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Vol 464j11 March 2010jdoi:10.1038/nature08898 PERSPECTIVES

Immunology and the elusive AIDS

Herbert W. Virgin1 & Bruce D. Walker2

Developing a human virus (HIV) vaccine is critical to end the global acquired immunodeficiency syndrome (AIDS) epidemic, but many question whether this goal is achievable. Natural is not protective, and despite of HIV vaccine candidates, human trials have exclusively yielded disappointing results. Nevertheless, there is an indication that success may be possible, but this will be dependent on understanding the antiviral immune response in unprecedented depth to identify and engineer the types of immunity required. Here we outline fundamental immunological questions that need to be answered to develop a protective HIV vaccine, and the immediate need to harness a much broader scientific community to achieve this goal.

nti-HIV have extended the lives of millions of Gaps in knowledge infected people, but the epidemic continues its course, Given the failure of empirical HIV vaccine approaches, understanding with high rates of new in regions least able to the nature of the immune response needed for protection is the essential A cope medically, socially and financially. For example, in missing ingredient. We still lack fundamental knowledge regarding the some particularly hard hit areas rates in young women soar nature, quality and quantity of immune responses that should be from less than 1% at age 15 to in excess of 50% by their mid-twenties. induced, the ideal to include, how to overcome the tremendous A vaccine will be required to ultimately conquer AIDS, but many sequence variability engendered by the error-prone HIV reverse tran- scientists believe that this is probably decades away, if even possible. scriptase, or even whether preventive vaccine strategies should focus on Thus far, HIV vaccine trials in humans have resulted in either no or protection from infection or protection from progression. relatively unimpressive protection despite measureable immuno- Classically, has depended on programming memory genicity of administered HIV antigens1–4. Indeed, natural immunity T and B cells to remember encounter with . The current itself is far from protective, inducing both humoral and cellular vaccine failures indicate that this is not sufficient, and that immuni- immune responses but not leading to spontaneous clearance or pro- zation strategies need now to be engineered with close attention to the tecting against HIV superinfection5. Therefore, simply generating an type and differentiation state of the immune response generated, immune response similar to what is generated in natural infection is the efficacy of vaccine-induced effector mechanisms against specific unlikely to immunize effectively against HIV. This conclusion sup- vulnerable steps in the pathogenesis of HIV infection, the capacity of ports the view that our fundamental approach to HIV the induced response to maintain activity at relevant body surfaces, the needs to be re-examined. influence of the virome and microbiome on vaccine responses, the We believe that the development of an effective HIV vaccine is an fitness cost of forced on HIV by specific immune responses, achievable goal, and that there are many ‘knowable unknowns’ that the capacity of vaccine-generated immune responses to avoid exhaus- can be addressed immediately to advance this effort. The goal of this tion, and the capacity of the different parts of the immune response Perspective is to outline progress from studies of HIV and the closely generated by vaccine to synergize with one another at the relevant sites. related AIDS-inducing monkey retrovirus simian immunodeficiency Because HIV initially establishes infection in a very small number of virus (SIV), together with insights derived from studies of other cells6, appropriate pre-existing immunity has the potential to prevent viruses, showing that some level of control of lentiviruses can be infection, clear infection, or limit replication so that the set point of achieved. This suggests that there are immune mechanisms that, if viraemia is low enough during chronic infection to prevent progres- induced by a vaccine, may prevent HIV infection or at least limit the sion to AIDS and inhibit spread from one person to another. A better level of viraemia in infected people. In addition to the critical value in understanding of the molecular mechanisms responsible for immune continuing and expanding current efforts to test vaccine candidates control of acute and chronic viral infections, and immune interactions in humans, we emphasize the importance of a long-term plan to with HIV at various stages of disease (Fig. 1 and ref. 6) will be needed to enhance understanding of basic immunological mechanisms that direct effective vaccine strategies to meet this desperate global need. may be able to prevent amplification and systemic spread from the limited initial nidus of HIV infection (see the accompanying paper6). Signals that vaccination may work We need better integration of novel concepts and information on There are, finally, weak signals from a human , and more new genes and mechanisms derived from work in robust findings from efforts to vaccinate against SIV, indicating that a model systems into vaccine science. These advances need to be measure of protective immunity to HIV or SIV can be induced. A recent rapidly translated into the HIV field to optimize chances for effective phase III trial combining an HIV envelope protein immunogen with a vaccination against AIDS. Achieving this goal will be greatly facili- recombinant canarypox-HIV vector suggested an encouraging (albeit tated by engagement of scientists from diverse but currently isolated insufficient) 30% reduction in acquisition1. In monkeys, induction of disciplines, who, by providing new insights, can accelerate progress virus-specific CD8 T- responses does not prevent infection, but if towards an effective vaccine. strong and broad enough, can curtail SIV replication after homologous

