Evaluation of glycoprotein B genotypes and load of CMV infecting blood leukocytes on prognosis of AIDS patients

BACKGROUND: Cytomegalovirus (CMV) remains an important pathogen to immunocompromised patients even in the era of HAART. The present study aimed at evaluating the influence of CMV viral load and its gB genotypes on AIDS patients' outcome. METHODS: Blood samples of 101 AIDS patients were collected and tested for HIV load, CD4 - cell count and opportunistic pathogens, including CMV. Semi-nested PCRs were run to detect CMV genome and in the positive samples, gB genotyping and CMV load were established using enzymatic restriction and real time PCR, respectively. All patients were clinically followed for four years. RESULTS: In thirty patients (31%) CMV was detected and all fatal cases (n = 5) occurred in this group of patients (p = 0.007), but only two patients had CMV disease (1.9%). However, viral load was not statistically associated with any analyzed parameter. The most frequently observed CMV genotype was gB2 (45.16%) followed by gB3 (35.48%). gB2 genotype was more frequently found in patients with CD4-cell counts under 200 cells/mm³ (p = 0.0017), and almost all fatal cases (80%) had gB2 genotype. CONCLUSIONS: Our study suggests that CMV and its polymorphisms in biologically relevant genes, such as the gB encoding ORF, may still influence the prognosis and outcome of AIDS patients. The gB2 genotype was associated to patient's bad outcome.

Impact of genetic polymorphisms in cytomegalovirus glycoprotein B on outcomes in solid-organ transplant recipients with cytomegalovirus disease.

BACKGROUND:It is unknown whether specific viral polymorphisms affect in vivo therapeutic response in patients with cytomegalovirus (CMV) disease. Polymorphisms in the CMV glycoprotein B (gB) gene allow discrimination of 4 distinct genotypes (gB1-gB4). We assessed the influence of gB genotypes on the clinical and virologic outcome of CMV disease. METHODS:Solid-organ transplant recipients enrolled in a multicenter trial of CMV disease treatment (VICTOR study) were included in this study. CMV gB genotyping was performed using quantitative real-time polymerase chain reaction at day 0 (start of antiviral therapy). RESULTS:Among 239 patients with CMV disease, the prevalence of gB strain types was 26% for gB1, 10% for gB2, 10% for gB3, and 5% for gB4, whereas mixed infections were present in 49%. Donor-seropositive/recipient-seropositive patients were more likely to have mixed gB infection than donor-seropositive/recipient-seronegative patients (40% vs. 12%; P = .001). Median baseline viral loads were higher and time to viral eradication was longer ( P = .006 and P = .026 , respectively) for mixed infection versus infection with a single genotype. In a multivariate model, mixed gB infection was a significant predictor of failure to eradicate virus by day 21 (mixed vs single genotype; odds ratio, 2.66; 95% confidence interval, 1.31-5.38; P = .007 ) after controlling for baseline viral load, CMV serostatus at baseline, ganciclovir resistance, and antiviral treatment. No effect of gB genotype was seen on virologic or clinical CMV recurrence. CONCLUSIONS:No specific gB genotype appears to confer a specific CMV virulence advantage. However, mixed gB genotype infections are associated with higher viral loads and delayed viral clearance.

Impact of genetic polymorphisms in cytomegalovirus glycoprotein B on outcomes in solid organ transplant recipients with cytomegalovirus disease

