Pathogenicity and immunogenicity of attenuated, nef-deleted HIV-1 strains in vivo

In efforts to develop an effective vaccine, sterilizing immunity to primate lentiviruses has only been achieved by the use of live attenuated viruses carrying major deletions in nef and other accessory genes. Although live attenuated HIV vaccines are unlikely to be developed due to a myriad of safety concerns, opportunities exist to better understand the correlates of immune protection against HIV infection by studying rare cohorts of long-term survivors infected with attenuated, nef-deleted HIV strains such as the Sydney blood bank cohort (SBBC). Here, we review studies of viral evolution, pathogenicity, and immune responses to HIV infection in SBBC members. The studies show that potent, broadly neutralizing anti-HIV antibodies and robust CD8+ T-cell responses to HIV infection were not necessary for long-term control of HIV infection in a subset of SBBC members, and were not sufficient to prevent HIV sequence evolution, augmentation of pathogenicity and eventual progression of HIV infection in another subset. However, a persistent T-helper proliferative response to HIV p24 antigen was associated with long-term control of infection. Together, these results underscore the importance of the host in the eventual outcome of infection. Thus, whilst generating an effective antibody and CD8+ T-cell response are an essential component of vaccines aimed at preventing primary HIV infection, T-helper responses may be important in the generation of an effective therapeutic vaccine aimed at blunting chronic HIV infection.


Introduction
Despite considerable effort, all attempts to develop an effective human immunodeficiency virus (HIV) vaccine based on subunit or prime-boost strategies have failed to elicit sterilizing immunity and protect against infection with wild type virus (reviewed in [1][2][3]). Current World Health Organization estimates indicate 42 million people are infected with HIV and approximately 20 million have died from AIDS. Approximately 5 million new infections occur annually. The overwhelming majority of these individuals live in developing countries with little or no access to potentially lifesaving antiretroviral therapies. Moreover, HIV is predicted to become the leading burden of disease in middle and low income countries by 2015 [4]. Thus, the need for an effective preventative and/or therapeutic HIV vaccine has never been more urgent.
Since the discovery of HIV nearly 25 years ago, there have been significant advances in our knowledge of HIV immunology (reviewed in [5][6][7]). As early as 1990 subunit vaccines based on the HIV envelope protein were developed, based on the observation that vaccinated chimpanzees were protected against homologous HIV challenge [8]. However, it is unlikely that such vaccines will ever be able to illicit immune responses sufficient for protection against heterologous HIV strains and, in fact, these approaches have failed repeatedly in animal models. In addition, HIV envelope protein-based vaccines were not efficacious in 2 phase III vaccine trials in humans [9][10][11][12]. More sophisticated vaccine approaches have targeted cellular immunity by the development of DNA prime-boost strategies, and have achieved strong stimulation of antibody and cytotoxic T-lymphocyte (CTL) responses in monkeys. However, despite robust immunological responses, these strategies have ultimately failed to protect against challenge infection. A better understanding of the correlates of immune protection against HIV infection would greatly assist efforts to develop an effective HIV vaccine [13,14].
In addition to envelope and DNA prime-boost vaccines, various other strategies have been adopted in HIV vaccine development including the use of recombinant viral and bacterial vectors, synthetic peptides, live attenuated virus, and whole inactivated HIV particles. These strategies have been reviewed in detail recently [1][2][3]15], and are summarized in Figure 1. Other innovative vaccine strategies that have been recently explored include the use of peptideloaded dendritic cells [16], and non-infectious viral particles surface-engineered to produce antigen presenting particles that mimic antigen presenting cells [17] to induce cellular immune responses. To date, sterilizing immunity to primate lentiviruses has only been achieved by the use of live attenuated simian immunodeficiency virus (SIV) and chimeric simian-HIV (SHIV) vaccines carrying major deletions in the nef gene and other accessory genes [18][19][20][21]. Passive infusion of broadly-neutralizing monoclonal antibodies in HIV animal models have also been shown to confer complete protection against challenge infection [22][23][24][25]. This provides proof of principle that protection against infection is possible with use of the appropriate antigen. However, nef-deleted virus is unlikely to be considered safe enough for use as a HIV vaccine, either because immunization may pose an immediate risk to individuals with weak immune systems, or because the attenuated vaccine strain could eventually evolve to a more pathogenic form [14]. Both of these outcomes have been demonstrated in macaque studies, whereby some animals vaccinated with nef-deleted SIV progressed to AIDS in the absence of wild type virus challenge infection [26,27]. Moreover, some individuals infected with nefdeleted HIV strains eventually experience CD4+ T-cell loss after many years of asymptomatic infection [28][29][30][31]. Nonetheless, studies of long-term survivors (LTS) naturally "vaccinated" with nef-deleted HIV, such as the Sydney blood bank cohort (SBBC) [32] and other rare cohorts [33-37], may provide unique insights into protective antibody and CTL responses, which may assist HIV vaccine development [14].

