Human immunodeficiency virus type 1 long-term non-progressors : the viral , genetic and immunological basis for disease non-progression

A small subset of human immunodeficiency virus type 1 (HIV-1)-infected, therapy-naive individuals – referred to as long-term non-progressors (LTNPs) – maintain a favourable course of infection, often being asymptomatic for many years with high CD4 and CD8 T-cell counts (.500 cells ml) and low plasma HIV-RNA levels (,10 000 copies ml). Research in the field has undergone considerable development in recent years and LTNPs offer a piece of the puzzle in understanding the ways that persons can naturally control HIV-1 infection. Their method of control is based on viral, genetic and immunological components. With respect to virological features, genomic sequencing has shown that some LTNPs are infected with attenuated strains of HIV-1 and harbour mutant nef, vpr, vif or rev genes that contain single nuclear polymorphisms, or less frequently, large deletions, in conserved domains. Studies have also shown that some LTNPs have unique genetic advantages, including heterozygosity for the CCR5-D32 polymorphism, and have been found with excitatory mutations that upregulate the production of the chemokines that competitively inhibit HIV-1 binding to CCR5 or CXCR4. Lastly, immunological factors are crucial for providing LTNPs with a natural form of control, the most important being robust HIV-specific CD4 and CD8 T-cell responses that correlate with lower viral loads. Many LTNPs carry the HLA class I B57 allele that enhances presentation of antigenic peptides on the surface of infected CD4 cells to cytotoxic CD8 T cells. For these reasons, LTNPs serve as an ideal model for HIV-1 vaccine development due to their natural control of HIV-1 infection.


Introduction
Who are long-term non-progressors?
Years after the initiation of the 1981 AIDS pandemic, a small subset of human immunodeficiency virus type 1 (HIV-1)-infected individuals emerged with a markedly slow disease progression to AIDS.These persons, referred to as HIV-1 long-term non-progressors (LTNPs), are a group of HIV-1 seropositive individuals who have never been on antiretroviral therapy and have been infected with HIV-1 for several years -in some cases up to 20-25 years (Mikhail et al., 2003).They make up between 2 and 5 % of all HIV-seropositive individuals (Okulicz et al., 2009).Throughout their course of infection LTNPs maintain low levels of viraemia and elevated CD4 + T-cell levels in contrast to rapid progressor HIV-1 subjects who succumb to AIDS after a few years of infection in the absence of therapy.
Normally in HIV-1 pathogenesis, chronic asymptomatic infection can span from 3 to 20 years depending on the person's rate of disease progression (Levy, 2009).The viral loads in most persons infected with HIV-1 decrease from several million RNA copies ml 21 in the acute phase to 11 000-50 000 copies ml 21 in the chronic asymptomatic phase (Gupta et al., 2007).Compared with rapid progressors who typically progress to AIDS within 3-5 years, LTNPs are often asymptomatic for 10-20 years with elevated CD4 + Tcell counts .500cells ml 21 (Fig. 1) (Pantaleo & Fauci, 1996).In comparison, LTNPs -sometimes referred to as viraemic controllers -maintain between 5000 and 15 000 RNA copies ml 21 during the course of their infection with the majority having fewer than ¡10 000 copies ml 21 and many having 50-2000 copies ml 21 (Hunt, 2009;Mikhail et al., 2003).Recent studies have shown that LTNPs and other controllers with undetectable viral loads control HIV-1 infection within 1-2 years of infection (Madec et al., 2005;Okulicz et al., 2009).
Individuals referred to as slow-progressors (SP) or longterm survivors are less conservatively defined in the literature as having lower CD4 + T-cell levels (,500 cells ml 21 ) and higher viral loads than other LTNPs (Betts et al., 1999;Levy, 2009).A small subset of LTNPs (¡1 %) referred to as elite controllers (ECs) or elite suppressors have undetectable levels of viral RNA (,50 copies ml 21 ) Downloaded from www.microbiologyresearch.orgby IP: 54.70.40.11 On: Thu, 25 Oct 2018 14:59:29 for an unspecified period of time that could be months rather than years (Table 1) (Piacentini et al., 2009;Saksena et al., 2007).The HIV Controller Consortium estimates that roughly 1 in 300 HIV-infected persons meet the criteria for being an EC and recently, in a cohort of 4586 HIV-infected subjects followed since 1986, Okulicz et al. (2009) showed that 0.55 % are ECs (Okulicz et al., 2009;Walker, 2007).In comparison, 3.32 and 2.04 % of individuals from the same cohort are classified as LTNPs with a CD4 + T-cell count .500cells ml 21 during 7 and 10 years of follow-up, respectively (Okulicz et al., 2009).
Though the majority of ECs and LTNPs have CD4 + T-cell counts within normal ranges, several can undergo immunodeficiency and opportunistic infections as a result of CD4 + T-cell depletion (Hunt, 2009).For instance, 7 % of controllers from the CASCADE cohort (Concerted Action on SeroConversion to AIDS and Death in Europe) eventually progressed to AIDS (Madec et al., 2005).Similarly, 15 and 7 % of individuals from an EC cohort (n530) infected for .16years and with very low viral loads (,75 RNA copies ml 21 ) had CD4 + T-cell counts ,350 cells ml 21 and met the clinical definition of AIDS (Hunt, 2009;Hunt et al., 2008).This evidence shows that elite control of HIV-1 infection as defined by nearly undetectable viral loads in the absence of therapy does not correlate with a preservation of CD4 + T-cell counts.Interestingly, viral load has been shown to be a likely determinant for disease progression among controllers who are defined based on levels of viraemia, but with no criteria for CD4 + T-cell counts.In their recent study, Okulicz et al. (2009) found that individuals they classified as viraemic controllers (50-2000 RNA copies ml 21 ) progressed to AIDS more rapidly and had low CD4 + T-cell counts (,350 cells ml 21 ) than ECs who maintained stable and more elevated CD4 + T-cell counts.
There is no official standard for defining the various controller subgroups and variations in clinical definitions can significantly alter clinical outcomes.For example, studies show that when comparing controller groups meeting criteria for low viral loads but not high CD4 + T-cell counts with those having high CD4 + T-cell counts but not low viral loads, the former are more likely to undergo continual CD4 + T-cell loss and progression to AIDS (Hunt et al., 2008;Madec et al., 2005).For the sake of simplicity, this review will refer to controller subgroups based on criteria most commonly used by investigators (Table 1).An important caveat that should not be overlooked is that EC mechanisms of control are not necessarily the same as LTNPs because of their differences in CD4 + T-cell counts (often higher in LTNPs) and viral loads (lower in ECs).Nevertheless, the two groups probably share some mechanisms of control due to their favourable clinical outcome, which will be discussed in further detail in this review.
Of interest is the pathogenesis of natural HIV-2 infection, which mirrors HIV-1 progression in LTNPs.HIV-2 is endemic to West Africa and shares the same route of  transmission as HIV-1, yet the majority of those infected with HIV-2 will not progress to AIDS (Leligdowicz & Rowland-Jones, 2008).Host HIV-2 immunology is remarkably similar to that of HIV-1: infected individuals have a slow CD4 + T-cell decline and low plasma viral load, maintained NK cell functioning, broad antibody neutralization and gag-specific cytotoxic CD8 + T cells directly correlated with lowering patient viraemia (Leligdowicz & Rowland-Jones, 2008).
This review focuses on the three central factors associated with disease non-progression -virology, genetics and immunology (Fig. 2).The principal viral factor believed to slow disease progression is HIV-1 strain attenuation arising from mutations in viral accessory genes that cause a reduction in virus infectivity and replication.In terms of host genetics, some LTNPs display polymorphisms in chemokine receptors (CCR5 and CXCR4) and chemokines (i.e.MIP-1a and RANTES).With respect to immunological factors, HLA class I alleles (i.e.HLA-B57 and -B27), which present antigenic virus peptides to cytotoxic T cells, are found overexpressed in LTNPs.Additionally, LTNPs commonly display an HIV-specific CD4 + and CD8 + Tcell response capable of inducing both cytotoxic and noncytotoxic activity against the virus.
