Replicating viral vectors as HIV vaccines: summary report from the IAVI-sponsored satellite symposium at the AIDS vaccine 2009 conference.

In October 2009, The International AIDS Vaccine Initiative (IAVI) convened a satellite symposium entitled 'Replicating Viral Vectors for use in AIDS Vaccines' at the AIDS Vaccine 2009 Conference in Paris. The purpose of the symposium was to gather together researchers, representatives from regulatory agencies, and vaccine developers to discuss issues related to advancement of replication-competent viral vector- based HIV vaccines into clinical trials. The meeting introduced the rationale for accelerating the development of replicating viral vectors for use as AIDS vaccines. It noted that the EMEA recently published draft guidelines that are an important first step in providing guidance for advancing live viral vectors into clinical development. Presentations included case studies and development challenges for viral vector-based vaccine candidates. These product development challenges included cell substrates used for vaccine manufacturing, the testing needed to assess vaccine safety, conducting clinical trials with live vectors, and assessment of vaccination risk versus benefit. More in depth discussion of risk and benefit highlighted the fact that AIDS vaccine efficacy trials must be conducted in the developing world where HIV incidence is greatest and how inequities in global health dramatically influence the political and social environment in developing countries.

Testing of saliva for antibodies to HIV-1.

To determine whether saliva is a potentially useful sample for screening for HIV infection when serum is not obtainable, saliva and serum samples from 50 HIV-infected and 50 uninfected subjects were tested for antibody to HIV-1 (anti-HIV-1) using a second-generation enzyme-linked immunosorbent assay (ELISA; Abbott) and prototype antibody-capture ELISA (Wellcome). Of saliva specimens from HIV-infected people, six gave negative results on the Abbott and one on the Wellcome assays; all specimens from uninfected people were negative by both assays. Sensitivity for the Abbott assay was therefore 88.0% [95% confidence interval (Cl) 76.2-94.4%], an unacceptable level for screening purposes. Sensitivity for the Wellcome assay was 98% (95% Cl 89.5-99.6%), a more satisfactory level for population screening. Further validation of this technique is necessary, and of methods for collection of saliva specimens in particular.

Detection of anti-HIV immunoglobulin M by particle agglutination following acute HIV infection.

In a study of 23 subjects infected with HIV, a modified particle agglutination assay was used to detect anti-HIV-specific immunoglobulin M (IgM). The presence of anti-HIV IgM was demonstrated in every subject, becoming detectable 1-2 weeks after the onset of acute symptoms, and showing a variable duration of 1-5 weeks. Anti-HIV immunoglobulin G (IgG) developed 1-2 weeks after anti-HIV IgM. Particle agglutination detected the presence of specific antibody up to 7-10 days earlier than the Abbott recombinant or Genetic Systems enzyme immunoassays. In this study, all subjects with acute infection became clearly positive by Western blot within 3 months of the onset of acute symptoms.

Use of a conserved immunodominant epitope of HIV surface glycoprotein gp41 in the detection of early antibodies.

An enzyme immunoassay (EIA) utilizing a synthetic peptide analogue of HIV gp41 (amino acids 579-599, RILAVERYLKDQQLLGIWGCS) as antigen was compared with two commercial assays (Genetic Systems, Abbott ENVACORE) for the ability to detect antibodies in the early stages of infection. Two panels, consisting of 96 sera from 15 people and 140 sera from 44 people seroconverting to HIV, were examined. In the first group the synthetic peptide assay (gp41 EIA) detected antibodies before the Genetic Systems EIA in seven out of 15 people and concurrently in the remaining eight. With the second panel the Abbott ENVACORE assay detected antibodies before the gp41 EIA in two out of 44 people while the gp41 EIA detected antibodies first in six out of 44. In the remaining 36 people antibodies were detected simultaneously by the two tests. The gp41 EIA usually detected anti-HIV antibodies before or concurrently with the two commercial assays examined suggesting that the epitope cluster represented by this peptide is recognized early in infection.

Recognition of envelope and tat protein synthetic peptide analogs by HIV positive sera or plasma.

