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Pseudotyped Viruses As a Molecular Tool to Monitor Humoral Immune Responses Against SARS-CoV-2 Via Neutralization Assay

Pseudotyped viruses (PVs) are molecular tools that can be used to study host-virus interactions and to test the neutralizing ability of serum samples, in ...

University of Verona

Induction of immune response after SARS-CoV-2 mRNA BNT162b2 vaccination in healthcare workers.

Although SARS-CoV-2 immunizations have started in most countries, children are not currently included in the vaccination programs; thus, it remains crucial to define their anti-SARS-CoV-2 immune response in order to minimize the risk for other epidemic waves. This study sought to provide a description of the virology ad anti-SARS-CoV-2 immunity in children with distinct symptomatology.

Virological and immunological features of SARS-COV-2 infected children with distinct symptomatology.

Mass SARS-Cov-2 vaccination campaign represents the only strategy to defeat the global pandemic we are facing. Immunocompromised patients represent a vulnerable population at high risk of developing severe COVID-19 and thus should be prioritized in the vaccination programs and in the study of the vaccine efficacy. Nevertheless, most data on efficacy and safety of the available vaccines derive from trials conducted on healthy individuals; hence, studies on immunogenicity of SARS-CoV2 vaccines in such populations are deeply needed. Here, we perform an observational longitudinal study analyzing the humoral and cellular response following the BNT162b2 mRNA COVID-19 vaccine in a cohort of patients affected by inborn errors of immunity (IEI) compared to healthy controls (HC). We show that both IEI and HC groups experienced a significant increase in anti-SARS-CoV-2 Abs 1 week after the second scheduled dose as well as an overall statistically significant expansion of the Ag-specific CD4+CD40L+ T cells in both HC and IEI. Five IEI patients did not develop any specific CD4+CD40L+ T cellular response, with one of these patients unable to also mount any humoral response. These data raise immunologic concerns about using Ab response as a sole metric of protective immunity following vaccination for SARS-CoV-2. Taken together, these findings suggest that evaluation of vaccine-induced immunity in this subpopulation should also include quantification of Ag-specific T cells.

Humoral and Cellular Response Following Vaccination With the BNT162b2 mRNA COVID-19 Vaccine in Patients Affected by Primary Immunodeficiencies.

Specific memory B cells and antibodies are a reliable read-out of vaccine efficacy. We analysed these biomarkers after one and two doses of BNT162b2 vaccine. The second dose significantly increases the level of highly specific memory B cells and antibodies. Two months after the second dose, specific antibody levels decline, but highly specific memory B cells continue to increase, thus predicting a sustained protection from COVID-19. We show that although mucosal IgA is not induced by the vaccination, memory B cells migrate in response to inflammation and secrete IgA at mucosal sites. We show that the first vaccine dose may lead to an insufficient number of highly specific memory B cells and low concentration of serum antibodies, thus leaving vaccinees without the immune robustness needed to ensure viral elimination and herd immunity. We also clarify that the reduction of serum antibodies does not diminish the force and duration of the immune protection induced by vaccination. The vaccine does not induce sterilizing immunity. Infection after vaccination may be caused by the lack of local preventive immunity because of the absence of mucosal IgA.

Highly Specific Memory B Cells Generation after the 2nd Dose of BNT162b2 Vaccine Compensate for the Decline of Serum Antibodies and Absence of Mucosal IgA.