The evidence shows that LTNPs are distinct from other HIV-infected individuals and their natural form of immunity against disease progression serves as an ideal model for the development of an HIV-1 vaccine.Much can be learned from LTNPs and their remarkable ability to overcome challenges like HIV-1's antigenic variation and immune evasion.A strong vaccine candidate should induce immune responses similar to those of LTNPs, which heavily rely on cell-mediated immunity (CMI) and Gag-specific CD8 + T cells.The most realistic approach to developing an HIV-1 vaccine is one that provides partial immune control against viral replication, while preventing clinical disease and not one that prevents virus infection.
Lessons have been learned from the failures of the recent STEP vaccine study (Buchbinder et al., 2008), which was a phase 2 CMI-based HIV-1 vaccine trial that failed to demonstrate a protective response in vaccinees, particularly in persons with pre-existing anti-vector immunity.By better understanding the problems associated with recombinant vector-based HIV-1 vaccine design, future HIV-1 vaccine trials should use natural controllers such as LTNPs as a model, particularly for their ability to induce crossreactive and polyfunctional CTLs that both proliferate and inhibit viral replication through granzyme B-mediated killing.

LTNP virus attenuation
Decreased HIV-1 strain evolution correlates with weaker viral fitness and the inability to evade the host immune system.Some studies have shown that virus strains from LTNPs are less evolved and thus less capable of evading the host immunological response when compared with progressor strains (Sandonı ´s et al., 2009;Wang et al., 2003).
The classic example of HIV-1 LTNP virus attenuation is the Sydney Blood Bank cohort (n58), whose members were infected from blood transfusions from the same donor infected with an HIV-1 strain that contained a large deletion in a long-terminal repeat from a conserved and biologically significant area in the nef gene (Crotti et al., 2006;Learmont et al., 1999).Six of the subjects went on to become LTNPs and maintained stable CD4 + T-cell levels and low viral loads for over 10 years.Experimental evidence shows compared with virus clones from progressors, LTNP clones replicate more slowly and infect CD4 + thymocytes less effectively in tissue culture (Choudhary et al., 2005).In their study, Choudhary et al. (2005) found that in the absence of interleukin-1 (IL-1) and transforming growth factor beta (TGF-b) -which upregulate C-C chemokine receptor type 5 (CCR5) expression -infectivity and cytopathic events were reduced in LTNP HIV-1 CCR5 clones compared with those of progressors.These findings suggest that these LTNPs harbour attenuating virus mutations that interfere with infection.However, reductions in viral fitness such as these are unlikely to fully explain LTNP control of viral replication, especially in light of other LTNPs that have replication competent viruses (Alexander et al., 2000;Blankson et al., 2007).
Viral attenuation results from mutations in viral structural and non-structural genes.Several studies have reported major attenuating gene deletions in LTNPs that are associated with the control of infection (Calugi et al., 2006;Deacon et al., 1995;Huang et al., 1998).However, this association is difficult to prove because attenuation could actually be the effect rather than the cause of longterm control.For example, following infection with a nonattenuated viral strain a strong T-cell response could induce mutations in the genome of HIV-1 that render the virus weaker.Miura et al. (2010) have recently shown that when constructing chimeric viruses expressing the gagprotease from the earliest post-infection samples from patient viruses, ECs (n518) had significantly lower replication capacities than progressors (P50.0003).These findings demonstrate that control of infection could be established very early and in the case of these patients could be the result of their infection with attenuated, poorly replicating viruses.
In the first study to sequence the entire genome of HIV-1 isolates from ECs (n54), Blankson et al. (2007) were only able to find minor mutations that did not affect the function of HIV-1's genes and thus could not explain the subjects' elite suppression of infection.Additionally, all of the viral isolates were replication competent and were capable of growing normally in primary CD4 + lymphoblasts.In a subsequent experiment (Bailey et al., 2007), the investigators sequenced the viral isolates of one of the ECs before and after virologic breakthrough in order to determine whether any mutations were associated with their disease non-progression.They found that the subject retained the same detectable mutations at all time points, which demonstrates that their mutations do not explain a mechanism for the control of infection and that other mechanisms must play a part in their long-term control of infection.If the same can be shown for the remaining three ECs from the original study (Blankson et al., 2007), this would potentially rule out viral attenuation as the cause of their disease non-progression, which should shift the focus to immunological and genetic modes of control.
LTNP recognition of HLA-B57-restricted peptides is strongly correlated with long-term control of infection and viral escape mutations in these epitopes are associated with eventual virologic breakthrough in controllers.Bailey et al. (2007) compared virus isolates from a therapy-naı ¨ve subject infected with a multiple drug-resistant X4-tropic virus before and after becoming viraemic and found that mutations in B57-restricted gag epitopes targeted by CTLs correlated with the viral escape in the subject, who underwent an increase in plasma viraemia after having an undetectable viral load for several months.Further research should determine whether the same is true for a wider range of HLA class I alleles and controller subjects.
Challenging their hypothesis that LTNPs may harbour virus isolates less capable of downregulating HLA class I alleles responsible for generating a robust CTL response, Nou et al. (2009) found that isolates from ECs (n55) and progressors (n58) equally downregulated HLA-A2 and HLA-B57 alleles.Furthermore, a recent longitudinal study (O'Connell et al., 2010) that examined proviral and plasma samples from six ECs for 5 years showed that there were no significant mutations in B57/58-and/or B27-restricted gag epitopes.In fact, much of the observed virus evolution was due to synonymous mutations rather than mutations in B57/58-and/or B27-restricted immunodominant gag epitopes.

Genomic sequence analysis of virus genes from controllers
Nef.One of the normal functions of the nef gene is to downregulate host antigen presentation.It does so by binding to HLA class I receptors with its binding motif thus interfering with intracellular HLA class I antigen processing (Tolstrup et al., 2006).Large deletions in the nef genome can disrupt the protein coding sequence and smaller mutations can lead to premature termination of the gene's protein synthesis.Evidence has shown that reduced inhibition of HLA class I antigen presentation on CD4 + T cells is seen in LTNPs but not progressors, and is due to different mutations outside of the conserved region of the nef gene (Casartelli et al., 2003).There were no patterns seen in mutations among LTNPs, making it unlikely that their nef genes were defective as a result of a common, nonrandom process.
Other studies have reported that LTNPs infected with HIV-1 from blood transfusions had very similar deletions in nef proviral DNA as members of the Sydney Blood Bank cohort (Brambilla et al., 1999;Deacon et al., 1995), which were statistically significant when compared with mutations found in progressors (Casartelli et al., 2003;Geffin et al., 2000).These attenuating deletions in nef have been shown to inhibit in vitro HIV-1 replication in PBMCs when compared with virus clones lacking the mutations (P,0.001).
A TAC point mutation at position 138 (T138Q) in nef has been reported to occur more frequently in LTNPs versus progressors and is cited as being a cause of decreased viral fitness and replication (Kirchhoff et al., 1999;Premkumar et al., 1996;Tolstrup et al., 2006).Kirchhoff et al. (1999) performed extensive sequence analysis to reveal the mutation was present in 8.5 % of 59 LTNPs/SPs, while it was completely absent in 32 progressors.In general, attenuating nef mutations such as T138Q are uncommon in most controllers, as has been shown in a large cohort of ECs (n563), in which only three subjects had deletions in their nef gene.The same has been shown in other cohorts, which rules out the possibility that defective nef genes are a cause of long-term control of HIV-1 infection (Blankson et al., 2007;Mologni et al., 2006).