Epidemiology and Clinical History of the Sydney blood bank cohort
The SBBC consists of 8 individuals (subjects C98, C54, C49, C64, C18, C135, C83 and C124) who became Various approaches for HIV vaccine development Figure 1 Various approaches for HIV vaccine development. The various approaches used in past and present HIV vaccine strategies that are summarized here have been described in detail previously [1][2][3]15].
infected with an attenuated strain of HIV via contaminated blood products from a common blood donor (subject D36) between 1981 and 1984 [30,32,38]. The SBBC blood transfusion recipients have been referred to as recipients 7, 13, 12, 9, 10, 4, 8, and 5, respectively, in one previous study [30] and subjects A (C18), B (C64), C (C98), D (C54), E (C49) and F (C83) in an earlier publication [38]. Viral attenuation has been attributed to gross deletions in the nef/long terminal repeat (LTR) region of the HIV genome [32]. The clinical history and laboratory studies of the subjects from the first identification as SBBC members through 1998 has been described previously [30], and a detailed update of the clinical and laboratory data through 2006 has been described recently [39]. Briefly, despite being infected from a single source, SBBC members now comprise slow progressors (SP) who have eventually experienced decline in CD4 T-cells after many years of asymptomatic infection (subjects D36, C98, C54), and "elite" long-term nonprogressors (LTNP) who have had stable CD4 T-cell counts and low or below detectable plasma HIV RNA levels for more than 20 years without antiretroviral therapy (ART) and remain asymptomatic (subjects C49, C64, C135) [28,30,31]. Five SBBC members have died of causes either unrelated to-or not directly related to HIV infection (C98, C54, C18, C83, C124) ( Table 1). The SBBC therefore provides a unique opportunity to study the pathogenesis of, and immune responses to nef-deleted HIV infection in a naturally occurring human setting.

HIV isolates and viral phenotypes
To determine whether changes in viral phenotype were occurring in SBBC members, HIV isolation was attempted from peripheral blood mononuclear cells (PBMC) collected longitudinally from all subjects except C124 and C83 [28,40]   When compared with wild type HIV isolates and isogenic controls with mutations in nef, replication capacity of SBBC isolates in PHA-activated PBMC was found to be consistent over time by viruses isolated from a particular subject, but heterogenous between subjects and fell into 3 distinct phenotypes [28,40] (  [61]), and loss of these cells ultimately leads to acquired immunodeficiency. Therefore, this result is consistent with the hypothesis that nef-deleted HIV has reduced pathogenicity in vivo.
Nevertheless, in the SBBC subjects studied with detectable plasma viral load, C54 and C98, there was concomitant elevation of CD8+ T cell activation, whereas the SBBC subjects with undetectable plasma viral load, C49, C64 and C135 had normal levels of CD8+ T cell activation [56]. Therefore, within the SBBC, the situation was similar to the strong correlation seen between plasma viral load and CD8+ T-cell activation in subjects infected with wild type HIV [62]. Furthermore, as described above, subjects D36, C98 and C54 exhibited a clear CD4+ T cell decline, albeit at a relatively slow rate [28][29][30]. This interesting finding argues that pathogenicity within the SBBC was more closely correlated with levels of viral replication (as assessed by plasma viral load) and CD8+ T-cell activation than with viral pathogenicity dictated by the presence or absence of nef. This finding likely represents the ability of host factors to modulate the pathogenicity of nef-deleted HIV-1 [63,64]. CD8+ T cell activation may reflect lymphocyte turnover during HIV infection, which has been proposed to lead to disruption of normal homeostasis and eventual consumption of both memory and naïve CD4+ T cells [65,66]. However, we did not find evidence of dramatically increased CD4+ T cell turnover in these subjects, as determined by expression of Ki-67 as a marker of cell proliferation [56].

