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For over two decades, nanomaterials have been employed to facilitate intracellular delivery of small interfering RNA (siRNA), both in vitro and in vivo, to induce post-transcriptional gene silencing (PTGS) via RNA interference. Besides PTGS, siRNAs are also capable of transcriptional gene silencing (TGS) or epigenetic silencing, which targets the gene promoter in the nucleus and prevents transcription via repressive epigenetic modifications. However, silencing efficiency is hampered by poor intracellular and nuclear delivery. Here, polyarginine-terminated multilayered particles are reported as a versatile system for the delivery of TGS-inducing siRNA to potently suppress virus transcription in HIV-infected cells. siRNA is complexed with multilayered particles formed by layer-by-layer assembly of poly(styrenesulfonate) and poly(arginine) and incubated with HIV-infected cell types, including primary cells. Using deconvolution microscopy, uptake of fluorescently labeled siRNA is observed in the nuclei of HIV-1 infected cells. Viral RNA and protein are measured to confirm functional virus silencing from siRNA delivered using particles 16 days post-treatment. This work extends conventional particle-enabled PTGS siRNA delivery to the TGS pathway and paves the way for future studies on particle-delivered siRNA for efficient TGS of various diseases and infections, including HIV.

A proportion of patients surviving acute coronavirus disease 2019 (COVID-19) infection develop post-acute COVID syndrome (long COVID (LC)) lasting longer than 12 weeks. Here, we studied individuals with LC compared to age- and gender-matched recovered individuals without LC, unexposed donors and individuals infected with other coronaviruses. Patients with LC had highly activated innate immune cells, lacked naive T and B cells and showed elevated expression of type I IFN (IFN-β) and type III IFN (IFN-λ1) that remained persistently high at 8 months after infection. Using a log-linear classification model, we defined an optimal set of analytes that had the strongest association with LC among the 28 analytes measured. Combinations of the inflammatory mediators IFN-β, PTX3, IFN-γ, IFN-λ2/3 and IL-6 associated with LC with 78.5–81.6% accuracy. This work defines immunological parameters associated with LC and suggests future opportunities for prevention and treatment.

Antiretroviral therapy (ART)-conferred suppression of HIV replication limits neuronal injury and inflammation. ART interruption tests efficacy in HIV cure trials and viral rebound after ART interruption may induce neuronal injury. We investigated the impact of protocol-defined ART interruption, commenced during primary HIV-1 infection (PHI) on a biomarker of neuro-axonal injury (neurofilament light protein (NfL)), and its associations with inflammation (D-dimer and interleukin-6 (IL-6)) and HIV-1 reservoir size (total HIV-1 DNA).

Measures of CD4 T-cell count and HIV-1 plasma viral load before antiretroviral therapy are proxies for virulence. Whether these proxies are changing over time has implications for prevention and treatment. The aim of this study was to investigate those trends.

Subject C135 is one of the members of the Sydney Blood Bank Cohort, infected in 1981 through transfusion with attenuated nef/3′ long terminal repeat (LTR)-deleted HIV-1, and has maintained undetectable plasma viral load and steady CD4 cell count, in the absence of therapy. Uniquely, C135 combines five factors separately associated with control of viraemia: nef/LTR-deleted HIV-1, HLA-B57, HLA-DR13, heterozygous CCR5 Δ32 genotype and vigorous p24-stimulated peripheral blood mononuclear cell (PBMC) proliferation. Therefore, we studied in detail viral burden and immunological responses in this individual.

A variety of interventions to induce a functional cure of HIV are being explored, with the aim being to allow patients to cease antiretroviral therapy (ART) for prolonged periods of time or for life. These interventions share the goal of inducing ART-free remission from HIV pathogenesis and disease progression but achieve this in quite different ways, by reducing the size of the latent reservoir (for example, small-molecule stimulation of latently infected cells), reducing the number of target cells available for the virus (for example, gene therapy) or improving immune responses (for example, active or passive immunotherapy). Here, we consider a number of these alternative strategies for inducing post-treatment control of HIV and use mathematical modelling to predict the scale of the challenge inherent in these different approaches. For many approaches, over 99.9% efficacy will likely be required to induce durable ART-free remissions. The efficacy of individual approaches is currently far below what we predict will be necessary, and new technologies to achieve lifelong functional cure are needed.

