br Materials and Methods br Results br Discussion

Materials and Methods
Results
Discussion Studies have suggested that dual-tropic strains evolve from R5 variants, and X4 viruses arise from the R5X4 strains (Pastore et al., 2004). It is unlikely that the emergence of a dual-tropic virus during the passage experiments occurred by chance because CXCR4-using variants were not detected in the absence of plasma and changes required for co-receptor switching are generally associated with diminished replication fitness (Pastore et al., 2004). The DM268 passage results were surprising because we observed no significant neutralization susceptibility difference to contemporaneous autologous plasma among co-existing R5 and dual-tropic strains (Fig. 1). Although previous studies have demonstrated that diverse glycosylation changes can render viruses neutralization resistant (Sagar et al., 2006, Wei et al., 2003), it remains unclear how the addition of PNGS at position 88 leads to low level CXCR4 usage. It has been shown that co-circulating R5 and R5X4 variants often have the same predicted V3 loop sequence (Huang et al., 2007; Lin et al., 2012), and thus it possible that modifications in other env segments lead to a structural change that can influence co-receptor usage. Interestingly, DM viruses did not emerge in the other 2 cases (DM8 and DM178) even though their X4 as compared to R5 envs were more neutralization resistant to autologous contemporaneous plasma. Our observations suggest that the passaged DM8 and DM178 env variants accommodated changes required for neutralization resistance to autologous plasma without co-receptor switching. Other R5 variants from these individuals, not examined in the passaging experiments, may acquire the ability to use the CXCR4 receptor as they become resistant to autologous antibodies. The earliest studies implied that lab-adapted SI, presumably CXCR4-using, strains were more neutralization sensitive compared to NSI–CCR5-utilizing variants (Mascola et al., 1996; Montefiori et al., 1998; D\'Souza et al., 1997; Sawyer et al., 1994). Subsequent studies showed that compared to CCR5-utilizing strains primary as opposed to lab-adapted CXCR4-using envs displayed a relatively similar range of neutralization susceptibilities to some mAbs and unrelated heterologous plasma (Trkola et al., 1998; Montefiori et al., 1998; Cecilia et al., 1998; Lacasse et al., 1998). This led to the notion that nAbs are unlikely to be the major selection pressure that drives co-receptor switching. Our results also show that X4 and co-existing R5 variants have equivalent sensitivity to SYN-117 targeting env domains not involved in co-receptor binding. More recent investigations with SHIV-1B and HIV-1B infected subjects argue that relatively neutralization sensitive CXCR4-utilizing variants emerge later in disease after HIV-1 induced immunological damage has led to a compromised humoral immune response (Bunnik et al., 2007; Ho et al., 2007). We, however, observed no case in which X4 or R5X4 as compared to co-circulating R5 envs were significantly more neutralization susceptible to autologous contemporaneous plasma, even though we sampled more envs from each subject and examined a greater number of individuals. Furthermore, the previous human study examined PBMC passaged variants (Bunnik et al., 2007), which may have led to the different results because passaging virus in PBMCs significantly impacts its neutralization properties in a unique manner for each virus antibody combination (Etemad et al., 2015; Provine et al., 2012). Our results also differ from the previous human study (Bunnik et al., 2007) potentially because we examined HIV-1C versus HIV-1B infected individuals. Our preliminary results, however, suggest this is not the case (Fig. S2). While HIV-1C constitutes the majority of worldwide infections, emergence of CXCR4 usage occurs at a lower frequency compared to HIV-1B (Lin et al., 2011; Ataher et al., 2012). Reasons for the difference in incidence remain uncertain. Although it has been reported that CCR5 and CXCR4-using viruses preferentially infect memory and naïve T cells respectively (Davenport et al., 2002; Blaak et al., 2000), there is no evidence that this preference differs among HIV-1B versus HIV-1C variants (Cashin et al., 2014; Ribeiro et al., 2006). While, it is known that the CCR5Δ32 allele frequency is higher among Europeans than Africans (Galvani and Novembre, 2005), the density of the CCR5 receptor on CD4+ T cells is not significantly different among people with European ancestry who constitute the majority of HIV-1B infections versus those with African heritage who are predominantly infected with HIV-1C (Picton et al., 2012). Thus, the higher incidence of DM virus population in HIV-1B as compared to HIV-1C infected individuals is unlikely because of lower availability of appropriate CCR5 expressing target cells. It has been suggested that HIV-1B and HIV-1C envs have structural differences, especially in the V3 loop (Patel et al., 2008). Potentially in response to these env structural differences, HIV-1C infected individuals develop antibodies targeting the C3-V4 region while those with HIV-1B generate responses against the V1-V2 domain and the base of the V3 loop (Rong et al., 2009, Moore et al., 2008; Tang et al., 2011). Interestingly, the main determinants for co-receptor switching are located in the V1 to V3 domains (O\'Brien et al., 1990; Chesebro et al., 1991; Shioda et al., 1992; Groenink et al., 1993; Fouchier et al., 1995). Greater prevalence of anti-V1-V2 or V3 loop antibodies may correlate with the greater frequency of DM virus populations in HIV-1B as compared to HIV-1C infected individuals.