Le cytomégalovirus (CMV) est le pathogène viral le plus important après transplantation d'organe. Le risque de développer une maladie à CMV chez les patients transplantés dépend d'une combinaison de facteurs de l'hôte et de facteurs viraux. Par exemple, il est bien établi que le status sérologique à CMV du donneur et du receveur est un facteur de risque très important pour développer une maladie à CMV, notamment chez le sous-groupe de patients donneurs positifs / receveurs négatifs (D+/R-). Par contre, il n'est pas complètement élucidé si des polymorphismes viraux spécifiques peuvent influencer l'évolution en la réponse thérapeutique chez des patients avec une infection à CMV. Nous avons évalué le rôle des différents génotypes de la glycoprotéine Β (gB) du CMV sur l'évolution clinique et virologique de la maladie à CMV chez des patients transplantés d'organe sous traitement antiviral.¦Pour ce faire, nous avons étudié 239 patients transplantés d'organe inclus dans une étude multicentrique évaluant deux médicaments antiviraux utilisés comme traitement de la maladie à CMV. Le génotypage de la gB du CMV a été réalisé en utilisant une PCR quantitative en temps réel au début du traitement antiviral. Les polymorphismes de la gB du CMV permettent la discrimination de quatre génotypes distincts (gBl, gB2, gB3 et gB4). Nous avons défini une infection mixte comme la présence simultanée de plus d'un génotype chez un patient avec maladie à CMV.¦La prévalence des différents génotypes de la gB a été 26% pour la gBl, 10% pour la gB2, 10% pour la gB3, et 5% pour la gB4, alors que les infections mixtes étaient présentes dans 49% des cas. Les patients D+/R+ présentaient plus fréquemment une infection mixte que les patients D+/R- (40% vs 12%, ρ <0.001). Les patients avec une infection mixte présentaient une médiane de la charge virale à CMV plus élevée et un temps d'éradication virale plus long comparé à des patients avec une infection par un génotype unique (p=0.005 et p=0.026, respectivement). Dans un modèle multivarié, les infections mixtes étaient un prédicteur important de l'échec de l'éradication de virus au jour 21 du début du traitement antiviral (rapport de côtes entre l'infection mixte vs. infection par un génotype unique = 2.66, intervalle de confiance à 95%= 1.31 à 5.38, p= 0.007). Aucun effet du génotype gB sur le développement d'une récidive clinique ou virologique de l'infection à CMV a été observé.¦Ces résultats indiquent qu'aucun génotype spécifique de la gB ne semble conférer un avantage de virulence au CMV. Cependant, les infections mixtes avec plusieurs génotypes de la gB sont associées à une charge virale plus élevée et à un retard de l'éradication virale suite au traitement antiviral.

Development of two potential diagnostic monoclonal antibodies against human cytomegalovirus glycoprotein B

Human cytomegalovirus glycoprotein B (gB) represents a target for diagnosis and treatment in view of the role it plays in virus entry and spread. Nevertheless, to our knowledge, rare detection of a gB antigen has been reported in transplant patients and limited information is available about diagnostic gB monoclonal antibodies (mAbs). Our aim was to develop gB mAbs with diagnostic potential. Hydrophilic gB peptides (ST: amino acids 27-40, SH: amino acids 81-94) of favorable immunogenicity were synthesized and used to immunize BALB/c mice. Two mAbs, named ZJU-FH6 and ZJU-FE6, were generated by the hybridoma technique and limited serial dilution and then characterized by indirect ELISA, Western blotting, immunoprecipitation, and immunohistochemical staining. The mAbs displayed high titers of specific binding affinities for the ST and SH synthetic peptides at an mAb dilution of 1:60,000 and 1:240,000, respectively. Western blotting and immunoprecipitation indicated that these mAbs recognized both denatured and native gB of the Towne and AD169 strains. The mAbs, when used as the primary antibody, showed positive staining in cells infected with both Towne and AD169 strains. The mAbs were then tested on patients submitted to allogeneic hematopoietic stem cell transplantation. The gB antigen positivity rates of the patients tested using ZJU-FH6 and ZJU-FE6 were 62.0 and 63.0%, respectively. The gB antigen showed a significant correlation with the level of pp65 antigen in peripheral blood leukocytes. In conclusion, two potential diagnostic gB mAbs were developed and were shown to be capable of recognizing gB in peripheral blood leukocytes in a reliable manner.

Global modulation of cellular transcription by human cytomegalovirus is initiated by viral glycoprotein B

Human cytomegalovirus (HCMV) infection alters the expression of many cellular genes, including IFN-stimulated genes (ISGs) [Zhu, H., Cong, J.-P., Mamtora, G., Gingeras, T. & Shenk, T. (1998) Proc. Natl. Acad. Sci. USA 95, 14470–14475]. By using high-density cDNA microarrays, we show that the HCMV-regulated gene expression profile in fibroblasts does not differ substantially from the response generated by IFN. Furthermore, we identified the specific viral component triggering this response as the envelope glycoprotein B (gB). Cells treated with gB, but not other herpesviral glycoproteins, exhibited the same transcriptional profile as HCMV-infected cells. Thus, the interaction of gB with its as yet unidentified cellular receptor is the principal mechanism by which HCMV alters cellular gene expression early during infection. These findings highlight a pioneering paradigm for the consequences of virus–receptor interactions.

