Short communication: HIV-1 gag genetic variation in a single acutely infected participant defined by high-resolution deep sequencing.

Acute HIV-1 infection is characterized by the rapid generation of highly diverse genetic variants to adapt to the new host environment. Understanding the dynamics of viral genetic variation at this stage of infection is critical for vaccine design efforts and early drug treatment. Here, using a high-resolution deep sequencing approach targeting the HIV-1 gag region, we reveal very early immune pressure with dramatic subpopulation shifts in a single acutely infected participant providing further insight into the genetic dynamics of acute HIV-1 infection.

HIV-1 quasispecies delineation by tag linkage deep sequencing.

Trade-offs between throughput, read length, and error rates in high-throughput sequencing limit certain applications such as monitoring viral quasispecies. Here, we describe a molecular-based tag linkage method that allows assemblage of short sequence reads into long DNA fragments. It enables haplotype phasing with high accuracy and sensitivity to interrogate individual viral sequences in a quasispecies. This approach is demonstrated to deduce ∼ 2000 unique 1.3 kb viral sequences from HIV-1 quasispecies in vivo and after passaging ex vivo with a detection limit of ∼ 0.005% to ∼ 0.001%. Reproducibility of the method is validated quantitatively and qualitatively by a technical replicate. This approach can improve monitoring of the genetic architecture and evolution dynamics in any quasispecies population.

Ineffectual targeting of HIV-1 Nef by cytotoxic T lymphocytes in acute infection results in no functional impairment or viremia reduction.

The human immunodeficiency virus type 1 (HIV-1) accessory protein Nef is heavily targeted by CD8(+) T lymphocytes (CTLs) during acute infection and therefore is included in many candidate vaccines. We investigated whether CTL targeting of Nef during acute infection contributes to immune control by disrupting the function of Nef. The sequence and function of Nef in parallel with CTL responses were assessed longitudinally from peak viremia until the viremia set point in a cohort of six subjects with acute infection. All but one individual had a single founder strain. Nef-specific CTL responses were detected in all subjects and declined in magnitude over time. These responses were associated with mutations, but none of the mutations were detected in important functional motifs. Nef-mediated downregulation of CD4 and major histocompatibility complex (MHC) class I molecules was better preserved in acute infection than in chronic infection. Finally, Nef-specific CTL responses were not associated with a reduction in viremia from its acute-phase peak. Our results indicate that CTLs targeting Nef epitopes outside critical functional domains have little effect on the pathogenic functions of Nef, rendering these responses ineffective in acute infection. Importance: These data indicate that using the whole Nef protein as a vaccine immunogen likely allows immunodominance that leads to targeting of CTL responses that are rapidly escaped with little effect on Nef-mediated pathogenic functions. Pursuing vaccination approaches that can more precisely direct responses to vulnerable areas would maximize efficacy. Until vaccine-induced targeting can be optimized, other approaches, such as the use of Nef function inhibitors or the pursuit of immunotherapies such as T cell receptor gene therapy or adoptive transfer, may be more likely to result in successful control of viremia.

HIV-1 Nef sequence and functional compartmentalization in the gut is not due to differential cytotoxic T lymphocyte selective pressure.

The gut is the largest lymphoid organ in the body and a site of active HIV-1 replication and immune surveillance. The gut is a reservoir of persistent infection in some individuals with fully suppressed plasma viremia on combination antiretroviral therapy (cART) although the cause of this persistence is unknown. The HIV-1 accessory protein Nef contributes to persistence through multiple functions including immune evasion and increasing infectivity. Previous studies showed that Nef's function is shaped by cytotoxic T lymphocyte (CTL) responses and that there are distinct populations of Nef within tissue compartments. We asked whether Nef's sequence and/or function are compartmentalized in the gut and how compartmentalization relates to local CTL immune responses. Primary nef quasispecies from paired plasma and sigmoid colon biopsies from chronically infected subjects not on therapy were sequenced and cloned into Env(-) Vpu(-) pseudotyped reporter viruses. CTL responses were mapped by IFN-γ ELISpot using expanded CD8+ cells from blood and gut with pools of overlapping peptides covering the entire HIV proteome. CD4 and MHC Class I Nef-mediated downregulation was measured by flow cytometry. Multiple tests indicated compartmentalization of nef sequences in 5 of 8 subjects. There was also compartmentalization of function with MHC Class I downregulation relatively well preserved, but significant loss of CD4 downregulation specifically by gut quasispecies in 5 of 7 subjects. There was no compartmentalization of CTL responses in 6 of 8 subjects, and the selective pressure on quasispecies correlated with the magnitude CTL response regardless of location. These results demonstrate that Nef adapts via diverse pathways to local selective pressures within gut mucosa, which may be predominated by factors other than CTL responses such as target cell availability. The finding of a functionally distinct population within gut mucosa offers some insight into how HIV-1 may persist in the gut despite fully suppressed plasma viremia on cART.

