Impaired human immunodeficiency virus type 1 replicative fitness in atypical viremic non-progressor individuals

Infection with human immunodeficiency virus type 1 (HIV-1)—in the absence of antiretroviral therapy—usually follows a well-defined path of virological and immunological changes; however, progression rates to advanced AIDS vary significantly among infected individuals [1, 2]. Based on plasma HIV-1 RNA (viral) load, CD4⁺ T-cell counts, and symptomatic or asymptomatic HIV-1 infection adult patients have been classified into four groups: (i) rapid progressors (RP), (ii) typical progressors (TP), and two groups of long-term non-progressors (LTNP) namely (iii) elite controllers and (iv) viremic controllers [3, 4]. Among the latter, a rare subgroup of HIV-1-infected individuals has been described who do not seem to progress to AIDS, maintaining high CD4⁺ T-cell counts despite high levels of viremia (i.e., over 2000 HIV-1 copies RNA/ml of plasma) for many years, also called viremic non-progressors (VNPs) [5‐8].

Multiple studies have tried to associate these differences in HIV-1 disease progression with a multitude of host (HIV-1-specific immune response and genetic markers) and viral factors [9‐13]. Among the immunological factors, strong HIV-1-specific cytotoxic T cell (CTL) responses have been shown to control HIV-1 infection right after transmission, perhaps determining viral set point in chronic stages of infection, which may help reduce the rate of HIV-1 disease progression [14, 15]. Similarly, a series of host genetic factors that seem to influence HIV-1 progression rates have been well described, including human leukocyte antigen (HLA) class I alleles such as HLA-B*27 and HLA-B*57 [16, 17] and a 32 bp deletion in the CCR5 chemokine receptor gene [18‐22]. On the other hand, the impact of restriction cellular factors like APOBEC3, TRIM5α, tetherin, and SAMHD1 on HIV-1 progression is still debated [10, 23]. Altogether, it has been long recognized that immune activation is one of the major contributors to HIV-1 disease and pathogenesis [6, 24‐27].

Viral factors—such as impaired HIV-1 replicative fitness—have been associated with slow or limited HIV-1 disease progression [11, 28‐31], particularly in patients infected with heavily mutated drug resistant viruses [28, 30, 32‐35]. High fitness of transmitted HIV-1 has been related to rapid disease progression [36], while some individuals carrying viruses with decreased replication capacity during acute/early infection have been classified as HIV controllers [37]. Although isolating replication competent HIV-1 strains from elite controllers has been difficult, two studies failed to identify any major defect on the replication ability of these viruses [38, 39]. In contrast, recombinant viruses constructed with HIV-1 gag, pol, and/or env genes from elite controllers exhibited reduced replication capacity compared to recombinant viruses constructed from typical progressors [40‐42]. On the other hand, very limited information about the replicative fitness of viruses infecting viremic non-progressor individuals is currently available [6, 43]. If a decrease in HIV-1 replicative fitness is associated with slower/no disease progression, what is causing the virus to have this reduced replication capacity—in the absence of drug resistance mutations—in a certain number of non-progressor patients?

To date, no common pattern or polymorphism(s) shared among HIV-1 variants infecting non-progressor individuals have been clearly defined. Several studies have described changes in the LTR, e.g., in the Sp1 binding site [44] or large deletions [11, 45] that could be associated with slow disease progression. Detrimental mutations in Gag have been associated with viral attenuation [46, 47], which seem to play an important role in HIV-1 disease [48, 49]. Significant replicative fitness loss was observed in recombinant viruses encoding pol genes with mutations affecting HLA-A, but surprisingly not HLA-B, binding [50], although the most attenuated viruses were those constructed from HIV-1 elite controllers expressing HLA-B*57 and HLA-B*51 [40]. Changes in the V2 region of gp120 [51, 52] and a single amino acid deletion in gp41 [53] in the env gene have also been associated with slow disease progression. However, most of the changes in the HIV-1 genome linked to altered pathogenesis have been identified in accessory genes. Single amino acid substitutions [54, 55] and large deletions [56, 57] in Nef have been associated with a decrease in replicative fitness and slow disease progression. Mutations at positions 72 and 77 in Vpr, disrupting the viral protein function, were frequently detected in non-progressors [58, 59]. Finally, single or large insertions in Vpu [60] and amino acid substitutions in Vif [61, 62] have also been associated with non-progression in HIV-1 disease.

