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Opinion

Dispelling myths and focusing on notable concepts in HIV pathogenesis Jay A. Levy Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA

Since the discovery of HIV over three decades ago, major efforts have been made to control and perhaps eliminate HIV infection worldwide. During these studies, certain myths or misconceptions about this infectious disease have been emphasized and other potentially beneficial concepts have received less attention. A true long-term solution to HIV infection merits an appreciation of alternative ideas and findings that could be beneficial in the ultimate control of HIV/AIDS. Here, I discuss six issues and call for more attention to the science of HIV and well-designed clinical trials. Overview and new considerations Over three decades ago, a new human disease syndrome that initially appeared in Africa, at least 10 years earlier, was recognized in industrialized nations [1]. It continued to spread and has now affected all countries in the world. The cause, HIV, was discovered within 2 years of the first published report of the clinical condition: AIDS [2]. Some time later, it was realized that individuals could be infected with HIV for an average of 10 years before developing symptoms of the disease [3]. Notably, a clinically healthy state can allow the virus to spread from individuals who are unaware that they are infected. Modern technology and advancements in science have enabled the development of highly reliable diagnostic tools (e.g., flow cytometry, and viral load measurements) to detect HIV infection [4,5]. Additionally, effective antiretroviral therapies (ART) now ensure that most infected treated individuals will live a long period of time without life-threatening conditions (http://aidsinfo.nih.gov/ ContentFiles/AdultandAdolesentGL.pdf). HIV itself is highly heterogeneous, existing as two types (HIV-1 and HIV-2), and in several groups and subtypes or clades (Table 1) [6,7]. The immune system, both innate and adaptive, can respond to HIV and, in some situations, keep the pathogen controlled without a need for therapy. In this case, the antiviral activity of the immune system inhibits virus replication and enables the carrier to have a relatively normal life for several years, perhaps for a lifetime [8]. This clinical state is found particularly in long-term-survivors (LTS), also known as long-term nonprogressors (LTNP), and elite controllers (EC) Corresponding author: Levy, J.A. ([email protected]). Keywords: HIV misconceptions; innate and adaptive immunity; vaccine; cure. 1471-4914/ ß 2015 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.molmed.2015.03.004

[8,9]. LTS are individuals infected for more than 10 years who remain healthy without therapy and maintain low plasma viral loads and normal CD4+ cell numbers. EC are those LTS with no detectable viral load for many years. Fortunately, for others who are infected and have symptoms, HIV can be controlled with ART, with nearly 30 different drugs currently used with different combinations and in several distinct protocols [10,11] (http://aidsinfo. nih.gov/ContentFiles/AdultandAdolesentGL.pdf). An important observation with HIV infection is that, in some individuals, the innate immune system appears particularly responsible for preventing infection despite multiple exposures to the virus [12–14]. This finding offers encouragement for the development of a vaccine. Moreover, in at least one case, a cure has been achieved via hematopoietic stem cell transplantation [15,16]. Thus, current studies are notably focused on the prevention of HIV infection through a vaccine and on directions toward a cure. Unfortunately, specific attention and access to immune-based therapies are still limited. As HIV investigators continue the campaign to control and conceivably eradicate HIV worldwide, certain strategies reflecting myths or misconceptions about this infectious disease have been embraced, while other concepts with merit have been left relatively unexplored. These issues in HIV pathogenesis threaten to prevent a true long-term solution to HIV infection and, perhaps, infections by other human pathogens. The issues include: a focus on individuals who have warded off the disease and even infection; the primary emphasis on adaptive immunity and not also innate immunity; the specific antiviral role of CD8+ cells; the time for initiating and prescribing ART; and recent approaches toward a vaccine and a cure. Moreover, the specific activities and interactions of both cellular and humoral immune responses in innate, as well as adaptive, immunity need to be better defined and appreciated [14,17]. Here, I consider six issues dealing with HIV infection and argue for more attention to the science of HIV and well-designed clinical trials. Consideration should be given to alternative explanations and approaches in the ongoing pursuit toward finding the best directions for handling HIV infection and AIDS. Is HIV infection a universally fatal diagnosis? When AIDS first appeared, it was considered a ‘death sentence’ because individuals with the diagnosis did not survive very long [1]. With time, we have learned that certain individuals infected by HIV can control the virus and ward off signs of illness without therapy. It is Trends in Molecular Medicine xx (2015) 1–13