Evolution of nef/LTR sequence
To determine whether an evolving nef/LTR sequence contributed to HIV progression in D36 and C98, we undertook a detailed longitudinal analysis of nef and LTR sequence changes occurring in D36, C98, C49, C54 and C64 over a 4 to 10 year period [29]. Sequential analysis of nef/LTR demonstrated a gradual loss of nef sequence that differed in magnitude between subjects. A large deletion of 128 bp emerged in D36 effectively removing the entire nef gene with the exception of the region surrounding the nef start codon, the polypurine tract which contains terminal signals for HIV integration, and a 90 bp region of the nef/LTR overlap region surrounding the negative regulatory element. The pattern of nef/LTR sequence loss in C98 was remarkably similar to that of D36. The pattern of nef /LTR sequence loss in C54 was also similar, but less extensive than that observed in D36 and C98. However, the additional loss of nef/LTR sequence in C64 was comparatively minimal. These data are illustrated in Figure 2, where the nef/LTR sequences cloned from the earliest available and most recent blood samples of these subjects are compared. A more detailed longitudinal analysis of nef/LTR sequences in these subjects has been reported in Churchill et al [29]. Thus, viruses harboured by D36, C54, C98 and C64 appeared to be evolving in a convergent fashion toward a highly deleted, minimal nef/LTR structure containing only sequence elements that are absolutely essential for HIV replication [29]. The convergent nature of the nef/LTR sequence changes implies the presence of strong selection pressures that maintains the ability of defective HIV genomes to persist in vivo. The highly evolved nef/LTR sequences harboured by D36, C54 and C98 were strikingly similar to those that remained dominant in C49 for at least 10 years (Fig. 2) [29]. Thus, the highly evolved nef/LTR structure appears to be stable, and in the case of C49 does not increase pathogenicity. However, taken together the results suggest the in vivo pathogenicity of nef-deleted HIV harboured by SBBC members is dictated by factors other than those that impose a unidirectional selection pressure on the nef/LTR region of the HIV genome. Due to the changes in the nef/LTR region, these other presumably host factors become more important in terms of disease outcome. This is exemplified by the marked variation from no disease progression with no detectable virus replication (C49 and C64), to no progression with a low viral load (C54), through to slow progression (D36 and C98).

Convergent evolution of SBBC nef/LTR sequences
Reversion to pathogenicity by nef-deleted SIV has been associated with restoration of a truncated Nef protein [26], acquisition of further deletions in the nef/LTR overlap region [67], and/or duplications of NF-κB binding sites in the LTR [67]. In contrast to the SIV studies, the in vivo evolution of nef-deleted HIV in SBBC members was unidirectional toward a smaller nef/LTR sequence and the majority of the additional sequence loss was within the nef-alone region [29]. Furthermore, none of the clones were capable of encoding Nef. Together, these results suggest improved viral replication by further deleting remnants of the nef gene. In addition, the presence of duplicated NF-κB binding sites in the LTR was not associated with clinical status of the SBBC subjects. Therefore, it is likely that viral factors that modulate the in vivo pathogenicity of nef-deleted HIV will be distinct from those in nef-deleted SIV. Interestingly, the unidirectional evolution toward the minimal nef/LTR sequence observed in SBBC members was strikingly similar to the pattern of evolution in a slow progressor infected with a nef/LTR-deleted variant of HIV circulating recombinant form 01_AE [34]. The convergent pattern of nef/LTR evolution among viruses harboured by SBBC members is therefore unlikely to be due to a unique property of the infecting strain, but rather a positive selection that is common across clades.

