Most of our current knowledge of HIV-1 drug susceptibility and resistance and interpretations of genotypic changes in HIV-1 reverse transcriptase (RT) and protease are based on data obtained from HIV-1 subtype B viruses prevalent in North America, Western Europe and Australia. Worldwide, however, the majority of people with HIV are infected with non-B subtypes, which differ from subtype B in the envelope gene by as much as 30%. Antigenic and epitope variation in non-subtype B viruses remains a major focus in the design of HIV vaccines.

With a growing demand for treatment and access to antiretroviral therapy in resource-limited settings, the susceptibility and resistance patterns of non-subtype B viruses to antiretroviral drugs is an important question. Both the Seville and Barcelona meetings included new information on non-subtype B resistance among drug naïve patients and relatively small numbers of patients who had failed treatment with HAART.

Are the current drug regimens, widely used in subtype B infection equally effective in other HIV subtypes? Will increased access to antiretroviral therapies in developing countries result in new patterns of antiretroviral drug resistance? While the answers to these questions will require large-scale clinical trials, monitoring drug responses and resistance in non-subtype B infected patients has begun.

Methodologic results
A theme of the presentations in Seville, which bears on the interpretation of subtype B as well as non-subtype B resistance genotype, was summed up by several statistical presentations that estimated sample sizes for rigorous methods to link genotypic resistance to clinical response. In abstract 84, DiRienzo et al estimated that thousands of sequences (even within subtype B viruses) were needed for the accurate estimation of virtual phenotype for specific drugs [1]. This was reinforced by Wang et al in abstract 119, who used neural network models to predict virologic response. The researchers found that large numbers of well-characterized genotypes, phenotypes, treatment histories and responses are needed to accurately predict the effectiveness of multidrug regimens [2].

At the two conferences there were 6 presentations at the 11th International HIV Drug Resistance Workshop in Seville, and 9 at the 14^th International AIDS conference in Barcelona focusing on drug resistance in non-B subtypes after antiretroviral therapy. Most of these were small groups of patients from clinics in Europe, Asia and Africa where mutations in RT and protease in the response to drug treatment were examined from patients with non-subtype B viruses who failed antiretroviral therapy.

Two presentations, which included work by several of the HIVresistanceweb board members, merged sequences from treated and untreated patients gathered by an international collaborative group spanning four continents. These summaries identified the underlying polymorphisms (in untreated patients) in different subtypes as well as responses of non-subtype B viruses to antiretroviral drugs among patients from Zimbabwe, South Africa, Thailand, Israel, United Kingdom, Belgium and the growing number of sequences in the literature. Kantor et al from Stanford University, representing the International non-B Workgroup, presented in abstract 171 (Seville) and TuPeB4614 (Barcelona), data from 1,240 persons infected with non-B HIV-1 subtypes-298 after treatment failure and 942 untreated subjects. These were compared to subtype B sequences from more than 1,000 untreated and treated persons (from the Stanford database). This comparison defined 3 main mutation categories including non-subtype-specific treatment-related mutations, subtype-specific polymorphisms and possible subtype-specific treatment-related mutations. Overall, nearly all drug resistance mutations known in subtype B occur in non-B isolates, although polymorphisms unique to non-B isolates, some in drug resistance positions, are common in non-subtype B isolates. In addition, a few treatment-related mutations, rarely seen in subtype B, were identified [3,4].

These data emphasize several important points regarding drug resistance in isolates from patients infected with non-B HIV-1. First and foremost, large amounts of data are required to study drug resistance in non-B isolates and worldwide efforts are needed to achieve this goal. Second, almost all drug resistance mutations, which are known in subtype-B infected patients, appear also in non-B subtype sequences, suggesting that similar drug therapy and genotypic interpretation techniques may be utilized in non-B infected patient care. Third, there were also clear differences between subtypes, including characteristic polymorphisms in untreated individuals with non-B viruses, some of which occur in drug-resistance positions. The identification of a small number of subtype-specific treatment-related mutations suggests that genotypic responses (resistance pathways) to drugs may differ among subtypes.

