HIV genetic variation: Clinical importance

Journal of Infection (i997) 34, 195-199

SCIENCE AND CLINICAL PRACTICE

HIV Genetic Variation: Clinical Importance A. M. L. Lever University of Cambridge Department of Medicine, Level 5, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 200_, U.I(.

Introduction One of the most striking (and exhaustively documented) characteristics of HIV is its variability at nucleic acid and amino acid level. 1-3 Intuitively this would appear to be a vital component of the virus's ability to survive both the onslaught of the immune system and of anti-viral chemotherapy. Following intervention with antiviral drugs, there is now good evidence that resistant mutants become dominant in the quasispecies. 4 The effect of immunological pressure on the appearance of mutants is less clearly defined. The immune system certainly would seem to exert influence in that the decline in plasma viraemia after seroconversion is associated with evidence of a cytotoxic T-cell response 5 and as the disease progresses the viral RNA load correlates inversely with the immunocompetence of the individual. In addition, long-term survival correlates with a vigorous immune response. 6 Whether the variation in viral quasispecies can be said to be driven by immunological pressure is less certain. It is certainly not the only cause of viral variation and the evidence that ' i m m u n e selection' is taking place is still largely circumstantial. Immunological escape appears to be well documented in other micro-organisms such as the gonococcus 7-9 and trypanosomes 1° in which antigenic variation occurs due to differential splicing or recombination or acquisition of new DNA and subsequent alteration of surface proteins. The major difference between HIV infection and most others is that in HIV infection alone an antigen specific CD4 cell recruited to a site of viral replication, where antigen presenting cells are exhibiting peptides for which that T-cell is specific, has a very high chance of being infected and killed by the very virus it is there to eliminate. This powerful extra influence on the dynamics of infection makes it difficult to draw conclusions regarding cause and effect in the interplay between viral variation and the immune response. In the related animal model of SIV, and to some extent in HIV, there is circumstantial evidence of difference in selective pressure on viruses in vitro and in vivo. The nef 0163-4453/97/030195 +05 $12.00/0

gene is one of the best examples of an immunogenic protein ~1 essential for high level replication in vivo 12 and in which stop codons appear in the gene in vitro correlating with increased replication. 13 In the SIV model in vitro mutations in nef repair back to wild type when the virus is passaged in vivo, suggesting that Nef has an important function in vivo yet is inessential/inhibitory to replication in vitro. 14 The antibody mapping studies of the gp120 envelope protein by Moore and Sodroski 1~ and others also tell a somewhat similar story with the (admittedly somewhat indirect) evidence suggesting the gp120 adopts an open, highly fusogenic structure on in vitro passage compared to wild type isolates in which immunoreactive epitopes are concealed within a more closed structure leading to reduced fusogenicity. The data with regard to cell mediated immunity dependent epitopes is less complete and, therefore, slightly less compelling. Although in vitro display of aberrant peptides can reduce the immune response to the native peptide, 16 this has not been proven as being influential in vivo. Certainly the scenario requiring co-infection of a cell by two viruses, one containing an inhibitory peptide sequence, one with a stimulatory peptide sequence has not been established in vivo. So HIV variation exists. It is an inevitable consequence of the remarkable ability of the virus to mutate, although the majority of mutations are probably neutral or deleterious to the virus. The significance of different viral variants found in different organs 17 has yet to be fully clarified but may have important therapeutic implications. Specific tropism for brain capillary endothelial cells has been demonstrated in some variants and viral sequences responsible for this have been identified. 18 This is one example where variants might profoundly influence disease manifestations. One particularly important piece of the jigsaw which has recently fallen into place is the variation in the envelope glycoprotein and its correlation with the use of cell specific receptors used by the different variants. Fusin, which has homology to the ~ and [3 chemokine receptor families, was identified as a co-receptor for T-cell tropic H1V strains. ~9 Subsequently a study of individuals exposed to HIV but uninfected led to the discovery that another chemokine receptor, CCR-5, can © 1997 The British Society for the Study of Infection

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act as a co-receptor for macrophage tropic strains of HIV1.2°-22 If, as has been suggested, mucosal transmission of HIV is only achieved by macrophage tropic strains 23 then development of T lymphocyte tropism m a y be a response to chemokine mediated inhibition of macrophage infection in which the virus modifies its coreceptor usage by envelope mutation. Is this immunologically 'driven' or simply a successful variant allowing colonization of a new population of cells.)