Changes in transcriptional activity
Viruses harbored by SBBC members contain unique alterations of NF-κB and Sp-1 binding sites in the LTR that may affect transcriptional activity and thus, replication capacity [28,29,32]. Therefore, we examined the nucleotide sequence and transcriptional activity of nef/LTR clones obtained sequentially from D36 blood samples and from D36 CSF obtained during HIVD, to determine whether changes in LTR activity may contribute to neuropathogen-esis of nef-deleted HIV-1 infection [68]. We found that the transcriptional activity of CSF-derived nef/LTR clones was up to 4.5-fold higher than blood-derived clones isolated before and during HIVD when tested under basal, PMAand Tat-activated conditions. The presence of duplicated NF-κB and Sp-1 binding sites or a truncated nef sequence in blood-derived nef/LTRs was not sufficient to mediate large increases in transcriptional activity. However, CSFderived nef/LTRs had duplicated NF-κB and Sp-1 binding sites coupled with a truncated nef sequence, which formed a regulatory unit that significantly enhanced LTR transcription [68].
Previous studies showed that LTR variants with augmented transcriptional activity enhance replication of HIV [69]. Therefore, to determine whether D36 nef/LTRs affect replication capacity of HIV in vitro, we produced and characterized full-length chimeric molecular clones of HIV NL4-3 carrying the nef/LTR nucleotide sequence of blood-derived D36 nef/LTRs or the CSF-derived D36 nef/ LTR [68]. We examined the capacity of chimeric NL4-3 viruses carrying D36 nef/LTRs to replicate in PBMC compared with wild type NL4-3 and the NL4-3∆Nef deletion mutant [70]. Compared to wild type NL4-3, chimeric HIV containing the nef/LTR sequence of blood derived D36 viruses had attenuated replication kinetics, similar to NL4-3∆Nef. In contrast, chimeric HIV containing the nef/ LTR of D36 CSF had enhanced replication capacity compared to wild type NL4-3. Thus, the nef/LTR derived from CSF of D36, which had augmented basal, PMA-and Tatactivated transcriptional activity compared to wild type and blood-derived D36 nef/LTRs, augmented replication of HIV in PBMC. Together, our results suggest unique features of the CSF-derived nef/LTR restore efficient replication capacity of nef-deleted HIV in PBMC by enhancing transcription. The results further suggest that nef and LTR mutations that augment transcription may contribute to neuropathogenesis of nef-deleted HIV.

Attenuation in other HIV genes
In addition to nef and LTR, mutations or polymorphisms in other HIV genes including gag, rev, tat, vif, vpr, vpu and env have been detected in SP or LTNP [71][72][73][74][75][76][77][78]. A previous study of HIV rev alleles isolated from a subject with longterm nonprogressive HIV infection showed a persistent Leu to Ile change at position 78 in the Rev activation domain which attenuated Rev function and HIV replication capacity [73], providing evidence that defective rev alleles may contribute to long-term survival of HIV infection in some patients. A subsequent study of naturally occurring rev alleles with rare sequence variations in the activation domain showed variable reductions in Rev activity [79], although it was unclear from this study whether the observed reductions in Rev activity would be sufficient to attenuate HIV replication capacity. Of note, CTL escape mutations in the second coding exon of Tat have been shown to attenuate virus in vivo, suggesting that in vivo sequence changes in other regulatory HIV-1 genes may potentially affect HIV-1 pathogenesis [80]. Since differences in HIV pathogenicity in SBBC members could not be fully explained by alterations in the nef/LTR region [29] or Env phenotype [40,50], we characterized dominant HIV-1 rev alleles that persisted in SBBC LTNP (C18, C64) and SP (C98, D36) [81]. We found that the ability of Rev derived from D36 and C64 to bind the Rev responsive element (RRE) in RNA binding assays was reduced by approximately 90% compared to Rev derived from NL4-3, C18 or C98. D36 Rev also had a 50-60% reduction in ability to express Rev-dependent reporter constructs in mammalian cells. In contrast, C64 Rev had only marginally decreased Rev function despite attenuated RRE binding. In D36 and C64, we found that attenuated RRE binding was associated with rare amino acid changes at 3 highly conserved residues; Gln to Pro at position 74 immediately N-terminal to the Rev activation domain, and Val to Leu and Ser to Pro at positions 104 and 106 at the Rev C-terminus, respectively. In D36, reduced Rev function and altered replication capacity was mapped to an unusual 13 amino acid extension at the Rev C-terminus. However, database analysis of rev sequence demonstrated that the presence of one or more of these rare amino acid changes was not able to discriminate between subjects with progressive or non-progressive HIV-1 infection. Moreover, none of these amino acid changes occurred in a previously identified LTNP with defective rev alleles [73]. Thus, our studies suggest the contribution of any or all of these mutations to decreased RRE binding and/or attenuated Rev function by SBBC Revs, and possibly to slow or absent HIV progression, is likely to be context dependent.
It is presently unclear whether attenuated D36 Rev function in vitro equates to attenuated Rev function in vivo, and indeed whether attenuated Rev function contributed to slow progression of HIV infection in this subject. Extrapolation of these in vitro findings to an in vivo role for attenuated D36 rev alleles is difficult, since this subject and other SBBC members are infected with virus containing gross nef/LTR deletions which have been shown to cause significant viral attenuation in this cohort [28,29,32]. Nonetheless, our findings raise the possibility that attenuated Rev function may contribute, at least in part, to viral attenuation and slow HIV progression in D36.

