Italy is a case in point. Sequencing the reverse transcriptase and protease regions of HIV’s pol gene, Chiara Riva (Institute of Infectious and Tropical Diseases, Milan) found that the non-B rate in a central Italian cohort climbed from 4% (67 of 1674 isolates) before 1997 to 26.6% (109 of 410 isolates) after 1997 [1]. In 2003, 13 of 24 newly diagnosed infections (54%) involved subtypes besides B.

Analyzing 2388 isolates from three Italian cohorts, Riva counted 219 non-Bs (9.1%). Of the 126 pure subtypes, 50.8% were subtype F1, 19.8% subtype A, 14.3% subtype C, and 11.1% subtype G. Among 93 isolates with recombinant patterns, she tallied 77 circulating recombinant forms (CRFs), of which 75.3% were CRF02_AG, 15.6% CRF01_AE, 3.9% CRF06_cpx, 1.3% CRF11_cpx, and 1.3% CRF13_cpx.

Do non-B subtypes, CRFs, and unique recombinant forms (URFs) differ from subtype B in ways other than name? Yes, said Ricardo Camacho (Hospital de Egas Moniz, Lisbon), in a talk reviewing the growing field of subtype research [2]. But whether those differences will have a big clinical impact remains uncertain:

• Non-B subtypes follow different pathways to resistance.
• Non-Bs differ in codon use at sites critical to resistance, for example, positions 106 and 210 in reverse transcriptase and 82 in protease.
• Mutations appear at sites not linked to resistance in subtype B, for example, M89I/V and D35N/G.
• Non-Bs differ from Bs in fitness (replicative capacity) assessed in vitro.
• Non-Bs differ from Bs in binding affinities for protease inhibitors (PIs) in vitro.

A single-center Belgian study of 56 subtype B-infected people and 119 with non-B viruses found no difference in time to undetectable viral load after they started their first regimen [5]. But in 24 months the mean CD4 gain in the non-B group (161 cells/µL) significantly lagged that in the B group (236 cells/µL).

Camacho surmised that responses to entry inhibitors may differ greatly from one HIV-1 subtype to the next because the gene encoding HIV-1’s envelope varies 25% from subtype to subtype. And some non-B isolates are naturally resistant to the fusion inhibitor enfuvirtide. But these differences may have little practical meaning because the high cost of enfuvirtide–and the likely high cost of entry inhibitors to come–puts them beyond the reach of most people with non-B virus.

One of the best-known subtype-dependent mutation quirks involves frequent selection of the D30N mutation by nelfinavir-treated subtype B virus, but a preference for L90M by nonBs [6]. One reason for this anomaly, proposed Zehava Grossman (National HIV Reference Lab, Tel Aviv) may be polymorphic meanderings at position 89, with 89L dominant in subtype B and 89M in other viral strains [7].

Pursuing this hypothesis, Grossman looked at virus from 502 subtype C-infected Israelis, 150 of them naive to antiretrovirals, 70 with antiretroviral experience but naive to PIs, and 282 with PI experience. L89M dominated in all three groups, outnumbering L89L 7 to 1 regardless of whether treatment provoked D30N or L90M. Grossman charted other perigrinations at protease position 89 during continued treatment, with L89I the most frequent, followed by L89V and a mix of L89H and L89T.

Progressive replacement of L89M by other substitutions may mean L89M renders virus less fit, Grossman suggested, so HIV keeps evolving. But replacement of L89M, usually by L89I, proved equally common in PI-treated people with D30N (91% to 68%) and L90M (91% to 65%) (P < 0.001 for both). Despite this similar drop in L89M frequency, she proposed, "preferential selection of L90M in nelfinavir-treated [subtype] C patients suggests that L89M generates a higher barrier for selection of D30N."

Certain substitutions at protease positions may mean little in subtype B virus while conferring greater susceptibility to PIs among other viral strains. That conclusion arose from a study of 43 treatment-naive people, 19 infected with subtype G, 11 with CRF02_AG, 6 with subtype F, and 7 with subtype C by Ana Abecasis (Hospital de Egas Moniz, Lisbon) [8].

Using Virco’s phenotyping test, Abecasis found that the CRF02_AG viruses were hypersusceptible to nelfinavir (P = 0.0004). This enhanced susceptibility correlated with a K70R genotype (P < 0.01). While K is wild-type (nonmutant) at position 70 in subtype B virus, she observed, 70R is wild-type in CRF02_AG. On the other hand, the E35D substitution correlated with decreased susceptibility of CRF02_AG to nelfinavir (P = 0.011).

