IgG2a endpoint titers as early as 2 weeks, and the induced antibodies were long lasting. I.N. and S.C. administration of Ad5.SARS‐CoV‐2‐S1 produced S1‐specific GC B cells in cervical and axillary LNs, respectively. Moreover, I.N. and S.C. immunization evoked significantly greater antigen‐specific T‐cell responses compared to unimmunized control groups with indications that S.C. injection was more effective than I.N. delivery in eliciting cellular immune responses. Mice vaccinated by either route demonstrated significantly increased virus‐specific neutralization antibodies on weeks 8 and 12 compared to control groups, as well as BM antibody forming cells (AFC), indicative of long‐term immunity. Thus, this Ad5‐vectored SARS‐CoV‐2 vaccine candidate showed promising immunogenicity following delivery to mice by S.C. and I.N. routes of administration, supporting the further development of Ad‐based vaccines against COVID‐19 and other infectious diseases for sustainable global immunization programs., Vaccine development against SARS‐CoV‐2 addresses the ongoing challenges posed by this pathogen and ensures preparedness for future outbreaks of coronaviruses. Subcutaneous or intranasal administration of Ad5.SARS‐CoV‐2 S1 results in multifaceted immune responses in mice, including GCs and long‐lived antibody forming cells, representing an attractive COVID‐19 vaccination strategy.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has severely impacted on public health, mainly on immunosuppressed patients, including solid organ transplant recipients. Vaccination represents a valuable tool for the prevention of severe SARS-CoV-2 infection, and the immunogenicity of mRNA vaccines has been evaluated in transplanted patients. In this study, we investigated the role of a third dose of the BNT162b2 vaccine in a cohort of kidney transplant recipients, analyzing both humoral and cell-mediated responses. We observed an increased immune response after the third dose of the vaccine, especially in terms of Spike-specific T cell response. The level of seroconversion remained lower than 50% even after the administration of the third dose. Mycophenolate treatment, steroid administration and age seemed to be associated with a poor immune response. In our cohort, 11/45 patients experienced a SARS-CoV-2 infection after the third vaccine dose. HLA antibodies appearance was recorded in 7 out 45 (15.5%) patients, but none of the patients developed acute renal rejection. Further studies for the evaluation of long-term immune responses are still ongoing, and the impact of a fourth dose of the vaccine will be evaluated.

Phleboviruses transmitted by phlebotomine sandflies are endemic in the Mediterranean basin. Toscana phlebovirus (TOSV), Sicilian phlebovirus (SFSV), and Naples phlebovirus (SFNV) are responsible of summer fever, with well-known pathogenic potential for humans ranging from asymptomatic to mild fever, in addition to neuro-invasive infections during summer. Although TOSV, in particular, is a significant and well-known human pathogen, SFVs remain neglected, with many gaps in the relevant knowledge. Sero-epidemiological studies and case reports recently showed a geographical wider distribution than previously considered, although the real incidence of phleboviruses infections in the Mediterranean area is still unknown. Here we retrospectively evaluated the circulation of phleboviruses during summer seasons between 2007 and 2019 in 649 patients showing neurological symptoms using both molecular and serological approaches. We found that 42/649 (6.5%) subjects experienced phlebovirus infection and only 10/42 cases were detected by molecular assays, whereas the other 32/42 were identified using serological approaches, including neutralization assays. During the 2013 summer, an outbreak in the Lombardy region is described because the prevalence of phlebovirus infection reached 37.2% (19/51 subjects). Interestingly, only 5/19 (26.5%) reported traveling in endemic areas. Of note, no cross-neutralization was observed between different strains tested, showing the possibility to be reinfected by newly discovered phlebovirus strains. In conclusion, phlebovirus infections are still inadequately considered by physicians and are generally underestimated. However, based on our results, sandfly fever viruses should be routinely included in diagnostic panels during summer period, including in Northern Italy.