Anti-HIV Ig responses
To better understand the humoral immune response to nef-deleted HIV infection, we measured the total IgG responses in longitudinal plasma samples of SBBC members by Western blotting, and compared these with total IgG responses in a control group of LTNP with intact nef genes [40]. We found a good correlation between total IgG responses in SBBC members and a detectable plasma VL, with plasma from C18, C54, D36 and C98 all being strongly reactive. Subjects C49 and C64, who consistently maintained undetectable HIV RNA copy numbers [29,30], had significantly reduced total IgG responses. Furthermore, subject C135, who has also had consistently undetectable HIV RNA levels has not fully seroconverted after more than 20 years of infection [40,42]. These studies highlight the importance of adequate antigenic stimulation by nef-deleted HIV to drive antibody production. In contrast, we found that total IgG responses in the control group were uniformly potent, reflecting the fact that all these individuals had detectable VLs. Among SBBC members, the strongest antibody responses were observed in individuals with low but detectable VL set points, less than approximately 10,000 RNA copies/ml. This is consistent with a recent observation of an undetectable VL and weak, delayed antibody responses in an unrelated individual with nef-deleted HIV [34].
Recent studies have highlighted the change in HIV antibody responses with respect to antibody isotype switching after initial infection, in particular IgG 3 reactivity to p24 [82,83]. Preliminary WB testing of p24 IgG 3 responses in the SBBC LTNP indicated there was reactivity for those individuals with the most potent total IgG responses (C18 and C54) (E. Verity, D. McPhee, K. Wilson, and D. Zotos, unpublished data). This hints at delayed isotype switching and hence delayed maturation of immune responses for at least some members of the SBBC.

Neutralization studies of nef-deleted HIV
In additional studies, we determined whether plasma from SBBC members had differences in ability to neutralize nef-deleted HIV strains. We did this by comparing the ability of longitudinally collected plasma from SBBC members or from control LTNP cohort members to neutralize the infectivity of HIV isolated from D36 and C18 [40]. We found that, for SBBC plasmas, neutralization of D36 or C18 viruses strongly correlated with VL, replication capacity of the isolated virus, and the strength of anti-HIV IgG responses. Plasma from SBBC members with undetectable VL was unable to neutralize the infectivity of these viruses. In contrast, plasmas from the control LTNP cohort were able to neutralize the infectivity of SBBC viruses with titres generally higher than that seen for SBBC members, but there was no correlation between neutralization and HIV RNA copy number or IgG responses in the control LTNP group.

