Author(s): Soares, Rui S.
Date: 2011
Persistent ID: http://hdl.handle.net/10451/3581
Origin: Repositório da Universidade de Lisboa
Subject(s): Alergia e imunologia; HIV; HIV-2; Carga viral; Replicação viral; Infecções por HIV; RNA mensageiro; Teses de doutoramento - 2011
Tese de doutoramento, Ciências Biomédicas (Imunologia), Universidade de Lisboa, Faculdade de Medicina, 2011
The acquired immunodeficiency syndrome (AIDS), first described in 1981, has caused more than 25 million deaths worldwide with an estimated 2 million deaths related to disease in 2008, representing one of the most important public health problems worldwide. AIDS is caused by infection with human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2). HIV-1 infection is pandemic, with more than 33 million people estimated to be infected, whilst HIV-2 infection remains relatively contained to a few West African countries, with Portugal representing the only non-African country with a significant HIV-2 prevalence (3.2% of infection cases reported in 2009). HIV-2 infection is characterized by a slower progression to AIDS as compared to HIV-1 infection, with a limited impact on the survival of the majority of infected adults. The rate of CD4 T cell decline is much slower than in HIV-1 and the levels of circulating virus (viremia) are significantly lower irrespective of disease stage. The low levels of viremia in patients infected with HIV-2 suggest a lower replicative activity. However, despite the lower viremia in HIV-2, our results, as well as studies from other groups, showed that levels of proviral DNA in peripheral blood mononuclear cells are similar in both infections, suggesting that a similar number of cells are infected. The cell tropism of HIV-2 and HIV-1 is determined by the expression of chemokine receptors on target cells in addition to the expression of the CD4 molecule. CCR5 and CXCR4 are the major co-receptors that are used for HIV-1 entry, and their levels of expression were shown to be directly related to the susceptibility to infection and to the rate of disease progression in HIV-1 infected individuals. Several studies suggest that, although HIV-2 can use a larger number of co-receptors as compared to HIV-1 in vitro, the major co-receptors used in vivo are also CCR5 and CXCR4. It is possible that different expression levels of these co-receptors lead to different patterns of infection in T cells populations determining the reduced production of virus and disease progression that is observed in individuals infected with HIV-2. We compared the expression of CCR5 and CXCR4 in patients infected with HIV-2 and HIV-1 that had similar levels of CD4 T cell depletion and found that patients infected with HIV-2 had an increased frequency of cells expressing CCR5 in the CD4 memory-effector population as compared to seronegative subjects. We also found a significant correlation between the frequency of cells expressing CCR5 in total CD4 T cells and the level of CD4 T cell depletion in individuals infected with HIV-2, but not in the ones infected with HIV-1. The expression of CCR5 is induced by cell activation and is usually restricted to the memory-effector population. We found the existence of a direct correlation between the frequency of cells expressing CCR5 and the levels of immune activation in individuals infected with HIV-2. These results demonstrate an association between the expansion of cells expressing CCR5 and immune activation in HIV-2. The lower frequency of cells expressing CCR5 in HIV-1 infected individuals, as compared with HIV-2+ individuals may be related to a continuous depletion of these cells in the presence of the higher levels of viremia associated with HIV-1 infection. Regarding the frequency of cells that express CXCR4 within CD4 T cell population, we found that this frequency was similar in patients infected with HIV-2 or HIV-1 and HIV-negative individuals. We also investigated the possibility that the similar levels of proviral DNA in the presence of different levels of viremia could be due to differences in the target cells. The levels of proviral DNA were quantified in purified naive and memory CD4 T cells from patients infected with HIV-2 and found to be very low in naive CD4 T cells, in marked contrast to the levels found in memory CD4 T cells. These results are in agreement with what is observed in individuals infected with HIV-1, suggesting that the memory CD4 T cell population is the main target for HIV-2, and further supporting the notion that CCR5 is the main co-receptor used by HIV-2 for infection in vivo. These data suggest that the lower viremia observed in patients infected with HIV-2 is not related to co-receptor availability on target cell populations. The low to undetectable levels of viremia in the presence of significant levels of proviral DNA may be due to the regulation of HIV-2 at the transcriptional level. Therefore, we proceeded with the study of viral mRNA expression through the development of methodologies based on real-time RT-PCR for the quantification of unspliced (gag) and multiply spliced (tat) mRNA transcripts of HIV-2 and HIV-1. The optimization of these approaches allowed the development of assays with high sensitivity, specificity and reproducibility, which were used to investigate the transcriptional activity in cohorts of HIV-1 and HIV-2 infected patients. We found that HIV-2 infected patients expressed significantly lower levels of tat mRNA as compared to patients infected with HIV-1. However, gag mRNA expression was found to be similar in the two cohorts. Given that tat mRNA seems to be mainly expressed in recently infected cells, our data suggest that the rate of new infections at the cellular level is lower in individuals infected with HIV-2 as compared to their HIV-1 infected counterparts. On the other hand, the observation of similar levels of gag mRNA in the two infections suggest that there is significant ongoing viral transcription in individuals infected with HIV-2, despite the low levels of viremia that characterize this infection. We further investigated the role of viremia, as well as levels of proviral DNA and tat and gag viral transcripts on the chronic immune hyperactivation observed in HIV-2 and HIV-1 infections. We found significantly higher levels of CD4 and CD8 T cell activation in HIV-2 infected individuals with detectable viremia as compared with aviremic HIV-2+ individuals. These results suggest a contribution of the circulating virus, even at low levels, to the state of immune activation found in these individuals. Our results further revealed a direct association between the levels of gag mRNA and the activation of CD4 T cells, supporting the possibility that persistent viral replication contributes significantly to the elevated immune activation levels observed in patients infected with HIV-2 despite the reduced viremia. In addition, we found a direct association between the levels of tat mRNA and CD8 T cell activation, particularly in individuals infected with HIV-1, suggesting that the levels of tat, or the number of newly infected cells, may have a role in CD8 T cell activation. Next, we evaluated the impact of antiretroviral therapy on the levels of viral transcripts and proviral DNA levels in treated HIV-2 infected individuals. Studies in treated HIV-1 patients showed that antiretroviral therapy led to decreased levels of viral mRNA, as well as a progressive decrease in the levels of proviral DNA. However, there were no data regarding the effects of antiretroviral therapy on the levels of viral transcripts and proviral DNA in treated HIV-2 infected individuals. We found that these individuals featured significantly higher levels of tat mRNA than their untreated counterparts, suggesting that the regimens used were not effective in reducing the rate of infection of new cells. We also documented the presence of mutations in reverse transcriptase and protease in the majority of treated patients, which indirectly supports the existence of persistent viral replication despite antiretroviral therapy. We further noted that these patients showed elevated levels of CD4 and CD8 T cell activation that negatively correlated with the number of CD4 T cells in circulation, possibly contributing to the reduced CD4 T cell recovery observed in these patients. In conclusion, we provide evidence of significant levels of viral replication in patients infected with HIV-2, and of its relationship with the levels of immune activation in spite of the low levels of circulating virus. Our data reinforce the importance of monitoring levels of persistent viral replication, even at low levels and emphasize the need to pursue clinical trials and studies on the efficacy of antiretroviral therapy in HIV-2 infection.
POCI 2010 e o FSE. Bolsa de Doutoramento da Fundação para a Ciência e a Tecnologia (referência: SFRH/BD/23863/2005)
