Ethical considerations related to the provision of care and treatment in vaccine trials.

Ethical principles of beneficence and justice combined with international human rights norms and standards create certain obligations on researchers, sponsors and public health authorities. These include treatment provision for participants enrolled in clinical trials of vaccines, drugs and other new preventive and curative technologies and methods. However, these obligations are poorly defined in practical terms, inconsistently understood or inadequately applied. Vaccine clinical trial designs normally define standards of prevention applicable to the population where the trial is to take place. The present document addresses specifically the setting of standards applicable to care and treatment in vaccine trials. The lack of clear guidance on how to achieve the optimal synergy between the development of new health technologies, on the one hand, and the promotion and protection of ethical and human rights principles, on the other, is a barrier to the progress of health research and therefore to the advancement of public health. The World Health Organization and UNAIDS have engaged in a series of consultations in Africa, the Americas, Asia and Europe to reflect on how this aim could best be achieved. This document highlights the outcome of these consultations. It proposes a structured approach to consensual decision making in the context of the clinical trial of vaccines against such public health challenges as HIV and newly emerging or threatening epidemics. A structured approach involving investigators and sponsors in a consultative process with trial communities and other stakeholders in research will ensure that the needs and legitimate expectations of trial participants are appropriately met, obligations towards them are delivered and, as a result, ethical research is facilitated in the interest of public health.

Analysis of drug resistance-associated mutations in treatment-naïve individuals infected with different genetic forms of HIV-1 circulating in countries of the former Soviet Union.

There are few data on drug resistance-associated mutations in the former Soviet Union since, studies have usually been focused on the env or gag genes for subtype information. This study examines the prevalence and patterns of resistance-associated mutations to reverse transcriptase and protease inhibitors (RTI, PRI) in 278 HIV-1-infected treatment-naïve subjects from countries of Eastern Europe, and defines characteristic polymorphisms of RT and PR sequences in HIV-1 subtype A viruses. Blood samples were collected between 1997 and 2004. Plasma RNA was used for PR-RT amplification by reverse transcription coupled with nested PCR and sequencing. Phylogenetic analysis was done with neighbor-joining trees and bootscanning. Analysis of drug resistance mutations, with Stanford University HIV Drug Resistance Database's algorithm, resulted in an overall prevalence of 12.9% resistance to RTI and 3.9% to PRI. The most frequent substitutions in the RT region were at positions 62 and 236. V77I substitution in PR was found in 47.8% of samples. Polymorphisms in subtype A sequences were identified. This is the first study reporting the prevalence and patterns of both PRI and RTI resistance-associated mutations in naïve HIV-1 infected patients from the former Soviet Union. These data underline the importance of genotypic resistance testing of chronically HIV-1-infected patients before initiating treatment, in order to select the most suitable drug regimen.

Cost effectiveness and delivery study for future HIV vaccines.

Research teams from five countries, Brazil, China, Kenya, Peru and Thailand, have initiated a policy-maker survey on vaccine delivery, cost studies for future HIV vaccination programmes, and associated simulation modeling exercises analysing the relative cost-effectiveness of potential HIV vaccination strategies. The survey assesses challenges and opportunities for future country-level HIV vaccination strategies, providing data on the vaccine characteristics (e.g. vaccine efficacies for susceptibility, infectiousness and disease progression) and vaccination programme strategies to be considered in the cost-effectiveness modeling analyses. The study will provide decision-makers with modeling data on vaccination policy considerations that will assist in developing country-level capacities for future HIV vaccine policy adoption and effective delivery systems, and will help delineate the long-term financial requirements for sustainable HIV vaccination programmes. The WHO-UNAIDS HIV Vaccine Initiative and the collaborating researchers welcome comments or questions from policy makers, health professionals and other stakeholders in the public and private sectors about this effort to help advance policy and capacity related to future potential HIV vaccines.

HIV vaccines: a global perspective.

