Relationship of human cytomegalovirus-infected endothelial cells and circulating leukocytes in the pathogenesis of disseminated human cytomegalovirus infection: A narrative review.

Among the leucocyte subpopulations circulating in peripheral blood of immune-compromised patients with disseminated Human cytomegalovirus (HCMV) infection, polymorphonuclear leuckocytes (PMNL) and M/M may carry infectious virus. While only in PMNL early HCMV replicative events do occur, monocytes are susceptible to complete virus replication when they enter human organs, where as macrophages become a site of active complete virus replication. In vivo leucocytes and endothelial cells interact continuously, as suggested by several in vitro experimental findings showing the bidirectional HCMV transmission from leucocytes to and from endothelial cells with the critical aid of adhesion molecules. Recently, the neutralising antibody response in sera from subjects with primary HCMV infection was reported to be much higher and earlier than in human embryonic lung fibroblasts (HELF) cells when measured in endothelial cells and epithelial cells, where virus entry is mediated mostly by the pentamer complex gH/gL/pUL128/pUL130/pUL131, whereas it was much lower and delayed when determined in HELF, where virus entry is mediated mostly by the trimer complex gH/gL/gO. Thus, these results suggested that products of UL128L were the molecules primary responsible for the differential neutralising antibody response. This conclusion was confirmed by a series of polyclonal and monoclonal antibodies directed to the components of pUL128L. Very recently, based on two sets of experiments including inhibition and immunoblotting assays, the pentamer complex/trimer complex ratio has been finally identified as the main factor of the neutralising antibody response. This ratio may change with the virus suspension producer and target cell system as well as number of cell culture passages.

Prevalence, Outcome, and Prevention of Congenital Cytomegalovirus Infection in Neonates Born to Women With Preconception Immunity (CHILd Study).

BACKGROUND: Human cytomegalovirus (HCMV) is the leading infectious cause of congenital disabilities. We designed a prospective study to investigate the rate, outcome, and risk factors of congenital CMV (cCMV) infection in neonates born to immune women, and the potential need and effectiveness of hygiene recommendations in this population. METHODS: The study was composed of 2 sequential parts: an epidemiology (part 1) and a prevention (part 2) study. Performance of part 2 depended upon a cCMV rate >0.4%. Women enrolled in part 1 did not receive hygiene recommendations. Newborns were screened by HCMV DNA testing in saliva and cCMV was confirmed by urine testing. RESULTS: Saliva swabs were positive for HCMV DNA in 45/9661 newborns and cCMV was confirmed in 18 cases. The rate of cCMV was .19% (95% confidence interval [CI]: .11-.29%), and 3 out of 18 infants with cCMV had symptoms of CMV at birth. Age, nationality, occupation, and contact with children were similar between mothers of infected and noninfected newborns. Twin pregnancy (odds ratio [OR]: 7.2; 95% CI: 1.7-32.2; P = .037) and maternal medical conditions (OR: 3.9; 95% CI: 1.5-10.1; P = .003) appeared associated with cCMV. Given the rate of cCMV was lower than expected, the prevention part of the study was cancelled. CONCLUSIONS: Newborns from women with preconception immunity have a low rate of cCMV, which appears to be mostly due to reactivation of the latent virus. Therefore, serological screening in childbearing age would be pivotal to identify HCMV-seropositive women, whose newborns have a low risk of cCMV. CLINICAL TRIALS REGISTRATION: www.clinicaltrials.gov (NCT03973359).

Correlates of postnatal human cytomegalovirus transmission in term babies in the first year.

