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.

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.

Identifying high-confidence variants in human cytomegalovirus genomes sequenced from clinical samples.

Understanding the intrahost evolution of viral populations has implications in pathogenesis, diagnosis, and treatment and has recently made impressive advances from developments in high-throughput sequencing. However, the underlying analyses are very sensitive to sources of bias, error, and artefact in the data, and it is important that these are addressed adequately if robust conclusions are to be drawn. The key factors include (1) determining the number of viral strains present in the sample analysed; (2) monitoring the extent to which the data represent these strains and assessing the quality of these data; (3) dealing with the effects of cross-contamination; and (4) ensuring that the results are reproducible. We investigated these factors by generating sequence datasets, including biological and technical replicates, directly from clinical samples obtained from a small cohort of patients who had been infected congenitally with the herpesvirus human cytomegalovirus, with the aim of developing a strategy for identifying high-confidence intrahost variants. We found that such variants were few in number and typically present in low proportions and concluded that human cytomegalovirus exhibits a very low level of intrahost variability. In addition to clarifying the situation regarding human cytomegalovirus, our strategy has wider applicability to understanding the intrahost variability of other viruses.

Congenital Human Cytomegalovirus Infection: A Narrative Review of Maternal Immune Response and Diagnosis in View of the Development of a Vaccine and Prevention of Primary and Non-Primary Infections in Pregnancy.

Congenital cytomegalovirus infection (cCMV) may affect about 1% of all newborns all over the world as a result of either a primary or recurrent human cytomegalovirus (HCMV) infection. While about 90% of infants affected by cCMV are asymptomatic at birth, the remaining 10% are symptomatic often with neurodevelopmental impairment and sensorineural hearing loss. In view of identifying the best approach to vaccine prevention of cCMV, this review will examine the most important steps made in the study of the immune response to, and diagnosis of, HCMV infection. The maternal immune response and immune correlates of protection are being partially identified with a partial contribution given by our laboratory. The diagnosis of primary infection is often difficult to achieve in the first three months of pregnancy, which is the time primarily involved in virus transmission to the fetus in association with the most severe symptoms and sequelae. Prevention of cCMV is anticipated by prevention of primary infection in early pregnancy by means of different measures, such as (i) behavioral-educational measures, (ii) immunoglobulin administration, (iii) antiviral treatment with valaciclovir. However, the most promising approach to cCMV prevention appears to be the development of a non-living vaccine, including at least three viral antigens: gB, pentamer complex gHgLpUL128L, and pp65, which have been shown to be able to stimulate both the humoral and the cellular arms of the maternal immune response. Primary HCMV infection may be managed in pregnancy by counseling of the couples involved by a team of specialists that includes virologists, obstetricians, infectivologists and neonatologists.

Detection of Genotype-Specific Antibody Responses to Glycoproteins B and H in Primary and Non-Primary Human Cytomegalovirus Infections by Peptide-Based ELISA.

BACKGROUND: Strain-specific antibodies to human cytomegalovirus (HCMV) glycoproteins B and H (gB and gH) have been proposed as a potential diagnostic tool for identifying reinfection. We investigated genotype-specific IgG antibody responses in parallel with defining the gB and gH genotypes of the infecting viral strains. METHODS: Subjects with primary (n = 20) or non-primary (n = 25) HCMV infection were studied. The seven gB (gB1-7) and two gH (gH1-2) genotypes were determined by real-time PCR and whole viral genome sequencing, and genotype-specific IgG antibodies were measured by a peptide-based enzyme-linked immunosorbent assay (ELISA). RESULTS: Among subjects with primary infection, 73% (n = 8) infected by gB1-HCMV and 63% (n = 5) infected by gB2/3-HCMV had genotype-specific IgG antibodies to gB (gB2 and gB3 are similar in the region tested). Peptides from the rarer gB4-gB7 genotypes had nonspecific antibody responses. All subjects infected by gH1-HCMV and 86% (n = 6) infected by gH2-HCMV developed genotype-specific responses. Among women with non-primary infection, gB and gH genotype-specific IgG antibodies were detected in 40% (n = 10) and 80% (n = 20) of subjects, respectively. CONCLUSIONS: Peptide-based ELISA is capable of detecting primary genotype-specific IgG responses to HCMV gB and gH, and could be adopted for identifying reinfections. However, about half of the subjects did not have genotype-specific IgG antibodies to gB.

Early T cell reconstitution and cytokine profile may help to guide a personalized management of human cytomegalovirus infection after allogeneic hematopoietic stem cell transplantation.

