Efficient use of longitudinal CD4 counts and viral load measures in survival analysis.

CD4 counts and viral loads are dynamic quantities that change with time in HIV-infected persons. Commonly used single summary measures, such as viral load set point or early CD4 count, do not explicitly account for changes in viral load or CD4 counts or other features of the overall time course of these measures. However, the efficient use of all repeated measurements within each subject is often a challenge made more difficult by sparse and irregular sampling over time. Here, we illustrate how functional principal component (FPC) analysis provides an effective statistical approach for exploiting the patterns in CD4 count and viral load data over time. We demonstrate the method by using data from Kenyan women who acquired HIV-1 during follow-up in a cohort that practices high-risk activities and were subsequently followed up prospectively from early infection. The FPC scores for each woman obtained using this method served as informative summary statistics for the CD4 count and viral load trajectories. Similar to baseline CD4 count or viral set point, the first FPC score can be interpreted as a single-value summary measure of an individual's overall CD4 count or viral load. However, unlike most single-value summaries of CD4 count or viral load trajectories, the first FPC score summarizes the dynamics of these quantities and is seen to reveal specific features of the trajectories associated with mortality in this cohort. Moreover, the FPC scores are shown to be a more powerful prognostic factor than other common summaries when used in survival analysis.

Emerging cytopathic and antigenic simian immunodeficiency virus variants influence AIDS progression.

Genetic variants of human and simian immunodeficiency virus (HIV and SIV) that evolve during the course of infection and progression to AIDS are phenotypically and antigenically distinct from their progenitor viruses present at early stages of infection. However, it has been unclear how these late variants, which are typically T-cell tropic, cytopathic and resistant to neutralizing antibodies, influence the development of clinical AIDS. To address this, we infected macaques with cloned SIVs representing prototype variants from early-, intermediate- and late-stage infection having biological characteristics typical of viruses found at similar stages of HIV infection in humans. These studies demonstrate that sequential, phenotypic and antigenic variants represent viruses that have become increasingly fit for replication in the host, and our data support the hypothesis that emerging variants have increased pathogenicity and drive disease progression in SIV and HIV infection.

Gender differences in HIV-1 diversity at time of infection.

To develop an HIV-1 vaccine with global efficacy, it is important to identify and characterize the viruses that are transmitted, particularly to individuals living in areas of high incidence. Several studies have shown that virus from the blood of acutely infected adults was homogeneous, even when the virus population in the index case was genetically diverse. In contrast to those results with mainly male cohorts in America and Europe, in several cases a heterogeneous virus population has been found early in infection in women in Africa. Thus, we more closely compared the diversity of transmitted HIV-1 in men and women who became infected through heterosexual contact. We found that women from Kenya were often infected by multiple virus variants, whereas men from Kenya were not. Moreover, a heterogeneous virus was present in the women before their seroconversion, and in each woman it was derived from a single index case, indicating that diversity was most likely to be the result of transmission of multiple variants. Our data indicate that there are important differences in the transmitted virus populations in women and men, even when cohorts from the same geographic region who are infected with the same subtypes of HIV-1 are compared.

Virus entry via the alternative coreceptors CCR3 and FPRL1 differs by human immunodeficiency virus type 1 subtype.

Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding to CD4 and a chemokine receptor, most commonly CCR5. CXCR4 is a frequent alternative coreceptor (CoR) in subtype B and D HIV-1 infection, but the importance of many other alternative CoRs remains elusive. We have analyzed HIV-1 envelope (Env) proteins from 66 individuals infected with the major subtypes of HIV-1 to determine if virus entry into highly permissive NP-2 cell lines expressing most known alternative CoRs differed by HIV-1 subtype. We also performed linear regression analysis to determine if virus entry via the major CoR CCR5 correlated with use of any alternative CoR and if this correlation differed by subtype. Virus pseudotyped with subtype B Env showed robust entry via CCR3 that was highly correlated with CCR5 entry efficiency. By contrast, viruses pseudotyped with subtype A and C Env proteins were able to use the recently described alternative CoR FPRL1 more efficiently than CCR3, and use of FPRL1 was correlated with CCR5 entry. Subtype D Env was unable to use either CCR3 or FPRL1 efficiently, a unique pattern of alternative CoR use. These results suggest that each subtype of circulating HIV-1 may be subject to somewhat different selective pressures for Env-mediated entry into target cells and suggest that CCR3 may be used as a surrogate CoR by subtype B while FPRL1 may be used as a surrogate CoR by subtypes A and C. These data may provide insight into development of resistance to CCR5-targeted entry inhibitors and alternative entry pathways for each HIV-1 subtype.