Increased D-dimer concentrations are associated with poor cardiovascular and other clinical outcomes in people with treated HIV infection. Proteinase activated receptor-1 (PAR-1) is activated by thrombin and overexpressed by immune cells from HIV-infected people. We aimed to study the efficacy of vorapaxar, a licensed inhibitor of PAR-1, in reducing HIV-associated hypercoagulation and inflammation.

CD8+ T lymphocytes can recognize and eliminate cells infected by viruses. However, the human immunodeficiency virus (HIV-1) has developed mechanisms to evade CD8+ T-cell-mediated clearance. Here, we describe a protocol to assess the role of the HIV-1 protein Nef in immune evasion. The viral competition assay reveals the preferential killing of HIV-1-infected cells unable to express Nef. This methodology can be extended to study HIV-1 proteins involved in immune evasion and viral variants encoding cytotoxic T lymphocyte escape mutations.

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Supplemental Digital Content is available in the text

The initiation of antiretroviral therapy (ART) during primary infection may offer clinical benefits for HIV-infected individuals by reducing HIV DNA reservoir size and chronic T-cell activation. Current evidence for the advantages of early ART, however, are mostly derived from cross-sectional studies, with the long-term benefits yet to be ascertained.

WHO-recommended second-line antiretroviral therapy (ART) of a pharmacologically enhanced (boosted) protease inhibitor plus nucleoside or nucleotide reverse transcriptase inhibitors (NtRTIs) might be compromised by resistance. Results of the 96 week SECOND-LINE randomised trial showed that NtRTI-sparing ART with ritonavir-boosted lopinavir and raltegravir (raltegravir-group) provided non-inferior efficacy to ritonavir-boosted lopinavir and two or three NtRTIs (NtRTI-group) in participants with virological failure composed of a first-line regimen of a non-nucleoside reverse transcriptase inhibitor plus two NtRTIs. We report the relation of baseline virological resistance with virological failure and emergent resistance on study.

Our aim was to review the global disparities of transmitted HIV drug resistance (TDR) in antiretroviral-naive MSM, people who inject drugs (PWID) and heterosexual populations in both high-income and lowmiddle-income countries.

ABSTRACTThe Maraviroc Switchcollaborative study (MARCH) is a study in aviremic patients on stable antiretroviral therapy and utilizes population-based sequencing of proviral DNA to determine HIV tropism and susceptibility to maraviroc. An external quality assessment (EQA) program was implemented to ensure competency in assessing the tropism of clinical samples conducted by MARCH laboratories (n= 14). The MARCH EQA has three prestudy phases assessing V3 loop sequencing and tropism determination using the bioinformatic algorithm geno2pheno, which generates a false-positive rate (FPR). DNA sequences with low FPRs are more likely to be from CXCR4-using (X4) viruses. Phase 1 of the EQA involved chromatogram interpretation. Phases 2, 2/3, and 3 involved patient and clonal samples. Clinical samples used in these phases were from treatment-experienced HIV-infected volunteers; 18/20 had viral loads of <50 copies/ml, and 10/15 were CXCR4-tropic on prior phenotyping. All samples were tested in triplicate, and any replicate with a geno2pheno FPR of <10% was designated X4. Performance was deemed adequate if =2 R5 and =1 X4 specimens were miscalled. For several clinical samples in the EQA, triplicate testing revealed marked DNA variability (FPR range, 0 to 96.7%). Therefore, a consensus-based approach was employed for each sample, i.e., a median FPR across laboratories was used to define sample tropism. Further sequencing analysis showed mixed viral populations in the clinical samples, explaining the differences in tropism predictions. All laboratories passed the EQA after achieving predefined competence thresholds in either of the phase 2 rounds. The use of clinical samples from patients resembling those who were likely to be screened in the MARCH, coupled with triplicate testing, revealed inherent DNA variability that might have been missed if single or duplicate testing and/or clonal samples alone were used. These data highlight the importance of intensive EQA of tropism laboratories before embarking on clinical studies. (This study has been registered at ClinicalTrials.gov under registration no. NCT01384682 [http://www.clinicaltrials.gov/ct2/show/study/NCT01384682?term=NCT01384682&rank=1].)