Integrated HIV-1 DNA persists despite antiretroviral therapy and can fuel viral rebound following treatment interruption. Hence, methods to specifically measure the integrated HIV-1 DNA portion only are important to monitor the reservoir in eradication trials. Here, we provide an up-to-date overview of the literature on the different approaches used to measure integrated HIV-1 DNA. Further, we propose an implemented standard-curve free assay to quantify integrated HIV-1 DNA, so-called -5LTR PCR, which utilises novel primer combinations. We tested the -5LTR PCR in 20 individuals on suppressive ART for a median of nine years; the results were compared to those produced with the standard-free -gag assay. The numbers of median integrated HIV-1 DNA copies were 5 (range: 1-12) and 14 (5-26) with the -gag and -5LTR, respectively. The ratios between -gag vs -5LTR results were distributed within the cohort as follows: most patients (12/20, 60%) provided ratios between 2-5, with 3/20 (15%) and 5/20 (25%) being below or above this range, respectively. -5LTR assay sensitivity was also determined using an "integrated standard"; the data confirmed the increased sensitivity of the assay, i.e., equal to 0.25 proviruses in 10,000 genomes. This work represents an improvement in the field of measuring proviral HIV-1 DNA that could be employed in future HIV-1 persistence and eradication studies.

Measuring Proviral HIV-1 DNA: Hurdles and Improvements to an Assay Monitoring Integration Events Utilising Human  Repeat Sequences.

COVID-19 vaccines have demonstrated effectiveness in reducing SARS-CoV-2 mild and severe outcomes. In vaccinated subjects with SARS-CoV-2 history, RBD-specific IgG and pseudovirus neutralization titers were rapidly recalled by a single BTN162b2 vaccine dose to higher levels than those in naïve recipients after the second dose, irrespective of waning immunity. In this study, we inspected the long-term kinetic and neutralizing responses of S-specific IgG induced by two administrations of BTN162b2 vaccine in infection-naïve subjects and in subjects previously infected with SARS-CoV-2.

Different decay of antibody response and VOC sensitivity in naïve and previously infected subjects at 15 weeks following vaccination with BNT162b2.

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.

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

Despite a successful antiretroviral therapy (ART), adolescents living with perinatally acquired HIV (PHIV) experience signs of B-cell hyperactivation with expansion of 'namely' atypical B-cell phenotypes, including double negative (CD27-IgD-) and termed age associated (ABCs) B-cells (T-bet+CD11c+), which may result in reduced cell functionality, including loss of vaccine-induced immunological memory and higher risk of developing B-cells associated tumors. In this context, perinatally HIV infected children (PHIV) deserve particular attention, given their life-long exposure to chronic immune activation.

Determinants of B-Cell Compartment Hyperactivation in European Adolescents Living With Perinatally Acquired HIV-1 After Over 10 Years of Suppressive Therapy.

HIV-1 infection in the absence of treatment results in progression toward AIDS. Host genetic factors play a role in HIV-1 pathogenesis, but complete knowledge is not yet available. Since less-expressed HLA-C variants are associated with poor HIV-1 control and unstable HLA-C variants are associated with higher HIV-1 infectivity, we investigated whether there was a correlation between the different stages of HIV-1 progression and the presence of specific HLA-C allotypes. HLA-C genotyping was performed using allele-specific PCR by analyzing a treatment-naïve cohort of 96 HIV-1-infected patients from multicentric cohorts in the USA, Canada, and Brazil. HIV-1-positive subjects were classified according to their different disease progression status as progressors (Ps,  = 48), long-term non-progressors (LTNPs,  = 37), and elite controllers (ECs,  = 11). HLA-C variants were classified as stable or unstable according to their binding stability to β2-microglobulin/peptide complex. Our results showed a significant correlation between rapid progression to AIDS and the presence of two or one unstable HLA-C variants (-value: 0.0078, -value: 0.0143, respectively). These findings strongly suggest a link between unstable HLA-C variants both at genotype and at allele levels and rapid progression to AIDS. This work provides further insights into the impact of host genetic factors on AIDS progression.

Increased Prevalence of Unstable HLA-C Variants in HIV-1 Rapid-Progressor Patients.

