The impact of HIV-1 Nef on CD4+ T cells

The role of thiamine in HIV infection

(See the major article by Abbas et al on pages 224–34.) The development of, and globlal access to, effective antiretroviral medications revolutionized human immunodeficiency virus type 1 (HIV-1) care. In addition to their important life-saving treatment benefits, antiretrovirals have recently been demonstrated to be highly efficacious for HIV-1 prevention as well, when used as antiretroviral therapy (ART) to reduce the infectiousness of HIV-1–infected persons and pre-exposure prophylaxis (PrEP) for uninfected persons who have ongoing HIV-1 exposure. Antiretroviral-based prevention, both ART and PrEP, are among the most promising strategies for reducing the number of new HIV-1 infections globally. Consequently, policymakers are weighing the costs, benefits, and risks of public health implementation of ART and PrEP for HIV-1 prevention. One potential risk of both ART and PrEP is the selection and transmission of HIV-1 variants that are resistant to one or more antiretroviral medications, which can result in HIV-1 treatment failure with associated morbidity and mortality and increased costs (of more complex second- and third-line treatment regimens); thus, there has been considerable speculation about the potential risks of resistance from both ART and PrEP. In this issue of the Journal of Infectious Diseases, Abbas et al present a mathematical model to estimate the number of HIV-1 infections averted and the number of acquired and transmitted HIV-1 cases of resistance in a setting similar to South Africa and under several scenarios about coverage of ART and PrEP [1]. For PrEP, the authors assumed use of combination emtricitabine-tenofovir, for which efficacy has been demonstrated [2–4]. For ART, the authors modeled first-line regimens containing the same antiretrovirals and assumed that second-line drugs were not available, given limited availability of second-line medications in many resource-limited settings. A number of additional scenarios were analyzed, including having ART initiation at CD4 lymphocyte cell counts at either <200 or <350 cells/µL, reflecting evolving international guidelines on clinical benefits of earlier ART initiation, and allowing for “inappropriate” PrEP use by persons that are already infected with HIV-1, either through PrEP initiation occurring during unrecognized seronegative acute HIV-1 infection or PrEP initiation by persons with undocumented, chronic HIV-1 infection, which could occur if HIV-1 testing is not conducted prior to initiation or through “black market” availability of PrEP. The authors used optimistic scenarios for ART retention and PrEP effectiveness; notably, the model assumed general distribution of PrEP rather than risk-targeted delivery. Not surprisingly, the results of this mathematical modeling article underscore that population-level coverage and effectiveness (which is dependent on adherence) are the main determinants of the number of infections averted with both ART and PrEP, and that implementation of a combination of ART and PrEP prevents more infections in a population than a program that delivers exclusively either ART or PrEP. More interestingly, the model analysis also suggests that HIV-1 drug resistance in a population would be largely driven by ART, not PrEP, in all scenarios modeled, as a result of insufficient ART adherence or lack of viral load monitoring in ART programs, leading to selection of resistant variants during incomplete viral suppression. The model also finds that the population prevalence of resistance as a direct result of PrEP may be very low and that the greatest resistance risks related to PrEP would be from inappropriate use by persons already HIV-1 infected, rather than from PrEP being prescribed for HIV-1 prevention, even anticipating inadvertent prescribing for persons with unrecognized acute HIV-1 infection. What do these findings mean for potential implementation of PrEP for HIV-1 prevention? First, these results directly address the often-voiced concern that PrEP will lead to substantial HIV resistance in populations. Instead, because high PrEP adherence prevents most HIV-1 infections, it is unlikely to select for resistant variants, although low PrEP adherence does not prevent infection; the model presented by Abbas et al suggests that substantial population-level resistance is unlikely. Moreover, even with optimistic assumptions about ART continuation rates, the amount of resistance generated from ART failure greatly exceeds the resistance selected by PrEP. Indeed, emerging evidence from Africa has demonstrated increasing resistance accompanying ART roll-out over the past decade [5–7]. Nevertheless, if persons on PrEP alternated between periods of good and poor adherence in patterns that increased the risk of them becoming HIV-1 infected and then taking PrEP, the risk of resistance could be greater than predicted in this model. Implementation of PrEP will require ongoing complementary strategies to ensure high quality HIV-1 testing, reduce HIV-1 risk, and maximize PrEP-taking. A second message from the Abbas et al model is the importance of HIV-1 testing before PrEP initiation to avoid inadvertent exposure for an HIV-1–infected person to what is effectively suboptimal mono- or dual-agent ART. The model assumed that 2.5% of persons with undiagnosed chronic HIV-1 infection would initiate PrEP each year, which is arguably very high. Nevertheless, the model alerts us to the importance of strategies to monitor quality HIV-1 testing and PrEP pharmacovigilance during this period where PrEP is moving from efficacy trials to implementation. Third, a highly intuitive finding of the model is that less drug resistance could result if ART and PrEP regimens were used that did not include the same antiretroviral agents. The completed, first-generation PrEP trials used tenofovir, alone or in combination with emtricitabine, resting on the substantial body of clinical safety and experience with these agents for testing PrEP as a novel HIV-1 prevention strategy. New PrEP agents are in development, but their use would not be routine for several years. While, hypothetically, it is preferable to utilize PrEP regimens that do not overlap with antiretrovirals used for treatment, there is also a cost of inaction—missing the opportunity to prevent new HIV-1 infections with demonstrated effective tenofovir-based PrEP while waiting for new safe and effective PrEP regimens to be identified. While providing some new insights, there are also limitations to the Abbas et al model. For ART, the key benefit that was not included in this model was its health impact in terms of saving lives. As the primary benefit of ART is to prolong life, and the primary problem of resistance is the loss of efficacy of ART, this is an important gap in the model. For PrEP, a critical operational factor for maximizing impact in terms of infections averted will be “prioritization,” in which age, gender, and risk behaviors are incorporated into risk assessments for potential PrEP users to maximize the likelihood that PrEP is provided to those most at risk of HIV-1 infection. To optimally use resources for PrEP, programs will need to prioritize those who are at highest risk of HIV-1 acquisition and are motivated to take PrEP. Whereas the authors of the present model used a coverage level of 30% of the general population and included those that did not adhere to PrEP well, other models have suggested that if PrEP delivery programs can target delivery to those at greater HIV-1 risk and achieve higher adherence in a prioritized population, by reducing the total number of new HIV-1 infections, PrEP could even reduce the prevalence of drug resistance [8]. Thus, the complex mathematical model developed by Abbas et al helps identify some of the next steps for mathematical models and needs for empiric data to clarify policy considerations and implementation priorities for antiretroviral-based HIV-1 prevention through ART and PrEP. For both PrEP and ART for HIV-1 prevention, adherence is key to effectiveness. For ART, the result is adherence over a lifetime, or until a cure is available. Expanding implementation of ART for HIV-1 prevention will include persons initiating at higher CD4 lymphocyte counts and earlier in their disease course before they have experienced symptoms, and they may face heightened adherence challenges. PrEP adherence has different challenges than ART, namely requiring persons without HIV-1 to perceive their own risk sufficiently to initiate and adhere to PrEP. Thus, PrEP needs to be delivered in a different model than ART, as it is not a commitment to life-long medications, but specially directed to individuals during life periods of highest risk. While much can be learned from the Abbas et al model about the potential for generation and spread of HIV-1 antiretroviral resistance related to ART and PrEP, equally important is what this model can teach us about the public health impact of these prevention strategies. The authors have moved the discussions about PrEP forward from modeling simply the number of drug resistance cases with their public health perspective in which they present the ratio of cumulative HIV-1 infections averted to prevalent HIV-1 drug resistance, which puts the deleterious effect of drug resistance into context with the benefits of HIV-1 infection prevention. This model clearly demonstrates that both ART and PrEP, particularly when rolled out together, offer the potential for substantial HIV-1 prevention. Recognizing the potential risks of PrEP and ART, including antiretroviral resistance, is critical for developing mitigating strategies, because the potential benefits of these new prevention strategies are substantial and there is real public health risk in not implementing tools that we know work.

