Antibody-independent protection against heterologous SARS-CoV-2 challenge conferred by prior infection or vaccination.

Vaccines have reduced severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) morbidity and mortality, yet emerging variants challenge their effectiveness. The prevailing approach to updating vaccines targets the antibody response, operating under the presumption that it is the primary defense mechanism following vaccination or infection. This perspective, however, can overlook the role of T cells, particularly when antibody levels are low or absent. Here we show, through studies in mouse models lacking antibodies but maintaining functional B cells and lymphoid organs, that immunity conferred by prior infection or mRNA vaccination can protect against SARS-CoV-2 challenge independently of antibodies. Our findings, using three distinct models inclusive of a novel human/mouse ACE2 hybrid, highlight that CD8+ T cells are essential for combating severe infections, whereas CD4+ T cells contribute to managing milder cases, with interferon-γ having an important function in this antibody-independent defense. These findings highlight the importance of T cell responses in vaccine development, urging a broader perspective on protective immunity beyond just antibodies.

Memory CD8+ T cell diversity and B cell responses correlate with protection against SARS-CoV-2 following mRNA vaccination.

Understanding immune responses to SARS-CoV-2 messenger RNA (mRNA) vaccines is of great interest, principally because of the poor knowledge about the mechanisms of protection. In the present study, we analyzed longitudinally B cell and T cell memory programs against the spike (S) protein derived from ancestral SARS-CoV-2 (Wuhan-1), B.1.351 (beta), B.1.617.2 (delta) and B.1.1.529 (omicron) variants of concern (VOCs) after immunization with an mRNA-based vaccine (Pfizer). According to the magnitude of humoral responses 3 months after the first dose, we identified high and low responders. Opposite to low responders, high responders were characterized by enhanced antibody-neutralizing activity, increased frequency of central memory T cells and durable S-specific CD8+ T cell responses. Reduced binding antibodies titers combined with long-term specific memory T cells that had distinct polyreactive properties were found associated with subsequent breakthrough with VOCs in low responders. These results have important implications for the design of new vaccines and new strategies for booster follow-up.

Isolation and Characterization of Neutralizing Monoclonal Antibodies from a Large Panel of Murine Antibodies against RBD of the SARS-CoV-2 Spike Protein.

The COVID-19 pandemic, once a global crisis, is now largely under control, a testament to the extraordinary global efforts involving vaccination and public health measures. However, the relentless evolution of SARS-CoV-2, leading to the emergence of new variants, continues to underscore the importance of remaining vigilant and adaptable. Monoclonal antibodies (mAbs) have stood out as a powerful and immediate therapeutic response to COVID-19. Despite the success of mAbs, the evolution of SARS-CoV-2 continues to pose challenges and the available antibodies are no longer effective. New variants require the ongoing development of effective antibodies. In the present study, we describe the generation and characterization of neutralizing mAbs against the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein by combining plasmid DNA and recombinant protein vaccination. By integrating genetic immunization for rapid antibody production and the potent immune stimulation enabled by protein vaccination, we produced a rich pool of antibodies, each with unique binding and neutralizing specificities, tested with the ELISA, BLI and FACS assays and the pseudovirus assay, respectively. Here, we present a panel of mAbs effective against the SARS-CoV-2 variants up to Omicron BA.1 and BA.5, with the flexibility to target emerging variants. This approach ensures the preparedness principle is in place to address SARS-CoV-2 actual and future infections.

Pediatric Slow-Progressive, but Not Non-Progressive Cerebellar Ataxia Delays Intra-Limb Anticipatory Postural Adjustments in the Upper Arm.

