Effect of early anti-retroviral therapy on the pathogenic changes in mucosal tissues of SIV infected rhesus macaques.

BACKGROUND: The gastrointestinal tissue plays an important role in the pathogenesis of HIV/SIV infection and serves as a viral reservoir in infected individuals under antiretroviral therapy (ART). However, the effect of ART administration in the very early stage of infection on HIV/SIV replication and pathogenesis in gastrointestinal tissue has not been fully studied. In this current study, rhesus monkeys infected with SIV were treated with ART starting at day 7 post-infection. The effect of early ART on SIV replication and infection-related pathogenic changes in mucosal tissues of the infected monkeys was examined. METHODS: Nuclear acids were extracted from snap frozen ileum and colon tissues and mesentery lymph nodes from SIV infected monkeys with or without ART. SIV RNA and DNA loads as well as levels of CD3, CD4 and cytokine mRNA were measured by PCR and RT PCR from the isolated nuclear acids. Tissue sections were stained by immuno-fluorescence labeled antibodies for CD3 and CD4. RESULTS: Without ART treatment, these monkeys underwent a mild SIV infection with low viral loads and slightly decreased CD4+ T cell counts in peripheral blood. In ART treated monkeys, SIV RNA loads were undetectable in blood with normal CD4+ T cell counts, however, SIV RNA and DNA were detected in the intestinal tissues and mesentery lymph nodes although the levels were lower than those in untreated monkeys. The levels of CD3 and CD4 positive cells in the tissues were similar between the infected untreated monkeys and infected ART treated monkeys based on RT-PCR and immune-fluorescence staining of the tissue sections. Furthermore, compatible levels of IL-6, TNF-a, IL-1b and MyD88 mRNAs were detected in most of intestinal tissues and mesentery lymph nodes of infected ART treated and infected untreated monkeys. CONCLUSIONS: These results suggest that early ART administration could not effectively inhibit SIV replication in intestinal tissues and mesentery lymph nodes and could not reduce the immune activation induced by SIV infection in the intestinal tissues.

An optimized sensitive method for quantitation of DNA/RNA viruses in heparinized and cryopreserved plasma.

Sodium heparin, an anticoagulant used widely for blood collection, has been known to inhibit DNA polymerase activity in polymerase chain reaction (PCR) assays. However, all cryopreserved plasma samples collected in the 1980s and early 1990s at the Multicenter AIDS Cohort Study were from heparin-treated blood, which poses a problem in quantifying the target nucleic acids contained in those samples by PCR assay. In this study, a nucleic acid extraction procedure was optimized to remove the heparin from extracted nucleic acids. Using this optimized method, similar human immunodeficiency virus 1 (HIV-1) and cytomegalovirus (CMV) loads of these viruses that were added to normal donor blood from ethylenediaminetetraacetic acid (EDTA), acid citrate dextrose (ACD) or sodium heparin tubes were detected by reverse transcriptase (RT) real-time PCR and real-time PCR. Comparable HIV-1 and CMV loads were also detected in the blood of persons with active HIV-1 and CMV infections collected in EDTA-, ACD- or sodium heparin-treated tubes by RT real-time and real-time PCR. The findings showed that the optimized nucleic acid extraction procedure efficiently removes the heparin inhibition effect on the performance of real-time PCR. This method could be used to extract nucleic acids from archived, heparinized plasma for PCR based quantitation of target molecules.

Induction of strong anti-HIV cellular immunity by a combination of Clostridium perfringens expressing HIV gag and virus like particles.

The lower gastrointestinal tract is a major mucosal site of HIV entry and initial infection. Thus, the induction of strong cellular immune responses at this mucosal site will be an important feature of an effective HIV vaccine. We have used a novel prime-boost vaccination approach to induce immune responses at mucosal sites. Orally delivered recombinant Clostridium perfringens expressing HIV-1 gag (Cp-Gag) was evaluated for induction of HIV-1 Gag specific T cell responses in a prime-boost model with intranasal inoculation of HIV-1 virus like particles (VLP). HIV-1 specific cellular immune responses in both the effector (Lamina propria) and inductive sites (Peyer's patches) of the gastrointestinal (GI) tract were significantly higher in mice immunized using Cp-Gag and VLPs in a prime-boost approach compared to mice immunized with either Cp-Gag or VLPs alone. Such cellular immune response was found to be mediated by both CD8(+) and CD4(+) T cells. Such a strong mucosal immune response could be very useful in developing a mucosal vaccine against HIV-1.

Induction of SIV p27-specific multifunctional T cells in the gut following prime-boost immunization with Clostridium perfringens and adenovirus vaccines expressing SIV p27.

A vaccine-induced cellular immune response to simian immunodeficiency virus (SIV) in the gut mucosal tissue may prevent the establishment or severity of new SIV infection. An oral Clostridium perfringens expressing SIV p27 (Cp-p27) vaccine that delivers SIV p27 to the gut was evaluated for its ability to prime multifunctional cellular immunity in the gut mucosa. Gut Peyer's patches dendritic cells matured in response to in vitro exposure to Cp-p27 and stimulated production of p27-specific IFN-gamma. In mice, the oral vaccination with the Cp-p27 vaccine and systemic immunization with adenovirus expressing SIV p27 (Ad-p27) induced robust systemic and mucosal immune responses. Furthermore, the prime-boost regimen induced p27-specific multifunctional CD8+ T cells in the gut. These results indicate that priming gut tissue with Cp-p27 can enhance the gut mucosal cellular immune response generated via systemic immunization with Ad-p27.