Infants with late breast milk acquisition of HIV-1 generate interferon-gamma responses more rapidly than infants with early peripartum acquisition.

Infants infected with HIV-1 after the first month of life have a lower viral set-point and slower disease progression than infants infected before 1 month. We investigated the kinetics of HIV-1-specific CD8(+) T lymphocyte secretion of interferon (IFN)-gamma in infants infected before 1 month of life compared with those infected between months 1 and 12 (late infection). HIV-1 infection was assessed at birth and at months 1, 3, 6, 9 and 12 and timing of infection was determined by HIV-1 gag DNA from dried blood spots and verified by plasma HIV-1 RNA levels. HIV-1 peptide-specific IFN-gamma responses were measured by enzyme-linked immunospot at months 1, 3, 6, 9 and 12. Timing of development of IFN-gamma responses was compared using the log-rank test and Kaplan-Meier survival curves. Infants infected late developed HIV-1-specific CD8(+) T cell responses 2.8 months sooner than infants infected peripartum: 2.3 versus 5.1 months after HIV-1 infection (n = 52, P = 0.04). Late-infected infants had more focused epitope recognition than early-infected infants (median 1 versus 2 peptides, P = 0.03); however, there were no differences in the strength of IFN-gamma responses. In infants infected with HIV-1 after the first month of life, emergence of HIV-1-specific CD8(+) IFN-gamma responses is coincident with the decline in viral load, nearly identical to what is observed in adults and more rapid than in early-infected infants.

A prospective study of risk factors for herpes simplex virus type 2 acquisition among high-risk HIV-1 seronegative women in Kenya.

OBJECTIVES: Several studies have demonstrated an association between herpes simplex virus type 2 (HSV-2) and HIV-1, but available data on risk factors for HSV-2 acquisition are limited. The objective of this analysis was to determine the incidence and risk factors for HSV-2 acquisition among HIV-1-seronegative female sex workers in Kenya. METHODS: Between February 1993 and December 2006, HIV-1-seronegative women attending a municipal sexually transmitted infection (STI) clinic were invited to enroll in a prospective cohort study. Screening for HIV-1 and STIs were done at monthly follow-up visits. Archived blood samples were tested for HSV-2. RESULTS: Of 1527 HIV-1-seronegative women enrolled, 302 (20%) were HSV-2 seronegative at baseline of whom 297 had at least one follow-up visit. HSV-2 incidence was high (23 cases/100 person-years; 115 cases). In multivariate analysis, HSV-2 was significantly associated with more recent entry into sex work, workplace and higher number of sex partners per week. Condom use was protective, although this was statistically significant only for the intermediate strata (25-75% condom use; HR 0.43; p = 0.05). There were statistical trends for bacterial vaginosis to increase HSV-2 risk (HR 1.56; p = 0.07) and for oral contraceptive use to decrease risk (HR 0.50; p = 0.08). The 23% annual HSV-2 incidence in this study is among the highest reported anywhere in the world. CONCLUSIONS: Women were at increased risk if they had recently entered sex work, had a higher number of sex partners or worked in bars. HSV-2 risk reduction interventions are urgently needed among high-risk African women.

Selection forces and constraints on retroviral sequence variation.

All retroviruses possess a highly error-prone reverse transcriptase, but the extent of the consequent sequence diversity and the rate of evolution differ greatly among retroviruses. Because of the high mutability of retroviruses, it is not the generation of new viral variants that limits the extent of diversity and the rate of evolution of retroviruses, but rather the selection forces that act on these variants. Here, we suggest that two selection forces--the immune response and the limited availability of appropriate target cells during transmission and persistence--are chiefly responsible for the observed sequence diversity in untreated retroviral infections. We illustrate these aspects of positive selection by reference to specific lentiviruses [human and simian immunodeficiency viruses (HIV and SIV)] and oncoviruses [feline leukemia virus (FeLV) and human T cell leukemia virus (HTLV)] that differ in their extent of variation and in disease outcomes.

