Haematological values from a Gambian cohort--possible reference range for a West African population.

The objective of this study was to establish haematological reference ranges for the West African subregion using a Gambian cohort. We analysed full blood counts from 1279 subjects aged > or =1 year. Anthropometric and body composition measurements were performed. Haematological mean values, medians and 90% reference values were calculated and related to malnutrition in children and thinness and/or obesity in adults. Haemoglobin (Hb) and mean corpuscular volume (MCV) significantly increased with age (P < 0.00001). There were gender-related changes in Hb from 15 years of age (P = 0.001) and for MCV only in adults (P = 0.0002). Hb was significantly reduced in underweight and stunted children (P = 0.0001 and 0.0002, respectively) but was unaffected by thinness or obesity in adults. White blood cell (WBC) and platelet counts were highest under 5 years and declined significantly with age (P < 0.0001 and 0.0001). While, there were no gender-related differences with WBC, there were higher WBC counts in underweight (P = 0.0001) and stunted (P < 0.0001) children. Adult females had significantly higher mean platelet counts compared with males (P = 0.006). The mean and median values of haematological parameters in The Gambia are similar to other standards but the 90% reference range for each parameter encompasses lower values when compared with Western standards.

FOXP3 gene expression in a tuberculosis case contact study.

Regulatory T lymphocytes (T(regs)) that express FOXP3 are involved in the beneficial attenuation of immunopathology, but are also implicated in down-regulation of protective responses to infection. Their role in tuberculosis (TB) is unknown. We classified 1272 healthy TB contacts according to their tuberculin skin test (TST) and interferon (IFN)-gamma enzyme-linked immunospot (ELISPOT) results and 128 TB cases, and studied the expression of FOXP3 and interleukin (IL)-10 in blood samples. Compared to the uninfected contact group (TST(-), ELISPOT(-)), we observed higher levels of FOXP3 mRNA in blood from TB patients (< 0.001), but IL-10 expression was slightly lower (P = 0.04). In contrast, FOXP3 expression levels were significantly lower (P = 0.001) in the recently infected contacts (TST(+), ELISPOT(+)) but there was no difference for IL-10 (P = 0.74). We hypothesize that during early/subclinical TB, most of which will become latent, FOXP3(+) T(regs) may be sequestered in the lungs, but when TB becomes progressive, FOXP3 reappears at increased levels in the periphery. While these findings do not reveal the role, beneficial or harmful, of T(regs) in TB, they emphasize the probable importance of these cells.

Clinical presentation and outcome of tuberculosis patients infected by M. africanum versus M. tuberculosis.

SETTING: A tuberculosis (TB) case contact study in the Gambia. OBJECTIVE: To test whether Mycobacterium africanum, which has lost around 68 kb compared with M. tuberculosis sensu stricto, causes less severe TB disease. DESIGN: We genotyped mycobacterial isolates and compared clinical and radiological characteristics as well as outcome data of M. africanum-infected TB patients with those infected with M. tuberculosis. RESULTS: Of 317 index cases, 301 had a mycobacterial isolate available, 290 of which had an interpretable spoligotype pattern. Of these, 110 isolates (38%) were M. africanum and 180 (62%) were M. tuberculosis. M. africanum cases had lower body mass indices (17 vs. 17.45 for M. tuberculosis-infected patients, P = 0.029) and their radiographic disease was more extensive (96% vs. 89% had at least moderately severe radiographic changes, P = 0.031). Outcome on treatment was similar (2.8% of human immunodeficiency virus [HIV] negative M. africanum patients died on treatment vs. 3.0% of M. tuberculosis patients, P = 0.95). CONCLUSION: M. africanum causes sputum smear-positive tuberculosis disease that is at least as severe as that caused by M. tuberculosis sensu stricto. Further clinical comparisons may be helpful in smear-negative patients and HIV-TB co-infected patients, and to identify whether there is any difference in time to develop disease.

Identifying ELISPOT and skin test cut-offs for diagnosis of Mycobacterium tuberculosis infection in The Gambia.

