Paul K. Farmer

Transmission of HIV-1 Gag immune escape mutations is associated with reduced viral load in linked recipients

In a study of 114 epidemiologically linked Zambian transmission pairs, we evaluated the impact of human leukocyte antigen class I (HLA-I)–associated amino acid polymorphisms, presumed to reflect cytotoxic T lymphocyte (CTL) escape in Gag and Nef of the virus transmitted from the chronically infected donor, on the plasma viral load (VL) in matched recipients 6 mo after infection. CTL escape mutations in Gag and Nef were seen in the donors, which were subsequently transmitted to recipients, largely unchanged soon after infection. We observed a significant correlation between the number of Gag escape mutations targeted by specific HLA-B allele–restricted CTLs and reduced VLs in the recipients. This negative correlation was most evident in newly infected individuals, whose HLA alleles were unable to effectively target Gag and select for CTL escape mutations in this gene. Nef mutations in the donor had no impact on VL in the recipient. Thus, broad Gag-specific CTL responses capable of driving virus escape in the donor may be of clinical benefit to both the donor and recipient. In addition to their direct implications for HIV-1 vaccine design, these data suggest that CTL-induced viral polymorphisms and their associated in vivo viral fitness costs could have a significant impact on HIV-1 pathogenesis.

Human Leukocyte Antigens and HIV Type 1 Viral Load in Early and Chronic Infection: Predominance of Evolving Relationships

Background During untreated, chronic HIV-1 infection, plasma viral load (VL) is a relatively stable quantitative trait that has clinical and epidemiological implications. Immunogenetic research has established various human genetic factors, especially human leukocyte antigen (HLA) variants, as independent determinants of VL set-point. Methodology/Principal Findings To identify and clarify HLA alleles that are associated with either transient or durable immune control of HIV-1 infection, we evaluated the relationships of HLA class I and class II alleles with VL among 563 seroprevalent Zambians (SPs) who were seropositive at enrollment and 221 seroconverters (SCs) who became seropositive during quarterly follow-up visits. After statistical adjustments for non-genetic factors (sex and age), two unfavorable alleles (A*3601 and DRB1*0102) were independently associated with high VL in SPs (p<0.01) but not in SCs. In contrast, favorable HLA variants, mainly A*74, B*13, B*57 (or Cw*18), and one HLA-A and HLA-C combination (A*30+Cw*03), dominated in SCs; their independent associations with low VL were reflected in regression beta estimates that ranged from −0.47±0.23 to −0.92±0.32 log10 in SCs (p<0.05). Except for Cw*18, all favorable variants had diminishing or vanishing association with VL in SPs (p≤0.86). Conclusions/Significance Overall, each of the three HLA class I genes had at least one allele that might contribute to effective immune control, especially during the early course of HIV-1 infection. These observations can provide a useful framework for ongoing analyses of viral mutations induced by protective immune responses.

Role of NFκB in the regulation of macrophage colony stimulating factor by tumor necrosis factor-α in ST2 bone stromal cells

Expression of MCSF in bone is important to the regulation of osteoclastogenesis. We show here that tumor necrosis factor‐α (TNFα) increases the production of both soluble (sMCSF) and membrane‐bound (mMCSF) macrophage colony stimulating factor by ST2 bone stromal cells. Treatment of ST2 cells with TNFα caused sMCSF levels to increase by 394 ± 5% from basal; mMCSF rose by 316 ± 66% from 30 ± 10 per 100,000 cells in the same time. These increases were consistent with increased expression of mRNAs encoding both isoforms. Increases in MCSF mRNA are also seen after stimulation with dexamethasone. To investigate the potential role of NFκB in this TNFα effect, we treated cells with sodium salicylate (NaS), an inhibitor of NFκB translocation. NaS decreased TNFα‐stimulated NFκB activation by 50% as assessed by EMSA. Despite inhibition of NFκB signaling, NaS enhanced TNFα‐stimulated MCSF secretion and did not prevent TNFα‐stimulated increases in sMCSF mRNA, suggesting that NFκB was not involved in TNFα effect on the gene. TNFα failed to stimulate transcription of a 774 nucleotide MCSF promoter‐luciferase reporter transfected into ST2 cells which contained the NFκB consensus sequence. Deletion of the seven nucleotides containing the NFκB homology response sequence from the MCSF promoter increased basal gene transcription by twofold. TNFα thus contributes to an osteoclastogenic environment through upregulation of bone expression of both MCSF isoforms. Our data suggests that NFκB is not the major signaling pathway through which this occurs. J. Cell. Physiol. 179:193–200, 1999.