We recently investigated the role of the cerebellum during development, reporting that children with genetic slow-progressive ataxia (SlowP) show worse postural control during quiet stance and gait initiation compared to healthy children (H). Instead, children with genetic non-progressive ataxia (NonP) recalled the behavior of H. This may derive from compensatory networks, which are hindered by disease progression in SlowP while free to develop in NonP. In the aim of extending our findings to intra-limb postural control, we recorded, in 10 NonP, 10 SlowP and 10 H young patients, Anticipatory Postural Adjustments (APAs) in the proximal muscles of the upper-limb and preceding brisk index finger flexions. No significant differences in APA timing occurred between NonP and H, while APAs in SlowP were delayed. Indeed, the excitatory APA in Triceps Brachii was always present but significantly delayed with respect to both H and NonP. Moreover, the inhibitory APAs in the Biceps Brachii and Anterior Deltoid, which are normally followed by a late excitation, could not be detected in most SlowP children, as if inhibition was delayed to the extent where there was overlap with a late excitation. In conclusion, disease progression seems to be detrimental for intra-limb posture, supporting the idea that inter- and intra-limb postures seemingly share the same control mechanism.

Structural and connectivity parameters reveal spared connectivity in young patients with non-progressive compared to slow-progressive cerebellar ataxia.

Introduction: Within Pediatric Cerebellar Ataxias (PCAs), patients with non-progressive ataxia (NonP) surprisingly show postural motor behavior comparable to that of healthy controls, differently to slow-progressive ataxia patients (SlowP). This difference may depend on the building of compensatory strategies of the intact areas in NonP brain network. Methods: Eleven PCAs patients were recruited: five with NonP and six with SlowP. We assessed volumetric and axonal bundles alterations with a multimodal approach to investigate whether eventual spared connectivity between basal ganglia and cerebellum explains the different postural motor behavior of NonP and SlowP patients. Results: Cerebellar lobules were smaller in SlowP patients. NonP patients showed a lower number of streamlines in the cerebello-thalamo-cortical tracts but a generalized higher integrity of white matter tracts connecting the cortex and the basal ganglia with the cerebellum. Discussion: This work reveals that the axonal bundles connecting the cerebellum with basal ganglia and cortex demonstrate a higher integrity in NonP patients. This evidence highlights the importance of the cerebellum-basal ganglia connectivity to explain the different postural motor behavior of NonP and SlowP patients and support the possible compensatory role of basal ganglia in patients with stable cerebellar malformation.

B cell analyses after SARS-CoV-2 mRNA third vaccination reveals a hybrid immunity like antibody response.

The continuous evolution of SARS-CoV-2 generated highly mutated variants able to escape natural and vaccine-induced primary immunity. The administration of a third mRNA vaccine dose induces a secondary response with increased protection. Here we investigate the longitudinal evolution of the neutralizing antibody response in four donors after three mRNA doses at single-cell level. We sorted 4100 spike protein specific memory B cells identifying 350 neutralizing antibodies. The third dose increases the antibody neutralization potency and breadth against all SARS-CoV-2 variants as observed with hybrid immunity. However, the B cell repertoire generating this response is different. The increases of neutralizing antibody responses is largely due to the expansion of B cell germlines poorly represented after two doses, and the reduction of germlines predominant after primary immunization. Our data show that different immunization regimens induce specific molecular signatures which should be considered while designing new vaccines and immunization strategies.

DNA-Vaccine-Induced Immune Response Correlates with Lower Viral SARS-CoV-2 Titers in a Ferret Model.

The COVID-19 pandemic is entering a new era with the approval of many SARS-CoV-2 vaccines. In spite of the restoration of an almost normal way of life thanks to the immune protection elicited by these innovative vaccines, we are still facing high viral circulation, with a significant number of deaths. To further explore alternative vaccination platforms, we developed COVID-eVax-a genetic vaccine based on plasmid DNA encoding the RBD domain of the SARS-CoV-2 spike protein. Here, we describe the correlation between immune responses and the evolution of viral infection in ferrets infected with the live virus. We demonstrate COVID-eVax immunogenicity as means of antibody response and, above all, a significant T-cell response, thus proving the critical role of T-cell immunity, in addition to the neutralizing antibody activity, in controlling viral spread.

Anatomy of Omicron BA.1 and BA.2 neutralizing antibodies in COVID-19 mRNA vaccinees.