SETTING: An urban area, The Gambia. OBJECTIVE: To identify ELISPOT and PPD skin test cut-offs, targeting sensitivity and specificity equivalence. DESIGN: Tuberculosis cases >5 years of age and their household contacts underwent ELISPOT, HIV and PPD skin tests. Cases and contacts sleeping in a different house were used to estimate sensitivity and specificity, providing two planes for estimating cut-offs. Specificity was adjusted for infection from previous exposure using a multivariate discrimination algorithm. RESULTS: The point on the line of intersection of the planes that maximised sensitivity and specificity equivalence occurred at 4 spots (95% confidence interval [CI] 3.5-5, multiplier=0 ) for CFP-10 and 5.5 spots (4.5-8, multiplier=0 for ESAT-6), yielding a sensitivity and specificity of 76% for both antigens. Combining ESAT-6 and CFP-10 using an 'or' statement yielded a maximum equivalence sensitivity and specificity of 76.5% at 6 spots for ESAT-6 and 11.5 spots for CFP-10. For the PPD skin test sensitivity and specificity, an equivalence of 78% occurred at 11 mm induration (9-13 mm). CONCLUSION: An ELISPOT cut-off for ESAT-6 or CFP-10 could be set at 4-8 spot forming units (20-40 spots per million), with little benefit from combining the results. A cut-off of 9-13 mm for the PPD skin test is reasonable when comparing with the ELISPOT.

Design and implementation of relational databases relevant to the diverse needs of a tuberculosis case contact study in the Gambia.

The data requirements of a large multidisciplinary tuberculosis case contact study are complex. We describe an ACCESS-based relational database system that meets our rigorous requirements for data entry and validation, while being user-friendly, flexible, exportable, and easy to install on a network or stand alone system. This includes the development of a double data entry package for epidemiology and laboratory data, semi-automated entry of ELISPOT data directly from the plate reader, and a suite of new programmes for the manipulation and integration of flow cytometry data. The double entered epidemiology and immunology databases are combined into a separate database, providing a near-real-time analysis of immuno-epidemiological data, allowing important trends to be identified early and major decisions about the study to be made and acted on. This dynamic data management model is portable and can easily be applied to other studies.

Respiratory viruses, symptoms, and inflammatory markers in acute exacerbations and stable chronic obstructive pulmonary disease.

The effects of respiratory viral infection on the time course of chronic obstructive pulmonary disease (COPD) exacerbation were examined by monitoring changes in systemic inflammatory markers in stable COPD and at exacerbation. Eighty-three patients with COPD (mean [SD] age, 66.6 [7.1] yr, FEV(1), 1.06 [0.61] L) recorded daily peak expiratory flow rate and any increases in respiratory symptoms. Nasal samples and blood were taken for respiratory virus detection by culture, polymerase chain reaction, and serology, and plasma fibrinogen and serum interleukin-6 (IL-6) were determined at stable baseline and exacerbation. Sixty-four percent of exacerbations were associated with a cold occurring up to 18 d before exacerbation. Seventy-seven viruses (39 [58.2%] rhinoviruses) were detected in 66 (39.2%) of 168 COPD exacerbations in 53 (64%) patients. Viral exacerbations were associated with frequent exacerbators, colds with increased dyspnea, a higher total symptom count at presentation, a longer median symptom recovery period of 13 d, and a tendency toward higher plasma fibrinogen and serum IL-6 levels. Non-respiratory syncytial virus (RSV) respiratory viruses were detected in 11 (16%), and RSV in 16 (23.5%), of 68 stable COPD patients, with RSV detection associated with higher inflammatory marker levels. Respiratory virus infections are associated with more severe and frequent exacerbations, and may cause chronic infection in COPD. Prevention and early treatment of viral infections may lead to a decreased exacerbation frequency and morbidity associated with COPD.

Nosocomial spread of viral disease.