Combination antiretroviral therapy suppresses but does not eradicate human immunodeficiency virus type 1 (HIV-1) in infected persons, and low-level viremia can be detected despite years of suppressive antiretroviral therapy. Short-course (28-day) intensification of standard antiretroviral combination therapy is a useful approach to determine whether complete rounds of HIV-1 replication in rapidly cycling cells contribute to persistent viremia. We investigated whether intensification with the integrase inhibitor raltegravir decreases plasma HIV-1 RNA levels in patients receiving suppressive antiretroviral therapy. Subjects (n = 10) with long-term HIV-1 suppression receiving combination antiretroviral regimens had their regimens intensified for 4 weeks with raltegravir. Plasma HIV-1 RNA level was determined before, during, and after the 4-week intensification period, using a sensitive assay (limit of detection, 0.2 copies of HIV-1 RNA/mL of plasma). A 4-week intensification course was chosen to investigate potential HIV-1 replication in cells with relatively short (approximately 1-14-day) half-lives. There was no evidence in any subject of a decline in HIV-1 RNA level during the period of raltegravir intensification or of rebound after discontinuation. Median levels of HIV-1 RNA before (0.17 log10 copies/mL), during (0.04 log10 copies/mL), and after (0.04 log10 copies/mL) raltegravir intensification were not significantly different (P > .1 for all comparisons in parametric analyses). High-performance liquid chromatography and mass spectroscopy experiments confirmed that therapeutic levels of raltegravir were achieved in plasma during intensification. Intensification of antiretroviral therapy with a potent HIV-1 integrase inhibitor did not decrease persistent viremia in subjects receiving suppressive regimens, indicating that rapidly cycling cells infected with HIV-1 were not present. Eradication of HIV-1 from infected persons will require new therapeutic approaches. ClinicalTrials.gov identifier: NCT00618371.