A new system to measure and compare hepatitis C virus replication capacity using full-length, replication competent viruses.

Measuring the in vitro replication capacity of viruses is an important tool for assessing the effects of selective pressure of immune responses and drug therapy. Measuring hepatitis C virus (HCV) replication capacity utilizing primarily sub-genomic reporter constructs is limited. To overcome some of these limitations a quantitative reverse transcriptase PCR (RT-qPCR) was designed to measure simultaneously the growth rate of 2 whole genome HCV variants under identical culture conditions. The assay demonstrates 100% specificity of detection of each variant and a linear detection range from 200 to 2×10(8) copies. The system was validated using a panel of HCV mutants, including the NS3 protease inhibitor drug resistance mutants R155K and T54A. The creation of a unique sequence tag results in highly sensitive and specific discrimination of parental JFH-FNX and modified clones using distinct probes in a RT-qPCR allowing for comparison of the effect of drug resistance or immune escape mutations on HCV replication. This system has advantages over existing methods both by permitting direct comparison of the replication capacity of fully replication-competent HCV mutants under identical culture conditions and by measuring effects on replication capacity due to mutations affecting all stages of the viral life cycle including entry and egress.

Combination of allele-specific detection techniques to quantify minority resistance variants in hepatitis B infection: a novel approach.

Detection of minor variant viral quasispecies of the rtV173L+rtL180M+rtM204V combination mutation in the hepatitis B virus (HBV) polymerase mediating both lamivudine resistance and vaccine escape is potentially important for tracking the development and evolution of resistance within both individuals and populations. A highly sensitive and specific assay to quantitate HBV genomes was developed with this mutation combination directly from viral DNA in serum using allele-specific quantitative PCR with locked nucleic acid primers and a minor groove binder probe. This combination of primers and probe yields a linear detection range down to 150 copies. This strategy has 100% specificity even in mixtures of predominately wild type genomes. The assay accurately detected 3×10² copies of the triple mutant spiked into 3 × 108 copies of the wild-type genomes (0.0001%), while maintaining 100% specificity. This approach was validated using serum from a subject infected with known lamivudine-resistant HBV. The triple mutant viral population was quantitated at 2.86 × 108 copies/ml within a total viral concentration of 1.03 × 10¹° copies/ml of serum (2.8%). This quantitative allele-specific PCR strategy therefore is a useful method for highly sensitive and specific detection of point mutation combinations that are clinically important in the pathogenesis of drug resistance and/or immune escape.

Immune selection in vitro reveals human immunodeficiency virus type 1 Nef sequence motifs important for its immune evasion function in vivo.

Human immunodeficiency virus type 1 (HIV-1) Nef downregulates major histocompatibility complex class I (MHC-I), impairing the clearance of infected cells by CD8(+) cytotoxic T lymphocytes (CTLs). While sequence motifs mediating this function have been determined by in vitro mutagenesis studies of laboratory-adapted HIV-1 molecular clones, it is unclear whether the highly variable Nef sequences of primary isolates in vivo rely on the same sequence motifs. To address this issue, nef quasispecies from nine chronically HIV-1-infected persons were examined for sequence evolution and altered MHC-I downregulatory function under Gag-specific CTL immune pressure in vitro. This selection resulted in decreased nef diversity and strong purifying selection. Site-by-site analysis identified 13 codons undergoing purifying selection and 1 undergoing positive selection. Of the former, only 6 have been reported to have roles in Nef function, including 4 associated with MHC-I downregulation. Functional testing of naturally occurring in vivo polymorphisms at the 7 sites with no previously known functional role revealed 3 mutations (A84D, Y135F, and G140R) that ablated MHC-I downregulation and 3 (N52A, S169I, and V180E) that partially impaired MHC-I downregulation. Globally, the CTL pressure in vitro selected functional Nef from the in vivo quasispecies mixtures that predominately lacked MHC-I downregulatory function at the baseline. Overall, these data demonstrate that CTL pressure exerts a strong purifying selective pressure for MHC-I downregulation and identifies novel functional motifs present in Nef sequences in vivo.

Partial escape of HIV-1 from cytotoxic T lymphocytes during chronic infection.

Viral mutational escape from CD8(+) cytotoxic T lymphocytes (CTLs) is typically considered to be a dichotomous process and uncommon during chronic HIV-1 infection. Ex vivo passaging of HIV-1 from persons with chronic infection, however, revealed the evolution of many fixed substitutions within and around CTL-targeted regions, with an associated increase in replicative capacity. This indicates an evolution of mutations during chronic HIV-1 infection that trade replicative fitness for incomplete evasion of CTLs, or "partial escape."