Viruses are important causes of nosocomial infection, but the fact that hospital outbreaks often result from introduction(s) from community-based epidemics, together with the need to initiate specific laboratory testing, means that there are usually insufficient data to allow the monitoring of trends in incidences. The most important defenses against nosocomial transmission of viruses are detailed and continuing education of staff and strict adherence to infection control policies. Protocols must be available to assist in the management of patients with suspected or confirmed viral infection in the health care setting. In this review, we present details on general measures to prevent the spread of viral infection in hospitals and other health care environments. These include principles of accommodation of infected patients and approaches to good hygiene and patient management. They provide detail on individual viral diseases accompanied in each case with specific information on control of the infection and, where appropriate, details of preventive and therapeutic measures. The important areas of nosocomial infection due to blood-borne viruses have been extensively reviewed previously and are summarized here briefly, with citation of selected review articles. Human prion diseases, which present management problems very different from those of viral infection, are not included.

Sputum and plasma endothelin-1 levels in exacerbations of chronic obstructive pulmonary disease.

BACKGROUND: Endothelin (ET)-l is a bronchoconstrictor peptide produced in the airways. It has been implicated in the pathogenesis of asthma and virally mediated airway inflammation and may play a role in exacerbations of chronic obstructive pulmonary disease (COPD). METHODS: Seventy one patients with COPD were followed prospectively and sampled for plasma and sputum ET-1 levels when stable and during an exacerbation. Sputum was also examined for cytokines, human rhinovirus, and Chlamydia pneumoniae. RESULTS: Plasma ET-1 levels were available for 67 patients with stable COPD (mean (SD) 0.58 (0.31) pg/ml); 28 pairs of stable-exacerbation plasma samples had a mean stable ET-1 level of 0.54 (0.30) pg/ml rising to 0.67 (0.35) pg/ml at exacerbation (mean difference 0.13, 95% confidence interval (CI) 0.04 to 0.21, p = 0.004). Plasma ET-1 levels in the 67 patients with stable COPD were inversely correlated with baseline forced expiratory volume in one second (FEV(1); r = -0. 29, p = 0.022) and forced vital capacity (FVC; r = -0.38, p = 0.002). The change in plasma ET-1 levels during an exacerbation correlated with the change in oxygen saturation (SaO(2); r = -0.41, p = 0.036). In 14 stable-exacerbation pairs of sputum samples median stable ET-1 levels were 5.37 (0.97-21.95) pg/ml rising to 34.68 (13.77-51.95) pg/ml during an exacerbation (mean difference 25.14, 95% CI 3.77 to 46.51, p = 0.028). This increase in sputum ET-1 levels correlated with the increase in plasma ET-1 levels (r = 0.917, p = 0.001) and sputum interleukin (IL)-6 levels (r = 0.718, p = 0.013). CONCLUSIONS: Sputum levels of ET-1 rise in COPD patients during an exacerbation and this is reflected by a smaller rise in plasma ET-1 levels. ET-1 may have a role in mediating airway inflammatory changes during exacerbations of COPD.

Detection of rhinovirus in induced sputum at exacerbation of chronic obstructive pulmonary disease.

Common colds are associated with exacerbations of chronic obstructive pulmonary disease (COPD). However, the role of the common cold virus (human rhinovirus) in the production of symptoms and lower airway inflammation at COPD exacerbation is unknown. Thirty three patients with moderate-to-severe COPD were seen at baseline, when the number of chest infections in the previous year was noted, and acutely at COPD exacerbation. Within 48 h after the onset of the exacerbation and at baseline, nasal aspirates and induced sputum were taken for rhinovirus reverse transcriptase polymerase chain reaction (RT-PCR) analysis and determination of cytokine levels. Symptoms, recorded on diary cards, were noted and forced expiratory volume in one second (FEV1) and forced vital capacity (FVC) measured. At exacerbation, mean FEV1 and FVC fell significantly from baseline (p<0.001). Ten of 43 exacerbations were associated with rhinovirus infection, detected in induced sputum. In four of these, nasopharyngeal samples contained no detectable rhinovirus. All baseline samples were negative for rhinovirus. The simultaneous presence of increased nasal discharge/nasal congestion (in 26 of the 43 exacerbations) and increased sputum (29 exacerbations) was strongly associated with the presence of rhinovirus (odds ratio 6.15; p=0.036). Total symptom scores were greater for rhinovirus as compared to nonrhinovirus exacerbations (p=0.039). Median baseline sputum interleukin-6 levels rose from 90.2 to 140.3 pg x mL(-1) at exacerbation (p=0.005); the change was greater in the presence of rhinovirus infection (p=0.008). Rhinovirus infection can be detected at chronic obstructive pulmonary disease exacerbation. This is associated with elevation of lower airway interleukin-6 levels, which may mediate lower airway symptom expression during chronic obstructive pulmonary disease exacerbations.