SARS-CoV-2 vaccines, administered to billions of people worldwide, mitigate the effects of the COVID-19 pandemic, however little is known about the molecular basis of antibody cross-protection to emerging variants, such as Omicron BA.1, its sublineage BA.2, and other coronaviruses. To answer this question, 276 neutralizing monoclonal antibodies (nAbs), previously isolated from seronegative and seropositive donors vaccinated with BNT162b2 mRNA vaccine, were tested for neutralization against the Omicron BA.1 and BA.2 variants, and SARS-CoV-1 virus. Only 14.2, 19.9 and 4.0% of tested antibodies neutralize BA.1, BA.2, and SARS-CoV-1 respectively. These nAbs recognize mainly the SARS-CoV-2 receptor binding domain (RBD) and target Class 3 and Class 4 epitope regions on the SARS-CoV-2 spike protein. Interestingly, around 50% of BA.2 nAbs did not neutralize BA.1 and among these, several targeted the NTD. Cross-protective antibodies derive from a variety of germlines, the most frequents of which were the IGHV1-58;IGHJ3-1, IGHV2-5;IGHJ4-1 and IGHV1-69;IGHV4-1. Only 15.6, 20.3 and 7.8% of predominant gene-derived nAbs elicited against the original Wuhan virus cross-neutralize Omicron BA.1, BA.2 and SARS-CoV-1 respectively. Our data provide evidence, at molecular level, of the presence of cross-neutralizing antibodies induced by vaccination and map conserved epitopes on the S protein that can inform vaccine design.

A Novel Viewpoint on the Anticipatory Postural Adjustments During Gait Initiation.

Anticipatory postural adjustments (APAs) are the coordinated muscular activities that precede the voluntary movements to counteract the associated postural perturbations. Many studies about gait initiation call APAs those activities that precede the heel-off of the leading foot, thus taking heel-off as the onset of voluntary movement. In particular, leg muscles drive the center of pressure (CoP) both laterally, to shift the body weight over the trailing foot and backward, to create a disequilibrium torque pushing forward the center of mass (CoM). However, since subjects want to propel their body rather than lift their foot, the onset of gait should be the CoM displacement, which starts with the backward CoP shift. If so, the leg muscles driving such a shift are the prime movers. Moreover, since the disequilibrium torque is mechanically equivalent to a forward force acting at the pelvis level, APAs should be required to link the body segments to the pelvis: distributing such concentrated force throughout the body would make all segments move homogeneously. In the aim of testing this hypothesis, we analyzed gait initiation in 15 right-footed healthy subjects, searching for activities in trunk muscles that precede the onset of the backward CoP shift. Subjects stood on a force plate for about 10 s and then started walking at their natural speed. A minimum of 10 trials were collected. A force plate measured the CoP position while wireless probes recorded the electromyographic activities. Recordings ascertained that at gait onset APAs develop in trunk muscles. On the right side, Rectus Abdominis and Obliquus Abdominis were activated in 11 and 13 subjects, respectively, starting on average 33 and 54 ms before the CoP shift; Erector Spinae (ES) at L2 and T3 levels was instead inhibited (9 and 7 subjects, 104 and 120 ms). On the contralateral side, the same muscles showed excitatory APAs (abdominals in 11 and 12 subjects, 27 and 82 ms; ES in 10 and 7 subjects, 75 and 32 ms). The results of this study provide a novel framework for distinguishing postural from voluntary actions, which may be relevant for the diagnosis and rehabilitation of gait disorders.

Dual-Hemisphere Transcranial Direct Current Stimulation on Parietal Operculum Does Not Affect the Programming of Intra-limb Anticipatory Postural Adjustments.