Molecular cloning of a feline leukemia virus that induces fatal immunodeficiency disease in cats.

A replication-defective variant of feline leukemia virus was molecularly cloned directly from infected tissue and found to induce a rapid and fatal immunodeficiency syndrome in cats. Studies with cloned viruses also showed that subtle mutational changes would convert a minimally pathogenic virus into one that would induce an acute form of immunodeficiency. The data suggest that acutely pathogenic viruses may be selected against by current methods for isolation of the human and simian immunodeficiency viruses.

Identification of a cellular cofactor required for infection by feline leukemia virus.

Retroviral infection involves continued genetic variation, leading to phenotypic and immunological selection for more fit virus variants in the host. For retroviruses that cause immunodeficiency, pathogenesis is linked to the emergence of T cell-tropic, cytopathic viruses. Here we show that an immunodeficiency-inducing, T cell-tropic feline leukemia virus (FeLV) has evolved such that it cannot infect cells unless both a classic multiple membrane-spanning receptor molecule (Pit1) and a second coreceptor or entry factor are present. This second receptor component, which we call FeLIX, was identified as an endogenously expressed protein that is similar to a portion of the FeLV envelope protein. This cellular protein can function either as a transmembrane protein or as a soluble component to facilitate infection.

Receptors and entry cofactors for retroviruses include single and multiple transmembrane-spanning proteins as well as newly described glycophosphatidylinositol-anchored and secreted proteins.

In the past few years, many retrovirus receptors, coreceptors, and cofactors have been identified. These molecules are important for some aspects of viral entry, although in some cases it remains to be determined whether they are required for binding or postbinding stages in entry, such as fusion. There are certain common features to the molecules that many retroviruses use to gain entry into the cell. For example, the receptors for most mammalian oncoretroviruses are multiple membrane-spanning transport proteins. However, avian retroviruses use single-pass membrane proteins, and a sheep retrovirus uses a glycosylphosphatidylinositol-anchored molecule as its receptor. For some retroviruses, particularly the lentiviruses, two cell surface molecules are required for efficient entry. More recently, a soluble protein that is required for viral entry has been identified for a feline oncoretrovirus. In this review, we will focus on the various strategies used by mammalian retroviruses to gain entry into the cell. The choice of receptors will also be discussed in light of pressures that drive viral evolution and persistence.

flvi-2, a target of retroviral insertional mutagenesis in feline thymic lymphosarcomas, encodes bmi-1.

LC-FeLV is a myc-containing strain of feline leukemia virus which induces thymic lymphosarcoma in the domestic cat with short latency. A locus in feline DNA, termed flvi-2, is commonly interrupted in naturally occurring and experimentally induced thymic lymphosarcomas containing LC-FeLV; thus, interruption of a gene encoded by flvi-2 may cooperate with the myc oncogene in the induction of T-cell tumors by LC-FeLV. Clones homologous to flvi-2 have been isolated from a normal human thymus cDNA library. Nucleotide sequence analysis of the cDNA clones demonstrates that flvi-2 encodes bmi-1, a gene previously identified as a target for MoMuLV integration and as a myc-collaborator in retrovirally-induced B-cell lymphomas in E mu-myc transgenic mice. In feline thymic lymphomas, retroviral integrations occur downstream of the gene, and result in enhanced expression of a bmi-1 transcript of normal size. These findings demonstrate the interruption of bmi-1 in natural as well as experimentally induced tumors, implicate the activation of bmi-1 in the induction of T-cell as well as B-cell lymphoma, and support the premise that bmi-1 functions as a myc collaborator.

Viral genetic variation, AIDS, and the multistep nature of carcinogenesis: the feline leukemia virus model.

