Isolation of human monoclonal antibodies that potently neutralize human cytomegalovirus infection by targeting different epitopes on the gH/gL/UL128-131A complex

Human cytomegalovirus (HCMV) is a widely circulating pathogen that causes severe disease in immunocompromised patients and infected fetuses. By immortalizing memory B cells from HCMV-immune donors, we isolated a panel of human monoclonal antibodies that neutralized at extremely low concentrations (90% inhibitory concentration [IC90] values ranging from 5 to 200 pM) HCMV infection of endothelial, epithelial, and myeloid cells. With the single exception of an antibody that bound to a conserved epitope in the UL128 gene product, all other antibodies bound to conformational epitopes that required expression of two or more proteins of the gH/gL/UL128-131A complex. Antibodies against gB, gH, or gM/gN were also isolated and, albeit less potent, were able to neutralize infection of both endothelial-epithelial cells and fibroblasts. This study describes unusually potent neutralizing antibodies against HCMV that might be used for passive immunotherapy and identifies, through the use of such antibodies, novel antigenic targets in HCMV for the design of immunogens capable of eliciting previously unknown neutralizing antibody responses

Fetal human cytomegalovirus transmission correlates with delayed maternal antibodies to gH/gL/pUL128-130-131 complex during primary infection

Primary human cytomegalovirus (HCMV) infections during pregnancy are associated with a high risk of virus transmission to the fetus. To identify correlates of intrauterine HCMV transmission, serial serum samples from HCMV transmitter and non-transmitter pregnant women with primary HCMV infection were analyzed for the presence of neutralizing antibodies against different glycoproteins and glycoprotein complexes, which are known to mediate entry into distinct types of host cells. Neutralizing activity was detected in the sera early after primary infection; absorption with a soluble pentameric complex formed by gH/gL/pUL128-131, but not with gH/gL dimer or with gB, abolished the capacity of sera to neutralize infection of epithelial cells. Importantly, an early, high antibody response to pentamer antigenic sites was associated with a significantly reduced risk of HCMV transmission to the fetus. This association is consistent with the high in vitro inhibition of HCMV infection of epithelial/endothelial cells as well as cell- to-cell spreading and virus transfer to leukocytes by anti-pentamer antibodies. Taken together, these findings indicate that the HCMV pentamer complex is a major target of the antibody-mediated maternal immunity

Mechanism of baricitinib supports artificial intelligence-predicted testing in COVID-19 patients

Baricitinib, is an oral Janus kinase (JAK)1/JAK2 inhibitor approved for the treatment of rheumatoid arthritis (RA) that was independently predicted, using artificial intelligence (AI)-algorithms, to be useful for COVID-19 infection via a proposed anti-cytokine effects and as an inhibitor of host cell viral propagation. We evaluated the in vitro pharmacology of baricitinib across relevant leukocyte subpopulations coupled to its in vivo pharmacokinetics and showed it inhibited signaling of cytokines implicated in COVID-19 infection. We validated the AI-predicted biochemical inhibitory effects of baricitinib on human numb-associated kinase (hNAK) members measuring nanomolar affinities for AAK1, BIKE, and GAK. Inhibition of NAKs led to reduced viral infectivity with baricitinib using human primary liver spheroids. These effects occurred at exposure levels seen clinically. In a case series of patients with bilateral COVID-19 pneumonia, baricitinib treatment was associated with clinical and radiologic recovery, a rapid decline in SARS-CoV-2 viral load, inflammatory markers, and IL-6 levels. Collectively, these data support further evaluation of the anti-cytokine and anti-viral activity of baricitinib and supports its assessment in randomized trials in hospitalized COVID-19 patients

Human Cytomegalovirus and Human Umbilical Vein Endothelial Cells: Restriction of Primary Isolation to Blood Samples and Susceptibilities of Clinical Isolates from Other Sources to Adaptation

In immunocompromised patients with disseminated infection, human cytomegalovirus (HCMV) is widespread in the microvascular endothelium of multiple organs. Human umbilical vein endothelial cells (HUVEC) were used in parallel to human embryonic lung fibroblasts (HELF) to recover HCMV from blood samples of immunocompromised patients. Using the shell vial technique, comparable median numbers of p72-positive HUVEC and HELF cells were found with the 26 HCMV-positive buffy coat samples out of 150 examined. Analysis of other clinical samples inoculated as controls revealed, in the presence of highly infected HELF monolayers, either the presence of very few infected HUVEC with urine specimens (n = 10 samples) or the lack of infected HUVEC with throat washes (n = 3) or amniotic fluid samples (n = 2). Thus, peripheral blood leukocytes (PBL) appear essential for primary isolation of HCMV in HUVEC. In this respect, HCMV strains, recovered from clinical samples other than buffy coats in HELF only, could be readily adapted to growth in HUVEC by coculturing PBL from healthy blood donors with infected HELF and then inoculating infected PBL onto HUVEC. Recently elucidated mechanisms of interaction of leukocytes and HUVEC with bidirectional transfer of virus seem to provide the basis for the restriction of HCMV primary isolation in HUVEC to blood samples. However, virus strains recovered from only HELF could be adapted to growth in HUVEC when inoculated with HELF-derived (either cell-associated or cell-free) HCMV strains upon primary isolation. In conclusion, due to the in vitro selection of virus variants provided with both PBL tropism and HUVEC tropism, HCMV recovery in HUVEC is PBL mediated and substantially restricted to blood samples. Lack of HCMV recovery in HUVEC from clinical samples other than blood leads to the assumption that epithelial cells, such as urinary, amniotic, or pharyngeal cells, do not possess adequate adhesion molecules to establish close contacts with HUVEC