Postnatal human cytomegalovirus (HCMV) infection in newborns is well characterized for preterm infants but less so for term infants. We sought to analyze the rates and routes of HCMV transmission in full-term infants during the first year of life. A cohort of 120 HCMV seropositive mothers and their 122 newborns were tested after delivery for HCMV-DNA shedding in different bodily fluids. Postnatal HCMV infection was defined as the detection of >2.5 × 102 HCMV-DNA copies/mL in infants' saliva swabs. Maternal neutralizing antibody serum titer, HCMV-specific T-cell response, and HCMV glycoprotein B immunoglobulin G on breastmilk were analyzed. HCMV shedding was detected in 67 of 120 mothers (55.8%), and 20 of 122 infants (16.4%) developed HCMV infection within the first 3 months of life. Six additional infants were infected during the first year, for a postnatal infection rate of 21.3%. Viral shedding was more frequent in breastmilk than saliva, urine, and vaginal secretions, and the mothers of infected infants showed higher levels of HCMV-DNA in milk. No association was found between the antibody levels in serum or milk and maternal viral shedding, whereas a slightly lower frequency of HCMV-specific CD4+ T-cells with long-term memory phenotype was observed in women with HCM-DNA-positive milk. About one out of five infants develop HCMV infection within the first year of life. Breastmilk appears the major route of transmission of the infection, maternal saliva has a minor role whereas the role of vaginal secretions is negligible.

ELISPOT assays with pp65 peptides or whole HCMV antigen are reliable predictors of immune control of HCMV infection in seropositive kidney transplant recipients.

Human cytomegalovirus (HCMV) infection represents a major complication for solid organ transplant recipients. The aim of this study was to verify if the measurement of HCMV-specific T-cells could help to identify patients protected against HCMV disease cytokine flow cytometry using infected dendritic cells as stimulus (CFC-iDC, which discriminates between CD4+ and CD8+ T cells), and ELISPOT, using infected cell lysate (ELISPOT-iCL) or pp65 (ELISPOT-pp65) as stimulus, were adopted. Among the 47 kidney transplant recipients (KTR) enrolled, 29 had a self-resolving HCMV infection (Controllers) and 18 required antiviral treatment (Non-Controllers). HCMV-specific T-cell frequency at the peak of HCMV infection identified Controllers and Non-Controllers, although the diagnostic performance of CD8+ CFC-iDC (area under the curve [AUC] of the receiver-operator characteristic curve: 0.65) was lower than that of CD4+ CFC-iDC (AUC: 0.83), ELISPOT-iCL (AUC: 0.83) and ELISPOT-pp65 (AUC: 0.80). CFC-iDC detected a protective immune reconstitution significantly earlier (median time: 38 days) than ELISPOT-iCL and ELISPOT-pp65 (median time: 126 and 133 days, respectively). Time to protective immune reconstitution in Non-Controllers was significantly longer than in Controllers with the ELISPOT and the CD4+ CFC-iDC assays, but not with CD8+ CFC-iDC. The majority of patients did not require antiviral treatment after protective immune reconstitution, with the exception of five patients according to CFC-iDC assay, one patient according to ELISPOT-iCL assay and three patients according to ELISPOT-pp65 assay. Monitoring the HCMV-specific immunological reconstitution with is effective in discriminating KTR at risk of or protected from HCMV disease and the ELISPOT assays are suitable for implementation in the clinical setting.

Characterization of immune response against monkeypox virus in cohorts of infected patients, historic and newly vaccinated subjects.

Monkeypox virus (MPXV) is a zoonotic disease endemic in the rainforest countries of Central and West Africa. Understanding the immune response in zoonosis is fundamental to prevent and contrast viral spreading. MPXV is a close relative of Variola (smallpox) virus and vaccination with vaccinia virus gives approximatively 85% of protection against MPXV. With the emergence of the recent MPXV outbreak, JYNNEOS vaccine has been proposed to individuals at high-risk of exposure. Comparative data on MPXV immune response in vaccinated or infected subjects are still limited. Here we set-up an immunofluorescence method for the evaluation of humoral response elicited by natural infection and healthy vaccinated subjects, including historically smallpox-vaccinated individuals and newly vaccinated subjects. Neutralization assay was also included, and in vaccinated subjects, cell-mediated response was evaluated. We observed that the natural infection produces a strong immune response that can control the disease. In naïve subjects, a second dose boosts the serological response to levels similar to those of the MPXV patients. Last, smallpox-vaccinated controls retain a degree of protection, even after years from vaccination, most visible in the t-cellular response.

Immunosuppressive treatments selectively affect the humoral and cellular response to SARS-CoV-2 in vaccinated patients with vasculitis.