Human cytomegalovirus (HCMV) infection is one of the major causes of mortality and morbidity after allo-hematopoietic stem cell transplantation (HSCT). Antiviral therapies are associated with toxicity and high economic burden. The aim of this retrospective study was to identify allo-HSCT HCMV-seropositive recipients at low risk of clinically significant HCMV infection who could avoid antiviral therapies. Sixty adult patients who underwent allo-HSCT were clustered in two groups: i) 22 (37%) spontaneously controlling HCMV reactivation (Controllers); ii) 38 (63%) developing clinically significant HCMV infection and receiving pre-emptive therapy (Non-Controllers). We analyzed several patient baseline characteristics, total/HCMV-specific CD4+ and CD8+ T-cell counts and their cytokine production (IFNγ, TNFα, IL2). Controllers presented a higher number of total/HCMV-specific CD4+ and CD8+ T-cells (P=0.001 and P=0.017 for total CD4+ and CD8+ T-cells respectively; P<0.001 for HCMV-specific T-cells) and a lower percentage of mono-functional IFNγ-producing HCMV-specific CD8+ T-cells (P=0.002). In bi-variable models, the prognostic impact of the percentage of mono-functional HCMV-specific CD8+ T-cells on treatment-free survival, adjusted for total/HCMVspecific CD4+ and CD8+ T-cells, was confirmed. An HCMV-seronegative donor was the only baseline characteristic associated with a clinically significant infection. These data, when confirmed by a larger prospective study, may provide information for guiding the personalized management of HCMV infection in allo-HSCT recipients.

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+ .

Analysis of the SARS-CoV-2 epidemic in Italy: The role of local and interventional factors in the control of the epidemic.

Containment measures have been applied in several countries in order to limit the diffusion of the SARS-CoV-2 epidemic. The scope of this study is to analyze the evolution of the first wave of the SARS-CoV-2 epidemic throughout Italy and factors associated to the different way it spread in the Italian Regions, starting from the day that the first indigenous cases were detected through day 81 (6 days after the end of the strict lockdown). Data were obtained from daily reports and are represented as number (and percentage) of cases/100,000 persons. A lockdown with movement restrictions, especially across Regions, was declared at day 20. At day 81, 219,070 cases (363/100,000 persons) were diagnosed. A regression analysis based on the Gompertz model predicts a total number 233,606 cases (386/100,000 persons) at the end of the epidemic. The 21 areas, divided into Italian Regions and autonomous Provinces, showed a wide range in the frequency of cases at day 81 (58-921, median 258/100,000 persons) and total predicted cases (58-946, median 267/100,000 persons). Similarly, the predicted time for the end of the wave of the epidemic (considering as surrogate marker the time at which 99% of the total cases are predicted to occur) was highly variable, ranging from 64 to 136 (median 99) days. We analyzed the impact of local and interventional variables on the epidemic curve in each Region. The number of cases correlated inversely with the distance from the area in which first cases were detected and directly also with the gross domestic product pro capite (as a marker of industrial activity) of the Region. Moreover, an earlier start of the lockdown (i.e. in the presence of a lower number of cases) and wider testing were associated with a lower final number of total cases. In conclusion, this analysis shows that population-wide testing and early lockdown enforcement appear effective in limiting the spreading of the SARS-CoV-2 epidemic.

Human Cytomegalovirus Congenital (cCMV) Infection Following Primary and Nonprimary Maternal Infection: Perspectives of Prevention through Vaccine Development.

Congenital cytomegalovirus (cCMV) might occur as a result of the human cytomegalovirus (HCMV) primary (PI) or nonprimary infection (NPI) in pregnant women. Immune correlates of protection against cCMV have been partly identified only for PI. Following either PI or NPI, HCMV strains undergo latency. From a diagnostic standpoint, while the serological criteria for the diagnosis of PI are well-established, those for the diagnosis of NPI are still incomplete. Thus far, a recombinant gB subunit vaccine has provided the best results in terms of partial protection. This partial efficacy was hypothetically attributed to the post-fusion instead of the pre-fusion conformation of the gB present in the vaccine. Future efforts should be addressed to verify whether a new recombinant gB pre-fusion vaccine would provide better results in terms of prevention of both PI and NPI. It is still a matter of debate whether human hyperimmune globulin are able to protect from HCMV vertical transmission. In conclusion, the development of an HCMV vaccine that would prevent a significant portion of PI would be a major step forward in the development of a vaccine for both PI and NPI.

Determination of anti-p52 IgM and anti-gB IgG by ELISA as a novel diagnostic tool for detection of early and late phase of primary human cytomegalovirus infections during pregnancy.