Vpr.The vpr gene has one of the most highly conserved sequences in the HIV-1 genome and a loss of vpr function significantly impairs HIV-1 infection because it impedes many of the gene's functions (i.e.G2 cell cycle arrest and apoptosis, T-cell depletion, nuclear localization of the HIV preintegration complex) (Caly et al., 2008;Lum et al., 2003).Whereas some studies have shown that LTNPs have increased frequencies of inhibitory mutations in the vpr gene compared with progressors (Caly et al., 2008;Lum et al., 2003;Mologni et al., 2006;Wang et al., 2003), others have not found genotypic differences in vpr functional domain sequences or HLA-binding motifs between LTNPs and progressors (Lai & Chen, 2006;Shen et al., 2008).
Vpr naturally induces G2 cell cycle arrest from the activity of a highly conserved motif at aa 71-82 in the gene's Cterminal region (Macreadie et al., 1995).Genomic analysis of this motif shows that some LTNPs have a mutation at position 77, called the R77Q mutation, which interferes with vpr-mediated cell cycle arrest and has been positively selected in some LTNPs (Lum et al., 2003;Mologni et al., 2006).For instance, Lum et al. (2003)  Vif.Vif is an accessory gene that normally promotes HIV-1 infectivity by enhancing viral replication and inducing the degradation of the endogenous anti-retroviral factor, apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3G (APOBEC3G) (Zhang et al., 2008).The vif-mediated degradation of APOBEC3G occurs through the recruitment of the Cullin5-ElonginB-ElonginC E3 ubiquitin ligase, which induces polyubiquitination and proteasome-mediated degradation of APOBEC3G (Zhang et al., 2008).The relationship between vif mutations and disease progression is unclear due to conflicting study results; some have shown that LTNPs have fully intact vif genes (Huang et al., 1998), while others have failed to find inhibitory mutations in the gene that could be an explanation for a reduction in LTNP virus isolate in vitro replicative capacities (Alexander et al., 2002;Rangel et al., 2009).
Rev. Defects are rarely reported, and in one study (Papathanasopoulos et al., 2003) sequence analysis from two vertically infected LTNP siblings with HIV-1 subtype C virus revealed that both subjects had ORF and full-length sequences for all genes except rev.Instead of exon 2 having a 16 aa truncation in the 39 end, which is typically found in subtype C viruses, the exon had a 3 aa insertion.Interestingly, the same mutation was found in subjects from the Sydney Blood Bank LTNP cohort (Oelrichs et al., 1998).Another study (Iversen et al., 1995) found an inhibitory mutation (L78I) in the activation domain of the rev gene from an LTNP, which was previously associated with slowing HIV-1 disease progression by impairing cell growth (Malim et al., 1991).
Gag. Single and double amino acid deletions have been identified in LTNPs in the C terminus of gag's p17 and p6 peptides albeit they have not been shown to correlate with lower viral loads in these subjects (Alexander et al., 2000;Miura et al., 2008).Miura et al. (2008) analysed viral sequences at the single-codon level and found only three codon differences in the gag sequences from more than 50 ECs and 80 progressors, which did not correlate with elite control of infection.Miura et al. (2010) have also recently shown that gag-protease chimeras constructed from acutely infected individuals (who later went on to become controllers) had significantly reduced in vitro replication capacities compared with chimeras constructed from acutely infected individuals that went on to become noncontrollers (n580) (P50.0003).Controllers also had a significantly higher prevalence of antiretroviral resistance mutations (P50.018) that interfered with viral replication.These results demonstrate the importance of identifying instances of reduced viral fitness in association with defective Gag proteins during early primary infection, which can be predictive of an individual's subsequent control of infection.
Env.The Env protein mediates HIV-1 entry into host CD4 + T cells.By using a novel cell line that allowed for inducible surface expression of CD4 and CCR5 receptors, Lassen et al. (2009) found that env clones from ECs (n57) were significantly less able to perform CD4-and CCR5mediated entry compared with env clones from chronically infected progressors.The reduction in env fitness resulted in significantly slower infection of EC CD4 + T cells compared with acutely infected subjects and progressors (P,0.0001).

Significance of viral factors associated with control: implications on vaccine design
There is not enough evidence to demonstrate patterns in attenuating mutations from LTNP and EC virus isolates that could contribute in any meaningful way to their control.Interesting are the studies that follow controllers at early time points during their acute phase of infection, before and after they have gone on to establish long-term control of infection.However, these studies are restricted to only a handful of controllers at a time and do not provide strong enough reason to believe that similar immune-induced mutations would be present in all controllers.While the nef and vpr mutations cause viral attenuation in some LTNPs and explain their infection with a lowly pathogenic virus, screening of larger cohorts of LTNPs has demonstrated that they are infrequently present (i.e. the T138Q nef mutation).Instead of being solely responsible for HIV-1 disease non-progression, inhibitory mutations in HIV-1 genes probably act in combination with other virological and immunological factors to facilitate control of infection.
Perhaps the most helpful lessons of viral mutations associated with long-term disease control have come from monkey models rather than human models.Interesting data from Mansfield et al. (2008) who vaccinated rhesus macaques with attenuated simian immunodeficiency virus (SIV) containing 100 aa deletions in the V1-V2 region of the env gene had significantly lower viral loads (,200 RNA copies ml 21 ) after 8 weeks of infection compared with macaques infected with parental SIV.No such attenuating mutations in env have been detected in LTNPs.Despite exciting findings from the macaque vaccinations, there are many caveats that must be addressed before proceeding with similar experiments in humans, including whether the attenuated vaccine is capable of broad, cross-reactive humoral immunity against the frequently mutating env gene and whether it continues to provide near complete inhibition against virus infection for periods longer than 8 weeks.

Genetic factors
Chemokine receptor polymorphisms.CCR5-D32 polymorphism.CCR5 is normally expressed at very low levels on the surface of naı ¨ve CD4 + T cells and at higher levels in activated CD4 + memory T cells as well as in monocytes and macrophages (Potter et al., 2007).A number of studies (Table 2) show that LTNPs carry a copy of the 32 bp deletion in the gene, called the CCR5-D32 polymorphism, which introduces a premature stop codon in the CCR5 gene that causes it to be produced as a truncated protein that is trapped in the endoplasmic reticulum.The frequency of the polymorphism varies with different ethnicities and it is estimated that among populations where the polymorphism is found more frequently, such as northern Europe and western Asia, roughly 1 % of Caucasians are homozygous and between 9 and 20 % are heterozygous for the CCR5-D32 polymorphism (Lucotte & Mercier, 1998;Pereyra et al., 2008).The polymorphism is nearly absent among native Africans, East Asians and American Indians (Stephens et al., 1998).Studies that find LTNPs are more frequently heterozygous for the polymorphism than progressors and uninfected controls have shown that possessing one copy of the polymorphism results in a reduction of CCR5 expression on CD4 + T cells and is associated with slowing progression to AIDS (Dean et al., 1996;Eugen-Olsen et al., 1997;Romiti et al., 2000).Individuals homozygous for this deletion are resistant to R5tropic HIV-1 infection (accounting for the majority of transmitted HIV-1 strains) due to the abrogated CCR5 surface expression on CD4 + T cells (Dean et al., 1996).
Study results are conflicting with regard to CCR5-D32's association with control of HIV-1 infection.Those that argue that it is associated with disease non-progression show that HIV-infected subjects heterozygous for the allele have a significantly slower progression to AIDS, whereas several more recent studies have found that the polymorphism is absent in their LTNP cohorts (Table 2).In some larger controller cohorts, LTNPs and SPs carrying the allele have a slower progression to AIDS and more stable CD4 + T-cell counts than progressors or uninfected controls (Eugen-Olsen et al., 1997;Misrahi et al., 1998;Morawetz et al., 1997;Romiti et al., 2000).For instance, Romiti et al. (2000) demonstrated that a group of therapynaı ¨ve HIV-1-infected children (n5229) (with at least 2 years of asymptomatic infection) that included 22 perinatally infected LTNPs had a 7.59-fold higher likelihood than progressors (n572) for being heterozygous for CCR5-D32.Specifically, 27.3 % of LTNPs and 1.4 % of progressors were heterozygous for CCR5-D32 (P50.0288).