Background Biomarkers of inflammation, coagulation, lipids and vitamin D have been associated with cardiovascular and mortality risk in HIV-infected individuals. Scarce data exist on changes in these markers from pre-to post-HIV seroconversion.Methods The study participants were drawn from the Health in Men Study, which recruited HIV-negative homosexual men. Participants with incident HIV infection (n=26) were compared with HIV-negative controls (n=52) matched on age at enrolment, date of visit and reported intravenous drug use. Levels of metabolic (lipids and vitamin D), inflammatory (C-reactive protein and interleukin-6) and coagulation (D-dimer and fibrinogen) biomarkers were measured at pre- and post-HIV seroconversion visits and corresponding visits for controls. Random-effect models were used to compare changes in markers between cases and controls.Results The median gap between pre- and post-seroconversion or matched first and second visits in controls was 12 months. HIV seroconversion was associated with decline in high density lipoprotein (HDL-C; difference in mean change between cases and controls -0.14 mmol/l; 95% CI -0.22, -0.01; P=0.035). There were no significant differences in changes in other lipids, markers of inflammation, coagulation or vitamin D.Conclusions Decline in HDL-C seems to be the main proatherogenic change within 1–1.5 years after HIV seroconversion. HIV seroconversion was not associated with profound changes in other lipids, or markers of inflammation, coagulation and vitamin D. Longitudinal assessment of these markers in comparable population needs further assessment.

Natural control of HIV infection is associated with CD8T-cell responses to Gag-encoded antigens of the HIV core and carriage of ‘protective’ human leukocyte antigen (HLA)-B alleles, but some HIV controllers do not possess these attributes. As slower HIV disease progression is associated with high levels of antibodies to HIV Gag proteins, we have examined antibodies to HIV proteins in controllers with and without ‘protective’ HLA-B alleles.

There has been a paradigm shift in our understanding of how protein regulation occurs within mammalian cells in the last 15 years. Our current understanding is that small, noncoding RNA molecules called microRNAs (miRNAs) play a vital role in modulating the translation of mRNAs into protein. Important studies suggest that HIV-1 replication may be restricted by certain host cellular miRNAs, and this in turn may play pivotal roles in host defense and in maintaining latency within resting CD4T cells. Conversely, host cellular miRNAs have also been demonstrated to be essential for certain viruses to establish infection and the altered expression of cellular miRNAs in the setting of HIV-1 may also be a factor favoring viral replication. The differential expression of important protective histocompatability locus antigen (HLA) alleles in HIV-1 infection has recently been shown to be regulated by miRNAs. To date, most efforts into finding an effective vaccine to combat HIV-1 have not been successful. Understanding the role that miRNAs may play in HIV-1 pathogenesis may allow a different approach to targeting key small RNAs or the identification of new important protein targets regulated by miRNAs, which may result in a better vaccine construct. The purpose of this review is to look at our current state of understanding of how HIV-1 and the miRNA pathway interact and the possible therapeutic interventions that this knowledge may entail.

We investigated the dynamics of HIV RNA and HIV DNA levels after the commencement of raltegravir-based antiretroviral therapy (ART) in primary (PHI) and chronically HIV-infected (CHI) individuals (the PINT study).

We have previously reported induction of transcriptional gene silencing (TGS) of HIV-1 by short hairpin RNA (shRNA) expressed in MOLT-4 cells. The shRNA (termed shPromA) targets the highly conserved tandem NF-kB binding sequences of the HIV-1 promoter. Recent articles have reported that TGS mediated by promoter-targeted siRNAs was exclusively the result of sequence non-specific off-target effects. Specifically, several mismatched siRNAs to the target promoter sequences were reported to also induce significant TGS, suggesting TGS was a consequence of off-target effects. Here, following extensive investigation, we report that shPromA induces sequence specific transcriptional silencing in HIV-1 infection in MOLT4 cells, while four shRNA variants, mismatched by 2-3 nucleotides, fail to suppress viral replication. We confirm similar levels of shRNA expression from the U6 promoter and the presence of processed/cleaved siRNAs for each construct in transduced MOLT-4 cells. HIV-1 sequence specific shPromA does not suppress HIV-2, which has an alternate NF-kB binding sequence. As a result of the unique sequence targeted, shPromA does not induce down-regulation of other NF-kB driven genes, either at the mRNA or protein level. Furthermore, we confirmed shPromA does not have sequence non-specific off-target effects through unaltered expression of CD4, CXCR4, and CCR5, which are used for viral entry. Additionally, shPromA does not alter PKR, IFN levels, and three downstream mediators of IFN-a response genes. Our data clearly shows that shPromA achieved highly specific TGS of HIV-1, demonstrating that effective TGS can be induced with minimal off-target effects.

