Steve Kaye

Mature CD8(+) T lymphocyte response to viral infection during fetal life.

Immunization of newborns against viral infections may be hampered by ineffective CD8(+) T cell responses. To characterize the function of CD8(+) T lymphocytes in early life, we studied newborns with congenital human cytomegalovirus (HCMV) infection. We demonstrate that HCMV infection in utero leads to the expansion and the differentiation of mature HCMV-specific CD8(+) T cells, which have similar characteristics to those detected in adults. High frequencies of HCMV-specific CD8(+) T cells were detected by ex vivo tetramer staining as early as after 28 weeks of gestation. During the acute phase of infection, these cells had an early differentiation phenotype (CD28(-)CD27(+)CD45RO(+), perforin(low)), and they acquired a late differentiation phenotype (CD28(-)CD27(-)CD45RA(+), perforin(high)) during the course of the infection. The differentiated cells showed potent perforin-dependent cytolytic activity and produced antiviral cytokines. The finding of a mature and functional CD8(+) T cell response to HCMV suggests that the machinery required to prime such responses is in place during fetal life and could be used to immunize newborns against viral pathogens.

Failure to detect the novel retrovirus XMRV in chronic fatigue syndrome.

Background In October 2009 it was reported that 68 of 101 patients with chronic fatigue syndrome (CFS) in the US were infected with a novel gamma retrovirus, xenotropic murine leukaemia virus-related virus (XMRV), a virus previously linked to prostate cancer. This finding, if confirmed, would have a profound effect on the understanding and treatment of an incapacitating disease affecting millions worldwide. We have investigated CFS sufferers in the UK to determine if they are carriers of XMRV. Methodology Patients in our CFS cohort had undergone medical screening to exclude detectable organic illness and met the CDC criteria for CFS. DNA extracted from blood samples of 186 CFS patients were screened for XMRV provirus and for the closely related murine leukaemia virus by nested PCR using specific oligonucleotide primers. To control for the integrity of the DNA, the cellular beta-globin gene was amplified. Negative controls (water) and a positive control (XMRV infectious molecular clone DNA) were included. While the beta-globin gene was amplified in all 186 samples, neither XMRV nor MLV sequences were detected. Conclusion XMRV or MLV sequences were not amplified from DNA originating from CFS patients in the UK. Although we found no evidence that XMRV is associated with CFS in the UK, this may be a result of population differences between North America and Europe regarding the general prevalence of XMRV infection, and might also explain the fact that two US groups found XMRV in prostate cancer tissue, while two European studies did not.

Short-course antiretroviral therapy in primary HIV infection.

BACKGROUND Short-course antiretroviral therapy (ART) in primary human immunodeficiency virus (HIV) infection may delay disease progression but has not been adequately evaluated. METHODS We randomly assigned adults with primary HIV infection to ART for 48 weeks, ART for 12 weeks, or no ART (standard of care), with treatment initiated within 6 months after seroconversion. The primary end point was a CD4+ count of less than 350 cells per cubic millimeter or long-term ART initiation. RESULTS A total of 366 participants (60% men) underwent randomization to 48-week ART (123 participants), 12-week ART (120), or standard care (123), with an average follow-up of 4.2 years. The primary end point was reached in 50% of the 48-week ART group, as compared with 61% in each of the 12-week ART and standard-care groups. The average hazard ratio was 0.63 (95% confidence interval [CI], 0.45 to 0.90; P=0.01) for 48-week ART as compared with standard care and was 0.93 (95% CI, 0.67 to 1.29; P=0.67) for 12-week ART as compared with standard care. The proportion of participants who had a CD4+ count of less than 350 cells per cubic millimeter was 28% in the 48-week ART group, 40% in the 12-week group, and 40% in the standard-care group. Corresponding values for long-term ART initiation were 22%, 21%, and 22%. The median time to the primary end point was 65 weeks (95% CI, 17 to 114) longer with 48-week ART than with standard care. Post hoc analysis identified a trend toward a greater interval between ART initiation and the primary end point the closer that ART was initiated to estimated seroconversion (P=0.09), and 48-week ART conferred a reduction in the HIV RNA level of 0.44 log(10) copies per milliliter (95% CI, 0.25 to 0.64) 36 weeks after the completion of short-course therapy. There were no significant between-group differences in the incidence of the acquired immunodeficiency syndrome, death, or serious adverse events. CONCLUSIONS A 48-week course of ART in patients with primary HIV infection delayed disease progression, although not significantly longer than the duration of the treatment. There was no evidence of adverse effects of ART interruption on the clinical outcome. (Funded by the Wellcome Trust; SPARTAC Controlled-Trials.com number, ISRCTN76742797, and EudraCT number, 2004-000446-20.).

