25-Hydroxyvitamin D concentrations do not affect the humoral or cellular immune response following SARS-CoV-2 mRNA vaccinations.

BACKGROUND: To improve effectiveness of vaccination against SARS-CoV-2, it is important to identify factors that influence the immune response induced by vaccination. Evidence for the role of vitamin D in immune response against SARS-CoV-2 is contradictory. It is therefore of interest whether 25-hydroxyvitamin D (25[OH]D) concentrations affect the humoral and/or cellular response following SARS-CoV-2 vaccination. METHODS: In this prospective cohort study, blood samples were collected from 98 SARS-CoV-2 naive health care workers (HCW) receiving the first two doses of either BNT162b2 or mRNA-1273 in 2021. Wild-type spike (S) protein binding and neutralizing antibodies were determined approximately three weeks after the first dose and four to five weeks after the second dose. Antigen specific T-cells and functionality (proliferative response and interferon gamma [IFN-γ] release) were determined in 18 participants four weeks after the second dose of BNT162b2. We studied the association between 25(OH)D concentrations, which were determined prior to vaccination, and humoral and cellular immune responses following vaccination. RESULTS: We found no association between 25(OH)D concentrations (median 55.9 nmol/L [IQR 40.5-69.8]) and binding or neutralizing antibody titers after complete vaccination (fold change of antibody titers per 10 nmol/L 25(OH)D increase: 0.98 [95% CI 0.93-1.04] and 1.03 [95% CI: 0.96-1.11], respectively), adjusted for age, sex and type of mRNA vaccine. Subsequently, continuous 25(OH)D concentrations were divided into commonly used clinical categories (<25 nmol/L [n = 6, 6%], 25-49 nmol/L [n = 33, 34%], 50-75 nmol/L [n = 37, 38%] and ≥75 nmol/L [n = 22, 22%]), but no association with the humoral immune response following vaccination was found. Also, 25(OH)D concentrations were not associated with the SARS-CoV-2 specific T cell response. CONCLUSION: No association was found between 25(OH)D concentrations and the humoral or cellular immune response following mRNA vaccination against SARS-CoV-2. Based on our findings there is no rationale to advise vitamin D optimization preceding SARS-CoV-2 vaccination in HCW with moderate vitamin D status.

HIV-associated gut dysbiosis is independent of sexual practice and correlates with noncommunicable diseases.

Loss of gut mucosal integrity and an aberrant gut microbiota are proposed mechanisms contributing to chronic inflammation and increased morbidity and mortality during antiretroviral-treated HIV disease. Sexual practice has recently been uncovered as a major source of microbiota variation, potentially confounding prior observations of gut microbiota alterations among persons with HIV (PWH). To overcome this and other confounding factors, we examine a well-powered subset of AGEhIV Cohort participants comprising antiretroviral-treated PWH and seronegative controls matched for age, body-mass index, sex, and sexual practice. We report significant gut microbiota differences in PWH regardless of sex and sexual practice including Gammaproteobacteria enrichment, Lachnospiraceae and Ruminococcaceae depletion, and decreased alpha diversity. Men who have sex with men (MSM) exhibit a distinct microbiota signature characterized by Prevotella enrichment and increased alpha diversity, which is linked with receptive anal intercourse in both males and females. Finally, the HIV-associated microbiota signature correlates with inflammatory markers including suPAR, nadir CD4 count, and prevalence of age-associated noncommunicable comorbidities.

Systemic and intrathecal immune activation in association with cerebral and cognitive outcomes in paediatric HIV.

Despite treatment, immune activation is thought to contribute to cerebral injury in children perinatally infected with human immunodeficiency virus (HIV). We aimed to characterize immune activation in relation to neuroimaging and cognitive outcomes. We therefore measured immunological, coagulation, and neuronal biomarkers in plasma and cerebrospinal fluid (CSF) samples of 34 perinatally HIV-infected children aged 8-18 years, and in plasma samples of 37 controls of comparable age, sex, ethnicity, and socio-economic status. We then compared plasma biomarker levels between groups, and explored associations between plasma/CSF biomarkers and neuroimaging and cognitive outcomes using network analysis. HIV-infected children showed higher plasma levels of C-reactive protein, interferon-gamma, interferon-gamma-inducible protein-10, and monocyte chemoattractant protein-1 than controls. In HIV-infected participants, plasma soluble CD14 was positively associated with microstructural white matter (WM) damage, and plasma D-dimer was negatively associated with WM blood flow. In CSF, IL-6 was negatively associated with WM volume, and neurofilament heavy-chain (NFH) was negatively associated with intelligence quotient and working memory. These markers of ongoing inflammation, immune activation, coagulation, and neuronal damage could be used to further evaluate the pathophysiology and clinical course of cerebral and cognitive deficits in perinatally acquired HIV.