OBJECTIVES: To analyse humoral and cellular immune response to mRNA COVID-19 vaccines in patients with GCA. METHODS: Consecutive patients with a diagnosis of GCA receiving two doses of BNT162b2 vaccine were assessed at baseline and 3 weeks from the second vaccine dose. Healthy subjects (n = 51) were included as controls (HC). Humoral response was assessed with Spike-specific IgG antibody response (S-IgG) and neutralizing antibodies (NtAb). Specific T cell response was assessed by enzyme linked immunosorbent spot (ELISpot). RESULTS: Of 56 included patients with GCA, 44 were eligible after exclusion of previous evidence of COVID-19 and incomplete follow-up. A significant proportion of patients with GCA (91%) demonstrated antibody (S-IgG) response, but this was significantly lower than HCs (100%); P < 0.0001. Neutralizing activity was not detected in 16% of patients with GCA. Antibody titres (S-IgG and NtAb) were significantly lower compared with HCs. Humoral response (S-IgG and NtAb) was significantly hampered by treatment with MTX. Cellular response was lacking in 30% of patients with GCA (vs 0% in HCs; P < 0.0001). Cellular response was significantly influenced by the levels of baseline peripheral T-lymphocytes and by glucocorticoid treatment. Treatment with tocilizumab did not affect any level of the immune response elicited by vaccination. CONCLUSIONS: Although patients with GCA apparently achieve a robust antibody seroconversion, there is a significant impairment of the neutralizing activity. MTX significantly reduced all levels of the humoral response. Up to one-third of patients do not develop a cellular immune protection in response to COVID-19 vaccination.

Fibroblast, Epithelial and Endothelial Cell-Derived Human Cytomegalovirus Strains Display Distinct Neutralizing Antibody Responses and Varying Levels of gH/gL Complexes.

In sequential sera from pregnant women with HCMV primary infection (PI), the serum neutralizing activity is higher against virions produced in epithelial and endothelial cells than in fibroblasts. Immunoblotting shows that the pentamer complex/trimer complex (PC/TC) ratio varies according to the producer cell culture type used for the virus preparation to be employed in the neutralizing antibody (NAb) assay, and is lower in fibroblasts and higher in epithelial, and especially endothelial cells. The blocking activity of TC- and PC-specific inhibitors varies according to the PC/TC ratio of virus preparations. The rapid reversion of the virus phenotype following its back passage to the original cell culture (fibroblasts) potentially argues in favor of a producer cell effect on virus phenotype. However, the role of genetic factors cannot be overlooked. In addition to the producer cell type, the PC/TC ratio may differ in single HCMV strains. In conclusion, the NAb activity not only varies with different HCMV strains, but is a dynamic parameter changing according to virus strain, type of target and producer cells, and number of cell culture passages. These findings may have some important implications for the development of both therapeutic antibodies and subunit vaccines.

Slow cytomegalovirus-specific CD4+ and CD8+ T-cell differentiation: 10-year follow-up of primary infection in a small number of immunocompetent hosts.

Differentiation of human cytomegalovirus specific T cells is a slow process requiring years. In the acute phase, EM predominate; subsequently, no contraction occurs (memory inflation) and TEMRA increase, especially among CD8+ T cells, while few LTM T cells appear. After some years, LTM stabilizes and predominate among CD4+ .

Impact of SARS-CoV-2 infection during pregnancy and persistence of antibody response.

BACKGROUND: Pregnant women may be at an increased risk of developing severe or critical disease associated with the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection causing severities during pregnancy. We performed a prospective study to describe the impact of SARS-CoV-2 infection on pregnancy outcomes and on the newborn, depending on the severity of the disease. The antibody response and persistence of SARS-CoV-2 anti-Spike (S) IgG, IgA and anti-Nu- cleocapsid (NCP) IgG, was investigated. METHODS: A total of 48 pregnant women with SARS-CoV-2 infection were enrolled, and sequential serum samples from 30 of them were collected until one year after infection. Outcomes of pregnan- cy and newborn parameters were evaluated in comparison with 200 uninfected controls. RESULTS: Asymptomatic infection was observed in 31/48 women (64.5%), mild COVID-19 in 12/48 women (25.0%), while 5/48 women (10.5%) developed pneumonia. Women with pneumonia mount- ed significantly higher levels of anti-S IgG, IgA and anti-NCP IgG between 1 and 3 months after onset of infection compared to asymptomatic women. Anti-S IgG persisted in the majority of women from 6 months to at least one year after infection, especially in those with symptomatic infection and pneumonia, while anti-S IgA and anti-NCP IgG declined earlier. Pregnancy complications and new- born parameters were not significantly different from those observed in uninfected controls. CONCLUSION: Anti-SARS-CoV-2 antibody development and persistence was not impaired in pregnant women, while SARS-CoV-2 infection did not cause major pregnancy or newborn complications in asymptomatic or symptomatic women, nor in women with pneumonia receiving prompt clinical care.