In June this year, it was 30 years since the identification of the first AIDS patient (see the review in this issue 1). Despite rapid responses by scientists and doctors to understand this disease in both clinical and experimental systems 2, 3, human immunodeficiency virus type 1 (HIV-1), the causative agent of AIDS (Fig. 1), continues to feature among world's three major killers destroying millions of lives, families and communities. More than 30 drugs have been developed just for HIV-1 and there have been three successful trials showing their impressive preventive potential. However, because of the drug unavailability, particularly in resource poor settings, side effects and potential development of resistance, the best hope for a profound fall in the incidence of HIV-1 infection remains the development of an effective prophylactic HIV-1 vaccine. Here, we discuss T-cell vaccine designs mainly, briefly mentioning antibody vaccines. HIV © PhotoDisc, Inc. Even if a vaccine that actively stimulates broadly neutralizing antibodies (bNAbs) can be made 4, it will be hard to stop some HIV-1 infection occurring (e.g. through cell–cell transmission) and T-cell-mediated immune responses to control infection will be required. T cells function by killing HIV-1-infected cells and producing soluble factors that can directly and indirectly control HIV-1 spread. While T cells cannot prevent the transmitted virus from infecting host cells, potent vaccine-induced HIV-1-specific T-cell responses could increase the dose of incoming virus necessary to establish infection (i.e. decrease acquisition) 5, limit the extent of viral replication during primary viremia (i.e. reduce tissue damage), lower the virus load at set point (i.e. reduce further virus transmission) and slow the rate of CD4+ T-cell decline (i.e. delay the development of AIDS). The simian immunodeficiency virus/macaque challenge model strongly supports this view, showing that potent T-cell responses alone can lower virus load and delay the development of AIDS 6-8. Thus, ideally, a successful HIV-1 vaccine will induce both T-cell and antibody responses; however, an effective T- or B-cell vaccine alone is nonetheless likely to impact the epidemic 9. Scientists developing HIV-1 vaccines face a long list of challenges. Although these differ for the induction of effective T-cell responses in comparison with induction of the desired bNAb specificity by active immunization, one major hurdle is common, namely the extreme HIV-1 variability. The main HIV-1 group has diversified into 15 major clades, subclades and interclade recombinant circulating forms, whereby individual HIV-1 variants, even within clades, may differ by up to 20% of their amino acid sequence, which is more than enough to severely impair broad T-cell and antibody recognition. CD8+ T-cell recognition of epitopes is usually highly sensitive to even a single amino acid deviation from the well-recognized sequence and this decreases T-cell recognition efficacy. Thus, a successful vaccine has to effectively recognize diverse infecting HIV-1 strains circulating in the population and then must deal with ongoing virus escape in infected individuals. Although in acute HIV-1 infection, the founding virus is usually single, the first T-cell responses tend to focus on immunodominant, but highly variable epitopes, in which mutations are selected very rapidly, escaping the early T-cell responses. NAbs develop much later in infection after the damage to the immune system is already done. HIV-1 has an enormous capacity to change. Some HIV-1 proteins such as the envelope are more variable than e.g. the internal structural proteins. On a sub-molecular level, some protein regions have to remain more-or-less constant to maintain their structural or biological functions and, therefore, even HIV-1 has its Achilles heel and this can be exploited. Focusing the vaccine-elicited responses on the functionally conserved regions of the HIV-1 proteome has a number of advantages. First, conserved regions are common to the diverse virus strains and clades to which vaccines are exposed. Second, targeting the conserved regions reduces the chance of virus escape in infected individuals. If escape mutations do occur, and some have been documented in conserved regions 10, they may often decrease virus fitness as shown e.g. for a B57-restricted epitope 11, or may require compensating mutation(s) as in the case of a B27-restricted Gag epitope 12. Therefore, escape mutations in the conserved regions may be good for patient's clinical prognosis or may be very delayed. Third, T-cell immunogens based on the functionally conserved parts of HIV-1 proteins redirect the naturally induced hierarchy of epitope responses, which is non-protective, towards invariable regions, which are arguably more likely to be protective. Finally, conserved immunogens can be designed as a simple single insert, representative of the major global clades A, B, C, and D equally. Therefore, vaccines based on the conserved regions of the HIV-1 proteome can be tested and potentially deployed in Europe, America, Asia, and Africa; they are universal. The first conserved region vaccine entered clinical evaluation in HIV-1 seronegative volunteers in Oxford, UK, and the results are expected in summer 2012. Most initial vaccine strategies focused on the breadth, i.e. the number of different epitopes of the HIV-1 proteome recognized by vaccine-induced responses, rather than the depth defined as the number of variants of the same epitopes. Therefore, early vaccines often incorporated into their formulations almost a whole set of virus proteins. The rationale behind this was that, if a multiplicity of epitopes were recognized at the same time, this would prevent HIV-1 from changing all of these epitopes at the same time and escaping immune surveillance. An example of such a single clade vaccine is MRKAd5 developed by the Merck Research Laboratories, which showed no efficacy in the first T-cell vaccine STEP trial in 2007 13, 14. When the power of the virus variability became more appreciated and respected, many vaccine designs mixed variants of the same protein derived from several different HIV-1 clades into a single formulation. One such vaccine is currently in a recently expanded phase IIb proof-of-concept trial designated the HIV Vaccine Trials Network (HVTN) protocol 505 15. More advanced T-cell-based vaccine strategies have taken full advantage of the Los Alamos National Laboratory (LANL) HIV Sequence Database, which has the most complete data set of known HIV-1 isolates. The first in silico approach that emerged computed centralized sequences 16. This approach uses either consensus (average) or centre-of-phylogenetic tree whole protein sequences or extrapolates individual amino acid positions in the whole proteins to common clade or group ancestors. This captures the intraclade variation, but is likely to be too stretched to comprehensively cover the whole main group of HIV-1 variants. The best coverage of the ‘non-conserved’ strategies computes mosaic proteins, which are artificial sequences assembled in silico using an iterative algorithm 17. Known 9-amino acid stretches were chosen because this is the most typical length of an epitope recognized by CD8+ T killer cells and by computing mosaic proteins the