Evidence shows that the postural and focal components within the voluntary motor command are functionally unique. In 2015, we reported that the supplementary motor area (SMA) processes Anticipatory Postural Adjustments (APAs) separately from the command to focal muscles, so we are still searching for a hierarchically higher area able to process both components. Among these, the parietal operculum (PO) seemed to be a good candidate, as it is a hub integrating both sensory and motor streams. However, in 2019, we reported that transcranial Direct Current Stimulation (tDCS), applied with an active electrode on the PO contralateral to the moving segment vs. a larger reference electrode on the opposite forehead, did not affect intra-limb APAs associated to brisk flexions of the index-finger. Nevertheless, literature reports that two active electrodes of opposite polarities, one on each PO (dual-hemisphere, dh-tDCS), elicit stronger effects than the "active vs. reference" arrangement. Thus, in the present study, the same intra-limb APAs were recorded before, during and after dh-tDCS on PO. Twenty right-handed subjects were tested, 10 for each polarity: anode on the left vs. cathode on the right, and vice versa. Again, dh-tDCS was ineffective on APA amplitude and timing, as well as on prime mover recruitment and index-finger kinematics. These results confirm the conclusion that PO does not take part in intra-limb APA control. Therefore, our search for an area in which the motor command to prime mover and postural muscles are still processed together will have to address other structures.

Transcranial Direct Current Stimulation on Parietal Operculum Contralateral to the Moving Limb Does Not Affect the Programming of Intra-Limb Anticipatory Postural Adjustments.

Recent data suggest that the parietal operculum acts as an integration center within a multimodal network, originating from different primary sensory and motor cortices and projecting to frontal, parietal and temporal cortical hubs, which in turn govern cognitive and motor functions. Thus, parietal operculum might also play a crucial role in the integrated control of voluntary movement and posture. As a first step to test this hypothesis, the Anticipatory Postural Adjustments (APAs) stabilizing the arm when the index-finger is briskly flexed were recorded, on the preferred side, in three groups of 10 healthy subjects, before, during and after CATHODAL or ANODAL transcranial Direct Current Stimulation (tDCS, 20 min at 2 mA) applied over the contralateral Parietal Operculum (coPO). Results were compared to those obtained in a SHAM group. In agreement with literature, in the SHAM group the activation of the prime mover Flexor Digitorum Superficialis was preceded by an inhibitory APA in Biceps Brachii and Anterior Deltoid, and almost simultaneous to an excitatory APA in Triceps Brachii. The same pattern was observed in both the CATHODAL and ANODAL groups, with no significant tDCS effects on APAs amplitude and timing. Index-finger kinematics were also unchanged. These negative results suggest that the coPO does not disturb the key network governing APAs in index-finger flexion. Since it has been well documented that such APAs share many features with those observed in trunk and limb muscles when performing several other movements, we suggest that coPO may not be crucial to the general APA control.

Aflatoxin B1 and M1: Biological Properties and Their Involvement in Cancer Development.

Aflatoxins are fungal metabolites found in feeds and foods. When the ruminants eat feedstuffs containing Aflatoxin B1 (AFB1), this toxin is metabolized and Aflatoxin M1 (AFM1) is excreted in milk. International Agency for Research on Cancer (IARC) classified AFB1 and AFM1 as human carcinogens belonging to Group 1 and Group 2B, respectively, with the formation of DNA adducts. In the last years, some epidemiological studies were conducted on cancer patients aimed to evaluate the effects of AFB1 and AFM1 exposure on cancer cells in order to verify the correlation between toxin exposure and cancer cell proliferation and invasion. In this review, we summarize the activation pathways of AFB1 and AFM1 and the data already reported in literature about their correlation with cancer development and progression. Moreover, considering that few data are still reported about what genes/proteins/miRNAs can be used as damage markers due to AFB1 and AFM1 exposure, we performed a bioinformatic analysis based on interaction network and miRNA predictions to identify a panel of genes/proteins/miRNAs that can be used as targets in further studies for evaluating the effects of the damages induced by AFB1 and AFM1 and their capacity to induce cancer initiation.

Disrupt of Intra-Limb APA Pattern in Parkinsonian Patients Performing Index-Finger Flexion.

