Filling the gaps: Towards improved surveillance and monitoring of immunological status relevant to long term co-morbidities in HIV infection

It is approaching 35 years since human immunodeficiency virus (HIV)-1 was first identified in Western Australia (WA) and 20 years since introducing highly active antiretroviral therapy (HAART) transforming HIV management, in the majority of cases, by maintaining ‘undetectable’ HIV-1 RNA and restoring CD4 T cell counts to healthy levels. Despite these advances patients are at increased risk of developing age-associated diseases. This thesis therefore investigates whether expanding laboratory approaches to incorporate aspects of the disease process, that are currently 'invisible' in clinical practice but may be prognostically important or which may inform treatment and prevention strategies at a population level, is warranted. The following aims were addressed; 1) analysing determinants of HIV-1 RNA residual viraemia; 2) investigating monocyte activation during chronic HIV-1 infection and; 3) assessing HIV-1 diversity from large scale sequences throughout WA and Australia, including the collaboration and formation of the Australian Molecular Epidemiology Network (AMEN). The main findings demonstrated HIV-1 residual viraemia was powerfully associated with the pre-treatment level of viraemia even after 10-15 years of starting HAART. Analysis of monocyte activation revealed multiple pathways involved in chronic immune activation responses to HIV-1, by altering CD16+ monocyte expression and elevated levels of sCD14 (previously associated with all-cause mortality), that were not corrected by HAART and therefore prognostically significant. In contrast, levels of other biomarkers (e.g. CXCL10 and sCD163) declined with HAART. Investigations of HIV-1 genetic diversity within the WA cohort (n=1021) and the Australian cohort (n=4873) revealed new challenges for HIV-1 prevention due to significant increases in HIV-1 non-B-subtypes, consistent with an increasing impact of migration, while local transmissions were predominantly HIV-1 B-subtypes. Overall, this thesis provides evidence for possible new approaches that may enhance HIV-1 laboratory practice that could aid clinical management, improve long-term health outcomes for those living with HIV-1 infection and guide effective preventative strategies

Elevated plasma soluble CD14 and skewed CD16+ monocyte distribution persist despite normalisation of soluble CD163 and CXCL10 by effective HIV therapy: a changing paradigm for routine HIV laboratory monitoring?

OBJECTIVE: We investigated plasma and flow cytometric biomarkers of monocyte status that have been associated with prognostic utility in HIV infection and other chronic inflammatory diseases, comparing 81 HIV+ individuals with a range of treatment outcomes to a group of 21 healthy control blood donors. Our aim is to develop and optimise monocyte assays that combine biological relevance, clinical utility, and ease of adoption into routine HIV laboratory practice. DESIGN: Cross-sectional evaluation of concurrent plasma and whole blood samples. METHODS: A flow cytometry protocol was developed comprising single-tube CD45, CD14, CD16, CD64, CD163, CD143 analysis with appropriately matched isotype controls. Plasma levels of soluble CD14 (sCD14), soluble CD163 (sCD163) and CXCL10 were measured by ELISA. RESULTS: HIV status was associated with significantly increased expression of CD64, CD143 and CD163 on CD16+ monocytes, irrespective of the virological response to HIV therapy. Plasma levels of sCD14, sCD163 and CXCL10 were also significantly elevated in association with viremic HIV infection. Plasma sCD163 and CXCL10 levels were restored to healthy control levels by effective antiretroviral therapy while sCD14 levels remained elevated despite virological suppression (p<0.001). CONCLUSIONS: Flow cytometric and plasma biomarkers of monocyte activation indicate an ongoing systemic inflammatory response to HIV infection, characterised by persistent alterations of CD16+ monocyte expression profiles and elevated sCD14 levels, that are not corrected by antiretroviral therapy and likely to be prognostically significant. In contrast, sCD163 and CXCL10 levels declined on antiretroviral therapy, suggesting multiple activation pathways revealed by these biomarkers. Incorporation of these assays into routine clinical care is feasible and warrants further consideration, particularly in light of emerging therapeutic strategies that specifically target innate immune activation in HIV infection

Model 2—Multivariate regression results showing significant associations of plasma biomarker with HIV clinical parameters, CVD risk age, gender, ethnicity and smoking after adjusting for CXCL10, sCD163 and sCD14.