A series of synthetic peptides corresponding to segments of HIV encoded proteins were selected using criteria described by Welling et al. [(1985) FEBS Lett. 188, 215]. Synthetic peptide analogs to gp120 (2-13), (55-65), gp41 (582-596) (659-670) and tatIII (71-83) were recognized by 41-67% of sera or plasma from individuals known to be infected with HIV on the basis of virus isolation or Western blot screening. The peptide which reacted with most sera or plasma was gp41 (582-596), a conserved region in the transmembrane glycoprotein. An extended peptide analog, gp41 (579-599), tested against the same samples showed almost 100% reactivity, confirming independent studies identifying a highly immunodominant region of gp41. There was an unexpected high prevalence of antibodies (25%) to the tatIII peptide.

The increased risk of fatal liver disease in renal transplant patients who are hepatitis Be antigen and/or HBV DNA positive.

To determine whether active viral replication is associated with increased morbidity and mortality in chronic carriers of hepatitis B virus (HBV) undergoing renal transplantation, we reviewed 23 years of experience at our hospital. Over the period 1966-1989, 42 chronic carriers of hepatitis B surface antigen (HBsAg) received renal transplants, 32 of whom had functioning grafts for 12 months or longer. Stored sera were tested for markers of hepatitis B virus, hepatitis C virus (HCV), and hepatitis delta virus (HDV) infection, and the serologic findings were correlated with clinical and biochemical data. The presence of HBV DNA and/or hepatitis Be antigen (HBeAg) in serum samples collected prior to transplantation was associated with an increased probability of death from liver disease. Whereas 5 of 10 patients in this group died of chronic liver disease, only 1 of 15 patients who were HBV DNA and/or HBeAg negative prior to transplantation died of liver disease. This difference is highly significant (P less than 0.02). No difference in outcome was attributable to age at transplantation, gender, country of birth, or the presence of abnormal hepatic transaminase levels prior to transplantation.

Comparison of electron microscopy, enzyme-linked immunosorbent assay, solid-phase radioimmunoassay, and indirect immunofluorescence for detection of human rotavirus antigen in faeces.

Four techniques were compared for their practicability, speed, and sensitivity for the detection of human rotavirus. Radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) were found to be the most sensitive means of identifying rotavirus, and, once processed, up to 40 specimens could be examined daily. Electron microscopy, although less sensitive than these techniques, had the advantage of being able to detect other viral agents present in faecal extracts. Indirect immunofluorescence failed to detect rotavirus as often as the other three methods. In laboratories where routine examination of faecal specimens from patients with gastroenteritis is required, ELISA and RIA are useful alternatives to electron microscopy.

Comparison of passive haemagglutination and haemagglutination-inhibition techniques for detection of antibodies to rubella virus.

Because of the technical problems and length of time involved in the satisfactory performance of the haemagglutination-inhibition test for detection of rubella-specific antibodies, a commercially available passive haemagglutination (PHA) kit utilising rubella antigen-sensitised human erythrocytes was tested for its suitability for use in diagnostic laboratories. The immune response to acute rubella infections as measured by PHA was considerably delayed compared to the response measured by haemagglutination-inhitition. Titres of rubella-specific antibody only became comparable six months after the infection. The commercially available PHA kits is a useful addition to diagnostic laboratories for the determination of immune status and, in conjunction with the haemagglutination-inhibition test, can be an indicator of recent rubella infection.

Studies on hepatitis B surface antigen and antibody in Nauru. I. Distribution amongst Nauruans.

The age specific prevalence of hepatitis B surface antigen and antibody was studied among a random sample of Nauruans over the age of 10 years. Overall, 79% of subjects showed evidence of past infection with hepatitis B virus with the highest prevalence among young people. While there was no difference in the infection rate between males and females, the carrier rate in males was 50% higher. In both sexes the carrier rate declined with increasing age. Significant differences in carrier rates were detected among people living in different parts of the island.

Studies on hepatitis B surface antigen and antibody in Nauru. II. Distribution amongst Gilbert and Ellice (Tuvalu) islanders.