SARS-CoV-2-specific IgG and NCP in vulnerable patients without symptoms.

Patients with severe COVID-19 both seroconvert earlier and develop higher concentrations of SARS- CoV-2-specific IgG than patients with mild symptoms. In this retrospective study we considered different categories of patients defined as "vulnerable" because affected by other pathologies, such as patients with genetic and cardiovascular diseases; patients with autoimmune dermatological dis- ease; kidney and lung transplant patients, and pregnant women because the prevalence of Covid-19 infection during pregnancy is not known. This study was performed at IRCCS San Matteo Hospital in Pavia, North Italy, a zone considered at high risk during the COVID-19 pandemic from June to December 2020. None of the positive screened patients had symptoms of COVID-19 infection at the time of inclusion in this study.

Asymptomatic SARS-CoV-2 infection is not associated with miscarriage in early pregnancy: a retrospective analysis.

The prevalence of SARS-CoV-2 infection during pregnancy is relatively unknown. In this study we report the potential impact of undiagnosed SARS-CoV-2 infection on pregnancy loss in the first half of pregnancy by comparing the prevalence of the infection in a retrospective group of pregnant women with miscarriage (n=62) and a prospective control group with no pregnancy loss in the first trimester (n=218). Of 62 women who had miscarriage, 2 (3.2%) resulted IgM for SARS-CoV-2 negative and IgG seropositive, while of 218 pregnant women, 5 (2.3 %) resulted IgM for negative and IgG seropositive. The SARS-CoV-2 seroprevalence was not significantly different in the two groups of women, therefore excluding a significant role of SARS-CoV-2 infection in pregnancy loss. Therefore, our data show that SARS-CoV-2 infection within the first trimester does not seem to predispose to early pregnancy loss and that the impact of asymptomatic or mildly symptomatic SARS-CoV-2 infection on pregnancy appears limited.

Kidney transplant rejection rate in screened patients for anti-SARS-CoV-2 antibodies, during COVID-19 pandemic in Northern Italy.

Coronavirus is a high-risk pathogen for kidney transplant recipients receiving immunosuppressive therapy; Coronavirus disease 2019 (COVID-19) is an infection causing severe acute respiratory syndrome (SARS-CoV2), and progressive withdrawal of immunosuppressive drugs has been suggested in transplanted patients. At IRCCS San Matteo Hospital in Pavia, Northern Italy, during the pandemic we performed a screening and all transplanted patients were evaluated for IgG anti SARS-CoV-2; 12 of 201 kidney transplant recipients (6%) screened for IgG anti SARS-CoV-2 (s) developed kidney transplant rejection; 10 (83%) were negative and 2 (17%) resulted positive for SARS-CoV-2 IgG, while among 189 patients without rejection, 162 (86%) resulted negative and 27 (14%) positive (P=0.69). COVID 19 course may be more severe in kidney transplant recipients but it does not significantly increase risk of kidney rejection.

Immune Correlates of Protection Against Human Cytomegalovirus Acquisition, Replication, and Disease.

Human cytomegalovirus (HCMV) is the most common infectious cause of infant birth defects and an etiology of significant morbidity and mortality in solid organ and hematopoietic stem cell transplant recipients. There is tremendous interest in developing a vaccine or immunotherapeutic to reduce the burden of HCMV-associated disease, yet after nearly a half-century of research and development in this field we remain without such an intervention. Defining immune correlates of protection is a process that enables targeted vaccine/immunotherapeutic discovery and informed evaluation of clinical performance. Outcomes in the HCMV field have previously been measured against a variety of clinical end points, including virus acquisition, systemic replication, and progression to disease. Herein we review immune correlates of protection against each of these end points in turn, showing that control of HCMV likely depends on a combination of innate immune factors, antibodies, and T-cell responses. Furthermore, protective immune responses are heterogeneous, with no single immune parameter predicting protection against all clinical outcomes and stages of HCMV infection. A detailed understanding of protective immune responses for a given clinical end point will inform immunogen selection and guide preclinical and clinical evaluation of vaccines or immunotherapeutics to prevent HCMV-mediated congenital and transplant disease.

