Molecular biological assessment methods and understanding the course of the HIV infection.

Abstract

Only some twenty years has passed since the first discovery of severe immunodeficiency among previously healthy homosexual men through the discovery of the causing virus and till the status today where the knowledge on the HIV virus and the pathogenic mechanisms induced by the virus are extensive, though still incomplete. Furthermore, steadily better treatments have been introduced at a paste that is probably without precedents. These processes have been fuelled by various molecular biological methods. The abilities to quantify viremia and to sequence virus and hence describe the evolution of the virus represent valuable tools for understanding the pathogenic processes. The current thesis describes some of the findings obtained. While it was initially thought that the virological profile mimicked the clinical with an acute infection followed for years by clinical latency and only after on average ten years signs of severe immunodeficiency, this understanding has been revised. There is no virological latency. The viral replication is on going throughout the infection. However, the virological profile does resemble the clinical. Viremia is high shortly after infection; hereafter declines, and stabilises around what has been termed the viral set point. This level of viremia is predictive of the clinical course of the infection. We have shown that the viremic levels, measured both as HIV RNA load and proviral DNA load, early in infection carry significant information about the course of the infection. It is; however, not only early viral loads that carry prognostic information, also viral load during late-stage infection is clinically informative. Viral load measurements have evolved as the major tool for monitoring the efficacy of antiretroviral therapy. HIV RNA has been shown to be a good surrogate marker for the clinical efficacy of antiretroviral treatment. How to use the measurements most optimally has however not been fully delineated. Various methods for describing virological response might yield different results, and it is recommended that the pros and cons of the various methods be investigated. In a cohort of patients who had obtained good virological suppression on antiretroviral therapy followed prospectively for two years we found that only few patients experienced high-grade viremia. Furthermore, baseline HIV DNA differed between the patients with various longitudinal HIV RNA profiles. The patients with the most pronounced HIV RNA suppression had lowest proviral load at baseline, with a clear gradient across the groups. The interplay between proviral load and treatment response deserves further investigations. Resistance can develop against all the available antiretrovirals. The high turnover rate of HIV along with the error-prone reverse transcriptase leads to the possibility of steady accumulation of resistance mutations if the viremic suppression is incomplete. While the interplay between viremia and resistance development is clear-cut for some antiretrovirals i.e. Lamivudine, the pattern is more complex for i.e. Zidovudine. With the availability of assays for resistances testing the knowledge on this issue has been ever evolving. How to use resistance testing in the clinical monitoring of patients remains to be clarified. Resistance testing can aid in the process of choosing salvage therapy for patients experiencing virological failure. Whether resistance testing will be of clinical benefit in other situations remains to be determined. Investigation of the viral sequences and evolution herein has not only been used for resistance analyses, but also for tracing the spread of the infection. HIV-1 exists in many subtypes, with various geographic distributions. Hence subtype analyses have been used to investigate the introduction and spread of the HIV infection into many countries. Phylogenetic analyses have also been used to investigate nosocomial transmission events. We used analyses of env and gag sequences to trace a case of nosocomial infection at the Department of Infectious Diseases, Rigshospitalet, Denmark. The study underlines the importance of steady awareness of the infection control precautions and possible breaks herein. The usefulness of this type of analyses was confirmed. In the early years of the AIDS epidemic various replicative patterns were described. Virus obtained from patients with late-stage infection often had virus that could induce syncytium formation (SI) when cultured, while virus obtained from patients in the early stages of infection did not have this ability. A correlation between the SI ability and the ability to yield high virus titres rapidly as well as the ability to establish infection in certain cell lines was found. Patients infected with SI virus experience more rapid clinical deterioration. We found that patients harbouring SI virus have HIV RNA loads no different from patients harbouring NSI virus. This is in line with the findings of other groups. Though patients harbouring SI virus had a more rapid development of resistance when treated with nucleoside reserve transcriptase inhibitor (NRTI's) monotherapy, this was not the case when treated with highly active antiretroviral therapy (HAART). HAART is today considered the treatment modality of choice; both for established HIV-infection and in cases where post exposure prophylaxis (PEP) is given in order to prevent establishment of infection after exposure. In a case of transfusion of HIV-contaminated though HIV antibody negative blood the recipient was treated with HAART. As the risk of infection is close to 100% under these circumstances the fact that the recipient remained uninfected is probably attributable to the prompt initiation and thorough maintenance of PEP. PEP is recommended to health care workers after percutaneous HIV exposure as well as after sexual exposure. Even with NRTI monotherapy PEP has been shown to be efficacious. While the explanation for the dichotomy (SI vs. NSI) was for many years unresolved, it is now known that this is due to the requirements of the virus for different co-receptors for cell entry. SI virus uses mainly CXCR4 while NSI virus uses CCR5. Being heterozygous for a 32 basepair deletion in the gene encoding CCR5 leads to slower disease progression. We have shown that heterozygotes have lower HIV RNA levels in the early years of the infection, possibly explaining the clinical advantage of having the deletion. HIV replicates in activated cells, and there is an intriguing interplay between HIV replication and immune activation. HIV-infected patients have elevated levels of immunoglobulins. HIV induces polygonal immunoglobulin production. We found that patients experiencing good virological suppression of HAART had lower IgA levels than patients with less complete viral suppression. Whether IgA can be used as a marker for imminent viral break-through remains to be determined. The full understanding of the interplay between immune activation and HIV replication awaits further studies. The finding of increased viremia in conjunction with acute bacterial or viral infection led to concerns about the safety of vaccinating HIV-infected patients against influenza and pneumococcal infection. We found no difference in HIV RNA levels measured before and median 42 days after anti-pneumococcal vaccination. This is in line with many other studies showing either no or only transient increases in viremia. In conclusion, the knowledge on HIV virology has expanded tremendously. This has led to significant improvements in treatments in the Western World leading to declines in HIV morbidity and mortality. The ability to quantify viral load and to perform sequence analyses represent valuable tools both for understanding the pathogenic actions of the virus and for the clinical monitoring of HIV-infected patients. The optimal usage of these tools in the clinical setting, however, still remains to be defined. The progresses obtained have unfortunately been restricted to the Western World and the calamities of HIV is spreading and worsening in the Developing World. The progress in the development of a vaccine has been disappointing and it is urgently necessary that the progresses obtained within the fields of prevention and treatment are translated into useful strategies in the parts of the world mostly affected by the HIV pandemic.