Management of Antiviral Resistance in Chronic Hepatitis B.

The primary goal of therapy for chronic hepatitis B (CHB) is to prevent liver disease progression. Hepatitis B surface antigen (HBsAg) seroclearance or seroconversion is regarded as an optimal endpoint to discontinue treatment. However, HBsAg seroclearance occurs very rarely with nucleos(t)ide analog (NUC) treatment, and long-term, almost indefinite, NUC treatment is required for the majority of patients. In patients with drug-resistant hepatitis B virus (HBV), a combination of tenofovir disoproxil fumarate (TDF) and entecavir (ETV), which is currently regarded as the strongest combination therapy against HBV, would be potentially safe to prevent the emergence of additional HBV resistance mutations. However, long-term tolerance data are lacking, and cost may be an issue for combination therapies. Several recent, well-designed, randomized controlled trials have shown that TDF monotherapy provides similar antiviral efficacy compared with the combination of TDF and ETV. Furthermore, no additional HBV resistance mutations emerged during TDF monotherapy for up to 96 weeks. Considering a comparable antiviral efficacy, extremely low risk of TDF-resistance, lower cost, and better safety potential, TDF monotherapy would be a reasonable choice for the treatment of drug-resistant patients with CHB.

Viral adaptation to an antiviral protein enhances the fitness level to above that of the uninhibited wild type.

Viruses often evolve resistance to antiviral agents. While resistant strains are able to replicate in the presence of the agent, they generally exhibit lower fitness than the wild-type strain in the absence of the inhibitor. In some cases, resistant strains become dependent on the antiviral agent. However, the agent rarely, if ever, elevates dependent strain fitness above the uninhibited wild-type level. This would require an adaptive mechanism to convert the antiviral agent into a beneficial growth factor. Using an inhibitory scaffolding protein that specifically blocks phiX174 capsid assembly, we demonstrate that such mechanisms are possible. To obtain the quintuple-mutant resistant strain, the wild-type virus was propagated for approximately 150 viral life cycles in the presence of increasing concentrations of the inhibitory protein. The expression of the inhibitory protein elevated the strain's fitness significantly above the uninhibited wild-type level. Thus, selecting for resistance coselected for dependency, which was characterized and found to operate on the level of capsid nucleation. To the best of our knowledge, this is the first report of a virus evolving a mechanism to productively utilize an antiviral agent to stimulate its fitness above the uninhibited wild-type level. The results of this study may be predictive of the types of resistant phenotypes that could be selected by antiviral agents that specifically target capsid assembly.

Management and prevention of drug resistance in chronic hepatitis B.

The management of hepatitis B virus resistance to antivirals has evolved rapidly in recent years. The definition of resistance is now well established, with the importance of partial response and the improvement of assays to detect genotypic resistance and virological breakthrough. Data on phenotypic resistance have allowed to define the cross-resistance profile for the main resistant mutants, providing a rationale for treatment adaptation. Clinical studies have shown that an early treatment intervention in case of a virological breakthrough or a partial response with the addition of a second drug having a complementary cross-resistance profile allows one to maintain the majority of patients in clinical remission. The prevention of resistance should rely on the use of the most potent antivirals with a high genetic barrier to resistance as a first-line therapy. The future perspectives are to design strategies to hasten the HBsAg clearance, which should become a new treatment endpoint, to prevent drug resistance and to decrease the incidence of complications of chronic hepatitis B.

Treatment strategies for highly treatment-experienced HIV-infected patients.

The management of highly treatment-experienced HIV-infected patients is often complicated by baseline antiretroviral drug resistance, patient intolerabilities, drug-drug interactions and quality-of-life issues; which are all factors that can limit the ability to construct a potent regimen. The mainstay of treatment has been to use new agents with activity against resistant virus. New agents, such as enfuvirtide and tipranavir/ritonavir, have shown promising results in highly active antiretroviral treatment regimens among patients with extensive treatment histories and resistance profiles, especially when used in combination with other active agents. Other strategies include mega-highly active antiretroviral treatment, double-boosted protease inhibitors, structured treatment interruptions and maintaining a replicative compromised virus. The future development of newer agents with activity against resistant virus is desperately needed, and many new compounds and classes of antiretrovirals are currently being investigated.

Suppression of multidrug-resistant HIV-1 reverse transcriptase primer unblocking activity by alpha-phosphate-modified thymidine analogues.

A dipeptide insertion between codons 69 and 70 together with the amino acid substitution T215Y in the reverse transcriptase (RT)-coding region of human immunodeficiency virus type 1 (HIV-1) strains are known to confer phenotypic resistance to zidovudine (AZT) and stavudine (d4T). Phenotypic resistance correlates with an increased ATP-dependent phosphorolytic activity. Nucleoside alpha-boranophosphate diastereoisomers derived from AZT and d4T were tested as substrates of a multidrug-resistant HIV-1 RT (designated as SS RT) bearing a Ser-Ser insertion at codons 69-70 and other drug resistance-related mutations, in DNA polymerization assays and ATP-mediated excision reactions. Using pre-steady-state kinetics, we show that SS RT can incorporate both R(p) and S(p) diastereoisomers, although R(p) is the preferred isomer. Chirality at the internucleotidic linkage formed upon incorporation of nucleoside alpha-boranophosphate did not affect ATP-mediated excision. As reported for AZT and d4T-terminated primers, substituting Thr, Asn or Ser for Tyr215 abrogates the ATP-dependent phosphorolytic activity on primers terminated with alpha-boranophosphate derivatives of thymidine analogues. However, unlike in the case of AZT, eliminating the dipeptide insertion in SS RT had no effect on the ATP-mediated excision of primers terminated with alpha-boranophosphate derivatives of d4T. Studies with ATP analogues showed that exchanging a non-bridging oxygen atom at the gamma-phosphate group for sulfur causes a significant reduction of the ATP-dependent phosphorolytic activity of SS RT. Interestingly, SS RT's excision activity is completely eliminated upon phosphorothioate substitution at the 3' end of primers terminated with AZT. These results suggest that phosphorothioate derivatives of currently approved drugs could be useful against excision-proficient HIV-1 strains.

