1. HIV interactions with coinfecting viruses. Over the past few years, we and others have identified several microbes that upon coinfection suppress HIV-1 replication. In particular we found that HHV-6 suppresses HIV in coinfected human tissues by upregulating CCL-5 (RANTES). However HHV-6 is also known as a co-factor in HIV disease progression. This apparent contradiction has now been resolved in our experiments with pig-tailed macaques (M. nemestrina) infected with SIVsmE660 or co-infected with SIVsmE660 andHHV-6AGS. SIV isolated were obtained from macaques after one year of infection and tested for their growth in human or macaque lymphoid tissue ex vivo. All HHV-6−co-infected animals progressed to AIDS within 2 years of infection and were found to harbor SIV variants with a reduced sensitivity to suppression by both HHV-6A and CCL5, despite maintaining an exclusive CCR5 specificity. Viruses derived from two of these animals replicated even more vigorously in the presence of exogenous HHV-6A or CCL5. In contrast, SIV isolates from singly SIV-infected macaques maintained CCL5 sensitivity. These results provide the first demonstration of SIV evolution towards CCL5 resistance under the influence of a coinfecting microbe, illustrating a potential mechanism for the accelerated progression to full-blown AIDS seen in HHV-6−coinfected macaques. CCL5 resistance may represent a common virulence factor allowing primate immunodeficiency retroviruses to evade a critical mechanism of host antiviral defense. In conclusion, our study illustrates a novel mechanism whereby coinfection with a putative AIDS-progression cofactor, the T-lymphotropic HHV-6A, may affect the in vivo evolution of SIV, leading to an accelerated development of AIDS. Understanding the complex interactions between HHV-6A and primate immunodeficiency viruses may provide important information not only on mechanisms of HIV pathogenesis, but also for the development of novel preventive and therapeutic strategies against HIV-1. 2. Acyclovir-derivatives as anti-HIV-1 drugs. Recently, we reported that phosphorylated acyclovir (ACV) inhibits HIV-1 reverse transcriptase (RT) in a cell free system. Also, ACV suppresses HIV-1 replication in human lymphoid tissues ex vivo co-infected with herpesviruses that are capable of phosphorylating this compound. In view of these data, we launched new studies and a new targeted clinical trial to understand the role of anti-HIV activity of the herpes-suppressive drugs in HIV-1−HSV co-infected patients. Also, we developed ACV based compounds that are active against HIV-1 independently of HHV-mediated phosphorylation. Since, ACV-phopshorylated itself can not be used as an efficient anti-HIV agent because of its instability in biological media and its poor penetration through plasma membranes, we applied the ProTide technology to deliver phosphorylated ACV into HIV−infected cells. This approach is based on the masking of negatively charged monophosphate with lipophilic groups (an aryl moiety and an amino acid ester). Once inside the cell, the masking groups of the prodrug are cleaved rendering the phosphorylated ACV available. We analyzed the effects of the aryl and ester moiety variation as well as that of variation of the amino acid of the ACV ProTides on their anti-HIV activity in isolated cells. L-alanine derivative-containing ProTides showed anti-HIV activity at concentrations far below those causing cytotoxicity. ACV ProTides with other amino acids with the exception of L-phenylalanine, were inactive against HIV in cell culture. Enzymatic and molecular modeling studies have been performed in order to better understand the antiviral behavior of these compounds. The next step of this project is to test ACV ProTides in human tissues. Meanwhile, we studied HIV-1 RT evolution under the pressure of ACV ProTides. We found that V75I was the dominant mutation emerging in ACV-resistant HIV-1. We studied the biochemical mechanism underlying this resistance and found that the in the V75I RT, the incorporated ACV-monophosphate is more vulnerable to excision in the presence of the pyrophosphate donor ATP. V75I mutation compromises binding of the next nucleotide, which can otherwise provide a protection from excision. Further studies of the development and mechanisms of HIV-1 resistance to ACV are necessary for assessing its potential clinical utility in combination with established NRTIs. 3. Development of experimental models for studying HIV pathogenesis in human tissues ex vivo. The study of human cell-cell and cell-pathogen interactions that occur in the context of tissue cytoarchitecture is critical for deciphering the mechanisms of many normal and pathogenic processes. Notwithstanding the complexity of HIV pathogenesis in vivo, HIV infection is usually studied in vitro in a relatively primitive system of isolated cells. Earlier, we developed a system of human tonsillar tissue ex vivo and studied the pathogenesis of HIV as well as that of other infectious agents in this system. Now we have extended this system to cervico-vaginal and rectosigmoid tissues, as these tissues are critical in HIV transmission and pathogenesis. Currently we are adapting ex vivo cervico-vaginal tissue in order to study HIV transmission through the cervical mucus. Also, we found that the developed system supports productive infection by HHV-6, HHV-7, HCMV (HHV-5), HSV-2 (HHV-2), vaccinia virus, measles virus, and West Nile virus as well by the parasite Toxoplasma gondii. In principle, this technique can be adapted to the study of other viruses as well as of various normal or pathophysiological processes. To compare various aspects of HIV-1 pathogenesis ex vivo as well as to combine data from different laboratories and to apply this system to evaluate microbicides, it is necessary to standardize the procedure of tissue culture and the assays for monitoring HIV. In collaboration with other laboratories, we undertook this task. We developed a novel soft endpoint method to provide an objective measurement of virus replication. The applicability of the soft endpoint is shown across several different ex vivo tissue types, cultured in different laboratories, and for a candidate microbicide. Statistical analysis showed that different laboratories can provide consistent measurements of anti-HIV-1 microbicide efficacy using the new assay. Finally, we developed a new methodology to measure HIV p24 in biological samples. This method relies on using beads coupled to a high affinity monoclonal antibody against HIV-1 p24, and a second complementary monoclonal antibody against another epitope of the same antigen. The developed bead-based assay is simple, sensitive and inexpensive offering a wide dynamic measurement range allowing the detection of p24 concentrations over 5 orders of magnitude. It is in the research community that we see the most promising application of this assay because of its low cost and its wide response range. However, this assay is as sensitive as current diagnostic test in select seroconversion panels, and may be adapted for diagnostic purposes, especially in resource-limiting settings.
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