BACKGROUND: Dating of primary human cytomegalovirus (HCMV) infection in pregnancy is crucial to define whether infection occurred before or during pregnancy and at which gestational age. OBJECTIVE: The aim of this study was to identify a diagnostic strategy for determination of early, intermediate and late phase of HCMV primary infection during pregnancy. STUDY DESIGN: Sequential serum samples from 40 pregnant women with defined onset of HCMV primary infection were tested retrospectively for IgM, IgG and IgG avidity against whole HCMV lysate, along with anti-p52 IgM and anti-gB IgG (Euroimmun AG). RESULTS: Anti-HCMV IgM were positive in all samples collected within the first 2 months, then decreased remaining weakly positive in about 40% of samples collected within 6-12 months after infection. Anti-p52 IgM followed similar kinetics but decreased earlier, remaining weakly positive only in 20% of late samples. Anti-HCMV IgG were positive in all samples and showed variable kinetics. Their avidity increased from low levels, observed within 2 months, to intermediate/high levels from 4 months onwards. Anti-gB IgG increased over time following kinetics similar to anti-HCMV IgG avidity. By combining results of anti-HCMV IgM plus IgG avidity, and confirming them with anti-p52 IgM plus anti-gB IgG as second-line assays, the early (within 2-3 months) and late (after 3 months) phases of HCMV infection were satisfactorily defined, whereas the intermediate phase overlapped with the beginning of the late phase. CONCLUSION: Anti-p52 IgM and anti-gB IgG provide additional tools besides classical anti-HCMV IgM, IgG and IgG avidity in dating HCMV primary infections.

Human Cytomegalovirus Cell Tropism and Host Cell Receptors.

In the 1970s-1980s, a striking increase in the number of disseminated human cytomegalovirus (HCMV) infections occurred in immunosuppressed patient populations. Autopsy findings documented the in vivo disseminated infection (besides fibroblasts) of epithelial cells, endothelial cells, and polymorphonuclear leukocytes. As a result, multiple diagnostic assays, such as quantification of HCMV antigenemia (pp65), viremia (infectious virus), and DNAemia (HCMV DNA) in patient blood, were developed. In vitro experiments showed that only low passage or endothelial cell-passaged clinical isolates, and not laboratory-adapted strains, could reproduce both HCMV leuko- and endothelial cell-tropism, which were found through genetic analysis to require the three viral genes UL128, UL130, and UL131 of the HCMV UL128 locus (UL128L). Products of this locus, together with gH/gL, were shown to form the gH/gL/pUL128L pentamer complex (PC) required for infection of epithelial cells/endothelial cells, whereas gH/gL and gO form the gH/gL/gO trimer complex (TC) required for infection of all cell types. In 2016, following previous work, a receptor for the TC that mediates entry into fibroblasts was identified as PDGFRα, while in 2018, a receptor for the PC that mediates entry into endothelial/epithelial cells was identified as neuropilin2 (Nrp2). Furthermore, the olfactory receptor family member OR14I1 was recently identified as a possible additional receptor for the PC in epithelial cells. Thus, current data support two models of viral entry: (i) in fibroblasts, following interaction of PDGFRα with TC, the latter activates gB to fuse the virus envelope with the cell membrane, whereas (ii) in epithelial cells/endothelial cells, interaction of Nrp2 (and OR14I1) with PC promotes endocytosis of virus particles, followed by gB activation by gH/gL/gO (or gH/gL) and final low-pH entry into the cell.

An overview of letermovir: a cytomegalovirus prophylactic option.

Introduction: Human cytomegalovirus (HCMV) or human herpesvirus 5 (HHV-5) is a ß-herpesvirus that causes widespread infection in nearly all members of the human population worldwide. Its persistence in humans after primary infection in a latent phase as well as a partial non-protective immune response is the basis for repeated re-activation/re-infection episodes occurring both in immunocompetent and immunocompromised subjects. In the latter patient populations, which include hematopoietic stem cell transplant (HSCT) recipients, HCMV reactivation episodes may be particularly severe, leading to both systemic and end-organ diseases. Since the 90s, at least four antiviral drugs targeting the DNA polymerase complex have been developed for the prevention and treatment of HCMV infections in transplant recipients, used as first-line (ganciclovir and valganciclovir) and second-line therapy (foscarnet and cidofovir). However, due to their toxicity and drug-resistance induction, new drugs with different targets were needed. Areas covered: In 2017, a new drug named letermovir (LTV), which targets the HCMV DNA terminase complex, was licensed for prophylaxis of HCMV infections in HSCT recipients. This is the focus of this review. Expert opinion: LTV safety and efficacy are promising. However, long-term adverse events and the emergence of drug-resistant HCMV strains must be investigated in extended clinical trials prior to drawing final conclusions.

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.

Comparison of the T-cell response to human cytomegalovirus (HCMV) as detected by cytokine flow cytometry and QuantiFERON-CMV assay in HCMV-seropositive kidney transplant recipients.