1Washington University School of and Midwest Regional Center of Excellence for Biodefense and Emerging Infectious Disease Research, Campus Box 8118, 660 South Euclid Avenue, Saint Louis, Missouri 63110, USA. 2Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Howard Hughes Medical Institute, 149 13th Street, Charlestown, Massachusetts 02129, USA. 224 ©2010 Macmillan Publishers Limited. All rights reserved NATUREjVol 464j11 March 2010 PERSPECTIVES

Figure 1 | HIV pathogenesis and outstanding What form(s) of HIV spreads? questions related to vaccination. Shown are the Free HIV? Cell-associated HIV? steps in HIV pathogenesis and ‘knowable HIV virus unknowns’ that, when defined, may alter how we Is HIV pathogenesis or immunity different at different mucosal sites? approach vaccination against HIV. APC, antigen- presenting cell.

Does compression of the quasispecies Dendritic cell associated with transmission have CD4 implications for ? Does HIV productively infect and/or impair the function of APCs? Can HIV latency be Do innate iimmune mechanisms How are APCs optimally licensed prevented or eliminated? affectc acquisition? for induction of immunity?

Replicate in Viraemia Can HIVHI be cleared by mucosa How does HIV evadede vaccine immunity?? immunityimmuninityty onceo infection isiitbs established?estab

Blood

What determines HIV fitness?ess? For transmission? Secondary infection For evasion of immunity?ty? lymphoid organs For establishment of set point? Are specific viral genotypes associated with elite controllers? CD4 T-cell depletion Lymph node

AIDS or heterologous virus challenge7,8. Use of simian vaccination (Fig. 2). For example, about one in 300 people maintain (CMV) as a vector to continuously deliver SIV antigens limits systemic extremely low HIV loads without treatment, some for more than 30 infection in some animals after intrarectal challenge with SIV9. These years14, and HIV-2 pathogenesis is similar to HIV-1 pathogenesis and and similar studies show that exposure to lentivirus antigens can lead to yet causes disease far less frequently15. Similarly, SIV can cause high- resistance to or control of infection and provide controlled models to level viraemia, infect and deplete CD4 cells, and yet not cause AIDS16. address substantial gaps in our knowledge of what immune mechan- Together these data indicate that there are as-yet-unidentified viral or isms are responsible for the observed effects. host factors that attenuate lentivirus pathogenesis. Identification of Encouragement afforded by these vaccine results is enhanced by these may define the type of immune responses needed to reach the studies of natural transmission that indicate that induction of even goal of vaccinating to prevent disease progression and spread within modest protective immunity at mucosal surfaces may tip the balance the population (Fig. 2). A key step in this direction would be iden- towards less efficient spread of HIV. Only one-third of born to tification of novel molecules involved in control of chronic virus infected acquire infection; sexual transmission occurs after infection in model systems, and rapid translation of such new findings between 1 out of 100 and 1 out of 1,000 exposures (although the risk for detailed analysis during HIV and SIV infection. may be far higher when the transmitting partner is acutely infected)10; exposure to infected products does not consistently lead to Fight using all the tools immunity offers infection11; commercial sex workers may remain uninfected despite Current evidence overwhelmingly argues for vaccines that simulta- extensive exposure12; and most infections are initiated by a single neously elicit effective CD4, CD8 and B-cell responses; however, an virus13. These data indicate that the induction of even modest pro- integrated understanding of the relationships between innate and tective immunity at mucosal surfaces before infection may be effec- adaptive immune responses to HIV antigens, and how this might tive at inhibiting spread of HIV. Indeed, this may be the mechanism lead to enhanced immunization, is yet to be achieved (Fig. 3). of apparent protection in the recent Thai vaccine trial, which limited Furthermore, although it is clear that innate immune mechanisms HIV acquisition but had no effect on set-point viral load once contribute to control of HIV17, it is not clear whether it is possible to systemic infection was established. Detailed studies of the earliest harness this response via vaccination. Indeed, the HIV vaccine field events in transmission in animal models and humans are needed to has been largely divided into B-cell approaches and CD8 T-cell define the mechanisms that explain resistance to infection in the vast approaches. There is little evidence that any vaccine achieves protec- majority of HIV exposures. tion through alone, or ever prevents initial infection entirely; the same holds for T-cell responses. Antibody functions De´tente teaches us how to go to war optimally in the setting of simultaneous CD4 T-cell responses that Not all HIV infections lead to AIDS, indicating that it is possible for provide help; CD4 and CD8 responses are probably needed to limit immunity to establish a very long-term de´tente with lentiviruses; or eliminate HIV replication at the initial site of infection through the mechanisms involved might provide insight into the goals for innate and adaptive mechanisms. Similarly, T-cell vaccines largely 225 ©2010 Macmillan Publishers Limited. All rights reserved PERSPECTIVES NATUREjVol 464j11 March 2010