While studies such as these strongly associate CCR5-D32 with reductions in viral load and elevated CD4 + T-cell counts, this does not make it an indicator of disease nonprogression.Only survival studies that follow subjects over many years and measure time to AIDS can make a more substantiated association with disease non-progression.
CCR5 promoter polymorphisms.Some studies have demonstrated that excitatory mutations in the promoter region of CCR5 mRNA transcripts increase the rate of disease progression to AIDS due to HIV-1 binding to the increased cell-surface expression of CCR5 (Piacentini et al., 2009).
Examples include the CCR5P1/P1 haplotype and 59029 A/ G polymorphism (Carrington et al., 1999).Additionally, the 59353C allele is found in higher frequency in some progressors compared with LTNPs (Jang et al., 2008).Perhaps the best-established association with disease nonprogression is the CCR5P1/P1 haplotype, which has a frequency greater than 40 % in Caucasians, Asians and persons of African descent (An et al., 2000;Carrington et al., 1999).It is believed that between 10 and 17 % of persons developing AIDS within 3.5 years of infection do so as a result of having the CCR5P1/P1 haplotype (Martin et al., 1998).
Other chemokine co-receptor polymorphisms.In the firstever genome wide study (Limou et al., 2010) of non-HLA-replicated association, recent data show that a polymorphism in CXCR6, known as the minor-HIV-1 co-receptor, was the strongest signal (P52.5610 27) in a large group of LTNPs (n5186) who were not ECs (viral load .100RNA copies ml 21 ), compared with uninfected controls.Investigators found that the rs2234358 polymorphism on chromosome 3 was independent from the CCR2-CCR5 locus, and was not linked to the control of viral load (LTNPs carrying the polymorphism had a similar mean viral load), exhibiting an overall high attributable risk of LTNP status of 12 % (Limou et al., 2010).
Polymorphisms in CCR2 and CX3CR1, which HIV-1 sometimes uses as co-receptors, have also been associated with slowing HIV-1 disease progression.For example, the CCR2-64I mutation has been shown to reduce CXCR4 expression on CD4 + T cells, thereby interfering with X4tropic virus infection (Kalinkovich et al., 1999).In one study's cohort of HIV-1 positive Kenyan sex workers (Anzala et al., 1998), the frequency of being positive for CCR2-64I was highest in LTNPs, which was three times greater than that for progressors.In a separate study, Vidal et al. (2005a) found the CX3CR1 V249I polymorphism significantly more frequently in LTNPs than progressors, but not healthy controls.
Variation in the gene encoding duffy antigen receptor for chemokines (DARC) has also been implicated in the rate of HIV-1 disease progression (He et al., 2008).DARC is located on red blood cells (RBCs) and binds to numerous chemokines, including RANTES, which leads to reduced RANTES plasma levels.The T-46C DARC polymorphism is prevalent in persons of African descent and causes reduced DARC RBC surface expression, associated with an increased risk of acquiring HIV-1 infection.Paradoxically, T-46C polymorphism has also been shown to slow the rate of disease progression and the rate of CD4 + T-cell loss in a cohort of HIV-infected African Americans (He et al., 2008).In fact, the survival advantage conferred by DARC T-46C in African Americans was shown to be just as protective as carrying a copy of the CCR5-D32 polymorphism in European Americans.
Chemokine variants.The natural chemokine ligands of CCR5 and CXCR4 (the two coreceptors that HIV-1 uses for infecting CD4 + T cells) act as competitive inhibitors of HIV-1 infection and have been associated with control of HIV-1 infection (Mikhail et al., 2003).CCR5 ligands include the b-chemokines macrophage inflammatory protein 1a and 1b (MIP-1a and MIP-1b), CCR5 ligand human CC chemokine ligand 3 like 1 gene (CCL3L1) and RANTES, and the CXCR4 ligand is SDF-1 (Kalinkovich et al., 1999).CD8 + T cells and macrophages secrete these soluble factors, which then inhibit HIV-1 replication in a non-cytolytic fashion (Gulzar & Copeland, 2004).
MIP-1a/CCL3 and MIP-1b/CCL4.The MIP-1a chemokine is believed to downregulate HIV-1 infection of CCR5bearing cells by facilitating chemokine receptor desensitization and lowering CCR5 cell surface expression (Piacentini et al., 2009).A few studies have shown that CD4 + T cells from LTNPs produce high levels of MIP-1a in comparison with AIDS subjects who produce hardly any measurable amount of the chemokine (Saha et al., 1998;Schinkel et al., 1999).A caveat to consider with these studies is that AIDS patients could have generalized reduced production of chemokines as an effect rather than cause of their severe immunosuppression.In fact, some studies show no difference in T-cell production levels of the chemokines between LTNPs and progressors (Potter et al., 2007).et al., 2005).
In one of the largest cohort studies to date looking at the relationship between CCL3L1 and HIV-1 disease progression, Dolan et al. (2007) monitored HIV-seropositive patients (n51132) and found that CCLC3L1 interfered with R5-tropic virus infection of host immune cells and also enhanced CMI against HIV-1 infection.
Overall, the study showed that in acute and chronic infection, the CCLC3L1-CCR5 genotype status was predictive of viral loads, CD4 + T-cell depletion and strength of CMI, demonstrating that host genetics could be used to determine the rate of HIV-1 disease progression in these subjects.Research has also shown that lower CCL3L1 copy numbers translate to an increased risk of infection without affecting rates of HIV-1 disease progression, which remains true even after stratifying according to CCR5-D32 (Lee et al., 2010;Nakajima et al., 2007;Urban et al., 2009).This could be due to differences in study population ethnicities, as it has been shown that Caucasians carry fewer CCL3L1 copies (Gonzalez et al., 2005).(Koning et al., 2003;McDermott et al., 2000).Some polymorphisms in RANTES' promoter region protect HIV-infected subjects against disease progression as a result of increased RANTES synthesis.G4 RANTES promoter region polymorphisms were studied in CCR5-D32-negative HIV-infected subjects (n5420) from the Multicenter AIDS cohort study and individuals with the G4 genotype significantly progressed more slowly to AIDS (median time to AIDS was 7.5 years) and had a slightly reduced CD4 + T-cell depletion than individuals with non-G4 genotypes (median time to AIDS was 5.6 years) (P50.007)(McDermott et al., 2000).Kaplan-Meier survival curves showed that elevated rates of survival in these subjects were comparable to subjects heterozygous for the CCR5-D32 polymorphism from the same study.
The RANTES 28G polymorphism found in the gene's promoter region has also been associated with protection against disease progression (Liu et al., 1999;Wichukchinda et al., 2006).One study's genotyping of RANTES from HIV-1 seroconverters (n5272) found that the mutation alone was sufficient to activate the gene's promoter region and that it correlated with increased levels of RANTES transcription and reduced rates of CD4 + T-cell depletion (Liu et al., 1999).Another study (Wichukchinda et al., 2006) demonstrated the mutation's protective effect by comparing disease progression in seroconverters carrying either the RANTES 28G polymorphism or the RANTES In.1.1Cpolymorphism, which is located in the gene's regulatory region and inhibits RANTES synthesis.Persons carrying the RANTES In.1.1Callele progressed significantly faster to AIDS compared with those carrying the RANTES 28G allele, along with those HIV-infected subjects without either mutation (P,0.02).
In a study of HIV-seropositive individuals from the Amsterdam Cohort of Homosexual Men (Koning et al., 2003), progressors (n56) had a significant reduction in virus sensitivity to RANTES-mediated neutralization compared with LTNPs (n57) (P,0.05).This could be the result of viral escape variants that are resistant to RANTES' antiviral effects.Results showed RANTES was the most potent inhibitor of R5-virus replication and had a considerably lower IC 50 against virus p24 antigen than MIP-1a and MIP-1b.This finding demonstrates that RANTES is better than the other chemokines in blocking R5-strain infection of CD4 + T cells.

SDF1 (CXCL12).