Immunization with DNA vaccines encoding glycoprotein D or glycoprotein B, alone or in combination, induces protective immunity in animal models of herpes simplex virus-2 disease.

DNA vaccines expressing herpes simplex virus type 2 (HSV-2) full-length glycoprotein D (gD), or a truncated form of HSV-2 glycoprotein B (gB) were evaluated for protective efficacy in two experimental models of HSV-2 infection. Intramuscular (i.m.) injection of mice showed that each construction induced neutralizing serum antibodies and protected the mice from lethal HSV-2 infection. Dose-titration studies showed that low doses (< or = 1 microgram) of either DNA construction induced protective immunity, and that a single immunization with the gD construction was effective. The two DNAs were then tested in a low-dosage combination in guinea pigs. Immune sera from DNA-injected animals had antibodies to both gD and gB, and virus neutralizing activity. When challenged by vaginal infection with HSV-2, the DNA-immunized animals were significantly protected from primary genital disease.

Cross-reactive recognition of human and primate cytomegalovirus sequences by human CD4 cytotoxic T lymphocytes specific for glycoprotein B and H

Although the importance of CD4(+) T cell responses to human cytonnegalovirus (HCMV) has recently been recognized in transplant and immunosuppressed patients, the precise specificity and nature of this response has remained largely unresolved. In the present study we have isolated CD4(+) CTL which recognize epitopes from HCMV glycoproteins gB and gH in association with two different HLA-DR antigens, DRA1*0101/DRB1*0701 (DR7) and DRA1*0101/DRB1*1101 (DR11). Comparison of amino acid sequences of HICMV isolates revealed that the gB and gH epitope sequences recognized by human CD4(+) T cells were not only conserved in clinical isolates from HCMV but also in CMV isolates from higher primates (chimpanzee, rhesus and baboon). Interestingly, these epitope sequences from chimpanzee, rhesus and baboon CMV are efficiently recognized by human CD4(+) CTL. More importantly, we show that gB-specific T cells from humans can also efficiently lyse pepticle-sensitized Patr-DR7(+) cells from chimpanzees. These findings suggest that conserved gB and gH epitopes should be considered while designing a prophylactic vaccine against HCMV. In addition, they also provide a functional basis for the conservation of MHC class 11 lineages between humans and Old World primates and open the possibility for the use of such primate models in vaccine development against HCMV.

Clinical correlations of human cytomegalovirus strains and viral load in kidney transplant recipients

Little is known about clinical differences associated with cytomegalovirus (CMV) infection by distinct strains in renal transplant patients. Different clinical pictures may be associated with specific viral genotypes. viral load, as well as host factors. The objective of this study was to identify CMV strains to determine viral load (antigenemia), and their correlation with clinical data in renal transplant recipients. Seventy-one patients were enrolled, comprising 91 samples. After selection, polymorphonuclear cells were used to amplify and sequence the gB region of CMV DNA. The sequences were analyzed to ascertain the frequency of different genotypes. Additionally, the results of this Study showed that the gB coding gene presents a great variability, revealing a variety of patterns: classical gB (1.4%), gB1V (46.4%), classical gB2 (35.2%), gB2V (2.8%), gB3 (1.4%), classical gB4 (4.9%) and gB4V (4.9%). The mean viral load in kidney transplant patient was 75.1 positive cells (1-1000). A higher viral load was observed in patients with genotype 4 infection. Statistically significant differences were detected between gB1 and gB4 (p=0.010), and between gB2 and gB4 (p=0.021). The average numbers of positive cells in relation to clinical presentation were: 34.5 in asymptomatic, 49.5 in CMV associated syndrome and 120.7 in patients with invasive disease (p=0.048). As a group, gB1 was the most frequent strain and revealed a potential risk for developing invasive disease. Viral load also seemed to be important as a marker associated with clinical presentation of the disease. (C) 2008 Elsevier B.V. All rights reserved.