HIV: Immune people unavailable, on SIV replication25, indicating that previous associations between Define correlates elite controllers limit progression 20 of immunity SIV: Protection from acquisition and CD4 T-cell activation and increased viraemia are correlative only . lowering of set point demonstrated, Furthermore, CD4 T cells can provide direct antiviral effects24,and mechanisms unknown the presence of strong virus-specific CD4 T-cell responsesdistinguishes Choosese desired HIV: Uncertain non-pathogenic HIV-2 infection from HIV-1 infection15.Possiblesup- immune responseses SIV: , CD8 T cells, CD4 T cells port for a role for immune CD4 T cells in vaccination may be found in the Thai HIV vaccine trial results, in which decreased HIV acquisition was observed with a vaccine that induces HIV-specific CD4 T cells1. Vaccine goal Prevent infection? Vaccine strategy Vaccine New data, A much greater emphasis on understanding the antiviral effector Limit set point? Vector? development assess correlates, properties of CD4 T cells is needed, and the role of these cells in control Prevent progression? Adjuvant? and trial revise approach Prevent spread? Dose? of HIV at mucosal sites and vaccination must be one of the highest Frequency? priorities for future studies.

Choose antigen HIV: Highly variable B cells and antibody come back into focus rapid targets Animal challenge studies indicate that a pre-existing neutralizing antibody response can prevent AIDS virus infection. Notably, a signifi- Define genome HIV: Well described, cant minority of HIV infections generate heterogeneous mixtures of and proteome moving target as epidemic expands polyclonal high-affinity broadly neutralizing to multiple 26 Figure 2 | Approach to vaccination and special challenges of HIV different envelope epitopes , giving hope that properly formulated vaccination. Shown are the steps in development of a vaccine strategy for vaccines might generate protective responses. However, it is unknown HIV. In blue are the key issues corresponding to each step that influence our whether vaccine-induced elicitation of such antibodies can counter capacity to immunize against HIV. enormous and growing HIV quasispecies diversity and prevent initial infection on exposure. So far, vaccine-elicited antibody responses have focused on CD8 T-cell responses7,8 are unlikely to succeed without not been protective and the form of the virus (free versus intracellular) antibody and HIV-specific CD4 T-cell responses9,18. Even if we knew that spreads is unknown (Fig. 1) so that epitopes exposed during what we wanted to induce in a vaccine, the science of engineering natural transmission are undefined. Moreover, the envelope glyco- specific immune responses to exceed the capacity of the natural protein is very difficult to produce in its folded trimeric form; this immune response is in its infancy. A key step in this important may be required to provide an effective immunogen. Furthermore, direction will be to define relationships and potential synergies because HIV spread between cells may occur by means of multiple between CD4 T-cell, CD8 T-cell, antibody and B-cell responses mechanisms, including virological synapse-mediated coupled to viral endocytosis, it is not clear that antibody will be able during vaccination and in situations in which long-term viral control 27 is achieved in infected people. to inhibit all forms of viral spread in vivo . As only a tiny subset of an infected person’s HIV quasispecies The case for re-embracing CD4 T cells transmits6, the relationship between the diversity of sequences of the surface glycoprotein gp120 worldwide and appropriate vaccine There is much controversy concerning the role of HIV-specific CD4 epitopes is unknown, but it is possible that much of this diversity is T cells in control versus promotion of HIV replication. HIV repli- irrelevant to immunogen design28,29. Recent results from large popu- cates best in activated CD4 T cells, there is evidence for lation screening studies have revealed the presence of novel broadly selective infection of HIV-specific CD4 T cells by HIV19, and a cor- neutralizing antibodies in some infected people, and provide important relation between SIV recrudescence and CD4 T-cell activation after 20 new avenues for antigen design through targeting these epitopes that depletion of CD8 T cells has been reported . Such observations are subdominant or entirely unrecognized in the majority of infected support concerns that induction of a large population of HIV- persons (for example, see refs 28, 30). Reasonable levels of one of the immune CD4 T cells might enhance rather than control HIV infec- few available broadly neutralizing monoclonal antibodies blocks low- tion. This view is countered by overwhelming evidence that CD4 T dose intravaginal challenge with SIV using the HIV gp120 (SHIV)31,32, cells are essential for antiviral responses in vivo including CD8 T-cell and encouraging recent studies show that complete protection from responses and effective B-cell responses in every system in which it SIV challenge has been achieved by adeno-associated virus (AAV)- has been studied (for example, see refs 21–23 and references therein). mediated continuous endogenous expression of immunoadhesins For example, CD8 T cells do not efficiently access mucosal sites to derived from neutralizing antibodies33. 18 combat viral infection without CD4 T-cell help , and the outcome of Even if protective B-cell epitopes are found, the mechanisms retroviral infection depends critically on speed and extent of virus- responsible for antibody action in vivo need to be much better under- 24 specific CD4 T-cell responses . The basic rules of immunology there- stood to foster induction of a polyfunctional humoral response fore indicate that it will be impossible to generate long-lived, stable, (Fig. 3)30,34,35,. Meeting this need is complicated by the lack of sur- high-level vaccine-induced immunity to HIV without inducing a rogate in vitro assays, other than virus neutralization, for protection. protective CD4 T-cell response, but most HIV vaccine strategies have We may be missing epitopes that generate antibodies that protect not specifically focused on inducing these responses. through antibody-dependent cell-mediated cytotoxicity, complement Current data strongly suggest that HIV-specific CD4 T cells are fixation, or the capacity to block infection without neutralizing, as beneficial rather than detrimental. The initial burst of HIV and SIV observed for other viruses (see refs 36–39 and references therein). replication during acute infection does not occur in HIV- or SIV- Antibodies may also be important through interaction with Fc recep- specific CD4 T cells6, and whereas infection of these cells could con- tors on granulocytes, , dendritic cells, or B cells to alter ceivably add fuel to this early fire, a strong effector-memory CD4 T-cell antigen presentation, tropism, or replication in vivo36. Furthermore, response is associated with diminished, not enhanced, SIV replication B cells are more than simply factories for making antibody, as B cells after intrarectal challenge9. Moreover, although HIV preferentially unable to make antiviral antibody exert T-cell-dependent control of infects HIV-specific CD4 cells, the overall magnitude of this increase chronic virus infection in mice40. Efficient antibody-mediated control is small, and the great majority of HIV-specific CD4 T cells are not of Friend leukaemia retrovirus requires CD4 and CD8 T-cell responses infected in vivo, even in the presence of high-level viraemia19.Blockade and is regulated by specific major complex (MHC) of CD4 T-cell activation after CD8 T-cell depletion using anti- alleles37,41. This may be particularly relevant to AIDS as specific MHC (IL)-15 antibody in SIV-infected macaques has no effect alleles are associated with more effective immune control of HIV 226 ©2010 Macmillan Publishers Limited. All rights reserved NATUREjVol 464j11 March 2010 PERSPECTIVES