Stromal cell-derived factor 1 (SDF1) or CXCL12 is CXCR4's only natural ligand and inhibits X4-tropic HIV-1 virus infection (Magierowska et al., 1999).The SDF1-39A polymorphism is a GAA mutation in the 39 untranslated region of the SDF1 gene that upregulates its transcription levels and has been associated with delaying disease progression in HIV-infected subjects who have been found to carry the polymorphism significantly more frequently than progressors (Hendel et al., 1998;Magierowska et al., 1999;Winkler et al., 1998).
However, several studies show a lack of relationship between SDF1-39A and HIV-1 disease non-progression (Vidal et al., 2005b;Ioannidis et al., 2001;Tresoldi et al., 2002).For instance, in their international meta-analysis, Ioannidis et al. (2001) measured the effects of subjects homozygous for the SDF1-39A polymorphism by reviewing studies that prospectively followed HIV-1-infected patients from seroconversion to AIDS diagnosis and death.Results showed that being homozygous for the polymorphism had no effect on disease progression and there was no significant difference in HIV-1 RNA levels among persons with and without the polymorphism.
Endogenous antiretrovirals.APOBEC3G.APOBEC3G is a cytidine deaminase that blocks HIV-1 integration into host DNA by inducing excessive GAA base mutations in the viral DNA that causes premature stop codons to form and makes HIV-1 replication-incompetent (Piacentini et al., 2009).HIV-infected individuals with low levels of APOBEC3G mRNA are associated with an increased risk of progression to AIDS, while the opposite is true for those with high levels of APOBEC3G mRNA (Jin et al., 2007).A recent study (Va ´zquez-Pe ´rez et al., 2009) that looked at APOBEC3G mRNA expression levels in PBMCs from a group of HIV-1-infected individuals at different disease stages found higher mRNA levels correlated with greater CD4 + T-cell counts and lower HIV-1 viral loads (P50.0004).Additionally, therapy-naı ¨ve subjects with lower viral loads (,10 000 copies ml 21 ) and greater than 3 years of infection had higher APOBEC3G expression levels than both progressors and uninfected healthy controls.APOBEC3G-induced hypermutations in vpu/env proviral DNA have been correlated with higher CD4 + Tcell counts (P50.042) in a large cohort of HIV-infected Kenyan women (n5208) (Land et al., 2008).Other APOBEC3G polymorphisms are associated with increased rates of disease progression, as demonstrated by An et al. (2004) in their study of 3073 HIV-1-infected subjects from six HIV-AIDS prospective cohorts.The study showed that the APOBEC3G exon 4 variant allele H186R correlated with faster progression to AIDS and a significant decline in CD4 + T-cell counts when compared with subjects that did not carry the polymorphism (P50.0009).
Tripartite motif protein 5a (Trim5a).Trim5a acts on HIV-1 in the post-cell-entry phase by attaching to the HIV-1 capsid protein to inhibit viral replication (van Manen et al., 2008).Physiological levels of Trim5a have been associated with slowing down the HIV-1 disease progression and the R136Q polymorphism is believed to be especially effective in blocking X4-tropic HIV-1 infection (van Manen et al., 2008).Javanbakht et al. (2006) examined the effect of 12 common Trim5a single nuclear polymorphisms (SNPs), including R136Q, in 939 HIV-1 seroconverters from five USA-based HIV-1 natural history cohorts and found that the SNPs were not related to patient progression to AIDS nor to low CD4 + T-cell counts (,200 cells ml 21 ).In a subsequent study, van Manen et al. (2008) found that the presence of the R136Q polymorphism in study subjects infected with X4-tropic but not R5-tropic HIV-1 correlated with their lower viral load.

Significance of genetic factors associated with control: implications on therapeutics
The main way in which host genetics has been experimentally associated with controlling HIV-1 progression is genetic polymorphisms that interfere with virus binding to susceptible target cells, either through impaired receptor expression on the cell-surface or an increase in production of competitive inhibitors.Experimental results have hardly been concordant, and among the most highly investigated aspects of host genetics relating to HIV-1 progression is the CCR5-D32 polymorphism.Nearly as many studies that relate the polymorphism to longer time to AIDS and elevated CD4 + T-cell counts also fail to establish a statistically significant difference between LTNPs and progressors.Other genetic mechanisms could play a more meaningful role albeit this is not certain because they have not been as widely studied as the CCR5-D32 polymorphism.
Although multiple protective effects have not been collectively observed in the same LTNP subject, the combinational effects of CCR5-D32 with excitatory mutations in the promoter region of CCL3L1 and RANTES would probably provide strong protection from CCR5tropic HIV-1 infection and should be considered for HIV-1 therapy.Though preventive and prophylactive vaccines cannot rely on genetics to confer protection from HIV-1 infection, gene therapy can be administered as antiretroviral therapeutics to control infection in HIV-1 seropositive individuals.An example of gene therapy is genetically engineered CD4 + T cells that are resistant to HIV-1 based on coreceptor expression or secretion of antiviral soluble factors, which are adoptively transferred to recipients (Varela-Rohena et al., 2008).Genetic correlates of protection that should be targets for gene therapy include antiviral factors like APOBEC3G and TRIM5a that restrict HIV-1 replication at the post-entry, pre-integration stage.
Another possible example of using adoptive immunotherapy to control HIV-1 infection involves the transfer of Tcell receptor genes from a T cell specific for HLA-B57restricted antigens that are inserted into a viral vector (Varela-Rohena et al., 2008).This would be especially useful in generating highly specific T-cell responses for conserved antigenic gag epitopes that are most strongly correlated with reducing viral load early in infection.

Innate immunity
Toll-like receptor (TLR) 9. Two studies (Bochud et al., 2007;Soriano-Sarabia et al., 2008) have found that SNPs in the TLR9 gene are associated with a rapid HIV-1 disease progression.The 1635A/G TLR9 and 1174G/A polymorphisms positively correlated with rapid disease progression, higher viral loads and low CD4 + T-cell counts in the studies' large cohorts of therapy-naı ¨ve HIV-infected individuals (Bochud et al., 2007;Soriano-Sarabia et al., 2008).
Mannose-binding lectin (MBL).Elevated MBL serum levels correlate with better survival in patients with AIDS and homozygosity for MBL variant alleles B, C and D have been associated with lower MBL serum levels and an increased susceptibility to HIV-1 infection (Garred et al., 1997;Piacentini et al., 2009).Studies that have looked at the effects of MBL genotype on the clinical outcome of HIV-1 infection in therapy-naı ¨ve subjects were unable to make a definitive correlation between MBL genotype (homozygous versus WT for MBL variants) and HIV-1 disease non-progression (Garred et al., 1997;Maas et al., 1998).For instance, in a cohort study (Garred et al., 1997) of HIV-infected homosexual seroconverters (n596) who were followed for 10 years, the correlation between MBL genotype and the time to AIDS was not statistically significant (P50.8).For subjects that went on to develop AIDS (n561), those who were carriers of the MBL variant alleles had a significantly shorter survival time after being diagnosed with AIDS versus those who were not carriers (11 versus 18 months, P50.007).While this suggests that higher MBL copy levels reduces AIDS mortality, it does not demonstrate a protective effect against the actual progression to AIDS.
NK cells.NK cells have many functions in innate and CMI due to their vast expression of surface receptors that enables them to both inhibit and activate target cells (Vieillard et al., 2010).NK cells from both viraemic and non-viraemic LTNPs have been shown to produce significantly higher levels of gamma interferon (IFN-c) than progressors and healthy uninfected subjects, which is the main mechanism by which NK cells mediate control of infection (O'Connor et al., 2007;Vieillard et al., 2010).NK cells are certainly important for controlling viral infection in HIV-infected subjects, albeit CD8 + T cells have been shown to exhibit superior control of HIV-1 replication in EC subjects (O'Connell et al., 2009).
Evidence shows that NK cells from LTNPs have an abnormal phenotype compared with both HIV-1-infected viraemic subjects and uninfected subjects (O'Connor et al., 2007).Recently, Vieillard et al. (2010) performed largescale analysis of 21 different phenotypic markers on NK cells to show that markers from LTNPs with undetectable viral loads were significantly different from viraemic controllers (50 000 RNA copies ml 21 ) and progressors.This indicates that for these subjects viral load influences receptor expression on NK cells.