Determination of human cytomegalovirus genetic diversity in different patient populations in Costa Rica

Seroprevalence of HCMV in Costa Rica is greater than 95% in adults; primary infections occur early in life and is the most frequent congenital infection in newborns. The objectives of this study were to determine the genetic variability and genotypes of HCMV gB gene in Costa Rica. Samples were collected from alcoholics, pregnant women, blood donors, AIDS patients, hematology-oncology (HO) children and HCMV isolates from neonates with cytomegalic inclusion disease. A semi-nested PCR system was used to obtain a product of 293-296 bp of the gB gene to be analyzed by Single Stranded Conformational Polymorphism (SSCP) and sequencing to determine the genetic polymorphic pattern and genotypes, respectively. AIDS patients showed the highest polymorphic diversity with 14 different patterns while fifty-six percent of HO children samples showed the same polymorphic pattern, suggesting in this group a possible nosocomial infection. In neonates three genotypes (gB1, gB2 and gB3), were determined while AIDS patients and blood donors only showed one (gB2). Of all samples analyzed only genotypes gB1, 2 and 3 were determined, genotype gB2 was the most frequent (73%) and mixed infections were not detected. The results of the study indicate that SSCP could be an important tool to detect HCMV intra-hospital infections and suggests a need to include additional study populations to better determine the genotype diversity and prevalence.

An assessment of donor-to-recipient transmission patterns of human cytomegalovirus by analysis of viral genomic variants.

BACKGROUND: We studied human cytomegalovirus (CMV) donor-to-recipient transmission patterns in organ transplantation by analyzing genomic variants on the basis of CMV glycoprotein B (gB) genotyping. METHODS: Organ transplant recipients were included in the study if they had CMV viremia, if they had received an organ from a CMV-seropositive donor, and if there was at least 1 other recipient of an organ from the same donor who developed CMV viremia. Genotypes (gB1-4) were determined by real-time polymerase chain reaction. RESULTS: Forty-seven recipients of organs from 21 donors developed CMV viremia. Twenty-three recipients had a pretransplant donor/recipient (D/R) CMV serostatus of D(+)/R(+), and 24 had a serostatus of D(+)/R(-). The prevalences of genotypes in recipients were as follows: for gB1, 51% (n = 24); for gB2, 19% (n = 9); for gB3, 9% (n = 4); for gB4, 0% (n = 0); and for mixed infection, 21% (n = 10). Recipients of an organ from a common donor had infection with CMV of the same gB genotype in 12 (57%) of 21 instances. Concordance between genotypes was higher among seronegative (i.e., D(+)/R(-)) recipients than among seropositive (D(+)/R(+)) recipients, although discordances resulting from the transmission of multiple strains were seen. In seropositive recipients, transmission of multiple strains from the donor could not be differentiated from reactivation of a recipient's own strains. CONCLUSION: Our analysis of strain concordance among recipients of organs from common donors showed that transmission of CMV has complex dynamic patterns. In seropositive recipients, transmission or reactivation of multiple CMV strains is possible.

Physiological parameters to select upland rice genotypes for tolerance to water deficit

Abstract: The objective of this work was to evaluate the feasibility of using physiological parameters for water deficit tolerance, as an auxiliary method for selection of upland rice genotypes. Two experiments - with or without water deficit - were carried out in Porangatu, in the state of Goiás, Brazil; the water deficit experiment received about half of irrigation that was applied to the well-watered experiment. Four genotypes with different tolerance levels to water stress were evaluated. The UPLRI 7, B6144F-MR-6-0-0, and IR80312-6-B-3-2-B genotypes, under water stress conditions, during the day, showed lower stomatal diffusive resistance, higher leaf water potential, and lower leaf temperature than the control. These genotypes showed the highest grain yields under water stress conditions, which were 534, 601, and 636 kg ha-1, respectively, and did not differ significantly among them. They also showed lower drought susceptibility index than the other genotypes. 'BRS Soberana' (susceptible control) was totally unproductive under drought conditions. Leaf temperature is a easy-read parameter correlated to plant-water status, viable for selecting rice genotypes for water deficit tolerance.