Can anybody prevent infection? Figure 3 | Immune responses to HIV and outstanding questions related to vaccination. Can the envelope be expressed as Is neutralization the only relevant an antigen in authentic form? assay for protective antibody Shown are the steps in the immune response to responses to HIV? HIV. For each component of the immune How do we generate broadly response, colour-matched ‘knowable unknowns’ Can the initial conditions of vaccination: cross-protective antibodies? are specified which, when defined, may alter how Enhance mucosal response? Antibody Enhance memory? Would a strong antibody response we approach vaccination against HIV. Deal with HIV diversity? improve the efficacy of T cells? Generate effector cells present constantly? How do we optimize antibody Prevent T-cell or B-cell exhaustion? responses at mucosal surfaces Generate relevant effector mechansims? where they are needed?

Essential help Can the immune system clear established infection by targeting replication and latently infected cells? APC CD4 Target T cell cell Are there undiscovered immune effector functions relevant to HIV? CD4?C CD8? Antibody? Innate immunity? Essential help Is it possible to target T cell and antibody Can APCs be altered epitopes that extract a high cost in viral or targeted to optimize fitness when they mutate? effective T-cell responses? CD8 T cell Can the most potent antiviral CD8 T-cell responses be defined? Are T-cell mechanisms operative to control set point relevant correlates of Would pre-exisiting immune CD4 T cells protection for vaccine development? help or hurt immune control of HIV infection?