In Vitro Transcription of the Rat Insulin-like Growth Factor-I Gene

Although the liver is the major source of circulating insulin-like growth factor-I (IGF-I), relatively little is known about the regulation of IGF-I gene transcription in this tissue. Since transcripts are initiated largely in exon 1, we established an in vitro transcription system to evaluate activation of transcription via the major exon 1 initiation site. Transcription of a G-free cassette reporter was directed by rat IGF-I genomic fragments, and the adenovirus major late promoter was used as an internal control. Tissue specificity was demonstrated by a 60-90% decrease in transcripts with spleen extracts as compared with liver. 54 base pairs (bp) of upstream sequence were sufficient to direct IGF-I gene transcription, and activity increased 5-fold with 300 bp of upstream sequence. DNase I footprinting revealed four protected regions between −300 and −60 bp; binding was confirmed by gel shift analysis, and tissue specificity was demonstrated by reduced shifts with spleen extracts. The necessity of transcription factor binding to such sites was established by competition analysis, which revealed a specific decrease in IGF-I transcription in the presence of a competing fragment. Use of this in vitro transcription system should permit analysis of the function of individual transcription factors involved in regulation of IGF-I gene expression.

HLA-B*57 versus HLA-B*81 in HIV-1 infection: slow and steady wins the race?

ABSTRACT Two human leukocyte antigen (HLA) variants, HLA-B*57 and -B*81, are consistently known as favorable host factors in human immunodeficiency virus type 1 (HIV-1)-infected Africans and African-Americans. In our analyses of prospective data from 538 recent HIV-1 seroconverters and cross-sectional data from 292 subjects with unknown duration of infection, HLA-B*57 (mostly B*57:03) and -B*81 (exclusively B*81:01) had mostly discordant associations with virologic and immunologic manifestations before antiretroviral therapy. Specifically, relatively low viral load (VL) in HLA-B*57-positive subjects (P ≤ 0.03 in various models) did not translate to early advantage in CD4+ T-cell (CD4) counts (P ≥ 0.37). In contrast, individuals with HLA-B*81 showed little deviation from the normal set point VL (P > 0.18) while maintaining high CD4 count during early and chronic infection (P = 0.01). These observations suggest that discordance between VL and CD4 count can occur in the presence of certain HLA alleles and that effective control of HIV-1 viremia is not always a prerequisite for favorable prognosis (delayed immunodeficiency). Of note, steady CD4 count associated with HLA-B*81 in HIV-1-infected Africans may depend on the country of origin, as observations differed slightly between subgroups enrolled in southern Africa (Zambia) and eastern Africa (Kenya, Rwanda, and Uganda).

Dynamics and frequency of Gag transmitted polymorphisms in Zambia

Methods Using 143 epidemiologically linked transmission pairs from a Zambian cohort we assessed: (1) the frequency of Gag polymorphisms circulating in the population, (2) if the polymorphisms could be associated with the infected individual’s HLA alleles, (3) the frequency at which polymorphisms are transmitted, and (4) the relevance of the transmitted polymorphisms (TP) to the newly infected individual’s HLA-I alleles.

Impact of transmitted CTL escape mutations on replicative capacity and HIV pathogenesis in early infection

Background Multiple HLA class I alleles have been shown to influence both HIV-1 transmission and viral load. In transmission pairs, viral loads of acutely infected partners correlate with viral loads (VL) of their chronically infected donors. This correlation becomes highly significant after adjustment for host factors known to modulate viral load. In addition, we have previously demonstrated that transmission of a virus containing multiple HLA-I associated polymorphisms resulted in a lower set point VL in Zambian linked recipients. These studies imply that transmitted viral characteristics play a role in defining early HIV-1 pathogenesis, and it will be important for vaccine development to understand which viral characteristics are responsible for this. Methods We investigated the role that the in vitro replicative capacity (RC) of the transmitted Gag plays in defining HIV-1 clinical parameters, by cloning gag genes from the founder virus in newly infected partners of 149 epidemiologically linked transmission pairs i nto the subtype C, R5 tropic MJ4 provirus. Results We observed a statistically significant positive correlation between the RC of Gag-MJ4 chimeras and set point VL in seroconverters (P=0.013). The RC of the transmitted Gag also correlated (P=0.025) to the viral load in the chronically infected donor, pointing to RC as the major viral characteristic responsible for the link between donor and linked recipient viral loads. The long term clinical benefit associated with the transmission of attenuated viruses was investigated by performing a Kaplan Meier analysis of time to CD4+ count less than 350. Individuals infected with attenuated gag sequences (RC 2) replicating viruses (P = 0.034).

OA06-03. Dynamics of CTL epitope escape and reversion in an African subtype C cohort

Background HIV immune escape follows a predictable mutational path in response to the HLA alleles carried by an individual. The kinetics of CTL epitope escape and reversion in subtype B HIV-1 infected individuals have recently been reported, however, the inferences drawn from them were limited by the absence of information about the transmitted sequence. To address these issues, we examined Gag, Pol and Nef sequences in both partners of 148 epidemiologically-linked transmission pairs from an African subtype C cohort.