Voluntary movements induce postural perturbations which are counteracted by anticipatory postural adjustments (APAs). These actions are known to build up long fixation chains toward available support points (inter-limb APAs), so as to grant whole body equilibrium. Moreover, recent studies highlighted that APAs also build-up short fixation chains, within the same limb where a distal segment is moved (intra-limb APAs), aimed at stabilizing the proximal segments. The neural structures generating intra-limb APAs still need investigations; the present study aims to compare focal movement kinematics and intra-limb APA latencies and pattern between healthy subjects and parkinsonian patients, assuming the latter as a model of basal ganglia dysfunction. Intra-limb APAs that stabilize the arm when the index-finger is briskly flexed were recorded in 13 parkinsonian patients and in 10 age-matched healthy subjects. Index-finger movement was smaller in parkinsonian patients vs. healthy subjects (p = 0.01) and more delayed with respect to the onset of the prime mover flexor digitorum superficialis (FDS, p < 0.0001). In agreement with the literature, in all healthy subjects the FDS activation was preceded by an inhibitory intra-limb APA in biceps brachii (BB) and anterior deltoid (AD), and almost simultaneous to an excitatory intra-limb APA in triceps brachii (TB). In parkinsonian patients, no significant differences were found for TB and AD intra-limb APA timings, however only four patients showed an inhibitory intra-limb APA in BB, while other four did not show any BB intra-limb APAs and five actually developed a BB excitation. The frequency of occurrence of normal sign, lacking, and inverted BB APAs was different in healthy vs. parkinsonian participants (p = 0.0016). The observed alterations in index-finger kinematics and intra-limb APA pattern in parkinsonian patients suggest that basal ganglia, in addition to shaping the focal movement, may also contribute to intra-limb APA control.

Evaluation of Aflatoxin M1 Effects on the Metabolomic and Cytokinomic Profiling of a Hepatoblastoma Cell Line.

Hepatoblastoma incidence has been associated with different environmental factors even if no data are reported about a correlation between aflatoxin exposure and hepatoblastoma initiation. Considering that hepatoblastoma develops in infants and children and aflatoxin M1 (AFM1), the aflatoxin B1 (AFB1) hydroxylated metabolite, can be present in mothers' milk and in marketed milk products, in this study we decided to test the effects of AFM1 on a hepatoblastoma cell line (HepG2). Firstly, we evaluated the effects of AFM1 on the cell viability, apoptosis, cell cycle, and metabolomic and cytokinomic profile of HepG2 cells after treatment. AFM1 induced: (1) a decrease of HepG2 cell viability, reaching IC50 at 9 µM; (2) the blocking of the cell cycle in the G0/G1 phase; (3) the decrease of formiate levels and incremented level of some amino acids and metabolites in HepG2 cells after treatment; and (4) the increase of the concentration of three pro-inflammatory cytokines, IL-6, IL-8, and TNF-α, and the decrease of the anti-inflammatory interleukin, IL-4. Our results show that AFM1 inhibited the growth of HepG2 cells, inducing both a modulation of the lipidic, glycolytic, and amino acid metabolism and an increase of the inflammatory status of these cells.

Identifying a panel of genes/proteins/miRNAs modulated by arsenicals in bladder, prostate, kidney cancers.

Arsenic and arsenic-derivative compounds, named as arsenicals, represent a worldwide problem for their effect on the human health and, in particular, for their capability to increase the risk of developing cancer such as kidney, bladder and prostate cancer. The main source of arsenical exposure is drinking water. Nowadays, it is well known that the chronic exposure to arsenicals leads to a series of epigenetic alterations that have a role in arsenic-induced effects on human health including cancer. Based on these observations, the aim of our study was to select by network analysis the genes/proteins/miRNAs implicated in kidney, bladder and prostate cancer development upon arsenical exposure. From this analysis we identified: (i) the nodes linking the three molecular networks specific for kidney, bladder and prostate cancer; (ii) the relative HUB nodes (RXRA, MAP3K7, NR3C1, PABPC1, NDRG1, RELA and CTNNB1) that link the three cancer networks; (iii) the miRNAs able to target these HUB nodes. In conclusion, we highlighted a panel of potential molecules related to the molecular mechanisms of arsenical-induced cancerogenesis and suggest their utility as biomarkers or therapeutic targets.