How can we generate the optimal CD4 helper response for protective antibody and CD8 responses? How do we get immune T cells to the Are there undiscovered mechanisms mucosal surfaces where they are needed? of T-cell antigen recognition? infection. Unexplored interactions between B cells, antibody res- the first critical hours of infection in the intestine or genitourinary ponses and T cells are knowable unknowns that are probably critical tract (Fig. 1 and ref. 6). A major opportunity to engineer vaccine to HIV vaccine strategies (Fig. 3). responses comes from studies in mice and non-human primates A major future challenge will be converting information on pro- showing that the immune system is dampened during persistent tective epitopes and antibody-mediated mechanisms of protection virus infection, generating ‘exhausted’ T cells to prevent tissue into protective vaccine antigens. Antibodies to HIV often target damage (reviewed in ref. 22). The key observation for vaccine design accessible gp120 epitopes that can mutate without fitness cost rather is that manipulation of the mechanisms responsible for exhaustion at than subdominant epitopes that may be more conserved and thus the time of first antigen exposure can fundamentally alter vaccine more targetable. A fundamental question is how to generate CD4 responses. T-helper cell activity for vaccine antibody responses to potentially Exhausted cells express downregulatory receptors, lack multiple protective subdominant epitopes. One approach to generating effector functions, and show weak proliferative responses. Exhaus- protective responses to subdominant epitopes is tion is best studied in T cells, but may also affect B cells50,51. For T cells, with viral vectors that express blocking antibody-like molecules33. exhaustion is mediated by factors including FOXP3-positive regula- Alternatively, the HIV glycoprotein may have to be engineered to tory T (Treg) cells, inhibitory receptors such as PD1 and CTLA4 and optimize responses to cross-protective subdominant epitopes. others22,48, and such as IL-10 (refs 52, 53) or transforming growth factor-b54. In mice infected with lymphocytic choriomenin- CD8 T cells are important yet insufficient gitis virus (LCMV) recent data show that IL-21, perhaps made by The failure of the first T-cell HIV vaccine in humans2,3 indicates that CD4 T cells, prevents exhaustion55–57. Blockade of IL-10 or PD1 more needs to be understood about the breadth, specificity and quality increases control of chronic LCMV and or SIV infection52,58,59. This of protective CD8 responses to HIV to optimize vaccine design is relevant to vaccine strategies as control of chronic LCMV infection (Fig. 3). Targeting of Gag epitopes is associated with lower viral loads is enhanced by either blockade of IL-10 during therapeutic DNA than targeting Env, a finding that needs to be evaluated in SIV models vaccination52 or blockade of PD1 during vaccinia vector vaccina- to assess whether this should inform vaccine design42. Some SIV- and tion60. These data support linking studies of immunoregulatory HIV-specific T cells are surprisingly ineffective at killing virus-infected mechanisms operating during chronic infection to vaccination targets43,44, and the relevance of killing to the protective activity of CD8 efforts. IL-10 and PD1 inhibit CD8 responses by independent T cells in vivo remains unknown. Immunoregulatory networks that mechanisms; simultaneous blockade of both may be advantageous53. exhaust adaptive CD4 and CD8 responses during chronic infection New data show that administration of the ‘immunosuppressive’ probably contribute to CD8 ineffectiveness45–48. Studies using assays rapamycin counterintuitively enhances memory CD8 T-cell differ- that more closely replicate the in vivo situation in which viral antigens entiation61, indicating that pharmacological manipulations at the time are processed and presented by primary cells rather than being pro- of vaccination may enhance differentiation of induced T or B cells. vided as synthetic peptides at supra-physiological doses are urgently Administration of antigen in specific contexts such as via dendritic needed, along with high-throughput single-cell analysis of T-cell phe- cells may induce immune responses that can enhance chronic notypes and gene expression, to define differences between effective immune control of HIV62. This striking finding indicates that similar and ineffective T-cell responses49. approaches to vaccination might induce immune responses that either eliminate the initial nidus of HIV infection or limit replication Tuning the immune system to fight AIDS and thus control set point viraemia. Similarly, one might argue to To vaccinate against HIV we need to induce B and T cells that have block induction of Treg cells during vaccination. However, counter- and retain very specific differentiated properties allowing them to intuitively, elimination of Treg cells can blunt the mucosal immune control replication of established infection and to function during response and control of vaginal infection with herpes simplex virus, a 227 ©2010 Macmillan Publishers Limited. All rights reserved PERSPECTIVES NATUREjVol 464j11 March 2010 finding potentially related to altering the balance between mucosal of correlates of HIV immunity, are yet to be defined. Translocated and lymphatic responses63. This provides a cautionary note as one intestinal products may not be the entire cause of systemic immune may not be able to translate all mechanisms of chronic control of HIV activation. It has been presumed that standard diagnostic tests rule to the vaccination effort. Another major issue is