In addition, the CTLs from the LTNPs made large amounts of perforin, a substance that is crucial for the ability of CTLs to kill infected cells [see note 2]. Dr. Connors stressed there was no defect in perforin per se, since the few CTLs from faster progressors that did proliferate were also able to produce perforin. In other words, the two functions are coupled, so CTLs that don't divide don't upregulate their expression of perforin, and consequently can't destroy infected cells.
His group further found that the non-dividing CTLs seem to be permanently stuck in this mode, since no matter how they tried to stimulate them in the laboratory, the cells would not divide and produce perforin. Dr. Connors presented two models that might explain the intransigence of the non-proliferating CTLs: first, the cells may not be receiving all of the necessary signals they need to differentiate and divide [see note 3]. Alternatively, the cells may have divided so many times they are "burnt out" and hence too tired or old to divide any more, a condition known as replicative senescence.
Contrary to some studies that have detected only small amounts of slowly developing anti-HIV antibodies, Dr. Shaw said his lab saw "massive amounts" of anti-HIV antibodies developing within only ten weeks after HIV infection. But although these anti-HIV antibodies were able to neutralize (prevent the virus from doing harm) HIV and lower viral load, viral mutations soon allowed HIV to "escape" the neutralizing antibodies (NAbs).
Surprisingly, Dr. Shaw found that few of the anti-HIV antibodies were directed to sites, or epitopes, on viral proteins that are the usual neutralizing antibody targets on other viruses, such as those involved with the attachment of the virus to cellular receptors. Instead, the anti-HIV antibodies were directed to parts of the virus that bind glycan (sugar) molecules to its surface. (The antibodies don't directly attack the sugar molecules since the same sugars also coat many of the body's natural proteins; such antibodies would cause an autoimmune reaction.)
Dr. Shaw also showed that antibody-containing blood plasma taken from an individual at later stages of infection was better able to neutralize virus collected from the same person at an earlier stage of infection than it was contemporary viral isolates or virus that developed months later. For example, virus from the 16th day after infection was neutralized by plasma from day 212 far more effectively than by plasma from day 16. Plasma from day 16 had no effect on virus from day 212. This indicates that vulnerable epitopes in early viral isolates had somehow become protected over time.
It's long been known that HIV is heavily coated with sugars and some have speculated that the sugars keep the viral binding sites covered until just before they're ready to attach to a cell. Yet Dr. Shaw found that none of the sugars on HIV were directly covering the virus' attachment proteins. Rather, many widely spread glycans somehow "cooperatively pack over the entire envelope spike to prevent" antibody access through steric (spatial) hindrance. Steric hindrance produces a kind of force field barrier that blocks antibodies from contacting their epitopes on the attachment proteins. It also means that the "glycan canopy" would only need to partially overlap those epitopes to prevent antibody access. Since most of the HIV mutations that Dr. Shaw observed were associated with its sugar binding regions and not its cell attachment sites, he proposed that the "cooperatively packed" glycans continually evolve (change) into ever-denser packs. In this way they would deter any newly arising antibodies, yet not prevent HIV from efficiently attaching to its target cells. Shaw dubbed this an "evolving glycan shield."
If this was the case, he suggested, then the evolved HIV resistance to antibody attack was not a generalized resistance, but rather the virus' adaptation to each individual's specific antibodies directed at the sugar binding sites [see note 6].
In his summation, Shaw noted the surprisingly large number of antibodies, as well as the unexpected occurrence of viral escape mutations at sites other than the common neutralizing epitopes. He concluded that vaccine-induced anti-HIV antibodies would likely not have much impact after acute infection; but he held out hope they might help prevent a new infection from being established since both the inoculum and efficiency of sexual transmission of HIV are low.
First he showed how his lab used a new biological tool called a microarray gene chip to compare which genes are expressed (turned on) in latently infected CD4 T cells, depending on whether the "latent" cells come from a person with undetectable viremia or high viremia. His research group found that high viremia resulted in latently infected cells expressing many genes whose protein products helped the cells "to be permissive for the production [and release from the cell] of HIV." He said the upregulated genes were not caused by broad, non-specific activation of the latently infected CD4 T cells, since they observed that genes upregulated when CD4 T cells are non-specifically activated are not the same genes that are expressed during high viremia. He therefore posited two other mechanisms by which high viremia could induce the expression of genes whose products help latent HIV start to replicate or reproduce.