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Table 1. The estimated infection rate of HIV groupsa

Table 2. Comparative immune systems

HIV group or subtype HIV-1 Group M Group N Group O HIV-2 Group A Group B Group C Group D Group E Group F Group G Group H

Feature

No. of global infections (% of total) 45 000 000 (99.6) 10 (0.000013) 10 000 (0.22) 50 000 (0.11) 25 000 (0.06) 1 (0.0000002) 1 (0.0000002) 1 (0.0000002) 1 (0.0000002) 1 (0.0000002) ?

a

2005 estimates. Adapted, with permission, from [6].

estimated that 5–8% of HIV-1-infected people can keep the virus in check for at least 10, and some for more than 35 years [3,7,8]. These untreated asymptomatic HIV carriers (LTS and EC) live with normal CD4+ cell counts, low viral loads, and no sign of infection. They offer a direction for the development of immune-based therapies because HIV appears to be controlled by their immunologic responses. In addition, HIV-2 infection is less pathogenic than HIV-1 infection [18], and many of these infected individuals can live a normal lifespan without therapy. Thus, we can gain insight from studying this seemingly attenuated virus infection. In addition, some people remain uninfected even after multiple exposures to HIV through known mechanisms of transmission, including mother–child, sexual activity, blood transfusions, and needle sharing. In some of these highly exposed seronegative (ESN) individuals, the lack of C-C chemokine receptor type 5 (CCR5) receptor expression and innate immune activities can be involved [7,12,13,19,20]. All of these observations indicate that HIV does not cause a universally fatal disease. Some infected individuals can live a normal asymptomatic life without intervention. Importantly, we can learn a great deal about the prevention of disease and infection by studying those exceptional people who have survived without AIDS or have warded off infection. Is anti-HIV innate immunity as important as adaptive immunity? The recognition of ESNs who have been exposed on many occasions to HIV without infection highlights the important role of the early innate immune response [12,14]. This activity occurs within minutes, without epitope specificity, and, therefore, can quickly act to prevent infection by incoming pathogens. If this type of antiviral activity is not successful, adaptive immunity, elicited by the innate immune system, responds within days to weeks to the HIV infection (Table 2). This later adaptive antiviral immune activity comprises humoral (e.g., antibody production) and cellular (e.g., CD8+ cell HIV-specific cytotoxic T lymphocyte; CTL) responses. The strength of innate immunity can determine whether HIV is controlled immediately and, if not, whether an 2

Innate immune system + Quick response (min–days) Delayed response (days–weeks) – – Antigen specific – Memory responses – Gene rearrangement ++ Conserved through evolution

Adaptive immune system – + + + + +

effective antiviral adaptive immune response can be induced [14]. Cytokines produced by innate immune cells can directly control the pathogen (e.g., interferon; IFN) or influence the extent of the immune response by cells of the adaptive immune system [e.g., interleukin (IL)-2, IL-4, IL-12; Figure 1)]. This latter immune activity, as a followup to the innate response, can determine the ultimate fate of the infection and whether disease develops. Thus, emphasis should be given to the innate immune response as the first line of defense against incoming microbes, such as HIV. Plasmacytoid dendritic cells (PDC) with interferon production [21] and natural killer (NK) cell cytotoxic activity [22], as well as the CD8+ cell noncytotoxic antiviral response (CNAR) [14,17], are noteworthy innate immune activities associated with an early response to HIV infection. They can protect against disease as well as infection as observed in ESN individuals [13,19,23]. Approaches to enhance these early innate immune responses through a vaccine with appropriate adjuvants would be important for obtaining effective prevention from HIV infection and the development of AIDS. What is the relative importance of CD8+ cell noncytotoxic and cytotoxic antiviral activities? The first immune response identified in HIV infection was an anti-HIV activity of CD8+ T cells that did not involve cell killing [24]. This CNAR correlates with a clinically healthy state [25–28]. The activity is mediated by secreted soluble cytokines, in particular the CD8+ cell antiviral

