Ronald C. Desrosiers

Importance of the nef gene for maintenance of high virus loads and for development of AIDS

When rhesus monkeys were infected with a form of cloned SIVmac239 having a premature stop signal at the 93rd codon of nef, revertants with a coding codon at this position quickly and universally came to predominate in the infected animals. This suggests that there are strong selective forces for open functional forms of nef in vivo. Although deletion of nef sequences had no detectable effect on virus replication in cultured cells, deletion of nef sequences dramatically altered the properties of virus in infected rhesus monkeys. Our results indicate that nef is required for maintaining high virus loads during the course of persistent infection in vivo and for full pathologic potential. Thus, nef should become a target for antiviral drug development. Furthermore, the properties of virus with a deletion in nef suggest a means for making live-attenuated strains of virus for experimental vaccine testing.

HIV vaccine design and the neutralizing antibody problem

Eliciting broadly neutralizing antibodies to human immunodeficiency virus could bring closer the goal of a successful AIDS vaccine. Here the International AIDS Vaccine Initiative Neutralizing Antibody Consortium discusses current approaches to overcome the problems faced.

Tat-specific cytotoxic T lymphocytes select for SIV escape variants during resolution of primary viraemia.

Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections are characterized by early peaks of viraemia that decline as strong cellular immune responses develop. Although it has been shown that virus-specific CD8-positive cytotoxic T lymphocytes (CTLs) exert selective pressure during HIV and SIV infection, the data have been controversial. Here we show that Tat-specific CD8-positive T-lymphocyte responses select for new viral escape variants during the acute phase of infection. We sequenced the entire virus immediately after the acute phase, and found that amino-acid replacements accumulated primarily in Tat CTL epitopes. This implies that Tat-specific CTLs may be significantly involved in controlling wild-type virus replication, and suggests that responses against viral proteins that are expressed early during the viral life cycle might be attractive targets for HIV vaccine development.

The familyHerpesviridae: an update

More than 10 years have elapsed since the first report of the International Committee for the Taxonomy of Viruses (ICTV) Herpesvirus Study Group [-187]. In that publication the Study Group designated Herpesviridae as the name of the herpesvirus family, erected three subfamilies, the Alphaherpesvirinae, Betaherpesvirinae, and Gammaherpesvirinae, and provided guidelines for the classification of herpesviruses into these subfamilies. Since that publication, the ICTV has approved the recommendation of previous Herpesvirus Study Groups to erect several genera. More important from the point of view of taxonomy and classification of herpesviruses, considerable amounts of new information regarding herpesviruses have accumulated in the past 10 years. Thus, (a) a large number of new herpesviruses has been discovered, (b) a few herpesviruses turned out to be contaminants, most likely derived from calf serum containing a bovine herpesvirus, (c) several herpesvirus genomes have been sequenced, (d) many highly conserved genes have been identified both within the family and in the individual subfamilies, and (e) we have a much better understanding at least of the problems facing the task of classifying herpesviruses. This paper updates the 1981 report. Definition

Epstein–Barr Virus MicroRNAs Are Evolutionarily Conserved and Differentially Expressed

The pathogenic lymphocryptovirus Epstein–Barr virus (EBV) is shown to express at least 17 distinct microRNAs (miRNAs) in latently infected cells. These are arranged in two clusters: 14 miRNAs are located in the introns of the viral BART gene while three are located adjacent to BHRF1. The BART miRNAs are expressed at high levels in latently infected epithelial cells and at lower, albeit detectable, levels in B cells. In contrast to the tissue-specific expression pattern of the BART miRNAs, the BHRF1 miRNAs are found at high levels in B cells undergoing stage III latency but are essentially undetectable in B cells or epithelial cells undergoing stage I or II latency. Induction of lytic EBV replication was found to enhance the expression of many, but not all, of these viral miRNAs. Rhesus lymphocryptovirus, which is separated from EBV by ≥13 million years of evolution, expresses at least 16 distinct miRNAs, seven of which are closely related to EBV miRNAs. Thus, lymphocryptovirus miRNAs are under positive selection and are likely to play important roles in the viral life cycle. Moreover, the differential regulation of EBV miRNA expression implies distinct roles during infection of different human tissues.

Reversion of CTL escape-variant immunodeficiency viruses in vivo

Engendering cytotoxic T-lymphocyte (CTL) responses is likely to be an important goal of HIV vaccines. However, CTLs select for viral variants that escape immune detection. Maintenance of such escape variants in human populations could pose an obstacle to HIV vaccine development. We first observed that escape mutations in a heterogeneous simian immunodeficiency virus (SIV) isolate were lost upon passage to new animals. We therefore infected macaques with a cloned SIV bearing escape mutations in three immunodominant CTL epitopes, and followed viral evolution after infection. Here we show that each mutant epitope sequence continued to evolve in vivo, often re-establishing the original, CTL-susceptible sequence. We conclude that escape from CTL responses may exact a cost to viral fitness. In the absence of selective pressure upon transmission to new hosts, these original escape mutations can be lost. This suggests that some HIV CTL epitopes will be maintained in human populations.