Detection of HIV-1 RNA/DNA and CD4 mRNA in feces and urine from chronic HIV-1 infected subjects with and without anti-retroviral therapy.

HIV-1 infects gut associated lymphoid tissues (GALT) very early after transmission by multiple routes. The infected GALT consequently serves as the major reservoir for HIV-1 infection and could constantly shed HIV-1 and CD4+ T cells into the intestinal lumen. To examine this hypothesis, we monitored HIV-1 RNA/DNA and CD4 mRNA in fecal samples of chronically infected subjects with and without antiretroviral therapy (ART). We compared this to levels of HIV-1 RNA/DNA in urine and blood from the same subjects. Our results show that HIV-1 DNA, RNA and CD4 mRNA were detected in 8%, 19% and 31% respectively, of feces samples from infected subjects with detectable plasma viral load, and were not detected in any of subjects on ART with undetectable plasma viral load. In urine samples, HIV-1 DNA was detected in 24% of infected subjects with detectable plasma viral load and 23% of subjects on ART with undetectable plasma viral load. Phylogenetic analysis of the envelope sequences of HIV-1 revealed distinct virus populations in concurrently collected serum, feces and urine samples from one subject. In addition, our study demonstrated for the first time the presence of CD4 mRNA in fecal specimens of HIV-1 infected subjects, which could be used to assess GALT pathogenesis in HIV-1 infection.

Biological properties of HIV-1 subtype B' isolates from infected Chinese blood donors at different disease stages.

Unsanitary blood/plasma collecting activities in central China during the 1990's caused a high prevalence of blood-borne HIV-1 infection. Although the genetic characterization of the proviral DNA of HIV-1 circulating in the infected former blood donors (FBDs) has been reported, there is little information about the biological characteristics of virus isolates in these FBDs. In this study, we have examined the biologic properties of HIV-1 isolates from AIDS patients and long-term non-progressors (LTNP) of FBDs. Our results indicate that the growth properties, co-receptor usage and syncitium inducing capabilities of the HIV-1 isolates are associated with the disease status of patients. The virus isolates from LTNPs replicated slower, used the CCR5 co-receptor and were of non-syncytium inducing phenotype. In contrast, HIV-1 isolates from AIDS patients showed high replication kinetics, used both CCR5 and CXCR4 co-receptors and induced syncytium formation. A higher level of cytopathicity was also detected in syncytium inducing virus compared to non-syncytium inducing isolates irrespective of patients' disease statuses. Although there was no significant differences in the binding and penetration of the target cells between the isolates from LTNPs and those from AIDS patients, viral DNA synthesis of viral isolates from LTNPs was much slower than the DNA synthesized by the isolates from AIDS patients, indicating a restriction at a post-entry step. Analysis of deduced amino acid sequences in C2-V5 regions of these isolates has provided a molecular basis for further identification of viral phenotypes of HIV-1 subtype B'. This study has provided valuable information on the biological properties of circulating HIV-1 strains among Chinese FBDs to better understand their viral characteristics and design more appropriate vaccine candidates for FBDs.

Lack of differences in HIV-1 nef functional domains in infected chinese blood donors at different stages of disease.

HIV-1 nef regions were amplified by polymerase chain reaction and sequenced from DNA samples of five asymptomatic subjects and five AIDS patients from a cohort of HIV-1-infected Chinese plasma and blood donors. Sequence analysis revealed that regardless of the stage of disease, each patient's HIV-1 nef sequences belonged to the clade B' subtype. Although there are some differences between the sequences from different patients, no significant differences have been detected in nef nucleotide sequences or functional motifs in the deduced amino acid sequences from patients at different stages of the disease. Furthermore, the predicted binding motifs of HLA-A2 and HLA-A11 were highly conserved among patient nef sequences. These results will contribute to a better understanding of the pathogenesis of circulating HIV-1 in infected Chinese former blood donors and may have important implications in developing an epitope-based vaccine suitable for Chinese blood donors.

Disruption of a toxin gene by introduction of a foreign gene into the chromosome of Clostridium perfringens using targetron-induced mutagenesis.

Clostridium perfringens (C. perfringens) has been developed as a potential oral delivery vehicle to deliver antigens or therapeutic compounds to Gut-Associated Lymphoid Tissue (GALT). However, this recombinant C. perfringens carries a plasmid-encoded expression system, which raises several safety concerns regarding possible horizontal plasmid transfer and spread of plasmid-associated antibiotic resistant genes. Furthermore, this bacterium produces the extracellular theta toxin, which poses a potential safety issue for general administration. Using a Clostridium-specific-targetron donor plasmid, we inserted the Simian Immunodefiency Virus (SIV) p27 gene into the theta toxin gene (pfoA) on the C. perfringens chromosome, which simultaneously inactivated the theta gene and introduced SIV p27 gene onto the bacterial chromosome. Such mutant C. perfringens without an input plasmid or antibiotic resistant gene stably produced a large amount of SIV p27 protein during sporulation and did not produce theta toxin. Upon oral feeding of the mutant bacteria to mice, intact p27 protein was detected in the lower GI tract. The re-engineered C. perfringens provides a biosafe efficient oral vehicle to deliver antigen to the gastrointestinal tract.