Broncho-alveolar inflammation in COVID-19 patients: a correlation with clinical outcome.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapidly reached pandemic proportions. Given that the main target of SARS-CoV-2 are lungs leading to severe pneumonia with hyperactivation of the inflammatory cascade, we conducted a prospective study to assess alveolar inflammatory status in patients with moderate to severe COVID-19. METHODS: Diagnostic bronchoalveolar lavage (BAL) was performed in 33 adult patients with SARS-CoV-2 infection by real-time PCR on nasopharyngeal swab admitted to the Intensive care unit (ICU) (n = 28) and to the Intermediate Medicine Ward (IMW) (n = 5). We analyze the differential cell count, ultrastructure of cells and Interleukin (IL)6, 8 and 10 levels. RESULTS: ICU patients showed a marked increase in neutrophils (1.24 × 105 ml- 1, 0.85-2.07), lower lymphocyte (0.97 × 105 ml- 1, 0.024-0.34) and macrophages fractions (0.43 × 105 ml- 1, 0.34-1.62) compared to IMW patients (0.095 × 105 ml- 1, 0.05-0.73; 0.47 × 105 ml- 1, 0.28-1.01 and 2.14 × 105 ml- 1, 1.17-3.01, respectively) (p < 0.01). Study of ICU patients BAL by electron transmission microscopy showed viral particles inside mononuclear cells confirmed by immunostaining with anti-viral capsid and spike antibodies. IL6 and IL8 were significantly higher in ICU patients than in IMW (IL6 p < 0.01, IL8 p < 0.0001), and also in patients who did not survive (IL6 p < 0.05, IL8 p = 0.05 vs. survivors). IL10 did not show a significant variation between groups. Dividing patients by treatment received, lower BAL concentrations of IL6 were found in patients treated with steroids as compared to those treated with tocilizumab (p < 0.1) or antivirals (p < 0.05). CONCLUSIONS: Alveolitis, associated with COVID-19, is mainly sustained by innate effectors which showed features of extensive activation. The burden of pro-inflammatory cytokines IL6 and IL8 in the broncho-alveolar environment is associated with clinical outcome.

Human Cytomegalovirus Genomes Sequenced Directly From Clinical Material: Variation, Multiple-Strain Infection, Recombination, and Gene Loss.

The genomic characteristics of human cytomegalovirus (HCMV) strains sequenced directly from clinical pathology samples were investigated, focusing on variation, multiple-strain infection, recombination, and gene loss. A total of 207 datasets generated in this and previous studies using target enrichment and high-throughput sequencing were analyzed, in the process enabling the determination of genome sequences for 91 strains. Key findings were that (i) it is important to monitor the quality of sequencing libraries in investigating variation; (ii) many recombinant strains have been transmitted during HCMV evolution, and some have apparently survived for thousands of years without further recombination; (iii) mutants with nonfunctional genes (pseudogenes) have been circulating and recombining for long periods and can cause congenital infection and resulting clinical sequelae; and (iv) intrahost variation in single-strain infections is much less than that in multiple-strain infections. Future population-based studies are likely to continue illuminating the evolution, epidemiology, and pathogenesis of HCMV.

Human cytomegalovirus (HCMV) infection/re-infection: development of a protective HCMV vaccine.