Acute infecon

Innate immune response

Cytokine release

Anmicrobial acvity

Adapve immune responses TRENDS in Molecular Medicine

Figure 1. Interaction of the innate and adaptive immune systems in response to acute infection. Following transmission and acute infection by a microorganism, components of the innate immune system respond rapidly, releasing cytokines that can have direct antimicrobial activity (e.g., interferon) or elicit antimicrobial activity by innate immune cells [e.g., neutrophils, dendritic cells, and natural killer (NK) cells]. These cytokines can also induce the subsequent responses of the adaptive immune system (T and B lymphocytes). Thus, the early response of the innate immune system can have an influence on both the innate and adaptive antimicrobial activities.

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Opinion Box 1. Major characteristics of the CD8+ cell noncytotoxic anti-HIV response*  Correlates directly with healthy clinical status and normal CD4+ cell counts; associated with long-term survival.  Early response to HIV infection; occurs before seroconversion.  Does not involve cell killing.  Active against all strains of HIV-1, HIV-2, and SIV tested; no resistant virus found.  Property of CD8+ T cells; not CD4+ cells, NK cells, or macrophages.  Not MHC restricted.  Suppresses HIV replication in naturally or acutely infected CD4+ cells.  Optimal activity with cell–cell contact; dose dependent.  Can inhibit HIV replication at low CD8+:CD4+ cell ratios (e.g., 350 cells/ml can have confounding factors that are not appreciated in the trials conducted thus far. How various ecological issues affect the observations should be considered, including the emergence of drug resistance [98]. Therefore, the decision on time of treatment can be misleading until the results from randomized control trials are available [99,100]. In the case of acute infection, we await the findings of the START and TEMPERANO clinical trials. Therapy within weeks after infection can reduce the virus spread, but also inhibit the natural immune response to HIV that could help control the virus, as in LTS. In this regard, studies indicate that recovery of CD4+ cells counts to at least 500 cells/ml will assure no increased risk of death compared with the general population [101–103]. 4