Non-B resistance patterns in Asia
Considering the data from specific geographic regions, there were 2 abstracts from the South East Asia region, where the CRF01_AE predominates (CRF-circulating recombinant form). In abstract TuPeB4605 (Barcelona), S. Sirivichayakul et al from Thailand, where double NRTIs were the most frequently used regimens due to cost constraints, found high prevalence of multi-drug resistance mutations in 54 CRF01_AE-infected Thai patients treated for as long as eight years with zidovudine/didanosine [5]. Interestingly, these patients' sequences also included an NNRTI mutation (G190A) without evidence of exposure to this drug class.

In abstract 182 (Seville), K Ariyoshi et al from Japan compared the mutation patterns in 45 CRF01_AE infected patients with those of subtype-B infected patients, and found significant differences in protease sequences from patients who failed a nelfinavir-containing regimen. They did not observe the nelfinavir-related mutations D30N or N88D in the CRF01_AE cases. Instead, they observed a strong correlation between N88S and nelfinavir-treatment failure in CRF01_AE, and concluded that the nelfinavir-resistant mutation patterns in CRF01_AE are different from those of subtype B [6].

Among subtype-C infected patients in Israel, investigators reached a similar conclusion in contrasting sequences from subtype C and B infected patients. In abstract 34 (Seville), Z Grossman et al reported that in their nelfinavir treated subtype-C infected patient population, D30N is not the preferred resistance pathway and that the frequency of D30N was 10 times higher in B-infected vs. C-infected individuals. They concluded that although the same major protease mutations develop in subtype B and C patients failing protease inhibitor (PI) therapy, the rates at which mutational pathways develop differ for certain drugs [7].

Within the RT, in abstract TuPeB4602 (Barcelona), this group studied the use of NRTIs and the development of resistance in subtype B and subtype C infected patients. They collected 94 samples from 92 drug-experienced patients and noted that as a group, the RT mutations at positions 67, 70, 215 and 219 were more common in B vs. C viruses [8]. In addition, in abstract 180 (Seville), the researchers reported a 6-base pair insertion at position 69 in the RT gene of a subtype C virus [9]. This insertion is known to induce multinucleoside resistance in subtype B HIV-1.

In an additional Israeli report in abstract TuPeB4599 (Barcelona), D Auerbuck et al studied resistance mutations in children infected with Non-Subtype B HIV-1 following HAART. They compared resistance in children to their parents, and to clade-C and clade-B adult patients. They analyzed 92 samples of 45 children and 34 samples of 22 parents. Their results pointed out different rates of resistance protease mutations, some higher in subtype B sequences (positions 10, 30 and 90) and some higher in subtype C sequences (positions 36, 41, 89 and 93). In the RT gene, both clades developed the key nucleoside associated mutations (NAMS) at positions 41, 67, 70, 210, 215, and 219, but these were more frequent in B vs. C-infected patients despite higher zidovudine usage by C patients. Interestingly, in this cohort as well, the D30N appeared at very low rates in subtype C isolates [10].

Non-B resistance patterns in Europe
Several European studies addressed these issues as well. In abstract TuPeB4592, Navaratne et al from the United Kingdom asked whether L90M is selected more frequently in non-B subtype HIV1 infected patients failing on Nelfinavir-containing HAART. This group looked at 13 patients infected with non-B HIV-1 (subtypes A, C, D, CRF01_AE and CRF02_AG), treated with nelfinavir as their initial and only PI. The researchers found that in this small cohort, L90M appears to be selected at least as frequently as D30N in non-B subtypes, as opposed to subtype B isolates [11]. In contrast, among subtype B patients receiving NFV as their first PI in the U.S., D30N was the predominant resistance mutation, followed by N88S, with the L90M noted in only one of 32 patients failing NFV. In abstract 175 (Seville), C. Loveday examined sequences from 42 individuals infected with subtype C, and although there were the expected polymorphisms, similar resistance mutations were seen in subtype C and subtype B, despite a significantly inferior virologic response in C vs. B-infected patients at 24 and 48 weeks of follow-up [12].