Identification of a nef allele that causes lymphocyte activation and acute disease in Macaque monkeys

Residues 17 and 18 in nef of SIVmac239 were changed from RQ to YE to create a translated sequence of SRPSGDLYERLLRARGETYGRLLGEVEDGYSQSP from residues 10-43. The YXXL motifs in this context match very well with consensus sequences for SH2 binding domains and are similar to ones present in nef of the acutely lethal pathogen SIVpbj14. The YE variant of SIVmac239, unlike SIVmac239 but like SIVpbj14, replicated well in resting peripheral blood mononuclear cell cultures, caused extensive T lymphocyte activation, and produced an acute disease in rhesus and pigtailed monkeys characterized by severe diarrhea, rash, and extensive lymphoid proliferation in the gastrointestinal tract. The YEnef gene transformed NIH 3T3 cells in culture. Both 239nef and YEnef were found to associate with src in cotransfected COS cells, and both 60 kDa src and 34 kDa nef were phosphorylated at tyrosine in these cells. The extent of tyrosine phosphorylation of 239nef was considerably less than that of YEnef in these assays. These findings identify an important determinant of the SIVpbj14 phenotype, and they provide evidence of a role for nef in signal transduction and cellular activation.

Role of CD8+ Lymphocytes in Control of Simian Immunodeficiency Virus Infection and Resistance to Rechallenge after Transient Early Antiretroviral Treatment

ABSTRACT Transient antiretroviral treatment with tenofovir, (R)-9-(2-phosphonylmethoxypropyl)adenine, begun shortly after inoculation of rhesus macaques with the highly pathogenic simian immunodeficiency virus (SIV) isolate SIVsmE660, facilitated the development of SIV-specific lymphoproliferative responses and sustained effective control of the infection following drug discontinuation. Animals that controlled plasma viremia following transient postinoculation treatment showed substantial resistance to subsequent intravenous rechallenge with homologous (SIVsmE660) and highly heterologous (SIVmac239) SIV isolates, up to more than 1 year later, despite the absence of measurable neutralizing antibody. In some instances, resistance to rechallenge was observed despite the absence of detectable SIV-specific binding antibody and in the face of SIV lymphoproliferative responses that were low or undetectable at the time of challenge. In vivo monoclonal antibody depletion experiments demonstrated a critical role for CD8+ lymphocytes in the control of viral replication; plasma viremia rose by as much as five log units after depletion of CD8+ cells and returned to predepletion levels (as low as <100 copy Eq/ml) as circulating CD8+ cells were restored. The extent of host control of replication of highly pathogenic SIV strains and the level of resistance to heterologous rechallenge achieved following transient postinoculation treatment compared favorably to the results seen after SIVsmE660 and SIVmac239 challenge with many vaccine strategies. This impressive control of viral replication was observed despite comparatively modest measured immune responses, less than those often achieved with vaccination regimens. The results help establish the underlying feasibility of efforts to develop vaccines for the prevention of AIDS, although the exact nature of the protective host responses involved remains to be elucidated.

HIV vaccine design: insights from live attenuated SIV vaccines

The International AIDS Vaccine Initiative has established a consortium to elucidate mechanisms of protection conferred by live attenuated simian immunodeficiency virus vaccines in monkeys. Here, the strategies defining key components of the protective immune response elicited by these vaccines are discussed.

Vector-mediated gene transfer engenders long-lived neutralizing activity and protection against SIV infection in monkeys

The key to an effective HIV vaccine is development of an immunogen that elicits persisting antibodies with broad neutralizing activity against field strains of the virus. Unfortunately, very little progress has been made in finding or designing such immunogens. Using the simian immunodeficiency virus (SIV) model, we have taken a markedly different approach: delivery to muscle of an adeno-associated virus gene transfer vector expressing antibodies or antibody-like immunoadhesins having predetermined SIV specificity. With this approach, SIV-specific molecules are endogenously synthesized in myofibers and passively distributed to the circulatory system. Using such an approach in monkeys, we have now generated long-lasting neutralizing activity in serum and have observed complete protection against intravenous challenge with virulent SIV. In essence, this strategy bypasses the adaptive immune system and holds considerable promise as a unique approach to an effective HIV vaccine.