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In another view on treatment, some epidemiologists advocate the use of antiretroviral therapies for all HIVinfected people to protect against HIV transmission [99,104,105]. This strategy reflects observations that HIV-infected individuals who have low levels of viremia are less likely to transmit the virus [106]. The concept of ‘Treatment as Prevention’ (TasP) has taken a front seat in a field that has had frustrating results with vaccine trials and approaches using barrier techniques and other behavior modifications [99,104]. Nevertheless, there is insufficient information from randomized clinical trials on the value of TasP. Notably, the results thus far do not indicate a total absence of transmission from individuals on longterm ART [107]. Importantly, in addition to toxicity, a major concern with widespread use of antiviral drugs should be the emergence of resistant isolates. Well-controlled trials with frequent visits over a short period of time (e.g., 3 years) are not sufficient to determine whether resistant viruses would appear with the universal distribution of ART. Long-term engagement in care is needed [108]. For example, individuals who are healthy will be reluctant to continue their drugs, particularly if they have even mild symptoms of the treatment, such as fatigue. Then, if the therapy is not taken consistently, resistant viruses can emerge. Adherence is a major reason for treatment failures. Some studies have indicated a marked decrease in adherence to treatment related to the amount of drugs and number of times they are taken a day, as well as age of the patient [109,110]. Moreover, even adherence does not seem to prevent drug resistance [111,112]. ART-resistant isolates, some multiresistant, have now been detected in locations with widespread use of ART [113–117]. This occurrence limits treatment options, increases clinical costs, and leads to a reservoir of resistant viruses that can spread in the population [118]. This lesson on the emergence of resistant organisms has been appreciated vividly in the loss of efficacy of many antibacterial agents [119,120]. It takes several years after introducing antimicrobials into a community to determine their efficacy and resistance [119]. Notably, this call for the universal use of ART is based on population effectiveness where the general result may look good with large numbers of people studied. However, the decision on treatment must also appreciate the physician’s responsibility to provide the individual with drugs that are not harmful and will not lose their efficacy. In this regard, a noteworthy benefit in reduction in transmission does appear to be achieved with the use of ART for prevention of new HIV infections among HIVnegative partners of infected subjects [121–124] and people who inject drugs [125]. Adherence is the key to the most successful pre-exposure prophylaxis (PreP) prevention program [99,126,127]. However, a day-before and/or day-after pill approach, which is being considered, seems to be exaggerating the observation. Prevention of virus infection with the effective drug, tenofovir, only gave the optimal result in SIV studies when treatment after virus exposure lasted for 1 month [128]. Also, PreP, even for sero-discordant couples, does not assure 100% protection. It ranges from 67% to 90% depending on adherence to ART

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Opinion Box 2. Challenges of developing an HIV vacciney  HIV integrates into the cellular genome.  Infected cells can transmit the infection.  Numerous HIV variants emerge because virus mutations are common.  HIV infects sanctuaries of the body (e.g., brain or testes).  HIV compromises immune function.  Autoimmune responses may be induced.

y

Modified, with permission, from [7].

[114,121]. PreP in injecting drug users has not reduced infection by over 50% [125] and, notably, in some studies, PreP has not been effective in preventing HIV infection of women [129,130]. There are also adverse effects of PreP, including nausea, dizziness, and reduced bone density [123]. More randomized studies with strategies for optimal adherence need be conducted before freely giving ART to anyone planning a high-risk exposure. The basic question is whether ART is the best global approach for HIV prevention. The results may look good over the short term, but a concern for harmful long-term effects must be appreciated. In a recent excellent group of commentaries on TasP (Clinical Infectious Diseases 59, Suppl.1, 2014), not one article specifically addressed these issues of drug toxicity and viral resistance. Aside from sero-discordant couples, the overall effectiveness of ART in protection from HIV infection is not high (40–50%) and that fact should influence a decision on PreP. Importantly, the use of ART for prevention also engenders a false sense of security. Moreover, TasP should not lead to complacency on this issue of protection from infection; other strategies need to be developed and encouraged [127,131]. Behavioral modification approaches (e.g., condom use and reduction in new partners) must continue to be emphasized [84,131–134] and further evaluation of diaphragms in women in a community-based environment should be considered [135]. Certainly, the observations cited above support the conclusion that ART cannot be advocated as a sole means of protection from HIV transmission. What strategies should be considered for an HIV vaccine? A vaccine is the optimal means of controlling an infectious disease by prevention, and certain unique challenges of HIV can be appreciated (Box 2). Attempts to develop an anti-HIV vaccine go back to the early 1980s, when a gp120 envelope vaccine was attempted without success [136]. Subsequently, several Phase I, II, and a few Phase III trials have been undertaken, with only one giving some limited successful (31%) protection [137]. In this latter case, prevention appeared to be linked to the presence of antibodies that participated in antibody-dependent cellular cytotoxicity (ADCC), rather than in virus neutralization [138]. This latter observation is noteworthy because, based on the success with established vaccines for other infectious diseases, several approaches directed at inducing neutralizing antibodies to protect from HIV infection have been encouraged and undertaken [139–143].