Objective: The protection from SARS-CoV-2 infection induced by SARS-CoV-2 anti-S1 and anti-S2 IgG antibody positivity resulting from natural infection was evaluated. Methods: The frequency of SARS-CoV-2 infection (as determined by virus RNA detection) was evaluated in a group of 1,460 seropositive and a control group of 8,150 seronegative healthcare workers in three Centres of Northern Italy in the period June-November 2020. Neutralizing serum titers were analyzed in seropositive subjects with or without secondary SARS-CoV-2 infection. Results: During the 6-month survey, 1.78% seropositive subjects developed secondary SARS-CoV-2 infection while 6.63% seronegative controls developed primary infection (odds ratio: 0.26; 95% confidence interval: 0.17-0.38). Secondary infection was associated with low or absent serum neutralizing titer (p

Objectives SARS-CoV-2 T-cell response characterization represents a crucial issue for defining the role of immune protection against COVID-19. The aim of the study was to assess the SARS-CoV-2 T-cell response in a cohort of COVID-19 convalescent patients and in a group of unexposed subjects. Methods SARS-CoV-2 T-cell response was quantified from peripheral blood mononuclear cells (PBMCs) of 87 COVID-19 convalescent subjects (range 7–239 days after symptom onset) and 33 unexposed donors by ex vivo ELISpot assay. Follow-up of SARS-CoV-2 T-cell response was performed in ten subjects up to 12 months after symptom onset. The role of SARS-CoV-2 specific CD4 and CD8 T cells was characterized in a group of COVID-19 convalescent subjects. Moreover, neutralizing antibodies were determined in serum samples. Results In 14/33 (42.4%) unexposed donors and 85/87 (97.7%) COVID-19 convalescent subjects a positive result for at least one SARS-CoV-2 antigen was observed. A positive response was observed up to 12 months after COVID-19 infection (median 246 days after symptom onset; range 118–362 days). Of note, SARS-CoV-2 T-cell response seems to be mainly mediated by CD4 T cells. A weak positive correlation was observed between Spike-specific T-cell response and neutralizing antibody titre (p 0.0028; r2 = 0.2891) and positive SARS-CoV-2 T-cell response was observed in 8/9 (88.9%) COVID-19 convalescent subjects with undetectable SARS-CoV-2 neutralizing antibodies. Discussion Cross-reactive SARS-CoV-2 T-cell response in uninfected patients may be due to previous infections with other common coronaviruses. Our data suggest that long-term SARS-CoV-2 T-cell response might accompany a waning humoral response.

SARS-CoV-2 still represents a global health burden, causing more than six million deaths worldwide. Moreover, the emergence of new variants has posed new issues in terms of vaccine efficacy and immunogenicity. In this study, we aimed to evaluate the neutralizing antibody response against SARS-CoV-2 variants in different cohorts of vaccinated and unvaccinated subjects. Four-fold diluted sera from SARS-CoV-2 naïve and recovered subjects vaccinated with two or three doses of the BNT162b2 vaccine were challenged against 14 SARS-CoV-2 variants, and the SARS-CoV-2 neutralizing antibody titer was measured. Results were compared with those obtained from unvaccinated COVID-19 recovered patients. Overall, a better SARS-CoV-2 NT Abs response was observed in recovered vaccinated subjects after three doses of the vaccine when compared to unvaccinated patients and vaccinated subjects with only two doses. Additionally, the lowest level of response was observed against the Omicron variant. In conclusion, third doses of BNT162b2 vaccine seems to elicit a sustained response against the large majority of variants.