As recent data have shown, polymorphic killer cell immunoglobulin-like receptors (KIR) in the presence of Mechanisms of control in HIV-1 LTNPs specific HLA-B alleles play a positive role in HIV-1 disease pathogenesis, particularly the inhibitory KIR3DL1 allotypes co-expressed with HLA class I alleles of the HLA-Bw4 family (Alter & Altfeld, 2009).Early in infection, strong inhibitory reactions between HLA-Bw480I alleles (i.e.HLA-B57) and KIR3DL1 have been postulated to activate developing NK cells and promote a robust cytotoxic cell response (Alter & Altfeld, 2009).In more than 1500 HIV-1infected individuals Martin et al. (2007) found that KIR3DL1 + Bw4 genotypes were protective against HIV-1 disease progression, with the KIR3DL1*004 allele being the most protective.Martin et al. (2002) were the first to show that HIV-infected individuals with co-expression of the KIR3DS1 allele -which is frequently found in Caucasians but not African-Americans -with HLA-Bw480I progressed significantly slower to AIDS than individuals with either one or neither of these two alleles.Slower progression to AIDS was related to rapid degranulation by KIR3DS1 + NK cells in response to HIV-infected HLA-Bw480I + CD4 + T cells (Alter & Altfeld, 2009).In a recent study, O'Connell et al. (2009) did not find either KIR3DS1 or KIR3DL1 overexpressed in a cohort of 20 ECs who were mostly African-American, and their KIR frequencies were similar to that seen in uninfected African-Americans.For functional analysis of NK cells for eight of the ECs, NK cell inhibition of viral replication did not correlate with KIR3DS1 or KIR3DL1 genotypes.
Other findings have implicated specific binding of KIR2DL2 to HLA-C alleles as being protective against HIV-1 progression, though KIR2DL2 has been shown to bind non-specifically to HLA-C1 and HLA-C2 molecules, indicating their mode of protection could be independent from a specific HLA-C subtype (Alter & Altfeld, 2009).

Adaptive immunity
HLA class I alleles.HLA class I alleles (A, B and C) present antigens from intracellular pathogens to CD8 + T cells.A heterozygous HLA class I genotype has been correlated with slowing HIV-1 disease progression and lowering HIV-1 viral load by enabling the presentation of a wider range of peptides and decreasing the risk of viral escape variants (Piacentini et al., 2009).In particular, HLA-B57 variantspredominantly B5701 and B5703 -strongly correlate with the control of HIV-1 infection by inducing a cross-reactive response against immunodominant Gag epitopes (Klein et al., 1998;Piacentini et al., 2009).HLA-B27 is also a wellstudied allele with respect to HIV-1 disease non-progression though the same relationship is not as statistically significant in large cohort studies (Bello et al., 2005;Klein et al., 1998).
Interestingly, an analogous relationship has been found between HLA-B27 and the MHC class I allele, Mamu-B08, in terms of disease non-progression; elite control of viral load in rhesus macaque monkeys infected with SIV is correlated with their carriage of the Mamu-B08 allele (Loffredo et al., 2009).Research shows that up to 50 % of Mamu-B08positive Indian rhesus macaques become elite controllers and that Mamu-B08-restricted epitopes nearly match the binding profile for HLA-B27-positive LTNPs (Loffredo et al., 2009).
Between 1 and 10 % of Caucasian, African and Asian populations carry a copy of the HLA-B57 allele and 1.4-8 % of persons in major continental populations carry a copy of the B27 allele (Di Lorenzo, 2008;Middleton et al., 2003).To date, at least 15 studies have measured the frequency of EC/LTNP/SP subjects carrying either HLA-B27 or -B57 in comparison to viraemic HIV-1 progressors or healthy uninfected controls (Table 3).B57's frequency in HIV-1 controller study groups varies considerably.In studies with cohorts of at least 30 subjects, between 8 and 63 % of either LTNPs or ECs have been found to carry the B57 allele with frequencies being the highest in ECs.These allelic frequencies are statistically significant when compared to frequencies in subjects with progressive HIV-1 infection that range from 2 to 24 %.In comparison, the B27 allelic frequencies in study cohorts larger than 30 subjects range from 2 to 20 % in controllers and 3 to 4 % in progressors, though comparisons of the two do not reach statistical significance.For this reason, there is no strong enough evidence to suggest that HLA-B27 provides any appreciable protection against HIV-1 disease progression.
Though the majority of studies find that the rates in controllers compared with progressors are statistically significant, there is a considerable amount of variation in study cohort size and duration of follow-up period for survival studies.One factor that is important to take into account when considering the significance of the B27 and B57 alleles is the viral load parameters applied to different LTNP cohorts.Studies that adhere to the most conservative definition of what constitutes an LTNP (viral load of ,75 RNA copies ml 21 , CD4 + T-cell counts .850cells ml 21 ) have high frequencies of subjects positive for B57 but negative for B27 (Migueles & Connors, 2010).Even more revealing and clinically important are studies that compare rates of B57 and B27 positivity in LTNPs to HIV-infected progressors -more studies report that B57 rather than B27 is present in LTNPs at statistically significant higher frequencies in both progressors and healthy seronegative controls.
Other protective HLA class I alleles include B13, B15, B44, B51 and B58 -in some cohorts as many as 90-95 % of LTNPs carry at least one of these alleles (Migueles & Connors, 2010).While individually B57 plays a role with HIV-1 disease non-progression, it is unclear what the protective effects of multi-allelic polymorphisms are in individuals that carry more than one of these protective alleles.Leslie et al. (2010) have recently demonstrated the power of additive effects of HLA-B class I alleles in a large cohort of HIV C-clade-infected individuals from South Africa (n51211).Even after excluding the alleles identified to have the strongest positive (B57 and B5801) and negative influence (B5802 and B18) on subject viral load and CD4 + T-cell count, HLA-B remained the strongest predictor of viral load (P50.0007) and CD4 + T-cell count (P,0.0001).This demonstrates that the role that HLA-B plays in controlling HIV-1 infection could be the additive effect of some or all of the HLA B alleles.
There are also HLA class I alleles associated with accelerating HIV-1 disease progression, which include A24, A29, B35, C4 and Cv4 (Piacentini et al., 2009;Saksena et al., 2007).Specifically, the B35-Px subtype allele has been linked to accelerated disease progression due to its reduced ability to bind HIV-1 peptides and activate a CTL response (Carrington & O'Brien, 2003).
CD4 + T cells.The role of CD4 + T cells in long-term control of HIV-1 infection is unclear, especially in terms of their ability to induce and maintain an HIV-specific CD8 +

Mechanisms of control in HIV-1 LTNPs
T-cell response.Some LTNPs have been found to have a robust, polyclonal and Gag-specific CD4 + T-cell response, which unlike progressors, is maintained at elevated levels for the duration of their infection and directly correlated with a lower viral load (Boaz et al., 2002;Migueles et al., 2000;Rosenberg et al., 1997).Compared with progressors and viraemic controllers, CD4 + T-cell responses from LTNPs have been shown to have the CD4 + IL-2 + IFN-c + phenotype with a large production of IL-2, indicative of a robust type 1 helper T-cell response (Migueles et al., 2000;Potter et al., 2007).However, it is difficult to assess the causal relationship that HIV-specific CD4 + T cells have with disease non-progression in these individuals because low levels of virus replication could in fact be inducing the production of virus-specific CD4 + T cells in LTNPs.So far, studies have not established causality of HIV-specific CD4 + T-cell levels and disease non-progression beyond reasonable doubt.