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Recently, a focus has been given to germ-line B cells for derivation of effective antibodies, particularly those with broad neutralizing activity [144], and this strategy may be more successful [145]. Unfortunately, because the few vaccine trials that were directed specifically at inducing anti-HIV cellular immunity were unsuccessful [146], attention to this arm of the immune system has been limited [147]. In these vaccine studies, the induction of CTLs was the major objective for the induction of T cell immunity. Notably, the innate CD8+ T cell noncytotoxic activity associated with control of the virus in primary infection, as well as in LTS and EC, was not evaluated. Results from some vaccine studies cannot be fully explained by CTLs. Currently, the most promising results in preclinical vaccine trials in monkeys have been obtained with a live cytomegalovirus (CMV) vaccine that has elicited CD8+ cell anti-SIV responses. Half of the immunized animals cleared plasma virus when challenged by virus [148,149]. The mechanism of prevention has not been fully elucidated, but an unusual MHC class II antiviral response of CD8+ cells [149] has been suggested as a possible explanation. The overall message in vaccine development is that investigators need to appreciate the value of basic science and place an emphasis on new ideas directed at innate as well as adaptive immunity–embracing both humoral and cellular immune responses. Vaccine trials toward this aim with appropriate (and novel) adjuvants need to be encouraged with specific clinical subgroups (e.g., clade C infection) as well as those targeting the general population [150]. More resources should be made available to try both classic and innovative strategies. Recent findings also support the conclusion that major efforts need to be directed at inducing effective innate and adaptive immune responses in the mucosae [151]. In this regard, two neglected observations merit attention in the development of an effective vaccine. First, further studies of ESN individuals are warranted. Some have been exposed to HIV in genital fluids and even blood on many occasions and have not become infected. What are the factors giving that protection? Second, a killed virus vaccine has been effective in preventing the cat lentivirus infection [152], and is showing promise for the equine infectious anemia lentivirus [153]. Would this approach work for HIV? For the first point, studies of ESN individuals have revealed that primarily innate immune responses are found, either reflected by an NK cell activity [23] or the CD8+ cell noncytotoxic activity described above [7,13,14]. No consistent observation of CTLs reflecting adaptive immune responses has been seen. The innate activities occur rapidly (within minutes to days) and apparently can ward off an early HIV infection in the exposed individuals and prevent the establishment of the infection. In this regard, recent studies suggest that often only one infectious virus is involved in the transmission process [154] and the transmitted founder virus can have a specific phenotype that could be targeted by a vaccine [155]. Thus, infection by the vaginal route could involve initially a few cells in the cervix and then entry of HIV through the cervical os into the uterus, which has many immune cell 5

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(A)

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(B)

(C)

Key: HIV Infected cells Innate cells TRENDS in Molecular Medicine

Figure 2. Early events in HIV transmission in the vaginal tract. (A) When viruses and HIV-infected cells enter the vaginal vault, the cervix is a major target (B). If innate cells [e.g., plasmacytoid dendritic cells (PDC), natural killer cells (NK), or CD8+ noncytotoxic cells] respond quickly, they can ‘wall off’ the infection (C), and suppress HIV replication. This response thereby prevents the establishment of HIV infection in the host.