Human cytomegalovirus (HCMV)-specific T-cell response in kidney transplant recipients (KTR) helps to identify patients at risk for severe infection. To assess the T-cell response, this study compared our in-house developed reference test, based on T-cell (both CD4+ and CD8+) stimulation by autologous HCMV-infected dendritic cells (iDC) and subsequent detection by cytokine flow cytometry (CFC-iDC), with the Quanti-FERON-CMV (QF-CMV) assay. Fifty-three HCMV-seropositive KTR were enrolled. At the DNAemia peak, 33 (62%) had low viral load (LVL, <3x105 DNA copies/mL) self-resolving infection, 19 (36%) high viral load (HVL, >3x105 DNA copies/mL) infection treated with antivirals, and one LVL patient (2%) tissue-invasive disease alone. Both assays showed a delayed recovery of HCMV-specific T-cell immunity in HVL vs LVL patients. Immune reconstitution kinetics did not significantly differ between the two assays in HVL patients. QF-CMV and CFC-iDC showed comparable sensitivities, but QF-CMV had a lower (although not significantly) specificity. Indeed, 7/19 HVL patients (37%) were erroneously considered protected from severe infection by QF-CMV, whereas CFC-iDC misidentified only 3/19 (16%) patients as protected. Although our reference test takes longer to complete, it appears slightly better at predicting patients at risk for severe HCMV infection. Moreover, QF-CMV may provide false negative results with some HLA types.

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.

Phenotype and specificity of T cells in primary human cytomegalovirus infection during pregnancy: IL-7Rpos long-term memory phenotype is associated with protection from vertical transmission.

Congenital human cytomegalovirus (HCMV) infection is the major cause of birth defects and a precise definition of the HCMV-specific T-cell response in primary infection may help define reliable correlates of immune protection during pregnancy. In this study, a high throughput method was used to define the frequency of CD4+ and CD8+ T cells specific for four HCMV proteins in the naïve compartment of seronegative subjects and the effector/memory compartments of subjects with primary/remote HCMV infection. The naïve repertoire displayed comparable frequencies of T cells that were reactive with HCMV structural (pp65, gB and the pentamer gHgLpUL128L) and non-structural (IE-1) proteins. Whereas, following natural infection, the majority of effector/memory CD4+ and CD8+ T cells recognized either gB or IE-1, respectively, and pp65. The pattern of T cell reactivity was comparable at early and late stages of infection and in pregnant women with primary HCMV infection transmitting or not transmitting the virus to the fetus. At an early stage of primary infection, about 50% of HCMV-reactive CD4+ T cells were long-term IL-7Rpos memory cells, while 6-12 months later, the frequency of these cells increased to 70%, approaching 100% in remote infections. In contrast, only 10-20% of HCMV-specific CD8+ T cells were long-term memory cells up to 12 months after infection onset, thereafter increasing to 70% in remote infections. Interestingly, a significantly higher frequency of HCMV-specific CD4+ T cells with a long-term IL-7Rpos memory phenotype was observed in non-transmitting compared to transmitting women. These findings indicate that immunodominance in HCMV infection is not predetermined in the naïve compartment, but is the result of virus-host interactions and suggest that prompt control of HCMV infection in pregnancy is associated with the rapid development of long-term IL-7Rpos memory HCMV-specific CD4+ T cells and a low risk of virus transmission to the fetus.

The pentameric complex of human Cytomegalovirus: cell tropism, virus dissemination, immune response and vaccine development.

Between the 1980s and 1990s, three assays were developed for diagnosis of human cytomegalovirus (HCMV) infections: leuko (L)-antigenemia, l-viremia and l-DNAemia, detecting viral protein pp65, infectious virus and viral DNA, respectively, in circulating leukocytes Repeated initial attempts to reproduce the three assays in vitro using laboratory-adapted strains and infected cell cultures were consistently unsuccessful. Results were totally reversed when wild-type HCMV strains were used to infect either fibroblasts or endothelial cells. Careful analysis and sequencing of plaque-purified viruses from recent clinical isolates drew attention to the ULb' region of the HCMV genome. Using bacterial artificial chromosome technology, it was shown by both gain-of-function and loss-of-function experiments that UL131-128 genes are indispensable for virus growth in endothelial cells and virus transfer to leukocytes. In addition, a number of clinical isolates passaged in human fibroblasts had lost both properties (leuko-tropism and endothelial cell-tropism) when displaying a mutation in the UL131-128 locus (referred to as UL128L). In the following years, it was shown that pUL128L was complexed with gH and gL to form the pentameric complex (PC), which is required to infect endothelial, epithelial and myeloid cells. The immune response to PC was studied extensively, particularly its humoral component, showing that the great majority of the neutralizing antibody response is directed to PC. Although anti-HCMV antibodies may act with other mechanisms than mere neutralizing activity, these findings definitely favour their protective activity, thus paving the way to the development of a potentially protective HCMV vaccine.

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.