Richard H. Glashoff

Functions of Tat: the versatile protein of human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1) Tat is a multifunctional protein that contributes to several pathological symptoms of HIV-1 infection as well as playing a critical role in virus replication. Tat is a robust transactivating protein that induces a variety of effects by altering the expression levels of cellular and viral genes. The functions of Tat are therefore primarily related to its role in modulation of gene expression. In this review the functions of HIV-1 Tat that have been well documented, as well as a number of novel functions that have been proposed for this protein, are discussed. Since some of the functions of Tat vary in different cell types in a concentration-dependent manner and because Tat sometimes exerts the same activity through different pathways, study of this protein has at times yielded conflicting and controversial results. Due to its pivotal role in viral replication and in disease pathogenesis, Tat and the cellular pathways targeted by Tat are potential targets for new anti-HIV drugs.

Rhinovirus Induction of the CXC Chemokine Epithelial-Neutrophil Activating Peptide-78 in Bronchial Epithelium

Epithelial-neutrophil activating peptide-78 (ENA-78) induces neutrophil migration, an early response to viral infection. Rhinovirus serotype 16 (RV16) was used to infect primary bronchial epithelial cells and a cell line (BEAS-2B). Release of ENA-78 protein was measured by enzyme-linked immunosorbent assay, ENA-78 mRNA production was quantified by reverse-transcription polymerase chain reaction, and ENA-78 promoter activity was assessed by use of a promoter construct. After infection with RV16, ENA-78 protein and mRNA increased significantly, and RV16 induced 3-fold increases in ENA-78 gene transcription. Nasal ENA-78 measured in patients with asthma with and without RV infection was more elevated in patients with RV infection present. Our study demonstrates that ENA-78 is produced in bronchial epithelial cells in response to RV16 infection. With other chemokines, it may be an important initiator of neutrophil airway inflammation during RV common colds and thus may play a role in the development of virus-associated airway pathologies.

Risk for HIV-1 infection associated with a common CXCL12 (SDF1) polymorphism and CXCR4 variation in an African population.

CXC chemokine ligand 12 (CXCL12), or stromal cell-derived factor 1 (SDF1), is the only known natural ligand for the HIV-1 coreceptor, CXC chemokine receptor 4 (CXCR4). A single nucleotide polymorphism (SNP) in the CXCL12 gene (SDF1-3′A) has been associated with disease progression to AIDS in some studies, but not others. Mutations in the CXCR4 gene are generally rare and have not been implicated in HIV-1/AIDS pathogenesis. This study analyzed the SDF1-3′A SNP and performed mutation screening for polymorphic markers in the CXCR4 gene to determine the presence or absence of significant associations with susceptibility to HIV-1 infection. The study consisted of 257 HIV-1-seropositive patients and 113 HIV-1-seronegative controls representing a sub-Saharan African population belonging to the Xhosa ethnic group of South Africa. The SDF1-3′A SNP was associated with an increased risk for HIV-1 infection (P = 0.0319) whereas no significant association was observed between the occurrence of the SDF1-3′A SNP and increased or decreased plasma levels of CXCL12. Comprehensive mutation analysis of the CXCR4 gene confirmed a high degree of genetic conservation within the coding region of this ancient population.

Role of inflammation in HIV-1 disease progression and prognosis.

Abstract Inflammation and immune activation have been thrust to center stage in the understanding of HIV-1 disease pathogenesis and progression. Early work demonstrated that heightened levels of immune activation correlated with the extent of CD4 + T cell death in lymphoid tissue; however, this concept was not incorporated into the general view of disease pathogenesis. Since these early studies, the extension of life for patients on combination antiretroviral therapies (cART) has heralded a new era of non-AIDS-related diseases and incomplete restoration of immune function. The common link appears to be ongoing inflammation and immune activation. Thus, despite good control of viral loads, persons living with HIV (PLWH) remain at increased risk of inflammatory-associated complications such as cardiovascular disease and certain cancers. HIV-specific mechanisms as well as non-specific generalized responses to infection contribute to ongoing activation of the immune system. An early loss of gastrointestinal (GI) tract mucosal integrity, the pro-inflammatory cytokine milieu, co-infections and marked destruction of lymph node architecture are all factors contributing to the ongoing activation of the immune system as well as impaired immune recovery. It is becoming increasingly evident that the CD4 count and viral load do not provide a complete picture of the underlying state of the immune system. Heightened levels of inflammatory markers have been shown to predict increased mortality and other adverse events. Therefore, it will be important to incorporate these markers into management algorithms as soon as possible. This is particularly relevant in resource-poor countries where difficulties in cART roll-out and access are still encountered and, therefore, a mechanism for prioritizing individuals for therapy would be of value. This review will focus on the closely inter-related concepts of immune activation and inflammation. Both are broad concepts involving the interaction of various key players in the immune system. Importantly, immune activation promotes inflammation and thrombosis and similarly, inflammation and thrombosis induce immune activation. These concepts are thus intricately linked. Studies highlighting the potentially harmful effects of ongoing inflammation/immune activation are reviewed and the contributions of the GI tract “damage” and other co-infections such as CMV are explored. The complications resulting from persistent immune activation include enhanced CD4 + T cell death, lymphoid tissue destruction, and various pathologies related to chronic inflammation. Ultimately, we envision that the long-term management of the disease will incorporate both the identification and the amelioration of the potentially harmful effects of ongoing immune activation and inflammation.