targets [7]. If innate immune responses are encouraged, this early infection could be curtailed by blocking the spread of HIV from a local cervical infection to the uterus, distant lymph nodes, and beyond (Figure 2). However, the factors that influence the responses leading to prevention of infection in the HIV-exposed individuals need better definition. For the second point, certain researchers do not believe that sufficient attention has been given to a killed virus vaccine that involves virus inactivation by several methods [156–158]. They argue that such an approach at first was not feasible because of the fear of an infectious virus emerging from the inactivation process. Also, initially, nonviral proteins were involved [159]. Current inactivation procedures provide more safety measures and the viral protein production methods focus on the native presentation of the HIV envelope for induction of neutralizing antibodies. Moreover, by using a mutated replication-defective virus and by having a drug such as tenofovir given at the same time, prevention of infection could be more assured. Tenofovir in monkey models has been effective in preventing infections both in pre-exposure and post-exposure situations [128,160] and, therefore, is used in TasP. With killed virus studies, as noted above, one could learn a great deal about immune responses against HIV even with a low number of people immunized. An immune activity specific for humans could be found that might not be surmised from studies in nonhuman primates [158]. One study by a Korean company is undertaking a virus inactivation approach and is in Phase II trials [161,162]. Those results will be important in assessing the value of a killed virus vaccine. In terms of experimental design, a concern to be appreciated with vaccine trials conducted in monkeys is whether they truly reflect the variables in a transmission event. The viruses used for challenge are given in culture medium and at larger quantities than found in genital fluids. Moreover, investigators neglect the fact that virus-infected cells may be a major source of HIV, particularly macrophages [163,164], but challenges with infected cells are rarely conducted. In addition, for the challenge, cells should be placed in seminal or vaginal fluid to recreate the environment of a natural transmission when inoculated into the 6

vaginal or rectal canals or onto the foreskin. Some proteins in seminal fluid can enhance HIV infection [165]. Another important concern raised by trials involving other viral vaccines is the possible induction of antibodies that will enhance, rather than limit, virus infection [166– 168]. This result is dramatically shown with dengue virus. Prior exposure to one virus subtype can lead to the production of antiviral antibodies that enhance infection and cause harmful clinical effects following infection by a different dengue virus subtype [169]. This enhancement is mediated by low-affinity antibodies that attach to the virus without inactivating it [170]. The virus–antibody complex permits infection of cells through the cellular Fc or complement receptor. Such enhancement of HIV infection by antibodies has been associated in vitro with disease progression [171], but has not been evaluated in human vaccine trials. Most recently, the possible induction of cellular immune responses that enhance HIV infection after vaccination has been proposed [146,172] and suggested by monkey studies [173]. The STEP trial evaluating the induction of cellular immunity against HIV indicated that prior infection by adenovirus was linked to a higher infection rate in those receiving the vaccine [146]. Monkey trials mimicking these results suggested that the cellular immune responses against the adenovirus alone were not the cause; instead, the adenovirus as a vector for antigens of HIV (or SIV) enhanced infection after vaccination [174]. The mechanism is considered to be related to an increase in CD4+ cell activation making the cells more susceptible to HIV infection [175]. No conclusion on the specific process has been reached and still needs to be elucidated. In this regard, one potentially important study using an inactivated SIV vaccine in conjunction with the oral administration of lactobacillus has suggested a preventive effect linked to a CD8+ Treg cell reduction in cell inflammation [176]. If confirmed, CNAR may also be involved. What approaches should be encouraged for an HIV cure? Great attention was given toward the possible cure of HIV infection when the ‘Berlin patient’ was reported [15]. After receiving two stem cell transplants for his leukemia from a homologous CCR5D32 donor, he emerged with no evidence

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Opinion Box 3. Approaches to a cure  Activate HIV from cellular reservoirs leading to cell death.  Activation of HIV replication concomitant with post-infection immunization.  Genetic procedures to derive CD34+ hematopoietic stem cells lacking CCR5 expression for transplantation into the autologous host.  Enhance anti HIV-immune responses that prevent virus replication and effect a ‘functional’ cure