Acute exacerbations of chronic obstructive pulmonary disease are accompanied by elevations of plasma fibrinogen and serum IL-6 levels.

BACKGROUND: Respiratory tract infections may acutely increase risk from coronary heart disease (CHD), though the mechanisms have not been defined. Patients with chronic obstructive pulmonary disease (COPD) are prone to repeated exacerbations that are often associated with respiratory infections. These patients also have increased cardiovascular morbidity and mortality. We hypothesized that transient acute increases in plasma fibrinogen, an independent risk factor for CHD, could occur at COPD exacerbation (mediated through a rise in IL6) and thereby provide a mechanism linking respiratory infection to risk of coronary heart disease. METHODS: 93 COPD patients [mean (SD) age 66.8 (8.1) years] were followed regularly over one year, with daily diary card monitoring of respiratory symptoms and peak expiratory flow rate (PEFR); 67 patients [mean FEV1 1.06 (0.44) l, FVC 2.43 (0.79) l] were seen during 120 exacerbations. At each visit spirometry was measured and blood samples taken for plasma fibrinogen and Interleukin-6 (IL-6) levels. RESULT: At baseline, the mean (SD) plasma fibrinogen was elevated at 3.9 (0.67) g/l in the 67 patients with exacerbations during the study and the median (IQR) IL-6 at 4.3 (2.4 to 6.8) pg/ml. Plasma fibrinogen increased by 0.36 (0.74) g/l at exacerbation (p <0.001). with IL-6 levels rising by 1.10 (-2.73 to 6.95) pg/ml (p = 0.008). There was a relation between the changes in fibrinogen at exacerbation and IL-6 levels (r = 0.348, p <0.001). Multiple regression revealed significantly greater rises in fibrinogen when exacerbations were associated with purulent sputum (b = 0.34 g/l; p = 0.03), increased cough (b = 0.31 g/l, p = 0.019) and symptomatic colds (b = 0.228; p = 0.024). CONCLUSIONS: Plasma fibrinogen levels were elevated in stable patients with COPD and may contribute to the increased cardiovascular morbidity and mortality in these patients. COPD exacerbations increased serum IL-6 levels, leading to a rise in plasma fibrinogen. Thus acute rather than chronic infection may have a role in predisposing to coronary heart disease or stroke.

Time course and recovery of exacerbations in patients with chronic obstructive pulmonary disease.

Although exacerbations of chronic obstructive pulmonary disease (COPD) are associated with symptomatic and physiological deterioration, little is known of the time course and duration of these changes. We have studied symptoms and lung function changes associated with COPD exacerbations to determine factors affecting recovery from exacerbation. A cohort of 101 patients with moderate to severe COPD (mean FEV(1) 41.9% predicted) were studied over a period of 2.5 yr and regularly followed when stable and during 504 exacerbations. Patients recorded daily morning peak expiratory flow rate (PEFR) and changes in respiratory symptoms on diary cards. A subgroup of 34 patients also recorded daily spirometry. Exacerbations were defined by major symptoms (increased dyspnea, increased sputum purulence, increased sputum volume) and minor symptoms. Before onset of exacerbation there was deterioration in the symptoms of dyspnea, sore throat, cough, and symptoms of a common cold (all p < 0.05), but not lung function. Larger falls in PEFR were associated with symptoms of increased dyspnea (p = 0.014), colds (p = 0.047), or increased wheeze (p = 0.009) at exacerbation. Median recovery times were 6 (interquartile range [IQR] 1 to 14) d for PEFR and 7 (IQR 4 to 14) d for daily total symptom score. Recovery of PEFR to baseline values was complete in only 75.2% of exacerbations at 35 d, whereas in 7.1% of exacerbations at 91 d PEFR recovery had not occurred. In the 404 exacerbations where recovery of PEFR to baseline values was complete at 91 d, increased dyspnea and colds at onset of exacerbation were associated with prolonged recovery times (p < 0.001 in both cases). Symptom changes during exacerbation do not closely reflect those of lung function, but their increase may predict exacerbation, with dyspnea or colds characterizing the more severe. Recovery is incomplete in a significant proportion of COPD exacerbations.