Feline leukemia virus (FeLV) infection in cats serves as a valuable animal model system for understanding the mechanisms of human diseases such as cancer and immunodeficiency. We have used experimental infection with molecularly cloned viruses to isolate and characterize novel FeLV variants that evolved in vivo and that were associated with the development of thymic lymphoma. One variant, FeLV-81T, contained a mutated envelope gene that conferred cytopathicity, enhanced replication rate, and syncytium induction in feline T cells, and is reminiscent of immunodeficiency-inducing strains of FeLV. Another variant transduced a portion of the feline Notch2 gene, which was expressed as a novel truncated protein in the cell nucleus and which we believe functioned as an oncogene in the development of T cell malignancy. Understanding how FeLV variants that either stimulate or destroy lymphocytes evolve and interrelate during disease progression will help elucidate the mechanisms of retroviral pathogenicity.

Effect of intrauterine device use on cervical shedding of HIV-1 DNA.

OBJECTIVE: Hormonal contraception has been associated with an increased prevalence of cervical shedding of HIV-1 DNA among infected women. We conducted this study to evaluate the effect of the use of an intrauterine device (IUD) on the detection of HIV-1 DNA in cervical secretions. DESIGN: A prospective study of HIV-1-seropositive women undergoing IUD insertion at two public family planning clinics in Nairobi, Kenya. METHODS: Cervical swab samples were collected before IUD insertion and approximately 4 months thereafter for the detection of HIV-1-infected cells using polymerase chain reaction (PCR) amplification of HIV-1 gag DNA sequences. RESULTS: Ninety-eight women were enrolled and followed after IUD insertion. The prevalence of HIV-1 DNA cervical shedding was 50% at baseline and 43% at follow-up [odds ratio (OR) 0.8, 95% confidence interval (CI) 0.5-1.2]. There was no statistically significant difference between the baseline and follow-up shedding rates in a multivariate model that controlled for previous hormonal contraceptive use, condom use, cervical ectopy, friable cervix, cervical infections at an interim visit, and CD4 lymphocyte levels (OR 0.6, 95% CI 0.3-1.1). CONCLUSION: The insertion of an IUD did not significantly alter the prevalence of cervical shedding of HIV-1-infected cells. The use of IUDs, in conjunction with condoms, may be an appropriate method of contraception for HIV-1-infected women from the standpoint of potential infectivity to the male partner through exposure to genital HIV-1.

Treatment of cervicitis is associated with decreased cervical shedding of HIV-1.

OBJECTIVE: To determine whether cervical mucosal shedding of HIV-1 RNA and HIV-1 infected cells decreases following successful treatment of cervicitis. DESIGN: Prospective interventional study. SETTING: Sexually Transmitted Infections Clinic, Coast Provincial General Hospital, Mombasa, Kenya. PARTICIPANTS: Thirty-six HIV-1 seropositive women with cervicitis: 16 with Neisseria gonorrhoeae, seven with Chlamydia trachomatis, and 13 with non-specific cervicitis. INTERVENTIONS: Treatment of cervicitis. MAIN OUTCOME MEASURES: Levels of total (cell-free and cell-associated) HIV-1 RNA and presence of HIV-1 DNA (a marker for infected cells) in cervical secretions before and after resolution of cervicitis. RESULTS: After treatment of cervicitis, the median HIV-1 RNA concentration in cervical secretions was reduced from 4.05 to 3.24 log10 copies/swab (P = 0.001). Significant decreases in cervical HIV-1 RNA occurred in the subgroups with N. gonorrhoeae (3.94 to 3.28 log10 copies/swab; P = 0.02) and C. trachomatis (4.21 to 3.19 log10 copies/swab; P = 0.02). Overall, the prevalence of HIV-1 infected cells in cervical secretions also decreased after treatment, from 67% to 42% (odds ratio, 2.8; 95% confidence interval, 1.3-6.0; P = 0.009). Detection of infected cells was associated with higher mean HIV-1 RNA levels (4.04 versus 2.99 log10 copies/swab; P< 0.0001). CONCLUSIONS: Effective treatment of cervicitis resulted in significant decreases in shedding of HIV-1 virus and infected cells in cervical secretions. Treatment of sexually transmitted diseases may be an important means of decreasing the infectivity of HIV-1 seropositive women by reducing exposure to HIV-1 in genital secretions.

Lentiviral genomes with G-to-A hypermutation may result from Taq polymerase errors during polymerase chain reaction.