Designing anti-AIDS drugs targeting the major mechanism of HIV-1 RT resistance to nucleoside analog drugs.

HIV reverse transcriptase (RT) is the target of a number of important anti-AIDS drugs. Drugs that inhibit RT are either nucleoside reverse transcriptase inhibitors (NRTIs) or non-nucleoside reverse transcriptase inhibitors (NNRTIs). Combinations of various anti-AIDS drugs (highly active anti-retroviral therapies or HAART) can reduce the viral load to non-detectable levels. However, the development of drug resistance leads to the emergence of HIV strains that are resistant to multiple anti-AIDS drugs. The nucleotide analogs that are used as anti-HIV-1 drugs lack the normal 3'-OH and as a consequence act as chain-terminators when incorporated into DNA. One mechanism of nucleoside analog resistance involves ATP-based excision to unblock chain-terminated primers and allow HIV replication to continue. There is an urgent need for new drugs and for new therapies that can overcome the excision mechanism of resistance. Compounds that disrupt the binding of the excision reaction substrate(s) (the blocked primer and/or ATP and/or pyrophosphate), or mimic the dinucleoside tetraphosphate product of the ATP-based excision reaction are potential inhibitors of excision. Detailed understanding of drug resistance mechanisms can reveal novel targets for anti-viral agents.

Multidrug-resistant HIV-1 reverse transcriptase: involvement of ribonucleotide-dependent phosphorolysis in cross-resistance to nucleoside analogue inhibitors.

Human immunodeficiency virus type 1 (HIV-1) strains having a dipeptide insertion between codons 69 and 70 of the viral reverse transcriptase (RT) have been observed in isolates from patients treated with 3'-azido-3'-deoxythymidine (AZT) and other nucleoside analogues. These viruses contain additional mutations related to drug resistance and display reduced susceptibility to most nucleoside analogue inhibitors, including AZT. The mechanism of AZT resistance implies an increased ability of the multidrug-resistant (SS) RT to remove AZT-monophosphate (AZTMP) from blocked primers through a nucleotide-dependent reaction. We show that its higher ATP-dependent phosphorolytic activity is also detectable with primers terminated with 2',3'-didehydro-3'-deoxythymidine-5'-monophosphate (d4TMP) or 2',3'-dideoxythymidine-5'-monophosphate (ddTMP), but is significantly reduced when the dipeptide insertion is deleted. Removal of AZTMP, d4TMP and ddTMP can be inhibited by the next complementary deoxynucleoside triphosphate (dNTP). AZTMP removal reactions catalysed by SS RT were highly resistant to dNTP inhibition (IC(50)>0.25mM), while unblocking of d4TMP- and ddTMP-terminated primers was around tenfold more sensitive to inhibition by the next complementary dNTP. Both SS and mutant 2S0S RTs were able to unblock and extend primers terminated with 2',3'-dideoxycytidine-5'-monophosphate (ddCMP) in the presence of ATP, albeit very poorly. Under these conditions, none of the RTs was able to remove 2',3'-dideoxy-3'-thiacytidine-5'-monophosphate (3TCMP) from a terminated DNA primer. Resistance mediated by ATP-dependent phosphorolysis depends on the intracellular levels of dNTP. High levels as found in transformed cell lines (i.e. H-9, CEM lymphoblasts, SupT1 cells, etc.) may prevent repair of primers terminated with d4TMP. However, ATP-dependent phosphorolysis could be relevant for d4T resistance in cells having low levels of dNTPs. This proposal could explain why insertion-containing HIV-1 variants have been detected in the absence of AZT, during d4T treatment.

Condyloma acuminatum presenting as a dorsal tongue lesion in a patient with AIDS.

Oral lesions have been recognized as a prominent feature of HIV infection and AIDS since the beginning of the epidemic. This report describes the case of a man with advanced AIDS and a nonpainful but enlarging dorsal tongue soft tissue growth of 6 months' duration. Incisional biopsy showed a red, papillary lesion with koilocytosis consistent with condyloma acuminatum. In situ hybridization and molecular techniques were used to identify human papillomavirus (HPV)-31 sequences in warty tissue. Eighteen months later, the lesion recurred and was reexcised without complication. This case is reported to illustrate that venereal transmission may not be as important in warts of the oral cavity as in HIV-associated anogenital warts, because warts of the oral cavity are rarely associated with HPV types 6, 11, 16, and 18. Instead, they may be present as a result of activation of latent HPV infection or perhaps autoinfection from skin and facial lesions. The carcinogenic potential of oral warts in HIV disease is undefined, as is the role of antiretroviral therapy in controlling HPV-associated oral lesions.

Structural pharmacogenomics, drug resistance and the design of anti-infective super-drugs.

Large-scale comparative analysis of drug-target polymorphism structures enables the rational design of next generation 'super drugs'--drugs that are less prone to development of drug resistance or that work for the largest possible fraction of the patient population. Furthermore, knowledge of the drug-target-shape repertoire that exists within the patient population enables predictions of likely clinical trial outcomes and response rates for drug efficacy. This gives information on the optimal drug candidates before the initiation of clinical trials. The economic impact of incorporating pharmacogenomics insights early on in the drug discovery process will be substantial and will afford significant competitive advantages to companies that successfully incorporate this technology.