Needlestick injury in clothing industry workers and the risks of blood-borne infection.

This paper identifies the hazard of a hollow needle device used extensively in the clothing industry and assesses the risk of transmission for HIV, Hepatitis B and Hepatitis C. A substantial risk of transmission is suggested and measures have been advised for its control. Occupational Health Physicians are advised to be aware of hollow needles in other industrial processes and where risks of cross-infection exist, the same safety considerations should be applied as in clinical medicine and veterinary work to avoid needlestick injuries. Needle sharing must be avoided.

Ability of yeast Ty-VLPs (virus-like particles) containing varicella-zoster virus (VZV)gE and assembly protein fragments to induce in vitro proliferation of human lymphocytes from VZV immune patients.

Yeast Ty virus-like particles (VLPs) containing viral protein inserts have previously been shown to be potent immunogens, inducing both humoral and cell mediated immunity (CMI). The antigenicity of hybrid VLPs containing fragments of the varicella-zoster virus (VZV) gE protein or the assembly protein (AP) was assessed by lymphocyte proliferation. Peripheral blood mononuclear cells (PBMCs) from patients with a recent natural VZV infection were stimulated in vitro with VZV-VLPs together with control antigens. PBMC samples from both varicella (85%) and zoster (75%) patients proliferated in responses to at least one of the gE VZV-VLPs. As reported for the first time, VZV specific lymphocyte responses were also identified towards the VZV AP in two varicella and two zoster patient samples. The results demonstrate specific CMI recognition of the VZV gE fragments tested and the VZV AP delivered in the form of recombinant Ty-VLPs, and highlights their potential use as a recombinant antigen delivery system for vaccination.

Effect of temperature on lung function and symptoms in chronic obstructive pulmonary disease.

The present study investigated whether falls in environmental temperature increase morbidity from chronic obstructive pulmonary disease (COPD). Daily lung function and symptom data were collected over 12 months from 76 COPD patients living in East London and related to outdoor and bedroom temperature. Questionnaires were administered which asked primarily about the nature of night-time heating. A fall in outdoor or bedroom temperature was associated with increased frequency of exacerbation, and decline in lung function, irrespective of whether periods of exacerbation were excluded. Forced expiratory volume in one second (FEV1) and forced vital capacity (FVC) fell markedly by a median of 45 mL (95% percentile range: -113-229 mL) and 74 mL (-454-991 mL), respectively, between the warmest and coolest week of the study. The questionnaire revealed that 10% had bedrooms <13 degrees C for 25% of the year, possibly because only 21% heated their bedrooms and 48% kept their windows open in November. Temperature-related reduction in lung function, and increase in exacerbations may contribute to the high level of cold-related morbidity from chronic obstructive pulmonary disease.

Molecular biology of varicella-zoster virus. A review prepared for the UK Advisory Group on Chickenpox.

Varicella-zoster virus (human herpesvirus 3; VZV) is one of eight herpes viruses that routinely infect humans. It is classified as a member of the genus Varicellovirus, subfamily Alphaherpesvirinae, family Herpesviridae. Of the other human herpes viruses it is most closely related to the herpes simplex viruses (also members of the Alphalerpesvirinae). Like all herpes viruses, the virus has a large double-stranded DNA genome within an icosahedral nucleocapsid. This is surrounded by a proteinaceous tegument and a trilaminar membrane derived from host-cell membranes into which the viral glycoproteins are inserted. The structure of the virion is summarized in Fig. 1.

Effect of exacerbation on quality of life in patients with chronic obstructive pulmonary disease.