Twenty years after its recognition, HIV/AIDS has become the most important infectious disease globally and the leading cause of death in Africa. A preventive vaccine represents the best long-term hope for its control. The development of such a vaccine, however, has encountered a number of scientific challenges, including the lack of information on immune correlates of protection, the limitations in our understanding of the relevance of primate protection experiments in relation to vaccine-induced protection in humans, and the significance of genetic and immunologic variability of HIV strains for potential vaccine efficacy. Despite these uncertainties, the first phase I trial of an HIV vaccine was conducted in the United States in 1987. Since then more than 30 candidate vaccines have been tested in over 70 phase I/II clinical trials in both industrialized and developing countries. The first HIV vaccine trial in a developing country was conducted in 1993, six years after the first trial in the United States. Since then eighteen phase I/II trials and one phase III trial have been or are being conducted in developing countries, and additional phase II and III trials are planned to start in 2003. Most of these initial trials have been conducted in Thailand, but more recently trials have been initiated in Africa, Latin America and the Caribbean. Over the past years, the HIV vaccine development effort has followed three major overlapping paradigms. The first "wave" of candidate vaccines aimed at inducing neutralizing antibodies. The second wave focused on stimulation of CD8+ T-cell responses. The current "wave" of HIV vaccine research is aimed at optimizing both humoral and cell-mediated immune responses. The first generation of candidate vaccines (based on the HIV envelope protein) entered phase III efficacy evaluation in 1998, and definitive results from these trials will become available in 2003. Plans to ensure wide access to future HIV vaccines must be developed well in advance.

Analysis of HIV type 1 protease and reverse transcriptase sequences from Venezuela for drug resistance-associated mutations and subtype classification: a UNAIDS study.

We report the first study on prevalence of antiretroviral drug-associated resistance mutations in Venezuela. Protease and reverse transcriptase (RT) coding regions were analyzed in DNA samples obtained from 100 HIV-1-infected individuals. Primary resistance mutations to RT inhibitors were identified in 26% of patients treated with these drugs. Transmission of HIV-1-resistant strains was detected in a drug-naive patient (3%). Primary resistance mutations to protease inhibitors (PIs) were present in 9% of the 44 PI-treated patients and in 1 PI-naive individual. Phylogenetic analysis of these samples has resulted in the most extensive survey, to date, of HIV-1 genetic forms circulating in Venezuela. Ninety-nine samples clustered with subtype B, and 1 individual harbored the first B/F recombinant virus reported in Venezuela, with protease clustering with subtype F and RT with subtype B. In addition, this isolate had a new insertion (Glu-34 duplication) in the protease gene.

Detection and quantification of multiple drug resistance mutations in HIV-1 reverse transcriptase by an oligonucleotide ligation assay.

OBJECTIVES: To develop an assay for the early detection and quantification of minor human immunodeficiency virus-1 populations bearing multiple drug resistance (MDR) mutations. STUDY DESIGN/METHODS: The oligonucleotide ligation assay (OLA) is based on ligation of probe and detector oligonucleotides annealed to a polymerase chain reaction amplicon strand with detection by an enzyme immunoassay. In OLA-MDR, oligonucleotides were designed to detect MDR mutations. The method was validated with wild-type and MDR mutant clones mixed at different proportions. RESULTS: K103N mutants were detected as minor populations (5%-30%) by OLA in 6 of 18 samples from patients treated with nonnucleoside reverse transcription inhibitors and classified as wild type by sequencing. In one patient, the kinetics of the increase of MDR mutants could be followed in sequential samples, with K103N being detected earlier by OLA than by sequencing. Q151M mutants were detected as minor populations (13%-24%) by OLA but not by sequencing in 4 samples. CONCLUSIONS: Oligonucleotide ligation assay MDR exhibits higher sensitivity than sequencing for detection of minor MDR mutant populations.

Identification of highly conserved and broadly cross-reactive HIV type 1 cytotoxic T lymphocyte epitopes as candidate immunogens for inclusion in Mycobacterium bovis BCG-vectored HIV vaccines.