coverage of all common variants of these sequences is maximized. For example, a tetravalent mosaic protein of Gag optimized on the main group sequences covers about 74% of the main group Gag-derived 9-mers as a perfect match. Both computed designs described are supported by a strong rationale; nevertheless, they do not refocus the immune responses away from the dominant, hypervariable regions towards the subdominant but invariant regions of HIV-1 18, 19. This means that the induced T-cell responses, although increased in depth, are just as likely to focus on variable regions and this opens the possibility of selecting novel escape variants not yet included in the LANL database. Recent deep sequencing of natural T-cell escape mutations showed that a very large number of alternative amino acids were generated by mutation during infection and ‘tested’ in these variable epitope positions 20. In essence, perhaps the best solution to a T-cell vaccine immunogen is one that consists of conserved regions made of mosaic sequences. The first mosaic vaccine is scheduled to enter clinical evaluation in year 2012. Even the most conserved regions of the HIV-1 proteome are not immunologically inert. By inspecting the LANL HIV-1 database, it can be seen that conserved regions contain their fair share of CD8+ T-cell epitopes, which are proportional to their amino acid length. These epitopes were identified mostly in chronically infected individuals, who had mounted T-cell responses against them. Moreover, preliminary immunogenicity results from the first trials of the conserved vaccines show encouraging immunogenicity. Nevertheless, as with any approach, vaccines based on the conserved regions have their theoretical caveats. First, conserved immunogens are chimeric proteins assembled from protein sub-regions and, as such, have sequence junctions where the sub-regions meet. These junctions may create novel irrelevant epitopes (not present in HIV-1), which could, for certain HLAs, be immunodominant and suppress induction of protective responses. However, based on the likelihood of creating such immunodominant interfering junctional epitopes, these will almost certainly be the exception rather than the rule. Second, CD4+ T cells, the main natural target cells for HIV-1 replication, do not have co-stimulatory molecules on their surface and, therefore, are not potent primers of T-cell responses. Thus, in natural HIV-1 infection, many or most T-cell responses are primed either by direct infection of ‘professional’ antigen-presenting cells or through cross-priming, for instance via the uptake of HIV-1-infected apoptotic cell debris by ‘professional’ antigen-presenting cells. While it is known that most immunodominant epitopes are expressed on HIV-1-infected cells, this has not been explored in great detail for subdominant epitopes such as those derived from the HIV-1 conserved regions. Thus, it is not guaranteed that HIV-1-infected cells express conserved epitopes on their surface in sufficient amounts for effective and timely killing by cytotoxic T cells, i.e. before the infected cells produce HIV-1 progeny, which is key for the success of conserved T-cell vaccines (Fig. 2). Both of these caveats are being investigated in the on-going clinical trials of the conserved vaccines by e.g. in vitro virus suppression assays utilising vaccine-induced T-cell effectors 21. Are subdominant epitopes from conserved regions of the HIV-1 proteome presented efficiently on the surface of HIV-1-infected cells? The strategy for controlling HIV-1 by the use of conserved T-cell epitopes has been proposed on several occasions 22-24. However, an actual T-cell vaccine employing conserved regions (rather than epitopes) of HIV-1, thus preserving the natural epitope adjacent sequences and also the possibility of inducing responses to as yet unidentified epitopes, was first reported by Letourneau et al., who employed the 14 most conserved regions of the proteome as 27- to 128-amino acid-long consensus sequences alternating the four major main global clades A, B, C, and D 25. At about the same time, such an approach was theoretically proposed by Rolland et al., who suggested the use of 45 conserved elements (CEs) at least 8 amino acids long that fulfilled stringent conservation criteria 26. Since then, there has been a flurry of papers addressing and discussing various aspects of universal vaccines based on conserved regions of HIV-1, including for example: conserved region vaccines being tested in rhesus macaques and found to be highly immunogenic 27; proposal of a universal peptide vaccine based on conserved regions of HIV-1 28; recognition of conserved and variable CD8+ T-cell epitopes with similar probabilities during both primary and chronic infections, whereby the conserved epitopes generally elicited subdominant responses 29; detection of an association between responses to conserved T-cell epitopes and lower virus loads 30; skewing of vaccine-elicited T-cell responses away from more conserved epitopes to the more variable and therefore possibly less protective epitopes, which did not match the infecting viruses, as detected in the failed STEP trial 31; a novel analysis of controllers who durably control HIV-1 without medications that revealed preferential targeting of a conserved sector in Gag and concluded that targeting regions with higher order evolutionary constraints provides a novel approach to immunogen design 32; Thus, support for T-cell vaccine strategies employing conserved regions of the HIV-1 proteome is growing. Many pathogens use antigenic variability of the most immunogenic regions on their surface to avoid host antibody-based defences. Thus, antibody-inducing vaccines have a much longer tradition in focusing on conserved regions 33. Indeed, even the most variable protein, Env, of HIV-1 has invariable regions, of which the most conserved is the CD4 receptor-binding site 34. Recently, there has been tremendous progress in understanding the mechanisms underlying potent and broad HIV-1 neutralization 35, 36. The roadblock of efficiently inducing such specificity by active vaccination remains, but conserved regions are once again at the centre of attention. This article has mainly concentrated on the theoretical arguments for and against the various HIV-1 immunogen platforms currently under evaluation; it provides only limited experimental evidence because this is only just starting to emerge. Vaccine success will depend significantly, but not exclusively on immunogens; it will also be critical to factor in how these immunogens are presented to the immune system, i.e. the choice of vaccine vectors and vector combinations, adjuvantation and routes of delivery 37. Which vaccine strategy is the best can be only decided by protection of humans against HIV-1 infection and/or AIDS and this, in turn, can only be answered in efficacy trials. These are expensive, but highly informative. Moreover, the very last one, RV144 38, even provided a moderate reason for optimism. Last but not least, vaccines will not be discovered without continued financial and political support, new scientific discoveries and human will and persistence. World AIDS day (http://www.worldaidsday.org/) on 1 December offers the perfect opportunity to ensure that such issues are highlighted globally.