how to use adjuvants, out a role for pathogens in HIV- and SIV-related enteropathy, but which may, in addition to enhancing the overall immunogenicity of this is not tenable as metagenomic analyses reveal many novel enteric an antigen, be used to direct and optimize the differentiation of cells viruses in humans and primates (for example, see refs 73, 74) whose responding to vaccine antigens. Here again are knowable unknowns: role in HIV/AIDS has not been assessed. Furthermore, monkeys it is important to determine how to engineer augmented vaccine harbour widely variant populations of intestinal bacteria that may responses via manipulation of immunoregulatory pathways during influence progression to AIDS and systemic vaccine responses75. The vaccination (Fig. 3). virome and microbiome of the host have profound effects on the nature of the mucosal and systemic immune response to new patho- Battling HIV in mucosal tissues gens, including the basal level of activation of innate immune cells and HIV delivers a near-lethal hit to the host immune system by killing a the development of T-cell subtypes in the intestinal mucosa22,69,76. huge proportion of T cells in the (ref. 6 and Future studies of immune cell function in HIV-infected individuals Fig. 1), and persistent viraemia may feed off of systemic immune and of HIV vaccine responses need to be designed while bearing in activation associated with mucosal damage during HIV infection. mind the potential role of the microbiome and virome in determining Mucosal events may contribute to HIV and SIV progression, vaccine responses. variation in vaccine responses, and variations in measurements of surrogate markers of protection. Continuous activation of mucosal Exploiting evolution in a vaccine trap HIV-immune cells may be required for protective vaccine responses HIV sequences have tremendous diversity; circulating HIV isolates given the speed of mucosal HIV pathogenesis6. The immune effector exhibit up to 35% sequence variation in gp120 (ref. 77) and tremendous mechanisms that target HIV in the mucosa are unknown, and rela- variability is generated in an individual after HIV passes through the tionships between mucosal and systemic immunity are poorly under- transmission bottleneck6,13,78. The HIV quasispecies probably has stood, leading to uncertainty over the benefits of mucosal versus pathogenetic properties independent of the capacity to mutate to evade systemic vaccination. Tissue-specific immune responses have a key immunity. For example, studies show that a quasispecies can role in control of viral infections22,64,65, and thus differences among confer pathogenetic properties such as neurotropism and virulence the vagina, penis, intestine and oropharynx might determine vaccine during viral pathogenesis79. efficacy. Furthermore, emerging evidence from humans and mice The tremendous variability of HIV also exposes some weaknesses. suggests that specific mechanisms maintain local immunity in skin In simultaneously infected adult identical twins, the earliest CD8 and genital tissues64,65. As studies of HIV or SIV immunity typically epitope escape mutations arose at the same nucleotide in both indi- analyse peripheral blood cells, we may be working from an inaccurate viduals, indicating that there are constraints on HIV evolution, and view of immunity to HIV and SIV. An increased focus on the role of that some escape pathways are detrimental to viral fitness80. Popula- mucosal events in HIV vaccination is therefore needed. tion studies show predictable pathways to escape within epitopes Although appropriate emphasis has been placed on local res- presented by specific HLA alleles81,82. These mutations, particularly ponses, there is nevertheless convincing evidence of effective induc- in Gag, can impair viral fitness83,84. It is perhaps not surprising that tion of mucosal responses to enteric murine norovirus infection and CD8 T cells targeting epitopes in Gag are associated with lower viral human papilloma virus after subcutaneous vaccination66,67, for load than CD8 T cells targeting the more mutable gp120 (ref. 42). mucosal protection against SIV after subcutaneous vaccination with Part of the role of HLA B*57 in people able to control HIV replication a CMV-based vector9, and for generation of substantial mucosal to undetectable levels without (‘elite controllers’) seems responses after systemic immunization in mice and macaques68. to be generation of fitness-impairing epitope mutations that cannot Thus, systemic vaccination can elicit effective mucosal immunity, easily be compensated83,85. Such CD8 T-cell-induced mutations and systemic immunoglobulin-G (IgG) responses, rather than associated with lower viral load can revert after transmission, pro- mucosal IgA responses, may be a reasonable vaccine goal. If lympho- viding in vivo evidence of impaired fitness86,87. The ability to define cytes with specific differentiated properties are essential for mucosal the fitness landscape of HIV now exists, and this information could immunity to HIV they must be better defined to optimize systemic have a significant impact on vaccine development. induction of mucosal immunity. The mucosal immune responses Careful observation of HIV evolution under selective pressure in associated with continuous antigen and potential adjuvant effects69 vivo also has the potential to provide key new insights into the nature of live vaccine vectors such as cytomegalovirus9 or live attenuated of potent immune responses. Clear signatures of selection have been SIV70, and with the immunization scheme used in the Thai vaccine identified that are not accounted for by known CD4 