One of the fundamental goals of current strategies to develop an efficacious vaccine for AIDS is the elicitation of cytotoxic T lymphocyte (CTL) reactivities capable of recognizing cells infected with different subtypes of the human immunodeficiency virus type 1 (HIV-1). In efforts to explore new vaccine candidates by the UNAIDS/WHO Vaccine Committee, we review the most recent data concerning CTL epitopes that are conserved among the different HIV-1 subtypes. Moreover, we examine HLA allelic frequencies in several different populations, to determine those that could contribute to the goal of a cumulative phenotype frequency (CP) of at least 80%. By analyzing conserved epitopes in the context of HLA restricting alleles, we define a set of HIV-1 gene regions that may have the greatest potential to induce cross-clade reactive CTLs. The absence of well-defined correlates of immune protection that link CTL epitopes to delayed disease progression and/or prevention of infection does not permit an assignment of rank order of the most relevant component of a candidate vaccine. Thus far, most of the studies conducted in clade B-infected patients to define conserved and immunodominant epitopes indicate gag and pol gene products to be the most conserved among the HIV-1 subtypes. Moreover, anti-Pol and -Gag CTL responses appear to correlate inversely with disease progression, suggesting that they should be among the first choice of antigens to be included in a candidate vaccine construct aimed at induction of broad CTL responses. The impact of a clade B-based vaccine as a worldwide candidate capable of inducing protective immune responses can be determined only after "in vivo" studies. Meanwhile, extensive parallel studies in populations infected with non-clade B HIV-1 subtypes should define the patterns of immunodominant epitopes and HLA for comparison with the data already collected in clade B-infected subjects.

Testing the hypothesis of a recombinant origin of human immunodeficiency virus type 1 subtype E.

The human immunodeficiency virus type 1 (HIV-1) epidemic in Southeast Asia has been largely due to the emergence of clade E (HIV-1E). It has been suggested that HIV-1E is derived from a recombinant lineage of subtype A (HIV-1A) and subtype E, with multiple breakpoints along the E genome. We obtained complete genome sequences of clade E viruses from Thailand (93TH057 and 93TH065) and from the Central African Republic (90CF11697 and 90CF4071), increasing the total number of HIV-1E complete genome sequences available to seven. Phylogenetic analysis of complete genomes showed that subtypes A and E are themselves monophyletic, although together they also form a larger monophyletic group. The apparent phylogenetic incongruence at different regions of the genome that was previously taken as evidence of recombination is shown to be not statistically significant. Furthermore, simulations indicate that bootscanning and pairwise distance results, previously used as evidence for recombination, can be misleading, particularly when there are differences in substitution or evolutionary rates across the genomes of different subtypes. Taken jointly, our analyses suggest that there is inadequate support for the hypothesis that subtype E variants are derived from a recombinant lineage. In contrast, many other HIV strains claimed to have a recombinant origin, including viruses for which only a single parental strain was employed for analysis, do indeed satisfy the statistical criteria we propose. Thus, while intersubtype recombinant HIV strains are indeed circulating, the criteria for assigning a recombinant origin to viral structures should include statistical testing of alternative hypotheses to avoid inappropriate assignments that would obscure the true evolutionary properties of these viruses.

An AB recombinant and its parental HIV type 1 strains in the area of the former Soviet Union: low requirements for sequence identity in recombination. UNAIDS Virus Isolation Network.

In the former Soviet Union (SU) increasing numbers of HIV-1 infections among injecting drug users (IDU) have been reported, especially in the Ukraine. The main subtype transmitted among the IDUs seems to be subtype A, but limited numbers of subtype B cases have also been reported. In Kaliningrad, Russia, an AB recombinant strain was earlier shown to be responsible for the local outbreak. Here we describe the genetic relationship of HIV-1 strains circulating among IDUs in the former SU. For subtype A and the AB recombinant strains nearly full-length genomes were sequenced, and for one subtype B strain the entire envelope gene was cloned. The relationship between the AB recombinant strain and the subtype A and subtype B strains and the mosaic structure of the recombinant was studied by phylogenetic analysis. Ukrainian A and B strains were shown to be the probable parental viruses of the Kaliningrad AB recombinant strain. In the envelope gene the recombination breakpoint could also be precisely mapped to a region of similarity of only 14 base pairs. This suggests that only short stretches of absolute sequence identity may be needed for efficient RNA recombination between HIV-1 subtypes.