Antiviral roles of APOBEC proteins against HIV-1 and suppression by Vif

Apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like (APOBEC) proteins are members of a protein family sharing the common characteristic of cytidine deaminase activity. The antiviral activity of APOBEC3G and APOBEC3F has been studied more extensively than that of the other members of this family. The antiviral activity of APOBEC3B and APOBEC3DE has also been described. Studies of other APOBEC proteins have not revealed any antiviral activities against HIV-1; however, further investigation is required. In the absence of human immunodeficiency virus type 1 (HIV-1) virion infectivity factor (Vif), APOBEC3G and APOBEC3F are incorporated into HIV-1 virions and hypermutate the viral genomic DNA by their cytidine deaminase activity. HIV-1 Vif protein suppresses the antiviral role of APOBEC proteins by several mechanisms that lead to inhibition of incorporation of APOBEC3G/3F into HIV-1 virions. The detailed mechanisms involved in the suppression of APOBEC proteins by Vif are still being elucidated. Novel studies in which as yet undefined aspects of the suppression of APOBEC proteins are investigated could reveal important and potentially exploitable information for addressing HIV-1 infection in humans.

High HIV, HPV, and STI prevalence among young Western Cape, South African women: EVRI HIV prevention preparedness trial.

Background:This study sought to assess the feasibility of conducting a phase III HIV prevention trial using a multivalent human papillomavirus (HPV) vaccine (Gardasil; Merck, Whitehouse Station, NJ). Methods:A total of 479 sexually active women aged 16–24 years in the Western Cape, South Africa, were enrolled in the Efficacy of HPV Vaccine to Reduce HIV Infection (EVRI) Trial. Of these, 402 were HIV negative, nonpregnant, and randomized 1:1 to receive Gardasil or a saline placebo vaccine. Vaccine doses were administered at enrollment, month 2, and month 6, and participants were followed for 1 month after the third dose. Enrollment HIV, HPV, other sexually transmitted infections (STIs), and cervical cytology were evaluated. Rates of accrual, vaccine compliance, and adherence to protocol were monitored. Results:High rates of accrual of eligible females to study (93%) and completion of the 3-dose vaccine series (91%) were noted, with few protocol violations. Ineligibility due to reported HIV positivity was 19%, and another 12% of those enrolled tested HIV positive. STI prevalence was high, with 6.2%, 10.9%, and 32.8% testing positive for syphilis, gonorrhea, and chlamydia, respectively. Cervical prevalence of ≥1 of 37 HPV types was 71%. STI and HPV prevalence was highest among the youngest women (<19 years). Conclusions:Feasibility (successful accrual, retention, and vaccination) of conducting randomized placebo-controlled trials of HPV vaccines among HIV high-risk women in South Africa was demonstrated. This work demonstrates that phase III HIV prevention trials need to intervene at young ages and screen and treat multiple STIs concurrently to have a measurable impact on HIV acquisition.

Serum adenosine deaminase and total immunoglobulin G correlate with markers of immune activation and inversely with CD4 counts in asymptomatic, treatment-naive HIV infection