The age-specific prevalence of hepatitis B surface antigen (HBsAg) and antibody was studied in a random sample of Gilbert and Ellice islanders over the age of 10 years living in Nauru. While approximately 82% of each group showed evidence of past infection with hepatitis B virus (HBV) the carriage rate of HBsAg was significantly lower in the Polynesian Ellice islanders (7.5%) than in the Micronesian Gilbertese (26.3%) and indigenous Nauruans (14.7%). These findings suggest that Micronesian and Polynesian populations may differ in the response to infection with HBV.

Human rotavirus infection in Efate, Vanuatu.

Serum and fecal samples collected from children with gastroenteritis and healthy children and adults living in Efate, Vanuatu (formerly the New Hebrides), were tested for the presence of human rotavirus antigen or antibody by electron microscopy and enzyme-linked immunosorbent assay. Virtually every subject was found to have detectable levels of antibody and age-specific studies showed that primary infections occur early in life. Human rotavirus was demonstrated to be the cause of an outbreak of gastroenteritis among children which occurred between August and September 1980, although it had not been detected in the population in the preceding 13 months. Epidemics of human rotavirus-associated gastroenteritis appear to occur every 2nd year in this population.

Evaluation of enzyme-linked immunosorbent assays: a method of data analysis.

Diagnostic tests are usually evaluated in terms of simple qualitative measures of sensitivity and specificity. When comparing different quantitative assays such as ELISAs, it is often more useful to deal with actual values (sample optical density/cut-off optical density ratio (OD ratio] rather than the qualitative relationship to the cut-off, i.e. positive or negative. This allows for a statistical approach to the questions of sensitivity and specificity. The National HIV Reference Laboratory of Australia has developed an approach for determining statistical estimates of sensitivity and specificity in terms of delta (delta). Delta is defined as the distance of the mean OD ratio of the sample population from the cut-off measured in standard deviation units. This paper discusses the derivation of this measurement and its usefulness when evaluating ELISA tests.

Detection of delta infection using reagents obtained from the serum of patients infected with HBV.

A microtitre solid-phase radioimmunoassay (SPRIA) was developed for the detection of delta antigen (delta Ag) and antibody (anti-delta) using sera from subjects who had been infected with this agent as the source of antigen and antibody. The assay was compared with reference tests, which use delta antigen extracted from liver tissue obtained at autopsy, and found to be equally sensitive and specific.

Hepatitis C antibody testing: problems associated with non-specific binding.

The prevalence of antibodies to hepatitis C virus (anti-HCV) was measured in a number of groups known to be at increased risk of blood-borne viral infections, using an enzyme-linked immunosorbent assay (EIA) based on a nonstructural peptide generated by recombinant DNA technology. The assay was repeatably reactive in 75.6% of men with haemophilia, 61.9% of intravenous drug users, 34.1% of homosexual men who were regular attenders at a gay sauna and 30.8% of prisoners. A lower reactivity was detected in sera collected from female prostitutes (10.4%), patients undergoing maintenance haemodialysis (5.9%), or renal transplantation (6.9%) and patients attending a sexually transmitted diseases clinic (6.2%). We also measured reactivity among inmates of a large institution for the mentally handicapped in which hepatitis B is known to be endemic, and in panels of sera which had been stored for 25-35 years. The test was positive in 41.1% of mentally handicapped patients with Down's syndrome and 7% of subjects with other forms of mental retardation. Similarly some 23% and 20% of sera collected in 1954 and 1964 from patients with a variety of illnesses were found to be reactive. As most diagnostic assays suffer from some degree of non-specificity and confirmatory tests for the anti-HCV assay were not initially available in Australia, we analysed the distribution of optical density (OD) values in the different groups, in an attempt to obtain an insight into the specificity of the results being obtained. Whereas the ODs of sera collected from patients with haemophilia and IVDU had a bimodal pattern, with two well separated sets of results on either side of the cut-off.(ABSTRACT TRUNCATED AT 250 WORDS)

A preliminary evaluation of five HIV antigen detection assays.