HIV-1 diversity in Brazil: genetic, biologic, and immunologic characterization of HIV-1 strains in three potential HIV vaccine evaluation sites. Brazilian Network for HIV Isolation and Characterization.

The Brazilian Network for HIV Isolation and Characterization was established for the surveillance of HIV variability in Brazil. Here, we report characterization of HIV strains and virus-specific immune responses from 35 clinical samples collected from three potential HIV vaccine sites. Three genetic subtypes of HIV-1 were identified by heteroduplex mobility assay (HMA) B (in 82.9% of the samples), F (14.3%), and C (2.9%). Phylogenetic analysis based on the C2V3/env DNA sequence from all 25 specimens examined was 100% concordant with HMA results. Four variants of subtype B with different tetrapeptides at the tip of the V3 loop were found: the GPGR motif (North American), GWGR motif (Brazilian B"), and two minor variants, GFGR and GPGS, as previously detected. No significant association was found between HIV-1 subtypes and the mode of transmission or biologic properties of HIV-1 isolates (derived from 88.6% of the specimens). Only 5 of 16 isolates studied were neutralized by the autologous sera. Consistent with previous results, no relation between viral subtype and peptide enzyme-linked immunosorbent assay (ELISA) seroreactivity or neutralization was evident. This study also demonstrated the effectiveness of the collaborative approach followed by Brazilian scientists when addressing a complex subject such as HIV variability.

Recombinant bacillus Calmette-Guérin as a potential vector for preventive HIV type 1 vaccines.

In August 1997, the World Health Organization (WHO) and the Joint United Nations Programme on HIV/AIDS (UNAIDS) convened an expert working group to discuss current strategies for the development of HIV type 1 vaccines. Based on the recent findings of investigators from Japan's National Institute of Infectious Diseases (NIID) in Tokyo using recombinant bacillus Calmette-Guérin (rBCG) as a potential vectored vaccine for HIV, a recommendation was made that further work in this area is a priority. As a result, the working group reconvened in September 1998 to discuss the progress to date with this vaccine approach, as well as areas of related research to assess the feasibility of a BCG-vectored HIV vaccine. This report summarizes the discussions addressing the available scientific data on the potential use of rBCG as a vector for preventive HIV vaccines, the work necessary to move such candidate vaccines into Phase 1 clinical trials, and recommendations targeted at facilitating the long-term development of rBCG-vectored HIV vaccines.

Epidemic transmission of human immunodeficiency virus in renal dialysis centers in Egypt.

In 1993 an epidemic of human immunodeficiency virus (HIV) infection occurred among 39 patients at 2 renal dialysis centers in Egypt. The centers, private center A (PCA) and university center A (UCA) were visited, HIV-infected patients were interviewed, seroconversion rates at UCA were calculated, and relatedness of HIV strains was determined by sequence analysis; 34 (62%) of 55 patients from UCA and 5 (42%) of 12 patients from PCA were HIV-infected. The HIV seroconversion risk at UCA varied significantly with day and shift of dialysis session. Practices that resulted in sharing of syringes among patients were observed at both centers. The analyzed V3 loop sequences of the HIV strain of 12 outbreak patients were >96% related to each other. V3 loop sequences from each of 8 HIV-infected Egyptians unrelated to the 1993 epidemic were only 76%-89% related to those from outbreak strains. Dialysis patients may be at risk for HIV infection if infection control guidelines are not followed.

Serotyping of HIV type 1 infections: definition, relationship to viral genetic subtypes, and assay evaluation. UNAIDS Network for HIV-1 Isolation and Characterization.