or CD8 T-cell, or trial, need to be defined. B-cell, epitopes82; known epitopes do not explain all CD4 and CD8 One of the key sources of data for immune correlates of HIV T-cell responses observed88. CD8 T-cell control of chronic infection immunity that might be converted into vaccine strategies is the nature with herpesviruses and polyomaviruses can involve non-classical of immunity to HIV during chronic infection (Fig. 2). Mucosal- MHC molecules, and similar responses have been reported in damage-associated translocation of intestinal contents, or immune- humans22. Thus, there are additional epitopes, and also potentially suppression-associated intestinal infection, may lead to systemic other mechanisms of innate or adaptive immune control, that await immune activation that fosters HIV disease progression16,71,. Further- discovery. more, SIV-infected non-progressors show fewer signs of intestinal These findings define a weakness in HIV’s most potent defence, the barrier dysfunction than progressors16. Studies in mice deficient for capacity to mutate, and indicate the potential for effective immuni- innate immune genes also show that alterations in mucosal barrier zation via an ‘evolutionary trap’, driving HIV into a fitness dead end function result in systemic activation of adaptive immunity72. by targeting epitopes in fitness-critical regions of viral proteins. Mucosal damage is therefore a new factor to be considered with Experiments in both HIV and in animal models in which immune regards to immune effects of HIV infection, and might well alter pressure on viruses can be easily controlled will be needed to develop measurements of HIV-immune cells or antibody and thus contribute the methods for evolutionary trap vaccination. This will require to false conclusions about the effects of HIV on immunity, and vice integration of computational biology, evolutionary biology and versa, during chronic infection. high-throughput sequencing to determine the sequence space within The antigens, pathogens, or microbial products that induce sys- which a ‘fit’ virus must reside. Full-length genomes will have to be temic immune activation, and may therefore complicate assessment analysed to define escape mutations and linkages between these 228 ©2010 Macmillan Publishers Limited. All rights reserved NATUREjVol 464j11 March 2010 PERSPECTIVES mutations and intragenic and extragenic suppressor mutations that additional animal models in which mice are adapted to HIV98,or restore fitness. Studies have already shown that immune-induced HIV is adapted to additional primates99. mutations in gp120 are more frequent yet less likely to confer a fitness cost to HIV84 than those in Gag that impair fitness83,84. However, even Tough choices ahead then compensatory mutations can sometimes rapidly restore viral There is hope for HIV vaccination. There are many knowable fitness89. Combining an evolutionary trap with mosaic vaccines based unknowns (Figs 1 and 3) that can guide this effort, and there is much on computationally derived sequences representing maximal coverage scientific knowledge yet to be applied. However, there are also major of HIV diversity is an attractive approach90. scientific challenges that must be met to optimize chances of generating an effective vaccine. Although a successful vaccine might be around the Weapons deployed by vaccination against HIV corner in the next human trial, instead the path to an effective vaccine In the end, inducing immune responses to HIV will be effective only if might be many years long and require novel discoveries and insights potent effector mechanisms are deployed against the virus. Because into the functioning of the immune system. Thus, the scientific issues viral integration and establishment of a pool of latently infected cells addressed in this Perspective are best considered in the broader context occurs very rapidly after transmission (Fig. 1 and ref. 6), these res- of how to structure the research effort to ensure success. Because many ponses will have to be present continually or arise extremely rapidly to of the basic immunological issues that need to be solved to conquer prevent establishment of chronic infection. Unfortunately, we still do AIDS are similar to those for C, , malaria, tuber- not know what an effective immune response to HIV includes, and culosis and , enhancing basic understanding of HIV immunity therefore are compromised in our quest for a vaccine (Figs 1–3). will have broad impacts. Calls for a greater focus on basic science to Recent focus has been on inducing polyfunctional T-cell responses facilitate HIV vaccine research were expressed 16 years ago101. We, and as a surrogate for effective responses, but which functions are truly many others102, make the same call today, as the kind of effort com- relevant remains unknown. Significant effort needs to be directed to mensurate with the problem is far from being realized. defining vaccine-relevant effector mechanisms that can eliminate the So what are the barriers, in addition to the complexity of the initial nidus of HIV infection, prevent systemic spread from an initial immunology, to rapid progress in basic science relevant to HIV vac- localized mucosal infection, or block the establishment of latency. The cination? Most important among these barriers, and most controversial prevention of latency might enhance vaccination by preventing the because it has important practical, philosophical, political, scientific virus from accessing a quiescent state of infection invisible to the and financial implications, is the separation of most HIV-related immune system (see below). In this