NK cells in self-limited HCV infection exhibit a more extensively differentiated, but not memory-like, repertoire.

Natural killer (NK) cells have long been thought of as a purely innate immune cell population, but increasing reports have described developmental and functional qualities of NK cells that are commonly associated with cells of the adaptive immune system. Of these features, the ability of NK cells to acquire functional qualities associated with immunological memory and continuous differentiation resulting in the formation of specific NK cell repertoires has recently been highlighted in viral infection settings. By making use of a unique cohort of monitored, at-risk intravenous drug users in this study, we were able to dissect the phenotypic and functional parameters associated with NK cell differentiation and NK cell memory in patients 3 years after acute HCV infection and either the subsequent self-clearance or progression to chronicity. We observed increased expression of cytolytic mediators and markers CD56bright and NKp46+ of NK cells in patients with chronic, but not self-limited HCV infection. Patients with a self-limited infection expressed higher levels of differentiation-associated markers CD57 and KIRs, and lower levels of NKG2A. A more extensively differentiated NK cell phenotype is associated with self-clearance in HCV patients, while the NK cells of chronic patients exhibited more naïve and effector NK cell phenotypic and functional characteristics. The identification of these distinct NK cell repertoires may shed light on the role NK cells play in determining the outcome of acute HCV infections, and the underlying immunological defects that lead to chronicity.

Intrahepatic IP-10 mRNA and plasma IP-10 levels as response marker for HBeAg-positive chronic hepatitis B patients treated with peginterferon and adefovir.

INTRODUCTION: Interferon-y-inducible protein-10 (IP-10), also called CXCL10, is produced by different types of cells such as monocytes, neutrophils and hepatocytes. IP-10 functions as an inflammatory cytokine, which after binding to its receptor CXCR3, expressed on T-lymphocytes, leads to immune activation. We aimed to establish if IP-10 expression in liver tissue and in plasma of chronic hepatitis B (CHB) patients correlated with each other and further to investigate if IP-10 levels before and during therapy with peginterferon and adefovir could predict treatment outcome in CHB patients. PATIENTS AND METHODS: A total of 86 CHB patients (41 HBeAg-positive and 45 HBeAg-negative) received combination therapy of peginterferon and adefovir for 48 weeks. Combined Response (CR) (HBeAg-negativity, HBV-DNA ≤ 2000 IU/mL, ALT normalization) and non-response (NR) were assessed at Week 72. Plasma IP-10 levels were measured at baseline and during treatment at Day 3 (D3) and Week 1 (W1). Pre-treatment liver biopsies from 40 of 86 patients were obtained and stored in liquid nitrogen for the analysis of intrahepatic IP-10 mRNA expression. RESULTS: CR was achieved in 14/41 HBeAg-positive and 17/45 HBeAg-negative patients. Mean baseline plasma IP-10 levels were significantly higher in HBeAg-positive patients with CR than NR (3.20 vs 3.00 log pg/mL p = 0.03); but not in HBeAg-negative patients. Baseline IP-10 levels correlated with ALT-levels in HBeAg-positive and -negative patients (both p < 0.001), and with a decline of HBsAg-levels of ≥0.5 log IU/mL at Week 12 in HBeAg-positive patients (p = 0.001). Plasma IP-10 levels were associated with intrahepatic IP-10 mRNA expression, however, more strongly in HBeAg-positive (R = 0.79, p < 0.001) than in HBeAg-negative patients (R = 0.53, p = 0.011). IP-10 levels only correlated with HAI-scores in HBeAg-positive patients (R = 0.40 p = 0.025). Mean plasma IP-10 levels of both HBeAg-positive and -negative patients increased significantly at D3 compared to baseline (+0.30 log pg/mL p = 0.003), to then decline subsequently at W1 to a level still significantly higher than baseline (+0.14 log pg/mL p < 0.001). The increase of IP-10 was significantly higher in HBeAg-positive patients with NR than in those with CR (+0.35 versus +0.11 log pg/mL p = 0.003). CONCLUSIONS: Baseline plasma IP-10 levels and IP-10 mRNA expression in the liver are correlated with each other, suggesting that plasma IP-10 reflects intrahepatic immune activation. Higher IP-10 levels at baseline seem to be associated with CR in HBeAg-positive patients treated with peginterferon and adefovir, but not in HBeAg-negative patients.