Mapping the precise determinants of T-cell efficacy against viruses in humans is a public health priority with crucial implications for vaccine design. To inform this effort, we performed a comprehensive analysis of the effective CD8+T-cell clonotypes that constitute responses specific for the HIV p24 Gag-derived KK10 epitope (KRWIILGLNK; residues 263-272) restricted by HLA-B*2705, which are known to confer superior control of viral replication in HIV-infected individuals. Particular KK10-specific CD8+T-cell clonotypes, characterized by TRBV4-3/TRBJ1-3gene rearrangements, were found to be preferentially selected in vivo and shared between individuals. These “public” clonotypes exhibit high levels of TCR avidity and Ag sensitivity, which impart functional advantages and enable effective suppression of HIV replication. The early L268M mutation at position 6 of the KK10 epitope enables the virus to avoid recognition by these highly effective CD8+T-cell clonotypes. However, alternative clonotypes with variant reactivity provide flexibility within the overall KK10-specific response. These findings provide refined mechanistic insights into the workings of an effective CD8+T-cell response against HIV.

Mapping the precise determinants of T-cell efficacy against viruses in humans is a public health priority with crucial implications for vaccine design. To inform this effort, we performed a comprehensive analysis of the effective CD8+ T-cell clonotypes that constitute responses specific for the HIV p24 Gag-derived KK10 epitope (KRWIILGLNK; residues 263-272) restricted by HLA-B*2705, which are known to confer superior control of viral replication in HIV-infected individuals. Particular KK10-specific CD8+ T-cell clonotypes, characterized by TRBV4-3/TRBJ1-3 gene rearrangements, were found to be preferentially selected in vivo and shared between individuals. These “public” clonotypes exhibit high levels of TCR avidity and Ag sensitivity, which impart functional advantages and enable effective suppression of HIV replication. The early L268M mutation at position 6 of the KK10 epitope enables the virus to avoid recognition by these highly effective CD8+ T-cell clonotypes. However, alternative clonotypes with variant reactivity provide flexibility within the overall KK10-specific response. These findings provide refined mechanistic insights into the workings of an effective CD8+ T-cell response against HIV.

The mechanisms of CD4+T-cell count decline, the hallmark of HIV disease progression, and its relationship to elevated levels of immune activation are not fully understood. Massive depletion of CD4+T cells occurs during the course of HIV-1 infection, so that maintenance of adequate CD4+T-cell levels probably depends primarily on the capacity to renew depleted lymphocytes, that is, the lymphopoiesis. We performed here a comprehensive study of quantitative and qualitative attributes of CD34+hematopoietic progenitor cells directly from the blood of a large set of HIV-infected persons compared with uninfected donors, in particular the elderly. Our analyses underline a marked impairment of primary immune resources with the failure to maintain adequate lymphocyte counts. Systemic immune activation emerges as a major correlate of altered lymphopoiesis, which can be partially reversed with prolonged antiretroviral therapy. Importantly, HIV disease progression despite elite control of HIV replication or virologic success on antiretroviral treatment is associated with persistent damage to the lymphopoietic system or exhaustion of lymphopoiesis. These findings highlight the importance of primary hematopoietic resources in HIV pathogenesis and the response to antiretroviral treatments.

ABSTRACTStorage of high-quality cryopreserved peripheral blood mononuclear cells (PBMC) is often a requirement for multicenter clinical trials and requires a reproducibly high standard of practice. A quality assurance program (QAP) was established to assess an Australia-wide network of laboratories in the provision of high-quality PBMC (determined by yield, viability, and function), using blood taken from single donors (human immunodeficiency virus [HIV] positive and HIV negative) and shipped to each site for preparation and cryopreservation of PBMC. The aim of the QAP was to provide laboratory accreditation for participation in clinical trials and cohort studies which require preparation and cryopreservation of PBMC and to assist all laboratories to prepare PBMC with a viability of >80% and yield of >50% following thawing. Many laboratories failed to reach this standard on the initial QAP round. Interventions to improve performance included telephone interviews with the staff at each laboratory, two annual wet workshops, and direct access to a senior scientist to discuss performance following each QAP round. Performance improved substantially in the majority of sites that initially failed the QAP (P= 0.002 and P= 0.001 for viability and yield, respectively). In a minority of laboratories, there was no improvement (n= 2), while a high standard was retained at the laboratories that commenced with adequate performance (n= 3). These findings demonstrate that simple interventions and monitoring of PBMC preparation and cryopreservation from multiple laboratories can significantly improve performance and contribute to maintenance of a network of laboratories accredited for quality PBMC fractionation and cryopreservation.