Human immunodeficiency virus type 1 (HIV-1) eradication is prevented by the establishment on infection of cellular HIV-1 reservoirs that are not fully characterized, especially in genital mucosal tissues (the main HIV-1 entry portal on sexual transmission). Here, we show, using penile tissues from HIV-1-infected individuals under suppressive combination antiretroviral therapy, that urethral macrophages contain integrated HIV-1 DNA, RNA, proteins and intact virions in virus-containing compartment-like structures, whereas viral components remain undetectable in urethral T cells. Moreover, urethral cells specifically release replication-competent infectious HIV-1 following reactivation with the macrophage activator lipopolysaccharide, while the T-cell activator phytohaemagglutinin is ineffective. HIV-1 urethral reservoirs localize preferentially in a subset of polarized macrophages that highly expresses the interleukin-1 receptor, CD206 and interleukin-4 receptor, but not CD163. To our knowledge, these results are the first evidence that human urethral tissue macrophages constitute a principal HIV-1 reservoir. Such findings are determinant for therapeutic strategies aimed at HIV-1 eradication.

SummaryUnderstanding the initial events in the establishment of vaginal human immunodeficiency virus type-1 (HIV-1) entry and infection has been hampered by the lack of appropriate experimental models. Here, we show in an ex vivo human organ culture system that upon contact in situ, HIV-1 rapidly penetrated both intraepithelial vaginal Langerhans and CD4+ T cells. HIV-1 entered CD4+ T cells almost exclusively by CD4 and CCR5 receptor-mediated direct fusion, without requiring passage from Langerhans cells, and overt productive infection ensued. By contrast, HIV-1 entered CD1a+ Langerhans cells primarily by endocytosis, by means of multiple receptors, and virions persisted intact within the cytoplasm for several days. Our findings shed light on the very earliest steps of mucosal HIV infection in vivo and may guide the design of effective strategies to block local transmission and prevent HIV-1 spread.

The intestinal mucosa contains many cells targeted by human immunodeficiency virus type 1 (HIV-1), and high levels of HIV-1 DNA persist in this compartment under antiretroviral therapy (ART). While CD4+ T cells are the best-characterized reservoir of HIV-1, the role of long-lived intestinal macrophages in HIV-1 persistence on ART remains controversial. We collected duodenal and colonic biopsies from 12 people with HIV (PWH) on suppressive ART, enrolled in the ARNS EP61 GALT study. Total, integrated, intact and defective HIV-1 proviruses were quantified from sorted T cells and monocytes/macrophages. HIV-1 env quasispecies were analyzed by single-molecule next-generation sequencing and env-pseudotyped viruses were constructed to assess macrophage versus T-tropism. Total HIV-1 DNA levels in intestinal T cells were significantly higher than those in monocytes/macrophages (P < .0001). Unintegrated HIV-1 DNA was detected in one-third of T-cell and monocyte/macrophage-positive samples. Intact HIV-1 proviruses were detected using the intact proviral DNA assay in 4 of 16 T-cell samples and 1 of 6 monocyte/macrophage samples with detectable HIV-1 DNA. HIV-1 sequences were compartmentalized between intestinal monocytes/macrophages and T cells in some subjects. Phenotypic analysis of pseudotyped viruses expressing HIV-1 envelopes from colonic monocytes/macrophages revealed T-tropism rather than M-tropism. Our results show that monocytes/macrophages from the intestinal mucosa of PWH on ART can contain HIV-1 DNA, including intact or unintegrated forms, but at much lower levels than those found in T cells, and with some compartmentalization, although they do not exhibit a specific macrophage tropism, raising the question of the mechanisms of infection involved.