HLA-C and KIR combined genotype as new response marker for HBeAg-positive chronic hepatitis B patients treated with interferon-based combination therapy.

Current treatment for chronic hepatitis B infection (CHB) consists of interferon-based therapy. However, for unknown reasons, a large proportion of patients with CHB do not respond to this treatment. Hence, there is a pressing need to establish response markers to select patients who will benefit from therapy and to spare potential nonresponders from unnecessary side effects of antiviral therapy. Here, we assessed whether HLA-C and KIR genotypes were associated with treatment outcome for CHB. Twelve SNPs in or near the HLA-C gene were genotyped in 86 CHB patients (41 HBeAg positive; 45 HBeAg negative) treated with peginterferon alfa-2a + adefovir. Genotyping of killer immunoglobin-like receptors (KIRs) was performed by SSP-PCR. One SNP in HLA-C (rs2308557) was significantly associated with combined response in HBeAg-positive CHB patients (P = 0.003). This SNP is linked to the HLA-C group C1 or C2 classification, which controls KIR binding. The combination of KIR2DL1 with its ligand HLA-C2 was observed significantly more often in HBeAg-positive patients with a combined response (13/14) than in nonresponders (11/27, P = 0.001). Patients with the KIR2DL1/C2 genotype had significantly higher baseline ALT levels (136 vs 50 U/L, P = 0.002) than patients without this combination. Furthermore, KIR2DL1-C2 predicted response independent of HBV genotype and ALT at baseline. HLA-C and KIR genotype is strongly associated with response in HBeAg-positive CHB patients treated with interferon-based therapy. In combination with other known response markers, HLA-C/KIR genotype could enable the selection of patients more likely to respond to interferon-based therapy.

Miravirsen dosing in chronic hepatitis C patients results in decreased microRNA-122 levels without affecting other microRNAs in plasma.

BACKGROUND: MicroRNA-122 (miR-122) is an important host factor for hepatitis C virus replication. Administration of miravirsen, an anti-miR-122 oligonucleotide, resulted in a dose dependent and prolonged decrease in HCV RNA levels in chronic hepatitis C patients. AIM: To assess the plasma level of various miRNAs in patients dosed with miravirsen. METHODS: We included 16 of 36 chronic hepatitis C patients who received five injections of either 3 mg/kg (n = 4), 5 mg/kg (n = 4), 7 mg/kg (n = 4) miravirsen or placebo (n = 4) over a 4-week period in a double-blind, randomised phase 2a study. Plasma levels of 179 miRNAs were determined by qPCR and compared between patients dosed with miravirsen or placebo. RESULTS: Median plasma miR-122 level at baseline in patients receiving miravirsen was 3.9 × 10(3) compared to 1.3 × 10(4) copies/4 µL in placebo-dosed patients (P = 0.68). At week 1, 4, 6 and 10/12, patients dosed with miravirsen had respectively a median 72-fold, 174-fold, 1109-fold and 552-fold lower expression of miR-122 than at baseline (P = 0.001, as compared to patients receiving placebo). At week 4 of dosing, miRNA-profiling demonstrated a significant lower expression of miR-210 and miR-532-5p compared to baseline (3.0 and 4.7-fold lower respectively). However, subsequent longitudinal analysis showed no significant differences in miR-210 and miR-532-5p plasma levels throughout the study period. CONCLUSIONS: We demonstrated a substantial and prolonged decrease in plasma miR-122 levels in patients dosed with miravirsen. Plasma levels of other miRNAs were not significantly affected by antagonising miR-122.