Retroviral genomes with a high frequency of G-to-A mutations are thought to originate during reverse transcription. Here we show that bursts of G-to-A mutation may also occur during DNA synthesis by Taq polymerase on a simian immunodeficiency proviral template. These G-to-A changes tend to occur at GpA and, to a lesser extent, GpG dinucleotides. Because the resulting sequences are like previously reported hypermutant human and simian immunodeficiency virus (HIV and SIV) genomes, it is important to design experiments that can clearly discriminate between Taq and reverse transcripts errors in studies of lentiviral G-to-A hypermutation.

Distinct but related human immunodeficiency virus type 1 variant populations in genital secretions and blood.

For a HIV vaccine to be effective, it will be essential that it protect against the virus variants to which individuals are most frequently exposed. HIV-1 is predominantly a sexually acquired virus, thus, variants in genital secretions are a potentially important reservoir of viruses that are transmitted. Because there are no data available on variants in the genital mucosa, we analyzed this provirus population and compared it to the proviruses in the blood of individuals chronically infected with HIV-1. A major genetic difference between variants within a patient were insertions, which were apparently created by duplication of adjacent sequences, that resulted in acquisition of new potential glycosylation sites in V1 and V2. Comparisons of mucosal and PBMC variants suggest that these tissues harbor distinct, but related populations of HIV-1 variants. In two of three patients, the mucosal variants were most closely related to a minor variant genotype in blood. In a third individual, viruses in both tissues were surprisingly homogeneous, but the majority of variants in the cervix encoded a V1 sequence with a predicted glycosylation pattern similar to a minor variant in blood. The V3 sequence patterns of the mucosal isolates indicate they may be predominantly macrophage-tropic viruses.

Phylogenetic evaluation of Kenyan HIV type 1 isolates.

Diversity among global isolates of HIV-1 presents a formidable challenge for vaccine development. As distinct clades of the virus are recognized, it will be important to monitor their geographic distribution and divergence. In this study, we characterized HIV-1 subtypes from 17 seropositive individuals in Nairobi and Mombasa, Kenya. Seventy-one percent of viruses were clade A and 29% were clade D. The most divergent clade A isolate in our survey, Q45-CxA, grouped closely with two other taxa that were previously reported as having no distinct clade affiliation. Thus, these data may suggest the emergence of an outlier group of clade A variants or a new subtype of HIV-1. Phylogenetic relatedness of the 17 Kenyan isolates was determined separately for C2-V3 and V2 sequences of envelope and subtype designation for these isolates was independent of the region analyzed. However, evaluation of transitions, transversions, and specific character state changes indicated that mutations characterizing V2 differed from those in V3 for clade A and clade D isolates. Comparison of secondary structural characteristics of the V1-V3 region between a clade A and a clade D virus revealed conservation of motifs.

PIP: The authors characterized HIV-1 subtypes from 17 seropositive individuals in Nairobi and Mombasa, Kenya. 71% of the viruses were clade A and 29% were clade D. The most divergent clade A isolate identified in the study, Q45-CxA, grouped closely with two other taxa previously reported as having no distinct clade affiliation. These findings may therefore signal the emergence of an outlier group of clade A variants or a new subtype of HIV-1. The evaluation of transitions, transversions, and specific character state changes indicated that mutations characterizing V2 differed from those in V3 for clade A and clade D isolates. Comparison of the secondary structural characteristics of the V1-V3 region between a clade A and a clade D virus revealed conservation of motifs.

Lymphokines modulate the growth and survival of thymic tumor cells containing a novel feline leukemia virus/Notch2 variant.