Human leukocyte antigen class I haplotypes of human immunodeficiency virus-1-infected persons on Likoma Island, Malawi.

Human leukocyte antigen (HLA) allele frequencies vary between different human populations, with implications both for the evolutionary pressures shaping those populations as well as for the outcome of new infectious epidemics. We defined HLA class I types in a well-described cohort of persons on Likoma Island in Malawi, a population for which there are lacking data on allelic frequencies. The profile of HLA frequencies was similar but phylogenetically distinct from those of other sub-Saharan African populations in neighboring regions. The most common A alleles included A30, A23, A28 (A*68), and A2, and the most common B alleles included B15 (group), B53, B58, and B44. Notably, the frequency of B53, which is protective against malaria, was similar to that of other malaria-endemic African countries, and higher than that in countries with less malaria. This is the first reported significant dataset of HLA class I allelic frequencies in Malawians.

Risk factors for infection and colonization with community-associated methicillin-resistant Staphylococcus aureus in the Los Angeles County jail: a case-control study.

BACKGROUND: Community-associated methicillin-resistant Staphylococcus aureus (MRSA) infections and outbreaks occur in correctional facilities, such as jails and prisons. Spread of these infections can be extremely difficult to control. Development of effective prevention protocols requires an understanding of MRSA risk factors in incarcerated persons. METHODS: We performed a case-control study investigating behavioral risk factors associated with MRSA infection and colonization. Case patients were male inmates with confirmed MRSA infection. Control subjects were male inmates without skin infection. Case patients and control subjects completed questionnaires and underwent collection of nasal swab samples for culture for MRSA. Microbiologic analysis was performed to characterize recovered MRSA isolates. RESULTS: We enrolled 60 case patients and 102 control subjects. Of the case patients, 21 (35%) had MRSA nasal colonization, compared with 11 control subjects (11%) (P .001). Among MRSA isolates tested, 100% were the USA300 strain type. Factors associated with MRSA skin infection included MRSA nares colonization, lower educational level, lack of knowledge about "Staph" infections, lower rate of showering in jail, recent skin infection, sharing soap with other inmates, and less preincarceration contact with the health care system. Risk factors associated with MRSA colonization included antibiotic use in the previous year and lower rate of showering. CONCLUSIONS: We identified several risks for MRSA infection in male inmates, many of which reflected preincarceration factors, such as previous skin infection and lower educational level. Some mutable factors, such as showering frequency, knowledge about Staph, and soap sharing, may be targets for intervention to prevent infection in this vulnerable population.

Simultaneous assessment of CD4 and MHC-I downregulation by Nef primary isolates in the context of infection.

The HIV-1 Nef protein plays a key role in pathogenesis, as demonstrated by strong selective pressure to maintain its open reading frame, and disease attenuation when it is deleted. Among myriad cellular effects attributed to Nef, downregulation of cell surface CD4 and major histocompatibility complex class I (MHC-I) proteins are the best documented. However, few data regarding primary isolate Nef functions are available, and most studies have been performed using transient transfections to express Nef driven by a non-physiologic promoter. A novel assay system to measure simultaneously the downregulation of CD4 and MHC-I by primary HIV-1 nef in a more physiologic viral genomic context is presented. Examination of plasma nef mixtures allowed comprehensive profiling of these Nef functions within the quasispecies in vivo. Subsets within the circulating nef population were observed that are either fully functional or non-functional. These data demonstrated that this assay system allows rapid characterization of bulk and clonal Nef functional profiles that can be used in pathogenesis studies to define further its important role in pathogenesis.

Functional adaptation of Nef to the immune milieu of HIV-1 infection in vivo.

Nef-mediated down-regulation of MHC class I (MHC-I) molecules on HIV-1-infected cells has been proposed to enhance viral persistence through evasion of host CTLs. This conclusion is based largely on demonstrations that Nef from laboratory HIV-1 strains reduces the susceptibility of infected cells to CTL killing in vitro. However, the function and role of Nef-mediated MHC-I down-regulation in vivo have not been well described. To approach this issue, nef quasispecies from chronically HIV-1-infected individuals were cloned into recombinant reporter viruses and tested for their ability to down-regulate MHC-I molecules from the surface of infected cells. The level of function varied widely between individuals, and although comparison to the immunologic parameters of blood CD4(+) T lymphocyte count and breadth of the HIV-1-specific CTL response showed positive correlations, no significant correlation was found in comparison to plasma viremia. The ability of in vivo-derived Nef to down-regulate MHC-I predicted the resistance of HIV-1 to suppression by CTL. Taken together, these data demonstrate the functionality of Nef to down-regulate MHC-I in vivo during stable chronic infection, and suggest that this function is maintained by the need of HIV-1 to cope with the antiviral CTL response.