Identification of FDA-approved drugs that target hepatitis B virus transcription.

In the treatment of chronic hepatitis B virus (HBV) infection, polymerase inhibitors successfully suppress HBV DNA production. However, the production of viral proteins continues unhindered, which hampers viral clearance. Here, we screen for compounds that suppress HBV transcription, which would prevent viral protein production. A total of 640 FDA-approved drugs were evaluated for their ability to inhibit HBV transcription in a transfection-based HBV reporter assay. The assay was performed in the presence and absence of the HBV accessory protein X (HBx), which is essential for in vivo HBV RNA transcription. We observed that in the absence of HBx 47, and in the presence of HBx 24 compounds suppressed transcription by more than 20%. We selected the 24 most potent compounds in each condition for further analysis. On average, the selected compounds reduced transcription by 33.9% (range: 24.1-65.8%) in the absence of HBx expression, and 30.6% (range: 20.4-48.9%) in the presence of HBx. The two selections of 24 compounds had 12 compounds in common, resulting in a final selection of 36 compounds, which were evaluated for their capacity to suppress HBV replication in constitutively HBV-replicating HepG2.2.15 cells. Twenty-three of these compounds reduced HBV replication by interfering with RNA transcription. Further analysis revealed that one of the compounds, terbinafine, potently and specifically suppressed HBx-mediated HBV RNA transcription in HepG2 cells. Inhibition of HBV protein production is a promising step towards HBV clearance. In combination with an HBV polymerase inhibitor, the added suppression of HBV RNA transcription may markedly improve antiviral treatment outcome.

Polymorphism in IFI16 affects CD4(+) T-cell counts in HIV-1 infection.

Interferon-γ-inducible protein 16 (IFI16) plays a pivotal role in the death of bystander CD4(+) T-cells. Here, we demonstrate that SNP rs1417806 in IFI16 is associated with CD4(+) T-cell counts at set point and HIV-1 disease progression, indicating that IFI16 affects HIV-1 pathogenesis especially during the early phase of infection.

Comparison of the hepatitis B virus core, surface and polymerase gene substitution rates in chronically infected patients.

For phylogenetic comparison of hepatitis B virus (HBV) isolates, often a region of the HBV surface gene is analysed. Because the surface gene completely overlaps the polymerase gene, its evolution is constrained, and it may not be the best choice for genetic comparison of HBV isolates. Analysing serial sample pairs of 33 chronically HBV-infected, untreated patients, with a cumulative follow-up of 184 years, the synonymous and nonsynonymous substitution rates of a part of the overlapping HBV surface and polymerase genes were compared to those of a nonoverlapping part of the HBV core gene. The substitution rate of the HBV core gene was higher (8.15 × 10(-4) vs 4.57 × 10(-4) substitutions/site/year) than that of the surface gene. The difference was mainly due to a significantly lower synonymous substitution rate in the surface gene, with dN/dS ratios of 0.412 in the core gene and 0.986 in the surface gene. Contrary to the core gene, the number of substitutions in the surface gene was higher in low viraemic hosts, who control HBV infection by suppressing replication. The number of substitutions in the core gene correlated more strongly with the duration of follow-up. The overlapping HBV surface and polymerase genes experience strong negative selection, which limits the number of substitutions. Because the HBV core gene reflects the duration of infection more accurately, it is more suitable for the analysis of short-term viral evolution and of hepatitis B transmission chains.

Diminished human immunodeficiency virus type 1 reverse transcription and nuclear transport in primary macrophages arrested in early G(1) phase of the cell cycle.