Underquantification of plasma HIV-1 RNA levels in a cohort of newly-diagnosed individuals

Human immunodeficiency virus type 1 (HIV-1) is a chronically replicating lentivirus that must escape from adaptive immune responses that arise during the course of infection. Viral persistence is maintained by the rapid rate of HIV-1 replication and the error-prone reverse transcription of the viral genome, which produces viral variants that continually escape antibody and cytotoxic T cell responses [1]–[3]. Antibodies directed against the gp120 and gp41 components of the viral envelope glycoprotein (Env) develop within the first few weeks of infection [4],[5], but antibodies that can neutralize the infecting virus (NAbs) are usually not detected until more than 12 weeks after HIV-1 infection [6]. Thus, in natural HIV-1 infection, NAbs are not believed to play a major role in containing the acute phase of HIV-1 replication. However, several studies have shown that once NAbs arise, they exert immune selection pressure on the viral quasispecies [7]–[14]. Viral escape from autologous NAbs was first described in lentiviral infections of several animal species [15]–[17]. For example, the successive waves of viremia in horses caused by equine infectious anemia virus are thought to be due to the sequential development of viral variants that temporarily evade the host NAb response. HIV-1 escape from autologous NAbs was first described in the early 1990s [18]–[20]. Subsequently, numerous research groups showed that plasma antibodies from a time point contemporaneous with viral isolation did not neutralize the autologous virus, and that NAbs against the isolated virus developed only months later [7]–[14],[21],[22]. Thus, the NAb response continually lags behind viral replication. The initial studies of NAb escape were limited by the inefficiency of isolating replication competent HIV-1 from patient plasma or lymphocytes. The more recently performed studies used molecularly cloned Env-pseudoviruses to more robustly study the plasma viral quasispecies at sequential time points. These data confirmed that, at any given time point during the course of HIV-1 infection, the circulating quasispecies of viral variants is resistant to the circulating plasma NAb. At first glance, these findings might suggest that HIV-1 should become progressively more resistant to neutralization over time. Interestingly, this is not the case. HIV-1 isolates that are resistant to circulating autologous NAbs generally remain sensitive to neutralization by several known monoclonal antibodies (mAbs) or by heterologous plasma obtained for other individuals with HIV-1. This has led to several key questions related to autologous virus NAb escape: What are the Env epitopes targeted by early autologous NAbs and how does the virus escape from these NAbs? How does continuous neutralization escape occur without leading to global changes in viral neutralization sensitivity? Finally, what are the implications of NAb escape for HIV-1 vaccines? In this issue of PLoS Pathogens, two teams of investigators provide some initial answers to these questions [23],[24]. Both groups utilized clinical samples collected from seroconversion cohorts of individuals with subtype C HIV-1. The investigators studied the development of the autologous NAb response from the acute phase, though the first 2 years of infection. A limiting dilution PCR methodology was used to clone and study HIV-1 variants from sequential plasma samples over time. Moore and colleagues studied four individuals and found that the early NAb response was restricted to two epitopes on the HIV-1 Env. They used chimeric viral clones and site-specific mutagenesis to define an epitope composed of the first and second variable region (V12) of the HIV-1 Env. A second epitope was identified within a variable alpha-2 helix region of Env that is just past the V3 loop. The restricted nature of the autologous NAb response to variable Env regions is an important finding, because it helps to explain how the virus can readily mutate to evade the NAb response. The V12 region in particular can tolerate insertions and deletions of amino acid residues without sacrificing Env function. In addition, specific amino acid changes and alterations in glycosylation in these two epitopes were found to be associated with neutralization escape. In one individual, the development of a NAb response to the alpha-2 helix region was associated with a 7-fold drop in plasma viremia, and a 4-fold rebound as neutralization escape occurred. Rong and colleagues similarly studied longitudinal samples from two individuals and found a highly restricted set of NAbs. They also identified the V12 region as a key target of autologous NAbs. Mapping studies demonstrated that specific amino acid sequence alterations, as well as changes in the pattern of glycosylation, were important components of neutralization escape. Importantly, they were able to isolate two mAbs from one patient, and demonstrated that a single amino acid substitution affecting a glycosylation site in V2 was responsible for resistance to these mAbs. In some cases, mutations outside of the specific neutralization epitopes were also associated with neutralizing escape. Given the complex trimeric structure of the HIV-1 Env, it is well known that distant mutations can affect the conformational structure of Env and impact antibody recognition of an epitope [25]. While these two new studies have probably not described the full spectrum of autologous NAb responses, the consistent finding of an early dominant NAb response to one or two variable regions of Env that can vary without major cost to viral fitness does help explain how the virus is able to effectively evade the NAb response. The study of the early autologous NAb response adds to our understanding of the role of NAbs in natural HIV-1 infection, and has potential implications for HIV-1 vaccine design. We know that, over time, more broadly reactive NAbs develop in some individuals with HIV-1 [26]–[28]. These NAbs appear to target functionally conserved regions of Env such as the receptor or co-recpetor binding sites, or conserved regions of gp41 [27], [29]–[31]. Thus, immune escape from such NAbs would, in theory, be much more difficult [32]. In addition, these antibodies can protect against AIDS virus infection in non-human primate models [33],[34]. We still do not understand why such NAbs arise so late during the course of HIV-1 infection. Hence, investigators should continue to study the longer-term evolution of the NAb response in order to better understand the early epitope dominance of the autologous NAb response, and the clinical and virologic factors associated with the evolution from a type-restricted NAb response to a more broadly reactive response. While NAbs may arise too late during natural HIV-1 infection to have a major impact on HIV-1 replication, a major goal of vaccine researchers is to generate pre-existing NAb responses that can prevent initial HIV-1 infection, or contain the virus during the initial phase of viral dissemination [3],[26],[35],[36]. A better understanding of the evolution of the natural NAb response during natural infection, including the viral epitopes targeted, can provide insights for vaccine immunogen design.