Primary human immunodeficiency virus type 1 (HIV-1) infection during HIV-1 Gag vaccination.

Vaccination for human immunodeficiency virus type 1 (HIV-1) remains an elusive goal. Whether an unsuccessful vaccine might not only fail to provoke detectable immune responses but also could actually interfere with subsequent natural immunity upon HIV-1 infection is unknown. We performed detailed assessment of an HIV-1 gag DNA vaccine recipient (subject 00015) who was previously uninfected but sustained HIV-1 infection before completing a vaccination trial and another contemporaneously acutely infected individual (subject 00016) with the same strain of HIV-1. Subject 00015 received the vaccine at weeks 0, 4, and 8 and was found to have been acutely HIV-1 infected around the time of the third vaccination. Subject 00016 was a previously HIV-1-seronegative sexual contact who had symptoms of acute HIV-1 infection approximately 2 weeks earlier than subject 00015 and demonstrated subsequent seroconversion. Both individuals reached an unusually low level of chronic viremia (<1,000 copies/ml) without treatment. Subject 00015 had no detectable HIV-1-specific cytotoxic T-lymphocyte (CTL) responses until a borderline response was noted at the time of the third vaccination. The magnitude and breadth of Gag-specific CTL responses in subject 00015 were similar to those of subject 00016 during early chronic infection. Viral sequences from gag, pol, and nef confirmed the common source of HIV-1 between these individuals. The diversity and divergence of sequences in subjects 00015 and 00016 were similar, indicating similar immune pressure on these proteins (including Gag). As a whole, the data suggested that while the gag DNA vaccine did not prime detectable early CTL responses in subject 00015, vaccination did not appreciably impair his ability to contain viremia at levels similar to those in subject 00016.

Thymus-derived leukemia-lymphoma in mice transgenic for the Tax gene of human T-lymphotropic virus type I.

Adult T-cell leukemia-lymphoma (ATLL) is a group of T-cell malignancies caused by infection with human T-lymphotropic virus type I (HTLV-I). Although the pathogenesis of ATLL remains incompletely understood, the viral regulatory protein Tax is centrally involved in cellular transformation. Here we describe the generation of HTLV-I Tax transgenic mice using the Lck proximal promoter to restrict transgene expression to developing thymocytes. After prolonged latency periods, transgenic mice developed diffuse large-cell lymphomas and leukemia with clinical, pathological and immunological features characteristic of acute ATLL. Transgenic mice were functionally immunocompromised and they developed opportunistic infections. Fulminant disease also developed rapidly in SCID mice after engraftment of lymphomatous cells from transgenic mice. Flow cytometry showed that the cells were CD4(-) and CD8(-), but CD44(+), CD25(+) and cytoplasmic CD3(+). This phenotype is indicative of a thymus-derived pre-T-cell phenotype, and disease development was associated with the constitutive activation of NF-kappaB. Our model accurately reproduces human disease and will provide a tool for analysis of the molecular events in transformation and for the development of new therapeutics.

Comparison of CREB- and NF-kappaB-mediated transactivation by human T lymphotropic virus type II (HTLV-II) and type I (HTLV-I) tax proteins.

The function of the transactivator protein Tax from HTLV-II subtype A, subtype B, Brazilian subtype C, and African subtype D isolates was compared to that of Tax from an HTLV-I isolate. HTLV-II subtypes A, B, and C were less active in the transactivation of a NF-kappaB reporter compared to HTLV-I Tax in 293T but not Jurkat T cells. In both cell types there were no significant differences between the functions of HTLV-II B, C, and D and HTLV-I Tax proteins on either a full-length HTLV-I LTR or a 21-bp repeat reporter, suggesting that there is equivalent CREB-mediated transactivation between the viruses and these subtypes. In contrast, Tax of some but not all HTLV-II subtype A isolates, including the prototype Mo, had a greatly decreased ability to transactivate, and this could be directly correlated with a decrease in protein expression. Employment of cDNA clones encoding both Rex and Tax demonstrated that Rex was unable to rescue the expression or activity of the IIA Mo isolate. These studies demonstrate that with the exception of some HTLV-IIA subtypes there are no significant differences in Tax transactivation via the CREB and NF-kappaB pathways between the two viruses and suggest that the HTLV-II Tax may have a pathogenic potential equivalent to that of HTLV-I.