Exacerbations occur commonly in patients with moderate or severe chronic obstructive pulmonary disease (COPD) but factors affecting their severity and frequency or effects on quality of life are unknown. We measured daily peak expiratory flow rate (PEFR) and daily respiratory symptoms for 1 yr in 70 COPD patients (52 male, 18 female, mean age [+/- SD] 67.5 +/- 8.3 yr, FEV1 1.06 +/- 0.45 L, FVC 2.48 +/- 0.82 L, FEV1/FVC 44 +/- 15%, FEV1 reversibility 6.7 +/- 9.1%, PaO2 8.8 +/- 1.1 kPa). Quality of life was measured by the St. George's Respiratory Questionnaire (SGRQ). Exacerbations (E) were assessed at acute visit (reported exacerbation) or from diary card data each month (unreported exacerbation). In 61 (87%) patients there were 190 exacerbations (median 3; range, 1 to 8) of which 93 (51%) were reported. There were no differences in major symptoms (increase in dyspnea, sputum volume, or purulence) or physiological parameters between reported and unreported exacerbations. At exacerbation, median peak flow fell by an average of 6.6 L/min (p = 0.0003). Using the median number of exacerbations as the cutoff point, patients were classified as infrequent exacerbators (E = 0 to 2) or frequent exacerbators (E = 3 to 8). The SGRQ Total and component scores were significantly worse in the group that had frequent exacerbations: SGRQ Total score (mean difference = 14.8, p < 0.001), Symptoms (23.1, p < 0.001), Activities (12.2, p = 0.003), Impacts (13.9, p = 0.002). However there was no difference between frequent and infrequent exacerbators in the fall in peak flow at exacerbation. Factors predictive of frequent exacerbations were daily cough (p = 0.018), daily wheeze (p = 0.011), and daily cough and sputum (p = 0.009) and frequent exacerbations in the previous year (p = 0.001). These findings suggest that patient quality of life is related to COPD exacerbation frequency.

DNA viruses in the pathogenesis of sporadic chronic idiopathic intestinal pseudo-obstruction.

BACKGROUND: Hereditary forms of chronic idiopathic intestinal pseudo-obstruction (CIIP) are well described but the aetiology of most cases of sporadic CIIP is unknown. AIM: To determines whether herpes viruses can persist in the gastrointestinal tract, thereby implicating them in the pathogenesis of CIIP. METHODS: Twenty one specimens of small and large intestine from 13 patients with CIIP (eight visceral myopathy, three visceral neuropathy, two undifferentiated), and 12 patients operated on for colorectal cancer (controls) were examined for evidence of Herpesvirus DNA (cytomegalovirus, Epstein-Barr virus (EBV), herpes simplex virus type 1, and varicella zoster virus) by nested polymerase chain reaction (PCR) and in situ DNA hybridisation (ISH) to localise signal to the muscularis propria or myenteric plexus. RESULTS: Screening with nested PCR produced three patients with positive results. One patient with an inflammatory visceral neuropathy had EBV detected in the small intestine by PCR, and ISH demonstrated localisation to neurones in the myenteric plexus. A patient with a visceral myopathy had EBV DNA in both the small and large intestine; and one patient with a visceral neuropathy had small intestine positive for CMV DNA (both negative by ISH). No control tissue was positive for any virus. CONCLUSIONS: In individual patients there appears to be evidence linking a viral aetiology to sporadic CIIP. The role of neurotropic viruses in acute and chronic motility disturbances needs further study.

HIV antibody testing: Genito-Urinary Clinic or additional site same-day testing service.