Subtype B protease is polymorphic at position 37, sporting either D, N, or S substitutions. But 37N was the wild-type variant in the non-B subtypes Abecasis studied, and 37N correlated with heightened susceptibility to indinavir (P = 0.0035) in these non-Bs strains.

Does higher susceptibility of these HIV-1 strains compared with subtype B mean people infected with non-Bs will respond better to nelfinavir, indinavir, or other PIs? So far no clinical research supports that hypothesis, and studies like this must be interpreted with great caution. As session chair Jonathan Schapiro observed, the viral susceptibility test used in this analysis depends on a subtype B vector.

The workshop’s biggest subtype study involved almost 2000 isolates from the trans-European CATCH study of treatment-naive people, presented by David van de Vijver (University Medical Center Utrecht) [9]. Comparing B and non-B viruses for minor substitutions in protease, he found that the non-Bs usually (but not always) harbored more minor substitutions. The impact of these minor shifts remains uncertain, he noted. Although minor changes do not directly impair viral susceptibility to PIs, they may promote resistance by enhancing viral replication capacity.

The study involved 1299 subtype B viruses, 209 subtype Cs, 86 subtype Gs, and more than 50 subtype As, CRF01_AEs, and CRF02_AGs, plus sprinklings of subtypes D, F, and J. The G, J, and CRF02_AG viruses had 70% more minor protease substitutions overall than subtype Bs, while C, D, and CRF01_AE strains had 20% more (P < 0.001 for both).

All non-B viruses analyzed had more minor substitutions involved in resistance to atazanavir, indinavir, nelfinavir, and ritonavir (P < 0.001). On the other hand subtype B virus had more minor shifts potentially relevant to amprenavir, lopinavir/ritonavir, saquinavir, and tipranavir (P < 0.001). K20R and M36I proved significantly more common among non-Bs, and L63P and V77I among Bs (P < 0.001 for both).

These little wobbles in protease positions may lower the resistance barrier to certain PIs, van de Vijver suggested, by edging the viral genotype closer to a fully resistant mutation lineup. If clinical evidence confirms that hypothesis, subtype differences in minor substitutions may be another factor to consider when picking a first-line regimen.

Scrutinizing substitutions in protease and reverse transcriptase among treated and untreated people around Paris, Nasser Al Hawajri (Avicenne University Hospital, Paris) also found distinct mutation profiles in people infected with subtype B and those infected with other HIV-1s [10].

The study involved 49 treatment-naive people, 27 (55%) infected with non-Bs and 22 (45%) with B, and 530 people with multiple antiretroviral experience, 405 (76%) infected with subtype B and 125 (24%) with non-B virus.

In the treatment-naive group, three people with subtype B and two with other strains had mutations tied to PI resistance. But substitutions at eight protease positions–including two tied to subtype B resistance–differed significantly between the groups. Eight naive people with subtype B and one with a non-B virus had reverse transcriptase mutations conferring resistance to AZT, d4T, 3TC, efavirenz, or nevirapine. The between-group substitution rate was significantly different at four positions, including 184, 210, and 215.

Among treatment-experienced people, protease mutations proved significantly more frequent with subtype B (48%) than with non-B virus (36%) (P = 0.016). Specific substitutions identified differed significantly at 47 positions, including 13 involved in resistance of subtype B virus to PIs. Mutations conferring resistance to reverse transcriptase inhibitors also proved more common in people with subtype B (59.6% versus 50.4%), but that difference lacked statistical significance. Substitutions differed significantly between groups at 30 reverse transcriptase positions, including six that mediate resistance of subtype B virus to reverse transcriptase inhibitors.

But constructing such algorithms will be impossible, Ricardo Comacho said, without big multinational clinical trials. Although the first batch of studies in this field yields strong suggestions of potential subtype-specific response differences, he noted, most comparisons involve subtype B versus a non-B grab-bag. When individual subtypes, CRFs, and URFs are compared separately with subtype B, the samples are usually tiny. So at this juncture genotyping a non-B virus before treatment would be pointless.

Mark Mascolini writes about HIV infection (markmascolini@earthlink.net).

References
(To view slides and posters from the Third European HIV Drug Resistance Workshop, go to http://www.hivpresentation.com.)

3. Frater AJ, Dunn DT, Beardall AJ, et al. Comparative response of African HIV-1-infected individuals to highly active antiretroviral therapy. AIDS 2002;16:1139-1146.

6. Grossman Z, Paxinos EE, Averbuch D, et al. Mutation D30N is not preferentially selected by human immunodeficiency virus type 1 subtype C in the development of resistance to nelfinavir. Antimicrob Agents Chemother 2004;48:2159-2165.