<p>Model 2—Multivariate regression results showing significant associations of plasma biomarker with HIV clinical parameters, CVD risk age, gender, ethnicity and smoking after adjusting for CXCL10, sCD163 and sCD14.</p

sCD14 levels are significantly elevated in HIV patients irrespective of HIV viral load status (A).

<p>In contrast sCD163 levels are only elevated in viremic HIV patients (B) and a similar relationship is seen with CXCL10 levels (C). (Data presented as mean values and 95% confidence intervals; p-values are derived from log transformed data and ANOVA tests were adjusted for multiple comparisons).</p

Demographics and patient characteristics from 474 HIV positive patients who underwent CVD risk assessments in 2010.

<p>Demographics and patient characteristics from 474 HIV positive patients who underwent CVD risk assessments in 2010.</p

There was a significantly strong correlation between sCD14 and smoking where HIV-1 smokers have higher sCD14 levels than HIV-1 non-smokers while patients on an integrase inhibitor had significantly lower sCD14 levels than patients on an alternative treatment.

<p>There was a significantly strong correlation between sCD14 and smoking where HIV-1 smokers have higher sCD14 levels than HIV-1 non-smokers while patients on an integrase inhibitor had significantly lower sCD14 levels than patients on an alternative treatment.</p

The median cell surface expression and 95% confidence intervals are shown for CD64 (A), CD163 (B) and CD143 (C) on monocyte subsets in HIV and control subjects.

<p>Expression on classical monocytes is significantly different between HIV and control groups for CD64 (p<0.001) but not for CD163 or CD143. Expression of all cell surface markers on CD16⁺ monocytes, however, are significantly different between the HIV infected and control groups (* p<0.05, ** p<0.01 *** p<0.001).</p

Differing correlation outcomes between the three plasma biomarkers and HIV-1 RNA levels.

<p>A significant correlation was recognised between HIV-1 RNA levels with CXCL10 (A) and sCD163 (B) while there was no significance with sCD14 (C).</p

Long-Term Associations between Human Cytomegalovirus Antibody Levels with All-Cause Mortality and Cardiovascular Outcomes in an Australian Community-Based Cohort

Human cytomegalovirus (HCMV) infection has been shown to increase the risk of cardiovascular events and all-cause death among individuals with clinically apparent cardiovascular disease (CVD). Whether this association exists in individuals with no history of CVD remains unclear. Serum levels of HCMV IgG antibody were measured using an ELISA in 2050 participants aged 40&ndash;80 years from the 1994/1995 Busselton Health Survey who did not have CVD at baseline. Outcomes were all-cause death, cardiovascular death, acute coronary syndrome (ACS) and major adverse coronary and cerebrovascular events (MACCE, composite of all-cause death, ACS, stroke and coronary artery revascularisation procedures). Cox proportional hazards regression analysis was used to investigate HCMV antibody levels as a predictor of death and cardiovascular outcomes during follow-up periods of 5, 10 and 20 years. At baseline, participants had a mean age of 56 years and 57% were female. During the 20-year follow-up, there were 448 (21.9%) deaths (including 152 from CVD), 139 (6.8%) participants had ACS and 575 (28.0%) had MACCE. In the fully adjusted model, levels of HCMV antibody at 20 years was associated with all-cause death (HR 1.04; 95% CI 1.00, 1.07, p = 0.037) but not with CVD death, ACS or MACCE. Levels of HCMV antibody are associated with all-cause death but not with cardiovascular outcomes in adults without pre-existing CVD

HIV viral load status has different influences on monocyte cell surface expression.

<p>Expression of CD64 on intermediate (A) and non-classical (B) monocytes, CD143 on intermediate (C) and non-classical (D) monocytes, and CD163 on intermediate (E) and non-classical (F) monocytes are shown below. (Data presented as mean values and 95% confidence intervals; p-values derived from Kruskal-Wallis tests).</p