of his cancer or HIV infection. He remains free of any detectable virus after more than 7 years [16]. Given that he was initially infected with both CCR5-tropic (R5) and CXCR4-tropic (X4) viruses, how did he eliminate the X4 viruses that would be able to replicate in the CCR5-negative, HIV- resistant T cells? While recent evidence suggests that his virus with an X4 genotype has R5 tropism [177], it may well be an innate immune response by the transplant (e.g., CNAR) that maintained control of all HIV types. Among the approaches directed toward a cure (Box 3), a popular strategy is derived from concepts that are not based on well-established experimental facts. For example, some investigators advocate the activation of HIV from latent immune cell reservoirs in individuals on ART. They champion this strategy on the assumption that the activating agents, by causing virus replication, will lead to the death of the latently infected cells [178,179]. However, early work on HIV indicated that virus replication does not necessarily lead to cell death. Only a robustly replicating virus will cause the demise of the infected CD4+ cell, the usual target for this approach [180]. Moreover, the major reservoir targeted for this experimental reactivation of HIV has only been CD4+ T cells of the immune system. Macrophages can be reservoirs of HIV for years, and virus replication in these cells is not generally associated with cell death [164,181]. Furthermore, whereas a small number of CD4+ cells were initially considered present as a reservoir, a greater amount of these latently infected CD4+ cells has now been identified [182]. Some researchers expect that the immune system will recognize the activated virus and kill the infected cells. However, ART can reduce anti-HIV immune responses [183–185] and, thus, the immune system needs stimulation to respond to HIV [186]. Toward this objective, postinfection immunization at the same time as virus activation might improve the results [187] or immune-based therapies targeting the infected cells [188,189]. These strategies merit further evaluation. Moreover, several other cells in the body can be infected by HIV, including those in the brain, kidney, liver, and heart [190–192]. Can one activate virus in cells from these tissues and eliminate HIV? Virus replication is usually low and not cytopathic in cells from these organs. Killing all the infected cells by immune therapy could also be harmful to the tissues. Thus, the presence of many different cellular reservoirs, the difficulty in eliminating the virus, and the chance for reinfection makes the ‘cure’ approach by reactivating the virus, even combined with enhancing the immune anti-HIV response, unlikely to succeed. There are new genetic-editing strategies that could be used to mimic the treatment of the ‘Berlin patient’ [15].

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Genetic modification of the hematopoietic stem cell from an HIV-infected individual could be performed with the elimination of the CCR5 gene or its replacement with the naturally occurring D32 bp deletion. Approaches toward this objective have included the use of zinc finger and other nucleases linked to adenovirus or lentivirus vectors [193– 196]. Following autologous stem cell transplantation, this strategy would give rise to an immune system, including macrophages, that is resistant to R5 viruses. In recent studies, the CCR5 gene in mature CD4+ T cells from infected subjects was deleted via the zinc finger approach and the cells were then used for autologous transplantation [196]. Cells with deleted CCR5 were present in the recipients at low levels and, in one case, appeared to reduce virus replication when ART was stopped. In other studies, research groups are modifying additional genes, including CXCR4, to prevent replication of both R5 and X4 HIV isolates in immune cells [193,195]. Results from genetic editing are encouraging, and novel techniques, involving Transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas, are being developed that improve the efficiency of success with this gene modification approach [197] while avoiding the use of integrating virus vectors. However, effective engraftment of HIV-resistant cells may be a major challenge. Critics complain that this genetic approach can be expensive, labor intensive, and not immediately applicable worldwide. Nonetheless, success with a proof-of-concept study could encourage creative approaches that could eventually make stem cell therapies widely available [181]. Another direction toward a ‘cure’ could take advantage of the observations made with LTS and elite controllers, namely, boosting the immune response through therapies that enhance anti-HIV immune responses [14,30,181,187]. That strategy could bring long-term control of HIV to a clinical state in which virus replication and its potential detrimental effects will be avoided. The virus might remain in some reservoirs, but the individual (free of detectable HIV) is ‘functionally cured.’ The secret is an effective antiviral immune response. One other question to consider is the definition of cure, particularly in newborn children. When ART was not available, 40–90% of children born of infected mothers were not infected [19,198,199]. Nevertheless, as is common during all births, some of the mother’s immune cells and selected immunoglobulins cross the placenta and enter the child via the umbilical cord [200–202]. Thus, finding viremia in the child or even integrated DNA in some cells does not necessarily indicate an infected newborn who needs ART. Importantly, the virus found in an infant’s blood may not be infectious for the infant; HIV transmission seems to occur only with selected isolates [154,155,203]. A rise in viremia over time in the child, as was evident in the Mississippi infant [204] who received ART, is suggestive of virus transmission. That child was later confirmed to be infected [205]. Unfortunately, assuming that an HIV infection took place could lead to unnecessary treatment with antiviral drugs. Their toxicity may not be evident until many years after the unnecessary ART is administered [206–208]. 7