Five commercial assays for detecting HIV antigen were evaluated using a panel of 40 coded samples in six laboratories. All assays were capable of detecting HIV-1 antigen (HIV-1 Ag), and are likely to prove useful for monitoring supernatant fluids from a variety of cell cultures. The concentration of HIV-1 protein which the assays detected varied from 25 ng/ml down to 25 pg/ml. Three of the five assays were also able to detect HIV-2 Ag. More extensive evaluations are needed to determine the sensitivity and specificity of these assays on serum samples and body fluids.

Comparison of enzyme immunoassay and reverse transcriptase assay for detection of HIV in culture supernates.

An enzyme immunoassay (EIA) was developed for detection of human immunodeficiency virus antigen (HIV Ag) in tissue culture supernatants. The assay was found to be specific for HIV and cheaper, easier to perform and more sensitive than the generally used reverse transcriptase (RT) assay. Cultures of peripheral blood leucocytes (PBL) from 106 patients with acquired immune deficiency syndrome (AIDS), AIDS related complex (ARC), healthy anti-HIV positive subjects and healthy anti-HIV negative subjects were held for 35 days and the supernatant fluid tested at regular intervals by EIA and RT. Of these 106 cultures, the presence of HIV was detected by EIA in 27 and by RT in 21. While six cultures were positive by EIA alone, none were positive by RT alone; the specificity of the results in the six EIA positive RT negative cultures was confirmed by subculture. In the 21 cultures in which HIV was detected by both techniques, the EIA became positive first on 10 occasions; in the remaining cultures both tests became positive at the same time. The HIV Ag assay reduces the time taken to process specimens and thus increases the efficiency and reduces the cost of isolation procedures.

An evaluation of competitive and second generation ELISA screening tests for antibody to HIV.

Two competitive anti-HIV ELISA screening assays (Behring and Wellcozyme) and two second generation assays using antigens generated by recombinant DNA technology (Abbott) and synthetic peptides (Biochrom) were evaluated against common panels of anti-HIV positive sera and sera known or thought likely to give false positive reactions. The assays were also tested on fresh sequential blood donations. Conventional estimates of sensitivity and specificity did not reveal a significant difference between the assays. Statistical analyses using log10 transformed data to determine delta values (the distance of the mean optical density (OD) ratio from the cut-off measured in standard deviation units) showed the Abbott assays to have the highest probability (greater than 99.99%) of detecting anti-HIV positive samples and the Behring assay as having the highest probability (greater than 99.99%) of correctly identifying anti-HIV negative specimens. The combined data from conventional estimates of sensitivity and specificity and delta values suggests that the Abbott assay is the test of choice for screening purposes.

The detection of hepatitis B surface antigen by radioimmunoassay.

A comparative study was performed to assess the sensitivity and specificity of counterimmunoelectrophoresis (CIEP) and radioimmunoassay (RIA) for the detection of hepatitis B surface antigen. The 8,823 sera examined included selected reference panels and sera collected from populations with low, moderate and high rates of chronic antigen carriage. Overall, hepatitis B surface antigen was detected in 265 sera by CIEP and in 376 by RIA. As well as detecting 46.4% additional positives, the RIA test detected all CIEP-positive sera; i.e., there were no false negative results. However, 150 sera (1.8% of the total tested) gave a positive result by RIA which was not repeatable on retesting. The explanation for this phenomenon appeared to lie in inadequate washing of the antibody-coated tubes.

A solid-phase enzyme-linked immunosorbent assay (ELISA) for detection of antibody to rubella virus.

An enzyme-linked immunosorbent assay was established for detection of antibodies to rubella virus. In this system commercially available rubella antigen was attached to the wells of polystyrene microtitre plates after which sera were added and incubated to allow the formation of antigen-antibody complexes. The presence of bound antibody was detected by adding anti-human globulin coupled to horseradish peroxidase and visually observing the colour change produced after addition of an appropriate substrate. The test was reproducible and simple to perform and had a similar sensitivity to the widely used haemagglutination inhibition system.