What are the practical effects of viral variation and mutation rates for those seeking treatment for HIV infection and in the search for a vaccine? One observational piece of evidence from other RNA viruses concerns the 'fitness', i.e. the ability to survive and replicate, of a population of viruses and its relation to the size of the infectious inocula. Elegant studies by Holland and co-workers 24 have confirmed predictions that genetic bottlenecks, i.e. small infectious inocula, lead to a very significant reduction in fitness of the viral population whereas large inocula m a y lead to a fitness increase, the bottleneck effect being more pronounced. This m a y provide support for the commonsensical view that attacking the virus as soon as possible after infection, i.e. at the point when it is most vulnerable, and least 'fit', might have profound effects in minimizing the subsequent expansion of the virus population and maximizing the ability of the i m m u n e response to respond and control it. This has been most dramatically demonstrated by the use of AZT in HIV infected pregnant w o m e n reducing the rates of transmission to the child from 2 7% down to 7%. 2~It also provides theoretical support for antiretroviral treatment of patients at seroconversion/primary infection where, in the case of sexual transmission, viral selection has been shown to occur 26 and for which there is now clinical evidence of efficacy. 27

Variation and Humoral Immunity Most vaccine studies have concentrated on developing antibodies against the envelope glycoprotein as has been used successfully for a number of other virus infections. In hindsight a n u m b e r of these have perhaps used glycoproteins from inappropriate virus strains as immunogens. The IIIB strain being developed as a vaccine by some groups has since been shown to be relatively unrepresentative of wild type HIV envelope glycoproteins. The overwhelming variability of the envelope glycoprotein would at first suggest that this approach is doomed to failure in that no vaccine could ever be constructed in which immunological representation could be achieved

for all or even the majority of envelope protein variants currently existing in infected individuals. This m a y be unnecessarily bleak. Although alternative receptors for HIV entry have been described, 28-3° the major common ligand is CD4. CD4 is not variable. Thus, whatever molecular contortions the envelope goes through it must always be capable of binding to CD4 efficiently or the virus falls at the first hurdle. If in addition only a narrow range of macrophage tropic viruses is capable of mucosal transmission, the vaccine target is even smaller. 23 It is perhaps, thus, not impossible that display of a relatively limited range of peptide regions involved directly or indirectly in the gp120/CD4 interaction m a y protect individuals against all, or at least all of the important, viral variants. There is published evidence for naturally occurring CD4 imitating antibodies in infected individuals. 3I CD4 alone has been known to be insufficient for viral entry for m a n y years and in an analogous manner, a vaccine aimed at disrupting the interaction between gp120 and the co-receptors (fusin, CCR-5, etc) m a y be efficacious.

Variation and Cellular Immunity Again the situation is complex and not altogether encouraging, and it is worth remembering that in vivo in HIV 32 and S I V 33 infection a viral clone containing an immunogenic epitope can still be detected in the circulation long after the appearance of a specific T-cell response to it. How then can one go forward in looking for protective immunogenic epitopes? The study of those individuals who apparently remain symptom-free despite being infected with HIV, 34 or those individuals who are apparently repeatedly exposed to HIV 3s'36 yet remain uninfected, has provided intriguing evidence of relatively common mutations in the newly identified co-receptor molecules which can render cells partially or completely resistant to infection by certain HIV strains. However, the search for common HLA types associated with susceptibility or resistance to HIV has less chance of yielding definitive results t h a n with parasites which have had a longer period of interaction with the h u m a n race, such as malaria. HIV infection probably has not yet had time to 'select out' genetically resistant h u m a n s by reducing reproduction in more susceptible individuals.