In a report from Finland in abstract MoPeA3036, Ristola et al studied the genetic resistance pattern of non-B HIV-1 strains to NNRTIs among patients who have failed antiretroviral therapy. They looked at seven non-B infected, treated patients and found that mutations associated with positions other than 103 in the RT genome of HIV-1 (the most common NNRTI resistance mutation) seemed more common in patients infected with non-B subtypes than with subtype B, as either a result of natural genetic variability of non-B subtypes or from failed therapy [13].

Non-B resistance patterns in Africa and South America
C. Laurent et al from France described in abstract MoPeB3279 (Barcelona), the results of a 30-months follow-up of a pilot study of HAART in Senegal. Within 131 patients with limited drug availability, they found that 13 patients' viruses developed drug resistance during follow-up, and concluded that implementation of antiretroviral therapy is feasible in Africa, with clinical and biologic results comparable to those seen in western countries [14].

Two additional abstracts from Africa were presented. C. Pillay et al from South Africa, where HIV-1 subtype C predominates, studied in abstract 74 (Seville and Barcelona), the emergence of RT resistance mutations in 22 infants infected through mother-to-child-transmission (MTCT) and treated with didanosine plus stavudine. Samples from 12 children were available at nine-12 months. Six of 12 had drug resistance mutations in the RT. Five of these had the rare T69N RT mutation, and two had the multi-drug resistant Q151M mutation [15].

In abstract TuPeC4825 (Barcelona) from Botswana, where HIV-1 subtype C predominates as well, P. Bollyky et al described the genotypic variation in HIV-1 RT and protease in subtype C samples in treated and untreated patients. This group compared samples from 51 drug-naive patients and 34 patients failing antiretroviral therapy. They found that mutations (polymorphisms specific to subtype C) previously associated with treatment resistance at protease positions 20, 36, 63 and 77 were found in substantial numbers of drug-naive patients. In treated patients, there were significantly more mutations previously associated with drug resistance at RT positions 41, 67, 103, 184 and 215. Novel mutations at RT positions 20, 36, 48 and 214 were also associated with treatment. They concluded that mutations associated elsewhere with treatment resistance are found in substantial numbers of treatment-naive patients in Botswana, and that treatment was associated with certain new mutations [16].

In a report from Brazil, P. Brindeiro et al analyzed in abstract TuPeB4648 the genotypic variation of clade B and non-B HIV-1 in pediatric antiretroviral therapy failure. The researchers collected samples from 10 non-B vertically infected children failing different antiretroviral therapies. They reported significant differences in the prevalence of known drug-resistance mutations among B and non-B clades in the protease and RT and concluded that differences in resistance patterns exist between clades [17].

Implications for therapy
Collectively, all of these findings are important to the introduction of antiretroviral therapy in developing countries, and to the treatment of northern patients infected with non-subtype B viruses. While there are insufficient comparative data from comparable patients in clinical trials, observations across these studies provide evidence that polymorphisms in protease, associated with treatment failure in subtype B, are common in a number of non-B subtypes. There is little evidence that this impacts the effectiveness of the PI drugs in HAART therapies.

However, there are emerging differences in the resistance patterns of non-subtype B viruses which show a higher rate of primary protease mutations and potential PI cross resistance (specifically the L90M mutation), following nelfinavir treatment of patients with non-subtype B viruses.

With nucleoside drugs, multi-nucleoside resistance, via the Q151 pathway or the 68-69 insertion, may be more common in non-subtype B infections, particularly when treatment is limited to dual nucleosides. Resistance to NNRTIs (most often including the K103N mutation) is consistently seen in up to 20% of women who receive single-dose Nevirapine to prevent MTCT in subtypes A, B, C and D. More information is needed to determine whether these transient NNRTI resistance mutations arising after Nevirapine as well as polymorphic substitutions at positions 98 and 190 will impact the effectiveness of treatment with NNRTI drugs.

It is still too early to tell whether different subtypes will require unique drug treatment and salvage strategies. However, worldwide collaboration and careful analysis of the clinical trials getting underway in developing countries will be essential to collect non-B RT and protease sequences to understand the role of subtypes in drug resistance and the ultimate impact on clinical response to HAART therapies.

References