Previously, we and others have demonstrated that the process of reverse transcription of human immunodeficiency virus type 1 (HIV-1) is disturbed in nondividing macrophages and quiescent T lymphocytes. Here we analyzed which phase of the cell cycle in macrophages is crucial for early steps in the HIV-1 replication cycle. HIV-1 Ba-L-inoculated macrophages arrested early in the G(1) phase by n-butyrate contained incomplete products of reverse transcription. In gamma-irradiated macrophages, reverse transcription was successfully completed but proviral integration could not be detected. In these cells, nuclear import was disturbed as reflected by the absence of two-long-terminal-repeat circles. In macrophages arrested late in G(1) phase by aphidicolin or 5, 6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB), reverse transcription was unaffected. Proviral integration occurred efficiently in DRB-treated macrophages, whereas integrated proviral DNA could not be detected after aphidicolin treatment. Arrest at G(2) phase of the cell cycle by nocodazole did not affect reverse transcription or proviral integration. Treatment of macrophages with hydroxyurea (HU), which reduces the intracellular deoxynucleoside triphosphate (dNTP) pool by blocking the de novo synthesis of dNTP, resulted in a dose-dependent inhibition of HIV-1 reverse transcription. This could partially be restored by the addition of nucleoside precursors. Addition of nucleoside precursors enhanced both reverse transcription and cell proliferation. However, the disturbed reverse transcription observed in the nonproliferating and n-butyrate-treated macrophages could not be restored by addition of nucleoside precursors. Similar to observations in quiescent T lymphocytes, incomplete proviral DNA species were arrested in the cytoplasm of the macrophages. Our results indicate that also in primary macrophages the intracellular nucleotide pools and other cellular factors that coincide with late G(1) phase of the cell cycle may contribute to efficient reverse transcription and nuclear localization.

R5 strains of human immunodeficiency virus type 1 from rapid progressors lacking X4 strains do not possess X4-type pathogenicity in human thymus.

Some individuals infected with only R5 strains of human immunodeficiency virus type 1 progress to AIDS as quickly as individuals harboring X4 strains. We determined that three R5 viruses were much less pathogenic than an X4 virus in SCID-hu Thy/Liv mice, suggesting that R5 virus-mediated rapid disease progression is associated with host, not viral, factors.

Phenotype of HIV-1 lacking a functional nuclear localization signal in matrix protein of gag and Vpr is comparable to wild-type HIV-1 in primary macrophages.

Human immunodeficiency virus type 1 (HIV-1) is considered to infect nondividing cells because nuclear localization signals (NLS) in matrix (MA, p17(Gag)) and Vpr allow active nuclear transport of the preintegration complex. Previous studies demonstrated that HIV-1 reverse transcription is successful only in cells with proliferative potential, thus restricting HIV-1 replication to cycling cells. To sort out this apparent discrepancy we compared the phenotype of a chimeric HIV-1 variant lacking a functional Vpr and MA-NLS (R7. deltaVpr.deltaNLS), and previously described to lack replicative capacity in macrophages and growth-arrested cells, with a chimera lacking a functional Vpr (R7.deltaVpr). Both variants replicated efficiently in primary macrophages, with only minimal differences in the kinetics of reverse transcription, integration, or p24 production. In agreement with our previous observation, elongation of reverse transcription was restricted to the proliferating subpopulation of macrophages. Replication of R7.deltaVpr and R7.deltaVpr.deltaNLS could also be demonstrated in aphidicolin-treated macrophages, indicating efficient nuclear transport in G1/S phase-arrested cells. In conclusion, our results confirm the dependency of the process of HIV-1 reverse transcriptase on cell proliferation in primary macrophages and exclude an important role of MA-NLS and Vpr in macrophage infection.

Proliferation-dependent replication in primary macrophages of macrophage-tropic HIV type 1 variants.

We previously demonstrated that completion of reverse transcription in macrophages inoculated with the HIV-1 Ba-L variant was established only in the subpopulation of cells with proliferative capacity. In our present study we further extended this observation with three additional HIV-1 isolates, being the macrophage-tropic ADA strain and two primary macrophage-tropic HIV-1 variants isolated from cerebrospinal fluid and from bronchoalveolar lavage from AIDS patients. On inoculation, irrespective of the virus variant used, elongated reverse transcription products could be demonstrated only in macrophages that had proliferated during inoculation as evidenced by the incorporation of bromodeoxyuridine (BrdU), a thymidine analog. The presence of newly synthesized early products of reverse transcription also in the BrdU-negative fraction indicated that viral entry is not disturbed in nondividing cells. Our data indicate that the process of reverse transcription is dependent on cellular conditions that coincide with cell proliferation, and therefore that HIV-1 replication is restricted to cells with proliferative potential.

Infectious cellular load in human immunodeficiency virus type 1 (HIV-1)-infected individuals and susceptibility of peripheral blood mononuclear cells from their exposed partners to non-syncytium-inducing HIV-1 as major determinants for HIV-1 transmission in homosexual couples.