area, particular emphasis needs research from research in other areas of immunology and virology. to be placed on defining novel mechanisms as currently known Considering the many issues that need to be solved to give us the best effector responses do not consistently correlate with control of HIV. long-term chance of effective HIV vaccination (for example, Figs 1 and Clues to potential novel immune effector mechanisms come from 3), it becomes clear that not all of the answers can efficiently come from analysing the complex cellular factors that promote or restrict HIV studies of HIV. The history of research on HIV immunology is filled replication91,92. HIV has evolved mechanisms to thwart many host with paradigm shifts based on studies of viruses and species commonly proteins that limit viral replication. The importance of defining the defined as not AIDS related. Examples include T-cell exhaustion, Treg relationship between immune evasion strategies, host restriction and cells, PD1, CTLA4, inhibitory cytokines, and many others. Many were immunity is shown by the recent demonstration that Rfv3, which first defined in mice by studying LCMV, a virus unrelated to HIV, and determines the effectiveness of antibody against Friend retrovirus many have taken years to translate from murine to human studies. in mice, is encoded by Apobec3, which encodes a protein that is a Studies of other human pathogens are also highly relevant. For homologue of human APOBEC3G and APOBEC3F proteins targeted example, systems-based studies of human vaccines that do work, such by HIV vif 93–95. The task we face is defining mechanisms that are not as the yellow virus vaccine, provide new genes and pathways, effectively blocked by HIV and which can be deployed by vaccine- some entirely unexpected, that may contribute to highly functional generated immune cells. Cytotoxic killing of an infected cell requires CD8 T-cell and B-cell vaccine responses49. Studies of cytomegalovirus recognition that occurs optimally only after HIV is integrated into led to the development of an HIV vaccine approach effective against the genome and proteins are expressed. Breaking the cycle of replica- mucosal challenge9. There is no debate that HIV vaccine research has tion and latency may therefore require different or additional benefitted significantly from research in other areas, and vice versa. The mechanisms, in particular those that block early events in HIV patho- question is how best to move forward to realize the great potential genesis (Fig. 1 and ref. 6). offered by integrating currently isolated efforts. One potential weapon that might be used against HIV would be We need, as a scientific community, to break down barriers prevention or regulation of latency. It is possible that the capacity of between the AIDS effort and other relevant areas of science to access HIV to enter an immunologically silent latent state in which the previously untapped human and technological resources for HIV provirus is present but antigenic proteins are not expressed early after vaccination. But this needs to be done in context, such that those infection of the mucosal surface could be responsible for vaccine with expertise from other fields are able to effectively tap into the failures or inefficient vaccination. Thus, immune responses that pre- wealth of current knowledge on HIV pathogenesis. It is not enough to vent the establishment of latency might enhance vaccine effective- simply funnel more funding to current AIDS research efforts. The key ness. Latent reservoirs of HIV and SIV are incompletely defined96, point here is that there is no lack of enthusiasm among scientists and this knowledge is critical to the vaccine effort. Reactivation of outside the HIV field to get involved in HIV/AIDS related research; HIV from latency is regulated by multiple overlapping factors includ- rather there is merely a lack of a way in. The study section system and ing chromatinization of the provirus, methylation, T-cell activation, foundation efforts should be reformed with this principle in mind. NFkB and cytokines including tumour-necrosis factor-a97–99. Studies in multiple systems, especially those that define novel Notably, the immune system can regulate the nature of latency in cells mechanisms and pathways related to chronic viral infection, should infected with herpesviruses100, and thus the nature of latency is not be linked to ongoing HIV efforts and pursued with urgency propor- necessarily cell intrinsic—a potentially important clue that needs to be tionate to the severity of the global AIDS crisis and under the AIDS exploited. Latency in macrophages or other cells that may have a role umbrella. in persistent infection is less well understood than in T cells. Therefore, We propose that AIDS-related research be redefined to promote it is not known whether vaccine-induced effector mechanisms should the integrated study of all mechanisms that may facilitate under- target HIV in macrophages or dendritic cells, in addition to T cells. A standing HIV pathogenesis and immunity. The HIV/AIDS problem further profound advantage of defining immune and cell-intrinsic is too severe to allow the relevant science to be restricted by policies control of latency in more detail could be the development of that separate disciplines that could have a real impact on this deadly 229 ©2010 Macmillan Publishers Limited. All rights reserved PERSPECTIVES NATUREjVol 464j11 March 2010 human pathogen. Although we must, of course, study vaccine can- 22. Virgin, H. W., Wherry, E. J. & Ahmed, R. Redefining chronic viral infection. 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