Some evidence suggests that LTNPs have reduced CD4 + Tcell activation in comparison with subjects with rapid disease progression (Bello et al., 2009;Marchetti et al., 2009).Less CD4 + T-cell immune activation reduces the number of cells susceptible to HIV-1 infection and can potentially lead to a better disease prognosis.In one study, ECs (n54) that were examined longitudinally for 4 years had low levels of immune activation (measured as HLA-DR + CD38 + cells) and spontaneous CD4 + T-cell apoptosis similar to uninfected controls and less than progressors (Bello et al., 2009).
The same has been shown in primate models.Sootey mangabeys infected with SIV rarely progress to AIDS, maintaining low CD4 + T-cell counts despite the presence of highly replicating virus (Estes et al., 2008).Sootey mangabeys have been found to have lower rates of immune activation than their primate counterparts, rhesus macaques, who suffer a poorer clinical outcome.In one study, both primate species underwent initial immune activation, which eventually declined in sootey mangabeys due to increased PD-1 expression on their CD4 + T cells (Estes et al., 2008).Analogous findings have been shown for HIV-1 controller human subjects.When followed for shorter periods (i.e.1.5 years) they have elevated levels of immune activation and spontaneous T-cell apoptosis similar to treated HIV-infected subjects or untreated rapid progressors (Ronquillo et al., 2010;Goicoechea et al., 2009).Low levels of virus replication probably explain for the presence of T-cell activation in controllers and progressors earlier in infection and which has been shown to decline over time in controllers, but not in progressors (Bello et al., 2009).
Contrary to these results, there is evidence that has shown LTNP CD4 + T effector cells are in a chronic activated state based on their increased HLA-DR cell-surface expression, decreased IL-7a production, and increased IFN-c production (Potter et al., 2007).These LTNP CD4 + T cells do not show signs of the same kind of generalized immune activation seen in rapid progressors, but rather specific immune activation restricted to effector memory T cells.In an antibody microarray analysis of 135 T-cell surface antigens, Wu et al. (2007) showed that LTNP CD4 + T cells had an upregulated cell-surface expression of HLA-DR, CD71 and CD38 compared with viraemic patients on highly active antiretroviral therapy (HAART).
IL-7/IL-7 receptor (IL-7R) balance is important for the maintenance of CD4 + T-cell levels and disrupting this balance increases the risk of progressing to AIDS (Marchetti et al., 2009).Studies have found that LTNPs have a significantly elevated IL-7R expression on CD4 + T cells compared with progressors.These results support an IL-7-mediated model of CD4 + T-cell thymic output used to replenish low levels of peripheral CD4 + T cells (Marchetti et al., 2009;Potter et al., 2007).This raises the question of whether there is a correlation between reduced T-cell activation with elevated IL-7R-expressing CD4 + T cells.LTNP CD4 + memory T cells have also been found to express elevated levels of chemokine receptor CCR7 on their cell surface compared with progressors and uninfected controls, which may reflect a difference in their CD4 + central memory cell homing patterns into secondary lymphoid organs (Marchetti et al., 2009).
CD8 + T cells.CD8 + T cells and their conversion into CTLs -the effector cytotoxic cells responsible for killing HIV-infected host cells -are the most important mediators of lowering viral load in HIV-1 controllers and are most strongly correlated with facilitating immune control in LTNPs (Migueles & Connors, 2001).The CD8 + T-cell responses of many LTNPs have been shown to have a high CTL precursor frequency that gives rise to broadly reactive CTLs specific for conserved sequences in the env, gag and pol genes (Betts et al., 1999;Geldmacher et al., 2007;Migueles & Connors, 2001).Gag-specific CTL responses, which are activated after binding to antigenic p24 epitopes presented by HLA class I alleles, positively select for viral escape variants that are unable to replicate as effectively (Geldmacher et al., 2007).Geldmacher et al. (2007) isolated specific epitopes from the N-and C-terminal regions of Gag that most strongly correlated with low viral loads and elevated CD4 + T-cell counts in LTNPs, identifying epitopes from these regions as important immunological targets.Recently, it has been shown (Ferre et al., 2010) that polyfunctional HIV-specific CD8 + T cells responding to Gag p24 were more abundant in rectal mucosa of HIV controllers than in non-controllers or HAART-treated individuals.The same CD8 + T-cell response was seen in the sera from these individuals, which was directed against conserved regions of Gag that included HLA-B27-and -B57-restricted epitopes.
However, the same caveat that applies to LTNP CD4 + T cells also applies to their CD8 + T cells -it is unknown if high levels of virus-specific CD8 + T cells are the cause or effect of their low viral loads.Before major advances in a cell-mediated HIV-1 vaccine can be made, it is important to understand what particular aspects of a robust and virus-specific HIV-1 CTL response (qualitative versus quantitative) most strongly correlate with disease nonprogression.Quantitative factors (the frequency of an HIVspecific CTL response) are not found to differ significantly between LTNPs and progressor and have been ruled out as playing a strong role in disease non-progression (Betts et al., 2006).In contrast, studies have found that LTNP CD8 + Tcell responses differ significantly qualitatively (i.e.proliferation, production of cytotoxic molecules like IFN-c, IL-2 and granzyme B) (Betts et al., 2006;Migueles et al., 2002).Betts et al. (2006) showed that progressors had much lower CD8 + T-cell polyfunctional responses (based on cytokine and chemokine secretion) than LTNPs.Specifically, MIP-1b secretion was the most predominant HIV-specific CD8 + T-cell secretory phenotype to each of HIV's antigens.The total frequency of HIV-specific CD8 + T cells did not correlate with viral load in study subjects, whereas qualitative factors like the frequency and per cent of Gag-specific CD8 + T cells expressing all five functions (degranulation, IFN-c, MIP-1b, tumour necrosis factor-a and IL-2) correlated inversely with viral load in LTNPs (P,0.001).While CD8 + T cells from progressors were found to produce no more than three functions at once, LTNP CD8 + T cells produced four and five responses simultaneously, demonstrating their enhanced polyfunctional response.
In their experiments, Migueles et al. have shown that HIVspecific proliferating CD8 + T cells from LTNPs had greater secretion of effector molecules like perforin and granzyme B, required for CTL-mediated lysis of infected CD4 + T cells (Migueles et al., 2002(Migueles et al., , 2008)).Perforin secretion is normally tightly regulated and is not coordinately secreted with granzyme B. Other phenotypic differences between LTNP and progressor CD8 + T cells include high secretion levels of IFN-c and IL-2 and low levels of IL-4 seen in LTNPs, with the converse seen in progressors.The antiinflammatory IL-4 secretion probably provided some advantages to virus survival in progressor CD4 + T cells.Additionally, Wu et al. (2007) used antibody microarrays to show that LTNP, but not progressor CD8 + T cells, had upregulated CD16 and CD56 cell surface expression, which activated NK cells.
Humoral immunity.Broadly acting neutralizing antibodies (NAbs) are rarely found in HIV-1-infected individuals with conflicting consensus regarding their frequency in LTNP populations.Some studies have shown they are more frequently found in LTNPs than progressors (Cecilia et al., 1999;Pilgrim et al., 1997), while others have shown they are not (Doria-Rose et al., 2010;Pereyra et al., 2008).Several studies have demonstrated, albeit with small sample sizes, that LTNPs have a stronger virus-specific NAb response than progressors against both autologous and heterologous viral strains (Braibant et al., 2008;Cecilia et al., 1999;Mahalanabis et al., 2009;Pilgrim et al., 1997;Sreepian et al., 2004).Interestingly, studies have shown that NAb levels peak in LTNPs late in their infection as a result of low levels of viral replication and more heterogeneous virus populations.In contrast, progressors rapidly lost their NAb response over the course of their infection due to their steady decline in CD4 + T cells (Cecilia et al., 1999;Lopalco, 2004).