FoxP3 determines the development of CD4+CD25+ regulatory T (Treg) cells and represses interleukin-2 (IL-2) expression in Treg cells. However, human immunodeficiency virus type 1 (HIV-1) infects and replicates efficiently in FoxP3+ Treg cells. We report that, while inhibiting IL-2 gene expression, FoxP3 enhances gene expression from HIV-1 long terminal repeat (LTR). This FoxP3 activity requires both the N- and C-terminal domains and is inactivated by human IPEX (immunodysregulation, polyendocrinopathy, enteropathy, X-linked syndrome) mutations. FoxP3 enhances HIV-1 LTR via its specific NFkappaB binding sequences in an NFkappaB-dependent fashion in T cells but not in HEK293 cells. FoxP3 decreases level of histone acetylation at the interleukin-2 locus but not at the HIV-1 LTR. Although NFkappaB nuclear translocation is not altered, FoxP3 enhances NFkappaB-p65 binding to HIV-1 LTR. These data suggest that FoxP3 modulates gene expression in a promoter sequence-dependent fashion by modulating chromatin structure and NFkappaB activity. HIV-1 LTR has evolved to both highjack the T-cell activation pathway for expression and to resist FoxP3-mediated suppression of T-cell activation.

The efficacy of various antiretroviral (ARV) therapy regimens for human immunodeficiency virus type 2 (HIV-2) infection remains unclear. HIV-2 is intrinsically resistant to the nonnucleoside reverse-transcriptase inhibitors and to enfuvirtide and may also be less susceptible than HIV-1 to some protease inhibitors (PIs). However, the mutations in HIV-2 that confer ARV resistance are not well characterized. Twenty-three patients were studied as part of an ongoing prospective longitudinal cohort study of ARV therapy for HIV-2 infection in Senegal. Patients were treated with nucleoside reverse-transcriptase inhibitor (NRTI)- and PI (indinavir)-based regimens. HIV-2 pol genes from these patients were genotyped, and the mutations predictive of resistance in HIV-2 were assessed. Correlates of ARV resistance were analyzed. Multiclass drug-resistance mutations (NRTI and PI) were detected in strains in 30% of patients; 52% had evidence of resistance to at least 1 ARV class. The reverse-transcriptase mutations M184V and K65R, which confer high-level resistance to lamivudine and emtricitabine in HIV-2, were found in strains from 43% and 9% of patients, respectively. The Q151M mutation, which confers multinucleoside resistance in HIV-2, emerged in strains from 9% of patients. HIV-1-associated thymidine analogue mutations (M41L, D67N, K70R, L210W, and T215Y/F) were not observed, with the exception of K70R, which was present together with K65R and Q151M in a strain from 1 patient. Eight patients had HIV-2 with PI mutations associated with indinavir resistance, including K7R, I54M, V62A, I82F, L90M, L99F; 4 patients had strains with multiple PI resistance-associated mutations. The duration of ARV therapy was positively associated with the development of drug resistance (P = .02). Nine (82%) of 11 patients with HIV-2 with no [corrected] detectable ARV resistance had undetectable plasma HIV-2 RNA loads (<1.4 log(10) copies/mL), compared with 3 (25%) of 12 patients with HIV-2 with detectable ARV resistance (P = .009). Patients with ARV-resistant virus had higher plasma HIV-2 RNA loads, compared with those with non-ARV-resistant virus (median, 1.7 log(10) copies/mL [range, <1.4 to 2.6 log(10) copies/mL] vs. <1.4 log(10) copies/mL [range, <1.4 to 1.6 log(10) copies/mL]; P = .003). HIV-2-infected individuals treated with ARV therapy in Senegal commonly have HIV-2 mutations consistent with multiclass drug resistance. Additional clinical studies are required to improve the efficacy of primary and salvage treatment regimens for treating HIV-2 infection.

Defining the latent human immunodeficiency virus type 1 (HIV-1) burden in the human brain during progressive infection is limited by sample access. Human hematopoietic stem cells (hu-HSCs)-reconstituted humanized mice provide an opportunity for this study. The model mimics, in measure, HIV-1 pathophysiology, transmission, treatment, and elimination in an infected human host. However, to date, brain HIV-1 latency in hu-HSC mice during suppressive antiretroviral therapy (ART) was not studied. To address this need, hu-HSC mice were administered long acting (LA) ART 14days after HIV-1 infection was established. Animals were maintained under suppressive ART for 3months, at which time HIV-1 infection was detected at low levels in brain tissue by droplet digital polymerase chain reaction (ddPCR) test on DNA. Notably, adoptive transfer of cells acquired from the hu-HSC mouse brains and placed into naive hu-HSC mice demonstrated viral recovery. These proof-of-concept results demonstrate replication-competent HIV-1 reservoir can be established in hu-HSC mouse brains that persists during long-term ART treatment. Hu-HSC mice-based mouse viral outgrowth assay (hu-MVOA) serves as a sensitive tool to interrogate latent HIV-1 brain reservoirs.