Broad neutralizing antibody responses
A number of studies have suggested an increased frequency of LTNP and long term survivors (LTS) possess strong, cross-reactive neutralising antibody responses [84][85][86]. However, very few studies have investigated antibody responses in LTNP/LTS infected with nef-deleted HIV, despite a number of studies showing strong neutralising antibody responses in macaques infected with nef-deleted SIV [87][88][89][90][91][92]. However, studies by Greenough et al., [93] showed measurable neutralising antibody responses concurrent with a detectable VL for a single individual infected with nef-defective HIV. We therefore determined the breadth of neutralizing antibodies against HIV for SBBC members.
We found that plasma from C18, and to a lesser extent D36, C54 and C98 were able to potently neutralize the infectivity of a number of laboratory adapted and primary HIV strains, but little or no neutralizing activity was evident in plasma from C49, C64 and C135 In contrast, longitudinal analysis of HIV-specific CD4+ Tcells showed that all SBBC nonprogressors able to completely control plasma HIV RNA levels to below detectable levels (C49, C64 and C135) had persistent and strong Thelper proliferative responses to HIV p24 antigen, whereas these responses were absent in all progressors with persistent viremia (C54, C98 and D36) (Fig. 3). The notable exception was C18 who had detectable but low plasma HIV RNA levels without evidence of CD4+ T-cell loss, and had detectable p24 T-helper responses over a short period of approximately 12 months leading up to his non-HIV related death at 83 years of age. In this subject, the p24 T-helper responses coincided with an exponential increase in CTL responses against Pol antigens, measured by memory CTL precursor frequency assay [98], and IFN-gamma ELISPOT responses (W. Dyer, unpublished data). However, it is difficult to interpret the significance of the T-cell responses in C18 given the short window of analysis. Together, although derived from a small cohort of individuals, these results suggest that immune suppression of nef-deleted HIV-1 by SBBC LTNP may be dependent upon persistent T-helper responses irrespective of the CD8+ T-cell and neutralizing antibody response to viral antigens.

Conclusion
The development of an effective HIV vaccine has been hampered by the lack of defined correlates of immune protection against HIV infection. Although nef-deleted strains of HIV are not suitable as live attenuated HIV vaccines due to safety concerns, the only lentiviral vaccines to date that have generated sterilizing immunity in animals are those based on live, attenuated viruses. To this end, studies of SBBC members naturally "vaccinated" with nef-deleted HIV may provide unique insights into protective immune responses to HIV infection, which may assist the development of an effective HIV vaccine. As a consortium, we have been studying nef-deleted HIV infection in the SBBC for a number of years, linking changes in pathogenicity in vivo with viral evolution and immune responses to infection. The major aim of this review was to summarize our recently published and unpublished studies on the SBBC. Together, the studies show that potent, broadly neutralizing anti-HIV antibodies and robust CD8+ T-cell responses to HIV infection were not necessary for long-term control of HIV infection in a subset of SBBC members, and were not sufficient to prevent HIV sequence evolution, augmentation of pathogenicity and eventual progression of HIV infection in another subset. However, a strong T-helper proliferative response to HIV p24 antigen was associated with long-term control of infection. Variation in the outcome of HIV infection in this cohort appears to be strongly host-dependent, consistent with other studies with wild-type HIV. Dependence upon the host's unique immune environment also appears to be important in contributing to control of infection. This further complicates development of a successful vaccine. We hope that results gleaned thus far from studies of this unique cohort of individuals will provide HIV vaccine researchers with novel insights into immune mechanisms that may serve to prevent or control HIV infection. Figure 3 Longitudinal analysis of T-helper proliferative responses to HIV p24 antigen in SBBC progressors and nonprogressors. T-helper proliferative responses to HIV p24 antigen were determined by [ 3 H]-thymidine incorporation, as described previously [99,100]. T-helper responses were persistently detectable (stimulation index > 3) in all nonprogressing subjects with below detectable plasma HIV RNA levels (C135, C64, and C49), but absent in all progressors (D36, C98, and C54). Of note, T-helper responses were detectable over a short window in C18, who was a nonprogressing individual with detectable plasma HIV RNA levels.