Mouse DNA contamination in human tissue tested for XMRV

BackgroundWe used a PCR-based approach to study the prevalence of genetic sequences related to a gammaretrovirus, xenotropic murine leukemia virus-related virus, XMRV, in human prostate cancer. This virus has been identified in the US in prostate cancer patients and in those with chronic fatigue syndrome. However, with the exception of two patients in Germany, XMRV has not been identified in prostate cancer tissue in Europe. Most putative associations of new or old human retroviruses with diseases have turned out to be due to contamination. We have looked for XMRV sequences in DNA extracted from formalin-fixed paraffin- embedded prostate tissues. To control for contamination, PCR assays to detect either mouse mitochondrial DNA (mtDNA) or intracisternal A particle (IAP) long terminal repeat DNA were run on all samples, owing to their very high copy number in mouse cells.ResultsIn general agreement with the US prevalence, XMRV-like sequences were found in 4.8% of prostate cancers. However, these were also positive, as were 21.5% of XMRV-negative cases, for IAP sequences, and many, but not all were positive for mtDNA sequences.ConclusionsThese results show that contamination with mouse DNA is widespread and detectable by the highly sensitive IAP assay, but not always with less sensitive assays, such as murine mtDNA PCR. This study highlights the ubiquitous presence of mouse DNA in laboratory specimens and offers a means of rigorous validation for future studies of murine retroviruses in human disease.

HIV-1 viral load, phenotype, and resistance in a subset of drug-naive participants from the Delta trial

BACKGROUND The Delta trial showed that combination therapy (zidovudine plus didanosine and zidovudine plus zalcitabine) substantially lengthened life and reduced disease progression compared with zidovudine monotherapy. We did a nested virological study in three countries (France, the Netherlands, and the UK) to investigate changes in markers for viral load and antiretroviral-drug resistance during therapy. METHODS 240 zidovudine-naive HIV-1-infected patients were randomly assigned zidovudine only (n = 87), zidovudine plus didanosine (n = 80), or zidovudine plus zalcitabine (n = 73). Viral load in peripheral-blood mononuclear cells and plasma was measured by quantitative culture. Plasma HIV-1 RNA was measured by reverse-transcriptase PCR amplification, and serum p24 antigen by ELISA. Resistance to antiretroviral drugs was measured phenotypically by culture and genotypically by detection and quantification of drug-related point mutations in the pol gene. Analyses were done by intention to treat. FINDINGS The reduction in viral load was greatest 4-12 weeks after the start of therapy and was most pronounced in the combination-therapy study groups (median reductions of RNA at 4 weeks 1.58, 1.28, and 0.49 log10 copies/mL for zidovudine plus didanosine, zidovudine plus zalcitabine, and zidovudine only, respectively). RNA levels at 8 weeks were predictive of disease progression and death after allowance for baseline values. At 48 weeks, the proportion of participants with phenotypic zidovudine resistance was similar in all three groups: didanosine and zalcitabine resistance were rare; zidovudine genomic resistance correlated with phenotypic resistance (r = 0.54, p < 0.0001) and developed earlier in the combined-therapy groups. However, participants in the zidovudine monotherapy group had higher circulating loads of resistant virus than those in the combined-therapy groups. INTERPRETATION Combined antiretroviral therapy was more efficient at lowering virus load than monotherapy. Although zidovudine resistance was common in monotherapy and combined-therapy groups, circulating concentrations of resistant virus were substantially lower in the combination groups, which is likely to be a result of the continued antiviral activity of didanosine or zalcitabine.

Transient relapses ("blips") of plasma HIV RNA levels during HAART are associated with drug resistance.

Introduction: In a large number of patients on HAART who achieved plasma HIV RNA levels below the limit of detection (50 copies/ml), transient relapses of HIV RNA levels (“blips”) are observed. Objective: To determine whether relapses of plasma HIV RNA during HAART are associated with development of drug resistance. Methods: Plasma samples from 15 patients with a transient viral load relapse during HAART were studied. All regimens contained lamivudine (3TC). We used an ultrasensitive sequence approach to analyze the presence of drug resistance mutations during the relapse. Results: The median plasma HIV RNA load of the relapse was 76 copies/ml (range 50‐1239). In 11 of 15 cases, a genotype of HIV could be obtained. Mutations in the RT and protease gene conferring resistance to one or more drugs were observed in 8 of 11 patients, 6 of whom had the M184V substitution. During a median follow‐up of 27 months after the relapse, plasma HIV RNA levels remained undetectable in 13 of 15 patients. Conclusions: Plasma HIV RNA blips during HAART can be associated with selection of drug‐resistant HIV. This indicates that viral replication may occur during HAART, probably caused by a temporary decrease in active drug concentrations. A blip containing only wild‐type virus is not necessarily caused by viral replication. In this situation the raise of HIV RNA could also originate from release of wild‐type viruses, caused by activation of the latent virus reservoir. Independent of the mechanism, blips did not preclude successful inhibition of viral replication during 2‐year follow‐up in the majority of these cases.