Interleukin-7 (IL-7) is a cytokine produced predominantly by stromal cells of the thymus and bone marrow and is essential for lymphopoiesis. This paper reviews the importance of IL-7 and its receptor (IL-7R) in T-cell genesis, peripheral survival, expansion and memory T-cell development. IL-7 is of particular importance in lymphopenic conditions. Its expression is up-regulated in a number of lymphopenic conditions including: marrow ablation prior to bone marrow transplantation, marrow suppression following chemotherapy and human immuno-deficiency virus (HIV) infection. Plasma IL-7 levels inversely correlate with CD4+ T-cell counts in these conditions. Animal models suggest that IL-7 improves immune reconstitution through increasing thymic output and, perhaps more importantly, through antigen-independent homeostatic driven proliferation in the periphery. Given the promising preliminary data on the use of IL-7 adjuvant therapy in simian immuno-deficiency virus (SIV) infected non-human primates, IL-7 has recently moved into Phase I/II clinical trials of its role as a possible adjuvant therapy for cancer and HIV infection. This paper discusses important considerations such as the possible negative impacts of IL-7 on increased viral infectivity, the induction of autoimmunity and risk of neoplastic events. Successful use of IL-7 will rely on further understanding of the regulation of the component parts of the IL-7R system. Ultimately this understanding may lead to therapeutics that manipulate and optimise signalling through the IL-7/IL-7R system.

We investigated whether HIV-1 antigen-specific CD4+ T cells expressed the viral coreceptor CCR5 during primary HIV-1 infection (PHI). In the peripheral blood of subjects with very early PHI (< 22 days after onset of symptoms), there was a 10- to 20-fold increase in the proportion of highly activated (CD38+++) and proliferating (Ki-67+) CD4+ T cells that expressed CCR5+, and were mostly T-cell intracellular antigen-1 (TIA-1)+ perforin+ granzyme B+. Inthe same patient samples, CD4+ T cells producing interferon (IFN)–γ in response to HIV group-specific antigen (Gag) peptides were readily detected (median, 0.58%) by intracellular cytokine assay—these cells were again predominantly CD38+++, Ki-67+, and TIA-++, as well as Bcl-2low. On average, 20% of the Gag-specific CD4+ T cells also expressed interleukin-2 (IL-2) and were CD127 (IL-7R)+. Taken together, these results suggest that Gag-specific T-helper 1 (Th1) effector cells express CCR5 during the primary response and may include precursors of long-term self-renewing memory cells. However, in PHI subjects with later presentation, antigen-specific CD4+ T cells could not be readily detected (median, 0.08%), coinciding with a 5-fold lower level of the CCR5+CD38+++ CD4+ T cells. These results suggest that the antiviral response to HIV-1 infection includes highly activated CCR5+CD4+ cytotoxic effector cells, which are susceptible to both apoptosis and cytopathic infection with HIV-1, and rapidly decline.

We investigated whether HIV-1 antigen-specific CD4+T cells expressed the viral coreceptor CCR5 during primary HIV-1 infection (PHI). In the peripheral blood of subjects with very early PHI (< 22 days after onset of symptoms), there was a 10- to 20-fold increase in the proportion of highly activated (CD38+++) and proliferating (Ki-67+) CD4+T cells that expressed CCR5+, and were mostly T-cell intracellular antigen-1 (TIA-1)+perforin+granzyme B+. Inthe same patient samples, CD4+T cells producing interferon (IFN)–γ in response to HIV group-specific antigen (Gag) peptides were readily detected (median, 0.58%) by intracellular cytokine assay—these cells were again predominantly CD38+++, Ki-67+, and TIA-++, as well as Bcl-2low. On average, 20% of the Gag-specific CD4+T cells also expressed interleukin-2 (IL-2) and were CD127 (IL-7R)+. Taken together, these results suggest that Gag-specific T-helper 1 (Th1) effector cells express CCR5 during the primary response and may include precursors of long-term self-renewing memory cells. However, in PHI subjects with later presentation, antigen-specific CD4+T cells could not be readily detected (median, 0.08%), coinciding with a 5-fold lower level of the CCR5+CD38+++CD4+T cells. These results suggest that the antiviral response to HIV-1 infection includes highly activated CCR5+CD4+cytotoxic effector cells, which are susceptible to both apoptosis and cytopathic infection with HIV-1, and rapidly decline.