Mycobacterium tuberculosis and human immunodeficiency virus-1 (HIV-1) syndemic interactions are a major global health concern. Despite the clinical significance of coinfection, our understanding of the cellular pathophysiology and the therapeutic pharmacodynamic impact of coinfection is limited. Here, we use single-round infectious HIV-1 pseudotyped viral particles expressing green fluorescent protein alongside M. tuberculosis expressing mCherry to study pathogenesis and treatment. We report that HIV-1 infection inhibited intracellular replication of M. tuberculosis and demonstrate the therapeutic activity of antiviral treatment (efavirenz) and antimicrobial treatment (rifampicin). The described method could be applied for detailed mechanistic studies to inform the development of novel treatment strategies.

A Quantitative Method for the Study of HIV-1 and Mycobacterium tuberculosis Coinfection.

Human immunodeficiency virus type 1 (HIV-1) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have caused two major viral outbreaks during the last century. Two major aspects of HIV-1 and SARS-CoV-2 co-infection have been extensively investigated and deserve attention. First, the impact of the co-infection on the progression of disease caused by HIV-1 or SARS-CoV-2. Second, the impact of the HIV-1 anti-retroviral treatment on SARS-CoV-2 infection. In this review, we aim to summarize and discuss the works produced since the beginning of the SARS-CoV-2 pandemic ranging from clinical studies to in vitro experiments in the context of co-infection and drug development.

HIV and SARS-CoV-2 Co-Infection: From Population Study Evidence to In Vitro Studies.

We have previously shown that eliciting SARS-CoV-2-specific IgM after vaccination is associated with higher levels of SARS-CoV-2 neutralizing IgG. This study aims to assess whether IgM development is also associated with longer-lasting immunity.

Article	Year
Induction of immune response after SARS-CoV-2 mRNA BNT162b2 vaccination in healthcare workers. Journal of virus eradication\| PubMed ID: 34026245	2021
Virological and immunological features of SARS-COV-2 infected children with distinct symptomatology. Pediatric allergy and immunology : official publication of the European Society of Pediatric Allergy and Immunology\| PubMed ID: 34174102	2021
Humoral and Cellular Response Following Vaccination With the BNT162b2 mRNA COVID-19 Vaccine in Patients Affected by Primary Immunodeficiencies. Frontiers in immunology\| PubMed ID: 34671350	2021
Highly Specific Memory B Cells Generation after the 2nd Dose of BNT162b2 Vaccine Compensate for the Decline of Serum Antibodies and Absence of Mucosal IgA. Cells\| PubMed ID: 34685521	2021
Measuring Proviral HIV-1 DNA: Hurdles and Improvements to an Assay Monitoring Integration Events Utilising Human Repeat Sequences. Life (Basel, Switzerland)\| PubMed ID: 34947941	2021
Different decay of antibody response and VOC sensitivity in naïve and previously infected subjects at 15 weeks following vaccination with BNT162b2. Journal of translational medicine\| PubMed ID: 34998405	2022
SARS-CoV-2 vaccination elicits unconventional IgM specific responses in naïve and previously COVID-19-infected individuals. EBioMedicine\| PubMed ID: 35196644	2022
Determinants of B-Cell Compartment Hyperactivation in European Adolescents Living With Perinatally Acquired HIV-1 After Over 10 Years of Suppressive Therapy. Frontiers in immunology\| PubMed ID: 35432380	2022
Increased Prevalence of Unstable HLA-C Variants in HIV-1 Rapid-Progressor Patients. International journal of molecular sciences\| PubMed ID: 36499177	2022
A Quantitative Method for the Study of HIV-1 and Mycobacterium tuberculosis Coinfection. The Journal of infectious diseases\| PubMed ID: 36537213	2023
HIV and SARS-CoV-2 Co-Infection: From Population Study Evidence to In Vitro Studies. Life (Basel, Switzerland)\| PubMed ID: 36556453	2022
Subjects who developed SARS-CoV-2 specific IgM after vaccination show a longer humoral immunity and a lower frequency of infection. EBioMedicine\| PubMed ID: 36796232	2023

Alessandra Ruggiero

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