Tumorigenesis occurs through a multistep process initiated by genetic lesions and facilitated by endogenous and external growth/survival signals. In many malignancies, specific oncogenic mutations correlate with phenotypic characteristics, inferring lineage-specific pathogenic mechanisms. To characterize these relationships in a unique feline tumor, we studied primary cells and two-cell lines independently-derived from a thymic lymphoma that contained and actively expressed a novel feline leukemia virus (FeLV) recombinant with transduced host Notch2 sequences. All three tumor cell populations contained similar FeLV/Notch2 proviral variants and phenotypically resembled mature thymocytes. Multiple Notch2 transcripts were expressed in the cell lines, including species that correspond to viral genomes and spliced subgenomic viral mRNA. Tumor cell line FeLV/Notch2 virus was packaged into virions; however, the variant was not efficiently transmitted to feline cells in vitro. Primary tumor cells constitutively expressed mRNA for interleukin-4 (IL-4), IL-6 and the p40 subunit of IL-12. Lymphokine mRNA was not detected in established tumor cell lines nor was T-cell growth-promoting activity found in culture supernatants. Exogenous IL-4 enhanced primary tumor cell survival, but inhibited proliferation of the cell lines. Interleukin-4 abrogated hydrocortisone-induced apoptosis in all three populations and had divergent effects on cell line clonogenic colony formation. Exogenous IL-7 and, to a lesser degree, IL-6 also had variable positive effects on the growth and viability of the tumor cell populations. Collectively, these data suggest that thymocytes are susceptible to the transforming potential of dysregulated Notch2 and that thymopoietic factors could, through overlapping and distinct mechanisms, promote the survival and outgrowth of FeLV/Notch2-containing neoplastic cells.

FeLV-FAIDS-induced immunodeficiency syndrome in cats.

Findings are reviewed, relevant to elucidation of the pathogenic, genetic and biochemical properties of a single, genetically heterogeneous isolate of feline leukemia virus (FeLV-FAIDS) shown to induce fatal immunodeficiency disease in nearly 100% of inoculated cats. Hypotheses are suggested which pertain to the mechanism of T-cell killing by this virus, and which extrapolate findings in the FeLV-FAIDS animal model to AIDS induced in humans by human immunodeficiency virus (HIV).

Protection against feline leukemia virus infection by use of an inactivated virus vaccine.

The protective immunity induced by 3 experimental FeLV vaccines were evaluated: Prototype inactivated FeLV vaccine developed from a molecularly cloned FeLV isolate (FeLV-FAIDS-61E-A); a mixture of immunodominant synthetic peptides corresponding to regions of the FeLV-Gardner-Arnstein-B (FeLV-GA-B) envelope proteins; and an adjuvant-disrupted but non-activated virus prepared from a non-cloned FeLV field isolate comprised of subgroup A and B viruses (FeLV-05821-AB). Included as controls were parallel groups of cats inoculated with adjuvants alone or with an established commercial FeLV vaccine. After each inoculation and after virulent virus challenge exposure, sera from all cats were assayed for ELISA-reactive antibody against purified FeLV, FeLV neutralizing (VN) antibody, and FeLV antigenemia/viremia--viral p27 antigen in serum and within circulating leukocytes. Immunity was challenged by oral/nasal exposure of vaccinated and control cats with FeLV-FAIDS-61E-A or FeLV-05821-AB, an infective, noncloned, tissue-origin, FeLV field isolate containing subgroup-A and -B viruses. Vaccine-induced immunity was assessed by comparing the postchallenge-exposure incidence of persistent viremia and the pre- and postchallenge exposure titers of VN and ELISA antibody in cats of the control and vaccine groups. The percentage of cats, that resisted development of persistent viremia after FeLV challenge exposure and the preventable fraction (PF) for the vaccine groups (which adjusts for the severity of the challenge and the degree of innate resistance in the controls) were as follows: adjuvant controls, 26%; FeLV-FAIDS-61E-A inactivated virus vaccine, 95% (PF = 93.2%); FeLV-GA-B peptide vaccine, 5% (-28.4%); FeLV-05821-AB noninactivated vaccine, 67% (55.4%); and commercial FeLV vaccine, 35% (12.2%). The prechallenge exposure mean VN antibody titer for each group was: less than 1:8 in the adjuvant controls; 1:43 in the FeLV-FAIDS-61E-A-vaccinated cats; less than 1:8 in the peptide-vaccinated cats; 1:38 in the noninactivated virus-vaccinated cats group; and 1:12 in the cats vaccinated with the commercial vaccine. Thus, induction of VN antibody in the vaccinated cats, although modest, appeared to be correlated with induction of protective immunity as defined by resistance to FeLV challenge exposure. Results of these studies indicate that inoculation of cats with an experimental inactivated virus vaccine prepared from a molecularly cloned FeLV isolate was most effective in stimulating protective immunity against heterologous and homologous FeLV challenge exposure.