Antigen-specific CD4+ effector T cells primarily provide help for B-cell antibody responses and CD8+ cytotoxic T-lymphocyte (CTL) responses. We have found an expanded population of HIV-1 p24-specific, T-cell receptor Vbeta17+, CD4+ T lymphocytes, defined by in vitro proliferative and interferon-γ responses to a 15-mer Gag peptide, in the peripheral blood of an individual with long-term nonprogressive HIV-1 infection. Ex vivo, these cells were CCR5+ and CCR7-, consistent with an effector/memory function. Surprisingly, these cells highly expressed several proteins characteristic of cytotoxic lymphocytes, including TIA-1 (T-cell intracellular antigen 1; GMP-17/NKG7), granzymes A and B, CD161 (NKRP-1), and CD244 (C1.7/2B4). Following in vitro peptide stimulation, these cells produced interleukin 2 (IL-2) and intracellular CD40L, suggesting possible helper function, in addition to induction of perforin and cytotoxicity. A subset of cytomegalovirus (CMV)–specific CD4+ T cells in healthy adults similarly expressed these CTL markers and CCR5, ex vivo. Furthermore, this distinct subset of CD4+ T cells was significantly elevated in healthy CMV-seropositive adults, compared with CMV-seronegative individuals. These results suggest that CCR5+ CD4+ CTL may be a major effector mechanism of the immune response to viral infections in humans. Moreover, expression of CCR5 may render them particularly susceptible to cytopathic effects during progressive HIV-1 infection.

Antigen-specific CD4+effector T cells primarily provide help for B-cell antibody responses and CD8+cytotoxic T-lymphocyte (CTL) responses. We have found an expanded population of HIV-1 p24-specific, T-cell receptor Vbeta17+, CD4+T lymphocytes, defined by in vitro proliferative and interferon-γ responses to a 15-mer Gag peptide, in the peripheral blood of an individual with long-term nonprogressive HIV-1 infection. Ex vivo, these cells were CCR5+and CCR7-, consistent with an effector/memory function. Surprisingly, these cells highly expressed several proteins characteristic of cytotoxic lymphocytes, including TIA-1 (T-cell intracellular antigen 1; GMP-17/NKG7), granzymes A and B, CD161 (NKRP-1), and CD244 (C1.7/2B4). Following in vitro peptide stimulation, these cells produced interleukin 2 (IL-2) and intracellular CD40L, suggesting possible helper function, in addition to induction of perforin and cytotoxicity. A subset of cytomegalovirus (CMV)–specific CD4+T cells in healthy adults similarly expressed these CTL markers and CCR5, ex vivo. Furthermore, this distinct subset of CD4+T cells was significantly elevated in healthy CMV-seropositive adults, compared with CMV-seronegative individuals. These results suggest that CCR5+CD4+CTL may be a major effector mechanism of the immune response to viral infections in humans. Moreover, expression of CCR5 may render them particularly susceptible to cytopathic effects during progressive HIV-1 infection.

Since the introduction of highly active antiretroviral therapy (HAART), HIV-related deaths have declined dramatically in the developed world. However, HAART is neither able to eradicate the virus nor are its immunomodulatory effects sufficient to effect complete control of the virus. In addition, the long-term use of HAART is complicated by drug-related toxicities and compliance issues, both of which impact upon the development of viral resistance. The failure of structured treatment interruption strategies in those with chronic HIV-infection combined with the above limitations, has prompted renewed interest in immunotherapy. Cytokines and therapeutic vaccination have been proposed as HAART-adjunctive and HAART-sparing treatments in HIV-infection, and the current and future role of cytokine therapy in this disease will be the subject of this review.