To study risk factors for homosexual transmission of human immunodeficiency virus type 1 (HIV-1), we compared 10 monogamous homosexual couples between whom transmission of HIV-1 had occurred with 10 monogamous homosexual couples between whom HIV-1 transmission had not occurred despite high-risk sexual behavior. In the group of individuals who did not transmit virus, peripheral cellular infectious load was lower and the CD4+ T-cell counts were higher than in the group of transmitters. HIV-1 RNA levels in serum did not differ between transmitters and nontransmitters. Compared with peripheral blood mononuclear cells (PBMC) from normal healthy blood donors, 8 of 10 nonrecipients and only 3 of 8 recipients had PBMC with reduced susceptibility to in vitro infection with non-syncytium-inducing (NSI) HIV-1 variants isolated from either their respective partners or an unrelated individual. No difference in susceptibility was observed for infection with a syncytium-inducing variant. Among the individuals who had PBMC with reduced susceptibility, five nonrecipients and one recipient had PBMC that were equally or even less susceptible to NSI variants than PBMC that had low susceptibility and that were derived from healthy blood donors that were heterozygous for a 32-bp deletion in the CCR5 gene (CCR5 delta32). Three of these individuals (all nonrecipients) had a CCR5 delta32 heterozygous genotype themselves, confirming an association between low susceptibility to NSI variants and CCR5 delta32 heterozygosity. All three recipients with less susceptible PBMC had partners with a high infectious cellular load; inversely, both nonrecipients with normally susceptible PBMC had partners with a very low infectious cellular load. These results suggest that a combination of susceptibility of target cells and inoculum size upon homosexual exposure largely determines whether HIV-1 infection is established.

Analysis of the temporal relationship between human immunodeficiency virus type 1 quasispecies in sequential blood samples and various organs obtained at autopsy.

We studied the temporal relationship between human immunodeficiency type 1 (HIV-1) quasispecies in tissues and in peripheral blood mononuclear cells (PBMC) of infected individuals. Sequential PBMC and tissue samples from various organs obtained at autopsy from three patients who died of AIDS-related complications were available for analysis. Biological HIV-1 clones were isolated from PBMC samples, and cellular tropism and syncytium-inducing (SI) capacity were determined. Genomic DNA was isolated from 1 cm3 of organ tissue, and proviral DNA was amplified by means of PCR and cloned with the PGEM-T vector system. A 185-bp region encompassing the third variable domain of the virus envelope, known to influence HIV-1 biological properties, was sequenced. HIV-1 could be amplified from all PBMC and organ samples, except from liver tissue for two patients. Both SI and non-syncytium-inducing (NSI) genotypes could be detected in the different tissues. Tissue-specific quasispecies were observed in brain, lung, and testis. Lymphoid tissues, such as bone marrow, lymph node, and spleen, harbored several different variants similar to those detected in blood in the last PBMC samples. In general, only tissues in which macrophages are likely to be the main target cell for HIV-1 harbored NSI HIV-1 sequences that clustered separately. Both SI and NSI sequences that clustered with sequences from late-stage PBMC were present in other tissues, which may indicate that the presence of HIV-1 in those tissues is secondary to lymphocyte infiltration rather than to tissue tropism of HIV-1 itself. These data suggest that the viral reservoir may be limited, which will have important implications for the success of HIV-1 eradication.

Analysis of CD8+ T lymphocyte-mediated nonlytic suppression of autologous and heterologous primary human immunodeficiency virus type 1 isolates.