Diagnosis of cytomegalovirus infections by qualitative and quantitative PCR in HIV infected patients

A high incidence of cytomegalovirus (CMV) infections is observed in Brazil. These viruses are causatives of significant morbidity and mortality among patients with advanced human immunodeficiency virus (HIV) infection. This work, shows the application of a PCR on determination of CMV load in the buffy coat and plasma. We analyzed the samples of 247 HIV infected patients in order to diagnose CMV infection and disease. We developed a semi-quantitative PCR that amplifies part of the glycoprotein B (gB) gene of CMV. The semi-quantitative PCR was carried out only in positive clinical samples in a qualitative PCR confirmed by a nested-PCR. CD4 lymphocyte count, HIV viral load and CMV disease symptom were correlated with CMV load. CMV genome was detected in the buffy coat of 82 of 237 (34.6%) patients, in 10 of these the CMV load was determined varying between 928 and 332 880 viral copies/mug DNA. None of these 237 patients developed any suggestive manifestation of CMV disease. For the other 10 HIV infected patients selected based on the suspicion of CMV disease, CMV genome was detected in only one case. This patient presented a high CMV load, 8 000 000 copies/mug DNA, and developed a disseminated form of CMV disease including hepatitis and retinitis. Our results were greatly influenced by the impact of the highly active antiretroviral therapy that reduced incidence of CMV viremia and occurrence of CMV disease in the HIV infected patients.

Acquired antibody responses against Plasmodium vivax infection vary with host genotype for duffy antigen receptor for chemokines (DARC).

BACKGROUND: Polymorphism of the Duffy Antigen Receptor for Chemokines (DARC) is associated with susceptibility to and the severity of Plasmodium vivax malaria in humans. P. vivax uses DARC to invade erythrocytes. Individuals lacking DARC are 'resistant' to P. vivax erythrocytic infection. However, susceptibility to P. vivax in DARC+ individuals is reported to vary between specific DARC genotypes. We hypothesized that the natural acquisition of antibodies to P. vivax blood stages may vary with the host genotype and the level of DARC expression. Furthermore, high parasitemia has been reported to effect the acquisition of immunity against pre-erythrocytic parasites. We investigated the correlation between host DARC genotypes and the frequency and magnitude of antibodies against P. vivax erythrocytic stage antigens. METHODOLOGY/FINDINGS: We assessed the frequencies and magnitudes of antibody responses against P. vivax and P. falciparum sporozoite and erythrocytic antigens in Colombian donors from malaria-endemic regions. The frequency and level of naturally-acquired antibodies against the P. vivax erythrocytic antigens merozoite surface protein 1 (PvMSP1) and Duffy binding protein (PvDBP) varied with the host DARC genotypes. Donors with one negative allele (FY*B/FY*Bnull and FY*A/FY*Bnull) were more likely to have anti-PvMSP1 and anti-PvDBP antibodies than those with two positive alleles (FY*B/FY*B and FY*A/FY*B). The lower IgG3 and IgG1 components of the total IgG response may account for the decreased responses to P. vivax erythrocytic antigens with FY*A/FY*B and FY*B/FY*B genotypes. No such association was detected with P. falciparum erythrocytic antigens, which does not use DARC for erythrocyte invasion. CONCLUSION/SIGNIFICANCE: Individuals with higher DARC expression, which is associated with higher susceptibility to P. vivax infection, exhibited low frequencies and magnitudes of P. vivax blood-stage specific antibody responses. This may indicate that one of the primary mechanisms by which P. vivax evades host immunity is through DARC indirectly down-regulating humoral responses against erythrocytic invasion and development.

Disentangling human tolerance and resistance against HIV.