In this pilot study we quantified the replicative fitness of HIV-1 isolates obtained from extremely rare viremic non-progressor HIV-infected individuals and compared them to the fitness of those from patients with typical or rapid disease progression. Using deep sequencing we analyzed the full-length HIV-1 genomes and determined intrapatient HIV-1 diversity to investigate if these viral factors could be contributing to the maintenance of stable CD4⁺ T-cell counts despite persistence viremia for more than 10 years, in the absence of antiretroviral treatment.

Since the discovery of HIV-1 as the etiological agent of AIDS [79, 80], multitude of studies have attempted to understand the differences on HIV-1 disease progression, particularly host and/or viral factors that could be associated with HIV-1 pathogenesis [11, 12, 81]. The common (“normal”) inverse correlation between CD4⁺ T-cell count and plasma HIV RNA load usually governs HIV-1 pathogenesis, i.e., elevated plasma viremia is typically accompanied by a decrease in CD4⁺ T-cells, leading to disease progression [2]. Some HIV-infected individuals have shown uncommon rates of disease progression (slow, non-progressor or elite controllers) but all of them have reduced or undetected plasma HIV RNA loads [3, 4, 42, 82]. On the other hand, a rare group of patients termed viremic non-progressors (VNP) seem to resemble Simian Immunodeficiency Virus (SIV) infection in sooty mangabeys, i.e., active viral replication without (or very limited) disease progression [26, 82‐84]. Here we identified three atypically VNP HIV-infected individuals and proceeded to study their HIV-1 isolates, focusing on replicative fitness and viral genomic analysis to try to understand the maintenance of stable CD4⁺ T-cell counts despite persistence viremia for more than 10 years, in the absence of antiretroviral treatment.

As described above, viremic non-progressor (controller) patients do not progress to AIDS, maintaining high CD4⁺ T-cell counts with HIV-1 replication for many years [5‐7, 85]. The three VNP individuals described in this study had been infected with HIV-1 for at least 10 years at the time the blood sample was taken, with stable CD4⁺ T-cell counts and high plasma HIV RNA loads. When compared with three typical (TP) and one rapid progressor (RP) patients, the number of CD4⁺ T-cells barely declined in the VNPs over this period of time, similar to what have been described for other adult and children VNPs [43, 82‐84, 86]. No other significant differences were observed among the VNP and TP patients, including plasma viral load levels and basic virus characteristics such as HIV-1 subtype (all clade B) and coreceptor tropism (all CCR5-tropic viruses).

Most studies involving the characterization of viremic non-progressor (controller) patients have focused in host factors to try to understand their odd HIV-1 disease progression profile. For example, the presence of the HLA-B*27 allele was associated with the VNP phenotype [43], while a higher frequency of the protective single nucleotide polymorphism −35 CC in the HLA-C gene was found in VNP patients [5]. Preferential Gag-specific CD8⁺ T cell responses have also been observed in viremic controllers [84] and limited infection of CD4⁺ central memory cells [8], perhaps related to low CCR5 expression on these cells [86], seem to be associated with lack of disease progression in VNPs. Limited deregulation of a series of interferon-stimulated genes correlating with time to disease progression was also identified in VNPs [85]. However, and similar to the absence of immune activation during natural SIV infection in sooty mangabeys [27, 87, 88], healthy CD4⁺ T cell counts despite detectable viremia seem to be common in most viremic non-progressor patients [86]. Yet, just a limited number of studies have analyzed HIV-1 strains infecting VNPs [6, 43].

Here we showed that the replicative fitness of all three VNP-derived HIV-1 isolates was impaired compared to the fitness of the viruses obtained from the TP and RP patients. We and others have reported that HIV-1 replicative fitness is associated with disease progression [28‐30, 42], that is, viruses with decreased replication capacity influence the natural history of HIV-1 infection, correlating with diminished viral burden and HIV-1 pathogenesis. Viruses from long-term non-progressor [28], slow progressor [41, 42] and elite controller [37, 40, 42] patients have been shown to have impaired replication capacity; however, the fitness of viruses from VNP individuals have not been extensively described. Choudhary et al. [6] showed that HIV-1 isolates derived from VNP were as cytopathic as viruses isolated from typical progressor patients. On the other hand, O’Connell et al. [43] observed a slight decrease in the fitness of a virus from a VNP patient, transmitted from a chronic progressor partner, but failed to identify any amino acid substitution(s) potentially responsible for this phenotype.