HIV-specific NAbs predominantly target epitopes within the Gag and Env structural proteins, though these epitopes are poorly immunogenic (Sreepian et al., 2004).The strongest NAb-mediated response found in LTNPs has been against Env's gp120 and gp41 proteins (Lopalco, 2004).The C1 and C2 region of gp120, the V3 loop of gp120 (GPGRAF and NNNT motifs) and the KLIC motif of the gp41 are examples of immunogenic peptides LTNP NAbs target (Sreepian et al., 2004).In a large LTNP cohort (n567), mutations in gp120 induced a broad LTNP NAb response, defined as at least 90 % NAb-mediated reductions in infectivity of four heterologous virus primary isolates from different clades (Mahalanabis et al., 2009).Similar results have been found in other studies that looked at the breadth of NAb responses from LTNPs and progressors to over 20 viral isolates (Deeks et al., 2006;Doria-Rose et al., 2010).
For the first time ever, Simek et al. (2009) have utilized a novel high-throughput screening technique to identify a group of 'elite neutralizers', who make up a subset of HIVinfected individuals with broadly acting neutralizing antibodies against multiple HIV-1 isolates representing several clade groups.These elite neutralizers, who make up roughly 1 % of approximately 1800 HIV-1-infected individuals from five continents, were found to neutralize more than one pseudovirus at an IC 50 titre of 300 within a clade group and across at least four clade groups.These newly discovered antibodies, which include the VRC01 antibody that targets the CD4-binding site in gp120, offer renewed hope that a vaccine can elicit HIV-1-specific NAbs.Wu et al. (2010) found that this antibody neutralizes roughly 90 % of nearly 200 HIV isolates from multiple clades and is both broad and potent in its neutralization -a dual response that researchers have feared would not be found simultaneously in HIV-specific neutralizing antibodies.The next step forward is to determine whether these specific antibodies are found in LTNP and EC subjects and whether they directly correlate with reducing their viral loads.

Significance of immunological factors associated with control: implications on vaccine design
When taking into consideration all the immunological responses to HIV-1 (i.e.innate, humoral and cellmediated), Gag-specific CD8 + T cells are most positively associated with a lower viral load and promoting longterm control of infection (Walker, 2007).In comparison to Env antigens that undergo frequent mutations in their genome in order to evade the host's immune response, mutations in the gag gene do not occur as frequently because they render the virus inactive and unable to replicate.Furthermore, Env-specific CD8 + T cells have a shorter-lasting response compared with Gag-specific CD8 + T cells (Walker, 2007).In light of past HIV-1 vaccine trial failures, the most realistic approach to developing an HIV-1 vaccine is one that provides partial immune control against viral replication, while preventing clinical disease rather than developing a vaccine that prevents virus infection.An ideal vaccine would utilize a combination of immunological responses, most importantly being robust and cross-reactive T-cell responses against highly heterologous viruses in unison with broad NAb activity.Importantly, the T cell must have a wide breadth of coverage against HIV-1 epitopes in order to prevent viral escape.This will prove particularly challenging with CD8 + T cells that have the tendency to focus on specific epitopes.
Important lessons should be learned from the failure of recent efforts in developing a CMI vaccine that relied on generating a strong CTL response in order to protect vacinees against viral replication and disease progression.Merck's phase IIb STEP vaccine trial (Buchbinder et al., 2008), which failed to demonstrate efficacy in protection of HIV-1 infection in the vaccinated versus placebo groups, used a recombinant adenovirus type 5 (rAd5) vector presenting HIV-1's gag, pol and nef genes.The trial resulted in a higher HIV-1 incidence in vacinees versus placebo recipients among Ad5 seropositive men and uncircumsized men, and an incidence that was similar for vacinees versus placebo recipients among Ad5 seronegative men and circumsized men.The risk of becoming infected was the same between Ad5 seronegative vacinees and the placebo group and the risk was over two times greater for Ad5 seropositive vacinees compared with the placebo group.Male vacinees that became infected had a similar viral load set point as infected individuals from the placebo group (Buchbinder et al., 2008).
As subsequent analysis has shown (O'Brien et al., 2009), the vaccine's failure to show efficacy in the vaccinated group is probably not related to T-cell activation and enhanced HIV-1 infection susceptibility, but rather elevated baseline Ad5 NAb titres in vacinees, which interfered with the rAd5 vector and caused a lower effective dose of the vaccine in vacinees.In fact, vaccination with the rAd5 vector resulted in a lower vector-specific T-cell response in Ad5 seropositive vacinees compared with seronegative vacinees and this is highly suggestive of the fact that baseline levels of Ad5-specific NAbs in vaccine recipients are not surrogate markers for Ad5-specific T-cell responses (O'Brien et al., 2009).For this reason, inducing a strong HIV-1 CMI response should still be considered the major goal of any future vaccine candidates and that failures in the STEP study were not due to the absence of an HIVspecific CTL response -the vaccine elicited IFN-c ELISPOT responses in 75 % of vacinees (Buchbinder et al., 2008) -but rather the partial neutralization of the vaccine vector by baseline Ad5 vector-specific NAbs.
An HIV-1 vaccine must induce a strong HIV-specific and polyfunctional Th1 CD4 + cell response capable of secreting high levels of IL-2 and IFN-c and activating CD8 + T cells.However, a caveat to such a response is the potential for increased HIV-1 infectivity due to proliferation of CCR5expressing CD4 + T cells.O'Brien et al. (2009) found no increase in Ad5-specific CD4 + T-cell activation among STEP trial vacinee recipients, though other research (Benlahrech et al., 2009) has shown that vaccination of human sera with an Ad5 vector results in expansion of CCR5 + Ad5-specific CD4 + T cells that have a mucosalhoming phenotype, which are especially susceptible to HIV-1 infection.Future research should determine if it is possible to minimize activation of vector-specific CCR5-expressing CD4 + T cells, and if it were possible, these vectors would be attractive candidates for use in HIV-1 vaccine trials.Another important target for HIV-1 vaccine design is the induction of a cytotoxic virus-specific NK-cells response, which can be achieved by enhancing the binding of inhibitory KIRs to HLA class I molecules.A robust NK response would be important for connecting innate immunity to the CMI component of HIV-1 vaccine candidates.
Producing an HIV-1 vaccine that stimulates virus-specific NAbs is difficult because of the sequence diversity of the env gene and the N-glycosylations that hide many of its immunogenic and conserved regions (Baker et al., 2009).
Nevertheless, an important component of vaccines is to stimulate a long-lasting humoral response.Immunodominant peptides of the env gene that are recognized by LTNP NAbs are potential vaccine targets as long as they have been strongly correlated with lowering viral load and are capable of broad cross-neutralizing activity.
Immunogens targeted by elite neutralizers and efforts to identify broadly neutralizing mAbs capable of protection against HIV-1 infection are a valuable asset to HIV-1 vaccine development.Additionally, recent studies have discovered broad and potent neutralizing antibodies in separate donors with broad NAb responses to HIV-1 (Burton & Weiss, 2010).These broadly acting antibodies are ideal targets for vaccine design because when given in high doses they could potentially provide sterilizing immunity.However, developing an HIV-1 vaccine capable of sterilizing immunity has proven unsuccessful in the past and continued effort should be placed on inducing a strong CMI response with minimal pre-existing immunity against the recombinant vector and that prevents clinical disease progression.

Fig. 2 .
Fig. 2. Overview of the viral, genetic and immunological factors associated with longterm control of HIV-1 infection.

Table 1 .
Definitions of major controller populations IP: 54.70.40.11On: Thu, 25 Oct 2018 14:59:29 (Dolan et al., 2007;onsidered the most potent agonist of CCR5 and African populations are found with greater copy numbers of the gene compared to Caucasian populations(Dolan et al., 2007; Gonzalez  et al., 2005).CCL3L1 gene copy number has been shown to inversely correlate with the number of CD4 + T cells expressing thePiacentini et al., 2009)L3L1 levels are associated with a higher risk for HIV-1 R5-tropic virus infection, increased viral loads and accelerated disease progression, while high CCL3L1 copy numbers correlate with a slower HIV-1 disease progression(Gonzalez et al.,  2005;Piacentini et al., 2009).High CCL3L1 copy numbers are shown to play a greater role than CCR5 genotype in determining the rate of HIV-1 disease progression in a study of 57 global cohorts (Gonzalez