Nonlytic CD8+ T lymphocyte antiviral factor (CAF) activity has been described as having an important role in the clinical course of human immunodeficiency virus type 1 (HIV-1) infection. Testing of CAF activity against autologous viruses isolated at approximately the same time points showed that CD8+ T lymphocytes from long-term survivors indeed possessed high CAF activity. However, in four of six progressors to AIDS, we observed the same amount of CAF activity in the face of increasing cellular HIV-1 load. In the other two progressors, CAF activity seemed preserved over time whereas the susceptibility of the virus isolate obtained late in infection seemed to be diminished. In a heterologous system, CAF activity of CD8+ T lymphocytes from 13 HIV-1-positive individuals did not correlate with CD4+ T lymphocyte counts. In two of three patients, syncytium-inducing (SI) HIV-1 variants, which are associated with a progressive clinical course, appeared to have a somewhat reduced susceptibility to CAF activity as compared to their coexisting non-SI HIV-1 variants. In a large donor group, suppression of SI isolates (as compared to non-SI isolates) mediated by heterologous CD8+ T lymphocytes was reduced. CD8+ T lymphocytes from five of six HIV-1-positive individuals suppressed HIV-1 replication in macrophages. CD8+ T lymphocytes from noninfected donors, even from cord blood, already had high CAF activity, suggesting that induction of this activity is neither virus nor HIV-1 specific.

Changes in cellular virus load and zidovudine resistance of syncytium-inducing and non-syncytium-inducing human immunodeficiency virus populations under zidovudine pressure: a clonal analysis.

Zidovudine treatment preferentially benefits persons with only non-syncytium-inducing (NSI) human immunodeficiency virus type 1 (HIV-1) variants. To understand this differential efficacy, changes in cellular virus load, clonal composition of HIV-1 populations, and development of resistance-conferring reverse transcriptase mutations were studied in 17 persons initiating zidovudine therapy. Zidovudine treatment resulted in larger and more sustained decreases in cellular virus load in persons with NSI variants only compared with persons also carrying syncytium-inducing (SI) variants. Although the former group had a delayed emergence of resistance mutations, differences in initial responses between the 2 groups were independent of the emergence of resistance mutations. Changes in virus load in subjects also carrying SI variants were due mainly to loss of coexisting NSI virus. Resistance mutations emerged at similar rates in both coexisting variants. Data suggest that mechanisms other than drug resistance are necessary to completely explain the phenotype-dependent benefit of zidovudine.

Relation between changes in cellular load, evolution of viral phenotype, and the clonal composition of virus populations in the course of human immunodeficiency virus type 1 infection.

The relationship between the evolution of human immunodeficiency virus type 1 (HIV-1) biologic phenotype, changes in the proportion of infected peripheral blood mononuclear cells, and the relative contribution of non-syncytium-inducing (NSI) and syncytium-inducing (SI) HIV-1 variants to virus load was studied during the course of HIV-1 infection. In 65 HIV-1-infected subjects, the proportion of infected CD4 T cells was higher in persons who carried SI variants. Longitudinal studies revealed that the emergence of SI HIV-1 variants can occur at relatively low numbers of HIV-1-infected cells. Emergence of SI variants frequently coincided with an increase of virus load due to an expansion of both NSI and SI variants, although the contribution of SI viruses to the total virus population significantly increased with time after SI phenotype conversion. These data indicate that NSI to SI phenotype conversion, rather than resulting from high virus load, is part of the sequence of events that leads to increased virus load and CD4 cell depletion.

Productive HIV-1 infection of macrophages restricted to the cell fraction with proliferative capacity.

Retroviruses establish productive infection only in proliferating cells. Macrophages are often considered to be non-proliferating in vitro yet are susceptible to HIV-1 infection. This has led to the conclusion that HIV-1 can establish infection independent of host cell proliferation. We here report that a small proportion of macrophages does have proliferative capacity. A comparable small fraction of monocyte derived macrophages (MDM) supported productive HIV-1 infection as demonstrated in limiting dilution culture. Fluorescence activated cell sorting on the basis of incorporation of BrdUrd, a thymidine analog, and subsequent PCR analysis revealed the presence of proviral DNA only in the BrdUrd positive cell fraction with DNA synthesizing activity. To identify which phase of cell cycle is required for establishment of productive infection, growth arrest in G1 or G1/S phase prior to inoculation was performed. gamma-Irradiation, which arrests primary cells in G1, prevented both cell proliferation and establishment of productive infection in MDM. Treatment of MDM with aphidicolin, a specific inhibitor of DNA polymerase alpha and delta which arrests cells in G1/S phase of the cell cycle, also inhibited DNA synthesis but did not prevent establishment of productive infection which is completely analogous to observations in T cells. Our data thus indicate that not cell division itself but cellular conditions that coincide with cell proliferation are apparently indispensable for establishment of productive infection.