Reduced HIV-1 replicative fitness is usually the consequence of changes in the viral quasispecies following the evasion of key selective pressures, e.g., host immune response and/or antiretroviral treatment [30, 31]. These changes are accompanied by single or multiple mutations in the targeted HIV-1 genomic region, deviating the virus population from the wild-type quasispecies distribution and impairing their ability to replicate efficiently compared to the original virus [89, 90]. The effect of a multitude of drug resistance mutations (in the pol gene) in viral replicative fitness have been amply described [30, 74]. A number of CTL escape mutations have been identified in HIV and SIV that carry a concomitant replicative fitness cost [46, 91, 92], including HIV-1 mutant variants escaping Gag-specific CD8⁺ T cell responses—like those identified in viremic controllers [84]—that had reduced replicative fitness [40]. Other studies have shown how multiple mutations and/or deletions in the LTR and all HIV-1 coding regions have a range of effects on the ability of the virus to replicate (virus attenuation) [11, 31, 32, 40, 42, 44‐62, 78]. Here we used deep sequencing to analyze near full-length HIV-1 genomes from all VNP-derived viruses, and compared them with the sequences from the TP- and RP-derived viral genomes. Despite identifying a series of SNPs in different genomic regions, some of them previously associated with a reduction in replicative fitness and/or disease progression [11, 31, 32, 40, 42, 44‐62, 77, 78], no clear pattern of signature mutations was detected in the viruses from the VNPs that could explain the impairment in replication capacity. Interestingly, it was the number of these HIV-1 genetic polymorphisms that correlated significantly with the replicative fitness of each HIV-1 isolate. In fact, it is reasonable to think that viruses with a higher number of critical SNPs have a more marked decrease in replicative fitness, similar to what has been described for multidrug-resistant HIV-1 variants carrying multiple mutations in the protease, reverse transcriptase and/or integrase coding regions [30, 74]. These results were corroborated by calculating intra-patient HIV-1 population diversity, i.e., the highly replication impaired VNP viruses had a more complex virus quasispecies population, most likely as a consequence of trying to evade the host immune response [93], which was highlighted by the significant correlation between replicative fitness and HIV-1 genetic diversity in highly immunogenic regions such as Gag and Gp120 [31, 42, 92]. We and others have described similar results where drug resistant HIV-1 variants with more heterogeneous virus population had lower viral replicative fitness [94, 95]. It is possible that impaired (less replication competent) HIV-1 strains are capable to ascertain a limited host immune response in VNPs, enough to maintain high levels of viremia, resulting in viral evolution but limiting host immune activation that could exacerbate HIV disease, i.e., decrease CD4⁺ T-cell counts. It is also possible that the preferential replication of HIV-1 in effector memory T cells, and preservation of central memory T cells, in VNPs [8] could result in greater virus production per HIV-infected cell. All interesting possibilities to be addressed in further studies.

Although prevalence of elite controllers seems to be less than 1% of the HIV-infected population [96, 97], a recent study involving over 13,000 HIV-infected individuals identified 271 viremic controllers for a 2.03% prevalence [82]. Interestingly, viremic pediatric nonprogressor patients with high CD4⁺ T cell counts seem to be more common than adults VNP individuals [86]. Is it possible that VNP patients are more common than what we originally thought? If so, what can we learn from these individuals that could help monitor and control HIV-1 infection in less fortunate patients? Several studies have pointed to high levels of immune activation as the major cause of HIV-1 pathogenesis [24, 98] and low immune activation seems to be—at least in part- responsible for the lack of disease progression in VNP individuals [86]. Moreover, the VNP-like phenotype during natural SIV infections in sooty mangabeys is definitely not related to a low rate of viral production nor an impairment on replicative fitness of the virus [25, 87]. However, we cannot rule out that original infection with less replication-competent viruses (therefore seeding HIV-1 reservoirs with replication impaired variants) could contribute to the decrease in immune activation, leading to the viremic non-progressor phenotype in these patients. Here we observed that all three VNP patients carried HIV-1 isolates with decreased replicative fitness compared to typical and rapid progressor individuals, and that accumulation of single-nucleotide polymorphisms (mutations) across the HIV-1 genome may be contributing to the overall fitness impairment. Therefore, it is possible that similar to the phenomenon observed in patients with a discordant response to antiretroviral therapy, i.e., high CD4⁺ cell counts with detectable plasma HIV-1 RNA load [99, 100], reduced viral replicative fitness could be contributing to late disease progression in untreated viremic controller individuals. Additional studies involving host immunology and genetics, together with more detailed analysis of the HIV-1 strains infecting VNP patients, are necessary to better understand HIV-1 pathogenesis, including approaches to prevent HIV-1 disease and potentially eliminate HIV reservoirs.