In ecology, "disease tolerance" is defined as an evolutionary strategy of hosts against pathogens, characterized by reduced or absent pathogenesis despite high pathogen load. To our knowledge, tolerance has to date not been quantified and disentangled from host resistance to disease in any clinically relevant human infection. Using data from the Swiss HIV Cohort Study, we investigated if there is variation in tolerance to HIV in humans and if this variation is associated with polymorphisms in the human genome. In particular, we tested for associations between tolerance and alleles of the Human Leukocyte Antigen (HLA) genes, the CC chemokine receptor 5 (CCR5), the age at which individuals were infected, and their sex. We found that HLA-B alleles associated with better HIV control do not confer tolerance. The slower disease progression associated with these alleles can be fully attributed to the extent of viral load reduction in carriers. However, we observed that tolerance significantly varies across HLA-B genotypes with a relative standard deviation of 34%. Furthermore, we found that HLA-B homozygotes are less tolerant than heterozygotes. Lastly, tolerance was observed to decrease with age, resulting in a 1.7-fold difference in disease progression between 20 and 60-y-old individuals with the same viral load. Thus, disease tolerance is a feature of infection with HIV, and the identification of the mechanisms involved may pave the way to a better understanding of pathogenesis.

Insight in the factors associated with the presence of protective antiviral T-cell response.

T cells play a primordial role in antiviral immunity. Virus-specific T-cell responses can be characterized by a number of independent variables. These include the magnitude of the response; the functional quality of the response, i.e. the types of cytokines secreted after stimulation and the proliferative or lytic potential; the tissue distribution of the T cells; the breadth of the response; and the avidity of the response. All of these together constitute the T-cell response to antigen (Ag) and comprise potential variables that may correlate with antiviral protective immunity. Substantial advances have recently been obtained in the characterization of virus-specific T-cell responses. These studies have shown that the quality (in term of functional profile) rather than the quantity of Ag-specific T cells was associated with protection. Recently, the term polyfunctional has been used to define T-cell responses that, in addition to typical effector functions such as secretion of IFN-g, TNF-a and MIP-1b and cytotoxic activity, comprise distinct T-cell populations, also able to secrete IL-2 and retaining Ag-specific proliferation capacity. The term \only effector" defines T-cell responses/ populations able to secrete cytokines such as IFN-g, TNF-a and MIP-1b and endowed with cytotoxic activity but lacking IL-2 and proliferation capacity. Several models of virus infections (HIV-1, cytomegalovirus [CMV], Epstein Barr virus [EBV], influenza [Flu] and Herpes Simplex virus) exclusively differentiated on the basis of Ag exposure and persistence, were investigated: 1) antigen clearance, 2) protracted Ag exposure and persistence and low Ag levels, 3) Ag persistence and high Ag levels, and 4) acute Ag exposure/re-exposure. These analyses have demonstrated that polyfunctional and not \only effector" T-cell responses were associated with protective antiviral immunity. However, the factors and mechanisms governing the generation of functionally distinct T-cell populations remain to be elucidated. Recently, several studies have shown a major influence of HLA genotype in the evolution of HIV and the progression of HIV-associated disease. In particular, certain HLA-B alleles were most closely associated with non-progressive disease and low viral load or disease and had a dominant involvement on the clinical course of HIV-associated diseases. In this study, we have investigated the relationship between HLA restriction and the functional profile of Tcell responses in order to determine whether HLA-B influenced the generation of polyfunctional CD8 T-cell responses. To be able to address this issue, we studied CD8 T-cell responses against HIV-1, CMV, EBV and Flu in 128 subjects. These analyses enabled us to demonstrate that HLA-Arestricted epitopes were mostly associated with \only effector" T-cell responses while, in contrast, polyfunctional CD8 T-cell responses were predominantly driven by virus epitopes restricted by HLA-B alleles. We then characterized eventual differences in the responsiveness of CD8 T-cell populations restricted by different HLA-A and HLA-B alleles. For this purpose, we investigated the T-cell receptor (TCR) avidity for the cognate epitope of polyfunctional and \only effector" CD8 T-cell populations. Our results indicated that overall virus-specific CD8 T-cell populations recognizing virus epitopes restricted by HLA-B alleles were equipped with lower avidity TCR for the cognate epitopes when compared to those recognizing epitopes restricted by HLA-A alleles. In conclusion, these results provide the rationale for the observed protective role of HLA-B genotypes in HIV-1- infection and new insights into the relationship between TCR avidity and functional profile of virus-specific CD8 Tcells.