It has been suggested that different groups of individuals, possibly those at greater risk, may choose to attend anonymous HIV services not attached to Genito-Urinary Medicine Clinics. This paper reports the results of a comparative study of documented sociodemographic profile and risk category for individuals having HIV antibody tests at a Genito-Urinary Medicine Clinic and at an additional site same-day HIV antibody testing service not attached to a Genito-Urinary Medicine Clinic but within the same hospital. A retrospective case record analysis for 12 months from 1.10.93 to 30.9.94 was performed. During the 12-month study period 954 people had HIV testing at the additional site HIV antibody testing service and 803 had HIV antibody testing at the Genito-Urinary Medicine Clinic. Of those attending the additional site testing service 218 (22.9%) were homosexual/bisexual males as compared with 128 (15.9%) of the Genito-Urinary Medicine Clinic attendees (p < 0.01). A total of 22 (2.3%) of the individuals attending the additional site service gave a history of sexual contact in Africa as compared with 52 (65%) attending the Genito-Urinary Medicine Clinic (p < 0.001). There was no significant difference in the gender or age of those having HIV antibody tests at the two locations (p = 0.82 and p = 0.39 respectively). Four hundred and seventeen (52%) of the tests performed at the Genito-Urinary Medicine Clinic were performed on those who had not already decided to have a test before attending the clinic. Of those individuals who had decided to have a test before attending, 2.5 times as many attended the Same-Day Service as attended the Genito-Urinary Medicine Clinic (954 additional site attenders compared with 384 Genito-Urinary Medicine Clinic attenders). The results suggest that to optimize patient choice and benefit from a HIV antibody testing service this should be available at both Genito-Urinary Medicine Clinics and at additional site testing services.

Induction of neutralizing antibody and T-cell responses to varicella-zoster virus (VZV) using Ty-virus-like particles carrying fragments of glycoprotein E (gE).

During infection with Varicella-zoster virus (VZV), the envelope proteins are highly immunogenic and glycoprotein E (gE) is one of the most abundant and antigenic. We have previously identified the immunodominant regions of gE and mapped the B-cell epitopes. In this study, we have evaluated the immunogenicity of recombinant hybrid Ty-virus-like particles (VLPs) carrying amino acids (1-134) or (101-161) of gE which contain the immunodominant sequences. VZV-specific antibodies were detected by ELISA in sera from mice and guinea pigs immunized with either gE(1-134)-VLPs or gE (101-161)-VLPs. The dominant B-cell epitopes, mapped by pepscan analysis of the sera, were found in peptides spanning amino acids 41-60, 56-75, 101-120, 116-135, 131-150 and 141-161. These sera also showed neutralizing activity against VZV in vitro. Epitopes recognized by neutralizing MAbs were mapped to both gE sequences (3B3 MAb recognizing amino acids 141-161 and IFB9 MAb recognizing amino acids 71-90). Lymphocyte proliferative responses to VZV were detected in four different mouse strains immunized with either gE(1-134)-VLPs or gE(101-134)-VLPs in alum. All mouse strains immunized with gE(1-134)-VLPs recognized epitopes in amino acids 11-30 and 71-90 and all those immunized with gE(101-161)-VLPs recognized epitopes in amino acids 91-110 and 106-125. These results indicate that VLPs carrying these gE sequences can prime potent humoral and cellular anti-VZV responses in small animals and warrant further investigation as potential vaccine candidates against varicella-zoster infections.

Cloning, expression, and immunogenicity of the assembly protein of varicella-zoster virus and detection of the products of open reading frame 33.

Herpesviruses produce assembly proteins (AP) that act as scaffolding proteins for the assembly of the viral capsids. The products of the assemblin gene, which encodes both maturational protease and AP, have been established for herpes simplex virus type 1 (HSV-1) and human cytomegalovirus (CMV). We cloned an inframe ORF (encoding amino acids 304-605), found within the ORF 33 assemblin gene of VZV, into a yeast expression vector. The 34-kDa AP was expressed as a fusion protein with the particle-forming Ty p1 protein, resulting in high-level production of hybrid AP-virus-like particles (AP-VLPs). When AP-VLPs were injected into mice and rabbits, antibodies were produced that reacted with, but that did not neutralise, native VZV. Three of four inbred strains of mice immunised with AP-VLPs produced a VZV-specific T-cell response. The mouse and rabbit sera reacted with six bands on native VZV by Western blot analysis. The dominant bands were found at 34 and 38 kDa. Bands were also seen at 66, 63, 41, and 31 kDa. The 38-kDa protein may represent the mature AP derived from the 41-kDa precursor AP, itself the release product from the full-length 66-kDa assemblin. The 34-kDa protein probably represents the product of the inframe co-translational gene within ORF 33 encoding amino acids 304-605. The genetic organisation and proteolytic maturation of VZV assemblin are, therefore, analogous to those of other herpesviruses.