Mother-to-child HIV-1 transmission accounts for more than 700 000 new paediatric HIV-1 infections in developing countries each year [1]. This comprises less than one-third of the infants born to human immunodeficiency virus type 1 (HIV-1) infected mothers, the majority of whom remain uninfected despite recurrent risk for contact with the virus in utero, during delivery and through breastfeeding. A comprehensive approach to studying infant immunity against HIV-1 may provide insight into the determinants of HIV-1 acquisition, promote an understanding of resistance to infection in the setting of repeated exposure to the virus and contribute to the development of therapeutic interventions or vaccines against HIV-1 transmission. Several unique features of mother-to-child HIV-1 transmission provide advantages in determining correlates of HIV-1 acquisition and viral immunity when compared to sexual HIV-1 transmission models. Both HIV-1 infected mothers and their exposed infants can be evaluated for viral and immunological factors associated with transmission. HIV-1 exposure can be characterized by quantifying maternal HIV-1 viral load in plasma, breast milk and genital tract secretions, and infant immune responses can be defined simultaneously or near the time of exposure. Timing of transmission can be estimated using HIV-1 polymerase chain reaction (PCR) at birth and at regular intervals after exposure during delivery and breastfeeding. Vertical HIV-1 transmission risk is also higher than sexual transmission risk. Per sexual act, it is estimated that the risk of heterosexual transmission is approximately 0·1% in an antiretroviral naive population [2]. The risk of HIV-1 acquisition during delivery ranges from 10 to 20%, more than 100-fold higher than heterosexual transmission rates. Disparities between heterosexual and vertical HIV-1 transmission rates persist in the setting of antiretroviral therapy. This enables mother–child transmission studies to provide robust epidemiological data regarding specific immune mechanisms and combinations of immune responses that may constitute protective immunity against HIV-1. This review examines the spectrum of innate, humoral and cellular immune responses and genetic factors that have been studied in infants who are HIV-1 infected or HIV-1 exposed and uninfected (Fig. 1). Fig. 1 Immune responses and genetic factors associated with mother-to-child HIV-1 transmission and paediatric HIV-1 disease progression. *May be maternally acquired in utero or via breast milk. †Alloimmunity is dependent on degree of maternal–infant ... INNATE IMMUNITY Innate immune responses are generated rapidly and are important in preventing and containing infections with a variety of viral pathogens. Broad innate immunity may also be capable of protecting against immune-escape viruses generated by more narrow adaptive immune responses. In HIV-1 transmission and disease progression, relevant innate mechanisms of immunity include the activity of natural killer (NK) cells and antiviral proteins such as the CC chemokines, CD8+ antiviral factor (CAF) and secretory leucocyte protease inhibitor (SLPI). Natural killer (NK) cell activity Natural killer (NK) cells induce inflammation and lyse infected cells without prior sensitization and in a non-HLA restricted manner. NK cells from HIV-1 infected individuals release the CC chemokines MIP-1α, MIP-1β and RANTES, three factors that have been shown to inhibit HIV-1 independently in vitro by blocking the CCR5 HIV-1 coreceptor [3,4]. NK cells also act by lysing HIV-1 infected cells via antibody-dependent cellular cytotoxicity (ADCC). This is initiated by binding of NK cell Fc receptors (CD16) to target cells coated with HIV-specific antibodies of the subclass IgG1 [5–7]. HIV-specific ADCC antibodies are directed against the viral envelope glycoproteins gp120 and gp41 and are distinct from virus-neutralizing antibodies [8]. There is conflicting evidence regarding the role of NK cells in containing HIV-1 in chronically infected children and in preventing vertical HIV-1 transmission. Several studies have evaluated HIV-specific ADCC antibody titres in sera of infants born to HIV-1 infected mothers and found that these antibodies are transferred efficiently across the placenta from mother to fetus [9,10]. However, there was no significant correlation between antibody titres at birth and either HIV-1 disease progression during 2 years of follow-up or mother-to-child HIV-1 transmission [9,10]. Active production of HIV-specific ADCC antibodies was observed in the majority of HIV-infected infants only after 12 months of age [10] and effector cells from HIV-1 infected children appear unable to generate NK cell-mediated cytotoxicity [11]. Thus, an immature immune system may account for the absence of ADCC-mediated NK protection against HIV-1 infection in neonates and young infants, despite adequate levels of passively transferred ADCC antibodies. This may contribute to rapid HIV-1 progression in children infected with HIV-1 early in life [10,11].

Viral reservoirs, residual viremia, and the potential of highly active antiretroviral therapy to eradicate HIV infection

HIV-1 acquisition in a man with ulcerative colitis on anti-α4β7 mAb vedolizumab treatment.