Drug Therapy The one area in which there is unequivocal evidence of the dramatic effects on HIV genotype and phenotype of an external influence is the field of drug administration. Studies by Ho et al. 37 and Wei et al. 38 using protease

HIV Genetic Variation: Clinical Importance inhibitors have shown how rapidly these reduce the level of circulating virus and equally how rapidly variants resistant to the drugs (probably already present) multiply and become dominant in the population. Through the seductively powerful retrospectoscope this was all highly predictable. The mutation rate of RNA viruses is the subject of m a n y publications 3941 and the infidelity of reverse transcriptase is well known. 42 It now seems remarkably naive to expect drug monotherapy to have made any impact at all on the natural history of a disease in which virions are being produced at the rate of 109-101° per day. A virus population with an error rate of 10 4 per replication cycle at this rate of production will produce single and double mutants each day. It took only five mutations to produce a virus which is highly resistant to AZT43 and viruses resistant to _A_ZT can develop resistance to other anti-retrovirals. 44 Add to this the tiny therapeutic window involved--reverse transcription probably takes a brief fraction of the 24 h intracellular cycle of the v i r u s - - a n d the inadequacy of monotherapy becomes even more plain. A minor decrease in local level of drug during this brief period and the virus has escaped. Thus, although AZT monotherapy is associated with a rise in CD4 cells of 50 m m 3 and an approximate 0.5 log fall in HIV RNA in the circulation, the reduced progression of disease seen early after initiation of therapy in asymptomatic patients has disappeared by 3 years 4s and most importantly there is no associated survival benefit, and possibly even a survival penalty. Thus, it is not a surprise, but is nonetheless encouraging, that new studies of drug combinations are proving that the virus m a y be vulnerable to simultaneous attack by multiple anti-viral agents. In particular some dd[ resistant mutants display increased AZT sensitivity 46 and a single escape mutation at codon 184 in reverse transcriptase causing resistance to 3TC leads to increased susceptibility to AZT. 47 Data such as this has led to a concerted eflbrt to find drug combinations for which escape mutation only produces non-viable viruses. One early paper suggesting this phenomenon was achievable 48 unfortunately has not been validated, but the approach is still justified. Although resistance to more than one antiretroviral has been documented both in primary isolates of HIV49 and during therapy, 44 the clinical evidence is clear that combination therapy is better t h a n monotherapy, implying that dual resistance to two antivirals is not necessarily associated with loss of benefit.

Current Therapy in the Clinic We are limited by available pharmaceutical and economic resources. The Delta trial results showed a 3 7% relative

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reduction in mortality in previously untreated patients for two drug combination therapy (ddI/AZT and ddC/ AZT) versus AZT alone and 23% reduction for AZT/ddI versus AZT in previously treated patients. Together with evidence from other studies this means that there now is little doubt that combination therapy with at least two drugs should be the recommended initial treatment for those with HIV infection, e.g. ddI+AZT, ddC+AZT, 3TC+AZT. The stage at which the therapy should start is as yet not completely defined. Most evidence is available only for the use of combination reverse transcriptase inhibitors in patients with CD4 counts below 350. However, with the new protease inhibitors now being licensed there would seem to be strong circumstantial evidence for including these in an initial therapeutic cocktail of anti-retrovirals, s° The rapidity with which protease inhibitor resistance arises in patients sl should not be taken as indicating lack of utility, as they have been shown to produce prolonged inhibition of viral replication and appear to be remarkably free of side effects. 52'~3

The Future The adage that prevention is better than cure applies to HIV infection no less than to other diseases, and probably more. Not getting infected should still be the primary aim. However, in those who already have been infected, estimated at approaching 20 million worldwide, what can be offered? Treatment of opportunistic infections has undoubtedly made great strides in the last 10 years. For anti-retroviral therapy drug combinations must be the preferred option from the start. Treatment at seroconversion with combination antivirals m a y be logical. Development of therapy aimed at other parts of the viral replication process is urgently needed, bearing in mind that even with RT inhibitors and protease inhibitors only a tiny proportion of the virus lifecycle is as yet being targeted.

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