First, different combinations of "aberrantly-expressed cytokines" (hormone-like substances which cells release in order to communicate with neighboring cells) may be turning on genes that promote HIV replication. Second, perhaps the virus' envelope or other viral proteins were helping to trigger HIV replication. He added that this study demonstrates there really is no HIV latency during periods of high viremia. He said that a semblance of true HIV latency is approached only in aviremic individuals, since they were found to secrete little or no virus from their latently infected CD4 T cells.
Dr. Fauci next compared the effects of low and high viremia on B cells. He found that during high viremia, B cells, like the CD8 T cells described above, lost their ability to proliferate, which he attributed in part to their inability to upregulate a receptor on their surface called CD25. Before B cells can proliferate they need to receive a signal from CD4 T-helper cells that is transmitted through the B cell's CD25 receptor to the genes in its nucleus. The molecule that delivers this signal is a cytokine called IL-2 or Interleukin-2, and thus the CD25 receptor is also known as the IL-2R or IL-2 receptor. Since many of the B cells in viremic patients lack the IL2R, they rarely receive a signal to proliferate and consequently do not function properly. This B cell defect was not found in aviremic patients.
Finally, Dr. Fauci compared the functioning of NK cells in viremic and aviremic patients. NK cells can destroy infected cells in a manner similar to CTL killing, but they arise earlier during infection and never become as specific in their killing as CTLs. Immunologists have described NKs as a bridge between the innate and adaptive immune systems and consider them very important for virus control. Dr. Fauci's group found that, as with B cells, high viremia results in the aberrant expression of NK cell receptors, thus causing these cells to malfunction. On the other hand, the NK cell receptors in aviremic patients appeared much the same as those from uninfected controls.
Since Dr. Fauci noted that some of the patients experiencing high viremia were on HAART, he seemed to be emphasizing the importance of maintaining as low a viral load as possible while on therapy. While the viremia-caused defects he described don't cause the affected immune cells to die, Dr. Fauci said that they "likely play a cumulative role in the pathogenesis of HIV disease," and may serve as the basis for some future "strategic avenues of approach to therapy."
Note 1. CD8 T cells can also release various soluble factors that don't kill infected cells, but rather prevent the viruses inside those cells from reproducing. Whereas this function may be more important in early infection, it's generally believed that to maintain good long-term control the CD8 T cells must function primarily as killers.
Note 2. Perforin punctures holes in the membranes of HIV+ cells, enabling the CTLs to then discharge various toxins called granzymes into the infected cells. The granzymes then stimulate signals in the cell that tell it to apoptose, or commit cellular suicide.
Note 3. The lack of signals from CD4 T-helper cells that have been killed (either directly or indirectly) due to HIV may seem the obvious reason why CTLs are not getting adequate support to proliferate. However, for various reasons many scientists do not believe the lack of CD4 T cell help is the whole or even necessarily part of the story. For example, when Connors' group stimulated the non-proliferating CTLs with IL-2, which is the main soluble factor released by CD4 T-helper cells, the CTLs still refused to proliferate.
Note 4. Before the more specific adaptive immune system has time to kick in, another line of defense called innate immunity acts as a first responder to pathogens. Innate immune responders consist of cells and soluble factors that are not specific for any particular pathogen, but rather act in a general way to rid the body of pathogens. Scientists have recently come to appreciate that the type of innate immune response initially engendered helps determine the type of adaptive response that will subsequently occur. Unlike innate factors, each individual CTL and antibody has a unique shape that will only react with the specific parts of pathogens that match that shape, much as each lock has its own key.
Note 6. In the words of Shaw's paper in Nature, "a rapidly evolving, non-glycan reactive, effective NAb repertoire matched by a similarly evolving glycan shield that obstructs NAb from binding at neighboring sites."
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