V3 serotyping refers to a system based on binding of antibody in patient sera to V3-loop peptides derived from HIV-1 env genetic subtypes. The V3x serotype represents reactivity of serum from an HIV-1-infected patient (regardless of viral genetic subtype), which reacts preferentially to a V3 peptide derived from the X subtype sequence. We have classified HIV-1 serotypes, determined the relationship between the HIV-1 V3 serotypes and viral genetic subtypes in a large study (n = 125), and evaluated the performance of three different V3 peptide-binding assays. Seven HIV-1 V3 serotypes were identified: A, B, B-Br, B-Th, C, D, and E. Serotypes B-Br and B-Th represent sera that react specifically to peptides derived from Brazilian B (B-Br, GWGR) and Thai B (B-Th, GPGQ) strains. The HIV-1 V3 B, C, and E serotypes correlated closely with their viral env genetic subtypes; 19-26 of 32 B sera (59-79%), 3-4 of 4 C sera (75-100%), and 19-22 of 23 E sera (83-96%) were identified as serotypes B, C, and E, respectively. In contrast, two major V3 serotypes were classified in A sera: A (14-18 of 36 [40-50%]) and C (12-19 of 36 [33-54%]). Similarly, two major V3 serotypes were classified in D sera: B (6-10 of 20 [30-50%]) and D (9-12 of 20 [45-60%]). Serotyping of subtype E sera showed the best concordance with genetic subtypes by all assays. Overall, HIV-1 V3 serotyping produced consistent results among three laboratories. However, HIV-1 V3 serotypes do not distinguish all HIV-1 genetic subtypes. The relative biological significance of the V3 serotypes remains to be elucidated.

HIV preventive vaccines. Progress to date.

The major conceptual problem for HIV vaccine development has been the lack of information on immune responses known to correlate with protection against HIV infection in humans. In this regard, studies on the natural history of HIV infection and AIDS, especially of people with apparent resistance to HIV infection and of patients with HIV infection who have long term survival without disease progression, may provide important information for vaccine development. In addition, a major concern for the development of broadly effective vaccines has been the extensive genetic variability which is characteristic of HIV. In spite of these unknowns, the first generation of HIV candidate vaccines has been developed and evaluated. HIV candidate vaccines based on the subunit recombinant envelope concept (gp120 or gp160) have been shown to protect chimpanzees from HIV infection on challenge, and have now been evaluated in humans in phase I and phase II trials. These products are well tolerated, and capable of inducing neutralising antibodies, but not cytotoxic T lymphocytes. A second vaccine concept, currently in phase I trials, is based on live recombinant vectors, especially using poxvirus vectors followed by boosting with subunit recombinant envelope vaccines. This concept is theoretically very attractive because preliminary data suggest that these vaccines induce both humoral and cell-mediated immunity. However, no published information is available on the ability of live recombinant vector vaccines to protect chimpanzees from HIV infection. The next step in HIV vaccine development is to proceed carefully to expanded phase II and phase III trials to assess the protective efficacy of these candidate vaccines in humans. These trials will be extremely complex from the logistical, scientific and ethical points of view, and will require close collaboration between clinical, basic science and behavioural researchers, national and international organisations, and the pharmaceutical industry.

Molecular characterization of an HIV type 1 isolate from Burundi.

PIP: HIV infection is highly prevalent in Burundi. There are, however, few published reports on the envelope sequence of the prevailing HIV-1 strains in the country. The authors selected an isolate of HIV-1 from Burundi and characterize the envelope glycoprotein gp120 in detail. A sample of venous blood was taken from a 36-year-old HIV-1-positive female volunteer from Bujumbura classified as WHO stage IV, exhibiting clinical AIDS and pulmonary tuberculosis. Nine clones containing complete gp120 genes were derived from the isolate and were designated 91BU009D/1-9. The sequences have been submitted to the Los Alamos Database under accession numbers L35452-L35459 (two clones had a stop codon in their C1 regions and were not studied further). The viral sequences from the coculture closely reflect that circulating in the patient's peripheral blood mononuclear cells. That finding is in striking contrast to the rapid adaptation and evolution of HIV-1 passaged onto human T cell lines. The ability to isolate and culture virus in vitro without the rapid appearance and selection of mutants is important, because it enables relevant observations to be made with regard to the antigenic recognition of the virus by the host. The authors' study found that although 91BU009D clustered with the D subtype, it contained unique sequences noticeably divergent from other characterized proviruses of the D subtype. Further work to investigate and clarify the relationship between genotype and serotype of HIV-1 isolates is of the utmost importance if molecular epidemiology is to be of value in designing an effective AIDS vaccine.^ieng