Evaluation of Xpert® Norovirus Assay performance in comparison with real-time RT-PCR in hospitalized adult patients with acute gastroenteritis

Xpert® Norovirus Assay (Cepheid, Sunnyvale, CA) was compared with a laboratory-developed real-time RT-PCR assay for the detection of Norovirus GI and GII in hospitalized patients with acute gastroenteritis. The two assays showed a high level of concordance but Xpert® Norovirus Assay allowed faster detection of Norovirus and a simpler sample handling

Rapid Detection of Human Metapneumovirus Strains in Nasopharyngeal Aspirates and Shell Vial Cultures by Monoclonal Antibodies

Monoclonal antibodies to human metapneumovirus (hMPV) were developed for direct fluorescent antibody (DFA) staining of nasopharyngeal aspirates from 40 infants with respiratory infections, as well as for hMPV identification in shell vial cultures. With reference to reverse transcription-PCR, DFA staining showed sensitivity, specificity, and positive and negative predictive values of 73.9%, 94.1%, 94.4%, and 72.7%, respectively. Monoclonal antibodies are useful for direct hMPV detection

Characterization of Varicella-Zoster (VZV) Specific T Cell Response in Healthy Subjects and Transplanted Patients by Using Enzyme Linked Immunospot (ELISpot) Assays

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

Pitfalls in the Serological Diagnosis of Primary Human Cytomegalovirus Infection in Pregnancy Due to Different Kinetics of IgM Clearance and IgG Avidity Index Maturation

Primary infection occurs when seronegative women are infected by human cytomegalovirus (HCMV). Diagnosis of primary infection is based on the following: antibody seroconversion, presence of IgM and low IgG avidity index (AI), and presence of DNAemia. The kinetics of HCMV-specific IgM antibody and maturation of AI might be very rapid or long-lasting during primary infection, which makes serological diagnosis insidious. The aims of this study were as follows: (i) to report atypical kinetics of HCMV-specific IgM antibody and AI early after onset of primary HCMV infection in a population of pregnant women, and (ii) to assess the frequency of such results. Altogether, 1309 sequential serum samples collected from 465 pregnant women with primary HCMV infection were included in the study. As a general rule, using the LIAISON®CMVIgMII and LIAISON®CMVIgGAvidityII assays, virus-specific IgM antibody levels decreased, while IgG AI increased over time during the first three months after infection onset. However, early clearance of IgM antibody and/or early IgG AI maturation occurred in 46/426 (10.7%) women. In more details, 20/426 (4.7%) and 26/418 (6.2%) women had undetectable IgM antibody or high IgG AI, respectively, when tested within 1–3 months after well-defined infection onset. Twenty sera from as many women with high IgG AI by the LIAISON assay were further tested for IgG AI by VIDAS®CMVIgGAvidityII and Mikrogen recomLineCMVIgG Avidity assays. Comparable results were obtained with VIDAS, whereas 14/20 sera gave low AI with the Mikrogen assay. In conclusion, about 11% of pregnant women undergoing a primary HCMV infection showed misleading serological results. Additional and appropriate testing might help in reducing the risk of missing HCMV primary infection in pregnancy. Furthermore, preconceptional testing should be strongly recommended

In Vitro Model for the Study of the Dissociation of Increasing Antigenemia and Decreasing DNAemia and Viremia during Treatment of Human Cytomegalovirus Infection with Ganciclovir in Transplant Recipients

The paradox phenomenon (i.e., the dissociation of increasing antigenemia and decreasing DNAemia and viremia) that occurs during treatment of human cytomegalovirus (HCMV) infections with ganciclovir (Gcv), in transplant recipients, was investigated by use of an in vitro model for the study of interactions between polymorphonuclear leukocytes and endothelial cells. The paradox phenomenon was reproduced in vitro in the presence of Gcv and, to a much lesser extent, in the presence of cidofovir, but not in the presence of foscarnet. The pathogenetic basis for such a paradox response was found, by use of drug concentrations in the range of 90%-99% of the inhibitory dose, to rely on the partial synthesis of HCMV phosphoprotein 65. The opposite situation (i.e., the simultaneous increase of antigenemia, viremia, and DNAemia), which is observed in clinical conditions associated with inefficacy of treatment due to drug-resistant strains, was also reproduced in vitro by use of drug-resistant HCMV strains. The conclusion for clinicians is that antiviral therapy must be changed only in the latter case