Impact of Human Immunodeficiency Virus Type 1 Subtypes on Virologic Response and Emergence of Drug Resistance among Children in the Paediatric European Network for Treatment of AIDS (PENTA) 5 Trial

The association between virologic response and human immunodeficiency virus type 1 (HIV-1) subtype was investigated in 113 HIV-1-infected children randomly assigned to receive zidovudine plus lamivudine, zidovudine plus abacavir, or lamivudine plus abacavir in the Paediatric European Network for Treatment of AIDS (PENTA) 5 trial. Symptomatic children (n=68) also received nelfinavir; asymptomatic children (n=45) were randomly assigned to receive nelfinavir or placebo. HIV-1 subtypes A, B, C, D, F, G, H, A/E, and A/G were found in 15%, 41%, 16%, 9%, 5%, 2%, 1%, 5%, and 7% of the children, respectively. Resistance assay failure rates were higher for non-B subtypes than for B subtypes (genotype, P=.01; phenotype, P=.02). HIV-1 subtype was not associated with virologic response at 24 and 48 weeks after initiation of treatment. No differences were observed in the frequency of development of resistance mutations L90M (P=1.00) and D30N (P=.61) in B and non-B viruses. In conclusion, no evidence that subtype determined virologic response to therapy was found.

Antiretroviral therapy in Africa.

We should stop and think about the risks of resistance, and ways of minimising them, before increasing access to antiretroviral therapy in Africa Demands for the introduction of antiretroviral therapy into Africa have been growing over the past few years. On the face of it, the availability of antiretroviral therapy at what seems to be an affordable price is good news. The treatment can produce dramatic clinical improvements in people with symptomatic HIV disease and, when used optimally, can delay the progression of disease. However, the potential short term gains from reducing individual morbidity and mortality may be far outweighed by the potential for the long term spread of drug resistance if the experience of adherence to treatment for tuberculosis is repeated. Without due forethought and planning, antiretroviral therapy is likely to be introduced to Africa in a random and haphazard way, with inconsistent prescribing practices and poor monitoring of therapy and adherence. This risks the rapid development and transmission of drug resistance. Virus strains with reduced sensitivity to zidovudine, the first drug used to treat HIV infection, were first observed in 1989, three years after it was introduced.1 Subsequently, resistance to every currently licensed antiretroviral drug has been observed.2 Drug resistance within an individual patient is not confined to a single compound, and cross resistance between drugs of the same class is the rule rather than the exception.3 Drug resistance arises by natural selection, mutant strains being selected when the virus replicates in sub-limiting drug concentrations. The only way to prevent resistance is to use a drug regimen that reduces virus replication to virtually zero (commonly equated with a plasma virus load below 50 RNA copies/ml). In this circumstance, the probability of a mutant arising to all the drugs used in a highly active antiretroviral therapy …

Cytomegalovirus Infection in Gambian Infants Leads to Profound CD8 T-Cell Differentiation

ABSTRACT Cytomegalovirus (CMV) infection is endemic in Gambian infants, with 62% infected by 3 months and 85% by 12 months of age. We studied the CD8 T-cell responses of infants to CMV following primary infection. CMV-specific CD8 T cells, identified with tetramers, showed a fully differentiated phenotype (CD28− CD62L− CD95+ perforin+ granzyme A+ Bcl-2low). Strikingly, the overall CD8 T-cell population developed a similar phenotype following CMV infection, which persisted for at least 12 months. In contrast, primary infection was accompanied by up-regulation of markers of activation (CD45R0 and HLA-D) on both CMV-specific cells and the overall CD8 T-cell population and division (Ki-67) of specific cells, but neither pattern persisted. At 12 months of age, the CD8 T-cell population of CMV-infected infants was more differentiated than that of uninfected infants. Although the subpopulation of CMV-specific cells remained constant, the CMV peptide-specific gamma interferon response was lower in younger infants and increased with age. As the CD8 T-cell phenotype induced by CMV is indicative of immune dysfunction in the elderly, the existence of a similar phenotype in large numbers of Gambian infants raises the question of whether CMV induces a similarly deleterious effect.

HIV-1 RNA serum-load and resistant viral genotypes during early zidovudine therapy

The response of HIV-1 to initial zidovudine (ZDV) treatment was assessed in 11 patients with severe HIV disease. We quantified serum HIV-1 concentrations and mutations associated with ZDV resistance by culture-independent methods. There was a prompt fall in serum HIV-1 RNA within 1-2 days of treatment with maximum suppression by seven days, which was paralleled by changes in serum p24 antigen (p24 Ag). Serum RNA started to return to pretreatment levels within weeks. The HIV reverse transcriptase (RT) gene in most patients developed mutations associated with drug resistance within months and as early as 25 days on therapy in one patient. The codon changes were not sufficient to explain the early return of serum HIV-1 RNA levels and their patterns continued to evolve after patients stopped taking ZDV. The significance of these findings is discussed in relation to the limited long-term efficacy of ZDV. The dynamic time course of viral load and RT responses to ZDV is of particular importance in short-term interventions such as pregnancy.