PurposeHIV-infection is characterized by aberrant immune activation and ongoing inflammation. Markers of inflammation are now recognized to have prognostic value for adverse events, independent of viral loads and CD4 counts. This study aimed to delineate a panel of affordable markers of immune activation in untreated HIV-infection that may have an impact on the management of HIV in resource-limited settings.MethodsThis was a cross-sectional study of 86 untreated newly diagnosed HIV-infected patients and 54 matched controls attending a voluntary testing clinic in Cape Town, South Africa. Serum levels of adenosine deaminase (ADA), total immunoglobulin G (IgG), soluble CD14 and lipopolysaccharide-binding protein (LBP) were measured and correlated with CD4 counts, viral loads and expression of CD38 on CD8+ T cells.ResultsADA, IgG and LBP were all significantly increased in the HIV infected group (p < 0.0001) compared with uninfected controls. Soluble CD14 was also significantly increased (p = 0.0187). Furthermore, all these parameters correlated inversely with CD4 counts (r = −0.481 p < 0.0001; r = −0.561; p < 0.0001; r = −0.387 p = 0.0007 and r = −0.254 p = 0.0240, respectively). Only ADA correlated with viral load (r = 0.260 p = 0.0172). Importantly, ADA, IgG and LBP correlated directly with %CD38 on CD8+ T cells (r = 0.369 p < 0.0001; r = 0.284 p = 0.001; r = 0.408 p = 0.0006, respectively).ConclusionAffordable parameters such as serum ADA and IgG correlated significantly with immune activation levels and markers of disease progression in untreated HIV-infection and therefore may add value to the management of these patients in resource-limited settings.

Characterization of HIV type 1 genetic diversity among South African participants enrolled in the AIDS Vaccine Integrated Project (AVIP) study.

The genetic diversity of HIV-1 strains circulating among HIV-1-infected South Africans was investigated in a cohort of 420 individuals enrolled as part of the AIDS Vaccine Integrated Project (AVIP) study. Representative samples (10%) were randomly selected from treatment-naive participants. Viral RNA was extracted for reverse transcriptase-initiated amplification and population-based sequencing of partial pol (encompassing protease and reverse transcriptase) and full-length integrase. Overall, HIV-1 sequences confirmed that 97.1% and 96.9% were HIV-1 subtype C in pol and integrase, respectively. Two participants were infected with unique A1/C and C/A1 recombinants in pol/integrase. Further pol sequence analysis identified mutation patterns associated with high level resistance to NNRTIs in two participants, whereas no primary mutations conferring resistance to integrase inhibitors were detected. The predominance of HIV-1 subtype C in South African populations is therefore confirmed in the AVIP cohort finalized for testing preventive or therapeutic vaccines against HIV-1 infection.

Molecular and phylogenetic analysis of HIV type 1 vpr sequences of South African strains

HIV-1 subtype C is the prevalent subtype in South Africa, with non-C subtypes being sporadically detected. The accessory genes of subtype C have not been well studied in South Africa. In this study the HIV-1 vpr region of 58 strains was amplified, sequenced, and subtyped. Phylogenetic analysis characterized 54 strains as HIV-1 subtype C and 4 strains as HIV-1 subtype B. The amino acid sequence of the protein was also investigated. The overall amino acid sequence of Vpr was conserved as well as the motifs FPRPWL (34-39: IH1) and TYGDTW (49-54: IH2). The C-terminal was more variable. The importance of these motifs and variability needs to be addressed.

A new antigen scanning strategy for monitoring HIV-1 specific T-cell immune responses

Delineation of the immune correlates of protection in natural infection or after vaccination is a mandatory step for vaccine development. Although the most recent techniques allow a sensitive and specific detection of the cellular immune response, a consensus on the best strategy to assess their magnitude and breadth is yet to be reached. Within the AIDS Vaccine Integrated Project (AVIP http://www.avip-eu.org) we developed an antigen scanning strategy combining the empirical-based approach of overlapping peptides with a vast array of database information. This new system, termed Variable Overlapping Peptide Scanning Design (VOPSD), was used for preparing two peptide sets encompassing the candidate HIV-1 vaccine antigens Tat and Nef. Validation of the VOPSD strategy was obtained by direct comparison with 15mer or 20mer peptide sets in a trial involving six laboratories of the AVIP consortium. Cross-reactive background responses were measured in 80 HIV seronegative donors (HIV-), while sensitivity and magnitude of Tat and Nef-specific T-cell responses were assessed on 90 HIV+ individuals. In HIV-, VOPSD peptides generated background responses comparable with those of the standard sets. In HIV-1+ individuals the VOPSD pools showed a higher sensitivity in detecting individual responses (Tat VOPSD vs. Tat 15mers or 20mers: p≤0.01) as well as in generating stronger responses (Nef VOPSD vs. Nef 20mers: p<0.001) than standard sets, enhancing both CD4 and CD8 T-cell responses. Moreover, this peptide design allowed a marked reduction of the peptides number, representing a powerful tool for investigating novel HIV-1 candidate vaccine antigens in cohorts of HIV-seronegative and seropositive individuals.