Reliable, efficient systems for producing soluble HLA-DR molecules, suitable for multimerization and use as staining reagents, have proved elusive. We found that the addition of a flexible linker between peptide and N terminus of the DRB1*0101-chain (Crawford, F., Kozono, H., White, J., Marrack, P. and Kappler, J., Immunity 1998. 8: 675–682.), results in greater in vitro foldingefficiency of Escherichia coli-expressed α- and β-chains, and increases both the yield and stability of the DRA1*0101/DRB1*0101/peptide complexes. Although a 10-amino acid linker functioned efficiently for a 20mer epitope from HIV p24, a longer linker was required to produce a DR1 MHC class II tetramer with the influenza hemagglutinin epitope (HA306–318). The DR1-HA tetramer was able to stain positively over 98% of a specific clone (HA 1.7) with only a brief 30-min incubation. The tetrameric complexes detected clone cells diluted into PBMC, with high sensitivity, coupled with low background staining in CD4⁺ cells. It was possible to detect antigen-specific CD4⁺ T cells within a population of PBMC stimulated with the HA peptide. This demonstrates the potential to monitor CD4⁺ T cell responses in peripheral blood in a number of clinical scenarios.

The immune response to HIV-1 in patients who carry human histocompatibility leukocyte antigen (HLA)-B27 is characterized by an immunodominant response to an epitope in p24 gag (amino acids 263–272, KRWIILGLNK). Substitution of lysine (K) or glycine (G) for arginine (R) at HIV-1 gag residue 264 (R264K and R264G) results in epitopes that bind to HLA-B27 poorly. We have detected a R264K mutation in four patients carrying HLA-B27. In three of these patients the mutation occurred late, coinciding with disease progression. In another it occurred within 1 yr of infection and was associated with a virus of syncytium-inducing phenotype. In each case, R264K was tightly associated with a leucine to methionine change at residue 268. After the loss of the cytotoxic T lymphocyte (CTL) response to this epitope and in the presence of high viral load, reversion to wild-type sequence was observed. In a fifth patient, a R264G mutation was detected when HIV-1 disease progressed. Its occurrence was associated with a glutamic acid to aspartic acid mutation at residue 260. Phylogenetic analyses indicated that these substitutions emerged under natural selection rather than by genetic drift or linkage. Outgrowth of CTL escape viruses required high viral loads and additional, possibly compensatory, mutations in the gag protein.

A major goal of antiretroviral HIV-1 therapy is the reversal of HIV-1-associated immunological dysfunction. However, the pathogenetic mechanisms involved and their significance are largely unknown. On the basis of the life cycle of naive, activated, and memory CD4+ T cell subsets, a mathematical model of immune reconstitution was developed and applied to data for T cell subsets in individuals with acute or chronic HIV-1 infection receiving antiretroviral therapy. The final model that most accurately fitted the data, and resulted in realistic estimates for CD4+ T cell turnover, considered three pathways of immune reconstitution for naive cells, including thymic production, peripheral expansion, and redistribution of naive cells from lymphoid tissue. The reconstitution of the memory compartment was fitted through differentiation and expansion of naive cells and peripheral expansion of memory cells as well as redistribution of memory cells trapped in the lymphoid tissue. Estimated median half-lives for naive and memory CD4+ T cells were 114 and 21 days, while total production rates were 9.1 × 107 and 2.4 × 109 cells/day, respectively. Peripheral expansion and thymic production contributed equally to the regeneration of naive cells, but peripheral expansion of memory cells was larger than production of these cells by differentiation of naive cells. A comparison of immune reconstitution in acute and chronic HIV-1 infection revealed that, after adjustment for age, the main difference was the more rapid release of a larger number of naive cells in treated acute HIV-1 infection. Thymic function and peripheral expansion rates, however, were similar in both cohorts.

Introduction

This study evaluates the impact of therapeutic vaccination with p24-VLP and zidovudine on the induction or maintenance of HIV-specific cytotoxic lymphocyte activity in a cohort of asymptomatic patients with CD4 counts greater than 400 cells/mu l. In a dummy, randomized, phase II clinical trial of the therapeutic vaccine, participants were randomized to one of three arms for 6 months: p24-VLP (500 mu g) in alum monthly plus zidovudine 200 mg tds, alum adjuvant plus zidovudine, or p24-VLP plus placebo. Subjects were studied for a total of 52 weeks from baseline. Monitoring included viral load, CD4 and CD8 counts, markers of immune activation, delayed-type hypersensitivity (DTH) skin testing, and cytotoxic T lymphocyte (CTL) measurement. The nine subjects who received p24-VLP and zidovudine had an augmentation and/or broadening of their CTL response compared with baseline (p = 0.004). The eight subjects receiving p24-VLP and seven subjects receiving zidovudine did not have a statistically significant increase or broadening of CTL activity. The augmentation of the CTL response in the subjects who received p24-VLP and zidovudine was not associated with a decline in viral load or an increase in CD8 counts. This study suggests that HIV-specific CTL activity can be augmented in HIV-infected individuals receiving p24-VLP and zidovudine, supporting the hypothesis of therapeutic vaccination in the presence of antiretroviral therapy.