OBJECTIVES: To define the seroprevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in blood donors (referred to the first lockdown area (Lodi Red-Zone) of the Lombardy region and in a contiguous area that was not included in the first lockdown); to define the agreement between a commercial serological assay and a reference microneutralization assay; and to evaluate the persistence of SARS-CoV-2 neutralizing antibodies in a cohort of blood donors. METHODS: Blood donors referred to the first lockdown area in Lombardy Region and the neighbouring area were analysed for SARS-CoV-2 IgG-specific antibodies during the period 18 March to 24 June 2020. Serum samples were analysed using both a chemiluminescent immunoassay (LIAISON® SARS-CoV-2 S1/S2 IgG, DiaSorin) for the quantitative characterization of SARS-CoV-2 anti-S1 and anti-S2 IgG antibodies and a neutralizing antibodies (NT-Abs) assay. RESULTS: In the period from 18 March to 24 June, 1922 blood donors were tested for the presence of SARS-CoV-2 IgG showing a prevalence of 378/1922 (19.7%). A subgroup of 1139 blood donors were tested in parallel with a SARS-CoV-2 IgG assay and a microneutralization assay showing a prevalence of 22.2% and 21.6%, respectively. SARS-CoV-2 IgG quantification was correlated with NT-Abs titres. In 78.2% of participants the NT-Abs titre was maintained, but in 15.8% it decreased by one four-fold dilution and in 6.0% it increased by one four-fold dilution. CONCLUSIONS: The duration of immunity of SARS-CoV-2 is crucial for the course of the pandemic and for this reason the monitoring of NT Abs is important. Despite a stable NT-Abs titre being observed in the majority of blood donors, our findings need to be validated in a long-term period of follow up.

West Nile virus (WNV) and Usutu virus (USUV) are two related arboviruses (genus Flavivirus, family Flaviviridae), with birds as a reservoir and mosquitoes as transmitting vectors. In recent years, WNV epidemiology changed in many European countries with increased frequency of outbreaks posing the issue of virus transmission risks by blood transfusion. USUV emerged for the first time in birds of the Tuscany region (Italy) in 1996 and in 2001 in Austria. While WNV is responsible for both mild and neuroinvasive diseases, USUV infection is usually asymptomatic and neuroinvasive symptoms are rare. Since WNV and USUV co-circulate, the surveillance of WNV allows also the detection of USUV. Due to the great similarity in amino-acid sequence of major surface proteins of the two viruses, a high cross-reactivity can lead to misinterpretation of serological results. Here, we report the results obtained from 54 asymptomatic blood donors during a three-year follow-up showing an unexpected high positivity (46.3%) for USUV. The major obstacle encountered in the differential diagnosis between these two viruses was the high cross-reactivity found in neutralizing antibodies (NT Abs) and, in some cases, a long follow-up was mandatory for a correct diagnosis. Moreover, two new ELISpot assays were developed for a more rapid and specific differential diagnosis, especially in those cases in which NT Abs were not determinant. Using a combination of Enzyme-linked immunospot (ELISpot), molecular, and serological tests, we could identify 25 true positive WNV and 25 true positive USUV blood donors. Our data highlight the importance of raising awareness for increasing USUV infections in endemic countries involved in blood transfusion and organ donation.

Hepatitis B vaccine is the most effective strategy to control hepatitis B virus (HBV) infection and disease. It is considered that an anti-HBs (antibodies against HBV surface antigen) titer >10 mIU/ml, measured shortly after a complete vaccination schedule, provides protection against infection. Approximately 4–10% of healthy individuals fail to respond to 3-dose vaccination. Long-term HBV-specific memory T-cell response has not been fully investigated, mainly due to the lack of a suitable assay. We quantified HBV-specific expandable memory T cells by using a cultured IFN-γ enzyme-linked immunospot (ELISPOT) assay. Cultured ELISPOT response to an overlapping peptide pool representing the complete L (large) HBV envelope polypeptide was evaluated in 41 healthy subjects vaccinated 15–20 y earlier and 5 unvaccinated. Plasma samples were tested for anti-HBs. Vaccinated subjects had significantly higher HBV-specific T-cellular response than unvaccinated (p = 0.0002). HBV-specific T-cell response was mainly mediated by CD4+ T cells. No concordance was found between cultured ELISPOT and anti-HBs data in vaccinated subjects. Thirty-one (76%) vaccinated subjects were responders (anti-HBs >10 mIU/ml). Nineteen (46%) vaccinated subjects were considered to be responders in cultured ELISPOT. Twenty-two (54%) vaccinated subjects were considered non-responders in cultured ELISPOT; 5 of them (23%) were also humoral non-responders. About 12% of healthy HBV-vaccinated subjects are both humoral and cellular non-responders. Although the prognostic value of this assay has not been established in terms of predictability for susceptibility to de-novo HBV infection, ELISPOT data suggest that these subjects may be at risk for HBV infection and disease, especially health care workers.