In recent years, one of the main objectives in the field of medical virology has been the development of a human cytomegalovirus (HCMV) vaccine that can prevent congenital HCMV infection in the offspring of pregnant women as well as systemic and end-organ disease in immunocompromised (AIDS and transplanted) patients. Major obstacles to the development of an efficacious HCMV vaccine are lack of protection provided by immune memory cells against HCMV re-activation (replication relapse of a latent strain following primary infection) and HCMV re-infection (infection of a seropositive individual by a new virus strain). Thus, while initial efforts were directed at the development of a vaccine for the prevention of primary infection, in the last decade the primary vaccine development endpoint was the prevention of primary HCMV infection, as well as HCMV re-activation and re-infection. Along this line of research, both HCMV live (including Towne, AD169 and its derivatives, Towne/Toledo chimeras, Viral-Vectored vaccines, and Virus Replicon Particles) and non-living vaccines (including the recombinant gB subunit, DNA- and RNA-based vaccines, Virus-like particles, Dense bodies, Peptide vaccines, and the Pentamer Complex) have been developed. To date, Phase I, II, and III clinical trials have been variably conducted in humans, and experimental inoculation in different animal models has been performed with different vaccine formulations. Notwithstanding the variable research conditions, clinical and experimental results achieved thus far predict that the ideal HCMV vaccine should be able to elicit both robust humoral (both neutralizing and binding) and HCMV-specific CD4+ and CD8+ T-cell responses. This vaccine should hypothetically contain: 1) gB, inducing both humoral and T-cell responses; 2) the pentameric complex (PC), inducing the most potent neutralizing antibody response; 3) pp65, inducing the most potent HCMV-specific T-cell response. Although the protective role of cell-mediated immunity has been repeatedly documented, while the protective effect of (mostly neutralizing) antibodies remains partly to be documented, a vaccine stimulating both arms of the immune response would likely confer the highest level of protection against HCMV infection/disease in both HCMV-seronegative and -seropositive individuals.

Human cytomegalovirus (HCMV)-specific T cell but not neutralizing or IgG binding antibody responses to glycoprotein complexes gB, gHgLgO, and pUL128L correlate with protection against high HCMV viral load reactivation in solid-organ transplant recipients.

Immune correlates of protection against human cytomegalovirus (HCMV) infection are still debated. This study aimed to investigate which arm of the immune response plays a major role in protection against HCMV infection in kidney transplant recipients (n = 40) and heart transplant recipients (n = 12). Overall, patients were divided into 2 groups: one including 37 patients with low viral load (LVL), and the other including 15 patients with high viral load (HVL). All LVL patients resolved the infection spontaneously, whereas HVL patients were all treated with one or more courses of antivirals. In HVL patients, viral DNAemia, which was more than 100 times higher than LVL, appeared and peaked at significantly earlier times, but disappeared much later than in LVL patients. During a 1-year follow-up, all LVL patients had levels of HCMV-specific CD4+ (and CD8+ ) T cells significantly higher than HVL patients. On the contrary, titers of neutralizing antibodies and enzyme-linked immunosorbent assay-IgG antibodies to gB, gHgLgO, and pentamer gHgLpUL128L were overlapping in the 2 patient groups. In conclusion, while a valid HCMV-specific T-cell response was detected in more than 90% of LVL patients, >90% of HVL patients lacked an adequate T-cell response. Antibody responses did not appear to be associated directly or indirectly with protection.

Maternal immune correlates of protection from human cytomegalovirus transmission to the fetus after primary infection in pregnancy.

Immune control of primary human cytomegalovirus (HCMV) infection appears to depend on the interaction of humoral and T-cell responses. In this review, we have separately explored the 2 arms of the immune response to primary HCMV infection in HCMV-seronegative pregnant women transmitting (T) or not transmitting (NT) the infection to the fetus, with the objective of correlating the immune risk factors associated with vertical HCMV transmission. As for the humoral response, the following findings were documented: (i) in competitive binding assays, antibody titers to different antigenic sites of the gH pentamer complex were significantly lower in T compared with NT women; (ii) in addition, the number of neutralization sites recognized by T was significantly lower compared with NT women; (iii) the plaque formation inhibition assay showed a faster kinetics and a higher titer in NT women. As for T-cell immunity, the delayed expression of 3 immunological parameters (lymphoproliferative response, CD45RA reexpression in both CD4+ and CD8+ HCMV-specific T cells, and interleukin-2 production by HCMV-specific CD4+ T cells) were significantly associated with vertical transmission. This overview provides important information at the population level, which may help to inform the evaluation of interventions such as vaccination or treatments designed to interrupt intrauterine transmission of HCMV during primary infection. However, although we are waiting for an HCMV vaccination to become available, we emphasize that none of these parameters can be prognostically used on an individual basis because of the great variation in values among women.