Apply the science, use a team approach, help patients to promote their own health, ensure access to support and accept a public health role. These are the practical lessons of our experience in HIV medicine.

Vδ2+T cells play a critical role in immunity to micro-organisms and cancer but exhibit substantial heterogeneity in humans. Here, we demonstrate that CD26 and CD94 define transcriptionally, phenotypically, and functionally distinct Vδ2+T cell subsets. Despite distinct antigen specificities, CD26hiCD94loVδ2+cells exhibit substantial similarities to CD26himucosal-associated invariant T (MAIT) cells, although CD26−Vδ2+cells exhibit cytotoxic, effector-like profiles. At birth, the Vδ2+Vγ9+population is dominated by CD26hiCD94locells; during adolescence and adulthood, Vδ2+cells acquire CD94/NKG2A expression and the relative frequency of the CD26hiCD94losubset declines. Critically, exposure of the CD26hiCD94losubset to phosphoantigen in the context of interleukin-23 (IL-23) and CD26 engagement drives the acquisition of a cytotoxic program and concurrent loss of the MAIT cell-like phenotype. The ability to modulate the cytotoxic potential of CD26hiCD94loVδ2+cells, combined with their adenosine-binding capacity, may make them ideal targets for immunotherapeutic expansion and adoptive transfer.

Estimating effective doses of novel HIV vaccines is challenging. Dose-response analyses of DNA and fowlpoxvirus HIV vaccines showed that 1 mg of DNA vaccine and 5 x 107pfu of fowlpoxvirus booster was immunogenic in macaques. However, this dose was poorly immunogenic in humans. When adjusted for body surface area, the human dose studied was equivalent to a poorly immunogenic lower dose in monkeys. These data provide a rationale for guiding dosing in future trials of HIV vaccine technologies.

The immune response in HIV-infected individuals who carry HLA-B27 is characterized by an immunodominant cytotoxic T lymphocyte (CTL) response to a conserved epitope corresponding to amino acids 263––272 of HIV-1 p24 gag. The arginine at position 264 is a crucial anchor residue. Amino acid substitution at 264 from arginine (R) to glycine (G), lysine (K), or threonine (T) results in a low affinity peptide that binds to HLA-B27 inefficiently and is poorly recognized by T cells that respond to the wild-type peptide. These mutants have been characterized as CTL escape mutations. We studied the plasma virus of 20 HLA-B27 longterm nonprogressors: 14 were wild type and 6 were found to be mutant. Five of these carried known escape mutations coding for K or G at position 264. One patient demonstrated a previously undescribed R264Q mutation in 30/31 clones. This altered epitope failed to elicit an IFN-γγ response from PBMC isolated from any of four HLA-B27-positive individuals with strong responses to wild-type peptide. A peptide binding assay confirmed that the R264Q mutant peptide had 30-fold lower binding affinity to HLA-B27 compared to wild type. Therefore, the R264Q variant is a likely novel escape mutation in HLA-B27-positive individuals.

From the resolution of primary infection until the CD4 cell count falls below 500/μL, people with HIV are usually functioning at their normal capacity physically, mentally and socially However, HIV is replicating actively within lymph nodes during this period of early immune deficiency It is important to determine the rate of immune depletion and to assess what factors, if any may be hastening that progression.

This study used curve-fitting techniques to detail the dynamics of human immunodeficiency virus (HIV)-1 and its relationship to circulating T lymphocyte changes in a cohort of 41 male patients (mean age 36 ± 7 years) infected with HIV-1. The following characteristics of viral kinetics were obtained: virus load peak, 6.35 ± 0.71 log10 RNA copies/mL at 12.2 ±7.1 days; virus load drop from peak, 2.02 ± 0.93 log10 copies/mL; viral decay rate from peak, 0.071 ± 0.042 log10 RNA copies/mL/day; and steady state virus load, 4.57 ± 0.68 log10 copies/mL at 135 ± 81 days. Analysis of individual virus load curves revealed highly variable viral kinetics. Although these could be grouped into three distinct patterns, virus load and CD4 lymphocyte counts were similar in all patterns at 12 months, but the interval from infection to achievement of steady state virus load varied significantly.