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Table 3. Outstanding questions and suggested approachesa Issue Is HIV infection a universally fatal diagnosis?

General view People infected with HIV will not survive without therapy

Other view About 5–8% of HIV-1-infected individuals can survive many years without therapy. Many HIV-2 infected subjects do not develop disease. Others have a natural resistance to HIV infection. Protection is derived from effective antiviral immune responses Innate immunity is important to induce an effective adaptive immune response; innate immunity alone can ward off disease and prevent HIV infection Both CD8+ cell responses are valuable, but more attention should be given to the innate broadly reactive CD8+ cell noncytotoxic anti-HIV response

Is anti-HIV innate immunity as important as adaptive immunity?

Efforts must be made to enhance primarily adaptive antiviral immunity

What is the relative importance of CD8+ cell noncytotoxic and cytotoxic antiviral activities?

Most important to induce CD8+ cell cytotoxic responses in HIV infection

When should ART be given?

ART should be given to everyone as a means of preventing disease and HIV transmission; ART is important in reducing immune-based inflammation

Given the potential toxicity of ART and development of drug resistance, therapy should only be used if CD4+ T cell counts are /=500/mm3 compared with the general population: evidence from a large European observational cohort collaboration. Int. J. Epidemiol. 41, 433–445 102 Rodger, A.J. et al. (2013) Mortality in well controlled HIV in the continuous antiretroviral therapy arms of the SMART and ESPRIT trials compared with the general population. AIDS 27, 973–979 103 Anglemyer, A. et al. (2014) Early initiation of antiretroviral therapy in HIV-infected adults and adolescents: a systematic review. AIDS 28 (Suppl. 2), S105–S118 104 Cohen, M.S. and Gay, C.L. (2010) Treatment to prevent transmission of HIV-1. Clin. Infect. Dis. 50 (Suppl 3), S85–S95 105 Porco, T.C. et al. (2004) Decline in HIV infectivity following the introduction of highly active antiretroviral therapy. AIDS 18, 81–88 106 Quinn, T.C. et al. (2000) Viral load and heterosexual transmission of human immunodeficiency virus type 1. Rakai Project Study Group. N. Engl. J. Med. 342, 921–929 107 Supervie, V. et al. (2014) Heterosexual risk of HIV transmission per sexual act under combined antiretroviral therapy: systematic review and Bayesian modeling. Clin. Infect. Dis. 59, 115–122 108 Gardner, E.M. et al. (2011) The spectrum of engagement in HIV care and its relevance to test-and-treat strategies for prevention of HIV infection. Clin. Infect. Dis. 52, 793–800 109 Ghidei, L. et al. (2013) Aging, antiretrovirals, and adherence: a meta analysis of adherence among older HIV-infected individuals. Drugs Aging 30, 809–819 110 Parienti, J.J. (2014) The case of adherence in the youth: rebel without a cause? AIDS 28, 1983–1985 111 Bangsberg, D.R. et al. (2003) High levels of adherence do not prevent accumulation of HIV drug resistance mutations. AIDS 17, 1925–1932 112 Parienti, J.J. et al. (2009) Better adherence with once-daily antiretroviral regimens: a meta-analysis. Clin. Infect. Dis. 48, 484– 488 113 Jackson, J.B. et al. (2000) Identification of the K103N resistance mutation in Ugandan women receiving nevirapine to prevent HIV1 vertical transmission. AIDS 14, F111–F115

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